ABCMdb

ABCC7 p.Arg560Thr

Admin's notes:	Class II (maturation defect) Veit et al.
ClinVar:	c.1680A>C , p.Arg560Ser ? , not provided c.1679G>A , p.Arg560Lys D , Pathogenic c.1678A>G , p.Arg560Gly ? , not provided c.1679G>C , p.Arg560Thr D , Pathogenic
CF databases:	c.1680A>C , p.Arg560Ser D , CF-causing ; CFTR1: This mutation was identified by DGGE and direct sequencing. c.1679G>C , p.Arg560Thr D , CF-causing c.1679G>A , p.Arg560Lys D , CF-causing ; CFTR1: The nucleotide change was identified once among 87 non-[delta]F508 chromosomes. tTHe patientis a compound heterozygote with 1717-1G->A on the other chromosome. She is 12 years old, with at this time a mild form of the disease. The mutation abolishes a HphI site in exon 11. The mutatee allele is 425 bp and the normal is 211 +214 after hphI digestion but the enzyme digestion identifies both the R560T and R560K. The R560K is a missense mutation and also probably a splice mutation because that position subsitutes AA for AG immediately up stream of the splice acceptor site, postition that has been reported to alter splicing (VIDAUD, PNAS, 86, 1041-1045). c.1678A>G , p.Arg560Gly (CFTR1) ? , This change has been detected by DGGE analysis and direct sequencing. The mutation creates a Mnl I restriction site
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: N (66%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), S: D (59%), T: D (53%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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II (maturation defect) <a href="https://www.ncbi.nlm.nih.gov/pubmed/26823392">Veit et al.</a>
admin on 2016-08-19 15:16:22

Publications
[hide] ABCC6/MRP6 mutations: further insight into the mol... Eur J Hum Genet. 2003 Mar;11(3):215-24. Hu X, Plomp A, Wijnholds J, Ten Brink J, van Soest S, van den Born LI, Leys A, Peek R, de Jong PT, Bergen AA
ABCC6/MRP6 mutations: further insight into the molecular pathology of pseudoxanthoma elasticum.
Eur J Hum Genet. 2003 Mar;11(3):215-24., [PMID:12673275]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a hereditary disease characterized by progressive dystrophic mineralization of the elastic fibres. PXE patients frequently present with skin lesions and visual acuity loss. Recently, we and others showed that PXE is caused by mutations in the ABCC6/MRP6 gene. However, the molecular pathology of PXE is complicated by yet unknown factors causing the variable clinical expression of the disease. In addition, the presence of ABCC6/MRP6 pseudogenes and multiple ABCC6/MRP6-associated deletions complicate interpretation of molecular genetic studies. In this study, we present the mutation spectrum of ABCC6/MRP6 in 59 PXE patients from the Netherlands. We detected 17 different mutations in 65 alleles. The majority of mutations occurred in the NBF1 (nucleotide binding fold) domain, in the eighth cytoplasmatic loop between the 15th and 16th transmembrane regions, and in NBF2 of the predicted ABCC6/MRP6 protein. The R1141X mutation was by far the most common mutation identified in 19 (32.2%) patients. The second most frequent mutation, an intragenic deletion from exon 23 to exon 29 in ABCC6/MRP6, was detected in 11 (18.6%) of the patients. Our data include 11 novel ABCC6/MRP6 mutations, as well as additional segregation data relevant to the molecular pathology of PXE in a limited number of patients and families. The consequences of our data for the molecular pathology of PXE are discussed.

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128 Further alignment showed that the R765Q mutation in ABCC6/MRP6 is the positional equivalent of both the R560T mutation in ABCC7,28 and the R842G mutation in ABCC8.29 Similarly, additional possible positional equivalent clusters of conserved and mutated residues were found between ABCC6/ MRP6 and ABCC2 (R1114H and R1150H),30 ABCC6/MRP6 and ABCC7 (3775 del T and W1204X),31 ABCC6/MRP6 and ABCR (R1459C and H2128R, 4220InsAGAA and R2077W, R1141X and L1631P).32,33 Interestingly, for both ABCC7 and ABCR, models were postulated in which the severity of the disease shows an inverse correlation with the predicted transport activity of the ABC protein. Login to comment

[hide] Molecular genetics of pseudoxanthoma elasticum: ty... Hum Mutat. 2005 Sep;26(3):235-48. Miksch S, Lumsden A, Guenther UP, Foernzler D, Christen-Zach S, Daugherty C, Ramesar RK, Lebwohl M, Hohl D, Neldner KH, Lindpaintner K, Richards RI, Struk B
Molecular genetics of pseudoxanthoma elasticum: type and frequency of mutations in ABCC6.
Hum Mutat. 2005 Sep;26(3):235-48., [PMID:16086317]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder that affects the elastic tissue in the skin, eye, and cardiovascular system. Mutations in the ABCC6 gene cause PXE. We performed a mutation screen in ABCC6 using haplotype analysis in conjunction with direct sequencing to achieve a mutation detection rate of 97%. This screen consisted of 170 PXE chromosomes in 81 families, and detected 59 distinct mutations (32 missense, eight nonsense, and six likely splice-site point mutations; one small insertion; and seven small and five large deletions). Forty-three of these mutations are novel variants, which increases the total number of PXE mutations to 121. While most mutations are rare, three nonsense mutations, a splice donor site mutation, and the large deletion comprising exons 23-29 (c.2996_4208del) were identified as relatively frequent PXE mutations at 26%, 5%, 3.5%, 3%, and 11%, respectively. Chromosomal haplotyping with two proximal and two distal polymorphic markers flanking ABCC6 demonstrated that most chromosomes that carry these relatively frequent PXE mutations have related haplotypes specific for these mutations, which suggests that these chromosomes originate from single founder mutations. The types of mutations found support loss-of-function as the molecular mechanism for the PXE phenotype. In 76 of the 81 families, the affected individuals were either homozygous for the same mutation or compound heterozygous for two mutations. In the remaining five families with one uncovered mutation, affected showed allelic compound heterozygosity for the cosegregating PXE haplotype. This demonstrates pseudo-dominance as the relevant inheritance mechanism, since disease transmission to the next generation always requires one mutant allelic variant from each parent. In contrast to other previous clinical and molecular claims, our results show evidence only for recessive PXE. This has profound consequences for the genetic counseling of families with PXE.

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290 R760W in ABCC6 aligns with R768W in ABCC2, and R765Q in ABCC6 is comparable with R560T in ABCC7. Login to comment

[hide] Proportion of cystic fibrosis gene mutations not d... JAMA. 1999 Jun 16;281(23):2217-24. Mak V, Zielenski J, Tsui LC, Durie P, Zini A, Martin S, Longley TB, Jarvi KA
Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia.
JAMA. 1999 Jun 16;281(23):2217-24., 1999-06-16 [PMID:10376575]

Abstract [show]
CONTEXT: Infertile men with obstructive azoospermia may have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, many of which are rare in classic cystic fibrosis and not evaluated in most routine mutation screening. OBJECTIVE: To assess how often CFTR mutations or sequence alterations undetected by routine screening are detected with more extensive screening in obstructive azoospermia. DESIGN: Routine screening for the 31 most common CFTR mutations associated with the CF phenotype in white populations, testing for the 5-thymidine variant of the polythymidine tract of intron 8 (IVS8-5T) by allele-specific oligonucleotide hybridization, and screening of all exons through multiplex heteroduplex shift analysis followed by direct DNA sequencing. SETTING: Male infertility clinic of a Canadian university-affiliated hospital. SUBJECTS: Of 198 men with obstructive (n = 149) or nonobstructive (n = 49; control group) azoospermia, 64 had congenital bilateral absence of the vas deferens (CBAVD), 10 had congenital unilateral absence of the vas deferens (CUAVD), and 75 had epididymal obstruction (56/75 were idiopathic). MAIN OUTCOME MEASURE: Frequency of mutations found by routine and nonroutine tests in men with obstructive vs nonobstructive azoospermia. RESULTS: Frequency of mutations and the IVS8-5T variant in the nonobstructive azoospermia group (controls) (2% and 5.1% allele frequency, respectively) did not differ significantly from that in the general population (2% and 5.2%, respectively). In the CBAVD group, 72 mutations were found by DNA sequencing and IVS8-5T testing (47 and 25, respectively; P<.001 and P = .002 vs controls) vs 39 by the routine panel (P<.001 vs controls). In the idiopathic epididymal obstruction group, 24 mutations were found by DNA sequencing and IVS8-5T testing (12 each; P=.01 and P=.14 vs controls) vs 5 by the routine panel (P=.33 vs controls). In the CUAVD group, 2 mutations were found by routine testing (P=.07 vs controls) vs 4 (2 each, respectively; P=.07 and P=.40 vs controls) by DNA sequencing and IVS8-5T testing. The routine panel did not identify 33 (46%) of 72, 2 (50%) of 4, and 19 (79%) of 24 detectable CFTR mutations and IVS8-5T in the CBAVD, CUAVD, and idiopathic epididymal obstruction groups, respectively. CONCLUSIONS: Routine testing for CFTR mutations may miss mild or rare gene alterations. The barrier to conception for men with obstructive infertility has been overcome by assisted reproductive technologies, thus raising the concern of iatrogenically transmitting pathogenic CFTR mutations to the progeny.

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28 Analysis for 31 of the most common CFTR mutations found within the white CF population,60 consisting of ⌬F508, W1282X, G542X, G551D, N1303K, R553X, G85E, R117H, S549N, V520F, R334W, A455E, R347P, R1162X, Y122X, S549R, 621+1G→T, ⌬I507, R560T, R347H, 3659delC, Q493X, 1898+1G→T, 711+1G→T, 3849+10C→T, 1717-1G→A, 3849+4A→G, 3905insT, 1078delT, 2183AA→G, and 2789+5G→A. Briefly, the technique involved amplification by polymerase chain reaction61 of the relevant exons, followed by digestion with appropriate restriction endonucleases and acrylamide gel electrophoresis with ethidium bromide staining. Login to comment

[hide] Two buffer PAGE system-based SSCP/HD analysis: a g... Eur J Hum Genet. 1999 Jul;7(5):590-8. Liechti-Gallati S, Schneider V, Neeser D, Kraemer R
Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease.
Eur J Hum Genet. 1999 Jul;7(5):590-8., [PMID:10439967]

Abstract [show]
The large size of many disease genes and the multiplicity of mutations complicate the design of an adequate assay for the identification of disease-causing variants. One of the most successful methods for mutation detection is the single strand conformation polymorphism (SSCP) technique. By varying temperature, gel composition, ionic strength and additives, we optimised the sensitivity of SSCP for all 27 exons of the CFTR gene. Using simultaneously SSCP and heteroduplex (HD) analysis, a total of 80 known CF mutations (28 missense, 22 frameshift, 17 nonsense, 13 splicesite) and 20 polymorphisms was analysed resulting in a detection rate of 97.5% including the 24 most common mutations worldwide. The ability of this technique to detect mutations independent of their nature, frequency, and population specificity was confirmed by the identification of five novel mutations (420del9, 1199delG, R560S, A613T, T1299I) in Swiss CF patients, as well as by the detection of 41 different mutations in 198 patients experimentally analysed. We present a three-stage screening strategy allowing analysis of seven exons within 5 hours and analysis of the entire coding region within 1 week, including sequence analysis of the variants. Additionally, our protocol represents a general model for point mutation analysis in other genetic disorders and has already been successfully established for OTC deficiency, collagene deficiency, X-linked myotubular myopathy (XLMTM), Duchenne and Becker muscular dystrophy (DMD, BMD), Wilson disease (WD), Neurofibromatosis I and II, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, and defects in mitochondrial DNA. No other protocol published so far presents standard SSCP/HD conditions for mutation screening in different disease genes.

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20 The distribution of analysed known mutations is similar to that of the total number of mutations in the entire CFTR gene: missense mutations account for 35% (G27E, G85E, R117H, A120T, I148T, H199Y, R334W, T338I, R347P, R347H, A455E, M718K, S5449N, S5449I, G551D, R560T, R560S, S945L, S977P, I1005R, R1066C, R1070Q, M1101K, D1152H, S1235R, R1283M, N1303K, N1303H), followed by 28% of frameshift mutations (175delC, 394delTT, 457TAT- > G, 905delG, 1078delT, I507, F508, 1609delCA, 1677delTA, 2143delT, 2176insC, 218delA, 2184insA, 2869insG, 3659delC, 3732delA, 3821delT, 3905insT, 4016insT, 4172delGC, 4382delA), 21% of nonsense mutations (Q30X, Q39X, Q220X, W401X, Q525X, G542X, Q552X, R553X, V569X, E585X, K710X, R792X, Y1092X, R1162X, S1255X, W1282X, E1371X), and 16% of splice site mutations (621 + 1G- > T, 711 + 1G- > T, 711 + 5G- > A, 1717-1G- > A, 1898 + 1G- > A, 1898 + 5G- > T, 2789 + 5G- > A, 3271 + 1G- > A, 3272-26A- > G, 3601-17T- > C, 3849 + 4A- > G, 3849 + 10kbC- > T, 4374 + 1G- > T). Login to comment
34 Intron 19, all 27 exons and their exon-intron boundaries, including the 24 most common mutations worldwide (G85E, R117H, 621 + 1G- > T, 711 + 1G- > T, 1078delT, R334W, R347P, A455E, I507, F508, 1717-1G- > A, G542X, S549N, G551D, R553X, R560T, 1898 + 1G- > A, 2184delA, 2789 + 5G- > A, R1162X, 3659delC, 3849 + 10kbC- > T, W1282X, N1303K) (Cystic Fibrosis Genetic Analysis Consortium 1994), and the 15 most common mutations in our population (I148T, 1078delT, R334W, R347P, F508, 1717-1G- > A, G542X, R553X, 2347delG, D1152H, R1162X, 3849 + 10kbC- > T, 3905insT, W1282X, N1303K), were considered in this study. Login to comment
61 The slots C present wild type (wt) sequences, 1-8 present amplification products from CF patients with the following genotypes: 1 = R553X/R553X; 2 = 1717-1G- > A/wt; 3 = R553X/wt; 4 = G542X/wt; 5 = G542X/1717-1G- > A; 6 = G551D/wt; 7 = R560T/wt; 8 = S549N/wt. Login to comment

[hide] Analysis of 31 CFTR mutations by polymerase chain ... J Med Screen. 1999;6(2):67-9. Gasparini P, Arbustini E, Restagno G, Zelante L, Stanziale P, Gatta L, Sbaiz L, Sedita AM, Banchieri N, Sapone L, Fiorucci GC, Brinson E, Shulse E, Rappaport E, Fortina P
Analysis of 31 CFTR mutations by polymerase chain reaction/oligonucleotide ligation assay in a pilot screening of 4476 newborns for cystic fibrosis.
J Med Screen. 1999;6(2):67-9., [PMID:10444722]

Abstract [show]
OBJECTIVES: Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns. METHODS: The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA. RESULTS: 144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being delta F508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. CONCLUSIONS: PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).

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46 Table 1 Mutations analysed in the CFTR gene using polymerase chain reaction/oligonucleotide litigation assay/sequence coded separation Mutation Location Nucleotide Result F508 Exon 10 3 bp deletion Deletion of Phe-508 I507 Exon 10 3 bp deletion Deletion of Ile-507 (or -506) Q493X Exon 10 C-1609 →→ T Gln-493 → Stop V520F Exon 10 G-1690 → T Val-520 → Phe 1717-1G → A Intron 10 G-1717-1 → A 3`-splice site mutation G542X Exon 11 G-1756 → T Gly-542 → Stop G551D Exon 11 G-1784 → A Gly-551 → Asp R553X Exon 11 C-1789 → T Arg-553 → Stop R560T Exon 11 G-1811 → C Arg-560 → Thr S549R Exon 11 T-1779 → G Ser-549 → Arg S549N Exon 11 G-1778 → A Ser-549 → Asn 3849+10 kb C → T Intron 19 C-3849+10 kb → T Splice mutation 3849+4A → G Intron 19 A-3849+4 → G Splice mutation R1162X Exon 19 C-3616 → T Arg-1162 → Stop 3659delC Exon 19 1 bp deletion Frameshift W1282X Exon 20 G-3978 → A Trp-1282 → Stop 3905insT Exon 20 1 bp insertion Frameshift N1303K Exon 21 C-4041 → G Asn-1303 → Lys G85E Exon 3 G-386 → A Gly-85 → Glu 621+1G → T Intron 4 G-621+1 → T 5`-splice site mutation R117H Exon 4 G-482 → A Arg-117 → His Y122X Exon 4 T-498 → A Tyr-122 → Stop 711+1G → T Intron 5 G-711+1 → T 5`-splice site mutation 1078delT Exon 7 1 bp deletion Frameshift R347P Exon 7 G-1172 → C Arg-347 → Pro R347H Exon 7 G-1172 → A Arg-347 → His R334W Exon 7 C-1132 → T Arg-334 → Trp A455E Exon 9 C-1496 → A Ala-455 → Glu 1898+1G → A Intron 12 G-1898+1 → A 5`-splice site mutation 2184delA Exon 13 Deletion A-2184; A-2183 → G Frameshift 2789+5G → A Intron 14B G-2789+5 → A Splice mutation Table 2 Summary of cystic fibrosis screening results No of samples analysed Normal subjects Carriers Carrier frequency Turin 1574 1521 53 1/29.7 Pavia 1341 1299 42 1/31.9 San Giovanni Rotondo 1561 1512 49 1/31.8 Total 4476 4332 144 1/31.1 Table 3 Detailed list of mutations detected in the Italian population Centre F508 G542X R347P 2183-AG N1303K 711+1GT 1717-1A R347H R117H 1898+1G 2789+5G W1282X R1162X I507 Other TO 33 2 1 1 5 1 1 2 3 2 2 - - - PV 27 - - 1 2 - 1 - 5 - 1 2 1 1 SGR 30 14 2 1 1 1 - - - - - - - - TO, Dipartimento di Patologia Clinica, Ospedale Infantile "Regina Margherita, Torino; PV, Istituto di Anatomia Patologica, Sezione di Anatomia Patologica, Università di Pavia, Pavia; SGR, Servizio di Genetica Medica and Divisione di Neonatologia, IRCCS Casa Sollievo della SoVerenza, San Giovanni Rotondo, Foggia. Login to comment

[hide] Pulmonary outcome in cystic fibrosis is influenced... Infect Immun. 1999 Sep;67(9):4744-50. Parad RB, Gerard CJ, Zurakowski D, Nichols DP, Pier GB
Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype.
Infect Immun. 1999 Sep;67(9):4744-50., [PMID:10456926]

Abstract [show]
Whether allelic variants of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) independently contribute to pulmonary outcome in CF patients has not been resolved. We used both cross-sectional and mixed-model longitudinal analyses of data from CF patients that were at least 12 years old to determine the influence on pulmonary function (percent predicted forced expiratory volume [FEV(1)]) of the CFTR gene genotype, gender, mucoid Pseudomonas aeruginosa (MPA) infection status, presence of total opsonic antibody to MPA, and, separately, the opsonic antibody activity specific to the mucoid exopolysaccharide (MEP) surface antigen. Two different factors were independently associated with the lack of MPA infection: a high level of MEP-specific opsonic activity (MSOA), implicating an immunologically based mechanism of resistance to infection, and a lack of any type of opsonic antibody to MPA, indicative of no significant exposure or infection. This latter phenotype was found in a subset of CF patients who carried at least one uncommon CFTR gene allele suggestive of a genetic basis for resistance to infection in this group of older CF patients. For CF patients in whom both CFTR gene alleles were identified by screening for the 12 most common variants (75% of alleles), cross-sectional analysis showed that MPA infection was best correlated with lower percent predicted FEV(1), while genotype (two versus one DeltaF508 CFTR gene allele) and a low level of MSOA were associated with increased risk of infection. A mixed-model analysis of longitudinal spirometric measurements that considered multiple risk factors to derive regression equations was used to determine which clinical parameters had the greatest effect on the annual rate of decline in percent predicted FEV(1). This analysis showed that the CFTR gene genotype only modestly modified the constant (y intercept) of the derived equations, while gender and MPA infection status had the largest effects on annual rates of decline in percent predicted FEV(1). These results indicate that the CFTR genotype is usually not a primary determinant of pulmonary function in most CF patients, but gender and MPA infection status are. Infection status is potentially influenced by both immunologic (a high level of MSOA) and genetic factors, such as carriage of a CFTR gene allele that leads to a diagnosis of CF but still confers resistance to infection that is comparable to that of the wild-type CFTR gene.

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51 Genomic DNA isolated from each subject was evaluated for the presence of any of twelve CFTR gene mutations (⌬F508, G551D, G542X, 621ϩ1G3T, ⌬I507, 1717-1 G3A, R117H, N1303K, W1282X, R560T, R553X, and 3849ϩ10kb C3T) by one of three standard assays (10, 11, 32). Login to comment

[hide] Partial CFTR genotyping and characterisation of cy... Clin Genet. 2000 Jan;57(1):56-60. Zebrak J, Skuza B, Pogorzelski A, Ligarska R, Kopytko E, Pawlik J, Rutkiewicz E, Witt M
Partial CFTR genotyping and characterisation of cystic fibrosis patients with myocardial fibrosis and necrosis.
Clin Genet. 2000 Jan;57(1):56-60., [PMID:10733236]

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Myocardial necrosis and fibrosis is a rare complication of cystic fibrosis (CF) causing sudden and unexpected death in infancy due to cardiac arrest. Characteristic morphological lesions are recognisable postmortem. The 18 CF patients with this complication had varied clinical features including mild pulmonary involvement, early onset severe pancreatic insufficiency, and profound electrocardiogram (ECG) changes. In this group of patients, 5 were deltaF508 homozygotes, 1 was deltaF508/ N1303K and 1 was a deltaF508/M compound heterozygote. A pair of affected siblings (deltaF508 homozygotes) were fully concordant for myocardial involvement and for the general course of the disease. The co-existence of a genetic predisposition to myocardial lesions resulting most probably from severe cystic fibrosis transmembrane (CFTR) genotypes (such as deltaF508/deltaF508, deltaF508/N1303K) and deficiency of certain trophic factors necessary for metabolism of the myocardium, are postulated to cause myocardial complications in CF leading to circulatory failure and early death.

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59 The latter was negative for 14 other mutations: DI507, 1717-1GA, G542X, G551D, R553X, R560T, 3849+10kbCT, N1303K, W1282X, S549I, S549N, 621+1GT, 2789+5GA, R117H. Login to comment

[hide] Branch migration inhibition in PCR-amplified DNA: ... Nucleic Acids Res. 2000 May 1;28(9):E42. Lishanski A, Kurn N, Ullman EF
Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.
Nucleic Acids Res. 2000 May 1;28(9):E42., 2000-05-01 [PMID:10756209]

Abstract [show]
A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures. Spontaneous DNA branch migration results in dissociation of these structures when the test and reference sequences are identical. Any base substitution, deletion or insertion inhibits branch migration and produces stable cruciform structures. When suitable ligands are attached to the PCR primers, the cruciform structures can be detected by standard immunochemical methods. This approach was tested using several commonly occurring mutations within the human cystic fibrosis gene. New methods for increasing the specificity of PCR amplifications are described that were used for successful mutation analysis.

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126 Genotype Without reference DNA With reference DNA Wild-type homozygotes wt/wt 1.4 (1.2) 2.4 wt/wt 1.6 (1.3) 2.1 wt/wt 2.0 (1.5) 2.4 wt/wt 2.1 (1.4) 2.0 wt/wt 1.6 (1.2) 3.2 wt/wt 1.7 (1.4) 1.5 Heterozygotes G542X/wt G→T 41 (42) 38 G542X/wt G→T 73 (90) 66 G551D/wt G→A 83 (84) 93 G551D/wt G→A 99 (69) 76 R553X/wt C→T 92 (84) 57 R553X/wt C→T 105 (95) 61 R560T/wt G→C 130 (123) 70 R560T/wt G→C 109 (135) 111 G551D/R553X G→A/C→T 134 (134) 193 G551D/R553X G→A/C→T 134 (144) 235 Mutant homozygotes G542X/G542X G→T 1.5 (1.4) 174 G542X/G542X G→T 1.4 (1.5) 133 Blank (no target DNA) 1.6 Figure 3. Login to comment
139 Genotype Without εAεAG With εAεAG wt/wt 37 1.4 wt/wt 39 1.4 wt/wt 41 1. wt/wt 41 1.5 G542X/wt 121 126 G551D/wt 100 93 R553X/wt 82 85 R560T/wt 97 96 vi A PCR modification similar to the use of nested primers provided a powerful alternative method for reducing background signals. Login to comment

[hide] Prenatal detection by real-time quantitative PCR a... Clin Chem. 2000 Sep;46(9):1417-20. Costes B, Girodon E, Vidaud D, Flori E, Ardalan A, Conteville P, Fanen P, Niel F, Vidaud M, Goossens M
Prenatal detection by real-time quantitative PCR and characterization of a new CFTR deletion, 3600+15kbdel5.3kb (or CFTRdele19).
Clin Chem. 2000 Sep;46(9):1417-20., [PMID:10973878]

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51 The mutations tested were S549N, S549R, R553X, G551D, V520F, ⌬I507, ⌬F508, Q493X, 1717-1G3A, G542X, R560T, R347P, R347H, 3849ϩ4A3G, W1282X, R334W, 1078delT, 3849ϩ10kbC3T, R1162X, N1303K, 3659delC, 3905insT, A455E, R117H, Y122X, 2183AA3G, 2789ϩ5G3A, 1898ϩ1G3A, 621ϩ1G3T, 711ϩ1G3T, and G85E. Login to comment

[hide] Mutation in the gene responsible for cystic fibros... JAMA. 2000 Oct 11;284(14):1814-9. Wang X, Moylan B, Leopold DA, Kim J, Rubenstein RC, Togias A, Proud D, Zeitlin PL, Cutting GR
Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.
JAMA. 2000 Oct 11;284(14):1814-9., 2000-10-11 [PMID:11025834]

Abstract [show]
CONTEXT: Chronic rhinosinusitis (CRS) is a common condition in the US general population, yet little is known about its underlying molecular cause. Chronic rhinosinusitis is a consistent feature of the autosomal recessive disorder cystic fibrosis (CF). OBJECTIVE: To determine whether mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, which is responsible for CF, predispose to CRS. DESIGN: Case-control study conducted from 1996 to 1999 in which the DNA of CRS patients and controls was typed for 16 mutations that account for 85% of CF alleles in the general population. Chronic rhinosinusitis patients with 1 CF mutation were evaluated for a CF diagnosis by sweat chloride testing, nasal potential difference measurement, and DNA analysis for additional mutations. SETTING: Otolaryngology-head and neck clinic of a US teaching hospital. PARTICIPANTS: One hundred forty-seven consecutive adult white patients who met stringent diagnostic criteria for CRS and 123 CRS-free white control volunteers of similar age range, geographic region, and socioeconomic status. MAIN OUTCOME MEASURES: Presence of CF mutations by DNA analysis among CRS patients vs controls. RESULTS: Eleven CRS patients were found to have a CF mutation (DeltaF508, n = 9; G542X, n = 1; and N1303K, n = 1). Diagnostic testing excluded CF in 10 of these patients and led to CF diagnosis in 1. Excluding this patient from the analyses, the proportion of CRS patients who were found to have a CF mutation (7%) was significantly higher than in the control group (n = 2 [2%]; P =.04, both having DeltaF508 mutations). Furthermore, 9 of the 10 CF carriers had the polymorphism M470V, and M470V homozygotes were overrepresented in the remaining 136 CRS patients (P =.03). CONCLUSION: These data indicate that mutations in the gene responsible for CF may be associated with the development of CRS in the general population. JAMA. 2000;284:1814-1819.

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30 Analysis of CFTR Genes Genomic DNA samples extracted from the blood of participants were screened for 16 mutations (R117H, 621+1G→T, R334W, R347P, A455E, ⌬I507, ⌬F508, 1717-1 G→A, G542X, S549N, G551D, R553X, R560T, 3849+10 Kb C→T, W1282X, and N1303K) that account for 85% of CF alleles in the white population using the multiplex reverse dot hybridization system (Roche Molecular Systems, Alameda, Calif).16,17 This test also identified the 5T, 7T, and 9T variants of the splice acceptor site in intron 8 and F508C, I507V, and I506V (exon 10) polymorphisms of the CFTR gene. Login to comment

[hide] Polymorphism of cystic fibrosis gene in Japanese p... Dig Dis Sci. 2000 Oct;45(10):2007-12. Kimura S, Okabayashi Y, Inushima K, Yutsudo Y, Kasuga M
Polymorphism of cystic fibrosis gene in Japanese patients with chronic pancreatitis.
Dig Dis Sci. 2000 Oct;45(10):2007-12., [PMID:11117575]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and the 5T genotype of the polythymidine tract at the exon 9 splice branch/acceptor site are shown to be associated with chronic pancreatitis in Caucasian patients. In contrast to Western countries, cystic fibrosis is extremely rare in Japan. In this study, we investigated the association of mutations or polymorphisms of the CFTR gene with chronic pancreatitis in Japanese patients. Forty-seven patients with chronic pancreatitis (alcohol-related in 31, idiopathic in 14, and familial in 2) were examined for the deltaF508 and R117H mutations and polymorphisms of intron 8. DNA was extracted from leukocytes. Mutations and polymorphisms were examined by the allele-specific polymerase chain reactions and confirmed by direct sequencing. None of the patients had deltaF508 or R117H mutations in the CFTR gene. All of 47 healthy Japanese showed the homozygous 7T/7T genotype, whereas the frequencies of 5T, 7T, and 9T alleles were 0.043, 0.894, and 0.064 in the patients, respectively. The difference in allele frequency is statistically significant. Therefore, the present study indicates the association of polymorphism of the polythymidine tract in intron 8 of the CFTR gene with chronic pancreatitis in Japanese patients.

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127 Other mutations included R117H in two patients and Q493X, R553X, R560T, and 621 ϩ 1(G-to-T) in one patient each. Login to comment

[hide] Unilateral renal agenesis associated with congenit... Hum Reprod. 2001 Feb;16(2):282-8. McCallum T, Milunsky J, Munarriz R, Carson R, Sadeghi-Nejad H, Oates R
Unilateral renal agenesis associated with congenital bilateral absence of the vas deferens: phenotypic findings and genetic considerations.
Hum Reprod. 2001 Feb;16(2):282-8., [PMID:11157821]

Abstract [show]
An association between congenital bilateral absence of the vas deferens (CBAVD), normal renal anatomy and cystic fibrosis (CF) gene mutations is well established (CF/CBAVD). We postulate that unilateral renal agenesis (URA) and CBAVD (URA/CBAVD) may have a non-CF mutation-mediated genetic basis that leads to abnormal development of the entire mesonephric duct at a very early stage in embryo development (< or =7 weeks). The physical, laboratory and radiographic findings of men with URA/CBAVD (n = 17) and CF/CBAVD (n = 97) were compared; the fertilization and pregnancy rates in the URA/CBAVD population calculated, and the incidence of renal agenesis in immediate family members and offspring of men with URA/CBAVD analysed. No statistical differences could be identified within any of the above comparisons. The fertilization rate for the URA/CBAVD group was 58.2 +/- 26.3%. Eight infants and two fetuses had normal renal anatomy, while one terminated male fetus had bilateral renal and vasal agenesis. Thirty first-order relatives had normal renal units. Anatomical expression of the reproductive ductal derivatives in men with URA/CBAVD and CF/CBAVD was similar, but the phenotypic outcome of the renal portion of the mesonephric duct was different. The potential for transmission of this fatal anomaly reinforces the need for prenatal ultrasounds with all pregnancies involving URA/CBAVD men.

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60 Therereflects the 26 mutation/5T allele polymorphism assessment at the was no history of maternal diabetes, obvious teratogen exposurepresent time at Boston University Center for Human Genetics: or evidence of known congenital syndromes in themselves, orR117H; 1717-1G→A; G542X; 621ϩ1; S549N; R560T; I507; G551D; their families, that the men in either group could recall. Login to comment

[hide] Comprehensive mutation screening in a cystic fibro... Pediatrics. 2001 Feb;107(2):280-6. Wine JJ, Kuo E, Hurlock G, Moss RB
Comprehensive mutation screening in a cystic fibrosis center.
Pediatrics. 2001 Feb;107(2):280-6., [PMID:11158459]

Abstract [show]
OBJECTIVES AND BACKGROUND: The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established. STUDY DESIGN: We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping. RESULTS: For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G. CONCLUSION: When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.

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86 Mutations in the Stanford CF Mutation Database After Screening With the Genzyme70 Assay Mutation n % n % ⌬F508 353 67.11% 353 67.11% Splice mutations 16 3.04% 621ϩ1 G3T 5 0.95% 1717-1 G3A 5 0.95% 2789ϩ5 G3A 1 0.19% 1898ϩ1 G3A 1 0.19% 3849ϩ10 kb C3T 4 0.76% Stop mutations 31 5.89% Q493X 1 0.19% G542X 13 2.47% R553X 4 0.76% R1162X 1 0.19% W1282X 10 1.90% S1455X 2 0.38% Insertions/deletions 9 1.71% 681 del C 1 0.19% 2184 del A 2 0.38% 3859 del C 5 0.95% 3905 ins T 1 0.19% Missense mutations 33 6.27% G85E 4 0.76% R117H 3 0.57% R334W 6 1.14% G551D 14 2.66% R560T 3 0.57% N1303K 3 0.57% Unknown mutations 84 15.97% 84 15.97% Total 526 100.00% 526 100.00% ARTICLES tients with positive sweat tests were selected for SSCP/HA analysis based on clinical status, ethnicity, and previous screening with the Genzyme70 assay. Login to comment

[hide] An alpha1-antitrypsin enhancer polymorphism is a g... Eur J Hum Genet. 2001 Apr;9(4):273-8. Henry MT, Cave S, Rendall J, O'Connor CM, Morgan K, FitzGerald MX, Kalsheker N
An alpha1-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis.
Eur J Hum Genet. 2001 Apr;9(4):273-8., [PMID:11313771]

Abstract [show]
Lung disease is the direct cause of death in over 90% of cystic fibrosis (CF) patients. Excess neutrophil elastase is an important determinant of pulmonary disease in CF. alpha1-antitrypsin (AAT), also known as alpha1-proteinase inhibitor (alpha1PI) is a major modulator of elastase activity. We investigated the hypothesis that an enhancer polymorphism in the AAT gene would contribute to pulmonary prognosis in CF. Respiratory function, chest X-ray scores, bacterial colonisation and infective exacerbation were assessed to evaluate pulmonary disease severity in the CF group. Sixteen patients were found to have the 1237A allele, and 108 the more frequent G allele. Contrary to expectation, the patients with the 1237A allele were found to have better indices of pulmonary disease progression than those without, as indicated by less change in X-ray score (1237A: 0.2+/-0.1; 1237G: 1.2+/-0.1; P = 0.002) and fewer infective exacerbations (1237A: 2.8+/-0.6; 1237G: 4.6+/-0.3; P = 0.03) over the preceding 2 years. Also, a higher proportion of the 1237A (25%) than the 1237G (6.5%) were not colonised by Pseudomonas Aeruginosa (P = 0.04). Prospective monitoring of infections for a further 2 years confirmed a lesser propensity to infection in patients with the 1237A allele. These trends were also observed in a tightly matched sub-set of CF genotypes of similar age and sex, thus confirming that these effects were independent of the CF genotype. These results indicate that this AAT enhancer polymorphism is associated with better pulmonary prognosis in CF. Though the number of CF patients with the polymorphism is small, and these data need to be confirmed in larger studies, they suggest that a cautious approach should perhaps be taken to treatment of CF patients with supplemental AAT.

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65 Non DF 508 alleles in the two groups were: 1237A group: G551D (3); N1303K (2); R117H (1); R560T (1); Unknown (3). Login to comment
66 1237G group: G551D (10); R117H (3); R560T (3); D1507 (2); E60X (2); N1303K (1); 1717-1 (1); 621H (1); G542X (1); POL 400 (1); R352Q (1); RT0F (1); 621+G4T (1); Unknown (15). Login to comment
70 For subjects heterozygous for DF508, the second allele was matched as closely as possible and included the following: G551D, N1303K, R117H and R560T. Login to comment
81 infective exacerbations over 2 years 4.7+0.7 2.8+0.6 0.03 over 4 yearsb 10.5+1.8 4.5+1.1 0.006 FEV1 % predicted 55.5+7.4 67.5+5.5 NS at reference visit 2 years post 53.1+8.5 68.4+5.5 (n=14) 0.09 (NS) reference visitb a Non DF 508 alleles in the two groups were: 1237A group: G551D (3); R117H (1); R560T (1); N1303K (2); Unknown (3); 1237G group: G551D (4); R117H (1); R560T (1); 621+G4T (1); Unknown (3). Login to comment

[hide] Intron-8 polythymidine sequence in Australasian in... Eur Respir J. 2001 Jun;17(6):1195-200. Massie RJ, Poplawski N, Wilcken B, Goldblatt J, Byrnes C, Robertson C
Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C.
Eur Respir J. 2001 Jun;17(6):1195-200., [PMID:11491164]

Abstract [show]
Compound heterozygotes for a severe cystic fibrosis transmembrane conductance regulator (CFTR) mutation and the R117H or R117C mutation (R117H/C) have clinical presentations that vary from classic cystic fibrosis (CF) to an incidental genetic finding. The aim of this study was to assess the influence of the intron-8 polythvmidine sequence (IVS8) on the relationship between genotype and phenotype of individuals with R117H/C. All individuals with R117H/C known to CF clinics in Australia and New Zealand were retrospectively studied by collecting information on genotype, age, pancreatic status, sweat electrolytes, sputum microbiology and pulmonary function. Forty-one individuals (39 with R117H and two with R117C), 16 on an IVS8-5T background and 25 on an IVS8-7T background were identified. Twelve individuals presented clinically, four were siblings of known R117H/C compound heterozygotes and 25 were detected by newborn screening. Eleven of 14 of the IVS8-5T group (78%) with sweat chloride results available had sweat CI > 60 mmol x L(-1) compared to 5 (20%) of the R117H/7T group (Chi-squared=10.4, p=0.001). Two were pancreatic insufficient, both IVS8-5T. Two IVS8-5T individuals have recently died (aged 43 and 19) and of the 14 surviving IVS8-5T group, 11 (79%) are symptomatic compared to eight (32%) of the IVS8-7T individuals (Chi-squared=6.1, p=0.01). In conclusion, most individuals with R117H/C on a IVS8-5T background have an elevated sweat chloride and clinical cystic fibrosis, which in some cases is severe. Most individuals with R117H/C on an IVS8-7T background do not have clinical cystic fibrosis but should be followed for the development of clinical disease.

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41 Infants with a positive (w60 mmol?L-1 ) or borderline (40 - 60 mmol?L-1 ) sweat chloride and in whom there is an unidentified mutation are referred for an extended mutation analysis which includes: DF508, R117H, G551D, A455E, G542X, N1303K, W1282X, 1717-1, R560T, R347P, R334W, R1162X, S549N, 621z1, 3849z10CwT, and the IVS8 polythymidine sequence. Login to comment

[hide] A combined analysis of the cystic fibrosis transme... Mol Biol Evol. 2001 Sep;18(9):1771-88. Chen JM, Cutler C, Jacques C, Boeuf G, Denamur E, Lecointre G, Mercier B, Cramb G, Ferec C
A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models.
Mol Biol Evol. 2001 Sep;18(9):1771-88., [PMID:11504857]

Abstract [show]
Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires.

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100 Moreover, all of the 11 most common missense mutations or single-amino-acid deletions (i.e., F508del, G551D, N1303K, R117H, R347P, I507del, G85E, R560T, A455E, R334W, and S549N) identified in classic and atypical CF patients worldwide (http://www.genet.sickkids.on.ca/cftr) occur in stringently conserved residues across the 15 CFTR sequences. Login to comment

[hide] Mutations of the cystic fibrosis gene in patients ... Am J Gastroenterol. 2001 Sep;96(9):2657-61. Truninger K, Malik N, Ammann RW, Muellhaupt B, Seifert B, Muller HJ, Blum HE
Mutations of the cystic fibrosis gene in patients with chronic pancreatitis.
Am J Gastroenterol. 2001 Sep;96(9):2657-61., [PMID:11569691]

Abstract [show]
OBJECTIVE: Several studies have reported an increased frequency of cystic fibrosis gene mutations in idiopathic but not in alcoholic chronic pancreatitis. The impact of cystic fibrosis gene mutations on the long-term course of chronic pancreatitis has not been analyzed. The aim of our study was to determine the frequency of cystic fibrosis gene mutations in patients with chronic pancreatitis with long-term follow-up and to see whether patients with mutations have a clinically different natural course compared to those without mutations. METHODS: Eighty two patients with chronic pancreatitis and 11 patients with recurrent acute pancreatitis of our well defined pancreatitis cohort were screened for the 31 most common cystic fibrosis gene mutations. The impact of cystic fibrosis gene mutations on the long-term course of chronic pancreatitis was assessed. RESULTS: A cystic fibrosis gene mutation was detected in five of 49 patients with alcoholic chronic pancreatitis (10.2%; 2.3 times the expected frequency) and in three of 14 patients with idiopathic-juvenile chronic pancreatitis (21.4%; 4.8 times the expected frequency). No mutations were found in the remaining patients with chronic pancreatitis of rare causes, hereditary pancreatitis, and recurrent acute pancreatitis. The frequency of pancreatic calcifications was significantly higher in patients with alcoholic chronic pancreatitis without mutations. This result was not confirmed in patients with idiopathic-juvenile chronic pancreatitis. The duration of pain and the frequency of exocrine and endocrine insufficiency was comparable in both subgroups irrespective of the mutation status. CONCLUSION: Our data indicate a significantly increased frequency of cystic fibrosis gene mutations both in patients with alcoholic and idiopathic-juvenile chronic pancreatitis. The natural course was similar in patients with mutations compared to those without mutations.

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56 Using multiplex PCR, 15 genomic fragments were amplified which contain the following mutations: ⌬F508, ⌬I507, Q493X, V520F, 1717-1G3A, G542X, G551D, R553X, R560T, S549R, S549N, 3849 ϩ 10kbC3T, 3849 ϩ 4A3G, R1162X, 3659delC, W1282X, 3905insT, N1303K, G85E, 621 ϩ 1G3T, R117H, Y122X, 711 ϩ 1G3T; 1078delT, R347P, R347H, R334W, A455E, 1898 ϩ 1G3A, 2183AA3G, 2789 ϩ 5G3A. Login to comment

[hide] Cystic fibrosis phenotype evaluation and paternity... Hum Reprod. 2001 Oct;16(10):2093-7. Josserand RN, Bey-Omar F, Rollet J, Lejeune H, Boggio D, Durand DV, Durieu I
Cystic fibrosis phenotype evaluation and paternity outcome in 50 males with congenital bilateral absence of vas deferens.
Hum Reprod. 2001 Oct;16(10):2093-7., [PMID:11574497]

Abstract [show]
BACKGROUND: Most infertile males with congenital bilateral absence of vas deferens (CBAVD) carry mutations on the cystic fibrosis transmembrane conductance regulator gene and may express mild cystic fibrosis (CF) symptoms. Barriers to paternity for these men can now be overcome by assisted reproduction. Our aims were to investigate the CF-related phenotype and clinical outcome for 50 patients with CBAVD seen at a CF adult centre between 1992 and 1999. METHODS AND RESULTS: The investigation of the patients included screening for 22 CF mutations and identification of the poly-T variant of intron 8, sweat testing, clinical investigation for CF-related extra-genital manifestations, and genetic counselling. CFTR mutations were detected on 56 alleles of the 50 patients. A total of 15 (30%) was compound heterozygote and 26 (52%) heterozygote. In all, 38% of the patients had a positive sweat test. Four patients were diagnosed with typical CF not detected previously. Twenty-one patients became fathers following ICSI (eight cases), artificial insemination by donor or IVF with sperm donor (seven cases) or through adoption (six cases). A mail survey allowed the identification of CF-related clinical symptoms. Information on the occurrence of CF-related symptoms was obtained for 58.5% of patients: in the absence of initial symptoms, no new clinical signs were reported. CONCLUSION: Patients diagnosed with CBAVD need genetic counselling before assisted reproduction. Even when no wish for paternity is expressed, CF gene screening should be associated with at least a sweat test and clinical evaluation because of possible mild forms of CF disease. Medical follow-up did not reveal any new symptoms.

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30 Leukocytes samples were analysed for a series of 22 CF mutations including the five most frequently encountered in our region (The CF Genotype Consortium, 1994): ∆F508, G542X, N1303K, 1717-G-A, 885E; and 17 others: R117H, R334W, R347H, R347P, 556delA, S549N, S549I, S549R, G551D, R553X, R560T, G1244E, S1255X, W1282X, R1283K, 3898ins C, D1270N. Login to comment

[hide] Analysis of exocrine pancreatic function in cystic... Eur J Clin Invest. 2001 Sep;31(9):796-801. Walkowiak J, Herzig KH, Witt M, Pogorzelski A, Piotrowski R, Barra E, Sobczynska-Tomaszewska A, Trawinska-Bartnicka M, Strzykala K, Cichy W, Sands D, Rutkiewicz E, Krawczynski M
Analysis of exocrine pancreatic function in cystic fibrosis: one mild CFTR mutation does not exclude pancreatic insufficiency.
Eur J Clin Invest. 2001 Sep;31(9):796-801., [PMID:11589722]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common cause of exocrine pancreatic insufficiency in childhood. The aim of the present study is to evaluate the correlation between genotype and exocrine pancreatic insufficiency in CF patients. The special emphasis was put on the analysis of mild CFTR mutations. DESIGN: The study comprised 394 CF patients and 105 healthy subjects (HS). Elastase-1 concentrations were measured in all subjects. RESULTS: Severe pancreatic insufficiency was associated with the presence of two CFTR gene mutations (DeltaF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717-1G-A, R533X, W1282X, 621GT, 2183AAG, R560T, 2184insA and DeltaI507, G551D, 895T) and mild insufficiency with the presence of at least one mutation (R117H, 3171insC, A155P2, 138insL, 296 + 1G-A, E92GK, E217G, 2789 + 5G-A. 3849 + 1kbC-T/3849 + 1kbC-T) genotype resulted in high elastase-1-values. However, in case of patients with genotype DeltaF508/3849 + 10kbC-T, 1717-1GA/3849 + 10kbC-T as well as with DeltaF508/R334W, both high and low elastase-1 concentrations were found. Low E1 values were found in a patient with DeltaF508/R347P genotype. CONCLUSION: Patients who carry two 'severe' mutations develop pancreatic insufficiency, whereas those who carry at least one 'mild' usually remain pancreatic sufficient. However, the presence of one mild mutation does not exclude pancreatic insufficiency.

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5 Results Severe pancreatic insufficiency was associated with the presence of two CFTR gene mutations (DF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717±1G-A, R533X, W1282X, 621GT, 2183AAG, R560T, 2184insA and DI507, G551D, 895T) and mild insufficiency with the presence of at least one mutation (R117H, 3171insC, A155P2, 138insL, 296 1 1G-A, E92GK, E217G, 2789 1 5G-A. Login to comment
51 Results Among 394 genotyped CF patients, the following mutations on alleles were found (n): DF508 (464), 3849 1 10kbC-T (30), CFTR dele2,3(21 kB) (21), N1303K (15), G542X (12), 1717±1G-A (9), R533X (6), W1282X (6), 621 1 G-T (3), R117H (2), 3171insC (2), A155P2 (2), 2183AAG (2), R334W (2), 895T (2), 296 1 1G-A (2), E92GK (2), 138insL (1), E217G (1), 2789 1 5G-A (1), R347P (1), R560T (1), 2184insA (1), I507 (1), G551D (1). Login to comment
57 CFTR gene mutations were classified as `severe' (E1 , 96 mg g21 ) ± severely affecting pancreatic function (DF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717±1G-A, R533X, W1282X, 621 1 1G-T, 2183AAG, 895T, R560T, 2184insA, DI507, G551D) and `mild' (E1 . Login to comment
81 500 DF508/3849 1 10kbC-T (17) 1 4 1 6 5 DF508/CFTR dele2,3(21kb) (15) 9 4 2 DF508/N1303K (10) 7 3 DF508/1717±1G-A (7) 5 2 DF508/G542X (7) 4 2 1 DF508/W1282X (5) 4 1 DF508/R553X (3) 3 DF508/R334W (2) 1 1 DF508/2183AAG (2) 2 DF508/R117H (1) 1 DF508/621GT (1) 1 DF508/R347P (1) 1 DF508/2184insA (1) 1 DF508/DI507 (1) 1 3849 1 10kbC-T/3849 1 10kbC-T (3) 3 N1303K/CFTR dele2,3(21kb) (2) 1 1 1717±1G-A/3849 1 10kbC-T (2) 1 1 3171insC/A155P2 (2) 1 1 296 1 1G-A/E92GK (2) 2 R117H/138insL (1) 1 W1282X/3849 1 10kbC-T (1) 1 N1303K/3849 1 10kbC-T (1) 1 CFTR dele2,3(21kb)/3849 1 10kbC-T (1) 1 R553X/G542X (1) 1 621 1 1G-T/621 1 1G-T (1) 1 G542X/M (4) 2 2 CFTR dele 2,3(21kb)/M (1) 1 2 3849 1 10kbC-T/M (2) 1 1 R533X/M (2) 2 N1303K/M (2) 2 895T/M (2) 1 1 E217G/M (1) 1 G551D/M (1) 1 R560T/M (1) 1 2789 1 5G-A/M (1) 1 Total (109) 44 21 10 4 12 18 M, unidentified mutation. Login to comment
86 Kristidis et al. [10] reported that pancreatic insufficiency strongly correlates also with two alleles of DI507, Q493X, G542X, R553X, W1282X, 621 1 1G-T, 1717±1G-A, 556delA, 3659delC, I148T, G480C, V520F and R560T while one or two mutations such as R117H, R334W, A455E, and P574H were correlated with a pancreatic sufficient phenotype. Login to comment

[hide] Spectrum of mutations in the CFTR gene of patients... Genet Test. 2001 Fall;5(3):235-42. Strandvik B, Bjorck E, Fallstrom M, Gronowitz E, Thountzouris J, Lindblad A, Markiewicz D, Wahlstrom J, Tsui LC, Zielenski J
Spectrum of mutations in the CFTR gene of patients with classical and atypical forms of cystic fibrosis from southwestern Sweden: identification of 12 novel mutations.
Genet Test. 2001 Fall;5(3):235-42., [PMID:11788090]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CFTR gene. The spectrum of CFTR mutations varies between populations and depends on different factors, such as ethnic background and geographical location. The extensive CFTR mutation screening of 129 patients with classical or atypical CF from the south-western region of Sweden revealed the presence of 37 CFTR mutations, including 12 novel alleles. The overall mutation detection rate in this study population was 92%, the highest among all tested regions in Sweden. Eight mutations with a frequency above 1% (DeltaF508, 394delTT, R117C, 3659delC, E60X, 1112delT, R764X, and 621 + 1G --> T) accounted for 78% of CF chromosomes and have been recommended for inclusion in the CFTR mutation screening panel for molecular diagnosis of CF in this region. The multiple occurrence of specific CFTR alleles less common than the predominant DeltaF508 mutation (394delTT, R117C, 3659delC) allowed for genotype-phenotype comparisons and revealed consistent relationships between these mutations and disease severity.

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27 MUTATIONS IDENTIFIED IN 258 CHROMOSOMES IN THE CF POPULATION ATTENDING THE SOUTH-WESTERN SWEDISH CF CENTRE Location in the Frequency of Mutation gene, exon Number of mutations mutation (%) Homozygotes Heterozygotes DF508 10 161 62.4 56 49 394delTT 3 13 5.0 3 7 R117C 4 7 2.7 7 3659delC 19 5 1.9 5 E60X 3 4 1.6 4 1112delT 7 4 1.6 1 2 R764X 13 4 1.6 1 2 621 1 1G ® T 4 3 1.2 3 G551D 11 2 0.8 2 I506L 10 2 0.8 2 N1088D (R75Q) 17b 2 0.8 2 Q1238X 19 2 0.8 2 R117H (IVS8-5T) 4 2 0.8 2 V603F (IVS8-5T) 13 2 0.8 2 1716G ® A 10 2 0.8 2 R75Q 3 2 0.8 2 R533X 11 1 0.4 1 2329A ® G Promoter 1 0.4 1 297-3 C ® A 2 1 0.4 1 Y161D 4 1 0.4 1 994del9 Exon/intron 6b 1 0.4 1 1154insTC 7 1 0.4 1 W361R 7 1 0.4 1 T338I 7 1 0.4 1 1249-5A ® G Intron 7 1 0.4 1 1717-2A ® G Intron 10 1 0.4 1 R560T 11 1 0.4 1 E1401X 23 1 0.4 1 3126del4 17a 1 0.4 1 S945L 15 1 0.4 1 R668C 13 1 0.4 1 2622 1 2del6 Intron 13 1 0.4 1 R1162Q Exon 19 1 0.4 1 3849 1 10kbC ® T Intron 19 1 0.4 1 R74W Exon 3 1 0.4 1 2363C ® T Promoter 1 0.4 1 IVS8-5Ta Intron 8 1 0.4 1 Unidentified 20 7.8 Total 258 100 61 116 The new mutations are displayed in bold. Login to comment

[hide] Genetic and clinical features of false-negative in... Acta Paediatr. 2002;91(1):82-7. Padoan R, Genoni S, Moretti E, Seia M, Giunta A, Corbetta C
Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.
Acta Paediatr. 2002;91(1):82-7., [PMID:11883825]

Abstract [show]
A study was performed on the delayed diagnosis of cystic fibrosis (CF) in infants who had false-negative results in a neonatal screening programme. The genetic and clinical features of false-negative infants in this screening programme were assessed together with the efficiency of the screening procedure in the Lombardia region. In total, 774,687 newborns were screened using a two-step immunoreactive trypsinogen (IRT) (in the years 1990-1992), IRT/IRT + delF508 (1993-1998) or IRT/IRT + polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) protocol (1998-1999). Out of 196 CF children born in the 10 y period 15 were false negative on screening (7.6%) and molecular analysis showed a high variability in the genotypes. The cystic fibrosis transmembrane regulator (CFTR) gene mutations identified were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 + 10kbC --> T, 2789 + 5G --> A, 5T-12TG and the novel mutation D110E. In three patients no mutation was identified after denaturing gradient gel electrophoresis of the majority of CFTR gene exons. Conclusion: The clinical phenotypes of CF children diagnosed by their symptoms at different ages were very mild. None of them presented with a severe lung disease. The majority of them did not seem to have been damaged by the delayed diagnosis. The combination of IRT assay plus genotype analysis (1998-1999) appears to be a more reliable method of detecting CF than IRT measurement alone or combined with only the delF508 mutation.

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34 It was initially performed by polyacrylamide gel electrophoretic (PAGE) analysis for the delF508 mutation, and later by polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) (31 mutations: G85E, 621 ‡ 1G ® T, R117H, Y122X, 711 ‡ 1G ® T, 1078delT, R347P, R347H, R334W, A455E, 1898 ‡ 1G ® A, 2183-AA ® G, 2789 ‡ 5G ® A, DelF508, I507del, Q493X, V520F, 1717-1G ® A, G542X, G551D, R553X, R560T, S549R, S549N, 3849 ‡ 10kbC ® T, 3849 ‡ 4A ® G, R1162X, 3659delC, W1282X, 3905insT, N1303K) (14). Login to comment

[hide] The relationship between genotype and exercise tol... Am J Respir Crit Care Med. 2002 Mar 15;165(6):762-5. Selvadurai HC, McKay KO, Blimkie CJ, Cooper PJ, Mellis CM, Van Asperen PP
The relationship between genotype and exercise tolerance in children with cystic fibrosis.
Am J Respir Crit Care Med. 2002 Mar 15;165(6):762-5., 2002-03-15 [PMID:11897641]

Abstract [show]
The relationship between fitness and genotype in children with cystic fibrosis (CF) and at least one copy of the DeltaF508 mutation was examined. Genotype was classified according to the second CF mutation. Fitness was measured by peak aerobic capacity (using a modified Bruce protocol during treadmill exercise) and anaerobic power (using the Wingate test on a cycle ergometer). The class of cystic fibrosis transmembrane regulator proteins (CFTR) mutation was statistically related with aerobic capacity, peak anaerobic power, body mass index, lung function (forced expiratory volume in one second), and disease severity as measured by the Shwachman score. Patients with mutations causing defective CFTR production (Class I) or processing (Class II) had a significantly lower peak aerobic capacity (28.6 +/- 4.2 ml/kg/min and 31.7 +/- 5.4 ml/kg/min, respectively) than those with a mutation conferring defective regulation of CFTR (Class III) (43.9 +/- 6.4 ml/kg/min). The peak anaerobic power in subjects with mutations inducing decreased CFTR conduction (Class IV) or CFTR mRNA (Class V), were significantly higher (11.4 +/- 1.7 and 11.6 +/- 1.5 watts/kg, respectively) than children with Class I (9.7 +/- 1.4 watts/kg), Class II (9.8 +/- 1.4 watts/kg), or Class III (10.5 +/- 1.8 watts/kg) mutations. There were no statistically significant differences in the lung function of patients with the different mutations. These results indicate a relationship between CF genotype and some measures of fitness, the mechanisms of which remain to be determined.

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82 II ⌬F508 (36), W1282X (1) III G551D (10), N1303K (4), R560T (2), A559T (1) IV R117H (14), R347H (3) V 3849 ϩ 10KbC→T (7), 3120G→A (3) AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 165 2002 All patients were recruited from a single center, and the sample size of this study was large compared with previously published studies of exercise capacity in children with CF (21). Login to comment

[hide] DHPLC screening of cystic fibrosis gene mutations. Hum Mutat. 2002 Apr;19(4):374-83. Ravnik-Glavac M, Atkinson A, Glavac D, Dean M
DHPLC screening of cystic fibrosis gene mutations.
Hum Mutat. 2002 Apr;19(4):374-83., [PMID:11933191]

Abstract [show]
Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

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42 The following mutations have been studied: exon 3: W57G, R74W, R75Q, G85E, 394delTT, 405+ 1G>A; exon 4: E92X, P99L, 441delA, 444delA, 457TAT>G, D110H, R117C, R117H, A120T, 541delC, 544delCA, Q151X, 621+1G>T, 662- 2A>C; exon 7: 1078delT, F331L, R334W, I336K, R347C, R347P, A349V, R352Q, 1221delCT; exon 10: S492F, Q493X, 1609delCA, deltaI507, deltaF508; exon 11: G542X, S549N, G551D, R553X, A559T, R560K, R560T; exon 13: K716X, Q685X, G628R, L719X; exon 17b: H1054D, G1061R, 3320ins5, R1066H, R1066L, R1070Q, 3359delCT, L1077P, H1085R, Y1092X; exon 19: R1162X, 3659delC, 3662delA, 3667del4, 3737delA, I1234V, S1235R, 3849G>A; exon 20: 3860ins31,S1255X,3898insC,3905insT,D1270N, W1282X, Q1291R; and exon 21: N1303H, N1303K, W1316X. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Clin Exp Allergy. 2002 May;32(5):756-61. Eaton TE, Weiner Miller P, Garrett JE, Cutting GR
Cystic fibrosis transmembrane conductance regulator gene mutations: do they play a role in the aetiology of allergic bronchopulmonary aspergillosis?
Clin Exp Allergy. 2002 May;32(5):756-61., [PMID:11994102]

Abstract [show]
BACKGROUND: Previous work suggests that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations may be implicated in the aetiology of allergic bronchopulmonary aspergilosis (ABPA). OBJECTIVE: To compare the frequency of CF gene mutations in asthmatics with ABPA of varying severity with asthmatics who were skin prick test (SPT)-positive to Aspergillus fumigatus (Af) without evidence of ABPA and asthmatics SPT-negative to Af. METHODS: Thirty-one Caucasian patients with ABPA were identified, together with asthmatics SPT positive to Af without evidence of ABPA (n = 23) and SPT negative to Af (n = 28). Genomic DNA was tested for 16 CF mutations accounting for approximately 85% of CF alleles in Caucasian New Zealanders. RESULTS: Four (12.9%) ABPA patients were found to be carriers of a CF mutation (DeltaF508 n = 3, R117H n = 1), one (4.3%) asthmatic SPT positive to Af without ABPA (DeltaF508), and one (3.6%) asthmatic SPT negative to Af (R117H). All patients with a CF mutation had normal sweat chloride (< 40 mM). There was no significant difference between the frequency of CF mutations in the ABPA patients and asthmatics without ABPA. However, the frequency of CF mutations in the ABPA patients was significantly different (P = 0.0125) to the expected carrier rate in the general population. CONCLUSION: These results lend further support to a possible link between CF mutations and ABPA.

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53 Cystic ®brosis mutation analysis Genomic DNA samples were screened for 16 CF mutations utilizing allelic-speci®c oligonucleotide (ASO) hybridization; ÁF508, ÁI507, R117H, W1282X, 621 IG3T, R334W, R347P, A455E, 1717-IG3A, G542X, 5549N, G551D, R553X, R560T, N1303K and 3849 10KC3T. Login to comment

[hide] Cystic fibrosis: a worldwide analysis of CFTR muta... Hum Mutat. 2002 Jun;19(6):575-606. Bobadilla JL, Macek M Jr, Fine JP, Farrell PM
Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening.
Hum Mutat. 2002 Jun;19(6):575-606., [PMID:12007216]

Abstract [show]
Although there have been numerous reports from around the world of mutations in the gene of chromosome 7 known as CFTR (cystic fibrosis transmembrane conductance regulator), little attention has been given to integrating these mutant alleles into a global understanding of the population molecular genetics associated with cystic fibrosis (CF). We determined the distribution of CFTR mutations in as many regions throughout the world as possible in an effort designed to: 1) increase our understanding of ancestry-genotype relationships, 2) compare mutational arrays with disease incidence, and 3) gain insight for decisions regarding screening program enhancement through CFTR multi-mutational analyses. Information on all mutations that have been published since the identification and cloning of the CFTR gene's most common allele, DeltaF508 (or F508del), was reviewed and integrated into a centralized database. The data were then sorted and regional CFTR arrays were determined using mutations that appeared in a given region with a frequency of 0.5% or greater. Final analyses were based on 72,431 CF chromosomes, using data compiled from over 100 original papers, and over 80 regions from around the world, including all nations where CF has been studied using analytical molecular genetics. Initial results confirmed wide mutational heterogeneity throughout the world; however, characterization of the most common mutations across most populations was possible. We also examined CF incidence, DeltaF508 frequency, and regional mutational heterogeneity in a subset of populations. Data for these analyses were filtered for reliability and methodological strength before being incorporated into the final analysis. Statistical assessment of these variables revealed that there is a significant positive correlation between DeltaF508 frequency and the CF incidence levels of regional populations. Regional analyses were also performed to search for trends in the distribution of CFTR mutations across migrant and related populations; this led to clarification of ancestry-genotype patterns that can be used to design CFTR multi-mutation panels for CF screening programs. From comprehensive assessment of these data, we offer recommendations that multiple CFTR alleles should eventually be included to increase the sensitivity of newborn screening programs employing two-tier testing with trypsinogen and DNA analysis.

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No. Sentence Comment
110 Germany ∆F508 (71.8%) 1789+5G→A (0.9%) 87.6 76.7 17 5662/1316 Dörk et al. [1992]; Dörk et al. R553X (2.0%) 3272-26A→G (0.9%) [1994]; Tümmler et al. [1996]; N1303K (1.8%) W1282X (0.7%) Estivill et al. [1997]; Dörk et G542X (1.2%) 2143delT (0.7%) al. [2000] R347P (1.2%) 1078delT (0.6%) CFTRdele2,3 (1.2%) 2183AA→G (0.6%) 3849+10KbC→T (1.0%) 2184insA (0.6%) G551D (0.9% 3659delC (0.6%) 1717-1G→A (0.9%) Greece ∆F508 (52.9%) 3272-26A→G (0.8%) 82.2 67.6 22 2097/718 Kanavakis et al. [1995]; Estivill 621+1G→T (5.0%) R1070Q (0.8%) et al. [1997]; Tzetis et al. G542X (4.1%) W496X (0.7%) [1997]; Macek et al. [2002] N1303K (3.3%) 621+3A→G (0.7%) 2183AA→G (1.8%) ∆I507 (0.7%) 2789+5G→A (1.7%) W1282X (0.7%) E822X (1.6%) 574delA (0.7%) R117H (1.2%) 1677delTA (0.7%) R334W (1.1%) A46D (0.6%) R1158X (1.0%) 3120+1G→A (0.6%) G85E (1.0%) G551D (0.5%) Hungary ∆F508 (54.9%) W1282X (1.8%) 68.3 46.6 9 1133/976 CFGAC [1994]; Estivill et al. 1717-1G→A (1.9%) G542X (1.7%) [1997]; Macek et al. [2002] R553X (2.1%) N1303K (1.3%) Y1092X (1.8%) G551D (1.0%) S1196X (1.8%) Ireland ∆F508 (70.4%) G542X (1.0%) 82.1 67.4 7 801/509 CFGAC [1994]; Estivill et al. G551D (5.7%) 621+1G→T (0.8%) [1994] R117H (2.4%) 1717-1G→A (0.6%) R560T (1.2%) Italy ∆F508 (50.9%) ∆I507 (0.65%) 60.3 36.4 9 3524 Estivill et al. [1997] (total) G542X (3.1%) W1282X (0.62%) 1717-1G→A (1.6%) Y122K (0.59%) N1303K (1.4%) G551D (0.53%) R553X (0.94%) Italy ∆F508 (47.6%) R553X (1.3%) 87.1 75.9 15 225 Bonizzato et al. [1995] (Northeast) R1162X (9.8%) 2789+G→A (1.3%) 2183AA→G (9.3%) Q552X (1.3%) N1303K (4.0%) 621+1G→T (0.9%) G542X (2.7%) W1282X (0.9%) 711+5G→A (2.7%) 3132delTG (0.9%) 1717-1G→A (2.2%) 2790-2A→G (0.9%) G85E (1.3%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS583 Italy ∆F508 (56.4%) 711+1G→T (1.3%) 85.7 73.4 13 660/396 Castaldo et al. [1996]; Castaldo (southern) N1303K (6.8%) G1244E (1.3%) et al. [1999] G542X (5.7%) R1185X (1.3%) W1282X (3.8%) L1065P (1.3%) 1717-1G→A (2.3%) R553X (1.1%) 2183AA→G (1.9%) I148T (0.7%) 4016insT (1.8%) Latvia 1) DF508 (58.3%) 4) CFTRdele2,3 (2.8%) - - 6 36 Dörk et al. [2000]; Macek et al. 2) 3849+10KbC®T (8.3%) 5) W1282X (2.8%) [2002] 3) N1303K (5.6%) 6) 394delTT (2.8%) Lithuania ∆F508 (31.0%) N1303K (2.0%) 39.0 15.2 4 94 Dörk et al. [2000]; Macek et al. R553X (4.0%) CFTRdele2,3 (2.0%) [2002] Macedonia ∆F508 (54.3%) 711+3A→G (1.0%) 69.2 47.9 12 559/226 Petreska et al. [1998]; Dörk et G542X (4.2%) 3849G→A (1.0%) al. [2000]; Macek et al. N1303K (2.0%) 2184insA (0.9%) [2002] CFTRdele2,3 (1.3%) 457TAT→G (0.7%) 621+1G→T (1.3%) V139E (0.7%) 611-1G→T (1.2%) 1811+1G→C (0.6%) Netherlands ∆F508 (74.2%) R1162X (0.9%) 86.8 75.3 9 3167/1442 Gan et al. [1995]; Estiville et al. A455E (4.7%) S1251N (0.9%) [1997]; Collee et al. [1998] G542X (1.8%) N1303K (0.9%) 1717-1G→A (1.5%) W1282X (0.7%) R553X (1.2%) Norway ∆F508 (60.2%) G551D (1.2%) 69.8 48.7 6 410/242 Schwartz et al. [1994]; Estivill 394delTT (4.2%) G542X (0.6%) et al. [1997] R117H (3.0%) N1303K (0.6%) Poland ∆F508 (57.1%) CFTRdele2,3 (1.8%) 73.5 54.0 11 4046/1726 CFGAC [1994]; Estivill et al. 3849+10Kb C→T (2.7%) R560T (1.5%) [1997]; Dörk et al [2000]; G542X (2.6%) W1282X (0.7%) Macek et al. [2002] 1717-1G→A (2.4%) ∆I507 (0.5%) R553X (1.9%) G551D (0.5%) N1303K (1.8%) Portugal ∆F508 (44.7%) R334W (0.7%) 49.7 24.7 5 739/454 CFGAC [1994]; Estivill et al. G542X (1.6%) N1303K (0.7%) [1997] R1066C (2.0%) Romania ∆F508 (36.6%) G542X (1.4%) 51.5 26.5 11 224/74 CFGAC [1994]; Estivill et al. 2043delG (2.0%) R553X (1.4%) [1997]; Popa et al. [1997]; W1282X (1.7%) G576X (1.4%) Macek et al. [2002] 1717-2A→G (1.4%) 1898+1G→A (1.4%) I148T (1.4%) 2183AA→G (1.4%) 621+1G→T (1.4%) Russia ∆F508 (54.4%) 552insA (0.9%) 70.7 50.0 12 5073/2562 CFGAC [1994]; Estivill et al. CFTRdele2,3 (5.0%) G542X (0.9%) [1997]; Dörk et al. [2000]; R553X (3.5%) R334W (0.9%) Macek et al. [2002] 2183AA→G (1.3%) 1677delTA (0.8%) W1282X (1.0%) Y122X (0.5%) 394delTT (1.0%) 1367del5 (0.5%) (Continued) BOBADILLAETAL. Login to comment
111 Slovakia ∆F508 (57.3%) CFTRdele2,3 (1.2%) 82.7 68.4 14 908/254 CFGAC [1994]; Estivill et al. G542X (6.8%) 3849+10KbC→T (1.0%) [1997]; Dörk et al. [2000]; R553X (4.0%) S42F (0.9%) Macek et al. [2002] N1303K (3.4%) R75X (0.9%) 2143delT (1.8%) G85E (0.9%) R347P (1.4%) 605insT (0.9%) W1282X (1.3%) 1898+1G→A (0.9%) Slovenia ∆F508 (57.8%) R347P (1.1%) 79.7 63.5 16 455/132 CFGAC [1994]; Dörk et al. 2789+5G→A (4.1%) S4X (0.8%) [2000]; Macek et al. [2002] R1162X (3.2%) 457TAT→G (0.8%) G542X (1.9%) D192G (0.8%) Q552X (1.5%) R553X (0.8%) Q685X (1.5%) A559T (0.8%) 3905insT (1.5%) 2907delTT (0.8%) CFTRdele2,3 (1.5%) 3667ins4 (0.8%) Spain ∆F508 (52.7%) G85E (0.8%) 80.2 64.3 21 3608/1356 Chillón et al. [1994]; Casals et G542X (8.0%) R1066C (0.8%) al. [1997]; Estivill et al. [1997] N1303K (2.5%) 2789+5G→A (0.7%) 3601-111G→C (2.0%) 2869insG (0.7%) 1811+1.6Kb A→G (1.7%) ∆I507 (0.6%) R1162X (1.6%) W1282X (0.6%) 711+1G→T (1.3%) L206W (0.5%) R334W (1.2%) R709X (0.5%) Q890X (1.0%) K710X (0.5%) 1609delCA (1.0%) 3272-26A→G (0.5%) 712-1G→T (1.0%) Sweden ∆F508 (66.6%) E60X (0.6%) 85.9 73.8 10 1357/662 Schwartz et al. [1994]; Estivill et 394delTT (7.3%) Y109C (0.6%) al. [1997]; Schaedel et al. 3659delC (5.4%) R117H (0.6%) [1999] 175insT (2.4%) R117C (0.6%) T338I (1.2%) G542X (0.6%) Switzerland ∆F508 (57.2%) K1200E (2.1%) 91.3 83.4 9 1268/1173 Estivill et al. [1997]; R553X (14.0%) N1303K (1.2%) Hergersberg et al. [1997] 3905insT (9.8%) W1282X (1.1%) 1717-1G→A (2.7%) R347P (0.6%) G542X (2.6%) Ukraine ∆F508 (65.2%) CFTRdele2,3 (1.1%) 74.6 55.7 6 1055/580 Estivill et al. [1997]; Dörk et al. R553X (3.6%) G551D (1.8%) [2000]; Macek et al. [2002] N1303K (2.4%) W1282X (0.5%) United ∆F508 (75.3%) 621+1G→T (0.93%) 81.6 66.6 5 19622/9815 Schwartz et al. [1995b]; Kingdom G551D (3.1%) 1717-1G→A (0.57%) Estivill et al. [1997] (total) G542X (1.7%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS585 United ∆F508 (56.6%) 621+1G→T (1.8%) 69.1 47.7 7 456 CFGAC [1994] Kingdom G551D (3.7%) R117H (1.5%) (N. Ireland) R560T (2.6%) ∆I507 (0.9%) G542X (2.0%) United ∆F508 (19.2%) 621+2T→C (3.8%) 84.4 71.2 11 52 Malone et al. [1998] Kingdom Y569D (15.4%) 2184insA (3.8%) (Pakistani) Q98X (11.5%) R560S (1.9%) 1525-1G→A (9.6%) 1898+1G→T (1.9%) 296+12T→C (7.7%) R709X (1.9%) 1161delC (7.7%) United ∆F508 (71.3%) 1717-1G→A (1.0%) 86.4 74.6 9 1236/730 Shrimpton et al. [1991]; Kingdom G551D (5.5%) 621+1G→T (0.6%) Gilfillan et al. [1998] (Scotland) G542X (4.0%) ∆I507 (0.6%) R117H (1.4%) R560T (0.6%) P67L (1.4%) United ∆F508 (71.6%) 1717-1G→A (1.1%) 98.7 97.4 17 183 Cheadle et al. [1993] Kingdom 621+1G→T (6.6%) 3659delC (0.5%) (Wales) 1898+1G→A (5.5%) R117H (0.5%) G542X (2.2%) N1303K (0.5%) G551D (2.2%) E60X (0.5%) 1078delT (2.2%) S549N (0.5%) R1283M (1.6%) 3849+10KbC→T (0.5%) R553X (1.1%) 4016insT (0.5%) ∆I507 (1.1%) Yugoslavia ∆F508 (68.9%) 3849G→A (1.0%) 82.2 67.6 11 709/398 Dabovic et al. [1992]; Estivill et G542X (4.0%) N1303K (0.8%) al. [1997]; Macek et al. R1162C (3.0%) 525delT (0.5%) (submitted for publication) 457TAT→G (1.0%) 621+1G→T (0.5%) I148T (1.0%) G551D (0.5%) Q552X (1.0%) Middle East/Africa Algeria 1) DF508 (20.0%) 4) 1812-1G®A (5.0%) - - 5 20 Loumi et al. [1999] 2) N1303K (20.0%) 5) V754M (5.0%) 3) 711+1G®T (10.0%) Jewish W1282X (48.0%) 3849+10KbC→T (6.0%) 95.0 90.3 6 261 Kerem et al. [1995] (Ashkenazi) ∆F508 (28.0%) N1303K (3.0%) G542X (9.0%) 1717-1G→A (1.0%) Jewish 1) N1303K - - 1 6 Kerem et al. [1995] (Egypt) Jewish 1) Q359K/T360K - - 1 8 Kerem et al. [1995] (Georgia) Jewish 1) DF508 2) 405+1G®A - - 2 11 Kerem et al. [1995] (Libya) Jewish 1) DF508 (72.0%) 3) D1152H (6.0%) - - 3 33 Kerem et al. [1995] (Morocco) 2) S549R (6.0%) Jewish ∆F508 (35.0%) W1282X (2.0%) 43.0 18.5 4 51 Shoshani et al. [1992] (Sepharadim) G542X (4.0%) S549I (2.0%) (Continued) BOBADILLAETAL. Login to comment
112 Jewish 1) 405+1G®A (48.0%) 3) W1282X (17.0%) - - 4 23 Kerem et al. [1995] (Tunisia) 2) DF508 (31.0%) 4) 3849+10KbC®T (4.0%) Jewish 1) G85E 4) G542X - - 6 10 Kerem et al. [1995] (Turkey) 2) DF508 5) 3849+10KbC®T 3) W1282X 6) W1089X Jewish (Yemen) None - - 0 5 Kerem et al. [1995] Lebanon 1) DF508 (35.0%) 6) 4096-28G®A (2.5%) - - 9 40 Desgeorges et al. [1997] 2) W1282X (20.0%) 7) 2789+5G®A (2.5%) 3) 4010del4 (10.0%) 8) M952I (2.5%) 4) N1303K (10.0%) 9) E672del (2.5%) 5) S4X (5.0%) Reunion ∆F508 (52.0%) 1717-1G→A (0.7%) 90.4 81.7 9 138 Cartault et al. [1996] Island Y122X (24.0%) G542X (0.7%) 3120+1G→A (8.0%) A309G (0.7%) A455E (2.2%) 2789+5G→A (0.7%) G551D (1.4%) Saudi North: 3) H139L - - North 1 49 families El-Harith et al. [1997]; Arabia 1) 1548delG 4) L1177X Central 3 Kambouris et al. [1997]; Central: 5) DF508 South 4 Banjar et al. [1999] 1)I1234V 6) 3120+1G®A West 9 2)1548delG 7) 425del42 East 6 3)DF508 8) R553X South: 9) N1303K 1) I1234V East: 2) 1548delG 1) 3120+1G®A 3) 711+1G®T 2) H139L 4) 3120+1G®A 3) 1548delG West: 4) DF508 1) I1234V 5) S549R 2) G115X 6) N1303K Tunisia ∆F508 (17.6%) G85E (2.6%) 58.7 34.5 11 78 Messaoud et al. [1996] G542X (8.9%) W1282X (2.6%) 711+1G→T (7.7%) Y122X (1.3%) N1303K (6.4%) T665S (1.3%) 2766del8NT (6.4%) R47W+D1270N (1.3%) R1066C (2.6%) Turkeye ∆F508 (24.5%) 1066L (1.3%) 80.6 65.0 36 1067/670 Yilmaz et al. [1995]; Estivill et al. 1677delTA (4.1%) E822X (1.3%) [1997]; Onay et al. [1998]; 2789+5G→A (3.9%) 2183+5G→A+2184insA (1.3%) Macek et al. [2002] 2181delA (3.8%) D110H (0.8%) R347H (3.6%) P1013L (0.8%) N1303K (2.9%) 3172delAC (0.8%) 621+1G→T (2.6%) 1259insA (0.8%) G542X (2.6%) M1028I (0.8%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS587 E92K (2.6%) 4005+1G→A (0.7%) A96E (2.6%) W1282X (0.7%) M152V (2.6%) I148T (0.6%) 2183AA→G (2.5%) R1162X (0.6%) 296+9A→T (1.6%) D1152H (0.6%) 2043delG (1.4%) W1098X (0.6%) E92X (1.4%) E831X (0.6%) K68N (1.4%) W496X (0.6%) G85E (1.3%) F1052V (0.5%) R1158X (1.3%) L571S (0.5%) United Arab S549R (61.5%) ∆F508 (26.9%) 88.4 78.1 2 86/52 Frossard et al. [1988]; Emirates Frossard et al. [1999] North/Central/South Americas Argentina ∆F508 (58.6%) N1303K (1.8%) 69.1 47.7 5 326/228 CFGAC [1994]; Chertkoff et al. W1282X (3.9%) 1717-1G→A (0.9%) [1997] G542X (3.9%) Brazilf ∆F508 (47.7%) W1282X (1.3%) 66.8 44.6 10 820/500 CFGAC [1994]; Cabello et al. (total) G542X (7.2%) G85E (1.3%) [1999]; Raskin et al. [1999]; R1162X (2.5%) R553X (0.7%) Bernardino et al. [2000] R334W (2.5%) L206W (0.6%) N1303K (2.4%) 2347delG (0.6%) South East: >∆F508, G542X South: >N1303K Brazil ∆F508 (31.7%) N1303K (2.5%) 42.5 18.1 3 120 Parizotto and Bertuzzo [1997] (Sao Paulo) G542X (8.3%) Canada ∆F508 (59.0%) G542X (0.5%) 98.5 97.0 13 381/200 Rozen et al. [1992]; (Lac St. Jean) 621+1G→T (24.3%) N1303K (0.5%) De Braekeleer et al. [1998] A445E (8.2%) Q890X (0.5%) Y1092X (1.2%) S489X (0.5) 711+1G→T (1.0%) R117C (0.5%) I148T (1.0%) R1158 (0.5%) G85E (0.8%) Canada ∆F508 (71.4%) ∆I507 (1.3%) 90.9 82.6 7 77 Rozen et al. [1992] (Quebec City) 711+1G→T (9.1%) Y1092X (1.3%) 621+1G→T (5.2%) N1303K (1.3%) A455E (1.3%) Canada ∆F508 (70.9%) W1282X (0.9%) 82.0 67.2 10 632 Kristidis et al. [1992] (Toronto) G551D (3.1%) R117H (0.9%) G542X (2.2%) 1717-1G→A (0.6%) 621+1G→T (1.3%) R560T (0.6%) N1303K (0.9%) ∆I507 (0.6%) Chile ∆F508 (29.2%) R553X (4.2%) 33.4 11.2 2 72 Rios et al. [1994] Columbia 1) DF508 (35.4%) 3) N1303K (2.1%) - - 4 48 Restrepo et al. [2000] 2) G542X (6.3%) 4) W1282X (2.1%) Ecuador 1) DF508 (25%) - - 1 20 Paz-y-Mino et al. [1999] (Continued) BOBADILLAETAL. Login to comment
113 Mexico ∆F508 (41.6%) G551S (0.5%) 75.5 57.0 35 374/194 Orozco et al.[1993]; Villalobos- G542X (5.6%) 1078delT (0.5%) Torres et al. [1997]; Liang et al. ∆I507 (2.5%) Y1092X (0.5%) [1998]; Orozco et al. [2000] S549N (1.9%) R117H (0.5%) N1303K (1.7%) G85E (0.5%) R75X (1.5%) 1716G→A (0.5%) 406-1G→A (1.5%) W1204X (0.5%) I148T (1.5%) W1098C (0.5%) 3849+10KbC→T (1.5%) 846delT (0.5%) 621+1G→T (1.2%) P750L (0.5%) 2055del9→A (1.0%) V754M (0.5%) 935delA (1.0%) R75Q (0.5%) I506T (1.0) W1096X (0.5%) 3199del6 (1.0%) L558S (0.5%) 2183AA→G (1.0%) 4160insGGGG (0.5%) G551D (0.5%) 297-1G→A (0.5%) R553X (0.5%) H199Y (0.5%) 1924del7 (0.5%) United States ∆F508 (68.6%) R553X (0.9%) 79.7 63.5 10 25048 Cystic Fibrosis Foundation (total) G542X (2.4%) 621+1G→T (0.9%) [1998] G551D (2.1%) 1717-1G→A (0.7%) W1282X (1.4%) 3849+10KbC→T (0.7%) N1303K (1.3%) R117H (0.7%) United States ∆F508 (48.0%) S1255X (1.4%) 77.3 59.8 16 160/148 Carles et al. [1996]; Macek et al. (African 3120+1G→A (12.2%) 444delA (0.7%) [1997]; Dörk et al. [1998]; American) 2307insA (2.0%) R334W (0.7%) Friedman et al. [1998] A559T (2.0%) ∆I507 (0.7%) R553X (2.0%) 1717-1G→A (0.7%) ∆F311 (2.0%) G542X (0.7%) G480C (1.4%) S549N (0.7%) 405+3A→C (1.4%) G551D (0.7%) United States 1) L1093P - - 1 2 Yee et al. [2000] (Cherokee) United States Non-French: French: Non- Non- Non- Non- Bayleran et al. [1996] (Maine) ∆F508 (82.0%) ∆F508 (58%) French: French: French: French: G542X (2.6%) 711+1G→T (8.3%) 95.3 90.8 11 191 G551D (2.6%) I148T (4.2%) French: French: French: French: N1303K (2.1%) A455E (4.2%) 80.3 64.5 8 72 R560T (1.0%) 1717-1G→A (1.4%) Total: 621+1G→T (1.0%) G85E (1.4%) 263 711+1G→T (1.0%) 621+1G→T (1.4%) R117H (1.0%) Y1092X (1.4%) 1717-1G→A (1.0%) G85E (0.5%) W1282X (0.5%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS589 United States ∆F508 (46.0%) R334W (1.6%) 58.5 34.2 7 129 Grebe et al. [1994] (SW Hispanic) G542X (5.4%) W1282X (0.8%) 3849+10KbC→T (2.3%) R553X (0.8%) R1162X (1.6%) United States 1) R1162X - - 3 17 Mercier et al. [1992] (SW Native 2) D648V American) 3) G542X United States 1) R1162X 3) G542X - - 4 16 Mercier et al. [1994] (Zuni Pueblo) 2) 3849+10KbC®T 4) D648V Venezuela ∆F508 (29.6%) G542X (3.7%) 33.3 11.1 2 54 Restrepo et al. [2000] Other Regions Australia ∆F508 (76.9%) 621+1G→T (1.1%) 88.7 78.7 8 761/464 CFGAC [1994] G551D (4.5%) N1303K (0.9%) G542X (2.8%) W1282X (0.6%) R553X (1.3%) R117H (0.6%) East Asia 1) 1898+1G®T 2) 1898+5G®T - - 2 28 Suwanjutha et al. [1998] Hutterite 1) M1101K (69.0%) 2) DF508 (31.0%) - - 2 32 Zielenski et al. [1993] Brethren New Zealand ∆F508 (78.0%) N1303K (1.9%) 87.4 76.4 5 636 CFGAC [1994] G551D (4.4%) 621+1G→T (1.1%) G542X (2.0%) *This table presents the mutation panels for all regions investigated in this study. Login to comment
213 Ideal Recommended CFTR Mutation Screening Panel for 2001 Neonatal Screening in the USA* Location Estimated Mutation in CFTRa percentageb Reason for inclusion DF508 Exon 10 68.6% CFF registry, >1%, Pan-European G542X Exon 11 2.4% CFF registry, >1%, Mediterranean G551D Exon 11 2.1% CFF registry, >1%, Celtic W1282X Exon 20 1.4% CFF registry, >1%, Ashkenazi Jew N1303K Exon 21 1.3% CFF registry, >1%, Mediterranean R553X Exon 11 0.9% CFF registry, >0.5%, Hispanic 621+1G®T Intron 4 0.9% CFF registry, >0.5%, multi-ethnic 1717-1G®A Intron 10 0.7% CFF registry, >0.5%, Italian 3849+10KbC®T Intron 19 0.7% CFF registry, >0.5%, Hispanic R117Hc Exon 4 0.7% CFF registry, >0.5% 1898+1G→T Intron 12 0.4% CFF registry, >0.1%, East Asian DI507 Exon 10 0.3% CFF registry, >0.1%, Hispanic 2789+5G®A Intron 14b 0.3% CFF registry, >0.1% G85E Exon 3 0.3% CFF registry, >0.1% R347P Exon 7 0.2% CFF registry, >0.1% R334W Exon 7 0.2% CFF registry, >0.1%, multi-ethnic R1162X Exon 19 0.2% CFF registry, >0.1%, multi-ethnic R560T Exon 11 0.2% CFF registry, >0.1% 3659delC Exon 19 0.2% CFF registry, >0.1% A455E Exon 9 0.2% CFF registry, >0.1% 2184delA Exon 13 0.1% CFF registry, >0.1% S549N Exon 11 0.1% CFF registry, >0.1%, multi-ethnic 711+1G®T Intron 5 0.1% CFF registry, >0.1% R75X Exon 3 0.2% Hispanic 406-1G→A Intron 3 0.2% Hispanic I148T Exon 4 0.2% Hispanic, French 2055del9→A Exon 13 0.1% Hispanic 935delA Exon 6b 0.1% Hispanic I506T Exon 10 0.1% Hispanic 3199del6 Exon 17a 0.1% Hispanic 2183AA→G Exon 13 0.1% Hispanic 3120+1G®A Intron 16 1.5% African American, Arabian 2307insA Exon 13 0.2% African American A559T Exon 11 0.2% African American ∆F311 Exon 7 0.2% African American G480C Exon 10 0.2% African American 405+3A→C Intron 3 0.2% African American S1255X Exon 20 0.2% African American L1093P Exon 17b Undetermined Native American D648V Exon 13 Undetermined Native American I1234V Exon 19 Undetermined Arabian linkage S549R Exon 11 Undetermined Arabian linkage 1898+5G→T Intron 12 Undetermined East Asian linkage CFTRdele2,3 Exons 2,3 Undetermined Eastern European linkage (Slavic) Y1092X Exon 17b Undetermined French linkage 394delTT Exon 3 Undetermined Nordic linkage Y569D Exon 12 Undetermined Pakistani linkage 3905insT Exon 20 Undetermined Swiss linkage (also: Amish, Acadian, Mennonite) 1898+1G®A Intron 12 Undetermined Welsh linkage M1101k Exon 17b Undetermined Hutterite ancestry *This table presents the top 50 mutations in the USA based on the Cystic Fibrosis Foundation CF Registry data from 1997 [Cystic Fibrosis Foundation, 1998], and data generated during our investigation. Login to comment

[hide] Development and evaluation of a PCR-based, line pr... Clin Chem. 2002 Jul;48(7):1121-3. Wang X, Myers A, Saiki RK, Cutting GR
Development and evaluation of a PCR-based, line probe assay for the detection of 58 alleles in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Clin Chem. 2002 Jul;48(7):1121-3., [PMID:12089190]

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68 Amplicon Size, bp Mutations (polymorphisms) Exon 13 598 2307 insA Intron 8, exon 09 548 A455E, 5T (7/9 T polymorphism) Exon 10 482 G480C, ⌬I507, ⌬F508 (F508C, I507V, I506V polymorphisms) Intron 10, exon 11 433 1717-1G3A, G542X, G551D, R553X, A559T, R560T Exon 19 420 R1162X, 3659delC Exon 21 397 N1303K Exon 20 359 S1255X, W1282X Exon 07 328 1078delT, R334W, R347P Exon 04, intron 4 288 R117H, 621ϩ1G3T Intron 14b 248 2789ϩ5G3A Intron 19 237 3849ϩ10kbC3T Exon 03 210 G85E, 405ϩ3A3C Intron 5 166 711ϩ1G3T Intron 16 139 3120ϩ1G3A Clinical Chemistry 48, No. Login to comment
88 The genotypes of each sample are as follows: lane 1, ϩ/ϩ (ϩ is the wild type); lane 2, 5T, R117H/3659delC; lane 3, G542X/ϩ; lane 4, I506V/ϩ; lane 5, I507V/ϩ; lane 6, F508C/⌬F508; lane 7, G85E/⌬F508; lane 8, 405ϩ3A3C/3120ϩ1G3C; lane 9, R117H/ϩ; lane 10, 621ϩ1G3T/⌬F508; lane 11, 711ϩ1G3T/⌬F508; lane 12, 1078delT/ϩ; lane 13, R334W/⌬F508; lane 14, R347P/⌬F508; lane 15, A455E/ϩ; lane 16, G480C/⌬F508; lane 17, ⌬I507/ϩ; lane 18, ⌬F508/ϩ; lane 19, 1717-1G3A/ϩ; lane 20, G542X/ϩ; lane 21, G551D/⌬F508; lane 22, R553X/ϩ; lane 23, R560T/⌬F508; lane 24, G551D/A559T; lane 25, 2307insA/ϩ; lane 26, 2789ϩ5G3A/⌬F508; lane 27, 3120ϩ1G3A/⌬F508; lane 28, R1162X/R1162X; lane 29, 3659delC/⌬F508; lane 30, 3849ϩ10kbC3T/⌬F508; lane 31, S1255X/⌬F508; lane 32, W1282X/G542X; lane 33, N1303K/ϩ. Login to comment

[hide] Predicting the risk of cystic fibrosis with abnorm... Am J Med Genet. 2002 Jun 15;110(2):109-15. Muller F, Simon-Bouy B, Girodon E, Monnier N, Malinge MC, Serre JL
Predicting the risk of cystic fibrosis with abnormal ultrasound signs of fetal bowel: results of a French molecular collaborative study based on 641 prospective cases.
Am J Med Genet. 2002 Jun 15;110(2):109-15., 2002-06-15 [PMID:12116247]

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Hyperechogenic fetal bowel is prenatally detected by ultrasound during the second trimester of pregnancy in 0.1-1.8% of fetuses. It has been described as a normal variant but has often been associated with severe diseases, notably cystic fibrosis (CF). The aim of our study was to determine the risk of CF in a prospective study of 641 fetuses with ultrasonographically abnormal fetal bowel and the residual risk when only one mutation is detected in the fetus. Fetal cells and/or parental blood cells were screened for CFTR mutations. Two screening steps were used, the first covering the mutations most frequently observed in French CF patients (mutation detection rate of 70-90%) and, when a CF mutation was detected, a DGGE-sequencing strategy. We observed a 3.1% risk of CF when a digestive tract anomaly was prenatally observed at routine ultrasound examination. The risk was higher when hyperechogenicity was associated with bowel dilatation (5/29; 17%) or with the absence of gall bladder (2/8; 25%). The residual risk of CF was 11% when only one CF mutation was detected by the first screening step, thereby justifying in-depth screening. Mutations associated with severe CF (DeltaF508 mutation) were more frequently observed in these ultrasonographically and prenatally detected CF cases. However, the frequency of heterozygous cases was that observed in the normal population, which demonstrates that heterozygous carriers of CF mutations are not at increased risk for hyperechogenic bowel. In conclusion, fetal bowel anomalies indicate a risk of severe cystic fibrosis and justify careful CFTR molecular analysis.

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48 A, G542X, G551D, R553X, R560T, S549R, S549N, 3849 þ 10kbC ! Login to comment

[hide] Screening for cystic fibrosis in newborn infants: ... J Med Screen. 2002;9(2):60-3. Corbetta C, Seia M, Bassotti A, Ambrosioni A, Giunta A, Padoan R
Screening for cystic fibrosis in newborn infants: results of a pilot programme based on a two tier protocol (IRT/DNA/IRT) in the Italian population.
J Med Screen. 2002;9(2):60-3., [PMID:12133923]

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OBJECTIVE: To assess the performance of a two tier neonatal screening programme (IRT/DNA/IRT) for cystic fibrosis, based on immunoreactive trypsinogen (IRT) followed by direct cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis (based on a panel of up to 31 mutations) in hypertrypsinaemic newborn infants and to compare it with a previous screening protocol. SETTING: The study comprised all the newborn infants in the period 1 October 1998 to 31 December 1999 in the Lombardia region, north western Italy. METHODS: The screening strategy consisted of an immunoreactive trypsinogen assay from dried blood spots, a polymerase chain reaction (PCR) followed by an oligonucleotide ligation assay (PCR-OLA), and a sequence code separation. RESULTS: 104 609 newborn infants were screened. 1457 hypertrypsinaemic infants (1.39%) were analysed with the PCR-OLA assay. 18 newborn homozygotes or compound heterozygotes for CFTR mutations were identified and referred to the cystic fibrosis (CF) centre at a mean age of 3 weeks. 125 infants presenting only one mutation were recalled for a sweat test: a diagnosis of CF was made in 13 infants, and parents of 112 neonates identified as carriers (1:13) received genetic counselling. The remaining 1314 hypertrypsinaemic newborn infants were recalled for IRT retesting and 177 were referred for a sweat test because the second IRT measurement was above the cut off value. Among this group a further two infants were diagnosed with CF (1.1%) leading to a CF prevalence of 1:3170. CONCLUSIONS: This strategy resulted in an early and accurate diagnosis of CF. The IRT/DNA/IRT protocol with an OLA assay was shown to be useful in an Italian population with a genetic heterogeneity, leading to the identification of 94% of infants with CF.

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266 Mutations identified by the assay are G85E, 621+1G→T, R117H, Y122X, 711+1G→T, 1078delT, R347P, R347H, R334W, A455E, 1898+1G→A, 2183-AA→G, 2789+5G→A, delF508, I507del, Q493X, V520F, 1717-1G→A, G542X, G551D, R553X, R560T, S549R, S549N, 3849+10kbC→T, 3849+4A→G, R1162X, 3659delC, W1282X, 3905insT, and N1303K. Login to comment

[hide] Analysis by mass spectrometry of 100 cystic fibros... Hum Reprod. 2002 Aug;17(8):2066-72. Wang Z, Milunsky J, Yamin M, Maher T, Oates R, Milunsky A
Analysis by mass spectrometry of 100 cystic fibrosis gene mutations in 92 patients with congenital bilateral absence of the vas deferens.
Hum Reprod. 2002 Aug;17(8):2066-72., [PMID:12151438]

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BACKGROUND: Limited mutation analysis for congenital bilateral absence of the vas deferens (CBAVD) has revealed only a minority of men in whom two distinct mutations were detected. We aimed to determine whether a more extensive mutation analysis would be of benefit in genetic counselling and prenatal diagnosis. METHODS: We studied a cohort of 92 men with CBAVD using mass spectrometry and primer oligonucleotide base extension to analyse an approximately hierarchical set of the most common 100 CF mutations. RESULTS: Analysis of 100 CF mutations identified 33/92 (35.9%) patients with two mutations and 29/92 (31.5%) with one mutation, compound heterozygosity accounting for 94% (31/33) of those with two mutations. This panel detected 12.0% more CBAVD men with at least one mutation and identified a second mutation in >50% of those considered to be heterozygotes under the two routine 25 mutation panel analyses. CONCLUSION: Compound heterozygosity of severe/mild mutations accounted for the vast majority of the CBAVD patients with two mutations, and underscores the value of a more extensive CF mutation panel for men with CBAVD. The CF100 panel enables higher carrier detection rates especially for men with CBAVD, their partners, partners of known CF carriers, and those with 'mild' CF with rarer mutations.

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20 Given the frequency of CF mutations, especially in the Caucasian population ( in 25), and the common request by CBAVD men to sire their own offspring by using surgical Table I. The 100 most common cystic fibrosis mutations listed by exon Mutationa Exonb Frequency (%)c G85E 3 0.1 394delTT 3 Swedish E60X 3 Belgium R75X 3 405ϩ1G→A Int 3 R117H 4 0.30 Y122X 4 French 457TAT→G 4 Austria I148T 4 Canada (French Canadian) 574delA 4 444delA 4 R117L 4 621ϩ1G→T Int 4 0.72 711ϩ1G→T Int 5 Ͼ0.1 712-1G→T Int 5 711ϩ5G→A Int 5 Italy (Caucasian) L206W 6a R347P 7 0.24 1078delT 7 Ͼ0.1 R334W 7 Ͼ0.1 1154InsTC 7 T338I 7 Italy R347H 7 Turkey Q359K/T360K 7 Israel (Georgian Jews) I336K 7 R352Q 7 G330X 7 S364P 7 A455E 9 0.20 I507 10 0.21 F508 10 66.02 1609delCA 10 Spain (Caucasian) V520F 10 Q493X 10 C524X 10 G480C 10 Q493R 10 1717-1G→A Int 10 0.58 R553X 11 0.73 G551D 11 1.64 G542X 11 2.42 R560T 11 Ͼ0.1 S549N 11 Q552X 11 Italy S549I 11 Israel (Arabs) A559T 11 African American R553G 11 R560K 11 1812-1G→A Int 11 A561E 12 E585X 12 Y563D 12 Y563N 12 1898ϩ1G→A Int 12 0.22 1898ϩ1G→C Int 12 2183AA→G 13 Italian 2184delA 13 Ͻ0.1 K710X 13 2143delT 13 Moscow (Russian) 2184InsA 13 1949del84 13 Spain (Spanish) 2176InsC 13 2043delG 13 2307insA 13 2789ϩ5G→A Int 14b Ͼ0.1 2869insG 15 S945L 15 Q890X 15 3120G→A 16 2067 Table I. continued Mutationa Exonb Frequency (%)c 3120ϩ1G→A Int 16 African American 3272-26A→G Int 17a R1066C 17b Portugal (Portugese) L1077P 17b R1070Q 17b Bulgarian W1089X 17b M1101K 17b Canada (Hutterite) R1070P 17b R1162X 19 0.29 3659delC 19 Ͼ0.1 3849G→A 19 3662delA 19 3791delC 19 3821delT 19 Russian Q1238X 19 S1235R 19 France, South S1196X 19 K1177R 19 3849ϩ10kbC→T Int 19 0.24 3849ϩ4A→G Int 19 W1282X 20 1.22 S1251N 20 Dutch, Belgian 3905insT 20 Swiss, Acadian, Amish G1244E 20 R1283M 20 Welsh W1282R 20 D1270N 20 S1255X 20 African American 4005ϩ1G→A Int 20 N1303K 21 1.34 W1316X 21 aMutations were chosen according to their frequencies (Cystic Fibrosis Genetic Analysis Consortium, 1994; Zielenski and Tsui, 1995; Estivill et al., 1997). Login to comment
34 The mutations in the 25 mutation panel were: ∆F508, G542X, N1303K, G551D, W1282X, 1717-1G→A, R553X, 621ϩ1G→T, R1162X, 2183AA→G, R117H, ∆I507, R560T, 3849ϩ10kbC→T, S549N, S549I, S549R, R1283M, R1283K, R553G, R560K, R117L, 1774delCT, 1811ϩ1G→C, and 4006-61del14. Login to comment
35 ACMG 25 mutation panel (ACMG25): The following mutations are the recommended core mutations for general population CF carrier screening by American College of Medical Genetics (ACMG) (Grody, et al 2001): ∆F508, G542X, N1303K, G551D, W1282X, 1717-1G→A, R553X, 621ϩ1G→T, R1162X, R117H, ∆I507, 1898ϩ1G→A, G85E, R347P, A455E, R560T, R334W, 3849ϩ10kbC→T, 3659delC, 1078delT, 2789ϩ5G→A, 711ϩ1G→T, 2184delA, 3120ϩ1G→A and I148T. Login to comment

[hide] Demographics of the UK cystic fibrosis population:... Eur J Hum Genet. 2002 Oct;10(10):583-90. McCormick J, Green MW, Mehta G, Culross F, Mehta A
Demographics of the UK cystic fibrosis population: implications for neonatal screening.
Eur J Hum Genet. 2002 Oct;10(10):583-90., [PMID:12357328]

Abstract [show]
The objective was to determine the composition of the Cystic Fibrosis (CF) Population attending specialist UK CF centres in terms of age, gender, age at diagnosis, genotype and ethnicity. With the planned introduction of the national CF screening programme in the UK, cystic fibrosis transmembrane regulator (CFTR) mutations were compared between different ethnic groups enabling a UK-specific frequency of mutations to be defined. Data were analysed from the patient biographies held in the UK CF Database (see www.cystic-fibrosis.org.uk). The currently registered population of 5,274 CF patients is 96.3% Caucasian with a male preponderance that significantly increases with age. The majority of the 196 non-Caucasian CF patients are from the Indian Subcontinent (ISC), of which one in 84 UK CF patients are of Pakistani origin. The commonest CFTR mutation, deltaF508, is found in 74.1% of all CF chromosomes. In the Caucasian CF population, 57.5% are deltaF508 homozygotes but the UK ISC CF population with only 24.7%, has significantly fewer deltaF508 homozygotes patients (95% confidence interval (CI) 0.2-0.4). The distribution of Caucasian patients with deltaF508/deltaF508, deltaF508/Other and Other/Other does not fit the expected distribution with a Hardy-Weinberg model unless those patients without a detected mutation are excluded (P<0.001). The UK CF Database has shown the UK CF population to have distinct characteristics separate from the North American and European CF Registries. The ISC group contains many mutations not recognised by current genetic analysis, and one in four ISC patients have no CFTR mutations identified. The CFTR analysis proposed for the screening programme would detect 96% of patients registered in the database, but is unlikely to achieve the desired >80% detection rates in the ethnic minority groups. Screen-positive, non-Caucasian infants without an identifiable CFTR mutation should be referred for a sweat test and genetic counselling when serum trypsinogen concentrations remain elevated after birth.

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79 It is envisaged that the proposed screening programme will be based on a three-stage protocol.6 In Table 3 Genotypes of the UK CF Caucasian and ISC populations Percentage of Percentage of genotyped UK CF genotyped UK CF Caucasian population ISC population Genotype n=4753 (%) n=78 (%) DF508/DF508 57.5 24.7 DF508/Unknown 11.5 3.5 DF508/G551D 5.1 0.0 DF508/G542X 2.8 0.0 Unknown/Unknown 2.7 27.1 DF508/621+1G?T 2.0 1.2 DF508/R117H 2.0 0.0 DF508/1898+1G?A 1.0 0.0 DF508/1717-G?A 0.9 0.0 DF508/N1303K 0.8 0.0 DF508 DI507 0.8 0.0 DF508/R553X 0.6 0.0 DF508/R560T 0.6 0.0 DF508/Q493X 0.5 0.0 G551D/Unknown 0.4 0.0 Other/Other 2.8 15.3* DF508/Other 6.7 0.0 Y569D/Y569D 0.0 8.2 L218X/L218X 0.0 3.5 1161delC/1161delC 0.0 3.5 R709X/V456A 0.0 2.4 G542X/G542X 0.4 2.4 Other/Unknown 1.0 3.5 The shaded areas represent the commonest genotypes in the ISC population. Login to comment
85 Table 4 The commonest CFTR mutations in the UK Genotypes UK CF population Genotyped UK Caucasian CF Genotyped UK CF ISC (n=9866 chromosomes) population (n=9506 chromosomes) population (n=156 chromosomes) CFTR mutation gene frequency per 1000 genes gene frequency per 1000 genes gene frequency per 1000 genes DF508 741.0 752.0 294.9 G551D 33.7 34.3 12.8 G542X 18.5 18.4 25.6 R117H 12.5 12.7 0.0 621+1G?T 12.7 12.7 6.4 1717-1G?A 5.8 5.8 0.0 1898+1G?A 5.7 5.9 0.0 N1303K 5.6 5.4 0.0 DI507 4.8 5.0 0.0 R560T 4.2 4.3 0.0 R553X 3.3 3.4 0.0 1154insTC 3.2 3.3 0.0 Q493X 2.8 2.9 0.0 3659delC 2.8 2.9 0.0 E60X 2.4 2.4 0.0 W1282X 2.7 2.7 0.0 P67L 2.1 2.1 0.0 G85E 2.1 2.0 0.0 V520F 1.6 1.7 0.0 1078delT 1.3 1.4 0.0 Y569D 1.5 0.0 96.2 L218X 0.6 0.0 38.5 1161delC 0.7 0.1 38.5 R1162X 0.9 0.6 19.2 R709X 0.4 0.2 12.8 3849+10kbC?T 1.2 0.8 19.2 S549R* 0.6 0.0 0.0 *S549R mutations appear in the non-Caucasian but not the ISC subgroup. Login to comment

[hide] Standards and guidelines for CFTR mutation testing... Genet Med. 2002 Sep-Oct;4(5):379-91. Richards CS, Bradley LA, Amos J, Allitto B, Grody WW, Maddalena A, McGinnis MJ, Prior TW, Popovich BW, Watson MS, Palomaki GE
Standards and guidelines for CFTR mutation testing.
Genet Med. 2002 Sep-Oct;4(5):379-91., [PMID:12394352]

Abstract [show]
One mission of the ACMG Laboratory Quality Assurance (QA) Committee is to develop standards and guidelines for clinical genetics laboratories, including cytogenetics, biochemical, and molecular genetics specialties. This document was developed under the auspices of the Molecular Subcommittee of the Laboratory QA Committee by the Cystic Fibrosis (CF) Working Group. It was placed on the "fast track" to address the preanalytical, analytical, and postanalytical quality assurance practices of laboratories currently providing testing for CF. Due to the anticipated impact of the ACMG recommendation statement endorsing carrier testing of reproductive couples, it was viewed that CF testing would increase in volume and that the number of laboratories offering CF testing would also likely increase. Therefore, this document was drafted with the premise of providing useful information gained by experienced laboratory directors who have provided such testing for many years. In many instances, "tips" are given. However, these guidelines are not to be interpreted as restrictive or the only approach but to provide a helpful guide. Certainly, appropriately trained and credentialed laboratory directors have flexibility to utilize various testing platforms and design testing strategies with considerable latitude. We felt that it was essential to include technique-specific guidelines of several current technologies commonly used in laboratories providing CF testing, since three of the four technologies discussed are available commercially and are widely utilized. We take the view that these technologies will change, and thus this document will change with future review.

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307 ⌬F508 R553X R1162X 2184delA 3120ϩ1GϾA ⌬I507 G542X G551D W1282X N1303K 621ϩ1GϾT R117H 1717-1GϾA A455E R560T G85E R334W R347P 711ϩ1GϾT 1898ϩ1GϾA 1078delT 3849ϩ10kbCϾT 2789ϩ5GϾA 3659delC I148T CF 3.3.2 Inclusion of the common R117H mutation in the test panel screens for CBAVD as well as for CF: The phenotypic consequences of the R117H mutation are modulated in cis by the 5/7/9T polypyrimidine tract in intron 8 such that R117H/7T is associated with CBAVD and R117H/5T is associated with CF.34 Moreover, the 5T allele is associated as a trans mutation in CBAVD.35 It is recommended that the 5/7/9T variant be excluded from the routine carrier screen but tested as a reflex for carriers shown to be heterozygous for the R117H mutation. Login to comment

[hide] Survey of CF mutations in the clinical laboratory. BMC Clin Pathol. 2002 Nov 19;2(1):4. Huber K, Mirkovic B, Nersesian R, Myers A, Saiki R, Bauer K
Survey of CF mutations in the clinical laboratory.
BMC Clin Pathol. 2002 Nov 19;2(1):4., 2002-11-19 [PMID:12437773]

Abstract [show]
BACKGROUND: Since it is impossible to sequence the complete CFTR gene routinely, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations causing disease in a high percentage of patients. Thus, in a cohort of 257 persons that were referred to our laboratory for analysis of CF gene mutations, reverse line probe assays for the most common CF mutations were performed. These techniques were evaluated as routine first-line analyses of the CFTR gene status. METHODS: DNA from whole blood specimens was extracted and subjected to PCR amplification of 9 exons and 6 introns of the CFTR gene. The resulting amplicons were hybridised to probes for CF mutations and polymorphisms, immobilised on membranes supplied by Roche Molecular Systems, Inc. and Innogenetics, Inc. Denaturing gradient gel electrophoresis and sequencing of suspicious fragments indicating mutations were done with CF exon and intron specific primers. RESULTS: Of the 257 persons tested over the last three years (referrals based on 1) clinical symptoms typical for/indicative of CF, 2) indication for in vitro fertilisation, and 3) gene status determination because of anticipated parenthood and partners or relatives affected by CF), the reverse line blots detected heterozygote or homozygote mutations in the CFTR gene in 68 persons (26%). Eighty-three percent of those affected were heterozygous (47 persons) or homozygous (10 persons) for the DeltaF508 allele. The only other CF-alleles that we found with these tests were the G542X allele (3 persons), the G551D allele (3 persons), the 3849+10kb C-T allele (2 persons) the R117H allele (2 persons) and the 621+1G-T allele (1 person).Of the fifteen IVS8-5T-polymorphisms detected in intron 8, seven (47%) were found in males referred to us from IVF clinics. These seven 5T-alleles were all coupled with a heterozygous DeltaF508 allele, they make up 35% of the males with fertility problems (20 men) referred to us. CONCLUSIONS: In summary, the frequency of CF chromosomes in the cohort examined with these tests was 26%, with the DeltaF508 allele affecting 83% of the CF chromosomes. It is a substantial improvement for routine CF diagnostics to have available a test system for 30 mutations plus the polypyrimidine length variants in intron 8. Our results show that this test system allows a routine first-line analyses of the CFTR gene status.

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36 F508C, I507V, I506V polymorphism exon 11 1717-1G → A, G542X, S549N, G551D, R553X, R560T exon 20 W1282X exon 21 N1303K intron 19 3849+10kb C → T Innogenetics assay: exon 3 394delTT, G85E, E60X exon/intron 4 621+1G-T, R117H exon 7 1078delT, R347P, R334W exon 13 2143delT, 2183AA-G, 2184delA exon 19 R1162X, 3659delC intron 5 711+5G-A intron8/exon 9 A455E,, 5T,7T,9T intron 14b 2789+5G-A intron 19 3849+10kb C-T Table 2: Genotypes of patients with mutations, final results Group 1) (patients with symptoms typical for/indicative of CF) No. Login to comment

[hide] Cystic fibrosis mutation frequencies in an Irish p... Clin Genet. 2003 Feb;63(2):121-5. Devaney J, Glennon M, Farrell G, Ruttledge M, Smith T, Houghton JA, Maher M
Cystic fibrosis mutation frequencies in an Irish population.
Clin Genet. 2003 Feb;63(2):121-5., [PMID:12630958]

Abstract [show]
The incidence of cystic fibrosis (CF) at birth in Ireland is 1/1461. Neonate CF genetic testing is not routinely performed in Ireland. Currently, screening is only carried out where there is clinical evidence or a family history to suggest disease. Here we report the frequencies of common CF mutations occurring in an Irish population composed of samples collected from western, mid-western and southern regions of Ireland. Rarer CF mutations were also identified in a selected number of CF patients. In addition, a number of polymorphisms were identified, some of which are reported to be functionally and phenotypically important.

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17 Eight common mutations were screened using polymerase chain reaction-restriction enzyme analysis (PCR-REA): R117H, 1717±1G > A, DI507, DF508, G542X, G551D, R553X and R560T. Login to comment
26 PCR-REA An in-house PCR-REA procedure was used to screen for the eight common mutations (R117H, 1717±1G > A, DI507, DF508, G542X, G551D, R553X and R560T). Login to comment
65 Frequency of common CF mutations Mutation Numberof chromosomes Frequency (%) R117H 25 2.70 1717^1G >A 20 2.16 DI507 4 0.43 DF508 658 70.97 G542X 4 0.43 G551D 70 7.55 R553X 2 0.22 R560T 4 0.43 Total 788 85 Frequencypercentages areadjustedtorepresent 85%. Login to comment
76 The R560T mutation (0.43%) was found at a similar frequency to the UK (15) (0.42%) and Ireland (11) (0.8%) and was found at a lower frequency to that of Northern Ireland (13) (2.9%). Login to comment

[hide] Clinical characteristics and genotype analysis of ... Clin Otolaryngol Allied Sci. 2003 Apr;28(2):125-32. Cimmino M, Cavaliere M, Nardone M, Plantulli A, Orefice A, Esposito V, Raia V
Clinical characteristics and genotype analysis of patients with cystic fibrosis and nasal polyposis.
Clin Otolaryngol Allied Sci. 2003 Apr;28(2):125-32., [PMID:12680831]

Abstract [show]
The prevalence of nasal polyps in a group of paediatric patients with cystic fibrosis was prospectively studied in comparison with a control group with cystic fibrosis but without polyps. Clinical variables, including pulmonary function tests, skin testing and mucociliary transport, were carried out in both groups, as well as genotype analysis. Endoscopic intranasal evaluation identified polyps in 29 of 89 patients (33%). Statistical analysis revealed that patients with nasal polyposis had better pulmonary function, a higher rate of Pseudomonas aeruginosa colonization, more hospitalizations, and more prevalence of allergy to Aspergillus fumigatus than did the comparison group. We found no statistically different genotype distribution between the polyposis and the control group. However, it can be emphasized that the prevalence of the compound heterozygous genotype is higher in the nasal polyposis group than in controls. Our observations suggest that other genetic and environmental factors could play an important role in the development of nasal polyposis.

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47 Analysis of mutations in the CFTR gene as tested by the multiplex polymerase chain reaction (PCR), followed by the reverse dot-blot technique, which searches for 29 of the most frequent mutations (DF508, N1303K, G542X, W1282X, 1717±1 G-A, R553X, 2183 AA-G, DI507, G551D, R560T, 3849 10kbC > T, R1162X, 3659delC, 3905insT, G85E, 621 1GT, R117H, R347P, R334W, A455E, 2789 5GA, Q552X, S1251N, 3905insT, 394delTT, E60X, 2143delT, 2184delA, 711 5G > A), and by ASO dot-blot for the following mutations: I148T, R1158X, 4016 1T, G1244E G >A.26 Statistical analysis was performed using multivariate analysis, by forward stepwise comparison; it was done to ®nd out which of the examined characteristics could be associated (P < 0.01) to nasal polyposis. Login to comment

[hide] Analysis of cystic fibrosis transmembrane conducta... Am J Med Genet A. 2003 Jul 1;120A(1):72-6. Timmreck LS, Gray MR, Handelin B, Allito B, Rohlfs E, Davis AJ, Gidwani G, Reindollar RH
Analysis of cystic fibrosis transmembrane conductance regulator gene mutations in patients with congenital absence of the uterus and vagina.
Am J Med Genet A. 2003 Jul 1;120A(1):72-6., 2003-07-01 [PMID:12794695]

Abstract [show]
The relationship between cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations and congenital absence of the uterus and vagina (CAUV) was examined. CFTR mutations have previously been associated with congenital bilateral absence of the vas deferens (CBAVD). CBAVD is caused by a disruption in the vas deferens, a Wolffian duct derivative. Because the embryologic development of the Mullerian ducts directly depends on the prior normal development of the Wolffian ducts, the same gene products may be necessary for normal embryologic development of both ductal systems. This study evaluated the role of CFTR mutations in the development of CAUV. DNA samples from 25 patients with CAUV were tested for the presence of 33 of the most common CFTR mutations. Protein-coding DNA fragments from the CFTR gene were amplified in vitro by the polymerase chain reaction (PCR) and analyzed for mutations using allele-specific oligonucleotide (ASO) probes. Two patients were heterozygous for CFTR mutations. One was heterozygous for the W1282X mutation and the other was heterozygous for the DeltaF508 mutation. The incidence of the 33 CFTR mutations found in the patients with CAUV (8%) was twice that found in the general population (4%), but much less than the incidence of CFTR mutations in men with CBAVD (80%). This data suggests that it is unlikely for CFTR mutations to cause CAUV in females as they cause CBAVD in some males. Furthermore, the data suggest that CAUV in females may be the same disorder as CBAVD in males who do not have CFTR mutations.

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82 CFTR Gene Mutations Tested DF508 R334W Y1092X 5T variant Y122X R347H G542X S549R 3,849 þ 4 G551D 3,849 þ 10 kb 2,789 þ 5 W1282X R553X 711 þ 1 3,905 þ T 621 þ 1 1,898 þ 1 N1303K 1,717À1 R1162X R117H 1078dT A455E D1507 Q493X 218dA R347P V520F G85E R560T S549N 3659dC Wolffian duct must occur at a time when the Mu¨llerian duct is no longer dependent on the Wolffian duct for development. Login to comment

[hide] Comparison of the CFTR mutation spectrum in three ... Hum Mutat. 2003 Jul;22(1):105. Scotet V, Barton DE, Watson JB, Audrezet MP, McDevitt T, McQuaid S, Shortt C, De Braekeleer M, Ferec C, Le Marechal C
Comparison of the CFTR mutation spectrum in three cohorts of patients of Celtic origin from Brittany (France) and Ireland.
Hum Mutat. 2003 Jul;22(1):105., [PMID:12815607]

Abstract [show]
This study aims to compare the spectrum of the mutations identified in the gene responsible for cystic fibrosis in three cohorts of patients of Celtic origin from Brittany and Ireland. It included 389 patients from Brittany, 631 from Dublin and 139 from Cork. The CFTR gene analysis relied on the detection of the most common mutations, followed by a complete gene scanning using DGGE or D-HPLC. High mutation detection rates were obtained in each cohort: 99.6%, 96.8%, and 96.0% respectively. A high frequency of the c.1652_1655 del3 mutation (F508del: 74.8% to 81.3%) and of the "Celtic" mutation (c.1784G>A (G551D): 3.7% to 9.7%) was observed in each population. Apart from this, the mutation spectrums differed. In Brittany, the most common abnormalities were: c.1078delT (3.6%), c.4041C>G (N1303K: 1.4%), c.2670G>A (W846X(2): 1.0%) and c.1717-1G>A (1.0%), whereas in the cohort of Dublin, the main mutations were: c.482G>A (R117H: 3.0%), c.1811G>C (R560T: 2.4%) and c.621+1G>T (1.7%). Finally, in the Cork area, only the c.482G>A mutation (R117H) reached a frequency of 1%. Two previously-unreported mutations were identified in the Dublin cohort: c.2623-2A>G and c.3446T>G (M1105R). This collaborative study highlights the similarities of the CFTR alleles in the Breton and Irish populations, but also the disparities that exist between these populations, despite their common origin. Each population has its own history, with its mixture of founder effects and genetic drifts, which are at the origin of the current mutation distribution. The molecular study of the CFTR gene provides new tools for retracing European populations' histories.

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7 In Brittany, the most common abnormalities were: c.1078delT (3.6%), c.4041C>G (N1303K: 1.4%), c.2670G>A (W846X2: 1.0%) and c.1717-1G>A (1.0%), whereas in the cohort of Dublin, the main mutations were: c.482G>A (R117H: 3.0%), c.1811G>C (R560T: 2.4%) and c.621+1G>T (1.7%). Login to comment
44 Firstly, the National Centre for Medical Genetics, Dublin performed an analysis of the most common CFTR mutations, using the ARMS test (Ferrie et al., 1992), which enables the detection of the following mutations: F508del, R117H, I507del, G542X, G551D, R560T, N1303K, R352Q, 1717-1G>A and 621+1G>T. Login to comment
50 Dublin Centre 1262 CF alleles 35 mutations F508del 76.5% G551D 6.5% R117H 3.0% R560T 2.4% 621+1G>T 1.7% Cork Area 278 CF alleles 10 mutations F508del 81.3% G551D 9.7% R117H 1.4% Brittany 778 CF alleles 62 mutations F508del 74.8% G551D 3.7% 1078delT 3.6% N1303K 1.4% W846X2 1.0% 1717-1G>A 1.0% Statistical analysis We determined the spectrum of the CFTR mutations identified in the three cohorts of patients and compared their respective frequencies by a Chi square test. Login to comment
64 Spectrum of the CFTR Mutations Identified in the Cohorts from Brittany, Dublin Centre, and Cork Area Nucleotide Amino acid change * change Exon Number Frequency Number Frequency Number Frequency 211delG 2 1 0.1% 310G>T E60X 3 5 0.6% 4 0.3% 347C>A A72D 3 1 0.1% 368G>A W79X 3 1 0.1% 386G>A G85E 3 2 0.3% 3 0.2% 403G>A G91R 3 2 0.3% 482G>A R117H 4 4 0.5% 38 3.0% 4 1.4% 498T>A Y122X 4 1 0.1% 574delA 4 1 0.1% 577G>A G149R 4 1 0.1% 621+1G>T int 4 5 0.6% 21 1.7% 790C>T Q220X 6a 1 0.1% 875+1G>C int 6a 1 0.4% 905delG 6b 1 0.1% 1065C>G F311L 7 2 0.3% 1078delT 7 28 3.6% 1132C>T R334W 7 1 0.1% 1172G>A R347H 7 5 0.6% 1172G>T R347L 7 1 0.1% 1172G>C R347P 7 1 0.1% 1187G>A R352Q 7 3 0.2% 2 0.7% 1208A>G Q359R 7 1 0.1% 1154insTC 7 2 0.2% 1221delCT 7 2 0.3% 1248+1G>A int 7 1 0.1% 1249-27delTA int 7 1 0.4% 1334G>A W401X 8 1 0.1% 1461ins4 9 5 0.4% 1471delA 9 2 0.2% 1607C>T S492F 10 2 0.3% 1609C>T Q493X 10 1 0.1% 1648_1653delATC I507del 10 3 0.4% 10 0.8% 1 0.4% 1652_1655del 3 bp F508del 10 582 74.8% 966 76.5% 226 81.3% 1690G>T V520F 10 4 0.3% 1717-1G>A int 10 8 1.0% 9 0.7% 1756G>T G542X 11 5 0.6% 8 0.6% 1779T>G S549R 11 1 0.1% 1784G>A G551D 11 29 3.7% 82 6.5% 27 9.7% 1789C>G R553G 11 1 0.1% 1789C>T R553X 11 3 0.4% 1 0.1% 1806delA 11 1 0.1% 1811G>A R560K 11 2 0.3% 1811G>C R560T 11 30 2.4% 2 0.7% 1819T>A Y563N 12 1 0.1% 1853C>A P574H 12 1 0.1% 1898+1G>A int 12 1 0.1% 2184delA 13 1 0.1% 1 0.1% 2184insA 13 1 0.1% 2622+1G>A int 13 1 0.1% 2 0.2% 2622+1G>T int 13 1 0.1% 2623-2A>G ** int 13 1 0.1% 2670G>A W846X2 14a 8 1.0% 2752-1G>T int 14a 1 0.1% 2752-26A>G int 14a 2 0.2% 2789+5G>A int 14b 6 0.8% 2966C>T S945L 15 2 0.3% 3007delG 15 4 0.3% 3040G>C G970R 15 1 0.1% 3062C>T S977F 16 1 0.1% 3120+1G>A int 16 1 0.1% 3272-26A>G int 17a 4 0.5% 2 0.2% 2 0.7% 3320dupli(CTATG) 17b 1 0.1% 3329G>A R1066H 17b 1 0.1% 3340C>T R1070W 17b 1 0.1% 3408C>A Y1092X 17b 7 0.9% 3442G>T E1104X 17b 1 0.1% 3446T>G ** M1105R 17b 1 0.1% 3586G>C D1152H 18 1 0.1% 3601-17T>C + 1367delC int 18 + 9 1 0.1% 3616C>T R1162X 19 1 0.1% 2 0.2% 3659delC 19 2 0.2% 3832A>G I1234V 19 2 0.3% 3849+4A>G int 19 1 0.1% 3849+10kbC>T int 19 3 0.2% 3877G>A G1249R 20 1 0.1% 3884G>A S1251N 20 1 0.1% 3898insC 20 1 0.1% 3905insT 20 2 0.3% 3978G>A W1282X 20 3 0.4% 4005+1G>A int 20 6 0.8% 4016insT 21 1 0.1% 4041C>G N1303K 21 11 1.4% 5 0.4% 4136T>C L1335P 22 1 0.1% 1 0.4% 4279insA 23 1 0.1% Unidentified Unidentified - 3 0.4% 41 3.2% 11 4.0% Total 778 100.0% 1262 100.0% 278 100.0% * All nucleotide changes correspond to cDNA numbering. Login to comment
73 In the cohort from Dublin, the three mutations presenting a frequency greater or equal to 1% were different: R117H (3.0%), R560T (2.4%) and 621+1G>T (1.7%). Login to comment
75 Finally, the main differences observed between these cohorts were the high frequency of the 1078delT mutation in Brittany (3.6%) and of the R560T in the Dublin cohort (2.4%). Login to comment
83 The other most frequent genotypes were: F508del/G551D (9.5%), F508del/R117H (3.5%) and F508del/R560T (2.7%). Login to comment
98 Number Frequency Number Frequency 1652_1655del 3 bp F508del 966 76.5% 226 81.3% 1192 77.4% 1784G>A G551D 82 6.5% 27 9.7% 109 7.1% 482G>A R117H 38 3.0% 4 1.4% 42 2.7% 1811G>C R560T 30 2.4% 2 0.7% 32 2.1% 621+1G>T 21 1.7% 21 1.4% 1648_1653delATC I507del 10 0.8% 1 0.4% 11 0.7% 1717-1G>A 9 0.7% 9 0.6% 1756G>T G542X 8 0.6% 8 0.5% 1187G>A R352Q 3 0.2% 2 0.7% 5 0.3% 1461ins4 5 0.4% 5 0.3% 4041C>G N1303K 5 0.4% 5 0.3% 310G>T E60X 4 0.3% 4 0.3% 1690G>T V520F 4 0.3% 4 0.3% 3007delG 4 0.3% 4 0.3% 3272-26A>G 2 0.2% 2 0.7% 4 0.3% 386G>A G85E 3 0.2% 3 0.2% 3849+10kbC>T 3 0.2% 3 0.2% Unidentified Unidentified 41 3.2% 11 4.0% 52 3.4% Total Total 1262 100.0% 278 100.0% 1540 100.0% Dublin cohort Cork cohort Ireland Amino acid change Nucleotide change We noted similar high frequencies of the F508del and G551D mutations in the three cohorts studied. Login to comment
109 The R560T, frequent in this cohort, has also been found in Northern Ireland (2.6%) (Bobadilla et al., 2002), whereas the 621+1G>T has also been observed with an abnormally high frequency in Saguenay-Lac-Saint-Jean, Quebec (23%), giving evidence of a founder effect (Rozen et al., 1992). Login to comment

[hide] Molecular consequences of cystic fibrosis transmem... Gut. 2003 Aug;52(8):1159-64. Ahmed N, Corey M, Forstner G, Zielenski J, Tsui LC, Ellis L, Tullis E, Durie P
Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas.
Gut. 2003 Aug;52(8):1159-64., [PMID:12865275]

Abstract [show]
BACKGROUND AND AIMS: We tested the hypothesis that the actual or predicted consequences of mutations in the cystic fibrosis transmembrane regulator gene correlate with the pancreatic phenotype and with measures of quantitative exocrine pancreatic function. METHODS: We assessed 742 patients with cystic fibrosis for whom genotype and clinical data were available. At diagnosis, 610 were pancreatic insufficient, 110 were pancreatic sufficient, and 22 pancreatic sufficient patients progressed to pancreatic insufficiency after diagnosis. RESULTS: We identified mutations on both alleles in 633 patients (85.3%), on one allele in 95 (12.8%), and on neither allele in 14 (1.9%). Seventy six different mutations were identified. The most common mutation was DeltaF508 (71.3%) followed by G551D (2.9%), G542X (2.3%), 621+1G-->T (1.2%), and W1282X (1.2%). Patients were categorized into five classes according to the predicted functional consequences of each mutation. Over 95% of patients with severe class I, II, and III mutations were pancreatic insufficient or progressed to pancreatic insufficiency. In contrast, patients with mild class IV and V mutations were consistently pancreatic sufficient. In all but four cases each genotype correlated exclusively with the pancreatic phenotype. Quantitative data of acinar and ductular secretion were available in 93 patients. Patients with mutations belonging to classes I, II, and III had greatly reduced acinar and ductular function compared with those with class IV or V mutations. CONCLUSION: The predicted or known functional consequences of specific mutant alleles correlate with the severity of pancreatic disease in cystic fibrosis.

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309 Table 2 Genotype classification according to the functional consequences of CFTR gene mutations Pancreatic status Class I Class II Class III Class IV Class V PS F1 , 875+1G→C(2) F, F (1) F, G551D (1) F, R117H (11) F,3849+10kbC→T (5) F, G85E2 (1) F, R347H (3) F,3272-26A→G (4) F, S1251N (2) F,A445E (3) F, D614G (1) F,P574H (2) F, R347P (1) F,3120G>A (1) R117H,R117H (1) F, 5T (8) F, L1335P (1) F,2789+5G→A (1) F,P67L (1) F,R347P/R347H (1) F,V232D(2) R334W, R334W(1) PS→PI F,3659delC (1) F,F (15) F,G551D (1) F, I1234V (1) F,2184insA (1) F,R560T (1) PI F, G542X (27) F,F (365) F, G551D (28) F, 621+1G→T (13) F, R560T (7) F,R553X (7) F, N1303K (9) F, R1162X (6) F,L1077P (2) F, 3659delC (5) F, I48T (1) F, 1717-1G→A (5) F,A559T (1) F, W1282X (5) F, G85E2 (2) F, 711+1G→T (5) G551D,G551D(1) F,2184delA(4) F,H199R (1) W1282X,W1282X (4) F,I1072T(1) F,Y1092X (3) F,S549 (R75Q) (1) F,556delA (3) F, Q493X (3) F,4016InsT (3) F, 3120+1G→A (2) F, G551D/R553X (2) F,Q814X(2) F,1154insTC (2) F,441delA (1) F, 4326delTC (1) F,Q552X(1) F,3007delG (1) F,2184insA (1) F, 4010del4 (1) F,3905insT (1) F,1078delT(1) F,E1104X (1) F,3876delA (1) F,4374+1G→T (1) F,E585X (1) F, E60X (1) CFTR, cystic fibrosis transmembrane regulator; PI, pancreatic insufficiency; PS, pancreatic sufficiency. Login to comment

[hide] Detection of cystic fibrosis mutations by peptide ... Clin Chem. 2003 Aug;49(8):1318-30. Malehorn DE, Telmer CA, McEwen SB, An J, Kinsey AD, Retchless AC, Mason C, Vieta WM, Jarvik JW
Detection of cystic fibrosis mutations by peptide mass signature genotyping.
Clin Chem. 2003 Aug;49(8):1318-30., [PMID:12881448]

Abstract [show]
BACKGROUND: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis. METHODS: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149-54) were assessed using the PMSG test for the CFTR gene. RESULTS: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. CONCLUSIONS: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.

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138 ⌬b 3 R Y 9863.78 G85E SerϾPhe 9923.90 Y 60.12 4.1 R N 7047.69 R117H AlaϾVal 7075.76 N 28.07 4.2 R Y 11161.32 lI48T AsnϾSer 11134.32 Y -27.00 621ϩ1 GϾT TyrϾTAA 6513.09 N -4648.23 5 R Y 11081.45 711ϩ1 GϾT ThrϾAsn 11094.48 Y 13.03 7.1 R N 7383.08 1078⌬T frameshift 9201.10 Y 1818.02 7 R Y 12233.9 R334W ArgϾGln 12205.87 Y -28.03 R347P ArgϾGly 12134.79 Y -99.11 9 F Y 14049.68 A455E AlaϾGlu 14107.74 Y 58.06 10.2 R Y 10525.57 ⌬I507 ⌬ Asp 10410.50 Y -115.07 ⌬F508 ⌬ Asp & LysϾAsn 10396.43 Y -129.14 11.2 F Y 11173.32 1717-1 GϾA GlyϾArg 11272.46 Y 99.14 G542X TrpϾLeu 11100.27 Y -73.05 G551D no change 11173.32 Y 0.00 R553X ThrϾMet 11203.42 Y 30.10 R560T no change 11173.32 Y 0.00 11 F N 8465.27 1717-1 GϾA no change 8465.27 N 0.00 G542X GlyϾTGA 6584.17 N -1881.10 G551D GlyϾAsp 8523.33 N 58.06 R553X ArgϾTGA 7541.18 N -924.09 R560T ArgϾThr 8410.21 N -55.06 12 F Y 10372.51 1898ϩ1 GϾA GlyϾAsp 10430.57 Y 58.06 13.2A R Y 10103.23 2184⌬A frameshift 8726.91 N -1376.32 14B R Y 9291.17 2789ϩ5 GϾA LeuϾPhe 9325.21 Y 34.04 16 F N 9398.67 3120ϩ1 GϾA ValϾIle 9412.72 N 14.05 19 F Y 17455.96 R1162X ArgϾTGA 6280.13 N -11175.83 3659⌬C frameshift 9650.06 N -7805.90 19i F Y 9699.9 3849ϩ10kB CϾT ArgϾTGA 7131.04 N -2568.86 20 F N 11125.48 W1282X TrpϾTGA 9370.40 N -1755.08 21 F Y 11183.44 N1303K AsnϾLys 11197.54 Y 14.10 a Denotes the directionality of exonic sequence when expressed as peptide. Login to comment
181 The heterozygous mutations depicted are as follows: (A), exon 3 wt/G85E; (B), exon 4.1 wt/R117H; (C), exon 4.2 wt/I148T; (D), exon 4.2 wt/621 ؉ 1G>T; (E), exon 5 wt/711 ؉ 1G>T; (F), exon 7.1 wt/1078⌬T; (G), exon 7 wt/R334W; (H), exon 7 wt/R347P; (I), exon 9 wt/A455E; (J), exon 10.2 wt/⌬I507; (K), exon 10.2 wt/⌬F508; (L), exon 11.2 wt/1717-1G>A; (M), exon 11 wt/G542X; (N), exon 11 wt/G551D; (O), exon 11 wt/R553X; (P), exon 11 wt/R560T; (Q), exon 12 wt/1898 ؉ 1G>A; (R), exon 13.2A wt/2184⌬A; (S), exon 14B wt/2789 ؉ 5G>A; (T), exon 16 wt/3120 ؉ 1G>A; (U), exon 19 wt/R1162X; (V), exon 19 wt/3659⌬C; (W), intron 19 wt/3849 ؉ 10kbC>T; (X), exon 20 wt/W1282X; (Y), exon 21 wt/N1303K. typical yield of purified protein was 1-30 ␮g/test well, depending on the analyte species. Login to comment
229 Mutations predicted on the basis of their peptide mass Table 2. Summary of PMSG screening of putative compound heterozygous patient samples.a Exon Sample P154 P156 P158 P164 P165 P166 P168 P169 P175 P176 3.1 9871 9868 9872 9867 9863 9861 9866 9867 9861 9868 4.1 7054 7052 7057 7049 7048 7039 7048 7044 7047 7046 4.2 11172 11164 11175 11164 11157 11166 11159 11158 11163 11156 5 11096 11084 11098 11088 11088 11071 11084 11079 11076 11085 7.1 7386 7392 7382 7390 7382 7383 7379 7380 7387 7386 7 12232 12229 12234 12231 12237 12238 12239 12239 12240 12238 9 14064 14060 14065 14056 14062 14045 14050 14049 NAb 14051 10.2 10534 10531 10542 10533 No peakc 10525 10528 10527 10527 10524 Mutant 10404 10399 10409 10401 10400 10396 10398 10397 11.2 11186 11180 11182 11182 11179 11168 11175 11178 11075 11179 Mutant 11112 11205 11209 11105 11106 11 8477 8470 8477 8469 8467 8459 8468 8465 8465 ‫؍‬ supd 8459 ‫؍‬ supd Mutant 6591 8420 8427 7541 7539 8409 & 6581 8403 & 6576 12 10382 10376 10394 10379 10385 10365 10370 10370 10378 10366 13.2A 10103 10104 10103 10104 10105 10099 10099 10100 10098 10100 Mutant 8723 14B 9299 9294 9306 9300 9293 9283 9289 9291 9295 9294 16 9414 9403 9408 9402 9409 9391 9400 9396 9398 9396 19 17486 17476 17478 17481 17452 17447 17472 17453 17461 17448 Intron 19 9712 9709 9708 9709 9714 9696 9697 9704 9702 9700 20 11138 11128 11138 11135 11131 11117 NA 11122 11120 11116 Mutant 9372 21 11191 11189 11190 11187 11185 11181 11183 11185 11187 11183 Sequence result ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 G542X G542X G542X W1282X R560T G551D ⌬F508 2183AAϾG R553X R553X R560T R560T a Shaded boxes highlight test analytes revealing evidence of mutation. Login to comment
256 This pooling is more noticeable in the diminution of the signal for the mutations of exon 11 relative to the wild-type analyte (e.g., mutants G551D and R560T in Fig. 3). Login to comment

[hide] HFE alleles in an Irish cystic fibrosis population... Genet Test. 2003 Summer;7(2):155-8. Devaney J, Maher M, Smith T, Houghton JA, Glennon M
HFE alleles in an Irish cystic fibrosis population.
Genet Test. 2003 Summer;7(2):155-8., [PMID:12885340]

Abstract [show]
The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. Genetic and environmental factors that determine whether an individual will develop associated complications are still being determined. It has been proposed that the gene for hemochromatosis, HFE, may be a modifier locus for CF disease phenotype. Recent research has suggested a relationship between mutations to the HFE gene and the development of meconium ileus (MI) and liver disease in CF. This study aims to expand our knowledge of the HFE mutations C282Y and H63D carrier rate in an Irish population of CF allele carriers. PCR restriction enzyme analysis was performed on blood samples from CF patients to identify the C282Y and H63D mutations. HFE status of CF allele carriers and CF patients (Delta F508) homozygotes with and without meconium ileus was determined. The carrier frequency for C282Y was 30.8% for the Delta F508 homozygote MI positive group, as compared to 12.5% for the non-Delta F508 MI positive group but did not reach statistical significance (p = 0.27). Interestingly, no Delta F508 homozygote patients were homozygous for the C282Y mutation.

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57 Our non-DF508 CF patients were screened for the R117H, 1717-1G R A, DI507, G542X, G551D, R553X, R560K, and R560T CFTR mutations prior to inclusion in this study; it is interesting to note that the G542X and G551D alleles have positive and negative associations, respectively, with MI development (Schwarz et al., 1995; Feingold and Gailloud-Bataille, 1999). Login to comment

[hide] Mutation analysis of the cystic fibrosis transmemb... Eur J Hum Genet. 2003 Sep;11(9):687-92. Perri F, Piepoli A, Stanziale P, Merla A, Zelante L, Andriulli A
Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the serine protease inhibitor, Kazal type 1 (SPINK1) gene in patients with alcoholic chronic pancreatitis.
Eur J Hum Genet. 2003 Sep;11(9):687-92., [PMID:12939655]

Abstract [show]
Susceptibility to alcoholic chronic pancreatitis (ACP) could be genetically determined. Mutations in cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), and serine protease inhibitor, Kazal type 1 (SPINK1) genes have been variably associated with both the hereditary and the idiopathic form of chronic pancreatitis (CP). Our aim was to analyze the three genes in ACP patients. Mutational screening was performed in 45 unrelated ACP patients and 34 patients with alcoholic liver disease (ALD). No mutation of PRSS1 was found in ACP and ALD patients. Three mutations of CFTR were detected in four ACP patients with a prevalence (8.9%) not significantly different from that observed (3.0%) in ALD patients and from that expected (3.2%) in our geographical area. Neither compound heterozygotes for CFTR nor trans-heterozygotes for CFTR/SPINK1 were found. One ACP patient (2.2%) was found to carry the most common mutation (N34S) of SPINK1 compared to none of the ALD patients (P=NS). In five other patients (two with ACP and three with ALD) other rare variants, including P55S, were found. In contrast with the hereditary and the idiopathic forms of CP, in which mutations of PRSS1, CFTR, and SPINK1 genes may occur, ACP is still a "gene(s)-orphan" disease. The supposed genetic susceptibility to ACP relies on other yet unknown gene(s) which could affect the alcohol metabolism or modulate the pancreatic inflammatory response to alcohol abuse.

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33 Mutation screening of the CFTR gene The 31 most frequent mutations (F508del, I507del, G551D, G542X, N1303K, 1717-1G4A, W1282X, R553X, R347P, R347H, R334W, 3849+10kb C4T, R117H, 621+1G4T, A455E, S549N, R560T, S549R, V520F, Q493X, 3849+ 4A4G, 1078delT, R1162X, 3659delC, 3905insT, Y122X, 2183delAA4G, 2789+5G4A, 1898+1G4A, 711+1G4T, and G85E) were examined with the polymerase chain reaction (PCR) followed by an oligonucleotide ligation assay (OLA, Applied Biosystems, Foster City, CA, USA) and finally a sequence-coded separation. Login to comment

[hide] Genetic disorders of the pancreas. Gastroenterol Clin North Am. 2003 Sep;32(3):763-87. Morinville V, Perrault J
Genetic disorders of the pancreas.
Gastroenterol Clin North Am. 2003 Sep;32(3):763-87., [PMID:14562574]

Abstract [show]
The venues opened to all by the remarkable studies of the genome are just starting to become manifest; they can now distinguish different variants of a disease; they are given the tools to better understand the pathophysiology of illness; they hope to be able to provide better treatment alternatives to our patients. The examples described in this review demonstrate the applicability of these concepts to pancreatic disorders. Researchers may be just scratching the surface at this time, but the potential is enormous. Many philosophic and ethical questions need to be answered as physicians move along: Should all family members of an index case be screened? Who should pay for testing? Who should get results? But, without the participation of so many patients, their family members, and numerous volunteers, researchers would not have witnessed the bridging of so many gaps as they have so far. All of us may now look forward to the application of this incredible knowledge to the therapeutic solutions so eagerly awaited.

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30 The close monitoring of the families affected with this condition played an important role in the identification of their genetic anomaly; the S family, described by McElroy and Christiansen in 1972 [34], was to play a pivotal role in helping Whitcomb et al 25 years later to uncover the Table 1 Recent genetic information on pancreatitis in children Gene Chromosome Mutations References Cationic trypsinogen (protease, serine1; PRSSI) 7q35 R122H; N29I A16V; others [4,11-19] Pancreatic trypsin inhibitor (PSTI) (SPINK1-serine protease inhibitor, Kazal Type 1) 5 N34S [20-22] CFTR-cystic fibrosis transmembrane regulator 7 DF508; R117H; Q493X R560T; R553X; 5Tallele; 621 + 1(G!T) and others [23-27] Parathyroid cell receptor (CaR) 3 (3q21-24) N178D; R220Q; P221S; R648X; others [28-30] Lipoprotein lipase (LPL) 8 (8p22) N291S, S447X; G715A [31,32] Apolipoprotein C-II (apoC-II) 19 (19q13.2) Val 18, Gln 2 and others [31] chromosomal [11], then the genetic abnormality [1], while in France Le Bodic et al [12] identified a very similar anomaly in a family described in 1963 by Cornet et al [35]. Login to comment
77 Mutations, including delta F508, R117H, Q493X, 621 + 1 (G!T), R560T, R553X, were found at 2.5 times the frequency expected in the general population studied (600 controls included). Login to comment

[hide] Mutations of the CFTR gene in pancreatic disease. Pancreas. 2003 Nov;27(4):332-6. Pezzilli R, Morselli-Labate AM, Mantovani V, Romboli E, Selva P, Migliori M, Corinaldesi R, Gullo L
Mutations of the CFTR gene in pancreatic disease.
Pancreas. 2003 Nov;27(4):332-6., [PMID:14576497]

Abstract [show]
INTRODUCTION: An association has been found between CFTR gene mutations and chronic pancreatitis; however, there is a lack of information about the frequency of CFTR gene mutations in acute pancreatitis and in pancreatic cancer. AIM: To prospectively evaluate the prevalence of CFTR gene mutations in acute pancreatitis, chronic pancreatitis, and pancreatic cancer. METHODOLOGY: Ninety-eight consecutive patients were studied and divided into 3 groups: 34 patients with acute pancreatitis, 46 patients with chronic pancreatitis, and 18 patients with pancreatic cancer. The mutation analysis of the CFTR gene was carried out using diagnostic commercial kits for the simultaneous detection of 29 mutations and Tn polymorphism. RESULTS: Among the 98 patients studied, 12 (12.2%) had CFTR gene mutations: 2 of the 34 patients (5.9%) with acute pancreatitis, 9 of the 46 (19.6%) with chronic pancreatitis, and 1 of the 18 (5.6%) with pancreatic cancer. All the mutations were found in heterozygosis (2 DeltaF508, 1 W1282X, and 9 T5 allele). CONCLUSION: Our prospective study adds further information about the frequency of CFTR mutations in patients with a single episode of acute pancreatitis. Furthermore, our results suggest an association of CFTR gene mutations with chronic alcoholic pancreatitis and emphasize the need for a multicenter study, possibly multinational, to conclusively establish the role of CFTR mutations as a genetic susceptibility factor for this disease.

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59 The 29 Mutations and the Tn Polymorphism Which Can Be Detected by INNO-LiPA Assays Mutation Exon/Intron (i) E60X, G85E, 394delTT 3 621 + 1G > T, R117H (i) 4, 4 711 + 5G > A (i) 5 1078delT, R347P, R334W 7 A455E, Tn (i) 8, 9 ⌬F508, ⌬I507 10 G542X, 1717-1 G > A, G551D, R553X, R560T, Q552X (i) 10, 11 2183AA > G, 2184del A, 2143delT 13 2789 + 5G > A (i) 14b R1162X, 3659delC 19 3849 + 10kbC > T (i) 19 3905insT, W1282X, S1251N 20 N1303K 21 Group 3: pancreatic cancer CFTR gene mutations were identified only in 1 of the 18 patients (5.6%) with this cancer. Login to comment

[hide] High allelic heterogeneity between Afro-Brazilians... Genet Test. 2003 Fall;7(3):213-8. Raskin S, Pereira L, Reis F, Rosario NA, Ludwig N, Valentim L, Phillips JA 3rd, Allito B, Heim RA, Sugarman EA, Probst CM, Faucz F, Culpi L
High allelic heterogeneity between Afro-Brazilians and Euro-Brazilians impacts cystic fibrosis genetic testing.
Genet Test. 2003 Fall;7(3):213-8., [PMID:14641997]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 1,000 different mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To determine the frequency of 70 common worldwide CFTR mutations in 155 Euro-Brazilian CF patients and in 38 Afro-Brazilian CF patients, we used direct PCR amplification of DNA from a total of 386 chromosomes from CF patients born in three different states of Brazil. The results show that screening for seventy mutations accounts for 81% of the CF alleles in Euro-Brazilians, but only 21% in the Afro-Brazilian group. We found 21 different mutations in Euro-Brazilians and only 7 mutations in Afro-Brazilians. The frequency of mutations and the number of different mutations detected in Euro-Brazilians are different from Northern European and North American populations, but similar to Southern European populations; in Afro-Brazilians, the mix of CF-mutations is different from those reported in Afro-American CF patients. We also found significant differences in detection rates between Euro-Brazilian (75%) and Afro-Brazilian CF patients (21%) living in the same state, Minas Gerais. These results, therefore, have implications for the use of DNA-based tests for risk assessment in heterogeneous populations like the Brazilians. Further studies are needed to identify the remaining CF mutations in the different populations and regions of Brazil.

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63 FREQUENCIES OF 70 CFTR MUTATIONS IN DIFFERENT STATES OF BRAZIL, BY CONTINENTA L GROUP CFTR mutations SC PR MG detected n n n n % n % N % DF508 53 39 54 146 47.1 8 10.5 154 39.9 G542X 6 9 8 23 7.4 1 1.3 24 6.2 R1162X 9 2 4 15 4.8 2 2.6 17 4.4 N1303K 5 5 0 10 3.2 0 0 10 2.6 R334W 5 1 4 10 3.2 0 0 10 2.6 G85E 2 2 4 8 2.6 1 1.3 9 2.3 1717-1G®A 1 3 2 6 1.9 0 0 6 1.6 W1282X 4 1 1 6 1.9 0 0 6 1.6 3849110kbC®T 1 3 1 5 1.6 0 0 5 1.3 R553X 0 2 0 2 0.7 0 0 2 0.5 1812-1G®A 0 1 3 4 1.3 1 1.3 5 1.3 2183AA®G 2 1 0 3 1.0 0 0 3 0.8 312011G®A 0 0 2 2 0.7 2 2.6 4 1.0 Y1092X 0 1 1 2 0.7 1 1.3 3 0.8 G551D 0 0 0 0 0 0 0 0 0 W1089X 0 0 1 1 0.3 0 0 1 0.3 6211G®T 0 1 0 1 0.3 0 0 1 0.3 Q1238X 0 1 0 1 0.3 0 0 1 0.3 711-1G®T 0 1 0 1 0.3 0 0 1 0.3 R347P 1 0 0 1 0.3 0 0 1 0.3 189811G®A 1 0 0 1 0.3 0 0 1 0.3 I507 0 0 1 1 0.3 0 0 1 0.3 Subtotal 91 73 86 250 80.7 16 21.1 266 68.9 Alleles with CFTR 5 27 28 60 19.4 60 79.0 120 31.1 mutations not detected Total 96 100 114 310 100.0 76 100.0 386 100.0 Detection rate (%) 94.8 73.0 75.4 250 80.7 16 21.1 266 68.9 The following 70 CFTR mutations were selected and tested on the basis of frequency in various populations, known association with CF, or predicted deleterious effect on the CFTR protein product; DF508, G542X, N1303K, G551D, R553X, DI507, A455E, A559T, C524X, D1270N, E60X, G178R, G330X, G85E, 2307insA, I148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P 2307insA, I148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P 2307insA, 1148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P, R352Q, R560T, S1196X, S1255X, S364P, S549N, S549R, V520F, W1089X, W1282X, W1310X, W1316X, Y1092X, Y122X, Y563D, 1078delT,1677delTA,1717-1G-A,1812-1G-A,1898 1 1G-A, 2043delG,2183delAA-G, 2184delA, 2789 1 5G-A, 2869insG, 2909delT, 3120 1 1G-A, 3120G-A, 3358delAC, 3659delC, 3662delA, 3750delAG, 3791delC, 3821delT, 3849 1 10KbC-T, 3849 1 4A-G, 3905insT, 405 1 1G-A, 444delA, 556delA, 574delA, 621 1 1G-T, and 711 1 1G-T. aSC, Santa Catarina State; PR, Parana State; MG, Minas Gerais State; n, number of chromosomes. Login to comment

[hide] Structure of nucleotide-binding domain 1 of the cy... EMBO J. 2004 Jan 28;23(2):282-93. Epub 2003 Dec 18. Lewis HA, Buchanan SG, Burley SK, Conners K, Dickey M, Dorwart M, Fowler R, Gao X, Guggino WB, Hendrickson WA, Hunt JF, Kearins MC, Lorimer D, Maloney PC, Post KW, Rajashankar KR, Rutter ME, Sauder JM, Shriver S, Thibodeau PH, Thomas PJ, Zhang M, Zhao X, Emtage S
Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.
EMBO J. 2004 Jan 28;23(2):282-93. Epub 2003 Dec 18., 2004-01-28 [PMID:14685259]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

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216 CF mutations in NBD1 The majority of sites of CF-causing missense mutations occur in NBD1, primarily in its a-subdomain, and the locations in the mNBD1 structure of the most common of these (A455E, G480C, I506T, DI507, DF508, S549N, S549R, G551D, A559T, R560T, Y569D, and D648V; Bobadilla et al, 2002) are shown in Figure 3D. Login to comment

[hide] Association between serum oncofetal antigens CA 19... Acta Paediatr. 2003 Nov;92(11):1267-71. Gronowitz E, Pitkanen S, Kjellmer I, Heikinheimo M, Strandvik B
Association between serum oncofetal antigens CA 19-9 and CA 125 and clinical status in patients with cystic fibrosis.
Acta Paediatr. 2003 Nov;92(11):1267-71., [PMID:14696845]

Abstract [show]
In cystic fibrosis (CF), mucus plugging in the airways and in the gastrointestinal tract leads to severe morbidity and mortality. The mucin-associated antigens CA 19-9 and CA 125 are markers of gastrointestinal malignancy, and CA 19-9 has also been reported in association with pulmonary function in CF. AIM: To test whether these antigens might serve as markers for the severity of pulmonary and gastrointestinal disease in CF. METHODS: In 99 patients, aged 1 to 48 y, serum levels of CA 19-9 and CA 125 were measured by RIA and ELISA and related to clinical data. RESULTS: Patients with severe mutations had significantly increased serum levels of CA 125, indicating an association with a more severe CF phenotype. This was further supported by the association with lung function, chronic pulmonary colonization of Pseudomonas aeruginosa and pancreatic insufficiency. CA 19-9 was also shown to be associated with lung function and Ps. aeruginosa colonization. No gastrointestinal malignancy was found in our patients despite very high values of CA 19-9 in some patients. During a 5-y follow-up, the very high serum levels of CA 19-9 decreased along with improved general condition of the patients. CONCLUSION: Increased serum levels of CA 125 in CF patients were associated with severe cystic fibrosis transmembrane conductance regulator mutations and a severe phenotype. Both antigens were associated with pseudomonas colonization and lung function and CA 125 also with pancreatic insufficiency. The estimates of CA 19-9 are hampered by the influence of the Lewis histo-blood group system on the synthesis of CA 19-9.

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45 The remaining 23 patients had at least one mild (I506L, R117C, S945L, T338I, W301R, 3849 10KBC → T, 1249-5 → G, R117H, R75Q), moderate (G551D, R560T, V603F) or unknown mutation. Login to comment

[hide] Improved detection of cystic fibrosis mutations in... Hum Reprod. 2004 Mar;19(3):540-6. Epub 2004 Jan 29. Danziger KL, Black LD, Keiles SB, Kammesheidt A, Turek PJ
Improved detection of cystic fibrosis mutations in infertility patients with DNA sequence analysis.
Hum Reprod. 2004 Mar;19(3):540-6. Epub 2004 Jan 29., [PMID:14998948]

Abstract [show]
BACKGROUND: Accurate determination of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is critical for genetic counselling and treatment of obstructive azoospermia. Of concern is that detection rates with routine CFTR mutation panels vary widely depending on patient ancestry; and such panels have limited value for azoospermic patients, who are more likely to carry rare mutations. An alternative approach offers comprehensive, CFTR mutation analysis by a DNA sequence method. We investigated whether this method could improve CFTR detection rates in men with obstructive azoospermia in a prospective study of men with obstructive azoospermia and their partners who were referred for genetic counselling and testing at one of two institutions. METHODS: Sixteen patients with congenital absence of the vas deferens (CAVD, n = 14) or idiopathic obstructive azoospermia (n = 2) were studied. DNA from all patients was analysed for mutations by the DNA sequence method. In addition to this method, six men underwent CFTR analysis by a common 25 or 31 mutation panel coupled with poly T analysis. In 10 subjects, common mutation panel findings were inferred from DNA sequence method results. RESULTS: Overall, 12/16 (75%) azoospermic patients had one or more CFTR mutations and/or 5T alleles, including 12 mutations in 10 patients (two compound heterozygotes) and seven 5T alleles in six patients (one homozygote). The sequence method detected all mutations and three variants of unknown significance. By comparison, the common mutation panels detected only 3/12 mutations (25%) and 0/3 variants. CONCLUSION: The DNA sequence method detects more CFTR mutations than common mutation panels. Given the serious, clinical consequences of transmitting such mutations, this study underscores the importance of accurate, CFTR mutation detection in men with obstructive azoospermia and their partners.

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59 Polyacrylamide gels were analysed for the presence of mutations following staining in ethidium bromide (EtBr) and image capture under UV using the Gel Doc 1000 system Table I. List of CFTR mutations included in common mutation panels American College of Medical Genetics CF panel (25 mutations) DF508 G542X G551D R117H W1282X N1303K R1162X 3849+10kbC®T DI507 R553X 1717-1G®A 621+1G®T R560T 3659delC 3120+1G®A I148T G85E R334W A455E 1898+1G®A 2148delA 711+1G®T 2789+5G®A R347P 1078delT Six additional mutations and one polymorphism in UCSF panel (31 mutations) Y1092X R347H 3849+4 Q493X 3905insT S549N F508C (polymorphism) (BioRad). Login to comment

[hide] Direct visualization of cystic fibrosis transmembr... Clin Chem. 2004 May;50(5):836-45. Epub 2004 Mar 9. Strom CM, Clark DD, Hantash FM, Rea L, Anderson B, Maul D, Huang D, Traul D, Chen Tubman C, Garcia R, Hess PP, Wang H, Crossley B, Woodruff E, Chen R, Killeen M, Sun W, Beer J, Avens H, Polisky B, Jenison RD
Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting.
Clin Chem. 2004 May;50(5):836-45. Epub 2004 Mar 9., [PMID:15010427]

Abstract [show]
BACKGROUND: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. METHODS: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. RESULTS: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism CONCLUSIONS: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.

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178 The optimal spotting conditions for each probe are indicated by the boxes around spots in C. wild-type controls and heterozygotes for each ACMG mutation and polymorphism, DNA from 12 compound heterozygotes (⌬F508/1898 ϩ 1GϾA, 711 ϩ 1GϾT/⌬F508, G85E/621 ϩ 1GϾT, 3659delC/⌬F508, 3120 ϩ 1GϾA/ 621 ϩ 1GϾT, R347P/G551D, A455E/⌬F508, R560T/ dF508, R553X/⌬F508, 621 ϩ 1GϾT/⌬F508, 621 ϩ 1GϾT/ 711 ϩ 1GϾT, R117H/⌬F508, and I506V/⌬F508) and DNA from 4 homozygous patients (⌬F508 and 2789 ϩ 5GϾA, 3849 ϩ 10kbCϾT, and G542X) was used in validation experiments. Login to comment
199 In this series, there were 17 ⌬F508 heterozygous patient samples, 1 ⌬F508 homozygous sample, 2 R117H heterozygous samples, and 1 heterozygous patient sample each for I148T, G542X, R553X, R347P, and 2789 ϩ 5GϾA, for a total of 26 mutant alleles. Additional mutant alleles detected in the control samples included three fixed control samples (⌬F508 homozygous, 5T/WT, 3659delC/⌬F508) on every plate and two heterozygous samples (R560T and 1078delT) and one heterozygous sample each for R334W, A455E, R347P, R117H, ⌬I507, I507V, G551D, and 1717-1GϾA as rotating controls. Login to comment
203 In this comparison, there were 19 ⌬F508 heterozygous patient samples, 3 I148T heterozygous samples, 3 R117H heterozygous and 1 R117H homozygous samples, 2 W1282X heterozygous samples, and 1 heterozygous patient sample each for G551D, R553X, R1162X, and 3849 ϩ 10kBCϾT, for a total of 36 mutant alleles. Additional mutant alleles detected for this study included fixed controls ⌬F508 homozygous, 5T/WT, and a N1303K heterozygous sample on all plates, and one heterozygous sample each for R560T, G542X, R553X, W1282X, 2184delA, G85E, I148T, 621 ϩ 1GϾT, R334W, R117H, 1078delT, and 1717-1GϾA as rotating controls. Login to comment

[hide] Population-based newborn screening for genetic dis... Pediatrics. 2004 Jun;113(6):1573-81. Comeau AM, Parad RB, Dorkin HL, Dovey M, Gerstle R, Haver K, Lapey A, O'Sullivan BP, Waltz DA, Zwerdling RG, Eaton RB
Population-based newborn screening for genetic disorders when multiple mutation DNA testing is incorporated: a cystic fibrosis newborn screening model demonstrating increased sensitivity but more carrier detections.
Pediatrics. 2004 Jun;113(6):1573-81., [PMID:15173476]

Abstract [show]
OBJECTIVES: Newborn screening for cystic fibrosis (CF) provides a model to investigate the implications of applying multiple-mutation DNA testing in screening for any disorder in a pediatric population-based setting, where detection of affected infants is desired and identification of unaffected carriers is not. Widely applied 2-tiered CF newborn screening strategies first test for elevated immunoreactive trypsinogen (IRT) with subsequent analysis for a single CFTR mutation (DeltaF508), systematically missing CF-affected infants with any of the >1000 less common or population-specific mutations. Comparison of CF newborn screening algorithms that incorporate single- and multiple-mutation testing may offer insights into strategies that maximize the public health value of screening for CF and other genetic disorders. The objective of this study was to evaluate technical feasibility and practical implications of 2-tiered CF newborn screening that uses testing for multiple mutations (multiple-CFTR-mutation testing). METHODS: We implemented statewide CF newborn screening using a 2-tiered algorithm: all specimens were assayed for IRT; those with elevated IRT then had multiple-CFTR-mutation testing. Infants who screened positive by detection of 1 or 2 mutations or extremely elevated IRT (>99.8%; failsafe protocol) were then referred for definitive diagnosis by sweat testing. We compared the number of sweat-test referrals using single- with multiple-CFTR-mutation testing. Initial physician assessments and diagnostic outcomes of these screened-positive infants and any affected infants missed by the screen were analyzed. We evaluated compliance with our screening and follow-up protocols. All Massachusetts delivery units, the Newborn Screening Program, pediatric health care providers who evaluate and refer screened-positive infants, and the 5 Massachusetts CF Centers and their affiliated genetic services participated. A 4-year cohort of 323 506 infants who were born in Massachusetts between February 1, 1999, and February 1, 2003, and screened for CF at approximately 2 days of age was studied. RESULTS: A total of 110 of 112 CF-affected infants screened (negative predictive value: 99.99%) were detected with IRT/multiple-CFTR-mutation screening; 2 false-negative screens did not show elevated IRT. A total of 107 (97%) of the 110 had 1 or 2 mutations detected by the multiple- CFTR-mutation screen, and 3 had positive screens on the basis of the failsafe protocol. In contrast, had we used single-mutation testing, only 96 (87%) of the 110 would have had 1 or 2 mutations detectable by single-mutation screen, 8 would have had positive screens on the basis of the failsafe protocol, and an additional 6 infants would have had false-negative screens. Among 110 CF-affected screened-positive infants, a likely "genetic diagnosis" was made by the multiple-CFTR-mutation screen in 82 (75%) versus 55 (50%) with DeltaF508 alone. Increased sensitivity from multiple-CFTR-mutation testing yielded 274 (26%) more referrals for sweat testing and carrier identifications than testing with DeltaF508 alone. CONCLUSIONS: Use of multiple-CFTR-mutation testing improved sensitivity and postscreening prediction of CF at the cost of increased referrals and carrier identification.

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79 The 16-mutation panel included ⌬F508, R117H, G551D, G542X, W1282X, N1303K, R334W, 621 ϩ 1GϾT, R553X, ⌬I507, 1717-1GϾA, R347P, R560T, 3849 ϩ 10kbCϾT, A455E, and S549N. Login to comment
159 Genotypes and Frequencies Observed in 112 CF-Affected Infants First Mutation Second Mutation N ⌬F508 ⌬F508 55 ⌬F508 R117H 7* ⌬F508 G551D 4 ⌬F508 N1303K 3 ⌬F508 W1282X 3 ⌬F508 G542X 2 ⌬F508 1898 ϩ 1 G Ͼ A 2 G85E R117C 2 ⌬F508 1717-GϾA 1 ⌬F508 3849 ϩ 10kbC Ͼ T 1 ⌬F508 R1066C 1 ⌬F508 Y1092X 1 ⌬F508 L206W 1 ⌬F508 R560T 1 ⌬F508 1152H 1 ⌬F508 621 ϩ 1G Ͼ T 1 R117H G551D 1 R117H G85E 1 G551D 2789 ϩ 5GϾA 1 G551D R117C 1 G85E 711 ϩ 1GϾT 1 W1282X 3849 ϩ 10kbCϾT 1 R553X 2183AAϾG 1 A455E S549R 1 ⌬F508 Unknown† 13 N1303K Unknown 2 G542X Unknown 1 Unknown Unknown 2 * Includes 1 of the false-negative screens. Login to comment

[hide] Risk of pancreatitis with mutation of the cystic f... Am J Gastroenterol. 2004 Jul;99(7):1358-63. Choudari CP, Imperiale TF, Sherman S, Fogel E, Lehman GA
Risk of pancreatitis with mutation of the cystic fibrosis gene.
Am J Gastroenterol. 2004 Jul;99(7):1358-63., [PMID:15233679]

Abstract [show]
BACKGROUND: Between 5% and 15% of patients with recurrent pancreatitis have no identified etiology after routine investigation and advanced endoscopic evaluation. OBJECTIVE: To determine whether mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is a risk factor for idiopathic pancreatitis. METHODS: We compared the frequency of CFTR mutations as measured by DNA probe analysis in a case group of persons with idiopathic pancreatitis and a control group without pancreatitis, all of whom underwent endoscopic retrograde cholangiopancreatography. A separate analysis compared the prevalence of CFTR mutations between the case group and controls with pancreatitis of known etiology. A subgroup comparison was made between cases of pancreas divisum with pancreatitis and controls with pancreas divisum and no pancreatitis. RESULTS: CFTR mutations were present in 19 (19%) of 96 cases and 7 (3.5%) of 198 controls without pancreatitis (odds ratio, OR = 6.7; 95% CI, 2.8-16.3; p < 0.00001). Compared to the controls with a known cause of pancreatitis (N = 78), cases had a higher prevalence of CFTR mutations (19% vs 2.6%, OR = 9.4; CI, 2.1-41.7; p= 0.0005). Among subjects with pancreas divisum, CFTR mutations were present in 8 (22%) of 37 cases compared to 0 (0%) of 20 controls (OR = 11.8; CI, 8.9-14.7; p= 0.02). CONCLUSION: The risk of idiopathic pancreatitis is greater among persons with CFTR mutations as compared to persons without CFTR mutations. Among persons with pancreas divisum, CFTR mutations appear to increase the risk for pancreatitis.

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45 ( F508, G551D, R553X, W1282X, N1303K, R117H, Delta I507, 621+1G- >T, R560T, 1717-1G->A, 711+1G->T, and R1162X; Nichols Institute, Nichols Institute Reference Laboratories, California). Login to comment

[hide] Cystic fibrosis screening: lessons learned from th... Genet Med. 2004 May-Jun;6(3):136-40. Strom CM, Crossley B, Redman JB, Buller A, Quan F, Peng M, McGinnis M, Sun W
Cystic fibrosis screening: lessons learned from the first 320,000 patients.
Genet Med. 2004 May-Jun;6(3):136-40., [PMID:15354331]

Abstract [show]
PURPOSE: To examine the data from > 335,000 Cystic fibrosis (CF) tests to detect unsuspected findings and obtain clinical data when indicated to optimize genetic counseling. METHODS: A proprietary database containing 335,204 consecutive CF DNA tests and 445 CF prenatal diagnostic tests was queried. Clinical information was obtained for prenatal and selected nonprenatal cases by telephone contact with physician offices. RESULTS: The mutation 1078delT was found in much lower frequency than expected with rates of only 1:55,867 tests and 0.06% of CF mutations. This level is below the threshold set by the American College of Medical Genetics. Homozygosity was observed for 2789+5G>A in a 29-year-old women and compound heterozygosity with delta F408 in a 40-year-old woman with isolated chronic sinusitis. Many patients elected prenatal diagnosis when not at a 1:4 risk due to echogenic bowel or IVS-8 5T issues. CONCLUSIONS: With the exception of 1078delT, all CF mutations in the ACMG panel were detected with a frequency of > 0.1% of CF chromosomes. When ACMG guidelines are strictly adhered to, population-based CF carrier screening will accurately identify couples at risk for having children with CF.

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47 Frequency, all tests Frequency, CF mutations (%) delta F508 7610 1:44 75% R117H/7T or 9T 1030 1:325 NAb R117H/5T 103 1:3,254 0.51c W1282X 529 1:625 5.2 G542X 382 1:909 3.8 G551D 278 1:1,250 2.7 N1303K 201 1:1,668 2.0 3849ϩ10kb CϾT 167 1:2,007 1.6 1717-1 GϾA 102 1:3,286 1.0 R553X 102 1:3,286 1.0 621ϩ1 GϾT 98 1:3,420 0.97 2789ϩ5 GϾA 82 1:4,087 0.80 3120ϩ1 GϾA 73 1:4,591 0.72 R1162X 54 1:6,207 0.53 R334W 54 1:6,207 0.53 685E 52 1:6,446 0.51 R560T 52 1:6,446 0.51 Delta I507 51 1:6,572 0.50 711ϩ1 GϾT 40 1:8,380 0.39 1898ϩ1 GϾA 37 1:9,059 0.36 3659 del C 36 1:9,311 0.36 A455E 34 1:9,858 0.33 R347P 33 1:10,158 0.32 2184 del A 14 1:23,943 0.14 1078 del T 6 1:55,867 0.06 a I148T has been eliminated from these data. Login to comment

[hide] Cystic fibrosis population carrier screening: 2004... Genet Med. 2004 Sep-Oct;6(5):387-91. Watson MS, Cutting GR, Desnick RJ, Driscoll DA, Klinger K, Mennuti M, Palomaki GE, Popovich BW, Pratt VM, Rohlfs EM, Strom CM, Richards CS, Witt DR, Grody WW
Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.
Genet Med. 2004 Sep-Oct;6(5):387-91., [PMID:15371902]

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70 It has been ar- Table 1 CFTR mutation frequency among individuals with clinically diagnosed cystic fibrosis by racial/ethnic group and in a pan-ethnic U.S. population CFTR mutation Mutation frequency among individuals with clinically diagnosed cystic fibrosis (%) Non-Hispanic Caucasian Hispanic Caucasian African American Asian American Ashkenazi Jewish Pan-Ethnic Population5 delF508 72.42 54.38 44.07 38.95 31.41 66.31 G542X 2.28 5.10 1.45 0.00 7.55 2.64 W1282X 1.50 0.63 0.24 0.00 45.92 2.20 G551D 2.25 0.56 1.21 3.15 0.22 1.93 621ϩ1GϾT 1.57 0.26 1.11 0.00 0.00 1.30 N1303K 1.27 1.66 0.35 0.76 2.78 1.27 R553X 0.87 2.81 2.32 0.76 0.00 1.21 dell507 0.88 0.68 1.87 0.00 0.22 0.90 3849ϩ10kbCϾT 0.58 1.57 0.17 5.31 4.77 0.85 3120ϩ1GϾT 0.08 0.16 9.57 0.00 0.10 0.86 R117H 0.70 0.11 0.06 0.00 0.00 0.54 1717-1GϾT 0.48 0.27 0.37 0.00 0.67 0.44 2789ϩ5GϾA 0.48 0.16 0.00 0.00 0.10 0.38 R347P 0.45 0.16 0.06 0.00 0.00 0.36 711ϩ1GϾT 0.43 0.23 0.00 0.00 0.10 0.35 R334W 0.14 1.78 0.49 0.00 0.00 0.37 R560T 0.38 0.00 0.17 0.00 0.00 0.30 R1162X 0.23 0.58 0.66 0.00 0.00 0.30 3569delC 0.34 0.13 0.06 0.00 0.00 0.28 A455E 0.34 0.05 0.00 0.00 0.00 0.26 G85E 0.29 0.23 0.12 0.00 0.00 0.26 2184delA 0.17 0.16 0.05 0.00 0.10 0.15 1898ϩ1GϾA 0.16 0.05 0.06 0.00 0.10 0.13 l148T 0.09 0.09 0.05 0.00 0.10 0.08 1078delT 0.02 0.09 0.00 0.00 0.00 0.03 Total 88.40 71.90 64.51 48.93 94.14 84.00 gued that a laboratory is obligated to report any and all information that is gleaned from a test system, however, there is no regulatory requirement and practice varies. Login to comment

[hide] CFTR mutation distribution among U.S. Hispanic and... Genet Med. 2004 Sep-Oct;6(5):392-9. Sugarman EA, Rohlfs EM, Silverman LM, Allitto BA
CFTR mutation distribution among U.S. Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening populations.
Genet Med. 2004 Sep-Oct;6(5):392-9., [PMID:15371903]

Abstract [show]
PURPOSE: We reviewed CFTR mutation distribution among Hispanic and African American individuals referred for CF carrier screening and compared mutation frequencies to those derived from CF patient samples. METHODS: Results from CFTR mutation analyses received from January 2001 through September 2003, were analyzed for four populations: Hispanic individuals with a CF diagnosis (n = 159) or carrier screening indication (n = 15,333) and African American individuals with a CF diagnosis (n = 108) or carrier screening indication (n = 8,973). All samples were tested for the same 87 mutation panel. RESULTS: In the Hispanic population, 42 mutations were identified: 30 in the patient population (77.5% detection rate) and 33 among carrier screening referrals. Five mutations not included in the ACMG/ACOG carrier screening panel (3876delA, W1089X, R1066C, S549N, 1949del84) accounted for 7.55% detection in patients and 5.58% among carriers. Among African American referrals, 33 different mutations were identified: 21 in the patient population (74.4% detection) and 23 in the carrier screening population. Together, A559T and 711+5G>A were observed at a detection rate of 3.71% in CF patients and 6.38% in carriers. The mutation distribution seen in both the carrier screening populations reflected an increased frequency of mutations with variable expression such as D1152H, R117H, and L206W. CONCLUSIONS: A detailed analysis of CFTR mutation distribution in the Hispanic and African American patient and carrier screening populations demonstrates that a diverse group of mutations is most appropriate for diagnostic and carrier screening in these populations. To best serve the increasingly diverse U.S. population, ethnic-specific mutations should be included in mutation panels.

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35 87 mutation panel The following mutations were included in the panel: ⌬F508, ⌬F311, ⌬I507, A455E, A559T, C524X, D1152H, D1270N, E60X, G178R, G330X, G480C, G542X, G551D, G85E, G91R, I148T, K710X, L206W, M1101K, N1303K, P574H, Q1238X, Q359K/T360K, Q493X, Q552X, Q890X, R1066C, R1158X, R1162X, R117C, R117H, R1283M, R334W, R347H, R347P, R352Q, R553X, R560T, S1196X, S1251N, S1255X, S364P, S549I, S549N, S549R, T338I, V520F, W1089X, W1282X, Y1092X, Y563D, 1078delT, 1161delC, 1609delCA, 1677delTA, 1717-1GϾA, 1812-1GϾA, 1898ϩ1GϾA, 1898ϩ5GϾT, 1949del84, 2043delG, 2143delT, 2183delAAϾG, 2184delA, 2307insA, 2789ϩ5GϾA, 2869insG, 3120ϩ1GϾA, 3120GϾA, 3659delC, 3662delA, 3791delC, 3821delT, 3849ϩ10kbCϾT, 3849ϩ4AϾG, 3905insT, 394delTT, 405ϩ1GϾA, 405ϩ3AϾC, 444delA, 574delA, 621ϩ1GϾT, 711ϩ1GϾT, 711ϩ5GϾA, 712-1GϾT, 3876delA CFTR mutation analysis Genomic DNA was extracted from peripheral blood lymphocytes, buccal cell swabs, or bloodspots by Qiagen QIAmp 96 DNA Blood Kit. Specimens were tested for 87 mutations by a pooled allele-specific oligonucleotide (ASO) hybridization method as previously described.16,17 Two multiplex chain reactions (PCR) were used to amplify 19 regions of the CFTR gene. Login to comment

[hide] Clinical sensitivity of prenatal screening for cys... Genet Med. 2004 Sep-Oct;6(5):405-14. Palomaki GE, FitzSimmons SC, Haddow JE
Clinical sensitivity of prenatal screening for cystic fibrosis via CFTR carrier testing in a United States panethnic population.
Genet Med. 2004 Sep-Oct;6(5):405-14., [PMID:15371905]

Abstract [show]
PURPOSE: To estimate CFTR mutation frequencies, clinical sensitivities (proportions of carrier couples or affected fetuses detected), and birth prevalence estimates for broad racial/ethnic groups and for a panethnic U.S. population. METHODS: Published sources of information were identified, corrected when appropriate, and summarized. Combining racial/ethnic-specific mutation frequencies and birth prevalence estimates allowed the computation of panethnic estimates. RESULTS: Two of the 25 recommended mutations do not meet the 0.1% threshold in a panethnic population set by the American College of Medical Genetics. The clinical sensitivities are estimated to be 71.9%, 51.7%, 41.6%, 88.6%, and 23.4% for non-Hispanic Caucasians, Hispanic Caucasian, African American, Ashkenazi Jewish Caucasian, and Asian American couples, respectively. Birth prevalence estimates are 1:2,500, 1:13,500, 1:15,100, 1:2,270, and 1:35,100, whereas the number of couples needed to screen to detect an affected fetus are about 3,200, 26,120; 36,040; 2,600, and 129,600, respectively, for the same racial/ethnic groups. CONCLUSIONS: Overall, the panethnic estimates for CFTR mutation frequencies are similar to those for non-Hispanic Caucasians. However, large differences in both clinical sensitivity and birth prevalence exist between the broad racial/ethnic groups examined. Whether and how the differences in the numbers of couples needed to screen to detect an affected fetus are to be included in prenatal screening for cystic fibrosis needs to be more explicitly addressed.

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32 Data from the International Cystic Fibrosis Consortium were taken from Table 1 of its publication.4 Data from the Cystic Fibrosis Foundation National Patient Registry were taken from the year 1999 and stratified according to whether or not the patient was seen Table 1 CFTR mutation frequencies among Hispanic Caucasians with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) CF Consortiumb CF Foundationc Average Cumulative 1 delF508 45.51 63.25 54.38 54.38 2 G542X 5.11 5.09 5.10 59.48 8 delI507 0.59 5.02 2.81 62.29 22 R334W 2.25 1.31 1.78 64.07 6 N1303K 1.65 1.67 1.66 65.73 10 3849 ϩ 10kbC Ͼ T 1.60 1.53 1.57 67.30 7 R553X 0.63 0.73 0.68 67.98 5 W1282X 0.53 0.73 0.63 68.61 19 R1162X 0.57 0.58 0.58 69.19 3 G551D 0.31 0.80 0.56 69.75 12 1717 - 1G Ͼ T 0.10 0.44 0.27 70.02 4 621 ϩ 1G Ͼ T 0.00 0.51 0.26 70.28 14 711 ϩ 1G Ͼ T 0.10 0.36 0.23 70.51 18 G85E 0.10 0.36 0.23 70.74 11 2789 ϩ 5G Ͼ A 0.10 0.22 0.16 70.90 13 R347P 0.10 0.22 0.16 71.06 20 2184delA 0.10 0.22 0.16 71.22 24 3120 ϩ 1G Ͼ T 0.10 0.22 0.16 71.38 17 3569delC 0.10 0.15 0.13 71.51 9 R117H 0.00 0.22 0.11 71.62 23 I148T 0.10 0.07 0.09 71.71 25 1078delT 0.10 0.07 0.09 71.80 16 A455E 0.10 0.00 0.05 71.85 21 1898 ϩ 1G Ͼ A 0.10 0.00 0.05 71.90 15 R560T 0.00 0.00 0.00 71.90 All 25 59.95 83.77 71.90 a The order is based on that found for non-Hispanic Caucasians.3 b Based on between 178 and 958 chromosomes (International Cystic Fibrosis Genetic Analysis Consortium.4 c Based on 1374 chromosomes from clinically diagnosed persons registered in the Cystic Fibrosis Foundation National Patient Registry. Login to comment
54 Only three mutations were never identified (R560T, A455E, and 1898ϩ1GϾA). Login to comment
80 The larger data- Table 2 CFTR mutation frequencies among African American individuals with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) CF Consortiumb CF Foundationc Average Cumulative 1 delF508 35.50 52.63 44.07 44.07 24 3120 ϩ 1G Ͼ T 12.50 6.64 9.57 53.64 8 delI507 0.74 3.89 2.32 55.96 7 R553X 2.37 1.37 1.87 57.83 2 G542X 1.18 1.72 1.45 59.28 3 G551D 0.59 1.83 1.21 60.49 4 621 ϩ 1G Ͼ T 1.18 1.03 1.11 61.60 19 R1162X 0.74 0.57 0.66 62.26 22 R334W 0.74 0.23 0.49 62.75 12 1717 - 1G Ͼ T 0.74 0.00 0.37 63.12 6 N1303K 0.00 0.69 0.35 63.47 5 W1282X 0.00 0.47 0.24 63.71 10 3849 ϩ 10kbC Ͼ T 0.00 0.34 0.17 63.88 15 R560T 0.00 0.34 0.17 64.05 18 G85E 0.00 0.23 0.12 64.17 9 R117H 0.00 0.11 0.06 64.23 13 R347P 0.00 0.11 0.06 64.29 17 3569delC 0.00 0.11 0.06 64.35 21 1898 ϩ 1G Ͼ A 0.00 0.11 0.06 64.41 20 2184delA 0.10 0.00 0.05 64.46 23 I148T 0.10 0.00 0.05 64.51 11 2789 ϩ 5G Ͼ A 0.00 0.00 0.00 64.51 14 711 ϩ 1G Ͼ T 0.00 0.00 0.00 64.51 16 A455E 0.00 0.00 0.00 64.51 25 1078delT 0.00 0.00 0.00 64.51 All 25 56.46 72.42 64.51 a The order is based on that found for non-Hispanic Caucasians.3 b Based on between 79 and 169 chromosomes reported by the International Cystic Fibrosis Genetic Analysis Consortium.4 c Based on 874 chromosomes from clinically diagnosed persons registered in the Cystic Fibrosis Foundation National Patient Registry. Login to comment
107 An earlier article10 reported that 97% of mutations were identified in 90 chromosomes from Ashkenazi Jewish individ- Table 3 CFTR mutation frequencies among Ashkenazi Jewish Caucasian individuals with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) CF Consortiumb Cumulative 5 W1282X 45.92 45.92 1 delF508 31.41 77.33 2 G542X 7.55 84.88 10 3849 ϩ 10kbC Ͼ T 4.77 89.65 6 N1303K 2.78 92.43 12 1717 - 1G Ͼ T 0.67 93.10 7 R553X 0.22 93.32 3 G551D 0.22 93.54 24 3120 ϩ 1G Ͼ T 0.10 93.64 21 1898 ϩ 1G Ͼ A 0.10 93.74 20 2184delA 0.10 93.84 23 I148T 0.10 93.94 11 2789 ϩ 5G Ͼ A 0.10 94.04 14 711 ϩ 1G Ͼ T 0.10 94.14 8 delI507 0.00 94.14 19 R1162X 0.00 94.14 22 R334W 0.00 94.14 4 621 ϩ 1G Ͼ T 0.00 94.14 15 R560T 0.00 94.14 18 G85E 0.00 94.14 9 R117H 0.00 94.14 13 R347P 0.00 94.14 17 3569delC 0.00 94.14 16 A455E 0.00 94.14 25 1078delT 0.00 94.14 Sum 94.14 a The order is based on that found for non-Hispanic Caucasians.3 b Based on between 57 and 503 chromosomes reported by the International Cystic Fibrosis Genetic Analysis Consortium.4 uals with cystic fibrosis, using a panel of 11 mutations. Login to comment
115 In an- Table 4 CFTR mutation frequencies among Asian American individuals with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) Heim et al.1b CF Foundationc Average Cumulative 1 delF508 18.80 59.09 38.95 38.95 10 3849 ϩ 10kbC Ͼ T 0.00 10.61 5.31 44.26 3 G551D 6.30 0.00 3.15 47.41 6 N1303K 0.00 1.52 0.76 48.17 8 delI507 0.00 1.52 0.76 48.93 2 G542X 0.00 0.00 0.00 48.93 4 621 ϩ 1G Ͼ T 0.00 0.00 0.00 48.93 5 W1282X 0.00 0.00 0.00 48.93 7 R553X 0.00 0.00 0.00 48.93 9 R117H 0.00 0.00 0.00 48.93 11 2789 ϩ 5G Ͼ A 0.00 0.00 0.00 48.93 12 1717 - 1G Ͼ T 0.00 0.00 0.00 48.93 13 R347P 0.00 0.00 0.00 48.93 14 711 ϩ 1G Ͼ T 0.00 0.00 0.00 48.93 15 R560T 0.00 0.00 0.00 48.93 16 A455E 0.00 0.00 0.00 48.93 17 3569delC 0.00 0.00 0.00 48.93 18 G85E 0.00 0.00 0.00 48.93 19 R1162X 0.00 0.00 0.00 48.93 20 2184delA 0.00 0.00 0.00 48.93 21 1898 ϩ 1G Ͼ A 0.00 0.00 0.00 48.93 22 R334W 0.00 0.00 0.00 48.93 23 I148T 0.00 0.00 0.00 48.93 24 3120 ϩ 1G Ͼ T 0.00 0.00 0.00 48.93 25 1078delT 0.00 0.00 0.00 48.93 Sum 25.10 72.74 48.93 a The order is based on that found for non-Hispanic Caucasians.3 b Based on 20 chromosomes. Login to comment
173 For exam- Table 7 Estimated number of carriers of the 25 recommended CFTR mutations by racial/ethnic group and weighted average, representing the panethnic population in the United States for 2002 Order CFTR mutation Number of CFTR Mutation Carriers Panethnic frequency, % Non-Hispanic Caucasian Hispanic Caucasian African American Asian American Ashkenazi Jewish Total 1 delF508 64,779 8,207 4,272 886 796 78,940 66.31 2 G542X 2,039 770 141 0 191 3,141 2.64 5 W1282X 1,342 95 23 0 1,164 2,624 2.20 3 G551D 2,013 85 117 72 6 2,293 1.93 4 621 ϩ 1G Ͼ T 1,404 39 108 0 0 1,551 1.30 6 N1303K 1,136 251 34 17 70 1,508 1.27 7 R553X 778 424 225 17 0 1,444 1.21 8 delI507 787 103 181 0 6 1,077 0.90 10 3849 ϩ 10kbC Ͼ T 519 237 16 121 121 1,014 0.85 24 3120 ϩ 1G Ͼ T 72 24 928 0 3 1,027 0.86 9 R117H 626 17 6 0 0 649 0.55 12 1717 - 1G Ͼ T 429 41 36 0 17 523 0.44 11 2789 ϩ 5G Ͼ A 429 24 0 0 3 456 0.38 13 R347P 403 24 6 0 0 433 0.36 14 711 ϩ 1G Ͼ T 385 35 0 0 3 423 0.36 22 R334W 125 269 47 0 0 441 0.37 15 R560T 340 0 16 0 0 356 0.30 19 R1162X 206 88 64 0 0 358 0.30 17 3569delC 304 20 6 0 0 330 0.28 16 A455E 304 8 0 0 0 312 0.26 18 G85E 259 35 12 0 0 306 0.26 20 2184delA 152 24 5 0 3 184 0.15 21 1898 ϩ 1G Ͼ A 143 8 6 0 3 160 0.13 23 I148T 80 14 5 0 3 102 0.09 25 1078delT 18 14 0 0 0 32 0.03 All 79,072 10,856 6,193 1,113 2,389 99,684 84.00 Bolded numbers indicate mutations that are more likely to be found in a racial/ethnic group other than non-Hispanic Caucasians. Login to comment

[hide] Use of MALDI-TOF mass spectrometry in a 51-mutatio... Genet Med. 2004 Sep-Oct;6(5):426-30. Buyse IM, McCarthy SE, Lurix P, Pace RP, Vo D, Bartlett GA, Schmitt ES, Ward PA, Oermann C, Eng CM, Roa BB
Use of MALDI-TOF mass spectrometry in a 51-mutation test for cystic fibrosis: evidence that 3199del6 is a disease-causing mutation.
Genet Med. 2004 Sep-Oct;6(5):426-30., [PMID:15371908]

Abstract [show]
PURPOSE: We developed a 51-mutation extended cystic fibrosis (CF) panel that incorporates the 25 previously recommended CFTR mutations, plus 26 additional mutations including 3199del6, which was associated with I148T. METHODS: This assay utilizes an integrated matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system. RESULTS: CF testing was performed on over 5,000 individuals, including a 3-year-old Hispanic-American patient with a compound heterozygous G542X/3199del6 genotype. He is negative for I148T, or other mutations assessed by CFTR gene sequencing. CONCLUSION: These results demonstrate the successful implementation of MALDI-TOF mass spectrometry in CF clinical testing, and establish 3199del6 as a disease-causing CF mutation.

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77 This assay also demonstrated heterozygosity for the G542X mutation, and reflex testing for the 5T variant at CFTR intron 8 showed a genotype of 7T/9T in this patient (data not Table 3 Description of the 16 multiplex assays designed to analyze 51 CFTR mutations Multiplex Mutations Exon 1 1078delT, G314E, R352Q, G330X 7 2 R347H, R347P, R334W, 1717-1A 7, 11 3 R553X, S549N, R1162X 11, 19 4 A559T, R560T, G551D 11 5 G542X, S549R, 621ϩ1T, Y122X 4, 11 6 W1282X, 3876delA, 3905insT, D1152H 18, 20 7 3849ϩ4G, 3659delC, 1898ϩ1A 12, 19 8 405ϩ1A, 405ϩ3C, 3120A, 3120ϩ1A 3, 16 9 394delTT, E60X, G85E 3 10 A455E, ⌬F508a 9, 10 11 G480C, Q493X, V520F 10 12 711ϩ1T, G178R, 3199del6 5, 17a 13 2143delT, 2184delA, K710X, F316L 7, 13 14 I148T, R117H, R117C 4 15 N1303K, 2789ϩ5A, 3849ϩ10kbT 14b, intron19, 21 16 ⌬I507a 10 17 5Tb intron 8 a F508C and I507V, I506V, I506M variants are tested for concurrently with the ⌬F508 and ⌬I507 assays respectively. Login to comment

[hide] Cystic fibrosis carrier screening: validation of a... Genet Med. 2004 Sep-Oct;6(5):431-8. Edelmann L, Hashmi G, Song Y, Han Y, Kornreich R, Desnick RJ
Cystic fibrosis carrier screening: validation of a novel method using BeadChip technology.
Genet Med. 2004 Sep-Oct;6(5):431-8., [PMID:15371909]

Abstract [show]
PURPOSE: To validate a novel BeadChip assay system for cystic fibrosis (CF) mutation testing using the panel of 25 ACMG recommended mutations and D1152H. METHODS: DNA from 519 individuals originally tested for CF mutation status by allele specific oligonucleotide hybridization (ASOH) were blindly analyzed by the BeadChip assay and the results were compared. The elongation mediated multiplexed analysis of polymorphisms (eMAP) protocol, which combines multiplex amplification of genomic DNA and multiplex detection of mutations on color-coded bead arrays, was used to analyze 26 CF mutations in two separate groups. RESULTS: The system accurately distinguished the 26 CF genotypes and had 100% concordance with the ASOH technique with an assay failure rate of 1.7%. Benign variants of exon 10 codons 506, 507, and 508 did not interfere with mutation identification and reflex testing for the 5/7/9T IVS8 polymorphism was performed on a separate array. CONCLUSIONS: The BeadChip assay system provided accurate and rapid identification of the ACMG recommended CF mutations.

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35 Mutation controls included DNA from previously identified positive patient samples (I148T, D1152H, W1282X, R117H, G85E, A455E, delF508, N1303K) and DNA from NIGMS Human Genetic Cell Repositories (Coriell Cell Repositories) (delF508, delI507, G542X, R560T, 3849ϩ10kbCϾT, N1303K, G85E; G551D, R553X, 621ϩ1GϾT, 1717-1GϾA, A455E, R334W, R347P, R1162X, 3659delC; 711ϩ1GϾT, 2789ϩ5GϾA, 3120ϩ1GϾA). Login to comment
46 Mutant ASOs were end-labeled with ␥-32 P-ATP and pooled into three subgroups (IA-IC) for Group I and four subgroups (IIA-IID) for Group II mutations with the following breakdown of mutations: IA: delF508, delI507, W1282X, R117H; IB: G542X, R560T, 3849ϩ10kbCϾT, N1303K, G85E; IC: G551D, R553X, 621ϩ1GϾT, 1717-1GϾA, I148T; IIA: A455E, R334W, D1152H; IIB: R347P, 1078delT, R1162X, 3659delC; IIC: 711ϩ1GϾT, 1898ϩ1GϾA, 2789ϩ5GϾA, 3120ϩ1GϾA; IID: 2184delA. Login to comment
84 Certain mutations including 711ϩ1GϾA, R117H, G542X, R560T, and W1282X, required a heterozygous allelic ratio with an upper limit set at 2.50. Login to comment

[hide] Lack of association of common cystic fibrosis tran... Am J Gastroenterol. 2005 Apr;100(4):874-8. Gallegos-Orozco JF, E Yurk C, Wang N, Rakela J, Charlton MR, Cutting GR, Balan V
Lack of association of common cystic fibrosis transmembrane conductance regulator gene mutations with primary sclerosing cholangitis.
Am J Gastroenterol. 2005 Apr;100(4):874-8., [PMID:15784035]

Abstract [show]
BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic progressive cholestatic liver disease of uncertain etiology. However, the histologic features of PSC liver disease can resemble those in cystic fibrosis (CF), an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to determine if PSC patients have a higher frequency of common CF alleles than disease controls. METHODS: DNA was extracted from peripheral lymphocytes of patients with end-stage liver disease. Samples were obtained before liver transplantation from 59 PSC patients and from three groups of control patients (20 each with primary biliary cirrhosis, autoimmune hepatitis, or hepatitis C). DNA samples were genotyped for 32 common CF mutations, the intron 8 T tract variants, and the M470V variant. RESULTS: One of 59 PSC patients (1.7%) had the common CF mutation (DeltaF508) in one CFTR gene. Two controls (3.3%) carried a single CF mutation (DeltaF508 in one primary biliary cirrhosis patient; W1282X in one hepatitis C patient). These rates do not differ from expected in the general population. The frequency of CFTR variants (5T and M470V) was also similar between PSC patients and controls. CONCLUSIONS: Despite anatomical similarities between CF liver disease and PSC, we could not confirm that PSC patients carried common CF mutations or common CFTR variants in higher than expected frequencies. These data suggest that CFTR dysfunction does not influence the pathogenesis of PSC.

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55 CFTR Mutations and Associated Phenotype Classic Nonclassic Cystic Fibrosis Cystic Fibrosis Variant Normal 621 + 1G→T R117H G85E* 7T 711 + 1G→T R334W 5T† 9T 1078delT R347P M470V‡ F508C I507 A455E I507V F508 2789 + 5G → A I506V 1717 - 1G→A 3849 + 10kbC→T G542X G551D R553X R560T R1162X 3659delC W1282X N1303K * Classic cystic fibrosis and nonclassic cystic fibrosis. Login to comment

[hide] Parallel single nucleotide polymorphism genotyping... Anal Chem. 2005 Apr 15;77(8):2400-5. Chen Y, Shortreed MR, Olivier M, Smith LM
Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection.
Anal Chem. 2005 Apr 15;77(8):2400-5., 2005-04-15 [PMID:15828773]

Abstract [show]
Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.

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170 508 of the protein product.23 The CF mutations chosen in this study, ∆F508, G551D, W1282X, N1303K, R117H, R560T, 3849+10kbCT, V520F, R334W, and I148T, are a subset of the standard panel. Login to comment

[hide] Time-motion analysis of 6 cystic fibrosis mutation... Clin Chem. 2005 Jul;51(7):1116-22. Epub 2005 Apr 28. Krafft AE, Lichy JH
Time-motion analysis of 6 cystic fibrosis mutation detection systems.
Clin Chem. 2005 Jul;51(7):1116-22. Epub 2005 Apr 28., [PMID:15860566]

Abstract [show]
BACKGROUND: A dramatic increase in requests for routine cystic fibrosis (CF) carrier screening prompted us to conduct a time-motion analysis comparing commercially available CF testing platforms. Questions addressed in the study included: (a) How much time is required to perform each step involved in carrying out the assay procedure? (b) Which system requires the minimum number of manual manipulations to complete a typical run? (c) What workflow benefits can be achieved by automation? METHODS: We used a 96-sample run for comparisons and analyzed each of the 6 methods to determine the number of pipetting steps and manual manipulations, the labor and instrument time, and the total time required to perform the assay. The survey participants included a staff of 4 technologists who perform complex molecular assays regularly. Time required for each procedure was determined by direct observation and from work logs completed by the technologists. RESULTS: The total number of pipetting motions varied from 78 to 344. Labor time ranged from 2.6 to 8.4 h, and total assay time from 7.6 to 13.7 h. CONCLUSION: Time-motion analysis allowed identification of a method that minimized pipetting motions and thus reduced the risk of repetitive stress injury.

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43 These included 58 patient DNA samples initially characterized by CF Gold 1.0, of which 28 were wild type and 30 contained 1 of the following 16 mutant alleles: F508del, R553X, 2184delA, 3120 ϩ 1GϾA, I507del, G542X, G551D, W1282X, N1303K, 621 ϩ 1GϾT, R117H, 1717-1GϾA, R560T, R334W, R347P, and I148T. Login to comment

[hide] Screening of mutations in the CFTR gene in 1195 co... Eur J Hum Genet. 2005 Aug;13(8):959-64. Stuppia L, Antonucci I, Binni F, Brandi A, Grifone N, Colosimo A, De Santo M, Gatta V, Gelli G, Guida V, Majore S, Calabrese G, Palka C, Ravani A, Rinaldi R, Tiboni GM, Ballone E, Venturoli A, Ferlini A, Torrente I, Grammatico P, Calzolari E, Dallapiccola B
Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.
Eur J Hum Genet. 2005 Aug;13(8):959-64., [PMID:15870824]

Abstract [show]
Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

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64 of detected carriers Prevalence among detected CFTR mutations DF508 40 (3.34%) 65.58% DI507 0 0 G542X 6 (0.50%) 9.84% 1717-1G-A 1 (0.08%) 1.64% G551D 0 0 R553X 0 0 R560T 0 0 Q552X 0 0 W1282X 7 (0.58 %) 11.48% S1251N 0 0 N1303K 3 (0.20%) 4.91% 394delTT 0 0 G85E 3 (0.25%) 4.91% E60X 0 0 621+1G-T 0 0 R117H 0 0 1078delT 0 0 R347P 0 0 R334W 0 0 2143delT 0 0 2183AA-G 0 0 2184delA 0 0 711+5G-A 0 0 2789+5G-A 1 (0.08%) 1.64% R1162X 0 0 3659del5 0 0 3849+10kbC-T 0 0 A455E 0 0 5T 78 (6.52%) Table 2 Distribution of CFTR mutations and 5T allele according to phenotype for the 1195 individuals Phenotype CF/WT 5T/WT CF/5T WT/WT Infertile males (non-CBAVD), N ¼ 304 20 (6.58%) 30 (9.87%) 0 254 (83.55%) Infertile males (CBAVD), N ¼ 16 0 10 (62.50%) 6 (37.50 %) 0 Infertile females, N ¼ 93 5 (5.37%) 7 (7.53%) 0 81 (87.10%) Unexplained infertility, N ¼ 782 30 (3.84%) 31 (3.96%) 0 721 (92.20%) Total ¼ 1195 55 (4.60%) 78 (5.50%) 6 (0.50%) 1056 (88.40%) CFTR alteration was detected, including a mutation in three cases and the 5T polymorphism in the remaining six. Login to comment

[hide] Pharmacological induction of CFTR function in pati... Pediatr Pulmonol. 2005 Sep;40(3):183-96. Kerem E
Pharmacological induction of CFTR function in patients with cystic fibrosis: mutation-specific therapy.
Pediatr Pulmonol. 2005 Sep;40(3):183-96., [PMID:15880796]

Abstract [show]
CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. Mutations can be classified according to the mechanisms by which they disrupt CFTR function. This understanding of the different molecular mechanisms of CFTR dysfunction provides the scientific basis for the development of targeted drugs for mutation-specific therapy of cystic fibrosis (CF). Class I mutations are nonsense mutations that result in the presence of a premature stop codon that leads to the production of unstable mRNA, or the release from the ribosome of a short, truncated protein that is not functional. Aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperones such as sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or adenosine tri-phosphate (ATP) binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively, restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the splicing machinery and generate both aberrantly and correctly spliced transcripts, the levels of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increases of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that had to be reached in order to achieve normal function.

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58 C-D565G II DF508 D1507 S549R S549I S549N S549R S945D S945L H1054D G1061R L1065P R1066C R1066M L1077P H1085R N1303K G85E III G551D S492F V520F R553G R560T R560S Y569D IV R117H, R117C, R117P, R117L D1152H, L88S, G91R, E92K, Q98R, P205S, L206W, L227R, F311L, G314E, R334W, R334Q, I336K, T338I, L346P, R347C, R347H, R347L, R347P, L927P, R1070W, R1070Q V 3849 þ 10 kb C ! Login to comment

[hide] Multiple mutation analysis of the cystic fibrosis ... Mol Hum Reprod. 2005 Jun;11(6):463-8. Epub 2005 May 20. Sanchez-Garcia JF, Benet J, Gutierrez-Mateo C, Luis Seculi J, Monros E, Navarro J
Multiple mutation analysis of the cystic fibrosis gene in single cells.
Mol Hum Reprod. 2005 Jun;11(6):463-8. Epub 2005 May 20., [PMID:15908456]

Abstract [show]
PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.

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61 The mutations assayed are: DF508, DI507, Q493X, V520F, 1717-1G.A, G542X, G551D, R560T, S459R, S459N and R553X labelled with FAM (blue), 3849þ10kbC.T, 3849 þ 4A . Login to comment

[hide] Genetics of cystic fibrosis. Semin Respir Crit Care Med. 2003 Dec;24(6):629-38. Gallati S
Genetics of cystic fibrosis.
Semin Respir Crit Care Med. 2003 Dec;24(6):629-38., [PMID:16088579]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein expressed in the apical membrane of exocrine epithelial cells. CFTR functions principally as a cyclic adenosine monophosphate (cAMP)-induced chloride channel and appears capable of regulating other ion channels. Mutations affect CFTR through a variety of molecular mechanisms, which can produce little or no functional gene product at the apical membrane. More than 1000 different disease-causing mutations within the CFTR gene have been described. The potential of a mutation to contribute to the phenotype depends on its type, localization in the gene, and the molecular mechanism as well as on interactions with secondary modifying factors. Genetic testing can confirm a clinical diagnosis of CF and can be used for infants with meconium ileus, for carrier detection in individuals with positive family history and partners of proven CF carriers, and for prenatal diagnostic testing if both parents are carriers. Studies of clinical phenotype in correlation with CFTR genotype have revealed a very complex relationship demonstrating that some phenotypic features are closely determined by the underlying mutations, whereas others are modulated by modifier genes, epigenetic mechanisms, and environment.

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43 Mutations (missense, nonsense, frameshift, splice, small and large in-frame deletions or insertions) con- Table 1 Distribution of theWorldwide 24 Most Common Cystic Fibrosis Mutationsa Exon/ Northern Southern North South Austral- Relative Mutation Intron Europe Europe America America asia Africa Asia Frequency G85E E 03 30 14 16 n.a. n.a. 0 7 0.15 R117H E 04 62 3 61 n.a. 7 0 0 0.30 621+1G→T I 04 97 37 154 n.a. 27 0 0 0.72 711+1G→T I 05 15 13 21 n.a. n.a. n.a. 0 0.11 1078delT E 07 53 2 1 n.a. 1 n.a. 0 0.13 R334W E 07 18 21 12 n.a. 2 0 0 0.12 R347P E 07 55 24 26 n.a. 1 0 0 0.24 A455E E 09 35 0 27 n.a. n.a. n.a. 0 0.14 ⌬I507 E 10 57 5 20 2 9 0 0 0.21 ⌬F508 E 10 14,866 4007 6901 342 2309 351 173 66.02 1717-1G→A I 10 160 65 44 n.a. 12 0 3 0.65 G542X E 11 439 259 234 38 56 9 27 2.42 S549N E 11 18 2 5 1 3 1 0 0.07 G551D E 11 356 37 206 1 117 0 0 1.64 R553X E 11 165 44 96 5 11 1 0 0.73 R560T E 11 40 0 24 0 3 0 0 0.15 1898+1G→A I 12 41 10 2 n.a. n.a. n.a. 0 0.12 2184delA E 13 14 7 8 n.a. n.a. n.a. 0 0.07 2789+5G→A I 14b 27 10 17 n.a. n.a. n.a. 0 0.12 R1162X E 19 36 68 19 0 2 0 0 0.28 3659delC E 19 39 1 14 n.a. n.a. n.a. 0 0.12 3849+10kbC→T I 19 23 8 57 n.a. n.a. n.a. 16 0.24 W1282X E 20 120 43 245 n.a. 6 2 120 1.22 N1303K E 21 209 179 130 11 23 8 29 1.34 Chromosomes 21,154 7281 10438 758 3095 515 608 screened Detection rate 80.2 66.7 79.9 52.8 83.7 72.2 61.7 aAccording to the Cystic Fibrosis Genetic Analysis Consortium, http://www.genet.sickkids.on.ca/cftr/. Login to comment
67 SSCP analysis is one of the most popular methods for the detection of sequence variants in polymerase chain reaction (PCR) amplified DNA fragments.29 The princi- Table 3 Cystic Fibrosis Mutations Detected by Commercial Kits INNO-LiPA Mutations CF2 ⌬F508, ⌬I507, G542X, 1717-1G→A, G551D, R553X, W1282X, N1303K CFTR12 ⌬F508, ⌬I507, G542X, 1717-1G→A, G551D, R553X, W1282X, N1303K, S1251N, R560T, 3905insT, Q552X CFTR17+Tn 394delTT, G85E, 621+1G→T, R117H, 1078delT, R347P, R334W, E60X, 2183AA→G, 2184delA, 711+5G→A, 2789+5G→A, R1162X, 3659delC, 3849+10kbC→T, 2143delT, A455E, (5T/7T/9T) Elucigene CF4 ⌬F508, G542X, G551D, 621+1G→T CF12 ⌬F508, G542X, G551D, N1303K, W1282X, 1717-1G→A, R553X, 621+1G→T, R117H, R1162X, 3849+10kbC→T, R334W CF20 1717-1G→A, G542X, W1282X, N1303K, ⌬F508, 3849+10kbC→T, 621+1G→T, R553X, G551D, R117H, R1162X, R334W, A455E, 2183AA→G, 3659delC, 1078delT, ⌬I507, R345P, S1251N, E60X CF Poly-T 5T/7T/9T OLA CF OLA assay ⌬F508, F508C, ⌬I507, Q493X, V520F, 1717-1G→A, G542X, G551D, R553X, R560T, S549R, S549N, 3849+10kbC→T, 3849+4A→G, R1162X, 3659delC, W1282X, 3905insT, N1303K, G85E, 621+1G→T, R117H, Y122X, 711+1G→T, 1078delT, R347P, R347H, R334W, A455E, 1898+1G→A, 2183AA→G, 2789+5G→A b Figure 2 Mutation screening of exon 19 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using polymerase chain reaction (PCR) followed by single-strand conformation polymorphism/heteroduplex (SSCP/HD) analysis on a silver-stained polyacrylamide gel. Login to comment

[hide] Spliceosome-mediated RNA trans-splicing with recom... Hum Gene Ther. 2005 Sep;16(9):1116-23. Liu X, Luo M, Zhang LN, Yan Z, Zak R, Ding W, Mansfield SG, Mitchell LG, Engelhardt JF
Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator function to polarized human cystic fibrosis airway epithelial cells.
Hum Gene Ther. 2005 Sep;16(9):1116-23., [PMID:16149910]

Abstract [show]
We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity to polarized human DeltaF508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral infection from the basolateral surface was required because of inefficient infection from the apical membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with alternative, more clinically viable, vector systems. Furthermore, because recombinant adeno-associated virus (rAAV) vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT correction of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investigated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF airway epithelial cells, using a SMaRT approach. AAV vectors were generated to carry a PTM engineered to bind intron 9 of CFTR pre-mRNA and then trans-splice the normal sequence for human CFTR exons 10-24 into the endogenous pre-mRNA. Human CF polarized airway epithelia were infected from the apical membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (doxorubicin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short-circuit currents 2 weeks postinfection. Levels of CFTR correction seen with rAAV2 (1.07 +/- 0.24 microA) and rAAV5 (0.90 +/- 0.20 microA) CFTR-PTM vectors were similar, representing conductance equivalent to 14.2 and 13.6% of that observed in non-CF human polarized epithelia, respectively. RT-PCR analysis demonstrated the existence of wild-type CFTR transcript in CFTR-PTM-corrected epithelia, whereas only DeltaF508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provide evidence that rAAV vectors are capable of using SMaRT to correct CFTR function after apical infection of human CF airway epithelia. The ability of CFTR-PTM-mediated correction to maintain endogenous CFTR regulation of the transgene product may further improve the efficacy of gene therapy for CF.

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38 Generation of polarized human airway epithelia and rAAV infection Freshly isolated human airway epithelial cells from either non-CF bronchial airways, or CF bronchial airways with ⌬F508/⌬F508, ⌬F508/3659⌬C, ⌬F508/R560T, and ⌬F508/ N1303K mutations, were seeded into collagen-coated 0.6-cm2 Millicell culture inserts (Millipore, Bedford, MA) and grown at the air-liquid interface in 24-well tissue culture plates containing 0.5 ml of medium in the basolateral compartment as previously described (Karp et al., 2002). Login to comment
69 The polarized human CF airway epithelial cells used in this study included several genotypes, all of which contained at least one ⌬F508 allele (⌬F508/⌬F508, ⌬F508/3659⌬C, ⌬F508/R560T, and ⌬F508/N1303K). Login to comment

[hide] A comparison of high-resolution melting analysis w... Am J Clin Pathol. 2005 Sep;124(3):330-8. Chou LS, Lyon E, Wittwer CT
A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model.
Am J Clin Pathol. 2005 Sep;124(3):330-8., [PMID:16191501]

Abstract [show]
High-resolution melting analysis (HRMA) was compared with denaturing high-performance liquid chromatography (dHPLC) for mutation scanning of common mutations in the cystic fibrosis transmembrane conductance regulator gene. We amplified (polymerase chain reaction under conditions optimized for melting analysis or dHPLC) 26 previously genotyped samples with mutations in exons 3, 4, 7, 9, 10, 11, 13, 17b, and 21, including 20 different genotypes. Heterozygous mutations were detected by a change in shape of the melting curve or dHPLC tracing. All 20 samples with heterozygous mutations studied by both techniques were identified correctly by melting (100% sensitivity), and 19 were identified by dHPLC (95% sensitivity). The specificity of both methods also was good, although the dHPLC traces of exon 7 consistently revealed 2 peaks for wild-type samples, risking false-positive interpretation. Homozygous mutations could not be detected using curve shape by either method. However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. HRMA easily detected heterozygotes in all single nucleotide polymorphism (SNP) classes (including A/T SNPs) and 1- or 2-base-pair deletions. HRMA had better sensitivity and specificity than dHPLC with the added advantage that some homozygous sequence alterations could be identified. HRMA has great potential for rapid, closed-tube mutation scanning.

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18 Materials and Methods Sample Source and Study Design Eleven commercially genotyped samples were obtained from Coriell Cell Repositories, Coriell Institute for Medical Research, Camden, NJ (Y122X, R334W, R347P, A455E, I507del, F508del, F508C, G542X/G542X, R553X, R560T, and M1101K). Login to comment
31 ❚Table 1❚ Mutations Analyzed in the Study Position From 5' Exon (or Intron) Genotype* No. of Samples Nucleotide Change SNP Class† End/Amplicon Size (bp) 3 394delTT 1 Del‡ - 132/234 4 R117H 1 G→A 1 83/270 Y122X 1 T→A 4 99/270 I148T 2 T→C 1 176/270 Intron 4 621+1 2 G→T 2 233/270 7 R334W 1 C→T 1 208/345 R347P 1 G→C 3 248/345 9 A455E 2 C→A 2 155/263 10 I507del 1 Del‡ - 171/292 F508del 3 Del‡ - 174/292 F508del/F508del 1 Del - 174/292 F508C 1 T→G 2 175/292 11 G542X 1 G→T 2 90/175 G542X/G542X 1 G→T 2 90/175 G551D 1 G→A 1 118/175 R553X 2 C→T 1 123/175 R560T 1 G→C 3 145/175 13 2184delA 1 Del‡ - 356/458 17b M1101K 1 T→A 4 196/292 21 N1303K 1 C→G 3 175/250 bp, base pairs; SNP, single nucleotide polymorphism. Login to comment
75 Additional mutations in exons 9, 10, 11, and 21 included 7 heterozygous SNPs (A455E, F508C, G542X, G551D, R553X, R560T, and N1303K) and 2 heterozygous 3-base deletions (I507del and F508del). Login to comment

[hide] Newborn screening for cystic fibrosis in Wisconsin... J Pediatr. 2005 Sep;147(3 Suppl):S73-7. Rock MJ, Hoffman G, Laessig RH, Kopish GJ, Litsheim TJ, Farrell PM
Newborn screening for cystic fibrosis in Wisconsin: nine-year experience with routine trypsinogen/DNA testing.
J Pediatr. 2005 Sep;147(3 Suppl):S73-7., [PMID:16202788]

Abstract [show]
OBJECTIVE: To describe the development and follow-up confirmatory results of the routine cystic fibrosis (CF) newborn screening (NBS) program in Wisconsin. METHODS: CF NBS has been performed on a routine clinical basis in Wisconsin since July 1994. The 2-tiered immunoreactive trypsinogen (IRT)/DNA technique was used on dried blood on filter paper spots. From July 1994 to February 2002, mutation analysis was for the DeltaF508 allele. Beginning in March 2002, multimutation analysis of 25 CF mutations was performed. Infants with a positive result on NBS were seen in certified CF centers for sweat testing by means of quantitative pilocarpine iontophoresis, and families received genetic counseling. RESULTS: From July 1994 to February 2002, there were 120 cases of CF detected by means of NBS (509,794 infants screened), with 53 DeltaF508 homozygotes and 67 compound heterozygotes. There were 8 clinically diagnosed cases of CF (no DeltaF508 allele). The CF incidence was 1:3983 (95%CI, 1:3373-1:4774). From March 2002 to June 2003, multimutation analysis identified 21 cases of classic CF (90,142 infants screened). Sweat tests were successfully performed in infants younger than 1 month. CONCLUSIONS: Early diagnosis of CF through NBS was successfully performed, with an estimated sensitivity rate of 99% using the IRT/25 CFTR multimutation assay.

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30 Mutations included in this assay are 2184delA, A455E, DI507, DF508, G542X, G551D, R553X, R560T, 1717-1G>A, R1162X, 3659delC, N1303K, W1282X, R334W, R347P, 1078delT, R117H, I148T, 62111G>T, 278915G>A, 3849110kbC>T, G85E, 109811G>A, 71111G>T and 312011G>A. Login to comment

[hide] Cystic fibrosis transmembrane regulator gene carri... Gut. 2005 Nov;54(11):1661-2. McWilliams R, Highsmith WE, Rabe KG, de Andrade M, Tordsen LA, Holtegaard LM, Petersen GM
Cystic fibrosis transmembrane regulator gene carrier status is a risk factor for young onset pancreatic adenocarcinoma.
Gut. 2005 Nov;54(11):1661-2., [PMID:16227367]

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277 R McWilliams Department of Oncology and Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA W E Highsmith Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA K G Rabe, M de Andrade, L A Tordsen Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA Conflict of interest: None declared. Table 1 Comparison of CFTR mutation frequencies detected in the young onset pancreatic cancer cohort versus the clinical database Young onset pancreatic cancer cases (,60 y old at diagnosis, n = 166) Mayo Clinic clinical database reference group (n = 5349) No % No % CFTR mutation non-carriers 152 91.6 5132 95.9 CFTR mutation carriers 14 8.4 217 4.1 Mutation distribution DF508 12 85.7 155 71.4 R177H 1 7.1 28 12.9 G551D 6 2.8 2789+5G.A 6 2.8 G542X 4 1.8 N1303K 1 7.1 3 1.4 1717-1G.T 2 0.9 3849+10kbC.T 2 0.9 A455E 2 0.9 R1162X 2 0.9 R347H 1 0.5 R553X 1 0.5 3905insT 1 0.5 621+1G.T 1 0.5 W1282X 1 0.5 1898+1G.A 1 0.5 R560T 1 0.5 Young onset pancreatic cancer cases were more frequent carriers of the CFTR mutations compared with patients in the control database (odds ratio 2.18 (95% confidence interval 1.24-3.29); p = 0.006). Login to comment

[hide] Markedly elevated neonatal immunoreactive trypsino... Arch Dis Child. 2006 Mar;91(3):222-5. Epub 2005 Oct 21. Massie J, Curnow L, Tzanakos N, Francis I, Robertson CF
Markedly elevated neonatal immunoreactive trypsinogen levels in the absence of cystic fibrosis gene mutations is not an indication for further testing.
Arch Dis Child. 2006 Mar;91(3):222-5. Epub 2005 Oct 21., [PMID:16243854]

Abstract [show]
AIMS: To investigate the immunoreactive trypsinogen (IRT) values above the usual 99th centile laboratory cut-off and determine the value of offering further testing to those infants with a markedly elevated IRT but no cystic fibrosis transmembrane regulator (CFTR) gene mutation identified by the screening programme. METHODS: All babies born in Victoria, Australia, between 1991 and 2003, were screened by IRT followed by CF gene mutation analysis. RESULTS: Of the 806,520 babies born, 9268 with the highest IRT levels had CFTR mutation analysis. There were 123 DeltaF508 homozygotes and 703 heterozygotes (86 with CF, 617 carriers). A total of 8442 babies had no CFTR gene mutation, of whom 18 (0.21%) had CF. The total number of CF babies with IRT greater than the laboratory cut-off was 227 (2.4%). The IRT results of the CF patients were distributed normally, with the majority above the laboratory cut-off of newborn IRT results. There was no evidence of an excess of babies with CF in the very highest levels of IRT above the 99th centile. CONCLUSIONS: Only a small proportion of babies with a neonatal IRT >99th centile have CF. Additional CF testing for infants with an elevated IRT but no CFTR gene mutation has an extremely low yield, no matter how high the IRT result.

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222 *All patients underwent an extended CFTR mutation analysis for the following mutations in addition to DF508: G551D, R553X, G542X, R117H, N1303K, 621+1G-T, A455E, V520F, 1717-1G-A, W1282X, R1162X, 3849+10kbC-T, R347P, R334W, R560T, S549N. Login to comment

[hide] Mutations of the CFTR gene in idiopathic pancreati... Pancreas. 2005 Nov;31(4):350-2. Gullo L, Mantovani V, Manca M, Migliori M, Bastagli L, Pezzilli R
Mutations of the CFTR gene in idiopathic pancreatic hyperenzymemia.
Pancreas. 2005 Nov;31(4):350-2., [PMID:16258369]

Abstract [show]
OBJECTIVES: Idiopathic pancreatic hyperenzymemia is a new syndrome that is characterized by a chronic increase of serum pancreatic enzymes in the absence of pancreatic disease. The aim of this study was to assess whether mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may have a role in the etiology of this hyperenzymemia. METHODS: Seventy subjects with idiopathic pancreatic hyperenzymemia, 44 men and 26 women (mean age, 48 years; range, 8-74 years), were studied. Thirteen of these 70 subjects had the familial form of the syndrome. The mutation analysis of the CFTR gene was carried out using diagnostic commercial kits for the simultaneous detection of 29 mutations and Tn polymorphism. RESULTS: Among the 70 subjects studied, 7 (10.0%) had CFTR gene mutations. None of these 7 subjects had the familial form of pancreatic hyperenzymemia. These mutations were DeltaF 508 in 1 subject, 2789 + 5 G > A in another subject, and T5 allele in the remaining 5. All these mutations were heterozygous, with the exception of 1 T5 allele that was homozygous in 1 subject. CONCLUSIONS: The frequencies of the mutations of the CFTR gene found in these subjects are similar to the carrier frequencies in the general Italian population. This finding does not support a role for CFTR gene mutations in the etiology of idiopathic pancreatic hyperenzymemia.

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53 A, G551D, R553X, R560T, Q552X (i) 10, 11 2183AA . Login to comment

[hide] Association of common haplotypes of surfactant pro... Pediatr Pulmonol. 2006 Mar;41(3):255-62. Choi EH, Ehrmantraut M, Foster CB, Moss J, Chanock SJ
Association of common haplotypes of surfactant protein A1 and A2 (SFTPA1 and SFTPA2) genes with severity of lung disease in cystic fibrosis.
Pediatr Pulmonol. 2006 Mar;41(3):255-62., [PMID:16429424]

Abstract [show]
Most individual cystic fibrosis transmembrane conductance regulator (CFTR) mutations appear not to correlate directly with severity of lung damage in cystic fibrosis (CF). Components of innate immunity, namely, mannose-binding lectin (MBL2), and surfactant protein A1 and A2 genes (SFTPA1 and SFTPA2), were shown to be critical in pulmonary host defenses. A pilot association study was conducted to identify genetic modifiers of lung disease in adult patients with CF. The structural and promoter (-221x/y) variants of MBL2, variants at codons 19, 50, 62, and 219 of SFTPA1, and at codons 9, 91, and 223 for SFTPA2, were studied in 135 adults with CF and compared to their forced expired volume in 1 sec (FEV1), diffusion of CO (DLCO), and other pulmonary scores. Predicted FEV1 was significantly lower in adults with the SFTPA1 6A3 allele and SFTPA2 1A1) allele (P = 0.01 and 0.009, respectively). The extended haplotype 6A3/1A1, which includes SFTPA1 and SFTPA2, was associated with lower pulmonary function, using FEV1 (P = 0.005) and poor pulmonary scores which were determined by American Medical Association, American Thoracic Society, and modified Shwachman-Kulczycki scores. Lower FEV1 and DLCO values were associated with MBL2 coding variants in those who had the DeltaF508 CFTR mutation (P = 0.03 and 0.004, respectively). These results support the current hypothesis that variants in pulmonary host defense molecules are potentially genetic modifiers of pulmonary disease in CF. Further work in larger populations is required to provide important new insights into the pathogenesis of CF.

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33 Complementary mutations were identified in 51 CF subjects: R117H (4), R347H (1), R347P (1), G542X (7), G551D (4), 1717-1G-A (2), 2789 þ 5G > A(3), 3120 þ 1G > A (2), 3659delC (3), 3849 þ 10kbC>T (6), 394delTT (1), 621 þ 1G>T (4), 711 þ 1G > T (1), G85E (1), I507 (1), N1303K (2), R352Q (1), R553X (2), R560T (1), and W1282X (4). Login to comment

[hide] Detection of F508del mutation in cystic fibrosis t... Singapore Med J. 2006 Feb;47(2):129-33. Zilfalil BA, Sarina S, Liza-Sharmini AT, Oldfield NJ, Stenhouse SA
Detection of F508del mutation in cystic fibrosis transmembrane conductance regulator gene mutation among Malays.
Singapore Med J. 2006 Feb;47(2):129-33., [PMID:16435054]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is one of the common genetic disorders in the western world. It has been reported to be very rare in Asian populations. According to the Cystic Fibrosis Genetic Analysis Consortium, more than 1,000 mutations of the CF gene have been identified. The CF gene, named the cystic fibrosis transmembrane conductance regulator (CFTR), is located on chromosome 7 and composed of 27 exons. This study aims to detect possible CFTR gene mutations in Malays. METHODS: We analysed 50 blood samples from healthy Malays with no symptoms of CF. DNA was extracted from blood using commercially available extraction kits (Eppendorf, Germany). Identification of CFTR gene mutation was performed using the CF OLA (Oligonucleotide Ligation Assay) kit (Applied Biosystems, USA). The PCR-ligation products were electrophoresed on eight percent sequagel using an ABI PRISM 377 genetic analyser (Applied Biosystems, USA). Electrophoresis data was analysed using the Genotyper software and a report of the CF genotype for all loci tested was created using the CF Genotyper Template software. Out of 50, one sample (two percent) was detected to have the F508del mutation (3bp deletion at exon 10), which is one of the most common CFTR gene mutations in Caucasians. RESULTS: The F508del mutation allele was detected in one subject. This indicates that she was a CF carrier. CONCLUSION: We report the finding of a carrier of the F508del mutation of the CFTR gene in the Malay population. Our finding revealed that CF could also affect the Malay population. Larger studies are necessary to determine the exact gene frequency of this population.

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55 MUTATIONS R553X G551D 1507 del F508 del 1717-1 G>A G542X R560T R347P W1282X R334W 1078 Del T 3849 + 10KB C>T R1162X N1303K 3659 Del C A455E R117H 2183 AA>G 2789+5 G>A 1898 +1 G>A 621+1 G>T 711+1 G>T G85E S549N S549R V520F Q493X R347H 3849 +4 A>G 3905 INS T Y122X 4 software before running the gel electrophoresis in 1X TBE using ABI PRISM® 377 Genetic Analyzer (Applied Biosystems, USA) for 45 minutes. Login to comment

[hide] Association of improved pulmonary phenotype in Iri... Pediatr Pulmonol. 2006 Jun;41(6):584-91. Courtney JM, Plant BJ, Morgan K, Rendall J, Gallagher C, Ennis M, Kalsheker N, Elborn S, O'Connor CM
Association of improved pulmonary phenotype in Irish cystic fibrosis patients with a 3' enhancer polymorphism in alpha-1-antitrypsin.
Pediatr Pulmonol. 2006 Jun;41(6):584-91., [PMID:16617455]

Abstract [show]
Modifier genes other than CFTR are thought to influence lung disease phenotype in cystic fibrosis (CF). In this study, we investigated the relationship between a polymorphism (1237 G --> A) in the 3' enhancer region of the alpha-1-antitrypsin (AAT) gene and pulmonary disease severity in 320 CF patients recruited from two independent adult referral centers in Ireland, and evaluated the in vivo effect of the polymorphism on AAT levels during acute infection. When corrected for confounding variables, the polymorphism was found to make a small but significant contribution to variance in percent predicted forced expired volume in 1 sec (FEV1) (1.1%, P = 0.05), with possession of the A allele being associated with better pulmonary function (AA/AG genotype: percent predicted FEV1, 70.8 +/- 3.9; GG genotype: percent predicted FEV1, 62.0 +/- 1.4). As would be expected of a modifier effect, the influence of the polymorphism was more marked in patient groups traditionally associated with more severe lung disease, contributing 3.2% (P = 0.033) to the variance in percent predicted FEV1 in patients homozygous for DF508, 3.3% (P = 0.007) to those infected with Pseudomonas aeruginosa, and 3% (P = 0.024) in female patients. In each instance, a positive association between possession of the A variant and higher percent predicted FEV1 was observed. We did not, however, find any evidence that possession of the A allele effected upregulation of AAT during acute infection in vivo. This lack of a demonstrable functional effect in vivo suggests that the polymorphism is a marker for a modifying effect on pulmonary phenotype in the Irish CF population by a mechanism that is yet to be explained.

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80 T 1.2% 1.7% 1.4% Nonmild R560T 1.5% 1.3% 1.4% Nonmild G542X 0.7% 1.7% 1.1% Nonmild E60X 0.5% 0.9% 0.6% Mild R553X 0.0% 1.3% 0.5% Nonmild N103K 0.7% 0.0% 0.5% Nonmild 9DELTT 0.0% 0.9% 0.3% Mild 3849 þ 10 kb C > T 0.0% 0.9% 0.3% Mild R75Q 0.0% 0.9% 0.3% Mild 1717 þ 1 G > A/À 0.5% 0.0% 0.3% Mild D1507 0.5% 0.0% 0.3% Mild Minor alleles 9.5% 27.4% 15.9% Mild 1 Alleles listed individually occur at a frequency of !0.5% in either population. Login to comment

[hide] Variants in the glutamate-cysteine-ligase gene are... Am J Respir Crit Care Med. 2006 Aug 15;174(4):415-9. Epub 2006 May 11. McKone EF, Shao J, Frangolias DD, Keener CL, Shephard CA, Farin FM, Tonelli MR, Pare PD, Sandford AJ, Aitken ML, Kavanagh TJ
Variants in the glutamate-cysteine-ligase gene are associated with cystic fibrosis lung disease.
Am J Respir Crit Care Med. 2006 Aug 15;174(4):415-9. Epub 2006 May 11., 2006-08-15 [PMID:16690975]

Abstract [show]
BACKGROUND: Chronic progressive lung disease is the most serious complication of cystic fibrosis (CF). Glutathione plays an important role in the protection of the CF lung against oxidant-induced lung injury. OBJECTIVES: We hypothesized that a polymorphism in a novel candidate gene that regulates glutathione synthesis might influence CF lung disease. METHODS: In a cross-sectional study, subjects were recruited from CF clinics in Seattle and multiple centers in Canada. We tested for an association between CF lung disease and a functional polymorphism in the glutamate-cysteine ligase catalytic subunit (GCLC) gene. Multiple linear regression was used to test for association between polymorphisms of GCLC and severity of CF lung disease while adjusting for age, Pseudomonas aeruginosa infection, and cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Analysis was repeated for patients with CF stratified by CFTR genotype. MEASUREMENTS AND MAIN RESULTS: A total of 440 subjects with CF participated in the study (51% male; mean [+/- SD] age, 26 +/- 11 yr; mean FEV(1), 62 +/- 28% predicted). In the total population, there was a trend toward an association between GCLC genotypes and CF lung disease (linear regression coefficient [SEM], 1.68 [1.0]; p = 0.097). In the stratified analysis, there was a highly significant association between GCLC genotype and CF lung function in subjects with a milder CFTR genotype (linear regression coefficient [SEM], 5.5 (1.7); p = 0.001). CONCLUSIONS: In patients with CF with a milder CFTR genotype, there is a strong association between functional polymorphisms of the GCLC gene and CF lung disease severity.

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62 * Severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations (Class I-III) ϭ G542X, R553X, W1282X, R1162X, 621-1G→T, 1717-1G→A, 1078⌬T, 3659⌬C, ⌬F508, ⌬I507, N1303K, S549N, G551D, R560T. Login to comment

[hide] Molecular analysis of the IVS8-T splice variant 5T... Mol Hum Reprod. 2006 Jul;12(7):469-73. Epub 2006 May 19. Radpour R, Gilani MA, Gourabi H, Dizaj AV, Mollamohamadi S
Molecular analysis of the IVS8-T splice variant 5T and M470V exon 10 missense polymorphism in Iranian males with congenital bilateral absence of the vas deferens.
Mol Hum Reprod. 2006 Jul;12(7):469-73. Epub 2006 May 19., [PMID:16714368]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is responsible for 2-6% of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. To investigate CBAVD at the molecular level in Iran, we have characterized the mutations in the CFTR gene in 106 patients with this condition. None had clinical manifestations of cystic fibrosis (CF). We also analysed a DNA variant (the 5T allele) in a noncoding region of CFTR, which causes reduced levels of the normal CFTR protein and M470V exon 10 missense polymorphism. Five of the 106 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Eighty-five patients had a mutation in at least one copy of CFTR, and of these patients, 46 had one 5T allele (in 11 cases, two alleles and in 35 cases, just one allele of 5T was detected). In 21 patients, no CFTR and 5T mutations were found (19.81%). 5T/M470 genotype was found in 19 patients, 5T/V470 was found in 3 and 5T with heterozygote form of M470V was found in 24 CBAVD patients. In CBAVD patients, 28 F508del carriers were identified. Most of our patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in Iran. The 5T allele mutation has a wide range of clinical presentations and revealed a high frequency, occurring in patients with CBAVD or moderate forms of CF and infertile men.

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90 Mutation geno types IVS8-PolyT M470V n (%) Two mutations detected F508del/R117H 9T/9T M/M 1 (0.94) F508del/621+1G>T 7T/7T V/V 1 (0.94) 1540A/G/1540A/G 7T/7T M/M 2 (1.89) R347H/R117H 9T/7T M/V 1 (0.94) G551D/IVS8-5T 7T/5T M/V 2 (1.89) F508del/IVS8-5T 7T/5T M/V 8 (7.55) 9T/5T M/M 6 (5.67) 1717-1G>A/IVS8-5T 7T/5T M/V 4 (3.77) R117H/IVS8-5T 7T/5T M/V 2 (1.89) 621+1G>T/IVS8-5T 7T/5T M/V 3 (2.83) 9T/5T M/M 2 (1.89) 1540A/G/IVS8-5T 7T/5T M/V 2 (1.89) R553X/IVS8-5T 7T/5T M/V 1 (0.94) IVS8-5T/IVS8-5T 5T/5T V/V 3 (2.83) 5T/5T M/M 8 (7.55) One mutation detected G85E/- 7T/7T V/V 2 (1.89) G551D/- 9T/7T V/V 1 (0.94) 621+1G>T/- 7T/7T M/M 2 (1.89) 9T/7T M/V 1 (0.94) R334W/- 7T/7T M/V 1 (0.94) F508del/- 7T/7T M/V 7 (6.60) 9T/7T M/M 3 (2.83) 9T/9T M/V 2 (1.89) IVS8-5T/- 5T/7T M/M 3 (2.83) 5T/9T M/V 2 (1.89) 1717-1G>A/- 7T/7T M/V 3 (2.83) 9T/7T M/V 2 (1.89) R117H/- 7T/7T M/M 2 (1.89) 9T/7T M/V 1 (0.94) 2789+5G>A/- 7T/7T M/M 1 (0.94) 3120+1G>A/- 9T/7T M/V 2 (1.89) R560T/- 9T/7T M/V 1 (0.94) N1303K/- 9T/7T V/V 1 (0.94) 1651A/G/- 7T/7T M/V 1 (0.94) R553X/- 9T/7T M/V 1 (0.94) No mutation detected -/- 7T/7T M/M 12 (11.32) -/- 9T/9T M/M 3 (2.83) -/- 9T/7T M/V 6 (5.66) Table IV. Login to comment

[hide] Analysis of CFTR, SPINK1, PRSS1 and AAT mutations ... J Pediatr Gastroenterol Nutr. 2006 Sep;43(3):299-306. Sobczynska-Tomaszewska A, Bak D, Oralewska B, Oracz G, Norek A, Czerska K, Mazurczak T, Teisseyre M, Socha J, Zagulski M, Bal J
Analysis of CFTR, SPINK1, PRSS1 and AAT mutations in children with acute or chronic pancreatitis.
J Pediatr Gastroenterol Nutr. 2006 Sep;43(3):299-306., [PMID:16954950]

Abstract [show]
OBJECTIVES: Defects of PRSS1, SPINK1, CFTR and AAT are considered causative or predisposing to pancreatitis. The aim of this study was to evaluate the impact of these defects into molecular pathology of chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP). METHODS: Ninety-two children with CP or ARP, 55 family members and 50 controls were investigated. The subjects were screened for PRSS1 mutations: R122H, R122C, A16V, N29I; SPINK1 N34S variant; panel of 14 CFTR defects: INNOLiPA CFTR12, CFTRdele2,3 and IVS8-T variant or panel of 3 CFTR defects-F508del, CFTRdele2,3 and IVS8-T; AAT mutations: E264V, E342K. RESULTS: We identified 1 mutated allele in at least 1 of 4 genes in 31 of 92 patients and 12 of 50 controls (P = 0.157). Mutations in SPINK1 and PRSS1 were most frequent. PRSS1 mutations were identified mainly in CP patients (9.6% of CP vs 2.5% of ARP alleles, P = 0.094), whereas N34S SPINK1 mutation was present with comparable frequency in CP and ARP patients (7.7% vs 10.0%, P = 0.768). The frequency of mutations in CFTR alleles was similar to controls (4.9% vs 5%, P = 0.587). Overall frequency of AAT mutations was lower than in the controls. Family studies showed that defects in the examined genes did not always segregate with disease. CONCLUSIONS: PRSS1 defects seem to be causative for pancreatitis, whereas defects in SPINK1 are suggested to be associated with the disease. No association between CFTR mutations and pancreatitis was observed. The importance of AAT variants remains speculative.

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64 For the first 50 patients enrolled in this study, the CFTR mutations F508del, G542X, G551D, R553X, N1303K, W1282X, 1717-1G/A, I507del, S1251N, R560T, 3905insT, Q552X (INNO-LiPA CFTR12, Innogenetics, Gent, Belgium), CFTRdele2,3 (16) and polyT variant in intron 8 (IVS8-T) (17) were analyzed. Login to comment

[hide] Revertant mutants G550E and 4RK rescue cystic fibr... Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17891-6. Epub 2006 Nov 10. Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD
Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17891-6. Epub 2006 Nov 10., 2006-11-21 [PMID:17098864]

Abstract [show]
The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wt- and V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control.

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1 Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. Login to comment
2 G550E rescued the trafficking defect of A561E but not that of R560T. Login to comment
37 47 ͉ 17891-17896 and 4RK act by different mechanisms. To explore this possibility, we tested the effects of G550E and 4RK on three additional CF mutations within NBD1: R560T, A561E, and V562I.ʈ We selected for study these CF mutants because (i) these residues constitute a hot spot for disease-causing mutations (seven mutations are associated with these three residues࿣ ); (ii) A561E and R560T are the second and fourth most frequent mutations among Portuguese and Irish CF patients, respectively (28); (iii) like G550E and R555K (one of the 4RK mutants), these mutations affect residues located between the LSGGQ and Walker B motifs of NBD1, which are highly conserved across species; and (iv) they all lie within the same ␣-helix (H5; G550-Y563) within the ATP-binding cassette ␣-subdomain of NBD1 (29, 30). Login to comment
38 To test the hypothesis that G550E and 4RK act by different mechanisms, we used biochemical and functional assays to investigate how these revertants rescue F508del-, R560T-, A561E-, and V562I-CFTR. Login to comment
39 Results R560T, but Not V562I, Disrupts the Processing of CFTR. Like F508del-CFTR, many CF mutations disrupt the processing of CFTR and its delivery to the cell surface. Login to comment
41 For the reasons outlined in the Introduction, we chose to analyze the CF mutations R560T, A561E, and V562I. Login to comment
43 Therefore, we first investigated the processing of R560T and V562I by using Western blot (WB). Login to comment
44 Fig. 1A demonstrates that like F508del- and A561E-CFTR, R560T-CFTR generated only a discrete Ϸ145 kDa band (band B). Login to comment
46 These data suggest that R560T, disrupts the trafficking of CFTR, whereas V562I-CFTR is delivered to the cell surface. Login to comment
47 Next, we investigated whether the revertants G550E and 4RK rescue the defective biosynthesis of R560T- and A561E-CFTR. Login to comment
55 The mature form of CFTR (band C) was clearly observed for F508del- and A561E-CFTR (Fig. 1D) in the presence of G550E and 4RK, but G550E failed to correct the defective biosynthesis of R560T-CFTR (Fig. 1 A and D). Login to comment
70 (A) WB of total protein (30 ␮g) from BHK cells stably expressing wt-, F508del-, R560T-, A561E-, or V562I-CFTR, alone or in cis with 4RK and G550E. Login to comment
75 (D) BHK cells expressing F508del-, R560T-, or A561E-CFTR alone or in cis with the revertants 4RK and G550E were analyzed by CFTR IP after pulse-labeling for 3 h. Labeled arrows indicate the positions of bands A, B, and C. Thus, the higher steady-state levels of band C for 4RK variants of both wtand V562I-CFTR (Fig. 1A) are explained only in part by a slight (but not significant) increase in the efficiency of processing band B to band C. Surprised that the revertants did not exert stronger effects on the processing of CFTR, we wondered how they might influence CFTR Cl-channel function. Login to comment
79 Consistent with the biochemical data (Fig. 1), these agonists had no effect on F508del-, R560T-, or A561E-CFTR (Fig. 3 B-D) but evoked a striking efflux of I- from V562I-CFTR (Fig. 3E), which has a time course equivalent to that of wt-CFTR and 1.3-fold greater (Fig. 3F). Login to comment
84 In contrast to F508del-CFTR, G550E did not restore CFTR function to R560T and both revertants rescued only modestly the CFTR function of A561E (Fig. 3 C, D, and F). Login to comment
96 (A-E) Time courses of I-efflux from BHK cells stably expressing wt- (A), F508del- (B), R560T- (C), A561E- (D), and V562I- (E) CFTR in the absence and presence of the 4RK and G550E mutations. Login to comment
127 Like F508del, R560T and A561E disrupt CFTR processing, whereas V562I traffics normally to the cell surface, forming a Cl-channel with properties indistinguishable from those of wt-CFTR. Login to comment
128 The revertants 4RK and G550E rescue the cell surface expression of A561E, albeit not as effectively as F508del, whereas G550E is without effect on R560T. Login to comment
129 Of note, G550E, but not 4RK, rescues the defective channel gating of F508del, suggesting that G550E and 4RK rescue the expression and function of F508del by distinct mechanisms. To understand the structural basis by which R560T and A561E disrupt the processing of CFTR (refs. Login to comment
132 R560T likely disrupts the interactions of R560 with neighboring residues and, hence, the folding of NBD1. Login to comment
205 When compared with both wtand F508del-CFTR, clones expressing V562I- and R560T-CFTR expressed higher and lower levels of protein, respectively, precluding studies on cell lines with equivalent amounts of CFTR protein. Login to comment

[hide] CFTR genotype as a predictor of prognosis in cysti... Chest. 2006 Nov;130(5):1441-7. McKone EF, Goss CH, Aitken ML
CFTR genotype as a predictor of prognosis in cystic fibrosis.
Chest. 2006 Nov;130(5):1441-7., [PMID:17099022]

Abstract [show]
STUDY RATIONALE: Certain CFTR genotypes are associated with reduced mortality. The accuracy of using CFTR genotype as a predictor of survival and the mechanisms through which CFTR genotype influences survival are unknown. PARTICIPANTS: All patients with cystic fibrosis (CF) enrolled in the US Cystic Fibrosis Foundation national registry between 1993 and 2002. DESIGN: We examined the prognostic value of CFTR genotype, grouped into "high-risk" and "low-risk" categories based on the effect of their CFTR genotype on phenotype and protein production. MEASUREMENTS AND RESULTS: Clinical and genetic data were available from 15,651 patients with CF. Patients with a high-risk CFTR genotype had a greater than twofold increased risk of death compared to patients with a low-risk CFTR genotype (relative risk, 2.25; 95% confidence interval [CI], 1.77 to 2.84; p < 0.001). This association was partly explained by lung function, nutritional status, pancreatic insufficiency, and Pseudomonas aeruginosa colonization. Of the 1,672 patients who died, median age at death for the high-risk CFTR genotype was 24.2 years (interquartile range, 18.4 to 32.0 years) and for the low-risk CFTR genotype was 37.6 years (interquartile range, 28.8 to 47.9 years; p < 0.001). The positive predictive value of this classification method as a test to identify patients who died before or after their 30th birthday was 69% (95% CI, 67 to 72%) with a negative predictive value of 71% (95% CI, 60 to 80%). CONCLUSIONS: Grouping patients into high-risk and low-risk CFTR genotype categories is associated with significant differences in survival and median age at death. These differences are not fully explained by lung function, nutritional measures, pancreatic insufficiency, or P aeruginosa colonization. Modest reassurance about the likelihood of a milder than average course can be provided for CF patients with a low-risk CFTR genotype, although it should be acknowledged that substantial phenotypic variability exists.

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46 Alleles High-risk CFTR genotype Class I 2,131 G542X, R553X, W1282X, R1162X, 621-1G3T, 1717-1G3A, 1078⌬T, 3659⌬C Class II 11,231 ⌬F508, ⌬I507, N1303K, S549N, G85E Class III 783 G551D, R560T Low-risk CFTR genotype Class IV 391 R117H, R334W, R347P Class V 421 3849 ϩ 10KbC3T, 2789 ϩ 5G3A, A455E *Patients with both CFTR alleles in either class I, class II, or class III were grouped together as a high-risk genotype, while patients with at least one mutant allele in class IV and V were considered to have low-risk genotypes; 380 patients had both mutations in either class I, II, or III, while 314 patients had both mutations in either class IV or V (total, n ϭ 15,651). Login to comment

[hide] Molecular study of (TG)m(T)n polymorphisms in Iran... J Androl. 2007 Jul-Aug;28(4):541-7. Epub 2007 Feb 21. Radpour R, Gourabi H, Gilani MA, Dizaj AV
Molecular study of (TG)m(T)n polymorphisms in Iranian males with congenital bilateral absence of the vas deferens.
J Androl. 2007 Jul-Aug;28(4):541-7. Epub 2007 Feb 21., [PMID:17314234]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia. Nearly 75% of men with CBAVD have at least 1 detectable common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The different alleles at the (TG)(m)(T)(n) polymorphic locus at the 3' end of human CFTR intron 8 determine the efficiency of exon 9 splicing. To study the CFTR gene mutations and (TG)(m)(T)(n) polymorphisms in Iranian CBAVD patients with presumed low CF frequency and to better understand the complex regulation of exon 9 splicing among our study population, we analyzed CFTR mutations and (TG)(m)(T)(n) polymorphisms in 112 Iranian CBAVD, 7 congenital unilateral absence of the vas deferens males from Iran, and 84 fertile males as controls. Moreover, we compared the rate of CFTR transcripts with exon 9 (9+) with reduction of the (T)(n) repeat in our study population. Our study showed that the 5T mutation was present with high frequency in our patients. Longer (TG)(m) polymorphic tracts increase the proportion of exon 9 deletion transcripts but only when activated by the 5T allele. The combination of the 5T allele in 1 copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in the Iranian population. We also observed the highest level of exon 9+ splicing efficiency among the tested samples with the (TG)(12)(T)(7) allele, which represents the most common intron 8 splice variant allele in the general population. Our results support the idea that a putative role of the (T)(n) repeat is to distance the (TG)(m) repeat from the 3' splice site and that the different alleles at the (T)(n) locus affect the efficiency by which the splice acceptor consensus sequence is recognized.

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77 CFTR gene mutations in 112 CBAVD patients and 7 CBAVD patients* Samples Mutation genotype3 (TG)m(T)n n (%) CBAVD Two mutations detected (5 /112 5 4.46%) F508del / R117H (TG)10 9T / (TG)10 9T 1 (0.89) F508del / 621+1G.T (TG)11 7T / (TG)11 7T 1 (0.89) 1540A/G / 1540A/G (TG)11 7T / (TG)11 7T 2 (1.79) R347H / R117H (TG)10 9T / (TG)11 7T 1 (0.89) One mutation detected with one 5T allele (32 / 112 5 28.57%) G551D / - (TG)10 7T/ (TG)13 5T 2 (1.79) F508del / - (TG)12 7T/ (TG)13 5T 8 (7.14) (TG)11 9T/ (TG)13 5T 6 (5.36) 1717-1G.A / - (TG)11 7T/ (TG)12 5T 4 (3.57) R117H / - (TG)12 7T/ (TG)13 5T 2 (1.79) 621+1G.T / - (TG)11 7T/ (TG)13 5T 3 (2.68) 2 (1.79) 1540A/G / - (TG)11 7T/ (TG)13 5T 2 (1.79) R553X / - (TG)12 7T/ (TG)13 5T 1 (0.89) Y122H / -4 (TG)11 7T / (TG)13 5T 1 (0.89) T338A / -4 (TG)10 7T / (TG)13 5T 1 (0.89) No mutation detected with two 5T alleles (11 / 112 5 9.82%) - / - (TG)12 5T / (TG)13 5T 3 (2.68) - / - (TG)13 5T / (TG)13 5T 8 (7.14) One mutation detected without 5T allele (35 / 112 5 31.25%) G85E / - (TG)11 7T / (TG)11 7T 2 (1.79) G551D / - (TG)10 9T / (TG)12 7T1 1 (0.89) 621+1G.T / - (TG)11 7T / (TG)11 7T 2 (1.79) (TG)10 9T / (TG)11 7T 1 (0.89) R334W / - (TG)12 7T / (TG)10 7T 1 (0.89) F508del / - (TG)11 7T / (TG)11 7T 7 (6.25) (TG)11 9T / (TG)12 7T 3 (2.68) (TG)10 9T / (TG)10 9T 2 (1.79) 1717-1G.A / - (TG)11 7T / (TG)12 7T 3 (2.68) (TG)10 9T / (TG)11 7T 2 (1.79) R117H/- (TG)12 7T / (TG)12 7T 2 (1.79) (TG)10 9T / (TG)11 7T 1 (0.89) 2789+5G.A / - (TG)10 7T / (TG)11 7T 1 (0.89) 3120+1G.A / - (TG)10 9T / (TG)11 7T 2 (1.79) R560T / - (TG)10 9T / (TG)11 7T 1 (0.89) N1303K / - (TG)10 9T / (TG)11 7T 1 (0.89) 1651A/G / - (TG)11 7T / (TG)12 7T 1 (0.89) R553X / - (TG)10 9T / (TG)10 7T 1 (0.89) K536X / -4 (TG)10 9T / (TG)10 9T 1 (0.89) No mutation detected with one 5T alleles (7 / 112 5 6.25%) - / - (TG)13 5T / (TG)12 7T 3 (2.68) - / - (TG)13 5T / (TG)10 9T 4 (3.57) No mutation detected (22 / 112 5 19.64%) - / - (TG)11 7T / (TG)11 7T 12 (10.71) - / - (TG)11 7T / (TG)12 7T 1 (1.79) - / - (TG)10 9T / (TG)10 9T 3 (2.68) - / - (TG)10 9T / (TG)11 7T 6 (5.36) CUAVD One mutation detected without 5T allele (2 / 7 5 28.57%) R334W / - (TG)10 9T / (TG)11 7T 1 (14.29) R117H / - (TG)11 7T / (TG)11 7T 1 (14.29) No mutation detected with one 5T alleles (3 / 7 5 42.86%) - / - (TG)11 9T / (TG)13 5T 2 (28.57) - / - (TG)10 7T / (TG)13 5T 1 (14.29) No mutation detected (2 / 7 5 28.57%) - / - (TG)10 9T / (TG)12 7T 2 (28.57) * CBAVD indicates congenital bilateral absence of the vas deferens; CUAVD, congenital unilateral absence of the vas deferens. Login to comment

[hide] Heteropolymeric triplex-based genomic assay to det... PLoS One. 2007 Mar 21;2(3):e305. Daksis JI, Erikson GH
Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples.
PLoS One. 2007 Mar 21;2(3):e305., [PMID:17375191]

Abstract [show]
Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

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125 Sequence bglIR-WT25C 1 59-TATTTTGATTATAGGACATGAAGAT-39 DR01-WT15 2 59-GAGCCGAAGGGGCAG-39 CFTR delta F508-WT25C 3 59-TAGGAAACACCAAAGATGATATTTT-39 CFTR delta F508-MUT25C 4 59-ATAGGAAACACCA---ATGATATTTTCT-39 CFTR delta I507-WT25C 5 59-TAGGAAACACCAAAGATGATATTTT-39 CFTR delta I507-MUT25C 6 59-ATAGGAAACACCAAAGA---TATTTTCT-39 CFTR 3659delC-WT25C 7 59-TGGTTTGGTTGACTTGGTAGGTTTA-39 CFTR 3659delC-MUT25C 8 59-ATGGTTTGGTTGACTTG-TAGGTTTA-39 CFTR 3849+10kbCRT-WT25C 9 59-GTGTCTTACTCGCCATTTTAATACT-39 CFTR 3849+10kbCRT-MUT25C 10 59-GTGTCTTACTCACCATTTTAATACT-39 CFTR 2789+5GRA-WT25C11 59-AATAGGACATGGAATACTCACTTTC-39 CFTR 2789+5GRA-MUT25C 12 59-AATAGGACATGGAATATTCACTTTC-39 CFTR G551D-WT25C 13 59-ATTCTTGCTCGTTGACCTCCACTCA-39 CFTR G551D-MUT25C 14 59-ATTCTTGCTCGTTGATCTCCACTCA-39 CFTR 621+1GRT-WT25C 15 59-AAGTATTACCTTCTTATAAATCAAA-39 CFTR 621+1GRT-MUT25C16 59-AAGTATTAACTTCTTATAAATCAAA-39 CFTR R1162X-WT25C 17 59-AACTTAAAGACTCGGCTCACAGATC-39 CFTR R1162X-MUT25C 18 59-AACTTAAAGACTCAGCTCACAGATC-39 CFTR 1717-1GRA-WT25C 19 59-TGGAGATGTCCTATTACCAAAAATA-39 CFTR 1717-1GRA- MUT25C 20 59-TGGAGATGTCTTATTACCAAAAATA-39 CFTR A455E-WT25C 21 59-CCAGCAACCGCCAACAACTGTCCTC-39 CFTR A455E-MUT25C 22 59-CCAGCAACCTCCAACAACTGTCCTC-39 CFTR G542X-WT25C 23 59-ATTCCACCTTCTCCAAGAACTATAT-39 CFTR G542X-MUT25C 24 59-ATTCCACCTTCTCAAAGAACTATAT-39 CFTR N1303K-WT25C 25 59-TAGGGATCCAAGTTTTTTCTAAATG-39 CFTR N1303K-MUT25C 26 59-TAGGGATCCAACTTTTTTCTAAATG-39 CFTR R560T-WT25C 27 59-AGTTATTCACCTTGCTAAAGAAATT-39 CFTR R560T-MUT25C 28 59-AGTTATTCACGTTGCTAAAGAAATT-39 CFTR W1282X-WT25C 29 59-TTTCCTCCACTGTTGCAAAGTTATT-39 CFTR W1282X-MUT25C 30 59-TTTCCTTCACTGTTGCAAAGTTATT-39 MTHFR C677T-WT25C 31 59-TGATGATGAAATCGGCTCCCGCAGA-39 MTHFR C677T-MUT25C 32 59-TGATGATGAAATCGACTCCCGCAGA-39 FVL G1691A-WT25C 33 59-CCCTCTGTATTCCTCGCCTGTCCAG-39 FVL G1691A-MUT25C 34 59-CCCTCTGTATTCCTTGCCTGTCCAG-39 All 25-mer probes listed were antisense. Login to comment
704 Mutation Source Number of tests Percentage GC in probe sequence Percentage difference of mismatched TAF relative to perfect match TAF CFTR delta F508 blood 102 28% 2100% to 281% CFTR delta I507 blood 6 28% 2100% to 285% CFTR 3659delC blood 11 40% 2100% to 255% CFTR 3849+10kbCRT blood 9 36% 2100% to 282% CFTR 2789+5GRA blood 16 36% 2100% to 275% CFTR 2789+5GRA saliva 13 36% 2100% to 266% CFTR G551D blood 11 48% 2100% to 261% CFTR 621+1GRT blood 5 20% 2100% to 257% CFTR R1162X blood 6 44% 267% to 236% CFTR 1717-1GRA blood 12 32% 2100% to 258% CFTR A455E blood 9 60% 2100% to 289% CFTR G542X blood 6 36% 2100% to 260% CFTR N1303K blood 8 32% 2100% to 283% CFTR R560T blood 6 28% 2100% to 254% CFTR W1282X blood 14 36% 2100% to 274% MTHFR C677T blood 55 52% 2100% to 272% FVL G1691A blood 34 60% 2100% to 281% TAF indicates Triplex-Associated Fluorescence. doi:10.1371/journal.pone.0000305.t004 ..................................................................................... brighter when intercalated into complexes of identical short oligonucleotides, such as the probes used in our assay, than when a like number of YOYO-1 molecules were in the presence of genomic duplex DNA. Login to comment

[hide] Airway nitric oxide in patients with cystic fibros... Chest. 2007 Jun;131(6):1857-64. Epub 2007 Mar 30. Keen C, Olin AC, Edentoft A, Gronowitz E, Strandvik B
Airway nitric oxide in patients with cystic fibrosis is associated with pancreatic function, Pseudomonas infection, and polyunsaturated fatty acids.
Chest. 2007 Jun;131(6):1857-64. Epub 2007 Mar 30., [PMID:17400678]

Abstract [show]
BACKGROUND: Airway nitric oxide (NO) is low or normal in cystic fibrosis (CF) patients. This may affect bacterial status since NO has antimicrobial properties. Arachidonic acid (AA), which is increased in the serum and airways of CF patients, has been shown to reduce NO levels. The aim of this study was to investigate whether airway NO level correlates with genotype and pancreatic function, and whether low airway NO level is associated with bacterial infection and increased serum AA level in CF patients. METHOD: Nasal NO (nNO) and exhaled NO (eNO) were measured according to the European Respiratory Society/American Thoracic Society standard in 59 CF patients aged 7 to 55 years, 80% of whom were pancreatic insufficient (PI) and 51% were chronically infected with Pseudomonas aeruginosa. RESULTS: PI CF patients had significantly lower nNO levels than pancreatic-sufficient (PS) patients. Airway NO level did not correlate with lung function or inflammatory parameters. PI patients chronically infected with P aeruginosa had significantly lower nNO levels than noninfected PI patients. nNO level correlated inversely with the AA/docosahexaenoic acid ratio, and eNO with the essential fatty acid (FA) deficiency index, which is the ratio between mead acid and AA. CONCLUSIONS: CF patients with PI, which is associated with more severe genotypes, had lower airway NO levels than patients with PS. Low NO level was correlated to chronic P aeruginosa infection, and an association was found between airway NO level and the abnormal serum phospholipid FA pattern.

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30 Patients in group 3 were heterozygous for mutations dF508 and V603F, R560T, or 621 ϩ 1G-T; group 4 patients were heterozygous for mutations dF508, 3659del C, or 394delTT and a mutation linked to a "mild" phenotype (eg, N1088D, R117C, R117H, R75Q, R658X, S945L, 1154insTC, or T338I). Login to comment

[hide] Validation of cystic fibrosis mutation analysis us... Diagn Mol Pathol. 2007 Mar;16(1):57-9. Huang CK, Pan Q
Validation of cystic fibrosis mutation analysis using ABI 3130XL genetic analyzer.
Diagn Mol Pathol. 2007 Mar;16(1):57-9., [PMID:17471160]

Abstract [show]
Cystic fibrosis (CF) is one of the most common autosomal recessive diseases in the white population, with a prevalence estimate of 1 in 2500 to 3300 live births. CF is characterized by viscous mucus in the lungs with involvement of digestive and reproductive systems as well as sweat glands (excess salt loss). Treatment for CF patients is palliative. Over 1300 mutations have been identified in the CFTR gene. However, most of the mutations are at frequencies of <0.1% or represent private mutations. Although other methodologies are available for CF testing, the oligonucleotide ligation assay is a unique approach to mutation detection of point mutations, small deletions, and small insertions, and consists of 2 phases. Applied Biosystems 3130 Series Genetic Analyzers are the next-generation platform for low to medium throughput laboratories and deliver improved performance. One disadvantage of the Genetic Analyzers is that there is no template of instrument settings for POP-6 polymer using 36-cm array. The Abbott CF oligonucleotide ligation assay ASRs can be run only using POP-6 polymer. We are the first to have optimized the instrument settings for POP-6 polymer based on the template of Rapidseq36-POP6 for Abbott Diagnostics CF V3 ASRs. Several conditions were tried, and the conditions of sample injection voltage at 10,000 v and sample injection time at 5 seconds gave better results, which were with clearer peaks and lower background signals. Twenty cell line DNA samples from Coriell were analyzed, and the results were matched. In addition, Synthetic Controls from AcroMetrix were analyzed, and the results were same as expected. Also, about 1500 clinical samples were analyzed, and high-quality reportable results were obtained. In conclusion, our modified protocol is robust and reliable on this ABI 3130XL instrument.

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58 Mutation controls: to specifically assess the detection of CF mutations, 20 cell line DNA samples with mutations of R553X, 3659delC/delF508, delF508/Q493X, 711+ 1G>T/621+1G>T, 621+1G>T/delF508, G85E/ 621+1G>T, R560T/delF508, A455E/621+1G>T, N1303K, W1282X, G551D/R553X, 2789+5G>A/ 2789+5G>A, 3849+10C>T/3849+10C>T, 1717-1G>A, delF508/delF508, R347P/G551D, R334W, V520F, R117H/delF508/5T/9T, or G542X/G542X, respectively, from the Coriell Cell Repositories were analyzed. Login to comment

[hide] Analysis of cystic fibrosis gene mutations and ass... Genet Test. 2007 Summer;11(2):133-8. Knezevic J, Tanackovic G, Matijevic T, Barisic I, Pavelic J
Analysis of cystic fibrosis gene mutations and associated haplotypes in the Croatian population.
Genet Test. 2007 Summer;11(2):133-8., [PMID:17627383]

Abstract [show]
The aim of this study was to reveal the CFTR gene mutation status in the Croatian population as well as to establish the haplotypes associated with cystic fibrosis (CF) and those associated with specific gene mutations. A total of 48 unrelated CF patients from Croatia were examined. Among 96 tested alleles, we found nine different mutations: DeltaF508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 + 1G --> T; G85E; Y569C; E585X; and S466X, 1.04%. Analysis of three polymorphic loci revealed 15 different haplotypes. Two of them (21-23-13 and 21-17-13) occurred with a higher frequency (40% and 24%). Both of these haplotypes also carried a CFTR gene mutation (DeltaF508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Because there are still no published data on the distribution of polymorphic loci in Croatia, nor haplotypes associated with mutations in the CFTR gene, our results greatly contribute to knowledge regarding the genetic background of CF in this region.

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39 INNOGENETICS INNO-LIPA CFTR 12 and INNO-LIPA CFTR 7 ϩ Tn diagnostic kits were used to assess the presence of the 29 mutations in CF patients; ⌬F508, ⌬I507, G542X, N1303K, 1717-1G Ǟ A, W1282X, G551D, R553X, S1251N, R560T, 3905insT, Q552X, 394delTT, G85E, E60X, 621 ϩ 1G Ǟ T, R117H, 1078delT, R347P, R334W, 2143delT, 2183AA Ǟ G, 2184delA, 711 ϩ 5G Ǟ A, 2789 ϩ 5G Ǟ A, R1162X, 3659delC, 3849 ϩ 10kbC Ǟ T, and A455E. Login to comment

[hide] Scanning the cystic fibrosis transmembrane conduct... Clin Chem. 2007 Nov;53(11):1891-8. Epub 2007 Sep 21. Montgomery J, Wittwer CT, Kent JO, Zhou L
Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis.
Clin Chem. 2007 Nov;53(11):1891-8. Epub 2007 Sep 21., [PMID:17890437]

Abstract [show]
BACKGROUND: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping. METHODS: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner(R). We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants. RESULTS: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays. CONCLUSIONS: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.

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145 2 223CϾT R31C 3 355CϾT R75X 386GϾA G85E 4 482GϾA R117H 575TϾC I148T 621 ؉ 1GϾTb 5 711 ؉ 1GϾT 7 1078delT 1132CϾT R334W 1150delA 1172GϾC R347P 8 1341 ϩ 18AϾCc 9 1496CϾA A455E 10 1651-1653del I507del 1653-1655del F508deld 11 1717 - 1GϾA 1756GϾT G542Xe 1784GϾA G551Db 1789CϾT R553Xf 1811GϾC R560T 12 1898 ؉ 1GϾA 13 2184delA 14b 2789 ؉ 5GϾAe 16 3120 ؉ 1GϾA 18 3500 - 2AϾTg 19 3616CϾT R1162X 3659delC Intron 19 3849 ؉ 10kbCϾTe 20 3978GϾA W1282X 21 4041CϾG N1303K 22 4178GϾA G1349Dc a Disease-causing variants recommended for genotyping by the ACMG (4) are in bold. Login to comment

[hide] One multiplex control for 29 cystic fibrosis mutat... Genet Test. 2007 Fall;11(3):256-68. Lebo RV, Bixler M, Galehouse D
One multiplex control for 29 cystic fibrosis mutations.
Genet Test. 2007 Fall;11(3):256-68., [PMID:17949287]

Abstract [show]
A simple approach is described to synthesize and clone an inexhaustible supply of any homozygous and/or heterozygous controls diluted with yeast genomic DNA to mimic human genome equivalents for use throughout the entire multiplex mutation assay. As a proof of principle, the 25 cystic fibrosis mutation panel selected by the American College of Medical Genetics and four additional mutant sequences were prepared as a single control mixture. The 29 CFTR mutations were incorporated into 17 gene fragments by PCR amplification of targeted sequences using mutagenic primers on normal human genomic DNA template. Flanking primers selected to bind beyond all published PCR primer sites amplified controls for most assay platforms. The 17 synthesized 433-933-bp CFTR fragments each with one to four homozygous mutant sequences were cloned into nine plasmid vectors at the multiple cloning site and bidirectionally sequenced. Miniplasmid preps from these nine clones were mixed and diluted with genomic yeast DNA to mimic the final nucleotide molar ratio of two CFTR genes in 6 x 10(9) bp total human genomic DNA. This mixture was added to control PCR reactions prior to amplification as the only positive control sample. In this fashion >200 multiplex clinical PCR analyses of >4,000 clinical patient samples have been controlled simultaneously for PCR amplification and substrate specificity for 29 tested mutations without cross contamination. This clinically validated multiplex cystic fibrosis control can be modified readily for different test formats and provides a robust means to control for all mutations instead of rotating human genomic controls each with a fraction of the mutations. This approach allows scores of additional mutation controls from any gene loci to be added to the same mixture annually.

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103 For example, the Intron 10/Exon 11 fragment spans 5 common mutation sites: 1717-1G Ǟ A, G542X, G551D, R553X, and R560T, while the ⌬I507 and ⌬F508 mutations in Exon 10 overlap by one basepair and each delete three basepairs. Login to comment
105 Because the G551D and R553X mutations are within four basepairs, these mutations were also synthesized on independently cloned Intron 10/Exon 11 fragments, both of which carried three other mutations: 1717-1G Ǟ A, G542X, and R560T (Fig. 2, fragments 1 and 3). Login to comment
165 As part of this validation, two different Intron10/Exon11 fragments were sequenced and tested: both contain the 1717-1G Ǟ A, G542X, and R560T mutations, and the first also contains the G551D mutation (Fig. 2, clone 1; Fig. 3, f1), while the second also contains the R553X mutation (Fig. 2, clone 3; Fig. 3, f3). Login to comment
166 When tested individually, clone 1 hybridized uniquely to the G551D mutant allelic site as well as to the other three mutations (1717-1G Ǟ A, G542X, and R560T), but not to the wild-type (normal) R553 allelic site because the G551D mutation sequence interferes with the binding to the wild type R553 probe on the strip (Fig. 3, f1, left strip). Login to comment

[hide] Genetic determinants and epidemiology of cystic fi... Diabetes Care. 2008 Sep;31(9):1789-94. Epub 2008 Jun 5. Adler AI, Shine BS, Chamnan P, Haworth CS, Bilton D
Genetic determinants and epidemiology of cystic fibrosis-related diabetes: results from a British cohort of children and adults.
Diabetes Care. 2008 Sep;31(9):1789-94. Epub 2008 Jun 5., [PMID:18535191]

Abstract [show]
OBJECTIVE: Longer survival of patients with cystic fibrosis has increased the occurrence of cystic fibrosis-related diabetes (CFRD). In this study we documented the incidence of CFRD and evaluated the association between mutations responsible for cystic fibrosis and incident CFRD, while identifying potential risk factors. RESEARCH DESIGN AND METHODS: This was a population-based longitudinal study of 50 cystic fibrosis speciality clinics in the U.K. Subjects included 8,029 individuals aged 0-64 years enrolled in the U.K. Cystic Fibrosis Registry during 1996-2005. Of these, 5,196 with data and without diabetes were included in analyses of incidence, and 3,275 with complete data were included in analyses of risk factors. Diabetes was defined by physician diagnosis, oral glucose tolerance testing, or treatment with hypoglycemic drugs. RESULTS: A total of 526 individuals developed CFRD over 15,010 person-years. The annual incidence was 3.5%. The incidence was higher in female patients and in patients with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classes I and II. In a multivariate model of 377 cases of 3,275 patients, CFTR class (relative risk 1.70 [95% CI 1.16-2.49], class I or II versus others), increasing age, female sex, worse pulmonary function, liver dysfunction, pancreatic insufficiency, and corticosteroid use were independently associated with incident diabetes. CONCLUSIONS: The incidence of CFRD is high in Britain. CFTR class I and II mutations increase the risk of diabetes independent of other risk factors including pancreatic exocrine dysfunction.

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54 Genotypes associated with cystic fibrosis were coded into five established classes reflecting CFTR function of defective production, processing, regulation, conductance, and quantity of CFTR protein (12) as follows: I: G542X, R553X, W1282X, R1162X, 621-1G3T, 1717- 1G3 A, 1078⌬T, and 3659⌬C; II: ⌬F508, ⌬I507, N1303K, and S549N; III: G551Dand R560T; IV: R117H, R334W, G85E, and R347P; V: 3849ϩ5G3A, and A455E; and unknown: 711ϩIG3 T, 2184DA, and 1898ϩIG3 A. Login to comment

[hide] Guidelines for diagnosis of cystic fibrosis in new... J Pediatr. 2008 Aug;153(2):S4-S14. Farrell PM, Rosenstein BJ, White TB, Accurso FJ, Castellani C, Cutting GR, Durie PR, Legrys VA, Massie J, Parad RB, Rock MJ, Campbell PW 3rd
Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report.
J Pediatr. 2008 Aug;153(2):S4-S14., [PMID:18639722]

Abstract [show]
Newborn screening (NBS) for cystic fibrosis (CF) is increasingly being implemented and is soon likely to be in use throughout the United States, because early detection permits access to specialized medical care and improves outcomes. The diagnosis of CF is not always straightforward, however. The sweat chloride test remains the gold standard for CF diagnosis but does not always give a clear answer. Genotype analysis also does not always provide clarity; more than 1500 mutations have been identified in the CF transmembrane conductance regulator (CFTR) gene, not all of which result in CF. Harmful mutations in the gene can present as a spectrum of pathology ranging from sinusitis in adulthood to severe lung, pancreatic, or liver disease in infancy. Thus, CF identified postnatally must remain a clinical diagnosis. To provide guidance for the diagnosis of both infants with positive NBS results and older patients presenting with an indistinct clinical picture, the Cystic Fibrosis Foundation convened a meeting of experts in the field of CF diagnosis. Their recommendations, presented herein, involve a combination of clinical presentation, laboratory testing, and genetics to confirm a diagnosis of CF.

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142 Recommended panel of CF-causing mutations Missense, deletion, stop mutations Splicing, frameshift mutations G85E I507del R560T 621ϩ1GϾT 2789ϩ5GϾA R117H F508del R1162X 711ϩ1GϾT 3120ϩ1GϾA R334W G542X W1282X 1717-1GϾA 3659delC R347P G551D N1303K 1898ϩ1GϾA 3849ϩ10kbCϾT A455E R553X 2184delA Revised from the mutation panel for population screening for CF developed by the ACMG.77 Additional or alternative mutations present at significant frequencies in an ethnic population served by an NBS program may be added. Login to comment

[hide] Clinical practice and genetic counseling for cysti... Genet Med. 2008 Dec;10(12):851-68. Moskowitz SM, Chmiel JF, Sternen DL, Cheng E, Gibson RL, Marshall SG, Cutting GR
Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders.
Genet Med. 2008 Dec;10(12):851-68., [PMID:19092437]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator-related disorders encompass a disease spectrum from focal male reproductive tract involvement in congenital absence of the vas deferens to multiorgan involvement in classic cystic fibrosis. The reproductive, gastrointestinal, and exocrine manifestations of cystic fibrosis transmembrane conductance regulator deficiency are correlated with CFTR genotype, whereas the respiratory manifestations that are the main cause of morbidity and mortality in cystic fibrosis are less predictable. Molecular genetic testing of CFTR has led to new diagnostic strategies and will enable targeting of molecular therapies now in development. Older diagnostic methods that measure sweat chloride and nasal potential difference nonetheless remain important because of their sensitivity and specificity. In addition, the measurement of immunoreactive trypsinogen and the genotyping of CFTR alleles are key to newborn screening programs because of low cost. The multiorgan nature of cystic fibrosis leads to a heavy burden of care, thus therapeutic regimens are tailored to the specific manifestations present in each patient. The variability of cystic fibrosis lung disease and the variable expressivity of mild CFTR alleles complicate genetic counseling for this autosomal recessive disorder. Widespread implementation of newborn screening programs among populations with significant cystic fibrosis mutation carrier frequencies is expected to result in increasing demands on genetic counseling resources.

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56 Liver disease is second to pulmonary disease (plus organ transplantation complications) as a cause of mortality in CF (1.7% of deaths).26 Table 3 Core mutation panel carrier recommended by the ACMG for routine CF diagnostic testing and carrier screening of the general population7 Intronic mutations Exonic mutations Missense Nonsense In-Frame Deletion 621ϩ1GϾT G85E G542X ⌬I507 711ϩ1GϾT R117H R553X ⌬F508 1717-1GϾA R334W R1162X 1898ϩ1GϾA R347P W1282X 2184delA A455E 2789ϩ5GϾA G551D 3120ϩ1GϾA R560T 3659delC N1303K 3849ϩ10kbCϾT Endocrine manifestations of CF CF-related diabetes mellitus (CFRDM) may present in adolescence. Login to comment

[hide] Genetics of cystic fibrosis. Semin Respir Crit Care Med. 2009 Oct;30(5):531-8. Epub 2009 Sep 16. Lommatzsch ST, Aris R
Genetics of cystic fibrosis.
Semin Respir Crit Care Med. 2009 Oct;30(5):531-8. Epub 2009 Sep 16., [PMID:19760540]

Abstract [show]
Cystic fibrosis (CF) is a complicated disease involving many organ systems. Identification of the cystic fibrosis transmembrane regulator (CFTR) genetic code has not only enhanced our understanding of the mechanism of CF pathology but has also provided explanations for phenotypic variation. Additionally, genetic testing has refined our ability to identify patients with CF and CF-related illnesses. Genetic mutations may be grouped by class (I-VI) and are directly related to the quantity of CFTR protein produced. This has direct implications regarding the severity of disease and has suggested organ-specific sensitivity to the presence of normally functioning CFTR. Further, it has improved understanding of the mechanism behind seemingly organ-specific manifestations of CF, such as congenital bilateral absence of the vas deferens (CBVAD).

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99 They also found an association with ~F508 and R117H in addition to Q493X, R560T, R553X, and 621 þ 1(G!T).34 Noone et al found an association between chronic pancreatitis and the 5T allele associated with complex alleles or in CFTR compound heterozygotes, but no significantly increased frequency has been found with the 5T allele alone.36,39 Finally, there appears to be an additive effect with being a CFTR compound heterozygote and the presence of N34S mutations of the pancreatic secretory trypsin inhibitor (PSTI).36,39 These studies demonstrate the increased risk of chronic pancreatitis due to an abnormally functioning CFTR protein (but may be due to just one mutant CFTR allele37 ). Login to comment

[hide] Population-based carrier screening for cystic fibr... Aust N Z J Obstet Gynaecol. 2009 Oct;49(5):484-9. Massie J, Petrou V, Forbes R, Curnow L, Ioannou L, Dusart D, Bankier A, Delatycki M
Population-based carrier screening for cystic fibrosis in Victoria: the first three years experience.
Aust N Z J Obstet Gynaecol. 2009 Oct;49(5):484-9., [PMID:19780730]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common inherited, life-shortening condition affecting Australian children. The carrier frequency is one per 25 and most babies with CF are born to parents with no family history. Carrier testing is possible before a couple has an affected infant. AIMS: To report the outcomes of a carrier screening program for CF. METHOD: Carrier screening was offered to women and couples planning a pregnancy, or in early pregnancy, through obstetricians and general practitioners in Victoria, Australia. Samples were collected by cheek swab and posted to the laboratory. Twelve CFTR gene mutations were tested. Carriers were offered genetic counselling and partner testing. Carrier couples were offered prenatal testing by chorionic villous sampling (CVS) if pregnant. The number of people tested, carriers detected and pregnancy outcomes were recorded from January 2006 to December 2008. RESULTS: A total of 3200 individuals were screened (3000 females). One hundred and six carriers were identified (one per 30, 95% confidence interval one per 25, one per 36). All carrier partners were screened, and nine carrier couples identified (total carriers 115). Ninety-six individuals (83%) were carriers of the p.508del mutation. Of the nine carrier couples, six were pregnant at the time of screening (five natural conception and one in vitro fertilisation) and all had CVS (mean gestation 12.5 weeks). Two fetuses were affected, three were carriers and one was not a carrier. Termination of pregnancy was undertaken for the affected fetuses. CONCLUSION: Carrier screening for CF by obstetricians and general practitioners by cheek swab sample can be successfully undertaken prior to pregnancy or in the early stages of pregnancy.

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103 Although we have promoted the uptake of CF carrier screening to both partners in the relationship it is evident Table 1 Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations identified in 2006-2008 CFTR gene mutation n p.508del 96 W1282X 5 c.3718-2477C > T 5 p.G551D 3 p.G542X 1 p.N1303K 1 p.507del 1 p.R560T 1 p.R553X 1 c.489+1G > T 1 p.V520F 0 c.1585-1G > A 0 Total 115 Carrierscreeningforcysticfibrosis (c)2009TheAuthors487 Journalcompilation(c)2009TheRoyalAustralianandNewZealandCollegeofObstetriciansandGynaecologists;49:484-489 Table 2 Carrier couples detected by cystic fibrosis population screening program, Victoria 2006-2008 Subjects Timing of CF carrier test (gestation) Conception Parents genotype Counselling Prenatal diagnosis Status of pregnancy Future plans 1 Pre-pregnancy Natural Both Genetic counsellor and CF physician CVS 12 weeks Termination of pregnancy 2008: Second pregnancy: CVS: carrier p508delp.508del Affected (p.508del/p.508del) 2 10 weeks Natural Both Genetic counsellor and CF physician CVS 12 weeks Continued p.508del Unaffected (no mutations) 3 11 weeks Natural Both Genetic counsellor CVS 13 weeks Continued p.508del Carrier (p.508del/-) 4 10 weeks Natural Both Genetic counsellor CVS 13 weeks Continued p.508del Carrier (p.508del/-) 5 11 weeks Natural Both Genetic counsellor CVS 13 weeks Continued p.508del Unaffected (no mutations) 6* 9 weeks IVF Both Genetic counsellor and CF physician CVS 12 weeks Termination of pregnancy Currently undergoing IVF conception with PGD.p.508del Affected (p.508del/p.508del) 7 Pre-pregnancy Not applicable Both Genetic counsellor and CF physician CVS 12 weeks Continued Did not attend PGD, established natural pregnancy 2 months after seen by genetic counsellor and respiratory physician p.508del Carrier p.508del 8** Pre-pregnancy Not applicable Both Genetic counsellor Not applicable Not applicable Likely to pursue PGD p.508del 9*** Pre-pregnancy Not applicable c.3718-2477C > T, Genetic counsellor and CF physician Not applicable Not applicable Likely to pursue PGD p.W1282X *This couple had an IVF pregnancy but were not offered carrier screening until nine weeks gestation. Login to comment
38 The following 12 mutations were screened using a polymerase chain reaction multiplex: p.508del, p.G551D, p.G542X, p.N1303K, c.1585-1G > A, p.I507del, p.R560T, p.W1282X, p.V520F, c.489+1G > T, p.R553X and c.3718-2477C > T. Login to comment

[hide] Non-classic cystic fibrosis associated with D1152H... Clin Genet. 2010 Apr;77(4):355-64. Epub 2009 Oct 15. Burgel PR, Fajac I, Hubert D, Grenet D, Stremler N, Roussey M, Siret D, Languepin J, Mely L, Fanton A, Labbe A, Domblides P, Vic P, Dagorne M, Reynaud-Gaubert M, Counil F, Varaigne F, Bienvenu T, Bellis G, Dusser D
Non-classic cystic fibrosis associated with D1152H CFTR mutation.
Clin Genet. 2010 Apr;77(4):355-64. Epub 2009 Oct 15., [PMID:19843100]

Abstract [show]
BACKGROUND: Limited knowledge exists on phenotypes associated with the D1152H cystic fibrosis transmembrane conductance regulator (CFTR) mutation. METHODS: Subjects with a D1152H allele in trans with another CFTR mutation were identified using the French Cystic Fibrosis Registry. Phenotypic characteristics were compared with those of pancreatic insufficient (PI) and pancreatic sufficient (PS) cystic fibrosis (CF) subjects in the Registry (CF cohort). RESULTS: Forty-two subjects with D1152H alleles were identified. Features leading to diagnosis included chronic sinopulmonary disease (n = 25), congenital absence of the vas deferens (n = 11), systematic neonatal screening (n = 4), and genetic counseling (n = 2). Median age at diagnosis was 33 [interquartile range (IQR, 24-41)] years in D1152H subjects. Median sweat chloride concentrations were 43.5 (39-63) mmol/l in D1152H subjects and were markedly lower than in PI and PS CF subjects (p < 0.05). Bronchiectasis was present in 67% of D1152H subjects, but Pseudomonas aeruginosa colonization and pancreatic insufficiency were present in <30% of subjects. Estimated rates of decline in forced expiratory volume in 1 s (FEV(1)) were lower in D1152H subjects vs PI CF subjects (p < 0.05). None of the D1152H subjects identified since 1999 had died or required lung transplantation. CONCLUSIONS: When present in trans with a CF-causing mutation, D1152H causes significant pulmonary disease, but all subjects had prolonged survival.

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42 The CF genetic analysis panel used in France seeks for 32 mutations: G85E, 394delTT, 621+1G>T, 711+1G>T, R334W, R347P, R347H, 1078delT, 5T/7T/9T, A455E, F508del, I507del, V520F, 1717-1G>A, G542X, G551D, R553X, R560T, S549R (T>G), S549N, 1898+1G>A, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, R1162X, 3659delC, 3849+10kbC>T, W1282X, 3905insT, 3876delA, N1303K. Login to comment

[hide] Deletion of CFTR translation start site reveals fu... Cell Physiol Biochem. 2009;24(5-6):335-46. Epub 2009 Nov 4. Ramalho AS, Lewandowska MA, Farinha CM, Mendes F, Goncalves J, Barreto C, Harris A, Amaral MD
Deletion of CFTR translation start site reveals functional isoforms of the protein in CF patients.
Cell Physiol Biochem. 2009;24(5-6):335-46. Epub 2009 Nov 4., [PMID:19910674]

Abstract [show]
BACKGROUND/AIMS: Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening autosomal recessive disease affecting Caucasians. We identified a CFTR mutation (c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese CF patients. This study aims at functionally characterizing the effect of this novel mutation. METHODS: RNA and protein techniques were applied to both native tissues from CF patients and recombinant cells expressing CFTR constructs to determine whether c.120del23 allows CFTR protein production through usage of alternative internal codons, and to characterize the putative truncated CFTR form(s). RESULTS: Our data show that two shorter forms of CFTR protein are produced when the initiation translation codon is deleted indicating usage of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation. CONCLUSIONS: The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation.

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189 Functional studies of 120del23-CFTR Despite its intrinsic instability and the very low membrane levels of this mutant, we find here that c.120del23-CFTR, still has residual Cl-channel activity, in contrast to other CF-causing mutants like F508del-CFTR (this study) and R560T-, orA561E-CFTR [14, 15]. Login to comment

[hide] Aquagenic wrinkling of the palms in cystic fibrosi... Arch Dermatol. 2009 Nov;145(11):1296-9. Berk DR, Ciliberto HM, Sweet SC, Ferkol TW, Bayliss SJ
Aquagenic wrinkling of the palms in cystic fibrosis: comparison with controls and genotype-phenotype correlations.
Arch Dermatol. 2009 Nov;145(11):1296-9., [PMID:19917960]

Abstract [show]
OBJECTIVE: To determine the prevalence of aquagenic wrinkling of the palms (AWP) in patients with cystic fibrosis (CF) compared with control patients, and evaluate for genotype-phenotype correlations. Since its first description over 30 years ago, AWP has frequently been anecdotally associated with CF, but this association has not been confirmed in a rigorous prospective case-control study. DESIGN: Blinded comparison. SETTING: The CF and dermatology clinics at St Louis Children's Hospital. PARTICIPANTS: Forty-four individuals with CF from a CF clinic and 26 controls from a dermatology clinic. Intervention Participants were tested for AWP using 3 minutes of water immersion with room-temperature tap water. Main Outcome Measure The degree of AWP was scored from 0 (no wrinkling) to 4 (severe wrinkling) by 3 blinded physicians. For genotype-phenotype correlations, patients with CF were divided into those homozygous for the DeltaF508 mutation and those with other genotypes. RESULTS: The mean AWP score of the CF group was significantly higher than the mean score of the control group (1.5 vs 0.6; P < .001). Patients with CF who were homozygous for the DeltaF508 mutation (n = 27) had significantly higher scores than patients with CF who were not homozygous for the DeltaF508 mutation (n = 17) (1.7 vs 1.1; P = .02). The 17 patients with CF who were not homozygous for the DeltaF508 mutation still had higher scores than the control group (1.1 vs 0.6; P = .03). There was no correlation between sweat chloride concentrations measured at the time of diagnosis and AWP score. CONCLUSIONS: Our results confirm the association between AWP and CF. Among patients with CF, greater AWP occurs in those who are homozygous for the DeltaF508 mutation.

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57 (%) 12 (46) 23 (52) Mean age, y 9.3 11.5 CFTR genotype NA ⌬F508/⌬F508 27 ⌬F508/unidentified 4 ⌬F508/R553X 2 ⌬F508/1898 ϩ 1G ~ A 2 ⌬F508/G542X 1 ⌬F508/G551D 1 ⌬F508/W1282X 1 ⌬F508/1717 ϩ 1G ~ A 1 ⌬F508/3120 ϩ 1G ~ A 1 ⌬F508/3849 ϩ 10KBC→T 1 ⌬F508/S1251N 1 R560T/unidentified 1 2184insA/4357 2A→G 1 Abbreviations: CF, cystic fibrosis; NA, not applicable. Login to comment
59 (%) 12 (46) 23 (52) Mean age, y 9.3 11.5 CFTR genotype NA ⌬F508/⌬F508 27 ⌬F508/unidentified 4 ⌬F508/R553X 2 ⌬F508/1898 ϩ 1G ~ A 2 ⌬F508/G542X 1 ⌬F508/G551D 1 ⌬F508/W1282X 1 ⌬F508/1717 ϩ 1G ~ A 1 ⌬F508/3120 ϩ 1G ~ A 1 ⌬F508/3849 ϩ 10KBC→T 1 ⌬F508/S1251N 1 R560T/unidentified 1 2184insA/4357 2A→G 1 Abbreviations: CF, cystic fibrosis; NA, not applicable. Login to comment

[hide] Do common in silico tools predict the clinical con... Clin Genet. 2010 May;77(5):464-73. Epub 2009 Jan 6. Dorfman R, Nalpathamkalam T, Taylor C, Gonska T, Keenan K, Yuan XW, Corey M, Tsui LC, Zielenski J, Durie P
Do common in silico tools predict the clinical consequences of amino-acid substitutions in the CFTR gene?
Clin Genet. 2010 May;77(5):464-73. Epub 2009 Jan 6., [PMID:20059485]

Abstract [show]
Computational methods are used to predict the molecular consequences of amino-acid substitutions on the basis of evolutionary conservation or protein structure, but their utility in clinical diagnosis or prediction of disease outcome has not been well validated. We evaluated three popular computer programs, namely, PANTHER, SIFT and PolyPhen, by comparing the predicted clinical outcomes for a group of known CFTR missense mutations against the diagnosis of cystic fibrosis (CF) and clinical manifestations in cohorts of subjects with CF-disease and CFTR-related disorders carrying these mutations. Owing to poor specificity, none of tools reliably distinguished between individual mutations that confer CF disease from mutations found in subjects with a CFTR-related disorder or no disease. Prediction scores for CFTR mutations derived from PANTHER showed a significant overall statistical correlation with the spectrum of disease severity associated with mutations in the CFTR gene. In contrast, PolyPhen- and SIFT-derived scores only showed significant differences between CF-causing and non-CF variants. Current computational methods are not recommended for establishing or excluding a CF diagnosis, notably as a newborn screening strategy or in patients with equivocal test results.

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64 Mutations in the CFTR gene grouped by clinical category Cystic fibrosis CFTR-related disease No disease T338I D614G L320V V920L L90S M470V H199R S1251N I203M G550R P111A I148T Q1291H R560K L1388Q L183I R170H I1027T S549R D443Y P499A L1414S T908N R668C S549N A455E E1401K Q151K G27E I1234L Y563N R347P C866R S1118C P1290S R75Q A559T V520F P841R M469V E1401G P67L G85E S50Y E1409K R933G G458V G178R Y1032C R248T I980K G85V V392G L973P L137H T351S R334W I444S V938G R792G R560T R555G L1339F D1305E P574H V1240G T1053I D58G G551D L1335P I918M F994C S945L L558S F1337V R810G D1152H G1247R P574S R766M D579G W1098R H949R F200I R352Q L1077P K1351E M244K L206W M1101K D1154G L375F N1303K R1066C E528D D110Y R347H R1070Q A800G P1021S S549K A1364V V392A damaging` (is supposed to affect protein function or structure) and 'probably damaging` (high confidence of affecting protein function or structure). Login to comment

[hide] Incidence, prevalence, etiology, and prognosis of ... Dig Dis Sci. 2010 Oct;55(10):2988-98. Epub 2010 Jan 28. Joergensen M, Brusgaard K, Cruger DG, Gerdes AM, de Muckadell OB
Incidence, prevalence, etiology, and prognosis of first-time chronic pancreatitis in young patients: a nationwide cohort study.
Dig Dis Sci. 2010 Oct;55(10):2988-98. Epub 2010 Jan 28., [PMID:20108119]

Abstract [show]
BACKGROUND/AIMS: Publications on etiology of chronic pancreatitis (CP) are infrequent. Etiologies today encompass genetic disorders. We wanted to describe etiologies of today and identify patients with genetic disorders like hereditary pancreatitis (HP), mutations in Serine Protease Inhibitor Kazal type1 (SPINK1), and the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) among patients formerly considered to have idiopathic CP. METHODS: Data on patients diagnosed with first-time CP < 30 years of age in Denmark identified in the Danish National Registry of Patients were retrieved. Patients previously considered to have idiopathic pancreatitis were offered genetic counseling and evaluation for HP, SPINK1, and CFTR mutations. RESULTS: In the period 1980-2004, 580 patients < 30 years of age presented with CP, the standardized prevalence ratio of CP increased from 11.7 per 100,000 person years in 1980-1984 to 17.0 per 100,000 in 2000-2004 (p < 0.001). The odds ratio (OR) having gallstone-related CP increased in the latter time period, especially in women, that of alcohol-induced CP decreased over time. OR having idiopathic CP increased in the latter period; 50% of patients with idiopathic pancreatitis accepted genetic reevaluation; 28 patients had a genetic mutation that totally or partly could explain their pancreatitis, nine of these had two, and 11 patients had HP. CONCLUSION: The prevalence of CP, especially in women, increased over time. Genetic causes that partly or totally could explain the CP were found in 54.90% (95% CI (40.45-68.62)) of those with idiopathic CP, as a minimum estimation 1.9% (95% CI (1.00-3.47)) of the total cohort had HP.

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49 1G [ T, F508del, S549 N, I507del, S549R, 2184delA, G551D, G85E, N1303 K, R560T, R117H, R347H, R347P, R334 W, 2789 ? Login to comment

[hide] Restoration of domain folding and interdomain asse... FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PMID:20233947]

Abstract [show]
Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.

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89 To test this hypothesis, we introduced suppressor mutations into another NBD1-processing mutant, R560T (17). Login to comment
91 As shown in Fig. 1B, R560T-CFTR, although not rescued by G550E alone (17), was very effectively rescued by the 4S combination. Login to comment
92 Interestingly, while lowering the temperature alone did not promote R560T-CFTR maturation, it augmented the extent of rescue by 4S (Fig. 1C). Login to comment

[hide] All azoospermic males should be screened for cysti... Fertil Steril. 2010 Nov;94(6):2448-50. Epub 2010 Apr 9. Mocanu E, Shattock R, Barton D, Rogers M, Conroy R, Sheils O, Collins C, Martin C, Harrison R, O'Leary J
All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.
Fertil Steril. 2010 Nov;94(6):2448-50. Epub 2010 Apr 9., [PMID:20381036]

Abstract [show]
We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

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50 Of these, 83% were F508del, 3.7% R117H, G551D, and 621þ1G>T, and 1.85% R560T, G542X, and I507del. Login to comment

[hide] Impact of gene patents and licensing practices on ... Genet Med. 2010 Apr;12(4 Suppl):S194-211. Chandrasekharan S, Heaney C, James T, Conover C, Cook-Deegan R
Impact of gene patents and licensing practices on access to genetic testing for cystic fibrosis.
Genet Med. 2010 Apr;12(4 Suppl):S194-211., [PMID:20393308]

Abstract [show]
Cystic fibrosis is one of the most commonly tested autosomal recessive disorders in the United States. Clinical cystic fibrosis is associated with mutations in the CFTR gene, of which the most common mutation among Caucasians, DeltaF508, was identified in 1989. The University of Michigan, Johns Hopkins University, and the Hospital for Sick Children, where much of the initial research occurred, hold key patents on cystic fibrosis genetic sequences, mutations, and methods for detecting them. Several patents, including the one that covers detection of the DeltaF508 mutation, are jointly held by the University of Michigan and the Hospital for Sick Children in Toronto, with Michigan administering patent licensing in the United States. The University of Michigan broadly licenses the DeltaF508 patent for genetic testing with >60 providers of genetic testing to date. Genetic testing is now used in newborn screening, diagnosis, and for carrier screening. Interviews with key researchers and intellectual property managers, a survey of laboratories' prices for cystic fibrosis genetic testing, a review of literature on cystic fibrosis tests' cost-effectiveness, and a review of the developing market for cystic fibrosis testing provide no evidence that patents have significantly hindered access to genetic tests for cystic fibrosis or prevented financially cost-effective screening. Current licensing practices for cystic fibrosis genetic testing seem to facilitate both academic research and commercial testing. More than 1000 different CFTR mutations have been identified, and research continues to determine their clinical significance. Patents have been nonexclusively licensed for diagnostic use and have been variably licensed for gene transfer and other therapeutic applications. The Cystic Fibrosis Foundation has been engaged in licensing decisions, making cystic fibrosis a model of collaborative and cooperative patenting and licensing practice.

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182 The ACMG specifically indicated that "Asian-Americans and Native Americans without significant Caucasian admixture should be informed of Table 1 Recommended core mutation panel for cystic fibrosis carrier screening in the general population Standard mutation panel R560T, ⌬F508a , R553Xb , R1162X, ⌬I507, 2184delA, G542X, G551Db , W1282X, N1303K, 621ϩ1G⌬T, R117H, 1717-1G⌬A, A455E, G85E, R334W, R347P, 711ϩ1G⌬T, 1898ϩ1G⌬A, 3849ϩ10kbC⌬T, 2789ϩ5G⌬A, 3659delC, and 3120ϩ1G⌬A Additional testable mutations I506Vc , I507Vc , F508Cc , and 5T/ 7T/9Td a University of Michigan/HSC Patent No. US 5,776,677. b Johns Hopkins University, Patent No. US 5,407,796. c Benign variants. Login to comment

[hide] Carrier screening for cystic fibrosis. Obstet Gynecol Clin North Am. 2010 Mar;37(1):47-59, Table of Contents. Dungan JS
Carrier screening for cystic fibrosis.
Obstet Gynecol Clin North Am. 2010 Mar;37(1):47-59, Table of Contents., [PMID:20494257]

Abstract [show]
Cystic fibrosis is the first genetic disorder for which universal screening of preconceptional or prenatal patients became a component of standard prenatal care. The molecular genetics and mutation profile of the CFTR gene are complex, with a wide range of phenotypic consequences. Carrier screening can facilitate risk assessment for prospective parents to have an affected offspring, although there remains a small residual risk for carrying a mutation even with a negative screening result. There are ethnic differences with respect to disease incidence and effectiveness of carrier testing, which may complicate counseling.

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102 However, in instances of a positive family history of affected individuals, but with no known mutation, further Table 2 Mutation panel recommended by ACOG and ACMG (listed in order of decreasing frequency in non-Hispanic Caucasian population) F508 del delI507 R347P R1162X G542X R553X 71111G>T 2184delA G551D R117H R560T 189811G>A 62111G>T 3849110kbC>T 3569delC R334W W1282X 1717À1G>T A455E 312011G>T N1303K 278915G>A G85E Data from Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Login to comment

[hide] Identification of the second CFTR mutation in pati... Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26. Giuliani R, Antonucci I, Torrente I, Grammatico P, Palka G, Stuppia L
Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.
Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26., [PMID:20657600]

Abstract [show]
Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.

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58 INNO-LiPA CFTR19 INNO-LiPA CFTR17 INNO-LiPA CFTR Italian regional [delta]F508 621+1G>T 1259insA G542X 3849+10kbC>T 4016insT N1303K 2183AA>G 4382delA W1282X 394delTT 852del22 G551D 2789+5G> A R1162X D579G 1717-1G>A 3659delC G1244E R553X R117H G1349D CFTRdele2,3 (21 kb) R334W I502T [delta]I507 R347P L1065P 711+1G>T G85E R1158X 3272-26A>G 3905insT 1078delT T338I R560T A455E S549R(A>C) 1898+1G>A S1251N 2143delA 711+5G>A 991del5 I148T E60X D1152H 3199del6 3120+1G>A 2184delA 1898+3A>G, R1070Q Q552X Poli-T tract variations R1066H R347H 621+3A>G R334Q E217G Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Mutations that permit residual CFTR function delay... Respir Res. 2010 Oct 8;11:140. Green DM, McDougal KE, Blackman SM, Sosnay PR, Henderson LB, Naughton KM, Collaco JM, Cutting GR
Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients.
Respir Res. 2010 Oct 8;11:140., [PMID:20932301]

Abstract [show]
BACKGROUND: Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF. METHODS: Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function CFTR mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories. RESULTS: Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile). CONCLUSIONS: Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.

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74 For Pa, the hazard ratio Table 1 Classification of CFTR alleles Category Mutation Specific mutations Class I Defective Protein Synthesis (nonsense, frameshift, aberrant splicing) 1078delT, 1154 insTC, 1525-2A > G, 1717-1G > A, 1898+1G > A, 2184delA, 2184 insA, 3007delG, 3120+1G > A, 3659delC, 3876delA, 3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+1G > T, 441delA, 556delA, 621+1G > T, 621-1G > T, 711+1G > T, 875+1G > C, E1104X, E585X, E60X, E822X, G542X, G551D/R553X, Q493X, Q552X, Q814X, R1066C, R1162X, R553X, V520F, W1282X, Y1092X Class II Abnormal Processing and Trafficking A559T, D979A, ΔF508, ΔI507, G480C, G85E, N1303K, S549I, S549N, S549R Class III Defective Channel Regulation/Gating G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, S549 (R75Q) Class IV Decreased Channel Conductance A800G, D1152H, D1154G, D614G, delM1140, E822K, G314E, G576A, G622D, G85E, H620Q, I1139V, I1234V, L1335P, M1137V, P67L, R117C, R117P, R117H, R334W, R347H, R347P, R347P/ R347H, R792G, S1251N, V232D Class V Reduced Synthesis and/or Trafficking 2789+5G > A, 3120G > A, 3272-26A > G, 3849+10kbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, P574H was increased 3 fold for those with 'Minimal` function when compared to those with 'Residual` function. Login to comment

[hide] Combined bicarbonate conductance-impairing variant... Gastroenterology. 2011 Jan;140(1):162-71. Epub 2010 Oct 25. Schneider A, Larusch J, Sun X, Aloe A, Lamb J, Hawes R, Cotton P, Brand RE, Anderson MA, Money ME, Banks PA, Lewis MD, Baillie J, Sherman S, Disario J, Burton FR, Gardner TB, Amann ST, Gelrud A, George R, Rockacy MJ, Kassabian S, Martinson J, Slivka A, Yadav D, Oruc N, Barmada MM, Frizzell R, Whitcomb DC
Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis.
Gastroenterology. 2011 Jan;140(1):162-71. Epub 2010 Oct 25., [PMID:20977904]

Abstract [show]
BACKGROUND & AIMS: Idiopathic chronic pancreatitis (ICP) is a complex inflammatory disorder associated with multiple genetic and environmental factors. In individuals without cystic fibrosis (CF), variants of CFTR that inhibit bicarbonate conductance but maintain chloride conductance might selectively impair secretion of pancreatic juice, leading to trypsin activation and pancreatitis. We investigated whether sequence variants in the gene encoding the pancreatic secretory trypsin inhibitor SPINK1 further increase the risk of pancreatitis in these patients. METHODS: We screened patients and controls for variants in SPINK1 associated with risk of chronic pancreatitis and in all 27 exons of CFTR. The final study group included 53 patients with sporadic ICP, 27 probands with familial ICP, 150 unrelated controls, 375 additional controls for limited genotyping. CFTR wild-type and p.R75Q were cloned and expressed in HEK293 cells, and relative conductances of HCO(3)(-) and Cl(-) were measured. RESULTS: SPINK1 variants were identified in 36% of subjects and 3% of controls (odds ratio [OR], 18.1). One variant of CFTR not associated with CF, p.R75Q, was found in 16% of subjects and 5.3% of controls (OR, 3.4). Coinheritance of CFTR p.R75Q and SPINK1 variants occurred in 8.75% of patients and 0.38% of controls (OR, 25.1). Patch-clamp recordings of cells that expressed CFTR p.R75Q showed normal chloride currents but significantly reduced bicarbonate currents (P = .0001). CONCLUSIONS: The CFTR variant p.R75Q causes a selective defect in bicarbonate conductance and increases risk of pancreatitis. Coinheritance of p.R75Q or CF causing CFTR variants with SPINK1 variants significantly increases the risk of ICP.

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99 Total CFTR Sequencing Results of Patients With SPINK1 Mutations Diagnosis Age at diagnosis (y) CFTR mutations SPINK1 mutations 1 SP 12 -/- N34S/P55S 2 SP 46 -/- N34S/P55S 3 SP 13 -/- N34S/N34S 4 FP Infant -/- N34S/N34S 5 FP 8 R560T/- N34S/P55S 6 FP 15 M952T/- N34S/N34S 7 SP 19 R75Q/-a N34S/N34S 8 SP 3 F508del/-a P55S/- 9 SP 3 F508del/1584GtoAa N34S/- 10 SP 19 F508del/-a N34S/- 11 FP 12 F508del/I807M, 3139ϩ42AtoTa N34S/- 12 SP 14 D443YϩG576AϩR668Cb N34S/- 13 SP 1 F508C/-a N34S/- 14 SP 20 IVS8-T5-TG12/-a N34S/- 15 SP 16 R75Q/-a P55S/- 16 SP 9 R75Q/-a N34S/- 17 SP 9 R75Q/-a N34S/- 18 SP 16 R75Q/ϩ1584GtoAa N34S/- 19 FP 7 R75Q/-a N34S/- 20 FP 35 R75Q/-a N34S/- 21 FP 2 1584GtoA/-a N34S/- 22 FP Child 1584GtoA/-a N34S/- 23 SP 14 1584GtoA/-a N34S/- 24 FP 14 3139ϩ42AtoT/- N34S/- 25 FP 28 -/- N34S/- 26 FP 36 -/- N34S/- 27 SP 8 -/- N34S/- 28 SP 9 -/- N34S/- 29 SP 3 -/- N34S/- FP, familial pancreatitis; SP, sporadic pancreatitis. Login to comment
89 Two mutations, p.F508del and p.R560T, well characterized as class II CFTR mutations that are often associated with pancreatic-insufficient CF, were categorized as CF severe for statistical calculation. Login to comment
136 CFTR Mutation Class Types and Corresponding Disease Severity CFTR mutation Exon CF mutation class Disease association % Carriers, case (n) % Carriers, controls (n) p.R75Q 3 "CP" 16.2 (80) 5.3 (525) c.1584GtoA (p.E528E) 10 "CP" 8.7 (80) 3.3 (150) p.F508del 10 II CF severe 8.7 (80) 2.3 (525) p.R560T 11 II CF severe 3.4 (29) 0 (95) IVS8 T5/TG12or13 i8 V CF mild 5.0 (80) 2.7 (150) p.F508C 10 CF mild 1.2 (80) 0 (150) p.I807M 13 CF mild 3.4 (29) 0 (95) p.D443YϩG576AϩR668Ca 9;12;13 CF mild 3.4 (29) 0 (95) p.G576AϩR668Ca 12;13 CF mild 0 (29) 1 (95) p.M952T 15 CF mild 3.4 (29) 0 (95) p.R668C 13 Other 0 (29) 1 (95) c.3139ϩ42AtoT i17a Other 3.4 (29) 0 (95) p.N1432K 24 Other 0 (29) 1 (95) c.-9CtoT 1 Other 0 (29) 1 (95) p.C76W 3 Other 0 (80) 0.7 (150) p.I148T 4 Other 0 (29) 1 (95) c.2657ϩ22GtoA i14b Other 0 (29) 1 (95) p.T1086A 17b Other 0 (29) 1 (95) NOTE. Login to comment

[hide] Low abundance of sweat duct Cl- channel CFTR in bo... Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R605-15. Epub 2011 Jan 12. Brown MB, Haack KK, Pollack BP, Millard-Stafford M, McCarty NA
Low abundance of sweat duct Cl- channel CFTR in both healthy and cystic fibrosis athletes with exceptionally salty sweat during exercise.
Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R605-15. Epub 2011 Jan 12., [PMID:21228336]

Abstract [show]
To understand potential mechanisms explaining interindividual variability observed in human sweat sodium concentration ([Na(+)]), we investigated the relationship among [Na(+)] of thermoregulatory sweat, plasma membrane expression of Na(+) and Cl(-) transport proteins in biopsied human eccrine sweat ducts, and basal levels of vasopressin (AVP) and aldosterone. Lower ductal luminal membrane expression of the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) was observed in immunofluorescent staining of sweat glands from healthy young adults identified as exceptionally "salty sweaters" (SS) (n = 6, P < 0.05) and from patients with cystic fibrosis (CF) (n = 6, P < 0.005) compared with ducts from healthy young adults with "typical" sweat [Na(+)] (control, n = 6). Genetic testing of healthy subjects did not reveal any heterozygotes ("carriers") for any of the 39 most common disease-causing CFTR mutations in the United States. SS had higher baseline plasma [AVP] compared with control (P = 0.029). Immunostaining to investigate a potential relationship between higher plasma [AVP] (and sweat [Na(+)]) and ductal membrane aquaporin-5 revealed for all groups a relatively sparse and location-dependent ductal expression of the water channel with localization primarily to the secretory coil. Availability of CFTR for NaCl transport across the ductal membrane appears related to the significant physiological variability observed in sweat salt concentration in apparently healthy humans. At present, a heritable link between healthy salty sweaters and the most prevalent disease-causing CFTR mutations cannot be established.

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114 Mutations tested in this panel were ⌬F508, R334W, S549N, 3659delC, ⌬I507, I347P, A559T, S1255X, 1898ϩ1GϾA, R347H, N1303K, 1898ϩ5GϾT, 3876delA, A455E, 394delTT, 2183GGϾA, 3905insT, 3120ϩ1GϾA, V520F, 2184delA, G85E, Y1092X, 711ϩ1GϾT, 2307insA, Y122X, S549R, M1101K, 1078delT, 2789ϩ5GϾA, G551D, G542X, 621ϩ1GϾT, R560T, W1282X, 1717-1 GϾA, 3849 ϩ 10KbCϾT, R553X, R117H, and R1162X. Login to comment
119 Mutations tested in this panel were ⌬F508, R334W, S549N, 3659delC, ⌬I507, I347P, A559T, S1255X, 1898ϩ1GϾA, R347H, N1303K, 1898ϩ5GϾT, 3876delA, A455E, 394delTT, 2183GGϾA, 3905insT, 3120ϩ1GϾA, V520F, 2184delA, G85E, Y1092X, 711ϩ1GϾT, 2307insA, Y122X, S549R, M1101K, 1078delT, 2789ϩ5GϾA, G551D, G542X, 621ϩ1GϾT, R560T, W1282X, 1717-1 GϾA, 3849 ϩ 10KbCϾT, R553X, R117H, and R1162X. Login to comment

[hide] Distribution of CFTR mutations in Eastern Hungaria... J Cyst Fibros. 2011 May;10(3):217-20. doi: 10.1016/j.jcf.2010.12.009. Epub 2011 Feb 4. Ivady G, Madar L, Nagy B, Gonczi F, Ajzner E, Dzsudzsak E, Dvorakova L, Gombos E, Kappelmayer J, Macek M Jr, Balogh I
Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.
J Cyst Fibros. 2011 May;10(3):217-20. doi: 10.1016/j.jcf.2010.12.009. Epub 2011 Feb 4., [PMID:21296036]

Abstract [show]
BACKGROUND: The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. METHODS: We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. RESULTS: The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. CONCLUSION: We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program.

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34 3528delC), p.Phe316LeufsX12 (c.948delT), p.Ile507del (c.1519_1521delATC), p.Arg347Pro (c.1040 G NC), p.Arg553X (c.1657 C NT), p.Glu60X (c.178 GNT), c.2988+1 GNA, c.2657+5 GNA, c.1766+1 GNA, c.579+1 GNT, p.Gly85Glu (c.254 GNA), c.p.Lys684AsnfsX38 (c.2052delA), and p.Arg560Thr (c.1679 GNC). Login to comment

[hide] Association between genotype and pulmonary phenoty... J Cyst Fibros. 2011 May;10(3):187-92. doi: 10.1016/j.jcf.2011.01.005. Epub 2011 Feb 26. Geborek A, Hjelte L
Association between genotype and pulmonary phenotype in cystic fibrosis patients with severe mutations.
J Cyst Fibros. 2011 May;10(3):187-92. doi: 10.1016/j.jcf.2011.01.005. Epub 2011 Feb 26., [PMID:21354377]

Abstract [show]
BACKGROUND: Despite numerous studies a clear relationship between genotype and pulmonary phenotype has not been established within the group pancreatic insufficient cystic fibrosis (CF) patients. We studied the relationship between class I and class II mutations and pulmonary function in Swedish patients with known CFTR functional classification. METHODS: 170 CF patients with two class II mutations, 18 with two class I mutations and 78 with a combination of class I and II mutations were included in the study. Spirometry was performed when patients were in an optimal clinical condition. RESULTS: Patients with two class I mutations had lower lung function (FEV(1) and FVC) compared to the group with either a combination of class I and II mutations or two class II mutations. CONCLUSION: CF patients carrying two class I mutations risk developing more severe lung disease compared to patients with at least one class II mutation.

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98 Class I Class II Class III Class IV Class V 1717-1 G-NA F508del G551D 297 C-NA 2789+5 G-NA 3659delC S945L R560T R117C 3849+10 kb CNT 394delTT R347P A455E R553X T 3381 3849+10 kb C-T 621+1 G-NT E60X G542X W79R W1282X decline of pulmonary function was more rapid in patients with pancreatic insufficiency, mainly class II mutations, compared to CF patients with normal pancreatic function [4]. Login to comment

[hide] Optimal DNA tier for the IRT/DNA algorithm determi... J Cyst Fibros. 2011 Jul;10(4):278-81. Epub 2011 Mar 8. Baker MW, Groose M, Hoffman G, Rock M, Levy H, Farrell PM
Optimal DNA tier for the IRT/DNA algorithm determined by CFTR mutation results over 14 years of newborn screening.
J Cyst Fibros. 2011 Jul;10(4):278-81. Epub 2011 Mar 8., [PMID:21388895]

Abstract [show]
BACKGROUND: There has been great variation and uncertainty about how many and what CFTR mutations to include in cystic fibrosis (CF) newborn screening algorithms, and very little research on this topic using large populations of newborns. METHODS: We reviewed Wisconsin screening results for 1994-2008 to identify an ideal panel. RESULTS: Upon analyzing approximately 1 million screening results, we found it optimal to use a 23 CFTR mutation panel as a second tier when an immunoreactive trypsinogen (IRT)/DNA algorithm was applied for CF screening. This panel in association with a 96th percentile IRT cutoff gave a sensitivity of 97.3%, but restricting the DNA tier to F508del was associated with 90% (P<.0001). CONCLUSIONS: Although CFTR panel selection has been challenging, our data show that a 23 mutation method optimizes sensitivity and is advantageous. The IRT cutoff value, however, is actually more critical than DNA in determining CF newborn screening sensitivity.

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75 CFTR mutationa Proportion of allele Frequency of allele (%) Cumulative detection (%)b F508del 137/214 64.02 92.52 3849+10KbCNT 6/214 2.80 92.52c G542X 5/214 2.34 94.39 N1303K 4/214 1.87 98.13 R117H 4/214 1.87 99.07 R553X 3/214 1.40 99.07 1717-1GNA 2/214 0.93 99.07 G551D 1/214 0.47 100 R347P 1/214 0.47 100 A455E 1/214 0.47 100 W1282X 1/214 0.47 100 621+1GNT 1/214 0.47 100 a The other 11 mutations in ACMG 23 mutation panel are G85E, 711+1GNT, R334W, I507del, R560T, 1898+1GNA, 2184delA, 2789+5GNA, 3120+1GNA, R1162X and 3659delC. Login to comment

[hide] Implementation of the first worldwide quality assu... Clin Chim Acta. 2011 Jul 15;412(15-16):1376-81. Epub 2011 Apr 14. Earley MC, Laxova A, Farrell PM, Driscoll-Dunn R, Cordovado S, Mogayzel PJ Jr, Konstan MW, Hannon WH
Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening.
Clin Chim Acta. 2011 Jul 15;412(15-16):1376-81. Epub 2011 Apr 14., 2011-07-15 [PMID:21514289]

Abstract [show]
BACKGROUND: CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS: Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS: The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS: Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.

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129 Allele Allele Allele Allele p.Gly85Glu G85E (0.26) p.Arg117His R117H (0.54) c.489+1 GNT 621+1 GNT (1.3) p.Phe508del F508del (66.31) p.Arg347Pro R347P (0.36) p.lle507del I507del (0.90) p.Gly551Asp G551D (1.93) c.2052delA 2184delA (0.15) c.1585-1 GNA 1717-1 GNA (0.44) p.Gly542X G542X (2.64) c.3528delC 3659delC (0.28) p.Asn1303Lys N1303K (1.27) p.Arg553X R553X (1.21) p.Arg560Thr R560T (0.30) p.Arg1162X R1162X (0.30) c.2657+5 GNA 2789+5 GNA (0.38) c.3717+12191 CNT 3849+10kbCNT (0.85) c.2988+1 GNA 3120+1 GNA (0.86) p.Trp1282X W1282X (2.20) p.Ala455Glu A455E (0.26) c.1766+1 GNA 1898+1 GNA (0.13) c.579+1 GNT 711+1 GNT (0.35) p.Arg334Trp R334W (0.37) c.54-5940 _273+10250del21kb CFTR dele2,3 p.Ser549Asn S549N (0.14) c.1584 GNA 1716 G→A c.2051_2052delAAinsG 2183AANG (0.1) c.3140-26ANG 3272-26ANG c.262_263delTT 394delTT p.Arg1066Cys R1066C (0.03) p.Arg1066His R1066H c.1022_1023insTC 1154insTC c.2989-1 GNA 3121-1 GNA c.(?_2989)_(3139_? Login to comment

[hide] Clinical outcomes in infants with cystic fibrosis ... Pediatr Pulmonol. 2011 Apr 29. doi: 10.1002/ppul.21475. Ren CL, Desai H, Platt M, Dixon M
Clinical outcomes in infants with cystic fibrosis transmembrane conductance regulator (CFTR) related metabolic syndrome.
Pediatr Pulmonol. 2011 Apr 29. doi: 10.1002/ppul.21475., 2011-04-29 [PMID:21538969]

Abstract [show]
An unavoidable outcome of cystic fibrosis newborn screening (CF NBS) programs is the detection of infants with an indeterminate diagnosis. The United States CF Foundation recently proposed the term cystic fibrosis transmembrane conductance regulator related metabolic syndrome (CRMS) to describe infants with elevated immunoreactive trypsinogen (IRT) on NBS who do not meet diagnostic criteria for CF. The objective of this study was to describe the clinical outcomes of infants with CRMS identified through an IRT/DNA algorithm. We reviewed the records of all infants with CRMS diagnosed at our CF Center from 2002 to 2010. We identified 12 infants, and compared them to 27 infants diagnosed with CF by NBS. Compared to CF patients, CRMS patients were more likely to be pancreatic sufficient as assessed by fecal elastase measurement (100% vs. 8%, P < 0.01). Their weight for age percentile was normal from birth. A positive oropharyngeal (OP) culture for Pseudomonas aeruginosa (Pa) was found in 25% of CRMS patients. One patient with the F508del/R117H/7T genotype was reassigned the diagnosis of CF after he had a positive OP culture for Pa, and his follow up sweat Cl at 1 year of life was 73 mmol/L. CF patients were more likely to receive oral antibiotics and be hospitalized for pulmonary symptoms. Our results indicate that CRMS patients can develop signs of CF disease, but have a milder clinical course than CF infants. Close initial monitoring of these patients is warranted. Pediatr. Pulmonol. (c) 2011 Wiley-Liss, Inc.

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60 Infants in both groups received treatment with inhaled tobramycin if they had a positive Pa OP culture, and treatment in both groups was associated with eradication of TABLE 1-CFTR Gene Mutation Panel Used by New York CF NBS Program F508del I50e7del G542X G551D W1282X N1303K R553X 621þ1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711þ1G>T 1898þ1G>A 2184delA 1078delT 3849þ10kbC>T 2789þ5G>A 3659delC I148T 3120þ1G>A 3876delA V520F S549R S549N 3849þ4 A-G 3905insT R347H Reflex testing for 5T polymorphism is performed if R117H is detected. Login to comment
78 TABLE 3- Clinical Features and Outcomes of the CRMS Infants Patient number Gender Race/ ethnicity Mean age at 1st sweat (weeks) Mean sweat chloride (range) CFTR gene mutations identified Follow up time (months) Fecal elastase (mcg/gm stool) History of Pa infection History of hospitalization 1 Male Caucasian 4 46 (38-54) F508del/R117H/7T 36 303 Yes No 2 Male Caucasian 5 40 (40-43) F508del/R117H/7T 60 500 Yes No 3 Female Caucasian 3 29 (27-31) F508del/R117H/7T 60 488 No No 4 Male Caucasian 3 34 (33-38) F508del/none 26 383 No No 5 Male Caucasian 3 45 (40-50) F508del/none 72 424 No No 6 Female Caucasian 3 35 (32-38) F508del/none 9 454 No No 7 Male Caucasian 3 41 (36-46) F508del/none 39 462 No No 8 Female Caucasian 5 50 (46-52) F508del/none 72 440 No Yes 9 Male Caucasian 4 43 (41-45) F508del/Y1032C 14 401 No No 10 Male Caucasian 3 52 (50-54) G542X/none 21 500 No No 11 Female Caucasian 8 34 (30-38) R560T/none 9 433 No No 12 Female Hispanic 6 36 (32-40) R334W/R117H/7T 24 500 Yes No Mean sweat chloride levels represent the mean of all tests performed in the neonatal period. Login to comment
61 Infants in both groups received treatment with inhaled tobramycin if they had a positive Pa OP culture, and treatment in both groups was associated with eradication of TABLE 1- CFTR Gene Mutation Panel Used by New York CF NBS Program F508del I50e7del G542X G551D W1282X N1303K R553X 621þ1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711þ1G>T 1898þ1G>A 2184delA 1078delT 3849þ10kbC>T 2789þ5G>A 3659delC I148T 3120þ1G>A 3876delA V520F S549R S549N 3849þ4 A-G 3905insT R347H Reflex testing for 5T polymorphism is performed if R117H is detected. Login to comment
79 TABLE 3- Clinical Features and Outcomes of the CRMS Infants Patient number Gender Race/ ethnicity Mean age at 1st sweat (weeks) Mean sweat chloride (range) CFTR gene mutations identified Follow up time (months) Fecal elastase (mcg/gm stool) History of Pa infection History of hospitalization 1 Male Caucasian 4 46 (38-54) F508del/R117H/7T 36 303 Yes No 2 Male Caucasian 5 40 (40-43) F508del/R117H/7T 60 500 Yes No 3 Female Caucasian 3 29 (27-31) F508del/R117H/7T 60 488 No No 4 Male Caucasian 3 34 (33-38) F508del/none 26 383 No No 5 Male Caucasian 3 45 (40-50) F508del/none 72 424 No No 6 Female Caucasian 3 35 (32-38) F508del/none 9 454 No No 7 Male Caucasian 3 41 (36-46) F508del/none 39 462 No No 8 Female Caucasian 5 50 (46-52) F508del/none 72 440 No Yes 9 Male Caucasian 4 43 (41-45) F508del/Y1032C 14 401 No No 10 Male Caucasian 3 52 (50-54) G542X/none 21 500 No No 11 Female Caucasian 8 34 (30-38) R560T/none 9 433 No No 12 Female Hispanic 6 36 (32-40) R334W/R117H/7T 24 500 Yes No Mean sweat chloride levels represent the mean of all tests performed in the neonatal period. Login to comment

[hide] Buccal cell DNA mutation analysis for diagnosis of... Pediatrics. 1998 May;101(5):851-5. Parad RB
Buccal cell DNA mutation analysis for diagnosis of cystic fibrosis in newborns and infants inaccessible to sweat chloride measurement.
Pediatrics. 1998 May;101(5):851-5., [PMID:9565413]

Abstract [show]
OBJECTIVES: To assess the application of DNA-based cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis as a primary cystic fibrosis (CF) diagnostic test in preterm and term newborns and infants for whom the quantitative pilocarpine iontophoresis test (QPIT) cannot be used. DESIGN: Retrospective survey. SETTING: DNA Diagnostic Laboratory, Children's Hospital, Boston, Massachusetts. Buccal cell DNA samples were received from inpatients, outpatients, and three neonatal intensive care units. OUTCOME MEASURE: Detection of at least 1 of 12 CFTR mutations. PATIENTS: Between November 1, 1992, and April 30, 1994, 28 newborns and infants under 12 months of age at risk for CF had CFTR DNA mutation analysis performed because a sweat chloride (SC) value could not be obtained. QPIT was either not performed (infant weight <2 kg, QPIT not available at site of hospitalization, or infant not accessible to QPIT laboratory) or was inconclusive (sweat volume <75 mg or indeterminate SC [>/=40, <60 mEq/L]). The postnatal age at time of testing ranged from 1 day to 11 months, and gestational age at birth from 25 to 40 weeks. RESULTS: Six (21%) of 28 infants with unobtainable or indeterminate QPIT had 1 or 2 CFTR mutations detected. Immediate CF diagnosis by direct detection of 2 CFTR mutations was made in 5 of these 6 patients. Definitive CF diagnosis in the infant with 1 CFTR mutation was delayed until an elevation in SC could be documented. The patients with no CFTR mutations detected had a low likelihood of CF. CONCLUSIONS: For infants in whom CF is suspected but QPIT cannot be obtained, buccal cell DNA-based CFTR mutation analysis can be used as a rapid, noninvasive primary diagnostic test. This simple mode of DNA collection may aid in the diagnosis of other inherited disorders in newborns.

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58 The brushes were then discarded and 60 ␮L 1 M Tris, pH 8.0, was added to the tubes.7 CFTR mutation analysis was performed for 12 mutations (⌬F508, G551D, G542X, 621ϩ1G3T, ⌬I507, 1717-1G3A, R117H, N1303K, W1282X, R560T, R553X, and 3849ϩ10kb C3T). Login to comment

[hide] Alpha1-antitrypsin deficiency alleles and the Taq-... Eur Respir J. 1998 Apr;11(4):873-9. Mahadeva R, Westerbeek RC, Perry DJ, Lovegrove JU, Whitehouse DB, Carroll NR, Ross-Russell RI, Webb AK, Bilton D, Lomas DA
Alpha1-antitrypsin deficiency alleles and the Taq-I G-->A allele in cystic fibrosis lung disease.
Eur Respir J. 1998 Apr;11(4):873-9., [PMID:9623690]

Abstract [show]
Cystic fibrosis (CF) is characterized by progressive and ultimately fatal pulmonary disease although there are notable variations in clinical features. This heterogeneity is thought to lie outside the cystic fibrosis transmembrane regulator (CFTR) gene locus and may stem from deficiencies in the antiproteinase screen that protects the lung from proteolytic attack. One hundred and fifty seven patients were recruited from two UK CF centres. The serum concentrations of alpha1-antitrypsin, alpha1-antichymotrypsin and C-reactive protein (CRP) were determined and patients were screened for the common S and Z deficiency alleles of alpha1-antitrypsin and the G-->A mutation in the 3' noncoding region of the alpha1-antitrypsin gene (Taq-I G-->A allele). Alpha1-antitrypsin deficiency phenotypes were detected in 20 (16 MS, 1 S and 3 MZ) out of 147 unrelated tested CF patients and were, surprisingly, associated with significantly better lung function (adjusted mean forced expiratory volume in one second (FEV1) 62.5% of predicted for deficient group and 51.1% pred for normal alleles; p=0.043). The Taq-I G-->A allele was found in 21 out of 150 unrelated patients and had no significant effect on CF lung disease or on levels of alpha1-antitrypsin during the inflammatory response. We show here that, contrary to current thinking, common mutations of alpha1-antitrypsin that are associated with mild to moderate deficiency of the protein predict a subgroup of cystic fibrosis patients with less severe pulmonary disease. Moreover, the Taq-I G-->A allele has no effect on serum levels of alpha1-antitrypsin in the inflammatory response, which suggests that the previously reported association of the Taq-I G-->A allele with chronic obstructive pulmonary disease is not mediated by its effect on the serum level of alpha1-antitrypsin.

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54 In the deficient α1-AT phenotype group, the "other" mutations in the five patients in the 508/other group were R560T, 3659delC, 2711delT and two in whom the other mutations were unknown at the time of the study. Login to comment

[hide] Pathology of pancreatic and intestinal disorders i... J R Soc Med. 1998;91 Suppl 34:40-9. Wilschanski M, Durie PR
Pathology of pancreatic and intestinal disorders in cystic fibrosis.
J R Soc Med. 1998;91 Suppl 34:40-9., [PMID:9709387]

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152 A small number of more Table 1 Classification of cystic fibrosis gene mutation as severe, mild or indeterminate with respect to pancreatic function Severe Mild Variable (classes 1, I/ or 111) (classes IV or V) (classes IV or V) AF508 R117H G85E 1148T R334W 2789+5G-*A G480C R347P G551D A455E R560T P574H N1303K 3849+1 Okb C-+T G542X G551S W1282X P5748 621 +1 G-T R352Q 1717-1G-T T3381 556delA Adapted from Ref 20 with permission recently described mutations [G85E and 278+5G-÷AI are less clearly determinant with respect to the pancreatic sufficient and pancreatic insufficient phenotypes. Login to comment

[hide] Mutations of the cystic fibrosis gene in patients ... N Engl J Med. 1998 Sep 3;339(10):645-52. Sharer N, Schwarz M, Malone G, Howarth A, Painter J, Super M, Braganza J
Mutations of the cystic fibrosis gene in patients with chronic pancreatitis.
N Engl J Med. 1998 Sep 3;339(10):645-52., 1998-09-03 [PMID:9725921]

Abstract [show]
BACKGROUND: The pancreatic lesions of cystic fibrosis develop in utero and closely resemble those of chronic pancreatitis. Therefore, we hypothesized that mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may be more common than expected among patients with chronic pancreatitis. METHODS: We studied 134 consecutive patients with chronic pancreatitis (alcohol-related disease in 71, hyperparathyroidism in 2, hypertriglyceridemia in 1, and idiopathic disease in 60). We examined DNA for 22 mutations of the CFTR gene that together account for 95 percent of all mutations in patients with cystic fibrosis in the northwest of England. We also determined the length of the noncoding sequence of thymidines in intron 8, since the shorter the sequence, the lower the proportion of normal CFTR messenger RNA. RESULTS: The 94 male and 40 female patients ranged in age from 16 to 86 years. None had a mutation on both copies of the CFTR gene. Eighteen patients (13.4 percent), including 12 without alcoholism, had a CFTR mutation on one chromosome, as compared with a frequency of 5.3 percent among 600 local unrelated partners of persons with a family history of cystic fibrosis (P<0.001). A total of 10.4 percent of the patients had the 5T allele in intron 8 (14 of 134), which is twice the expected frequency (P=0.008). Four patients were heterozygous for both a CFTR mutation and the 5T allele. Patients with a CFTR mutation were younger than those with no mutations (P=0.03). None had the combination of sinopulmonary disease, high sweat electrolyte concentrations, and low nasal potential-difference values that are diagnostic of cystic fibrosis. CONCLUSIONS: Mutations of the CFTR gene and the 5T genotype are associated with chronic pancreatitis.

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32 DNA Studies We extracted DNA from buccal cells obtained by having the patients rinse their mouths with 10 ml of 4 percent sucrose.19 The CFTR locus was examined for the 22 mutations that together account for 95 percent of all such mutations in patients with cystic fibrosis in the northwest of England.20 The amplification- refractory mutation system Elucigene CF(4)m kit (Zeneca Diagnostics, Macclesfield, United Kingdom) was used to detect the four most common mutations: ∆F508, G551D, G542X, and 621+1(G→T)21; the polymerase chain reaction, restriction-enzyme analysis, and allele-specific oligonucleotide hybridization facilitated the detection of R560T, R117H, 1898+1(G→A), R553X, S549N, 1717¡1(G→A), N1303K, W1282X, E60X, 1154insTC, R347P, 3659delC, Q493X, V520F, R334W, ∆I507, 3849+10Kb(C→T), and 1078delT. Login to comment
66 * PATIENT NO.† SEX MUTANT ALLELE POLYT GENOTYPE AGE AT ONSET OF PANCREATITIS AGE AT STUDY ENTRY EXOCRINE STATUS AND CALCULI‡ ALCOHOLISM »10 CIGARETTES/ DAY SWEAT TESTING BASE-LINE NASAL POTENTIAL DIFFERENCE SODIUM CHLORIDE yr mmol/liter mV 1 M DF508 9T/7T 8 27 PS0 No No 43.5 32.0 12.5 2 F DF508 9T/5T 15 34 PS1 No No 55.0 47.5 ND 3 M R117H 7T/7T 18 21 PS0 No Yes 44.0 33.0 ¡9.7 4 M DF508 9T/7T 18 26 PI3 No No ND ND ND 5 M DF508 9T/7T 18 30 PI3 No Yes ND ND ND 6 F Q493X 7T/5T 19 21 PS3 No Yes 51.5 41.0 ND 7 F DF508 9T/7T 20 31 PS3 No No 35.0 23.0 ¡10.8 8 M 621+1(G→T) 9T/7T 21 37 PS3 Yes Yes 72.0 48.5 5.0 9 M R560T 7T/7T 21 39 PI0 Yes Yes 103.0 76.0 ¡4.4 10 M DF508 9T/5T 22 36 PI3 Yes No 53.0 34.0 ¡17.6 11 M DF508 9T/7T 31 45 PS3 No Yes 55.0 34.0 ¡11.5 12 M R117H 7T/7T 35 38 PI2 Yes No ND ND ND 13 F DF508 9T/7T 36 39 PS3 No Yes 60.0 39.0 ¡10.2 14 F R553X 7T/5T 37 56 PI3 No Yes ND ND ND 15 F DF508 9T/7T 45 47 PI3 Yes Yes 104.0 80.0 ¡8.3 16 M DF508 9T/7T 49 52 PS1 Yes Yes ND ND ND 17 F DF508 9T/7T 64 76 PI3 No No 69.0 50.0 ¡10.3 18 F DF508 9T/9T 75 79 PS3 No No 34.5 19.0 ¡14.7 or radiologic abnormalities in 133 patients. Login to comment

[hide] Relation between mutations of the cystic fibrosis ... N Engl J Med. 1998 Sep 3;339(10):653-8. Cohn JA, Friedman KJ, Noone PG, Knowles MR, Silverman LM, Jowell PS
Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis.
N Engl J Med. 1998 Sep 3;339(10):653-8., 1998-09-03 [PMID:9725922]

Abstract [show]
BACKGROUND: It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS: We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS: Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION: In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.

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34 Pancreatograms were assessed for the severity of chronic pancreatitis according to published criteria by a reviewer who was unaware of the patients` histories (Table 1).19 DNA Studies We extracted DNA from blood samples20 and tested for 16 CFTR mutations - ∆F508, W1282X, R117H, 621+1(G→T), R334W, R347P, A455E, ∆I507, 1717¡1(G→A), G542X, S549N, G551D, R553X, R560T, N1303K, and 3849+10Kb(C→T) - using reverse dot blot strips (Roche Molecular Systems, Alameda, Calif.). Login to comment

[hide] Pharmacology of CFTR chloride channel activity. Physiol Rev. 1999 Jan;79(1 Suppl):S109-44. Schultz BD, Singh AK, Devor DC, Bridges RJ
Pharmacology of CFTR chloride channel activity.
Physiol Rev. 1999 Jan;79(1 Suppl):S109-44., [PMID:9922378]

Abstract [show]
Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109-S144, 1999. - The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl- channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl-. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

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579 A second possibility is that the phosporylation state of the channel obtainedsurements, that NS004 similarly activated an additional CFTR mutant, P574H, while failing to activate the CF- during activation by forskolin is distinct from that after activation of the channel by exogenous PKA/ATP in ancausing R560T or DI507 CFTR mutants (70). Login to comment

[hide] Increased risk of idiopathic chronic pancreatitis ... Hum Mutat. 2005 Oct;26(4):303-7. Cohn JA, Neoptolemos JP, Feng J, Yan J, Jiang Z, Greenhalf W, McFaul C, Mountford R, Sommer SS
Increased risk of idiopathic chronic pancreatitis in cystic fibrosis carriers.
Hum Mutat. 2005 Oct;26(4):303-7., [PMID:16134171]

Abstract [show]
Cystic fibrosis (CF) is a recessive disease caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. The risk of idiopathic chronic pancreatitis (ICP) is increased in individuals who have CFTR genotypes containing a CF-causing mutation plus a second pathogenic allele. It is unknown whether the risk of ICP is increased in CF carriers who have one CF-causing mutation plus one normal allele. In this study, 52 sporadic cases of ICP were ascertained through the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer. Individuals with pathogenic cationic trypsinogen mutations were excluded. DNA was comprehensively tested for CFTR mutations using a robotically enhanced, multiplexed, and highly redundant form of single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing. Fifteen subjects had a total of 18 pathogenic CFTR alleles. Eight subjects had common CF-causing mutations. This group included seven CF carriers in whom the second CFTR allele was normal (4.3 times the expected frequency, P=0.0002). Three subjects had compound heterozygotes genotypes containing two pathogenic alleles (31 times the expected frequency, P<0.0001). A variant allele of uncertain significance (p.R75Q) was detected in eight of the 52 ICP subjects and at a similar frequency (13/96) in random donors. ICP differs from other established CFTR-related conditions in that ICP risk is increased in CF carriers who have one documented normal CFTR allele. Having two CFTR mutations imparts a higher relative risk, while having only one mutation imparts a higher attributable risk.

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93 Abnormal CFTR Genotypes Detected in 52 Patients with ICPa Genotype categorya ] Patients Genotypes detectedb Compound heterozygotes and homozygotes 3 p.F508del / p.L967S p.D1152H / p.D1152H p.V920M / p.L967S Heterozygotes, common mutation causing classic CFa 7 p.F508del /^ ('ve subjects)c p.R560T/^ p.G542X /^ Heterozygotes, uncommon mutation causing variable phenotype 3 p.S1235R /^ p.A209S /^ p.L997F/^ Heterozygotes, common CBAVD-associated mutation 2 IVS8(5T) /^ (two subjects) a Common CF-mutations consistently cause classic CF in compound heterozygotes and homozygotes [Rosenstein and Cutting, 1998]. Login to comment

[hide] Outcomes of a cystic fibrosis carrier testing clin... Med J Aust. 2009 Nov 2;191(9):499-501. Christie LM, Ingrey AJ, Turner GM, Proos AL, Watts GE
Outcomes of a cystic fibrosis carrier testing clinic for couples.
Med J Aust. 2009 Nov 2;191(9):499-501., 2009-11-02 [PMID:19883345]

Abstract [show]
OBJECTIVE: To review the outcomes of offering carrier testing for cystic fibrosis (CF) to couples considering pregnancy, and to women in early pregnancy and their partners. METHODS: An after-hours clinic was established in Newcastle for discussion of issues related to prenatal testing. Couples were offered CF carrier testing by extracting DNA from a mouthwash sample. An expanded one-step model was used with both partners being tested initially for the p.F508del cystic fibrosis transmembrane conductance regulator gene (CFTR) mutation. If one partner was a p.F508del carrier, the other partner was tested for an additional 28 CFTR mutations. RESULTS: Of 1000 individuals who were offered CF carrier testing, none declined. No re-collections of mouthwash samples were required, and results were available within 14 days. There were 730 individuals who had no family history of CF (73%); 27 were carriers (4%; 95% CI, 2.4%-5.3%), and there were two high-risk couples where both partners were carriers of p.F508del. There were 270 individuals who had an affected family member with CF or a child identified as a CF carrier through newborn screening; 126 were carriers (46%; 95% CI, 40.6%-52.8%), and there were two high-risk couples - one couple where both partners were carriers of p.F508del, and another couple where the woman was homozygous for p.F508del and the man was a p.F508del carrier. The information on carrier status led the four high-risk couples to change their reproductive decisions to avoid having a child with CF. CONCLUSION: CF carrier testing for couples using an expanded one-step model will detect about 80% of high-risk couples and enables various reproductive choices. We believe that all couples considering pregnancy, and women in early pregnancy and their partners, should be offered CF carrier testing.

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72 This provides each individual with information on their carrier status, and accurate residual risks of 1 CFTR mutations tested for in individuals whose partner was a carrier of p.F508del* p.F508del p.F316leufsX p.I507del p.R347P p.G542X p.S1251N p.G551D p.E60X p.N1303K p.W1282X c.1585-1G>A p.D1152H p.R553X c.2988+1G>A c.489+G>T c.2657+5G>A p.R117H c.1766+1G>A p.R1162X c.579+1G>A c.3717+10kbC>T p.G85E p.R334W p.K684fs p.A455E p.I148T p.K684fs p.R560T p.T1176fs CFTR = gene encoding cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Genetic, epidemiological, and clinical aspects of ... Am J Gastroenterol. 2010 Aug;105(8):1876-83. Epub 2010 May 25. Joergensen MT, Brusgaard K, Cruger DG, Gerdes AM, Schaffalitzky de Muckadell OB
Genetic, epidemiological, and clinical aspects of hereditary pancreatitis: a population-based cohort study in Denmark.
Am J Gastroenterol. 2010 Aug;105(8):1876-83. Epub 2010 May 25., [PMID:20502448]

Abstract [show]
OBJECTIVES: In a population-based, well-defined group of patients first regarded as having pancreatitis of unknown origin (PUO), we identified, described, and compared the clinical and genetic aspects of patients with hereditary pancreatitis (HP) and with cystic fibrosis transmembrane conductance regulator gene (CFTR) and serine protease inhibitor Kazal type 1 gene (SPINK1) mutations with patients who retained the diagnosis of true idiopathic pancreatitis (tIP) after genetic testing for HP, SPINK1, and CFTR mutations. METHODS: Patients with PUO were identified in the Danish National Registry of Patients or were referred by clinicians. DNA from blood was analyzed for cationic trypsinogen (PRSS1), SPINK1, and CFTR mutations. Considering the diagnosis of HP, a pedigree was drawn for each patient. RESULTS: A genetic mutation was found in 40% of 122 patients with PUO. After testing first-degree relatives of the 18 initially identified HP patients, 38 HP patients in total were identified, and 28 patients had SPINK1-CFTR mutations. Among HP patients, no p.N29I mutations were found and the p.A16V mutation was more frequent than previously reported, 45 and 32% had exocrine and endocrine insufficiency, respectively, and among tIP patients 9 and 12%, respectively. Pancreatic cancer was diagnosed in 5% of the HP families. CONCLUSIONS: The genotype of the Danish population with HP differs from that of previously described cohorts. The occurrence of exocrine and endocrine insufficiency is higher among patients with HP than in patients with SPINK1-CFTR mutations and tIP, and more HP families develop pancreatic cancer. Genetic testing thus helps to predict the prognosis of the pancreatitis.

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57 The samples were also tested for 33 CFTR mutations, and all 6 classeswererepresented:394delTT,p.R553X,621+1G>T,p.R1162X, 1717-1G>A,3659delC,p.G542X,2183AA>G,p.W1282X,1078delT, 711+1G>T, F508del, p.S549N, I507del, p.S549R, 2184delA, p.G551D, p.G85E, p.N1303K, p.R560T, p.R117H, p.R347H, p.R347P, p.R334W, 2789+5G>A, 3849+10kbC>T, p.A445E, 3120+1G>A, p.V520F,1898+1G>A,3876delA,3905insT,andIVS8-5T.DNAwas amplified by multiplex PCR (Hybaid 4 A62, Middlesex, UK). Login to comment

[hide] Cystic fibrosis in Chilean patients: Analysis of 3... J Cyst Fibros. 2011 Jan;10(1):66-70. Epub 2010 Oct 30. Lay-Son G, Puga A, Astudillo P, Repetto GM
Cystic fibrosis in Chilean patients: Analysis of 36 common CFTR gene mutations.
J Cyst Fibros. 2011 Jan;10(1):66-70. Epub 2010 Oct 30., [PMID:21036675]

Abstract [show]
BACKGROUND: CFTR gene mutations have worldwide differences in prevalence and data on Chilean patients is scarce. METHODS: We studied 36 of the most common CFTR mutations in Chilean patients from the CF National Program [Programa Nacional de Fibrosis Quistica (PNFQ)] of the Ministry of Health of Chile. RESULTS: Two hundred and eighty-nine patients were studied. Fourteen different mutations were identified with an overall allele detection rate of 42.0%. Mutations with frequencies greater than 1% were p.F508del (30.3% of alleles), p.R334W (3.3%), p.G542X (2.4%), c.3849+10Kb C>T (1.7%), and p.R553X (1.2%). A north to south geographical gradient was observed in the overall rate of detection. CONCLUSIONS: Southern European CFTR mutations predominate in the Chilean population, but a high percentage of alleles remain unknown. Geographical heterogeneity could be explained in part by admixture. Complementary analyses are necessary to allow for effective genetic counselling and improve cost-effectiveness of screening and diagnostic tests.

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81 Mutation This study Rios et al. [4] Molina et al. [5] Repetto et al. [6] Perez et al. [13] CFGAC [2] (n=578) (%) (n=72) (%) (n=36) (%) (n=100) (%) (n=4102) (%) (n=43,849) (%) Chile Chile Chile Chile Latin-Americaa Worldwide Unknown 58.0 66.6 61.1 34.0 36.7 22.7 p.F508del 30.6 29.2 30.6 45.0 47.1 66.0 p.R334W 3.1 - - 2.0 0.8 0.1 p.G542X 2.4 0 8.3 7.0 5.0 2.4 c.3849+10Kb CNT 1.7 - - 3.0 0.3 0.2 p.R553X 1.2 4.2 0 1.0 0.4 0.7 p.R1162X 0.9 - - 2.0 1.0 0.3 p.1078delT 0.5 - - 0 b0.1 0.1 p.G85E 0.5 - - - 0.8 0.2 p.W1282X 0.2 - - 5.0 1.0 1.2 c.3120+1 GNA 0.2 - - - 0.3 - c.711+1 GNT 0.2 - - - 0.1 0.1 p.R117H 0.2 - - 0 b0.1 0.3 p.A455E 0.2 - - 0 0 0.1 p.I148T 0.2 - - - - - p.G551D 0 0 0 1.0 0.1 1.6 p.N1303K 0 0 0 0 1.8 1.3 c.621+1 GNT 0 - - 0 0.2 0.7 c.1717-1 GNA 0 - - 0 0.3 0.6 p.I507del 0 - - 0 0.2 0.2 p.R347P 0 - - 0 0 0.2 c.2789+5 GNA 0 - - - 0.2 0.1 c.1898+1 GNA 0 - - - 0.1 0.1 c.2184delA 0 - - - b0.1 0.1 p.S549N 0 - 0 - 0.1 0.1 c.3659delC 0 - - 0 0.1 0.1 p.R560T 0 - - - 0 0.1 c.1811+1.6Kb ANG 0 - - - 0.4 - c.2183AANG 0 - - 0 0.1 - p.S549R 0 - - - 0.1 - c.3272-26 ANG 0 - - - 0.1 - c.3199del6 0 - - - b0.1 - p.E60X 0 - - 0 0 - c.3905insT 0 - - - 0 - p.S1251N 0 - - 0 - - CFTRdele2,3 0 - - - - - p.R347H 0 - - - - - p.V520F 0 - - - - - p.Q552X 0 - - - - - c.394delTT 0 - - - - - c.711+1 GNA 0 - - - - - c.2143delT 0 - - - - - c.3876delA 0 - - - - - a Data from Chilean patients published in Rios et al., Molina et al., and Repetto et al. [4-6] included in this publication were excluded in this table to avoid repetition. Login to comment

[hide] Functional hot spots in human ATP-binding cassette... Protein Sci. 2010 Nov;19(11):2110-21. Kelly L, Fukushima H, Karchin R, Gow JM, Chinn LW, Pieper U, Segal MR, Kroetz DL, Sali A
Functional hot spots in human ATP-binding cassette transporter nucleotide binding domains.
Protein Sci. 2010 Nov;19(11):2110-21., [PMID:20799350]

Abstract [show]
The human ATP-binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small alpha-helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease-associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily.

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50 Disease-associated nsSNPs at Three Structural Hotspots in Human ABC Transporter NBDs Gene Disease Position ARA motif ABCB11 BRIC2 A570T ABCD1 X-ALD A616V CFTR CF A559T ABCC6 PXE R765Q ABCC8 HHF1 R841G ABCC8 HHF1 R1493Q ABCC8 HHF1 R1493W ABCD1 X-ALD R617C ABCD1 X-ALD R617G ABCD1 X-ALD R617H CFTR CF R560K CFTR CF R560S CFTR CF R560T ABCA1 HDLD1 A1046D ABCB4 ICP A546D C-loop 1 motif ABCC8 HHF1 D1471H ABCC8 HHF1 D1471N CFTR CBAVD G544V ABCC8 HHF1 G1478R C-loop2 motif ABCA4 STGD1 H2128R ABCC8 HHF1 K889T ABCD1 X-ALD R660P ABCD1 X-ALD R660W ABCA1 HDLD2 M1091T ABCA4 STGD1 E2131K ABCA12 LI2 E1539K ABCA4 STGD1 and CORD3 E1122K CFTR CF L610S ABCC8 HHF1 L1543P ABCA1 Colorectal cancer sample; somatic mutation A2109T ABCC9 CMD1O A1513T ABCD1 X-ALD H667D CFTR CF A613T ABCA1 HDLD2 D1099Y ABCD1 X-ALD T668I CFTR CF D614G ABCA4 STGD1 R2139W ABCA4 STGD1 R1129C ABCA4 ARMD2, STGD1, and FFM R1129L Disease abbreviations are as follows: BRIC2, benign recurrent intrahepatic cholestasis type 2; X-ALD, X-linked adrenoleukodystrophy; CF, cystic fibrosis; PXE, Pseudoxanthoma elasticum; HHF1, familial hyperinsulinemic hypoglycemia-1; HDLD1, high density lipoprotein deficiency type 1; ICP, intrahepatic cholestasis of pregnancy; CBAVD, congenital bilateral absence of the vas deferens; STGD1, Stargardt disease type 1; HDLD2, high density lipoprotein deficiency type 2; LI2, ichthyosis lamellar type 2; CORD3, cone-rod dystrophy type 3; CMD1O, cardiomyopathy dilated type 1O; ARMD2, age-related macular degeneration type 2; FFM, fundus flavimaculatus. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2. Ooi CY, Durie PR
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis.
J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2., [PMID:22658665]

Abstract [show]
BACKGROUND: The pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized. RESULTS: The aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype-phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed. Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype-phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate-severe phenotypic consequences at any given time. CONCLUSIONS: The genotype-phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.

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855 CFTR mutation Total PI Total PI + PS PIP score CFTR mutation Total PI Total PI + PS PIP score 621+1G>T 96 96 1.00 G542X 74 75 0.99 711+1G>T 36 36 1.00 F508del 1276 1324 0.96 I507del 34 34 1.00 1717-1G>A 20 21 0.95 R553X 24 24 1.00 W1282X 19 20 0.95 Q493X 11 11 1.00 N1303K 45 48 0.94 S489X 11 11 1.00 R1162X 12 13 0.92 1154insTC 10 10 1.00 Y1092X 12 13 0.92 3659delC 9 9 1.00 I148T 10 11 0.91 CFTRdele2 7 7 1.00 V520F 9 10 0.90 4016insT 7 7 1.00 G551D 59 67 0.88 E60X 7 7 1.00 L1077P 5 6 0.83 R560T 7 7 1.00 R1066C 5 6 0.83 R1158X 7 7 1.00 2184insA 9 12 0.75 3905insT 6 6 1.00 2143delT 3 4 0.75 I148T;3199del6 5 5 1.00 1161delC 3 4 0.75 2183AA>G 5 5 1.00 3120+1G>A 3 4 0.75 1898+1G>A 5 5 1.00 S549N 3 4 0.75 2347delG 4 4 1.00 G85E 16 22 0.73 Q1313X 3 3 1.00 R117C 2 3 0.67 Q220X 3 3 1.00 M1101K 19 30 0.63 2184delA 3 3 1.00 P574H 3 5 0.60 1078delT 3 3 1.00 474del13BP 1 2 0.50 L1254X 3 3 1.00 R352Q 1 2 0.50 E585X 3 3 1.00 Q1291H 1 2 0.50 3876delA 2 2 1.00 A455E 18 37 0.49 S4X 2 2 1.00 R347P 6 15 0.40 R1070Q 2 2 1.00 2789+5G>A 6 16 0.38 F508C 2 2 1.00 L206W 6 18 0.33 DELI507 2 2 1.00 IVS8-5T 4 16 0.25 Q1411X 2 2 1.00 3272-26A>G 1 4 0.25 365-366insT 2 2 1.00 R334W 1 10 0.10 R709X 2 2 1.00 3849+10kbC>T 2 22 0.09 1138insG 2 2 1.00 P67L 1 14 0.07 CFTRdele2-4 2 2 1.00 R117H 1 25 0.04 3007delG 2 2 1.00 R347H 0 5 0.00 Q814X 2 2 1.00 G178R 0 3 0.00 394delTT 2 2 1.00 E116K 0 2 0.00 406-1G>A 2 2 1.00 875+1G>C 0 2 0.00 R75X 2 2 1.00 V232D 0 2 0.00 CFTRdel2-3 2 2 1.00 D579G 0 2 0.00 E193X 2 2 1.00 L1335P 0 2 0.00 185+1G>T 2 2 1.00 Mild mutations (based on PIP scores) are shaded in gray. Login to comment

[hide] Newborn screening for cystic fibrosis: Polish 4 ye... Eur J Hum Genet. 2012 Aug 15. doi: 10.1038/ejhg.2012.180. Sobczynska-Tomaszewska A, Oltarzewski M, Czerska K, Wertheim-Tysarowska K, Sands D, Walkowiak J, Bal J, Mazurczak T
Newborn screening for cystic fibrosis: Polish 4 years' experience with CFTR sequencing strategy.
Eur J Hum Genet. 2012 Aug 15. doi: 10.1038/ejhg.2012.180., [PMID:22892530]

Abstract [show]
Newborn screening for cystic fibrosis (NBS CF) in Poland was started in September 2006. Summary from 4 years' experience is presented in this study. The immunoreactive trypsin/DNA sequencing strategy was implemented. The group of 1 212 487 newborns were screened for cystic fibrosis during the programme. We identified a total of 221 CF cases during this period, including, 4 CF cases were reported to be omitted by NBS CF. Disease incidence in Poland based on the programme results was estimated as 1/4394 and carrier frequency as 1/33. The frequency of the F508del was similar (62%) to population data previously reported. This strategy allowed us to identify 29 affected infants with rare genotypes. The frequency of some mutations (eg, 2184insA, K710X) was assessed in Poland for the first time. Thus, sequencing assay seems to be accurate method for screening programme using blood spots in the Polish population.European Journal of Human Genetics advance online publication, 15 August 2012; doi:10.1038/ejhg.2012.180.

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57 Mutations D537N and P731L have not been Period of NBS CF Method The most frequent mutations in Polish population under analysis September 2006 - December 2007 Estonia Asper Biotech assay E60X, G85E, 394delTT, R117H, R117P, R117L, I148T, 621G>A, 711+1G>T, 711+5G>A, 1078delT, R334W, R347H, R347P, R347L, IVS8-T, A455E, I507del, F508del, 1717-1G>A, G542X, p.G551D, Q552X, R553X, R553G, R560T, R560K, 1898+1G>A, 1898+1G>T, 1898+1G>C, 2143delT, 2184delA, 2183AA>G, 2789+5G>A, 3120+1G>A, 3199del6, 3272-26A>G, R1162X, 3659delC, 3849+10kbC>T, 3905insT, S1235R, S1251N, W1282X, W1282C, N1303K, CFTRdele2,3 January 2007 - June 2009 Sanger sequencing of exons: 4, 7, 10, 11, 13, 21, fragment of intron 19 F508del, CFTRdele2,3, 3849+10kbC>T, R117H+IVS8-T*, R334W, R347P, 1717-1G>A, G542X, R553X, K710X, 2184insA, 2143delT, 2183AA>G, N1303K July 2009 - currently Sanger sequencing of exons: 7, 10, 11, 13, 17b, 20, 21, fragment of intron 19 F508del, CFTRdele2,3, 3849+10kbC>T, R334W, R347P, 1717-1G>A, G542X, R553X, K710X, 2184insA, 2143delT, 2183AA>G, N1303K, 3272-26A>G**, W1282X** * removed from DNA analysis since July 2009 , **added into DNA analysis since July 2009 Figure 1 NBS CF in Poland. Login to comment

[hide] Prospective and parallel assessments of cystic fib... Eur J Pediatr. 2012 Aug;171(8):1223-9. Epub 2012 May 12. Krulisova V, Balascakova M, Skalicka V, Piskackova T, Holubova A, Paderova J, Krenkova P, Dvorakova L, Zemkova D, Kracmar P, Chovancova B, Vavrova V, Stambergova A, Votava F, Macek M Jr
Prospective and parallel assessments of cystic fibrosis newborn screening protocols in the Czech Republic: IRT/DNA/IRT versus IRT/PAP and IRT/PAP/DNA.
Eur J Pediatr. 2012 Aug;171(8):1223-9. Epub 2012 May 12., [PMID:22581207]

Abstract [show]
Cystic fibrosis (CF) is a life-threatening disease for which early diagnosis following newborn screening (NBS) improves the prognosis. We performed a prospective assessment of the immunoreactive trypsinogen (IRT)/DNA/IRT protocol currently in use nationwide, versus the IRT/pancreatitis-associated protein (PAP) and IRT/PAP/DNA CF NBS protocols. Dried blood spots (DBS) from 106,522 Czech newborns were examined for IRT concentrations. In the IRT/DNA/IRT protocol, DNA-testing was performed for IRT >/= 65 ng/mL. Newborns with IRT >/= 200 ng/mL and no detected cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations were recalled for a repeat IRT. In the same group of newborns, for both parallel protocols, PAP was measured in DBS with IRT >/= 50 ng/mL. In PAP-positive newborns (i.e., >/=1.8 if IRT 50-99.9 or >/=1.0 if IRT >/= 100, all in ng/mL), DNA-testing followed as part of the IRT/PAP/DNA protocol. Newborns with at least one CFTR mutation in the IRT/DNA/IRT and IRT/PAP/DNA protocols; a positive PAP in IRT/PAP; or a high repeat IRT in IRT/DNA/IRT were referred for sweat testing. CONCLUSION: the combined results of the utilized protocols led to the detection of 21 CF patients, 19 of which were identified using the IRT/DNA/IRT protocol, 16 using IRT/PAP, and 15 using IRT/PAP/DNA. Decreased cut-offs for PAP within the IRT/PAP protocol would lead to higher sensitivity but would increase false positives. Within the IRT/PAP/DNA protocol, decreased PAP cut-offs would result in high sensitivity, an acceptable number of false positives, and would reduce the number of DNA analyses. Thus, we concluded that the IRT/PAP/DNA protocol would represent the most suitable protocol in our conditions.

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81 According to the protocol, this result indicated the sequencing of the Table 1 Parallel comparison of CF NBS protocols IRT/DNAa /IRT IRT/PAP IRT/PAP/DNAa Newborns screened (N) 106,522 106,522 106,522 IRT positives (N; %) 1,158 (1.09) 3,155 (2.96) 3,155 (2.96) PAP positives (N; %) - 260 (0.24) 260 (0.24) Median age (range) at the availability of DNA-testinga results (days) 36 (9-222b ) - 36 (9-222b ) 1 and/or 2 CF mutations detected (N; %) 76 (0.07) - 27 (0.03) Recalled newborns for repeated IRT examination (N; %) 47 (0.04) - - Positive CF NBS (N; %) 123 (0.12) 260 (0.24) 27 (0.03) Positive IRT in newborns recalled for repeated examination (N) 1 - - ST indicated (N; %) 77 (0.07) 260 (0.24) 27 (0.03) ST carried out (N; % of indicated ST) 72c (93.51) 204c (78.46) 24c (88.89) CF carriers (N) 55 - 12 Prevalence of CF carriers 1 in 21 - 1 in 22 Diagnosed CF patients (N) 19 16 15 False positives based on performed ST (N; % of all cases screened) 99d (0.09) 188 (0.18) 9 (0.01) Newborns with equivocal diagnosis [F508del/R117H-IVS-8 T(7) and ST<30 mmol/L; N] 2 - 0 False negatives (N) 2 5 6 Total of CF patients detected (N) 21e Median age (range) at diagnosis (days) 36 (9-57)e CF prevalence 1 in 5,072e Sensitivity (TP/TP+FN) 0.9048 0.7619 0.7142 Specificity (TN/TN+FP) 0.9991 0.9982 0.9999 PPV (TP/TP+FP) 0.1610 0.0784 0.625 N number, % of all cases screened, TP true positives, FN false negatives, TN true negatives, FP false positives, PPV positive predictive value, ST sweat test a CF-causing mutations covered by Elucigene assays ("legacy" nomenclature) with the CF-EU1Tm accounting for: p.Arg347Pro (R347P), c.2657+ 5G>A (2789+5G>A), c.2988+1G>A (3120+1G>A), c.579+1G>T (711+1G>T), p.Arg334Trp (R334W), p.Ile507del (I507del), p.Phe508del (F508del), c.3718-2477C>T (3849+10kbC>T), p.Phe316LeufsX12 (1078delT), p.Trp1282X (W1282X), p.Arg560Thr (R560T), p.Arg553X (R553X), p.Gly551Asp (G551D), p.Met1101Lys (M1101K), p.Gly542X (G542X), p.Leu1258PhefsX7 (3905insT), p.Ser1251Asn (S1251N), c.1585-1G>A (1717-1G>A), p.Arg117His (R117H), p.Asn1303Lys (N1303K), p.Gly85Glu (G85E), c.1766+1G>A (1898+1G>A), p.Lys684AsnfsX38 (2184delA), p.Asp1152His (D1152H), c.54-5940_273+10250del (CFTRdele2,3), p.Pro67Leu (P67L), p.Glu60X (E60X), p.Lys1177SerfsX15 (3659delC), c.489+1G>T (621+1G>T), p.Ala455Glu (A455E), p.Arg1162X (R1162X), p.Leu671X (2143delT), c.1210-12T[n] (IVS8-T(n) variant), including additional mutations in the CF-EU2Tm : p.Gln890X (Q890X), p.Tyr515X (1677delTA), p.Val520Phe (V520F), c.3140-26A>G (3272-26A>G), p.Leu88IlefsX22 (394delTT), p.Arg1066Cys (R1066C), p.Ile105SerfsX2 (444delA), p.Tyr1092X (C>A) (Y1092X(C>A)), p.Arg117Cys (R117C), p.Ser549Asn (S549N), p.Ser549ArgT>G (S549R T>G), p.Tyr122X (Y122X), p.Arg1158X (R1158X), p.Leu206Trp (L206W), c.1680-886A>G (1811+1.6kbA>G), p.Arg347His (R347H), p.Val739TyrfsX16 (2347delG) and p.Trp846X (W846X) b failed DNA isolation from DBS, including repetition of DNA-testing c deceased patient or non-compliance with referrals (five CF carriers in IRT/DNA/IRT, 56 newborns in IRT/PAP, three CF carriers in IRT/PAP/DNA) d comprising newborns with repeated IRT (47 newborns) e aggregate data from all protocols entire CFTR coding region in both newborns, and led to the identification of p.Ile336Lys (I336K) and p.Glu1104Lys (E1104K) mutations. Login to comment

[hide] Improving test properties for neonatal cystic fibr... J Inherit Metab Dis. 2012 Jul;35(4):635-40. Cornel MC, Gille JJ, Loeber JG, Vernooij-van Langen AM, Dankert-Roelse J, Bolhuis PA
Improving test properties for neonatal cystic fibrosis screening in the Netherlands before the nationwide start by May 1st 2011.
J Inherit Metab Dis. 2012 Jul;35(4):635-40., [PMID:22302635]

Abstract [show]
When new technical possibilities arise in health care, often attunement is needed between different actors from the perspectives of research, health care providers, patients, ethics and policy. For cystic fibrosis (CF) such a process of attunement in the Netherlands started in a committee of the Health Council on neonatal screening in 2005. In the balancing of pros and cons according to Wilson and Jungner criteria, the advantages for the CF patient were considered clear, even though CF remains a severe health problem with treatment. Nevertheless, screening was not started then, mainly since the specificity of the tests available at that time was considered too low. Many healthy infants would have been referred for sweat testing and much uncertainty would arise in their parents. Also the limited sensitivity for immigrants and the detection of less severe phenotypes and carriers were considered problematic. The Health Council recommended a pilot screening project which was subsequently performed in some provinces, leading to a 4-step protocol: IRT, PAP, screening for a CFTR mutation panel, and sequencing of the CFTR gene. This would lead to the identification of 23 cases of classical CF, two infants with less severe forms and 12 carriers per year in the Netherlands. Thus many CF patients can be diagnosed early, while limiting the number of referrals, the number of infants with less severe forms diagnosed and the number of carriers identified. Technical solutions were found to limit the ethical problems. A nationwide program using this four step protocol started by 1 May 2011.

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69 This protocol was expected to identify 25 CF patients on an annual basis, additional to four infants already diagnosed because of meconium ileus (Health Council of 1 Using the LiPA test (INNO-LiPA CFTR 19 en INNO-LiPA CFTR 17+Tn; Innogenetics, Gent, Belgium) the following CFTR mutations can be detected: exon 2-3del (21 kb), 394delTT, E60X, G85E, R117H, 621+1G>T, 711+1G>T, 711+5G>A, 1078delT, R334W, R347P, A455E, I507del, F508del, 1717-1G>A, G542X, G551D, Q552X, R553X, R560T, 1898+1G>A, 2143delT, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, 3199del6, 3272-26A>G, 3659delC, R1162X, 3849+10kbC>T, 3905insT, S1251N, W1282X en N1303K. Login to comment
70 This test also identifies the CFTR polymorphism Tn in intron 8 which is important in cases where the mutation R117H is detected. Login to comment

[hide] Link between CFTR mutations and ABPA: a systematic... Mycoses. 2012 Jul;55(4):357-65. doi: 10.1111/j.1439-0507.2011.02130.x. Epub 2011 Oct 17. Agarwal R, Khan A, Aggarwal AN, Gupta D
Link between CFTR mutations and ABPA: a systematic review and meta-analysis.
Mycoses. 2012 Jul;55(4):357-65. doi: 10.1111/j.1439-0507.2011.02130.x. Epub 2011 Oct 17., [PMID:21999194]

Abstract [show]
Summary There is a biological plausibility on the link between cystic fibrosis transmembrane conductance regulator (CFTR) mutations and allergic bronchopulmonary aspergillosis (ABPA). The aim of the systematic review was to investigate this link by determining the frequency of CFTR mutations in ABPA. We searched the PubMed and EmBase databases for studies reporting CFTR mutations in ABPA. We pooled the odds ratio (OR) and 95% confidence intervals (CI) from individual studies using both fixed and random effects model. Statistical heterogeneity was evaluated using the I(2) test and the Cochran-Q statistic. Publication bias was assessed using both graphical and statistical methods. Our search yielded four studies (79 ABPA, 268 controls). The odds of encountering CFTR mutation was higher in ABPA compared with the control group (OR 10.39; 95% CI, 4.35-24.79) or the asthma population (OR 5.53; 95% CI 1.62-18.82). There was no evidence of statistical heterogeneity or publication bias. There is a possible pathogenetic link between CFTR mutations and ABPA. However, because of the small numbers of patients, further studies are required to confirm this finding. Future studies should adopt a uniform methodology and should screen for the entire genetic sequence of the CFTR gene.

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59 (2002)[33] 31ABPAHealthycontrols (n=34) Asthma(n=51) Asthma,positiveSPTtoAf,totalIgE >1000ngml)1 ,elevatedAf-IgE,positive precipitinstoAf,bloodeosinophilia >350ll)1 ,pulmonaryinfiltratesonCXR orCBonCT/(NewZealand) 16CFmutations-F508del,I507del, R117H,W1282X,621+1G>T, R334W,R347P,A455E, 1717-1G>A,G542X,5549N, G551D,R553X,R560T,N1303Kand 3849+10kbC>T ASOhybridisationand DGGEwithDNA sequencing 4/31(F508del[n=3], R117H[n=1])vs.2/51 asthma(F508del[n=1], R117H[n=1])vs.1/34 healthycontrols ABPA,allergicbronchopulmonaryaspergillosis;ARMS,amplificationrefractorymutationsystem;ASO,allele-specificoligonucleotide;CB,centralbronchiectasis;CFTR,cysticfibrosis transmembraneconductanceregulator;DGGE,denaturinggradientgelelectrophoresis;OR,oddsratio CFTRmutationclass(classI--1717-1G>A,R1162X,G542X;classII--F508del,N1303K;classIV--R347H,R117H). Login to comment

[hide] Rapid detection of the ACMG/ACOG-recommended 23 CF... J Biomol Tech. 2012 Apr;23(1):24-30. Elliott AM, Radecki J, Moghis B, Li X, Kammesheidt A
Rapid detection of the ACMG/ACOG-recommended 23 CFTR disease-causing mutations using ion torrent semiconductor sequencing.
J Biomol Tech. 2012 Apr;23(1):24-30., [PMID:22468138]

Abstract [show]
Cystic fibrosis (CF) is one of the most frequently diagnosed autosomal-recessive diseases in the Caucasian population. For general-population CF carrier screening, the American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists (ACOG) have recommended a core panel of 23 mutations that will identify 49-98% of carriers, depending on ethnic background. Using a genotyping technology that can rapidly identify disease-causing mutations is important for high-throughput general-population carrier screening, confirming clinical diagnosis, determining treatment options, and prenatal diagnosis. Here, we describe a proof-of-concept study to determine whether the Ion Torrent Personal Genome Machine (PGM) sequencer platform can reliably identify all ACMG/ACOG 23 CF transmembrane conductance regulator (CFTR) mutations. A WT CF specimen along with mutant DNA specimens representing all 23 CFTR mutations were sequenced bidirectionally on the Ion Torrent 314 chip to determine the accuracy of the PGM for CFTR variant detection. We were able to reliably identify all of the targeted mutations except for 2184delA, which lies in a difficult, 7-mer homopolymer tract. Based on our study, we believe PGM sequencing may be a suitable technology for identifying CFTR mutations in the future. However, as a result of the elevated rate of base-calling errors within homopolymer stretches, mutations within such regions currently need to be evaluated carefully using an alternative method.

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26 Amplicons were then pooled together in equimolar concentrations and purified using the T A B L E 1 Data Generation from Three PGM Runs Run Total number of reads Total bases (Mbp) AQ17 total bases (Mbp) AQ17 avg. read length CF WT 101,211 8.5 6.5 68 CF 23 pooled mutants 222,247 18.6 12.52 64 CF mutant 135,000 11.7 8.8 72 T A B L E 2 CFTR Variant Coverage, Mutant Read Percentage, and Base-Call Accuracy from a WT Library Using PGM Sequencing Variant cDNA position Coverage Mutant read % Accuracy/base G85E c.254G Ͼ A 408 0 99.5 R117H c.350G Ͼ A 3627 0 99.9 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 245 0 99.6 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 2660 0 99.9 R334W c.1000C Ͼ T 5419 0 99.7 R347P c.1040G Ͼ C 3562 0 99.4 A455E c.1364C Ͼ A 10,340 0 99.9 ⌬I507 c.1519_1521delATC 6507 0 98.6 ⌬F508 c.1521_1523delCTT 6507 0 99.4 1717-1G Ͼ A c.1585-1G Ͼ A 2086 0 99.2 G542X c.1624G Ͼ T 854 0 97.8 G551D c.1652G Ͼ A 3901 0 99 R553X c.1657C Ͼ T 3915 0 99.9 R560T c.1679G Ͼ C 3924 0 99.6 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 1793 0 97.6 2184delAa c.2052delA 2001 35% 63.6 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 293 0 100 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 2408 0 100 R1162X c.3484C Ͼ T 9610 0 98.1 3659delC c.3528delC 9271 0 100 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 10,157 0 99.9 W1282X c.3846G Ͼ A 4789 0 95.6 N1303K c.3909C Ͼ G 3236 0 99.5 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not detected accurately as a result of homopolymer-length sequencing errors. Login to comment
67 For this data set, the PGM 314 chip output was 18.6 Mbp, with ϳ67% aligning to the CFTR T A B L E 3 PGM CFTR Variant Coverage and Mutant Read Percentage from a Pooled Mutant Library Representing All 23 ACMG/ACOG Mutations Variant cDNA position Coverage Mutant read % Predicted read % Genotype G85E c.254G Ͼ A 93 33 50 Het R117H c.350G Ͼ A 6228 39 50 Het 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 1243 46 50 Het 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 1352 29 50 Het R334W c.1000C Ͼ T 13,284 8 25 Het R347P c.1040G Ͼ C 9454 27 25 Het A455E c.1364C Ͼ A 19,527 43 50 Het ⌬I507 c.1519_1521delATC 15,587 14 25 Het ⌬F508 c.1521_1523delCTT 15,587 68 50 Homo 1717-1G Ͼ A c.1585-1G Ͼ A 3584 36 50 Het G542X c.1624G Ͼ T 610 41 50 Het G551D c.1652G Ͼ A 6714 16 17 Het R553X c.1657C Ͼ T 6670 15 17 Het R560T c.1679G Ͼ C 6395 22 17 Het 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 3293 49 50 Het 2184delAa c.2052delA 2256 63 50 Het 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 1765 54 50 Het 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 7447 40 50 Het R1162X c.3484C Ͼ T 19,060 54 50 Het 3659delC c.3528delC 28,321 30 50 Het 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 27,102 46 50 Het W1282X c.3846G Ͼ A 9219 48 50 Het N1303K c.3909C Ͼ G 4842 49 50 Het a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
86 Using samples characterized previously, we analyzed the PGM`s data out- T A B L E 4 PGM CFTR Variant Coverage and Mutant Read Percentage from an Individual Harboring Two Disease-Causing CFTR Mutations Variant cDNA position Coverage Mutant read % G85E c.254G Ͼ A 237 0 R117H c.350G Ͼ A 3774 0 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 936 0 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 2018 0 R334W c.1000C Ͼ T 10,899 0 R347P c.1040G Ͼ C 7720 0 A455E c.1364C Ͼ A 14,525 0 ⌬I507 c.1519_1521delATC 8855 0 ⌬F508 c.1521_1523delCTT 8855 47 1717-1G Ͼ A c.1585-1G Ͼ A 2216 0 G542X c.1624G Ͼ T 2035 41 G551D c.1652G Ͼ A 4581 0 R553X c.1657C Ͼ T 4545 0 R560T c.1679G Ͼ C 4774 0 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 2702 0 2184delAa c.2052delA 2837 18.5 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 860 0 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 4347 0 R1162X c.3484C Ͼ T 12,039 0 3659delC c.3528delC 7169 0 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 11,588 0 W1282X c.3846G Ͼ A 6187 0 N1303K c.3909C Ͼ G 4479 0 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
66 For this data set, the PGM 314 chip output was 18.6 Mbp, with b03;67% aligning to the CFTR T A B L E 3 PGM CFTR Variant Coverage and Mutant Read Percentage from a Pooled Mutant Library Representing All 23 ACMG/ACOG Mutations Variant cDNA position Coverage Mutant read % Predicted read % Genotype G85E c.254G b0e; A 93 33 50 Het R117H c.350G b0e; A 6228 39 50 Het 621 af9; 1G b0e; T c.489 af9; 1G b0e; T 1243 46 50 Het 711 af9; 1G b0e; T c.579 af9; 1G b0e; T 1352 29 50 Het R334W c.1000C b0e; T 13,284 8 25 Het R347P c.1040G b0e; C 9454 27 25 Het A455E c.1364C b0e; A 19,527 43 50 Het èc;I507 c.1519_1521delATC 15,587 14 25 Het èc;F508 c.1521_1523delCTT 15,587 68 50 Homo 1717-1G b0e; A c.1585-1G b0e; A 3584 36 50 Het G542X c.1624G b0e; T 610 41 50 Het G551D c.1652G b0e; A 6714 16 17 Het R553X c.1657C b0e; T 6670 15 17 Het R560T c.1679G b0e; C 6395 22 17 Het 1898 af9; 1G b0e; A c.1766 af9; 1G b0e; A 3293 49 50 Het 2184delAa c.2052delA 2256 63 50 Het 2789 af9; 5G b0e; A c.2657 af9; 5G b0e; A 1765 54 50 Het 3120 af9; 1G b0e; A c.2988 af9; 1G b0e; A 7447 40 50 Het R1162X c.3484C b0e; T 19,060 54 50 Het 3659delC c.3528delC 28,321 30 50 Het 3849 af9; 10kbC b0e; T c.3717 af9; 12191C b0e; T 27,102 46 50 Het W1282X c.3846G b0e; A 9219 48 50 Het N1303K c.3909C b0e; G 4842 49 50 Het a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
85 Using samples characterized previously, we analyzed the PGM`s data out- T A B L E 4 PGM CFTR Variant Coverage and Mutant Read Percentage from an Individual Harboring Two Disease-Causing CFTR Mutations Variant cDNA position Coverage Mutant read % G85E c.254G b0e; A 237 0 R117H c.350G b0e; A 3774 0 621 af9; 1G b0e; T c.489 af9; 1G b0e; T 936 0 711 af9; 1G b0e; T c.579 af9; 1G b0e; T 2018 0 R334W c.1000C b0e; T 10,899 0 R347P c.1040G b0e; C 7720 0 A455E c.1364C b0e; A 14,525 0 èc;I507 c.1519_1521delATC 8855 0 èc;F508 c.1521_1523delCTT 8855 47 1717-1G b0e; A c.1585-1G b0e; A 2216 0 G542X c.1624G b0e; T 2035 41 G551D c.1652G b0e; A 4581 0 R553X c.1657C b0e; T 4545 0 R560T c.1679G b0e; C 4774 0 1898 af9; 1G b0e; A c.1766 af9; 1G b0e; A 2702 0 2184delAa c.2052delA 2837 18.5 2789 af9; 5G b0e; A c.2657 af9; 5G b0e; A 860 0 3120 af9; 1G b0e; A c.2988 af9; 1G b0e; A 4347 0 R1162X c.3484C b0e; T 12,039 0 3659delC c.3528delC 7169 0 3849 af9; 10kbC b0e; T c.3717 af9; 12191C b0e; T 11,588 0 W1282X c.3846G b0e; A 6187 0 N1303K c.3909C b0e; G 4479 0 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment

[hide] CFTR, SPINK1, CTRC and PRSS1 variants in chronic p... Gut. 2012 Mar 17. Rosendahl J, Landt O, Bernadova J, Kovacs P, Teich N, Bodeker H, Keim V, Ruffert C, Mossner J, Kage A, Stumvoll M, Groneberg D, Kruger R, Luck W, Treiber M, Becker M, Witt H
CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated?
Gut. 2012 Mar 17., [PMID:22427236]

Abstract [show]
OBJECTIVE: In chronic pancreatitis (CP), alterations in several genes have so far been described, but only small cohorts have been extensively investigated for all predisposing genes. DESIGN: 660 patients with idiopathic or hereditary CP and up to 1758 controls were enrolled. PRSS1, SPINK1 and CTRC were analysed by DNA sequencing, and cystic fibrosis transmembrane conductance regulator (CFTR) by melting curve analysis. RESULTS: Frequencies of CFTR variants p.R75Q, p.I148T, 5T-allele and p.E528E were comparable in patients and controls. We identified 103 CFTR variants, which represents a 2.7-fold risk increase (p<0.0001). Severe cystic fibrosis (CF)-causing variants increased the risk of developing CP 2.9-fold, and mild CF-causing variants 4.5-fold (p<0.0001 for both). Combined CF-causing variants increased CP risk 3.4-fold (p<0.0001), while non-CF-causing variants displayed a 1.5-fold over-representation in patients (p=0.14). CFTR compound heterozygous status with variant classes CF-causing severe and mild represented an OR of 16.1 (p<0.0001). Notably, only 9/660 (1.4%) patients were compound heterozygotes in this category. Trans-heterozygosity increased CP risk, with an OR of 38.7, with 43/660 (6.5%) patients and 3/1667 (0.2%) controls being trans-heterozygous (p<0.0001). CONCLUSIONS: Accumulation of CFTR variants in CP is less pronounced than reported previously, with ORs between 2.7 and 4.5. Only CF-causing variants reached statistical significance. Compound and trans-heterozygosity is an overt risk factor for the development of CP, but the number of CFTR compound heterozygotes in particular is rather low. In summary, the study demonstrates the complexity of genetic interactions in CP and a minor influence of CFTR alterations in CP development.

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72 The following CFTR variants were analysed with specific FRET probes: p.E60X, p.R75Q, p.G85E, p.R117H, p.I148T, c.621 +1G>T (IVS4+1G>T), c.711+1G>T (IVS5+1G>T), c.1078delT, p.R334W, p.R347P, 9-13TG, 5-9T, p.A455E, p.M470V, p.F508del, c.1716G>A (p.E528E), c.1717-1G>A (IVS10-1G>A), p.G542X, p.S549N, p.R553X, p.R560T, c.1898+1G>A (IVS12 +1G>A), c.2143delT, c.2183AA>G, c.2562T>G, c.2657+5G>A (IVS14B+5G>A), p.L997F, p.I1005R, p.Y1092X, p.D1152H, p.R1162X, c.3659delC, p.S1235R, p.S1251N, p.W1282X, p.N1303K, and c.4389G>A. Login to comment
69 The following CFTR variants were analysed with specific FRET probes: p.E60X, p.R75Q, p.G85E, p.R117H, p.I148T, c.621 +1G>T (IVS4+1G>T), c.711+1G>T (IVS5+1G>T), c.1078delT, p.R334W, p.R347P, 9-13TG, 5-9T, p.A455E, p.M470V, p.F508del, c.1716G>A (p.E528E), c.1717-1G>A (IVS10-1G>A), p.G542X, p.S549N, p.R553X, p.R560T, c.1898+1G>A (IVS12 +1G>A), c.2143delT, c.2183AA>G, c.2562T>G, c.2657+5G>A (IVS14B+5G>A), p.L997F, p.I1005R, p.Y1092X, p.D1152H, p.R1162X, c.3659delC, p.S1235R, p.S1251N, p.W1282X, p.N1303K, and c.4389G>A. Login to comment

[hide] CFTR mutation analysis and haplotype associations ... Mol Genet Metab. 2012 Feb;105(2):249-54. doi: 10.1016/j.ymgme.2011.10.013. Epub 2011 Oct 26. Cordovado SK, Hendrix M, Greene CN, Mochal S, Earley MC, Farrell PM, Kharrazi M, Hannon WH, Mueller PW
CFTR mutation analysis and haplotype associations in CF patients.
Mol Genet Metab. 2012 Feb;105(2):249-54. doi: 10.1016/j.ymgme.2011.10.013. Epub 2011 Oct 26., [PMID:22137130]

Abstract [show]
Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies.

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104 Mutation N alleles c.966T>G(5'flanking) c.234T>A(5'flanking)a c.-8G>C(5'UTR) c.-4G>C(Exon1) c.274-179G>A(Intron3) c.743+40A>G(Intron6) c.744-31TTGA(5_7)(Intron6) c.869+11C>T(Intron7) c.869+88T>A(Intron7) c.1209+43T>G(Intron9) IVS8CA(15-23)(Intron9) TG(10-13)_T(5-9)(Intron9) c.1393-61A>G(Intron10) M470V(Exon11) F508del(Exon11) c.1766+152T>A(Intron13) c.1767-231T>C(Intron13) c.1767-136T>C(Intron13) c.1767-132A>G(Intron13) c.2562T>G(Exon15) c.2604A>G(Exon15) c.2619+86_2619+87del(Intron15) c.2619+106T>A(Intron15) c.2909-92G>A(Intron17) IVS17bCA(11-17)(Intron20) c.3368-140A>C(Intron20) c.3469-65C>A(Intron21) F508del 32 TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- GA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- A5- 55- 55- 55- 66- 66- 66- 66- 66- 66- 66- 66- 66- 66- 55- 55- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TC- TT- TT- TT- TC- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TG- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- T17- 10_9- G- F508del- TA- 13C F508del 29 G23- 10_9- G- F508del- TA- 13C F508del 1 G21- 10_9- G- GG- G-F508del- TA- 13C F508del 1 G17- 10_9- G- F508del- A- G- delTA- 17- C- A N1303K 6 G542X 6 3849+10kbC→T 1 del Ex17a, b, Ex18 1 GG- GG- GG- 23- 10_9- GG-F508- T- TA- 13- C A455E 1 G22- 10_9- G- F508- T- TA- 13- C 621+1G→T 5 G21- 10_9- G- GG- GG- F508C- TA- 13- C 711+1G→T 3 3272-26A→G 2 3659delC 2 R347P 2 G16- 11_7- A- A-F508- TA- 13C del Ex 2, 3 2 del Ex 17a,17b 2 Normal 1 R334W 2 G17- 11_7- A- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA-AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- A-AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- F508- TA- 13C 2183AA→G 2 G16- 10_7- F508- TATA- TATA- TATA- TATA- TATA- TATA- 13C del Ex 2 1 G16- 11_7- F508- 14C 1288insTA 1 G16- 12_7- F508- 13C Normal 1 G16- 12_7- F508- 13C R1162X 1 G17- 10_7- F508- 13C del Ex 2,3 1 G16- 11_7- F508- A17- C del Ex 17a,17b 1 GA- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT-16- 11_7- F508- 14- C G85E 1 G16- 11_7- F508- 15C 1898+1G→A 1 G16- 11_7- F508- G13- C no mut detected 1 GT- TT- T16- 10_7- F508- 13C no mut detected 1 G16- 10_7- F508- 17A W1282X 2 G17- 10_7- F508- 17A W1282X 4 GC- CC- C17- 10_7- F508- delTA- 17- A Q39X 1 I507del 1 3849+10kbC→T 1 R560T 2 1717-1G→A 2 G551D 3 G16- 10_7- F508- delTA- 17- A G551D 2 1154insTC 1 G16- 10_7- F508- delTA- 17- 1717- 17A 1717-1G→A 1 2789+5G→A 1 GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- G17- 10_7- F508- AdelTA- A R1066C 1 GG- 17- 10_7- F508- delTA- A R1066H 1 GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- G17- 9_7- F508- delTAC R553X 3 GG- GG- CA- AA- AA- AA- A17- 12_7- F508- delTA- 11- C 3121-1G→A 1 C17- 12_7- F508- delTA- 11- C R334W 1 G17- 12_7- F508- TA- 13- C (TG)13T5b 1 G17- 13_5- F508- delTA- 13- C CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- R117H 1 CA- 6C- TT- 15- 12_5- AG- F508- T- TT- AT- ATA- TG- 13A- C R117H1 1 CA- 6C- TT- 16- 12_5- AG- F508- T- TT- AT- ATA- TG- 13A- C 1717-1G→A 1 R117Hb 1 GA- 6C- TT- 16- 10_7- AA- F508- A- TC- AG- AdelTA- TG- 13A- C 144c a Variation found in a sample where the haplotype could not be predicted. Login to comment

[hide] Lessons learned from 20 years of newborn screening... Med J Aust. 2012 Jan 16;196(1):67-70. Massie RJ, Curnow L, Glazner J, Armstrong DS, Francis I
Lessons learned from 20 years of newborn screening for cystic fibrosis.
Med J Aust. 2012 Jan 16;196(1):67-70., [PMID:22256939]

Abstract [show]
OBJECTIVE: To compare three cystic fibrosis (CF) newborn screening strategies used in Victoria since 1989. DESIGN, SETTING AND PARTICIPANTS: Retrospective review of newborn screening and clinical records for people with CF born in Victoria between 1989 and 2008 to compare screening strategies: repeat immunoreactive trypsinogen (IRT) testing (IRT/IRT, 1989-1990), IRT and p.F508del mutation analysis (IRT/p.F508del, 1991-2006) and IRT with analysis of 12 CFTR mutations (IRT/12 mutations, 2007-2008). MAIN OUTCOME MEASURES: Total number of infants screened, people identified with CF (by screening or clinical diagnosis), number of CF-affected terminations of pregnancy, and number of carriers detected. RESULTS: There were 420 people born with CF (live-birth prevalence, 1/3139; 95% CI, 1/2853-1/3462) and 78 CF-affected pregnancy terminations (overall prevalence, 1/2647; 95% CI, 1/2425-1/2896). Of the babies born with CF, 283 (67.4%) were detected by newborn screening alone, 61 (14.5%) had meconium ileus, 33 (7.9%) had a family history of CF, nine (2.1%) were diagnosed antenatally, and 34 (8.1%) were missed by screening (17 missed because IRT level was < 99th percentile, two with repeat IRT level not elevated, 14 without a screened CFTR mutation, and one with missing data). The sensitivities of the protocols were 86.6% for IRT/IRT, 89.9% for IRT/p.F508del, and 95.8% for IRT/12 mutations. Including 12 mutations in the analysis detected one patient who would otherwise have been missed and, had this protocol been implemented from 1989, it would have detected four others. CONCLUSION: Most babies with CF without meconium ileus, a family history or antenatal diagnosis are detected by newborn screening. Despite improved sensitivity with the 12-mutation analysis, most infants detected would have been diagnosed using the IRT/p.F508del protocol.

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14 From 1991 to 2006, babies with an IRT level > 99th percentile had CFTR gene mutation analysis for p.F508del and, from 2007, for 12 CFTR mutations (p.F508del, p.G551D, p.G542X, p.N1303K, c.1585- 1G>A, p.I507del, p.R560T, p.W1282X, p.V520F, c.489+1G>T, p.R553X, c.3718-2477C>T). Login to comment

[hide] Cystic fibrosis mutations for p.F508del compound h... Clin Genet. 2012 Dec;82(6):546-551. doi: 10.1111/j.1399-0004.2011.01804.x. Epub 2011 Nov 29. Sebro R, Levy H, Schneck K, Dimmock D, Raby B, Cannon C, Broeckel U, Risch N
Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency.
Clin Genet. 2012 Dec;82(6):546-551. doi: 10.1111/j.1399-0004.2011.01804.x. Epub 2011 Nov 29., [PMID:22035343]

Abstract [show]
Sebro R, Levy H, Schneck K, Dimmock D, Raby BA, Cannon CL, Broeckel U, Risch NJ. Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency. Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype. Over 1500 mutations in the CFTR gene have been identified; however, the p.F508del mutation is most common. There has been limited correlation between the CFTR mutation genotype and the disease phenotypes. We evaluated the non-p.F508del mutation of 108 p.F508del compound heterozygotes using the biological classification method, Grantham and Sorting Intolerant from Tolerant (SIFT) scores to assess whether these scoring systems correlated with sweat chloride levels, pancreatic sufficiency, predicted FEV(1) , and risk of infection with Pseudomonas aeruginosa in the last year. Mutations predicted to be 'mild' by the biological classification method are associated with more normal sweat chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and decreased risk of infection with Pseudomonas in the last year (p = 0.014). Lower Grantham scores are associated with more normal sweat chloride levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT scores are associated with more normal sweat chloride levels (p < 0.001) and pancreatic sufficiency (p = 0.011). There was no association between pulmonary function measured by predicted FEV(1) and the biological classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62), which suggests the pulmonary disease related to CF may involve other modifier genes and environmental factors.

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64 CFTR mutation classification for compound heterozygotesa Mutations n (%) Biological classification Grantham score SIFT Q493X 3 (3) Ib - - G542X 21 (20) Ib,c,e - - R553X 4 (4) Ib,e - - Y1092X 2 (2) Ib - - R1158X 1 (1) NA - - W1282X 9 (9) Ib,e - - G85E 4 (4) IIIb 98 0.01 R117H 4 (4) IVb,c 29 0.60 R334W 1 (1) IVb 101 0.02 R347P 1 (1) IVb 103 0.05 R352Q 1 (1) NA 43 0.35 G551D 20 (19) IIIb,c 94 0.00 R560T 3 (3) IIIb 71 0.00 D1270N 1 (1) NA 23 0.01 N1303K 6 (6) IIg 94 0.00 I507del 3 (3) IId - - 394delTT 1 (1) NAc - - 621+1G>T 7 (7) Ib,f - - 711+1G>T 2 (2) Ib - - 1717-1G>A 5 (5) Ib,c,e,f - - 1898+1G>A 2 (2) NA - - 2789+5G>A 3 (3) Vb - - 3659delC 1 (1) Ib - - 3849+10kbC>T 2 (2) Vb,c,f - - 3905insT 1 (1) Ib - - NA, not applicable; SIFT, Sorting Intolerant from Tolerant. a The following mutations biological classification scores could not be verified: 1898+G-A, 394delTT, D1270N, R352Q, and R1158X. Login to comment

[hide] First study of the F508del mutation in Malaysian c... J Paediatr Child Health. 2011 Aug;47(8):573-5. doi: 10.1111/j.1440-1754.2011.02149.x. Nathan AM, Thong MK, deBruyne J, Ariffin H
First study of the F508del mutation in Malaysian children diagnosed with cystic fibrosis.
J Paediatr Child Health. 2011 Aug;47(8):573-5. doi: 10.1111/j.1440-1754.2011.02149.x., [PMID:21843195]

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48 Letters to the Editor Journal of Paediatrics and Child Health 47 (2011) 572-575 (c) 2011 The Authors Journal of Paediatrics and Child Health (c) 2011 Paediatrics and Child Health Division (Royal Australasian College of Physicians) Table1Summaryoftheclinicalcharacteristics,sweattestresultsandcysticfibrosistransmembraneconductanceregulatormutationstudiesofthepatientsdiagnosedwithcysticfibrosisinUniversity MalayaMedicalCenterfrom2000to2009 PatientAgeat presentation PresentingsymptomsOtherfindingsConsanguinityRaceSweatconductivity (mmol/l) KS score Mutations Rin3monthsRecurrentpneumoniaandFTTPseudo-Bartter`ssyndromeYesIndian13440†Nonedetected Nes8yearsSeverepersistentasthmaFTTNoIndian12450F508del/unknown Abd4monthsSeverepneumoniaandventilator dependent FTTYesYemeni11730F508del/F508del Ben7yearsCirrhosisoftheliverwithportal hypertension FTTUnknown(adopted)Unknown14080†Nonedetected(7T polymorphism) Sak3monthsRecurrentpneumoniaandFTTNDYesIndian11350F508del/F508del Ngan3yearsPseudo-Bartter`ssyndromeNDNoChinese13790Notdone(parentsrefused) LJH5monthsPseudo-Bartter`ssyndromeRecurrentpneumoniaNoChinese/Indonesian9465F508delnegative Josh5monthsPseudo-Bartter`ssyndromeandFTTNDNoIndian8585†Nonedetected Nur3monthsChronicdiarrhoeaandFTTPseudo-Bartter`ssyndromeNoMalay/Chinese13085‡†R553X/nonedetected Vin4monthsRecurrentpneumoniaandFTTNDNoChinese12260F508delnegative Muh5yearsPoorlycontrolledasthmaNDNoMalay10765F508delnegative Naz3monthsFTTandsteatorrhoeaRecurrentlunginfectionsand pseudo-Bartter`s NoMalay14675F508delnegative Additionalmutationsscreenedinthefourpatients:†F508del,I506/7del,G551D,G542X,R553X,R117C,R117H,621+1G>T,V520F,A455E,N1303K,3849+10kbC>T.‡R334W,R347P,A455E,S549N,R560T, 3659delC,W1282X.FTT,failuretothrive;KS,Schwachman-Kulczycki(KS)score;ND,nodata. Login to comment

[hide] The K+ channel opener 1-EBIO potentiates residual ... PLoS One. 2011;6(8):e24445. Epub 2011 Aug 31. Roth EK, Hirtz S, Duerr J, Wenning D, Eichler I, Seydewitz HH, Amaral MD, Mall MA
The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.
PLoS One. 2011;6(8):e24445. Epub 2011 Aug 31., [PMID:21909392]

Abstract [show]
BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K(+) channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl(-) secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl(-) secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl(-) secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl(-) secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl(-) secretion by 39.2+/-6.7% (P<0.001) via activation of basolateral Ca(2)(+)-activated and clotrimazole-sensitive KCNN4 K(+) channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl(-) secretion in tissues with residual CFTR function by 44.4+/-11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl(-) conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl(-)secretion in native CF tissues expressing CFTR mutants with residual Cl(-) channel function by activation of basolateral KCNN4 K(+) channels that increase the driving force for luminal Cl(-) exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

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46 CFabsent CFresidual CFTR genotype Number of individuals CFTR genotype Number of individuals F508del/F508del 10 F508del/Y161C 1 F508del/W57X 1 F508del/V232D 1 F508del/G85E 3 F508del/R334W 2 F508del/120del23 1 F508del/T338I 1 F508del/182delT 1 F508del/I1234V 1 F508del/G542X 1 F508del/3272-26 A.G 1 F508del/A561E 1 F508del/3849+10 kb C.T 1 F508del/Y1092X 1 F508del/4005 +5727 A.G 1 F508del/N1303K 1 F508del/G576A 1 F508del/1525-1 G.A 2 N1303K/R334W 1 F508del/Q39X 1 F1052V/M1137R 1 F508del/Q552X 1 1898+3 A.G/ 1898+3 A.G 1 G85E/G85E 1 R334W/3199del6 1 Q552X/R1162X 1 R334W/X 1 A561E/A561E 2 dele2,3/X 1 R764X/1717-1 G.A 1 R1158X/2183AA.G 1 R1158X/R560T 1 doi:10.1371/journal.pone.0024445.t001 luminal and basolateral surfaces of the epithelium were perfused continuously with a solution of the following composition (mmol/ L): NaCl 145, KH2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, Ca-gluconate 1.3, pH 7.4, at 37uC. Login to comment

[hide] The use of DHPLC (Denaturing High Performance Liqu... J Prenat Med. 2010 Jul;4(3):45-8. Mesoraca A, Di Natale M, Cima A, Di Giacomo G, Sarti M, Barone MA, Bizzoco D, Cignini P, Mobili L, D'emidio L, Giorlandino C
The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis.
J Prenat Med. 2010 Jul;4(3):45-8., [PMID:22439061]

Abstract [show]
OBJECTIVE: The aim of the study is to evaluate the role of Denaturing High Performance Liquid Chromatography (DHPLC) in the second level screening of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. METHODS: A 9-month prospective study, between June 2008 and March 2009 at Artemisia Fetal Medical Centre, included 3829 samples of amniotic fluid collected from women undergoing mid-trimester amniocentesis.The genetic diagnosis of CF was based on research of the main mutations of the CFTR gene on fetal DNA extracted from the amniocytes, (first level screening) using different commercial diagnostic systems. A second level screening using DHPLC, on the amniotic fluid and on a blood sample from the couple, was offered in case of fetuses heterozygous at first level screening. RESULTS: Of 3829 fetuses, 134 were found to be positive, 129 heterozygous and 5 affected. Of the 129 couples, following appropriate genetic counselling, 53 requested a second level screening. Through the use of DHPLC, 44 couples were found to be negative, and in nine couples, nine rare mutations were identified. CONCLUSIONS: The first level screening can be useful to evidence up to 75% of the CF mutations. The second level screening can identify a further 10% of mutant alleles. DHPLC was found to be a reliable and specific method for the rapid identification of the rare CFTR mutations which were not revealed in initial first level screening.

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100 48 Journal of Prenatal Medicine 2010; 4 (3): 45-50 Table III Mutations found with II level screening through DHPLC Mutations of mutated alleles DF508 29 W1282X 3 N1303K 8 1717-1G®A 2 3659delC 1 G85E 1 2789 +5G®A 2 R553X 2 R1162X 1 R117H 1 G542X 3 Total 53Table I Mutations found through I level screeningMutations analysed with I level screening through OLA CFTR Mutations Position on the CFTR gene DF508 Exon 10 3849+10KbC®T Intron 19 R334W Exon 7 W1282X Exon 10 V520F Exon 10 3905insT Exon 20 N1303K Exon 21 3876delA Exon 20 1717-1G®A Exon 11 3659delC Exon 19 DI507 Exon 10 A455E Exon 9 G85E Exon 3 2789 +5G®A Exon 14 / Intron 14 2183AA®G Exon 13 1898+1G®A Exon 12 / Intron 12 R347P Exon 7 R347H Exon 7 R560T Exon 11 1078delT Exon 7 R553X Exon 11 711+1G®T Exon 5 / Intron 5 G551D Exon 11 R1162X Exon 19 S549R Exon 11 R117H Exon 4 S549N Exon 11 621+1G®T Exon 4 G542X Exon 11 394delTT Exon 3 3120+1G®ðA Exon 16/ Intron 16 2184delA Exon 13 Table II Mutations found through I level screening Mutations Positions on CFTR gene R1066C Exon 17 b L1065P Exon 17 b A1006E Exon 19 R75Q Exon 3 D537E Exon 11 W1134X Exon 18 W1145X Exon 18 L1077P Exon 17b C524X Exon 11 Total 9 The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis Journal of Prenatal Medicine 2010; 4 (3): 45-50 49 tion was to provide the couple with adequate counselling in order to better understand the genotype-phenotype correlation in the various associations of mutations. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Hepatol. 2009 Jan;50(1):150-7. Epub 2008 Oct 7. Henckaerts L, Jaspers M, Van Steenbergen W, Vliegen L, Fevery J, Nuytten H, Roskams T, Rutgeerts P, Cassiman JJ, Vermeire S, Cuppens H
Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis.
J Hepatol. 2009 Jan;50(1):150-7. Epub 2008 Oct 7., [PMID:18992954]

Abstract [show]
BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disease commonly associated with inflammatory bowel disease (IBD) and characterized by fibrosing inflammatory destruction of bile ducts. The histological features in the liver of PSC patients are similar to those observed in cystic fibrosis (CF). Our aim was to study whether variants in the CFTR gene are associated with the occurrence and/or evolution of PSC. METHODS: PSC patients (n=140) were genotyped for F508del, the TGmTn variants, and four additional polymorphic loci (1001+11 C>T, M470V, T854T and Q1463Q), and compared to 136 matched healthy controls. RESULTS: The 1540G-allele, encoding V470, was less frequent in PSC (52%) than in controls (64%, p=0.003), and was associated with protection against PSC in individuals without IBD (OR 0.25, 95% CI 0.12-0.52, p=0.0002). Also TG11-T7 was less frequent in PSC (53%) than in controls (61%, p=0.04), this haplotype was associated with reduced risk for PSC (OR 0.34, 95% CI 0.17-0.70, p=0.003) in individuals without IBD. CONCLUSIONS: In this cohort of PSC patients, several CFTR-variants affecting the functional properties of the CFTR protein seem to offer protection against the development of PSC, confirming our hypothesis that CFTR might be implicated in the pathogenesis of PSC.

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91 There was Table 4 Summary of the 37 CFTR variants studied in the exploratory phase INNO-LiPA CFTR 19 INNO-LiPA CFTR17+Tn Update F508del 621+1GfiT G542X 3849+10kbCfiT N1303K 2183AAfiG W1282X 394delTT G551D 2789+5GfiA 1717-1GfiA R1162X R553X 3659delC CFTRdele2,3(21kb) R117H I507del R334W 711+1GfiT R347P 3272-26AfiG G85E 3905insT 1078delT R560T A455E 1898+1GfiA 2143delT S1251N E60X I148T 2184delA 3199del6 711+5GfiA 3120+1GfiA Tn Q552X Fig. 1. Login to comment

[hide] Cystic fibrosis carrier frequency and estimated pr... J Cyst Fibros. 2008 Sep;7(5):440-3. Epub 2008 Feb 1. Ratbi I, Genin E, Legendre M, Le Floch A, Costa C, Cherkaoui-Deqqaqi S, Goossens M, Sefiani A, Girodon E
Cystic fibrosis carrier frequency and estimated prevalence of the disease in Morocco.
J Cyst Fibros. 2008 Sep;7(5):440-3. Epub 2008 Feb 1., [PMID:18243066]

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BACKGROUND: The epidemiology of cystic fibrosis (CF) is poorly known in North African populations, in particular in Morocco and the CF carrier frequency in the general Moroccan population has never been evaluated. METHODS: To estimate the prevalence of CF mutations in Morocco, blood samples from 150 healthy Moroccans were tested for frequent CFTR mutations and the intron 8 polyT variant. RESULTS: Two subjects were heterozygous for F508del and eight others for the (T)5 variant. CONCLUSION: These findings indicate that the Moroccan population is at risk for CF and CFTR-related disorders. CF prevalence could be in the range of that found in European populations. Wider studies are necessary to identify the clinical pattern and accurately determine the prevalence and molecular basis of CF in Morocco.

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27 We screened for 32 CFTR gene mutations (G85E, 394delTT, R117H, 621+1GNT, 711+1GNT, R334W, R347P, R347H, 1078delT, A455E, I507del, F508del, V520F, 1717-1GNA, G542X, G551D, R553X, R560T, S549R(TNG), S549N, 1898+1GNA, 2183AANG, 2184delA, 2789+5GNA, 3120 + 1G NA, R1162X, 3659delC, 3849 + 10kbC NT, W1282X, 3905insT, 3876delA, N1303K) and the (T)5 splicing variant of intron 8, using a commercial kit (CF v3 Genotyping Assay, Abbott, Rungis, France). Login to comment

[hide] Consensus on the use and interpretation of cystic ... J Cyst Fibros. 2008 May;7(3):179-96. Castellani C, Cuppens H, Macek M Jr, Cassiman JJ, Kerem E, Durie P, Tullis E, Assael BM, Bombieri C, Brown A, Casals T, Claustres M, Cutting GR, Dequeker E, Dodge J, Doull I, Farrell P, Ferec C, Girodon E, Johannesson M, Kerem B, Knowles M, Munck A, Pignatti PF, Radojkovic D, Rizzotti P, Schwarz M, Stuhrmann M, Tzetis M, Zielenski J, Elborn JS
Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
J Cyst Fibros. 2008 May;7(3):179-96., [PMID:18456578]

Abstract [show]
It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

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1236 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment
1239 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment

[hide] Analysis of the CFTR gene in Iranian cystic fibros... J Cyst Fibros. 2008 Mar;7(2):102-9. Epub 2007 Jul 27. Alibakhshi R, Kianishirazi R, Cassiman JJ, Zamani M, Cuppens H
Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations.
J Cyst Fibros. 2008 Mar;7(2):102-9. Epub 2007 Jul 27., [PMID:17662673]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common inherited disorder in Caucasian populations, with over 1400 mutations identified in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Mutations in the CFTR gene may be also causative for CBAVD (Congenital Bilateral Absence of the Vas Deferens). The type and distribution of mutations varies widely between different countries and/or ethnic groups, and is relatively unknown in Iran. We therefore performed a comprehensive analysis of the CFTR gene in Iranian CF patients. METHODS: 69 Iranian CF patients, and 1 CBAVD patient, were analysed for mutations in the complete coding region, and its exon/intron junctions, of their CFTR genes, using different methods, such as ARMS (amplification refractory mutation system)-PCR, SSCP (single stranded conformation polymorphism) analysis, restriction enzyme digestion analysis, direct sequencing, and MLPA (Multiplex Ligation-mediated Probe Amplification). RESULTS: CFTR mutation analysis revealed the identification of 37 mutations in 69 Iranian CF patients. Overall, 81.9% (113/138) CFTR genes derived from Iranian CF patients could be characterized for a disease-causing mutation. The CBAVD patient was found to be homozygous for the p.W1145R mutation. The most common mutations were p.F508del (DeltaF508) (18.1%), c.2183_2184delAAinsG (2183AA>G) (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789+5G>A (4.3%), p.G542X (3.6%), c.3120+1G>A (3.6%), p.R334W (2.9%) and c.3130delA (2.9%). These 9 types of mutant CFTR genes totaled for 52% of all CFTR genes derived from the 69 Iranian CF patients. Eight mutations, c.406-8T>C, p.A566D, c.2576delA, c.2752-1_2756delGGTGGCinsTTG, p.T1036I, p.W1145R, c.3850-24G>A, c.1342-?_1524+?del, were found for the first time in this study. CONCLUSIONS: We identified 37 CFTR mutations in 69 well characterized Iranian CF patients, obtaining a CFTR mutation detection rate of 81.9%, the highest detection rate obtained in the Iranian population so far. These findings will assist in genetic counseling, prenatal diagnosis and future screening of CF in Iran.

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50 Mutations were detected as follows: In a first phase, all subjects were analyzed with an amplification refractory mutation system assay (ARMS-PCR), as described by Ferrie et al. [20], detecting the following mutations: p.F508del, p.N1303K, p.G542X, c.1717-1GNA, p.R553X, p.W1282X, p.G551D, c.621+1GNT, c.I507del and p.R560T. Login to comment

[hide] High incidence of the CFTR mutations 3272-26A-->G ... J Cyst Fibros. 2007 Nov 30;6(6):371-5. Epub 2007 May 3. Storm K, Moens E, Vits L, De Vlieger H, Delaere G, D'Hollander M, Wuyts W, Biervliet M, Van Schil L, Desager K, Nothen MM
High incidence of the CFTR mutations 3272-26A-->G and L927P in Belgian cystic fibrosis patients, and identification of three new CFTR mutations (186-2A-->G, E588V, and 1671insTATCA).
J Cyst Fibros. 2007 Nov 30;6(6):371-5. Epub 2007 May 3., [PMID:17481968]

Abstract [show]
We have analyzed 143 unrelated Belgian patients with a positive diagnosis of cystic fibrosis (CF) for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. An initial screening for 29 CFTR mutations led to mutation identification in 89.9% of the tested chromosomes. Subsequently an extensive analysis of the CFTR gene was performed by denaturating gradient gel electrophoresis (DGGE) in those patients with at least one unknown mutation after preliminary screening. In addition to 10 previously reported mutations we identified 2 new mutations 186-2A-->G and E588V. A third new mutation 1671insTATCA was identified during routine screening for DeltaF508. Two mutations were detected with a higher frequency than expected: 3272-26A-->G, which is the second most common mutation after DeltaF508 in our CF population with a frequency of 3.8%, and L927P (2.4%). The clinical data is presented for the mutations 186-2A-->G, E588V, 3272-26A-->G and L927P. The mutation data are useful for the Belgian population to supplement the initial screening set of mutations.

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31 The Inno Lipa™ CFTR12 assay contains normal and mutant probes for 12 different CFTR mutations (ΔF508, G542X, N1303K, 1717-1G→A, W1282X, G551D, R553X, S1251N, R560T, 3905insT, Q552X, ΔI507). Login to comment

[hide] Spectrum of mutations in CFTR in Finland: 18 years... J Cyst Fibros. 2005 Dec;4(4):233-7. Epub 2005 Jul 26. Kinnunen S, Bonache S, Casals T, Monto S, Savilahti E, Kere J, Jarvela I
Spectrum of mutations in CFTR in Finland: 18 years follow-up study and identification of two novel mutations.
J Cyst Fibros. 2005 Dec;4(4):233-7. Epub 2005 Jul 26., [PMID:16051530]

Abstract [show]
BACKGROUND: The incidence of cystic fibrosis (CF) is low in the isolated Finnish population and the Finnish CF mutation spectrum has differed from many European countries. METHODS: We have analyzed the mutation spectrum and the geographical distribution of CF mutations in Finland covering the last 18 years (1987-2004). RESULTS: A total of 14 mutations were identified; two of them new, 774insT and S589T (G>C at 1,898). The overall coverage of mutations was 97% (99/102 chromosomes). The most frequent mutations were F508del and 394delTT, found in 36% (37/102) and 35% (36/102) of the CF chromosomes respectively. Of the rare mutations, a mutation of presumable Slavic origin, CFTRdele2.3 (21 kb), was enriched in a rural isolate with a frequency of 5,9% (6/102), and a mutation that possibly indicates Swedish influence, 3659delC, was scattered throughout the country with a similar frequency of 5,9% (6/102). G542X, R1162X, R117H, 3732delA, 1,898 + 3A >C, S1196X, S945L, W57R, 774insT and S589T were each identified in a number of chromosomes from one to three. CONCLUSIONS: Our observations of the Finnish CF mutation spectrum fit well with the characteristics of Finland as a population of multiple local founder effects.

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36 The InnoLipa assay recognizes 36 mutations: E60X (c.178G>T, p.Glu60X), G85E (c.254G>A, p.Gly85- Glu), 394delTT, R117H (c.350G>A, p.Arg117His), I148T (c.443T>C, p.Ile148Thr), 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), 711+5G>A (c.579+5G>A), 1078delT (c.948delT, p.Phe316fs), R334W (c.1000C>T, p.Arg334Trp), R347P (c.1040G>C, p.Arg347Pro), A455E (c.1364C>A, p.Ala455Glu), I507del (c.1519_1521delATC, p.Ile507del), F508del, 1717À1G>A (c.1585À1G>A), G542X, G551D (c.1652G >A, p.Gly551Asp), Q552X (c.1654C > T, p.Gln552X), R553X (c.1657C > T, p.Arg553X), R560T (1679G>vC, p.Arg560Thr), 1898+ 1G > A (c.1766 + 1G > A), 2143delT (c.2012delT, p.Leu671fs), 2183AA > G (c.2051_2052delAAinsG, p.Lys684fs), 2184delA (c.2052delA, p.Lys684fs), 2789+ 5G>A (c.2657+5G>A), 3120+1G>A (c.2988+1G>A), 3199del6 (c.3067_3072del, p.Ile1023_Val1024del), 3272À 26A > G (c.3140 À26A > G), R1162X (c.3484C > T, p.Arg1162X), 3849+10kbCYT, 3659delC (c.3528delC, p.Lys1177fs), S1251N (c.3752G > A, p.Ser1251Asn), 3905insT (c.3773dupT, p.Leu1258fs), W1282X (c.3846G> A, p.Trp1282X), N1303K (c.3909C>G, p.Asn1303Lys), CFTRdele2,3(21kb) and Tn-polymorphism on intron 8. Login to comment
37 The InnoLipa assay recognizes 36 mutations: E60X (c.178G>T, p.Glu60X), G85E (c.254G>A, p.Gly85Glu), 394delTT, R117H (c.350G>A, p.Arg117His), I148T (c.443T>C, p.Ile148Thr), 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), 711+5G>A (c.579+5G>A), 1078delT (c.948delT, p.Phe316fs), R334W (c.1000C>T, p.Arg334Trp), R347P (c.1040G>C, p.Arg347Pro), A455E (c.1364C>A, p.Ala455Glu), I507del (c.1519_1521delATC, p.Ile507del), F508del, 1717 1G>A (c.1585 1G>A), G542X, G551D (c.1652G >A, p.Gly551Asp), Q552X (c.1654C > T, p.Gln552X), R553X (c.1657C > T, p.Arg553X), R560T (1679G>vC, p.Arg560Thr), 1898+ 1G > A (c.1766 + 1G > A), 2143delT (c.2012delT, p.Leu671fs), 2183AA > G (c.2051_2052delAAinsG, p.Lys684fs), 2184delA (c.2052delA, p.Lys684fs), 2789+ 5G>A (c.2657+5G>A), 3120+1G>A (c.2988+1G>A), 3199del6 (c.3067_3072del, p.Ile1023_Val1024del), 3272 26A > G (c.3140 26A > G), R1162X (c.3484C > T, p.Arg1162X), 3849+10kbCYT, 3659delC (c.3528delC, p.Lys1177fs), S1251N (c.3752G > A, p.Ser1251Asn), 3905insT (c.3773dupT, p.Leu1258fs), W1282X (c.3846G> A, p.Trp1282X), N1303K (c.3909C>G, p.Asn1303Lys), CFTRdele2,3(21kb) and Tn-polymorphism on intron 8. Login to comment

[hide] Hyperechogenic fetal bowel: counseling difficultie... Eur J Med Genet. 2005 Oct-Dec;48(4):421-5. Marcus-Soekarman D, Offermans J, Van den Ouweland AM, Mulder AL, Muntjewerff N, Vossen M, Kleijer W, Schrander-Stumpel C, Dooijes D
Hyperechogenic fetal bowel: counseling difficulties.
Eur J Med Genet. 2005 Oct-Dec;48(4):421-5., [PMID:16378926]

Abstract [show]
The detection of echodense fetal bowel on ultrasound examination in the second trimester of pregnancy justifies invasive procedures such as amniocentesis to detect an underlying cause. We present a case in which initial tests identified only one mutation in the cystic fibrosis transmembrane regulator (CFTR)-gene of the fetus, the family history being negative for CF. Strongly reduced intestinal enzyme activities suggested intestinal obstruction and further increased the estimated risk for CF. After the 24th gestational week, a second mutation was found, confirming cystic fibrosis in this child. Problems in counseling in this particular case are discussed.

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67 Routine CFTR-mutation analysis, using Table 1 CFTR-mutations screened for in the first step E60X 2143delT G542X G85E 2183AA-G G551D 394delTT 2184delA Q552X 621 + 1G-T 2789 + 5G-A R553X R117H 3849 + 10kbC-T R560T 711 + 5G-A R1162X S1251N 1078delT 3659delC 390insT R334W delta I507 W1282X R347P delta F508 N1303K A455E 1717-1G-A a panel of 29 CFTR-mutations, detects only 41.6% of CFTR-mutations in the Turkish population [1]. Login to comment

[hide] Genotyping microarray for the detection of more th... J Mol Diagn. 2005 Aug;7(3):375-87. Schrijver I, Oitmaa E, Metspalu A, Gardner P
Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
J Mol Diagn. 2005 Aug;7(3):375-87., [PMID:16049310]

Abstract [show]
Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.

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51 Complete List of Mutations Detectable with the CF APEX Assay CFTR location Amino acid change Nucleotide change 1 E 1 Frameshift 175delC 2 E 2,3 Frameshift del E2, E3 3 E 2 W19C 189 GϾT 4 E 2 Q39X 247 CϾT 5 IVS 2 Possible splicing defect 296 ϩ 12 TϾC 6 E 3 Frameshift 359insT 7 E 3 Frameshift 394delTT 8 E 3 W57X (TAG) 302GϾA 9 E 3 W57X (TGA) 303GϾA 10 E 3 E60X 310GϾT 11 E 3 P67L 332CϾT 12 E 3 R74Q 353GϾA 13 E 3 R75X 355CϾT 14 E 3 G85E 386GϾA 15 E 3 G91R 403GϾA 16 IVS 3 Splicing defect 405 ϩ 1GϾA 17 IVS 3 Possible splicing defect 405 ϩ 3AϾC 18 IVS 3 Splicing defect 406 - 1GϾA 19 E 4 E92X 406GϾT 20 E 4 E92K 406GϾA 21 E 4 Q98R 425AϾG 22 E 4 Q98P 425AϾC 23 E 4 Frameshift 444delA 24 E 4 Frameshift 457TATϾG 25 E 4 R117C 481CϾT 26 E 4 R117H 482GϾA 27 E 4 R117P 482GϾC 28 E 4 R117L 482GϾT 29 E 4 Y122X 498TϾA 30 E 4 Frameshift 574delA 31 E 4 I148T 575TϾC 32 E 4 Splicing defect 621GϾA 33 IVS 4 Splicing defect 621 ϩ 1GϾT 34 IVS 4 Splicing defect 621 ϩ 3AϾG 35 E 5 Frameshift 624delT 36 E 5 Frameshift 663delT 37 E 5 G178R 664GϾA 38 E 5 Q179K 667CϾA 39 IVS 5 Splicing defect 711 ϩ 1GϾT 40 IVS 5 Splicing defect 711 ϩ 1GϾA 41 IVS 5 Splicing defect 712 - 1GϾT 42 E 6a H199Y 727CϾT 43 E 6a P205S 745CϾT 44 E 6a L206W 749TϾG 45 E 6a Q220X 790CϾT 46 E 6b Frameshift 935delA 47 E 6b Frameshift 936delTA 48 E 6b N287Y 991AϾT 49 IVS 6b Splicing defect 1002 - 3TϾG 50 E 7 ⌬F311 3-bp del between nucleotides 1059 and 1069 51 E 7 Frameshift 1078delT 52 E 7 Frameshift 1119delA 53 E 7 G330X 1120GϾT 54 E 7 R334W 1132CϾT 55 E 7 I336K 1139TϾA 56 E 7 T338I 1145CϾT 57 E 7 Frameshift 1154insTC 58 E 7 Frameshift 1161delC 59 E 7 L346P 1169TϾC 60 E 7 R347H 1172GϾA 61 E 7 R347P 1172GϾC 62 E 7 R347L 1172GϾT 63 E 7 R352Q 1187GϾA 64 E 7 Q359K/T360K 1207CϾA and 1211CϾA 65 E 7 S364P 1222TϾC 66 E 8 Frameshift 1259insA 67 E 8 W401X (TAG) 1334GϾA 68 E 8 W401X (TGA) 1335GϾA 69 IVS 8 Splicing changes 1342 - 6 poly(T) variants 5T/7T/9T 70 IVS 8 Splicing defect 1342 - 2AϾC Table 1. Continued CFTR location Amino acid change Nucleotide change 71 E 9 A455E 1496CϾA 72 E 9 Frameshift 1504delG 73 E 10 G480C 1570GϾT 74 E 10 Q493X 1609CϾT 75 E 10 Frameshift 1609delCA 76 E 10 ⌬I507 3-bp del between nucleotides 1648 and 1653 77 E 10 ⌬F508 3-bp del between nucleotides 1652 and 1655 78 E 10 Frameshift 1677delTA 79 E 10 V520F 1690GϾT 80 E 10 C524X 1704CϾA 81 IVS 10 Possible splicing defect 1717 - 8GϾA 82 IVS 10 Splicing defect 1717 - 1GϾA 83 E 11 G542X 1756GϾT 84 E 11 G551D 1784GϾA 85 E 11 Frameshift 1784delG 86 E 11 S549R (AϾC) 1777AϾC 87 E 11 S549I 1778GϾT 88 E 11 S549N 1778GϾA 89 E 11 S549R (TϾG) 1779TϾG 90 E 11 Q552X 1786CϾT 91 E 11 R553X 1789CϾT 92 E 11 R553G 1789CϾG 93 E 11 R553Q 1790GϾA 94 E 11 L558S 1805TϾC 95 E 11 A559T 1807GϾA 96 E 11 R560T 1811GϾC 97 E 11 R560K 1811GϾA 98 IVS 11 Splicing defect 1811 ϩ 1.6 kb AϾG 99 IVS 11 Splicing defect 1812 - 1GϾA 100 E 12 Y563D 1819TϾG 101 E 12 Y563N 1819TϾA 102 E 12 Frameshift 1833delT 103 E 12 D572N 1846GϾA 104 E 12 P574H 1853CϾA 105 E 12 T582R 1877CϾG 106 E 12 E585X 1885GϾT 107 IVS 12 Splicing defect 1898 ϩ 5GϾT 108 IVS 12 Splicing defect 1898 ϩ 1GϾA 109 IVS 12 Splicing defect 1898 ϩ 1GϾC 110 IVS 12 Splicing defect 1898 ϩ 1GϾT 111 E 13 Frameshift 1924del7 112 E 13 del of 28 amino acids 1949del84 113 E 13 I618T 1985TϾC 114 E 13 Frameshift 2183AAϾG 115 E 13 Frameshift 2043delG 116 E 13 Frameshift 2055del9ϾA 117 E 13 D648V 2075TϾA 118 E 13 Frameshift 2105-2117 del13insAGAA 119 E 13 Frameshift 2108delA 120 E 13 R668C 2134CϾT 121 E 13 Frameshift 2143delT 122 E 13 Frameshift 2176insC 123 E 13 Frameshift 2184delA 124 E 13 Frameshift 2184insA 125 E 13 Q685X 2185CϾT 126 E 13 R709X 2257CϾT 127 E 13 K710X 2260AϾT 128 E 13 Frameshift 2307insA 129 E 13 V754M 2392GϾA 130 E 13 R764X 2422CϾT 131 E 14a W846X 2670GϾA 132 E 14a Frameshift 2734delGinsAT 133 E 14b Frameshift 2766del8 134 IVS 14b Splicing defect 2789 ϩ 5GϾA 135 IVS 14b Splicing defect 2790 - 2AϾG 136 E 15 Q890X 2800CϾT 137 E 15 Frameshift 2869insG 138 E 15 S945L 2966CϾT 139 E 15 Frameshift 2991del32 140 E 16 Splicing defect 3120GϾA interrogation: ACCAACATGTTTTCTTTGATCTTAC 3121-2A3G,T S; 5Ј-ACCAACATGTTTTCTTTGATCTTAC A GTTGTTATTAATTGTGATTGGAGCTATAG-3Ј; CAACAA- TAATTAACACTAACCTCGA 3121-2A3G,T AS. Login to comment
73 Genomic DNA Samples Used for Mutation Evaluation on the APEX Array Mutations validated with native DNA CFTRdel 2,3 (21 kb) 394delTT G85E R75X 574delA Y122X R117C R117H 621 ϩ 1GϾT 621 ϩ 3AϾG 711 ϩ 1GϾT I336K R334W R347P IVS8-5T IVS8-7T IVS8-9T A455E ⌬F508 ⌬I507 1677delTA 1717 - 1GϾA G542X G551D R553X R560T S549N 1898 ϩ 1GϾA 1898 ϩ 1GϾC 2183AAϾG 2043delG R668C 2143delT 2184delA 2184insA 2789 ϩ 5GϾA S945L 3120 ϩ 1GϾA I1005R 3272 - 26AϾG R1066C G1069R Y1092X (CϾA) 3500 - 2AϾT R1158X R1162X 3659delC S1235R 3849 ϩ 10 kb CϾT W1282X primer. Login to comment

[hide] Complete gene scanning by temperature gradient cap... J Mol Diagn. 2005 Feb;7(1):111-20. Chou LS, Gedge F, Lyon E
Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model.
J Mol Diagn. 2005 Feb;7(1):111-20., [PMID:15681482]

Abstract [show]
Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.

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75 Mutation Samples with Known Genotypes Scanned by TGCE* Exon Mutation† Amplicon size (bp) Location of mutation from 5Ј end (bp) Base change Detection‡ 3 G85E 234 124 G to A 1/1 3 394delTT 234 132 del TT 1/1 4 R117H 270 83 G to T 2/2 4 I148T 270 176 T to C 3/3 Intron 4 621 ϩ 1 G/T 270 233 G to T 1/1 5 663delT/663delT 186 75 del T 0/1 Intron 5 711 ϩ 1 G/T 186 124 G to T 1/1 7 R334W 345 208 C to T 1/1 7 R347P 345 248 G to C 1/1 9 A455E 263 155 C to A 2/2 10 I506V 292 168 A to G 1/1 10 ⌬I507 292 171 del ATC 2/2 10 ⌬F508 292 174 del TTT 2/2 10 ⌬F508/⌬F508 292 174 del TTT 0/1 10 F508C 292 175 T to G 1/1 10 V520F 292 210 G to T 1/1 Intron 10 1717-1 G/A 175 50 G to A 1/1 11 G542X 175 90 G to T 2/2 11 G542X/G542X 175 90 G to T 0/1 11 G551D 175 118 G to A 3/3 11 R553X 175 123 C to T 3/3 11 R560T 175 145 G to C 2/2 13 2184delA 834 356 del A 1/1 Intron 14b 2789 ϩ 5G/A 192 102 G to A 1/1 Intron 16 3120 ϩ 1G/A 216 111 G to A 1/1 19 R1162X 322 68 C to T 1/1 19 3659delC 322 111 del C 1/1 20 W1282X 206 154 G to A 1/1 21 N1303K 250 175 C to G 2/2 Total exon/intron Overall accuracy 17 93% *Samples were compared with their respective wild-type control (confirmed by sequencing). Login to comment

[hide] Rapid screening for 31 mutations and polymorphisms... Methods Mol Med. 2005;114:147-71. Dunbar SA, Jacobson JW
Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array.
Methods Mol Med. 2005;114:147-71., [PMID:16156102]

Abstract [show]
A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

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25 A 635-nm 10-mW red diode laser excites the two fluo- 148 Dunbar and Jacobson xMAP™ 149 Table 1 Recommended Core Mutation Panel for General Population Cystic Fibrosis (CF) Carrier Screening Standard mutation panel ΔF508 ΔI507 G542X G551D W1282X N1303K R553X 621+1G→T R117H 1717-1G→A A455E R560T R1162X G85E R334W R347P 711+1G→T 1898+1G→A 2184delA 1078delT 3849+10kbC→T 2789+5G→A 3659delC 1148T 3120+1G→A Reflex tests I506Va I507Va F508Ca 5T/7T/9Tb a Benign variants. Login to comment
94 Methods The methods described below include: (1) design of oligonucleotide capture probes, (2) design of PCR amplification primers and multiplexed PCR reactions, (3) preparation of the probe-conjugated microsphere sets, (4) verification 150 Dunbar and Jacobson xMAPTM Table 2 Oligonucleotide Capture Probesa Target Microsphere Probe sequence Modificationb Sequence 5' → 3' set Standard mutation panel 1c I507 & F508 5'-AmMC12 AACACCAAAGATGATATTTT 006 2B DI507 5'-AmMC12 ACACCAAAGATATTTTCTT 008 3B DF508 5'-AmMC12 AAACACCAATGATATTTTC 015 4B W1282 5'-AmMC12 CAACAGTGGAGGAAAGCC 012 5B W1282X 5'-AmMC12 CAACAGTGAAGGAAAGCC 020 6 1717-1G 5'-AmMC12 TTGGTAATAGGACATCTCCA 017 7 1717-1GÆA 5'-AmMC12 TTGGTAATAAGACATCTCCA 019 8B G542 5'-AmMC12 TATAGTTCTTGGAGAAGGTGGA 026 9B G542X 5'-AmMC12 TATAGTTCTTTGAGAAGGTGGA 028 10C G551 & R553 5'-AmMC12 AGTGGAGGTCAACGAGCAA 038 11B G551D 5'-AmMC12 GTGGAGATCAACGAGCAA 030 12C R553X 5'-AmMC12 GTGGAGGTCAATGAGCAA 032 13 R560 5'-AmMC12 CTTTAGCAAGGTGAATAACT 035 14 R560T 5'-AmMC12 CTTTAGCAACGTGAATAACT 039 15 R117 5'-AmMC12 AGGAGGAACGCTCTATCGCG 042 16 R117H 5'-AmMC12 AGGAGGAACACTCTATCGCG 025 17B I148 5'-AmMC12 CTTCATCACATTGGAATGCAGA 034 18B I148T 5'-AmMC12 CTTCATCACACTGGAATGCAGA 045 19C 621+1G 5'-AmMC12 TTTATAAGAAGGTAATACTTCCT 046 20E 621+1G→T 5'-AmMC12 ATTTATAAGAAGTTAATACTTCCTT 048 21 N1303 5'-AmMC12 GGGATCCAAGTTTTTTCTAA 051 22 N1303K 5'-AmMC12 GGGATCCAACTTTTTTCTAA 052 23B 1078T 5'-AmMC12 CACCACAAAGAACCCTGA 054 24C 1078delT 5'-AmMC12 ACACCACAAGAACCCTGA 061 25 R334 5'-AmMC12 ATATTTTCCGGAGGATGATT 063 26 R334W 5'-AmMC12 ATATTTTCCAGAGGATGATT 064 27B R347 5'-AmMC12 ACCGCCATGCGCAGAACAA 067 28B R347P 5'-AmMC12 ACCGCCATGGGCAGAACAA 053 29C 711+1G 5'-AmMC12 ATTTGATGAAGTATGTACCTAT 059 30C 711+1G→T 5'-AmMC12 ATTTGATGAATTATGTACCTAT 071 31 G85 5'-AmMC12 TGTTCTATGGAATCTTTTTA 066 32B G85E 5'-AmMC12 ATGTTCTATGAAATCTTTTTA 073 33 3849+10kbC 5'-AmMC12 GTCTTACTCGCCATTTTAAT 077 34 3849+10kbC→T 5'-AmMC12 GTCTTACTCACCATTTTAAT 075 35 A455 5'-AmMC12 CCAGCAACCGCCAACAACTG 011 36D A455E 5'-AmMC12 TCCAGCAACCTCCAACAACTG 036 37 R1162 5'-AmMC12 TAAAGACTCGGCTCACAGAT 060 38 R1162X 5'-AmMC12 TAAAGACTCAGCTCACAGAT 068 39B 3659C 5'-AmMC12 TTGACTTGGTAGGTTTAC 022 40C 3659delC 5'-AmMC12 TTGACTTGTAGGTTTACC 079 41B 2789+5G 5'-AmMC12 TGGAAAGTGAGTATTCCATGTC 074 42D 2789+5G→A 5'-AmMC12 TTGGAAAGTGAATATTCCATGTC 014 43E 2184A 5'-AmMC12 GAAACAAAAAAACAATC 007 44E 2184delA 5'-AmMC12 AGAAACAAAAAACAATC 018 45B 1898+1G 5'-AmMC12 TATTTGAAAGGTATGTTCTTTG 013 (Continued) of microsphere coupling, (5) direct hybridization of biotinylated PCR amplification products to the multiplexed probe-coupled microsphere sets, and (6) results and data analysis. Login to comment
106 Table 3 Reverse Complementary Oligonucleotide Targetsa Target Target sequence Modification Sequence 5' → 3' Standard mutation panel C1b I507 & F508 5'-Biotin AAAATATCATCTTTGGTGTT C2 ΔI507 5'-Biotin AAAGAAAATATCTTTGGTGT C3 ΔF508 5'-Biotin AGAAAATATCATTGGTGTTT C4 W1282 5'-Biotin GGCTTTCCTCCACTGTTGC C5 W1282X 5'-Biotin GGCTTTCCTTCACTGTTGC C6 1717-1G 5'-Biotin TGGAGATGTCCTATTACCAA C7 1717-1G→A 5'-Biotin TGGAGATGTCTTATTACCAA C8 G542 5'-Biotin CCACCTTCTCCAAGAACTAT C9 G542X 5'-Biotin CCACCTTCTCAAAGAACTAT C10 G551 & R553 5'-Biotin CTTGCTCGTTGACCTCCACT C11 G551D 5'-Biotin CTTGCTCGTTGATCTCCACT C12 R553X 5'-Biotin CTTGCTCATTGACCTCCACT C13 R560 5'-Biotin AGTTATTCACCTTGATAAAG C14 R560T 5'-Biotin AGTTATTCACGTTGCTAAAG C15 R117 5'-Biotin CGCGATAGAGCGTTCCTCCT C16 R117H 5'-Biotin CGCGATAGAGTGTTCCTCCT C17 I148 5'-Biotin CTGCATTCCAATGTGATGAA C18 I148T 5'-Biotin CTGCATTCCAGTGTGATGAA C19 621+1G 5'-Biotin GGAAGTATTACCTTCTTATA C20 621+1G→T 5'-Biotin GGAAGTATTAACTTCTTATA C21 N1303 5'-Biotin TTAGAAAAAACTTGGATCCC C22 N1303K 5'-Biotin TTAGAAAAAAGTTGGATCCC C23 1078T 5'-Biotin CTCAGGGTTCTTTGTGGTGT C24 1078delT 5'-Biotin TCTCAGGGTTCTTGTGGTGT C25 R334 5'-Biotin AATCATCCTCCGGAAAATAT C26 R334W 5'-Biotin AATCATCCTCTGGAAAATAT C27 R347 5'-Biotin ATTGTTCTGCGCATGGCGGT C28 R347P 5'-Biotin ATTGTTCTGCCCATGGCGGT C29 711+1G 5'-Biotin TAGGTACATACTTCATCAAA C30 711+1G→T 5'-Biotin TAGGTACATAATTCATCAAA C31 G85 5'-Biotin TAAAAAGATTCCATAGAACA C32 G85E 5'-Biotin TAAAAAGATTTCATAGAACA C33 3849+10kbC 5'-Biotin ATTAAAATGGCGAGTAAGAC C34 3849+10kbC→T 5'-Biotin ATTAAAATGGTGAGTAAGAC C35 A455 5'-Biotin CAGTTGTTGGCGGTTGCTGG C36 A455E 5'-Biotin CAGTTGTTGGAGGTTGCTGG C37 R1162 5'-Biotin ATCTGTGAGCCGAGTCTTTA C38 R1162X 5'-Biotin ATCTGTGAGCTGAGTCTTTA (Continued) Rapid CF Screening by xMAPTM 153 Table 3 (Continued) Target Target sequence Modification Sequence 5' → 3' C39 3659C 5'-Biotin GGTAAACCTACCAAGTCAAC C40 3659delC 5'-Biotin AGGTAAACCTACAAGTCAAC C41 2789+5G 5'-Biotin ACATGGAATACTCACTTTCC C42 2789+5G→A 5'-Biotin ACATGGAATATTCACTTTCC C43 2184A 5'-Biotin AAGATTGTTTTTTTGTTTCT C44 2184delA 5'-Biotin AAGATTGTTTTTTGTTTCTG C45 1898+1G 5'-Biotin AAAGAACATACCTTTCAAAT C46 1898+1G→A 5'-Biotin AAAGAACATATCTTTCAAAT C47 3120+1G 5'-Biotin TTTTTACATACCTGGATGAA C48 3120+1G→A 5'-Biotin TTTTTACATATCTGGATGAA Reflex panel CR2 I506V 5'-Biotin GAAAATGTCATCTTTGGTGT CR3 I507V 5'-Biotin GAAAATATCGTCTTTGGTGT CR4 F508C 5'-Biotin AAAATATCATCTGTGGTGTT CR5 5T 5'-Biotin TCCCTGTTAAAAACACACAC CR6 7T 5'-Biotin CCCTGTTAAAAAAACACACA CR7 9T 5'-Biotin CCTGTTAAAAAAAAACACAC a The position and sequence of the mutation or variation is indicated in bold type. b Target C1 (I507 & F508) is also used in the reflex panel. Login to comment
114 Using a small target DNA (approx 100-300 bp) minimizes the potential for steric hindrance to affect the xMAPTM Table 4 PCR Amplification Primers Size CFTR target Mutation(s) Primer 5' Modification Sequence 5' → 3' (bp) Exon 10 ΔI507, ΔF508, BE10U 5'-Biotin TTCTGTTCTCAGTTTTCCTGG 107 I506V, I507V, E10D None TTGGCATGCTTTGATGACG F508C Exon 20 W1282X E20U None TTGAGACTACTGAACACTGAAGG 126 BE20D 5'-Biotin TTCTGGCTAAGTCCTTTTGC Intron 10 1717-1G→A E11U None TCAGATTGAGCATACTAAAAGTGAC 89 BE11D2 5'-Biotin GAACTATATTGTCTTTCTCTGCAAAC Exon 11 G542X, G551D, E11U2 None AAGTTTGCAGAGAAAGACAATATAG 135 R553X, R560T BE11D 5'-Biotin GAATGACATTTACAGCAAATGC Exon 4 R117H E4U None TTTGTAGGAAGTCACCAAAGC 145 BE4D2 5'-Biotin GAGCAGTGTCCTCACAATAAAGAG Exon 4/intron 4 I148T, E4U2 None CTTCTCTTTATTGTGAGGACACTGC 169 621+1G→T BE4D 5'-Biotin ATGACATTAAAACATGTACGATACAG Exon 21 N1303K BE21U 5'-Biotin TGCTATAGAAAGTATTTATTTTTTCTGG 106 E21D None AGCCTTACCTCATCTGCAAC Exon 7 1078delT, BE7U 5'-Biotin GAACAGAACTGAAACTGACTCG 199 R334W, R347P E7D3 None CAGGGAAATTGCCGAGTG Intron 5 711+1G→T I5U None CAACTTGTTAGTCTCCTTTCC 99 BI5D2 5'-Biotin AGTTGTATAATTTATAACAATAGTGC Exon 3 G85E E3U None CTGGCTTCAAAGAAAAATCC 117 BE3D2 5'-Biotin TGAATGTACAAATGAGATCCTTACC Chromosome 7 3849+10kbC→T BC7U 5'-Biotin GACTTGTCATCTTGATTTCTGG 148 C7D None TTTGGTGCTAGCTGTAATTGC Exon 9 A455E BE9U 5'-Biotin TCACTTCTTGGTACTCCTGTCC 105 E9D None CAAAAGAACTACCTTGCCTGC Exon 19-I R1162X BE19U 5'-Biotin ATTGTGAAATTGTCTGCCATTC 167 E19Da None CAATAATCATAACTTTCGAGAGTTG Exon 19-II 3659delC BE19U2 5'-Biotin TTTAAGTTCATTGACATGCCAAC 91 E19Da None CAATAATCATAACTTTCGAGAGTTG Intron 14B 2789+5G→A I14BU None GTGTCTTGTTCCATTCCAGG 147 BI14BD 5'-Biotin TGGATTACAATACATACAAACATAGTGG Exon 13 2184delA E13U None AGATGCTCCTGTCTCCTGG 126 BE13D 5'-Biotin TGCACAATGGAAAATTTTCGTATAG Intron 12 1898+1G→A I12U None TTAGACTCTCCTTTTGGATACC 110 BI12D 5'-Biotin GTCTTTCTTTTATTTTAGCATGAGC Intron 16 3120+1G→A I16U None ATGACCTTCTGCCTCTTACC 118 BI16D 5'-Biotin ATGAAAACAAAATCACATTTGC Intron 8 5T/7T/9T I8U None TAATGGATCATGGGCCATGTGC 212 BI8D 5'-Biotin ACTGAAGAAGAGGCTGTCATCACC CFTR, cystic fibrosis transmembrane conductance regulator gene. Login to comment
118 Table 5 Genomic DNA Samples CFTR genotype Sourcea Normal/normal Sigma, D6537 ΔF508/normal Patient sample ΔF508/ΔF508 Coriell Cell Repositories, NA04540 ΔI507/normal Coriell Cell Repositories, NA11277 W1282/normal Coriell Cell Repositories, NA11723 1717-1G→A/normal Coriell Cell Repositories, NA12444 G542X/G542X Coriell Cell Repositories, NA11496B G542X/normal Coriell Cell Repositories, NA11497B ΔF508/G551D Coriell Cell Repositories, NA11274 ΔF508/R553X Coriell Cell Repositories, NA07469 G551D/R553X Coriell Cell Repositories, NA11761 ΔF508/R560T Coriell Cell Repositories, NA11284 ΔF508/R117H Coriell Cell Repositories, NA13591 I148T/normal Patient sample ΔF508/621+1G→T Coriell Cell Repositories, NA11281 N1303K/G1349D Coriell Cell Repositories, NA11472A ΔF508/1078delT Patient sample R334W/? Login to comment

[hide] Microsphere bead arrays and sequence validation of... J Mol Diagn. 2004 Nov;6(4):348-55. Hadd AG, Laosinchai-Wolf W, Novak CR, Badgett MR, Isgur LA, Goldrick M, Walkerpeach CR
Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms.
J Mol Diagn. 2004 Nov;6(4):348-55., [PMID:15507674]

Abstract [show]
The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.

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197 Intron 8 Genotype by Coriell Number, Characterized CF Mutation and Allele Fraction for 5/7/9T Intron 8 genotype Coriell sample Characterized mutation Allele fraction by probe 5T 7T 9T 7T/7T NA09947 Normal 0.04 0.93 0.03 NA11277 ⌬I507/normal 0.06 0.90 0.04 NA11761 G551D/R553X 0.06 0.92 0.02 NA11859 2789ϩ5GϾA/2789ϩ5GϾA 0.02 0.96 0.02 NA11860 3849ϩ10kbCϾT/3849ϩ10kbCϾT 0.03 0.94 0.03 NA12444 1717-1GϾT/normal 0.06 0.87 0.07 NA12585 R1162X/normal 0.07 0.86 0.08 NA12785 R347P/G551D 0.04 0.92 0.05 NA12960 R334W/normal 0.06 0.92 0.02 NA12961 V520F/normal 0.06 0.89 0.05 NA13033 F508C/normal 0.03 0.93 0.04 9T/9T NA01531 ⌬F508/⌬F508 0.14 0.04 0.82 NA11281 621ϩ1GϾT/⌬F508 0.14 0.04 0.82 NA11283 A455E/⌬F508 0.13 0.05 0.82 NA11290 A455E/621ϩ1GϾT 0.12 0.01 0.87 NA11496 G542X/G542X 0.14 0.05 0.81 5T/7T NA11723 W1282X/normal 0.53 0.44 0.03 NA13032 I506V/normal 0.58 0.39 0.03 5T/9T NA11279 129GϾC/⌬F508 0.51 0.00 0.49 NA13591 R117H/⌬F508 0.52 0.00 0.48 7T/9T NA07441 3120ϩ1GϾA/621ϩ1GϾA 0.08 0.41 0.51 NA07552 R553X/⌬F508 0.09 0.36 0.55 NA07830 556dA/⌬F508 0.11 0.37 0.52 NA11275 3659dC/⌬F508 0.10 0.37 0.53 NA11278 Q493X/⌬F508 0.09 0.38 0.53 NA11280 711ϩ1GϾT/621ϩ1GϾA 0.09 0.37 0.54 NA11282 G85E/621ϩ1GϾA 0.07 0.39 0.53 NA11284 R560T/⌬F508 0.08 0.39 0.52 NA11472 N1303K/G1349D 0.08 0.39 0.54 Figure 3. Login to comment

[hide] Pancreatitis in hispanic patients with cystic fibr... Clin Gastroenterol Hepatol. 2004 Jun;2(6):504-9. Maisonneuve P, Campbell P 3rd, Durie P, Lowenfels AB
Pancreatitis in hispanic patients with cystic fibrosis carrying the R334W mutation.
Clin Gastroenterol Hepatol. 2004 Jun;2(6):504-9., [PMID:15181620]

Abstract [show]
BACKGROUND & AIMS: Cystic fibrosis (CF) results from abnormal production of sticky mucus, which obstructs many organs. In most cases, the pancreas is severely compromised, but 10%-15% of patients with CF have pancreas sufficiency (PS) and are subject to develop pancreatitis. The aim of this study is to determine which specific genotypes lead to the development of pancreatitis in patients with CF. METHODS: We used prospective data collected by the Cystic Fibrosis Foundation and performed a nested case-control study with all patients who reported at least 1 episode of pancreatitis constituting the cases. We used logistic regression to assess the association between pancreatitis and genotype and the Kaplan-Meier method to estimate the cumulative incidence of pancreatitis for selected genotypes. RESULTS: Three hundred sixty-four of 17,871 genotyped patients with CF (2.0%) reported at least 1 episode of pancreatitis. Only 0.9% of 12,997 patients with genotypes generally associated with pancreas insufficiency reported pancreatitis against 11.9% of 868 patients carrying at least 1 mild CF mutation generally associated with PS. The greatest rate of pancreatitis (19.0%) was observed for patients carrying an R334W mutation: 48% of these 79 patients were Hispanic and 13 patients were living in Puerto Rico. CONCLUSIONS: Of all patients with CF, those carrying an R334W mutation have the greatest risk for developing pancreatitis. This mutation is found mostly in Hispanic patients with CF living in Puerto Rico. There are no current data to determine whether asymptomatic carriers of the R334W mutation are at greater risk for developing pancreatitis or whether this mutation is frequent in Hispanics with idiopathic pancreatitis.

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26 Of Ͼ1000 identified mutations in the CFTR gene, only 25 proven disease-causing mutant alleles (⌬F508, G551D, G542X, R553X, W1282X, R347P, NI303K, R560T, ⌬I507, 1717-1GϾA, A455E, 3120ϩ1GϾA, 621ϩ1GϾT, R117H, 711ϩ1GϾT, R1162X, 3849ϩ10kbCϾT, 2789ϩ5GϾT, R334W, G85E, 1078delT, 1898ϩ1GϾT, 2184delA, 3659delC, and I148T) are recommended by the American College of Medical Genetics for routine diagnostic and carrier testing.16 Most of these are routinely recorded in the CFF registry, but rarer mutations can be recorded if identified by more comprehensive testing. Login to comment
28 Patients were classified according to their genotype: those carrying 2 severe mutations (⌬F508, ⌬I507, G542X, G551D, N1303K, R553X, R560T, R1162X, W1282X, 621ϩ1GϾT, 711ϩ1GϾT, 1717-1GϾA, 2184delA, and 3659delC), which generally are associated with pancreas insufficiency (PI); and those carrying at least 1 mild mutation (3849ϩ10kbCϾT, R117H, 2789ϩ5GϾA, R347P, R334W, and A455E), which are generally associated with PS. Login to comment
67 aPatients with pancreas insufficiency (PI) carrying 2 PI mutations (⌬F508, ⌬I507, G542X, G551D, N1303K, R553X, R560T, R1162X, W1282X, 621ϩ1GϾT, 711ϩ1GϾT, 1717-1GϾA, 2184delA, 3659delC). Login to comment

[hide] Genotype and phenotype correlations in patients wi... Gastroenterology. 2002 Dec;123(6):1857-64. Durno C, Corey M, Zielenski J, Tullis E, Tsui LC, Durie P
Genotype and phenotype correlations in patients with cystic fibrosis and pancreatitis.
Gastroenterology. 2002 Dec;123(6):1857-64., [PMID:12454843]

Abstract [show]
BACKGROUND & AIMS: Pancreatitis is known to occur in some patients with cystic fibrosis (CF), but the prevalence, natural history, and genotypic basis are unclear. We examined a well-defined cohort of patients with CF to answer these questions. METHODS: Patients with CF were identified from a computerized database (1966-1996). Chart audit identified all patients with CF and pancreatitis. RESULTS: Among 1075 patients with CF, 937 (87%) were pancreatic insufficient at diagnosis, 28 (3%) were pancreatic sufficient but developed pancreatic insufficiency after diagnosis, and 110 (10%) have remained pancreatic sufficient. No patients with pancreatic insufficiency developed pancreatitis. Nineteen patients (17.3%) with pancreatic sufficiency experienced one or more attacks of pancreatitis. The mean age at diagnosis of pancreatitis was 22.7 +/- 10.3 years (range, 10-35 years), and pancreatitis was recognized before the diagnosis of CF in 6 patients (32%). The diagnosis of CF in pancreatic-sufficient patients, with and without pancreatitis, was established at a significantly older age than in those with pancreatic insufficiency (P < 0.0001). Genotyped patients with pancreatic insufficiency carried 2 severe mutant alleles. All genotyped patients with pancreatic sufficiency and pancreatitis carried at least one mild mutation. No specific genotype was predictive of pancreatitis. CONCLUSIONS: Patients with CF with pancreatic sufficiency carry at least one mild mutant allele and are at a significant risk of developing pancreatitis. Symptoms of pancreatitis may precede the diagnosis of CF. Pancreatitis is associated with an otherwise mild CF phenotype.

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105 CFTR Genotypes Among CF Patients With PS With and Without Pancreatitis Two mutations (n) ⌬F508/R117H (9) ⌬F508/(5T) (6) ⌬F508/3272-26A 3 G (4) ⌬F508/R347H (2) ⌬F508/P574H (2) ⌬F508/875 ϩ 1G Ͼ C (2) ⌬F508/3849 ϩ 10kb C 3 T (1) ⌬F508/A455E (1) ⌬F508/D614G (1) ⌬F508/G85E (1) ⌬F508/R347P (1) ⌬F508/S1251N (1) ⌬F508/⌬F508a (1) ⌬F508/3120G Ͼ A (1) ⌬F508/G551Da (1) G542X/R117H (1) R560T/L206W (1) R117H/R117H (1) R31L/P67L (1) 1461ins4 (AGAT)/G85E (1) G551D/(5T) (1) R1066C/3849 ϩ 10kb C Ͼ T (1) G551D/3849 ϩ 10kb C Ͼ T (1) R334W/R334W (1) R334W/681delC (1) W1282X/3489 ϩ 10kb C Ͼ T (1) One mutation (n) ⌬F508/- (18) L1077P/- (1) W1282X/- (1) M1137V/- (1) G551D/- (1) R347H/- (1) Q30X1/- (1) G1244E/- (1) R117H/- (1) 621 ϩ 2G621 ϩ 1G 3 T/- (1) NOTE. Login to comment

[hide] Cystic fibrosis and related diseases of the pancre... Best Pract Res Clin Gastroenterol. 2002 Jun;16(3):511-26. Naruse S, Kitagawa M, Ishiguro H, Fujiki K, Hayakawa T
Cystic fibrosis and related diseases of the pancreas.
Best Pract Res Clin Gastroenterol. 2002 Jun;16(3):511-26., [PMID:12079272]

Abstract [show]
The discovery of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), brought about a new era in the study of this disease. Identification of the molecular target has yielded a flood of data that add to our understanding of the pathogenesis, diagnosis and treatment of CF. The CFTR protein is a cAMP-regulated Cl(-) channel with multiple functions in epithelial cells. In the exocrine pancreas the CFTR plays a key role in the apical Cl(-), HCO(3)(-), and water transport in duct cells. The severe loss of functions, caused by mutations of the CFTR gene, leads to pathological lesions of the pancreas. Over 1200 CFTR mutations and polymorphisms have been identified and their diversity may explain the high level of heterogeneity in the CF phenotype. Mutation analyses of the CFTR gene have revealed a spectrum of CFTR-related diseases that do not fit the classical CF picture but are associated with dysfunction of CFTR, such as chronic pancreatitis.

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62 is observed only when normal CFTR function is less than 1%.13 In general, patients with pancreatic insuciency are homozygous or compound heterozygous for two severe mutations (class I, II or III in Figure 3), such as DF508, DI507, Q493X, G542X, R553X, W1282X, 621 1G 4 T, 1717-1G 4 A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T, whereas the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H (class IV or V).5,20 EXOCRINE PANCREAS IN CYSTIC FIBROSIS Pathology of the pancreas in CF There is a spectrum of pancreatic abnormalities in CF irrespective of age.21,22 Pancreatic lesions may be absent in an individual case, but in long-standing CF the pancreas is small, hard and nodular with increased fat and multiple cysts; hence the name `cystic ®brosis of the pancreas'. Login to comment
64 is observed only when normal CFTR function is less than 1%.13 In general, patients with pancreatic insuQciency are homozygous or compound heterozygous for two severe mutations (class I, II or III in Figure 3), such as DF508, DI507, Q493X, G542X, R553X, W1282X, 621 W 1G 4 T, 1717-1G 4 A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T, whereas the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H (class IV or V).5,20 EXOCRINE PANCREAS IN CYSTIC FIBROSIS Pathology of the pancreas in CF There is a spectrum of pancreatic abnormalities in CF irrespective of age.21,22 Pancreatic lesions may be absent in an individual case, but in long-standing CF the pancreas is small, hard and nodular with increased fat and multiple cysts; hence the name `cystic &#ae;brosis of the pancreas'. Login to comment

[hide] CFTR mutations in three Latin American countries. Am J Med Genet. 2000 Apr 10;91(4):277-9. Restrepo CM, Pineda L, Rojas-Martinez A, Gutierrez CA, Morales A, Gomez Y, Villalobos MC, Borjas L, Delgado W, Myers A, Barrera-Saldana HA
CFTR mutations in three Latin American countries.
Am J Med Genet. 2000 Apr 10;91(4):277-9., [PMID:10766983]

Abstract [show]
We analyzed 192 cystic fibrosis (CF) alleles in three Latin American countries: Mexico, Colombia, and Venezuela. Mutation screening was performed by polymerase chain reaction (PCR) and a reverse dot blot detection kit that enables determination of 16 of the most common CF mutations worldwide. Mutations were detected in 47.9% of the screened CF alleles. The most prevalent CF allele was DeltaF508 (39. 6%). The remaining 16 non-DeltaF508 detectable mutations represented 8.3% of the CF alleles. Among them, the G542X, N1303K, and 3849+10kb C>T were the most common. Although the frequency of DeltaF508 described here is lower than that reported for Caucasian populations, including in Spain, it is remarkable that mutation prevalences found in this study resemble those observed in Spain. Two of these mutations, G542X and 3849+10kb C>T, that were relevant in this analysis, have a particularly high incidence in Spanish communities. The low frequency of DeltaF508 described here may be explained by the Amerindian, Caucasian, and Black admixture that occurred in Latin America after the discovery of the New World, and also by the probable occurrence of mutations contributed by the original natives, which were undetectable in this analysis.

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34 The isolated DNA from each patient was amplified by polymerase chain reaction (PCR) using a kit for reverse dot blot detection of 16 common CF mutations: ⌬F508, R553X, G542X, G551D, N1303K, W1282X, R117H, R334W, R347P, A455E, ⌬I507, 1717-1 G>A, R560T, 3849+10kb C>T, 621+1 G>T, S549N [Villalobos-Torres et al., 1997]. Login to comment

[hide] A comparison of fluorescent SSCP and denaturing HP... Hum Mutat. 2000;15(6):556-64. Ellis LA, Taylor CF, Taylor GR
A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning.
Hum Mutat. 2000;15(6):556-64., [PMID:10862085]

Abstract [show]
We examined 67 different mutations in 16 different amplicons in a comparison of mutation detection by fluorescent single strand conformation polymorphism (F-SSCP) and by denaturing HPLC (DHPLC). F-SSCP was used to analyze fluorescent amplicons with internal size standards and automated fragment analysis (GeneScan, PE Applied Biosystems, Foster City, CA). In DHPLC, unlabelled amplicons were analyzed by reverse phase HPLC with fragment detection by absorbance at 260nm. Both methods had high sensitivity (95-100%) and specificity (100%). Overall, F-SSCP with external temperature control was the more sensitive method, but DHPLC was particularly useful for the rapid analysis of novel fragments.

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81 MDE alone failed to detect R560T, S549R, and R709X under any conditions described. Login to comment
97 Comparison of F-SSCP and DHPLC Using a Panel of ABCC7 Mutations Gel condition Location Location 49:1 49:1 49:1 49:1 MDE MDE MDE Capillary DHPLC °C from 5' (bp) from 3' (bp) 15 20 25 35 20 25 35 35 N/A Exon 3 (320bp) E60X 128 192 + + + + + + + + - P67L 150 170 + + + - + + + - + R75X 173 147 + + + + + + + + + R75Q 174 146 + + + - + + + + + G85E 204 116 + + + - + + + + + L88S 213 107 + + + + + + + + + Exon 4 (400bp) 441delA 135 265 + + + + + + + + + D110H 154 246 + + + + + + - + + R117H/H 176 224 + + + + + + + + N/A R117R/H 176 224 + + + + + + + + + L137H 236 164 + + + + + + + + + I148T 261 139 + + + + + + + + + 621+1 (G>T) 309 91 + + + + + + + + + Exon 7 (360bp) R334W 180 180 + + + + + + + - + 1058delC 105 255 + + + + + + + + + 1078delT 125 235 + + + - + + + + + 1138insG 226 134 - + + - + + + + + 1154insTC 202 158 + + + + + + + + + 1161delC 209 151 + + + + + + + + + R347H 220 140 + + + + + + - + + R347P 220 140 + + + - + + + - + A349V 226 134 + + + + + + + + + W356X 248 112 + + + + + + + + + Exon 10 (365bp) M470V 143 222 + + + + + + + + + Q493X 212 153 + + + + + + - + - DelF508 255 110 + + + + + + + + - Del I507 253 112 + + + + + + + + + V520F 293 72 + + - + + - + - + Exon 11 (190bp) 1717-1 (G>A) 54 136 + + + - + + - + + G542X 94 96 + + + - + + - + + S549N 116 74 + + + + + + + + - S549R 117 73 + + + + - - - + + G551D 122 68 + - - - + + + - + R553X 127 63 + + + + + + + + + G551D/R553X + + + + + + + + + R560T 149 41 + + + - - - - - + R560K 149 41 + + + - + + + - + 1811+1 (G>C) 150 40 + + + + + + + + + Exon 12 (250bp) 1898+1(G>A) 167 83 + + + + + + - + + Exon 13a (290bp) C590W 87 203 + + - - + - - + + Exon 13b (405bp) 2184insA 148 257 + + + + + + + - + R709X 220 185 - + - - - - - - + V754M 453 52 + + + + + + + - - Exon 13c (345bp) V754M 65 280 + + + + + + - - + R785X 158 187 + + - - + + - - + Exon 19 (370bp) 3601-17 (T>C) 29 341 - + + - + + + - + R1162X 61 309 + + - - + - - + + 3659delC 105 265 - - - + + + + + + Y1182X 123 247 - + + - + + + - + Exon 20 (370bp) W1282X 186 184 + + + + + + + + + % detected 90 96 86 66 94 88 74 72 90 remainder were detected using DGGE. Login to comment
135 Effect of Amplicon Size on Mutation Detection Rate in ABCC7 Exon 11 SSCP slab gels SSCP 310 DHPLC 49:1, 20°C MDE, 20°C 35°C Exon 11 ABCC7 190bp 490bp 190bp 490bp 190bp 490bp 190bp 490bp 1717-1G>A - - + + + + + + G542X + + + + + - + + S549N + + + + + + - - S549R + + - + + + + - G551D - + + + - - + - R553X + + + + + + + - G551D/R553X + + + + + + + - R560T + - - + - - + + R560K + + + + - - + + 1811+1G>C + + + + + + + + Sensitivity 9/10 8/10 8/10 10/10 7/10 6/10 9/10 5/10 ammonium acetate as an ion-pairing agent has the unintended consequence of modifying the stability of GC and AT base pairs in a similar manner to the effects of tetra-alkyl ammonium salts, tetraethyl ammonium chloride and tetramenthyl ammonium chloride. Login to comment

[hide] A mutation in the cystic fibrosis transmembrane co... Hum Mol Genet. 1998 Apr;7(4):729-35. Mickle JE, Macek M Jr, Fulmer-Smentek SB, Egan MM, Schwiebert E, Guggino W, Moss R, Cutting GR
A mutation in the cystic fibrosis transmembrane conductance regulator gene associated with elevated sweat chloride concentrations in the absence of cystic fibrosis.
Hum Mol Genet. 1998 Apr;7(4):729-35., [PMID:9499426]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to cause cystic fibrosis (CF) and male infertility due to congenital bilateral absence of the vas deferens. We report the identification of a 6.8 kb deletion (del14a) and a nonsense mutation (S1455X) in the CFTR genes of a mother and her youngest daughter with isolated elevated sweat chloride concentrations. Detailed clinical evaluation of both individuals found no evidence of pulmonary or pancreatic disease characteristic of CF. A second child in this family with classic CF was homozygous for the del14a mutation, indicating that this mutation caused severe CFTR dysfunction. CFTR mRNA transcripts bearing the S1455X mutation were stable in vivo , implying that this allele encoded a truncated version of CFTR missing the last 26 amino acids. Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. When expressed in CF airway cells, this mutant generated cAMP-activated whole-cell chloride currents similar to wild-type CFTR. Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. These data indicate that mutations in CFTR can be associated with elevated sweat chloride concentrations in the absence of the CF phenotype, and suggest a previously unrecognized functional role in the sweat gland for the C-terminus of CFTR.

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151 Mutation analysis Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G→T, R334W, R349P, A455E, 1717-1G→A, ∆I507, ∆F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C→T, W1282X, N1303K) by reverse dot-blot hybridization (46). Login to comment
152 Mutation analysis Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G࢐T, R334W, R349P, A455E, 1717-1G࢐A, ࢞I507, ࢞F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C࢐T, W1282X, N1303K) by reverse dot-blot hybridization (46). Login to comment

[hide] Analysis of the CFTR gene in Turkish cystic fibros... Hum Genet. 1998 Feb;102(2):224-30. Onay T, Topaloglu O, Zielenski J, Gokgoz N, Kayserili H, Camcioglu Y, Cokugras H, Akcakaya N, Apak M, Tsui LC, Kirdar B
Analysis of the CFTR gene in Turkish cystic fibrosis patients: identification of three novel mutations (3172delAC, P1013L and M1028I).
Hum Genet. 1998 Feb;102(2):224-30., [PMID:9521595]

Abstract [show]
In order to determine the spectrum of cystic fibrosis (CF) mutations in the Turkish population, a complete coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene including exon-intron boundaries, on 122 unrelated CF chromosomes from 73 Turkish CF families was analysed by denaturing gradient gel electrophoresis and multiplex heteroduplex analysis on MDE gel matrix. In addition to 15 previously reported mutations and 12 polymorphisms, three novel mutations, namely 3172delAC, P1013L and M1028I, were detected. DeltaF508 was found to be present on 18.8% of CF chromosomes. The second most common mutation was 1677delTA, with a frequency of 7.3%, followed by G542X and 2183AA-->G mutations, with frequencies of 4.9%. These four most common mutations in Turkish CF population account for approximately 36% of mutations. This study could only detect 52.5% of disease-causing mutations in this population; 47.5% of CF alleles remain to be identified, reflecting the high molecular heterogeneity of the Turkish population.

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26 Other frequent mutations, namely G542X, R560T, S549N or S549I and G551D or R553X, were analysed by the multiplex ARMS method combined with restriction enzyme analysis (Dean et al. 1990; Cutting et al. 1990; Ferrie et al. 1992). Login to comment

[hide] Modeling of nucleotide binding domains of ABC tran... J Bioenerg Biomembr. 1997 Oct;29(5):503-24. Bianchet MA, Ko YH, Amzel LM, Pedersen PL
Modeling of nucleotide binding domains of ABC transporter proteins based on a F1-ATPase/recA topology: structural model of the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR).
J Bioenerg Biomembr. 1997 Oct;29(5):503-24., [PMID:9511935]

Abstract [show]
Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) using the two similar domains of mitochondrial F1-ATPase. The models obtained, provide useful insights into the putative functions of these domains and their possible interaction as well as a rationale for the basis of Cystic Fibrosis causing mutations. First, the two nucleotide binding domains (folds) of CFTR are each predicted to span a 240-250 amino acid sequence rather than the 150-160 amino acid sequence originally proposed. Second, the first nucleotide binding fold, is predicted to catalyze significant rates of ATP hydrolysis as a catalytic base (E504) resides near the y phosphate of ATP. This prediction has been verified experimentally [Ko, Y.H., and Pedersen, P.L. (1995) J. Biol. Chem. 268, 24330-24338], providing support for the model. In contrast, the second nucleotide binding fold is predicted at best to be a weak ATPase as the glutamic acid residue is replaced with a glutamine. Third, F508, which when deleted causes approximately 70% of all cases of cystic fibrosis, is predicted to lie in a cleft near the nucleotide binding pocket. All other disease causing mutations within the two nucleotide binding domains of CFTR either reside near the Walker A and Walker B consensus motifs in the heart of the nucleotide binding pocket, or in the C motif which lies outside but near the nucleotide binding pocket. Finally, the two nucleotide binding domains of CFTR are predicted to interact, and in one of the two predicted orientations, F508 resides near the interface. This is the first report where both nucleotide binding domains of an ABC transporter and their putative domain-domain interactions have been modeled in three dimensions. The methods and the template used in this work can be used to analyze the structures and function of the nucleotide binding domains of all other members of the ABC transporter super-family.

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360 The CFTR NBD1 model that results (Fig. 6) gathers the disease causing mutations in three different clusters: (1) mutations affecting the nucleotide binding pocket and the putative general base: A455E, G458V, E504Q AI507 AF508 P574H; (2) mutations in motif C which are probably related to an interaction with region D: S549[R,N,I] G551[S,D], R553Q; and (3) mutations within or near motif B, L558S, A559T, R560T, Y563N and mutations S492F and G480C. Login to comment

[hide] Newborn screening for cystic fibrosis in Wisconsin... Pediatrics. 1997 Jun;99(6):819-24. Gregg RG, Simantel A, Farrell PM, Koscik R, Kosorok MR, Laxova A, Laessig R, Hoffman G, Hassemer D, Mischler EH, Splaingard M
Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods.
Pediatrics. 1997 Jun;99(6):819-24., [PMID:9164776]

Abstract [show]
OBJECTIVES: To evaluate neonatal screening for cystic fibrosis (CF), including study of the screening procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screening method with that of a two-tier method using IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DeltaF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol. METHODS: From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respectively, were screened for CF. For the IRT protocol, neonates with an IRT >/=180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT >/=110 ng/mL were tested for the presence of the DeltaF508 CFTR allele, and if the DNA test revealed one or two DeltaF508 alleles, a sweat test was obtained. RESULTS: Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statistically significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA population revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF infants were homozygous for DeltaF508 and 40% had one DeltaF508 allele. Adding analyses for 10 more CFTR mutations has only a small effect on the sensitivity but is likely to add significantly to the cost of screening. CONCLUSIONS: Advantages of the IRT/DNA protocol over IRT analysis include improved positive predictive value, reduction of false-positive infants, and more rapid diagnosis with elimination of recall specimens.

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52 Polyacrylamide gel electrophoresis of PCR-amplified DNA served to identify the ⌬F508 mutation.14 Mutations S549N, R553X, and G551D were screened by PCR amplification of exon 11, followed by restriction enzyme digests that are diagnostic of each mutation. Login to comment
57 Mutations, G542X, W1282X, R117H, R553X, N1303K, 1717-1G3A, R560T, and 621ϩ1G3T were analyzed by the ARMS procedure using published primers and conditions.18 A total of 360 patients were studied by multimutation analysis (80% of the Wisconsin CF population). Login to comment
123 Tested) Frequency (%) Theoretical Cumulative Detection† (%) Patients Missed in One Year‡ ⌬F508§ 513/720 71.25 91.74 1.32 G542X 17/162 3.02 93.38 1.05 W1282X 7/102 1.97 94.36 0.90 R117H 6/101 1.71 95.14 0.77 R553X 11/197 1.61 95.82 0.66 G551D 9/195 1.33 96.35 0.58 N1303K 3/100 0.86 96.67 0.53 1717 - 1G 3 A 2/99 0.58 96.88 0.49 R560T 2/106 0.54 97.06 0.47 621 ϩ 1G 3 T 3/163 0.53 97.25 0.44 S549N 0/196 0.0 97.25 0.44 * Chromosomes were analyzed on blood or cheek cell specimens obtained from 360 patients (80% of the total Wisconsin CF population), all of whom had a positive sweat test. Login to comment
152 DNA Analysis of Genotyped CF Patients in the US* n Percent ⌬F508 12701 67.7 G542X 403 2.2 G551D 357 1.9 W1282X 240 1.3 N1303K 223 1.2 R553X 157 0.8 3849 ϩ 10kbC 3 T 102 0.5 621 ϩ 1G 3 T 147 0.8 1717 - 1G 3 A 101 0.5 R117H 101 0.5 R334W 36 0.2 ⌬I507 42 0.2 R347P 37 0.2 R560T 23 0.1 R1162X 44 0.2 2789 ϩ 5G 3 A 25 0.1 A455E 16 0.1 3120 ϩ IG 3 A 14 0.0 S549N 12 0.0 711 ϩ IG 3 T 9 0.0 Other 178 0.9 Unidentified 3814 20.3 Total 18782 99.7† Patient Genotypes Allele 1/Allele 2 n % of Genotype ⌬F508/⌬F508 4573 48.7 ⌬F508/Known 1511 16.1 ⌬F508/Unknown 2044 21.8 Known/unknown 310 3.3 Known/known 223 2.4 Unknown/unknown 730 7.8 Total 9391 100.0 *Data from Cystic Fibrosis Registry, 1995; Annual Report. Login to comment
118 Tested) Frequency (%) Theoretical Cumulative Detectionߤ (%) Patients Missed in One Yearߥ DF508&#a7; 513/720 71.25 91.74 1.32 G542X 17/162 3.02 93.38 1.05 W1282X 7/102 1.97 94.36 0.90 R117H 6/101 1.71 95.14 0.77 R553X 11/197 1.61 95.82 0.66 G551D 9/195 1.33 96.35 0.58 N1303K 3/100 0.86 96.67 0.53 1717 2 1G 3 A 2/99 0.58 96.88 0.49 R560T 2/106 0.54 97.06 0.47 621 1 1G 3 T 3/163 0.53 97.25 0.44 S549N 0/196 0.0 97.25 0.44 * Chromosomes were analyzed on blood or cheek cell specimens obtained from 360 patients (80% of the total Wisconsin CF population), all of whom had a positive sweat test. Login to comment
147 DNA Analysis of Genotyped CF Patients in the US* n Percent DF508 12701 67.7 G542X 403 2.2 G551D 357 1.9 W1282X 240 1.3 N1303K 223 1.2 R553X 157 0.8 3849 1 10kbC 3 T 102 0.5 621 1 1G 3 T 147 0.8 1717 2 1G 3 A 101 0.5 R117H 101 0.5 R334W 36 0.2 DI507 42 0.2 R347P 37 0.2 R560T 23 0.1 R1162X 44 0.2 2789 1 5G 3 A 25 0.1 A455E 16 0.1 3120 1 IG 3 A 14 0.0 S549N 12 0.0 711 1 IG 3 T 9 0.0 Other 178 0.9 Unidentified 3814 20.3 Total 18782 99.7ߤ Patient Genotypes Allele 1/Allele 2 n % of Genotype DF508/DF508 4573 48.7 DF508/Known 1511 16.1 DF508/Unknown 2044 21.8 Known/unknown 310 3.3 Known/known 223 2.4 Unknown/unknown 730 7.8 Total 9391 100.0 *Data from Cystic Fibrosis Registry, 1995; Annual Report. Login to comment

[hide] Identification of common cystic fibrosis mutations... Am J Hum Genet. 1997 May;60(5):1122-7. Macek M Jr, Mackova A, Hamosh A, Hilman BC, Selden RF, Lucotte G, Friedman KJ, Knowles MR, Rosenstein BJ, Cutting GR
Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.
Am J Hum Genet. 1997 May;60(5):1122-7., [PMID:9150159]

Abstract [show]
Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans.

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39 Mutation Analysis All patients were screened for the AF508 mutation and 15 common Caucasian CF mutations using a reverse dot strip hybridization system (Kawasaki et al. 1993) (R117H, 621+1G--T, R334W, R347P, A455E, A1507, 1717-1G-+A, G542X, S549N, GSS1D, R553X, R560T, 3849+10kbC-+T, W1282X, and N1303K) (Welsh et al. 1995) and a deep intron 11 splice-site mutation, 1811+1.6kbA-+G (Chillon et al. 1995). Login to comment
86 The most common muta- Table 2 Distribution of CF Mutations in African-American and U.S.-Caucasian CF Patients African-American U.S. Caucasiana Mutation (n= 148) % (n = 8,714) % Caucasian mutations: AF508 71 48 5,769 66.2 R117H 0 0 47 .5 621+1 G--T 0 0 68 .8 R334W 1 .7 7 .1 R347P 0 0 24 .3 A455E 0 0 5 .1 AI507 1 .7 10 .1 1717-1 G-IA 1 .7 39 .5 G542X 1 .7 204 2.3 S549N 1 .7 4 .1 GS51D 1 .7 173 2.0 R553X (Caucasian)b 0 0 87 1.0 R560T 0 0 16 .2 3849+10kb C-T 0 0 51 .6 W1282X 0 0 235 2.7 N1303K 0 0 116 1.3 Subtotal 77 52 6,855 78.7 African-American mutations: 405+3 A-C 2 1.4 ... ... 444delA 1 .7 ... ... G480C 2 1.4 ... ... R553X (African)b 3 2.0 ... ... A559T 3 2.0 ... ... 2307insA 3 2.0 ... ... 3120+1 GC-A 18 12.2 ... ... S1255X 2 1.4 ... ... Subtotal 34 23 ... ... Total 111 75.0 6,855 78.7 NOTE.-Percentages are rounded. Login to comment
40 Mutation Analysis All patients were screened for the AF508 mutation and 15 common Caucasian CF mutations using a reverse dot strip hybridization system (Kawasaki et al. 1993) (R117H, 621+1G--T, R334W, R347P, A455E, A1507, 1717-1G-+A, G542X, S549N, GSS1D, R553X, R560T, 3849+10kbC-+T, W1282X, and N1303K) (Welsh et al. 1995) and a deep intron 11 splice-site mutation, 1811+1.6kbA-+G (Chillon et al. 1995). Login to comment
87 The most common muta- Table 2 Distribution of CF Mutations in African-American and U.S.-Caucasian CF Patients African-American U.S. Caucasiana Mutation (n= 148) % (n = 8,714) % Caucasian mutations: AF508 71 48 5,769 66.2 R117H 0 0 47 .5 621+1 G--T 0 0 68 .8 R334W 1 .7 7 .1 R347P 0 0 24 .3 A455E 0 0 5 .1 AI507 1 .7 10 .1 1717-1 G-IA 1 .7 39 .5 G542X 1 .7 204 2.3 S549N 1 .7 4 .1 GS51D 1 .7 173 2.0 R553X (Caucasian)b 0 0 87 1.0 R560T 0 0 16 .2 3849+10kb C-T 0 0 51 .6 W1282X 0 0 235 2.7 N1303K 0 0 116 1.3 Subtotal 77 52 6,855 78.7 African-American mutations: 405+3 A-C 2 1.4 ... ... 444delA 1 .7 ... ... G480C 2 1.4 ... ... R553X (African)b 3 2.0 ... ... A559T 3 2.0 ... ... 2307insA 3 2.0 ... ... 3120+1 GC-A 18 12.2 ... ... S1255X 2 1.4 ... ... Subtotal 34 23 ... ... Total 111 75.0 6,855 78.7 NOTE.-Percentages are rounded. Login to comment

[hide] Analysis of 16 cystic fibrosis mutations in Mexica... Am J Med Genet. 1997 Apr 14;69(4):380-2. Villalobos-Torres C, Rojas-Martinez A, Villareal-Castellanos E, Cantu JM, Sanchez-Anzaldo FJ, Saiki RK, Barrera-Saldana HA
Analysis of 16 cystic fibrosis mutations in Mexican patients.
Am J Med Genet. 1997 Apr 14;69(4):380-2., [PMID:9098486]

Abstract [show]
We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

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60 Mutation Frequency Data and Geographic Distribution of the Mutations Found in 80 Chromosomes From Mexican CF Patients Mutation Northeast n ‫ס‬ 54 Central n ‫ס‬ 16 Western n ‫ס‬ 10 Total n ‫ס‬ 80 CFGAC [1994] (%)n (%) n (%) n (%) n (%) ⌬F508 27 (50) 2 (12.5) 7 (70) 36 (45) 66 G542X 2 (3.7) 2 (12.5) 0 4 (5) 2.4 3849 + 10 kb C→T 1 (1.9) 0 1 (10) 2 (2.5) 0.2 N1303K 0 1 (6.25) 0 1 (1.25) 1.3 S549N 0 1 (6.25) 0 1 (1.25) 0.1 621 + 1 G→T 0 0 1 (10) 1 (1.25) 0.7 Othera 24 (44.4) 10 (62.5) 1 (10) 35 (43.7) Detected 30 (55.6) 6 (37.5) 9 (90) 45 (56.3) a Different from W1282X, R117H, R334W, R347P, A455E, ⌬I507, 1717 - 1 G→T, G551D, R553X, and R560T. Login to comment

[hide] Evidence for safety and efficacy of DOTAP cationic... Gene Ther. 1997 Mar;4(3):210-8. Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, Carothers AD, Wallace WA, Moralee S, Hoenes C, Kallmeyer G, Michaelis U, Naujoks K, Ho LP, Samways JM, Imrie M, Greening AP, Innes JA
Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.
Gene Ther. 1997 Mar;4(3):210-8., [PMID:9135734]

Abstract [show]
In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR:2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA-liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.

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32 Table 1 Anthropometric data Group Patient Gender Age Genotype FEV1 No (% predicted) Placebo 03 Female 33 ⌬F508/R117H 57 06 Male 27 ⌬F508/⌬F508 55 08 Female 29 ⌬F508/A455E 97 11 Female 42 ⌬F508/Q493X 24 15 Male 30 ⌬F508/R560T 20 16 Female 20 ⌬F508/⌬F508 70 18 Female 27 ⌬F508/⌬F508 21 21 Female 20 ⌬F508/⌬F508 20 Mean (s.d.) 6F, 2M 28.5 (7.1) 51.9 (28.1) Treated 01 Male 31 ⌬F508/G551D 45 05 Female 30 ⌬F508/⌬F508 91 09 Male 32 G551D/G551D 37 10 Female 29 ⌬F508/⌬F508 63 13 Male 16 ⌬F508/⌬F508 55 14 Female 37 ⌬F508/G551D 66 19 Male 23 ⌬F508/W1282X 37 23 Female 21 ⌬F508/G551D 53 Mean (s.d.) 4F, 4M 27.4 (6.8) 55.9 (17.8) and illustrative results shown in Figure 3. Login to comment

[hide] Sensitivity of the denaturing gradient gel electro... Hum Mutat. 1997;9(2):136-47. Macek M Jr, Mercier B, Mackova A, Miller PW, Hamosh A, Ferec C, Cutting GR
Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene.
Hum Mutat. 1997;9(2):136-47., [PMID:9067754]

Abstract [show]
More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.

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42 621 + 1 GÃT, R334W, R347P, A455E, aI507, aF508, 1717-1 GÃA, G542X, S549N, G551D, R553X, R560T, 3849 + 10kb CÃT, W1282X, and N1303K) was performed using the rapid multiplex reverse dot hybridization system, under conditions provided by Roche Molecular Systems (Alameda, CA) (Kawasaki et al., 1993; Welsh et al., 1995). Login to comment

[hide] Heterogeneity of phenotype in two cystic fibrosis ... J Med Genet. 1996 Aug;33(8):711-3. Parad RB
Heterogeneity of phenotype in two cystic fibrosis patients homozygous for the CFTR exon 11 mutation G551D.
J Med Genet. 1996 Aug;33(8):711-3., [PMID:8863168]

Abstract [show]
In the heterozygous state, the cystic fibrosis transmembrane conductance regulator (CFTR) exon 11 mutation G551D has been described as "severe," causing pancreatic insufficiency. Two cystic fibrosis (CF) patients homozygous for this mutation showed a mild rather than severe pancreatic phenotype and a variable pulmonary phenotype.

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10 R B Parad Received 27 December 1995 Revised version accepted for publication 15 March 1996 Methods Cheekbrush DNA for CFTR mutation analysis was collected and prepared according to Richards et al.1 CFTR mutation analysis was performed for 12 mutations (AF508, G551D, G542X, 621 + 1G->T, AI507, 1717-1G-4A, R117H, N1303K, W1282X, R560T, R553X, 3849 + 1Okb C-+T). Login to comment

[hide] Cystic fibrosis heterozygote screening in 5,161 pr... Am J Hum Genet. 1996 Apr;58(4):823-35. Witt DR, Schaefer C, Hallam P, Wi S, Blumberg B, Fishbach A, Holtzman J, Kornfeld S, Lee R, Nemzer L, Palmer R
Cystic fibrosis heterozygote screening in 5,161 pregnant women.
Am J Hum Genet. 1996 Apr;58(4):823-35., [PMID:8644747]

Abstract [show]
A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.

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69 Lab group A was screened for the 6 most common mutations (F508, G542X, G551D, R553X, W1282X, and N1303K); lab group B was screened for 12 mutations, including the 6 most common and an additional 6 less common alleles (R117H, 621+1, I507, 1717G-A, R560T, and S549N). Login to comment
142 Table 7 CFTR Mutations in Screened Women NUMBER (%) wITH MUTATIONa GROUP ETHNiciTY F508 G542X GS5lD R553X W1282X N1303K R117H 621+1 1507 1717G-A R560T S549N TOTAL A Caucasian 26 (81) 5 (16) 1 (3) NA NA NA NA NA NA 32 Hispanic 2 (100) NA NA NA NA NA NA 2 B Caucasian 63b (65) 4 (4) 2 (3) 1 (1) 2 (2) 4 (4) 16b (16) 4 (4) 1 (1) 97b Hispanic 7 (88) 1 (12) 8 Caucasian/ Hispanic 2 (50) 1 (25) 1 (25) 4 'NA = not applicable. Login to comment
150 Seven heterozygotes were identified: 4 F508s and 1 each of R117H, 1717G-A, and R560T. Login to comment

[hide] Survey of cystic fibrosis transmembrane conductanc... Dig Dis Sci. 1996 Mar;41(3):540-2. McGill JM, Williams DM, Hunt CM
Survey of cystic fibrosis transmembrane conductance regulator genotypes in primary sclerosing cholangitis.
Dig Dis Sci. 1996 Mar;41(3):540-2., [PMID:8617131]

Abstract [show]
A variety of cholestatic liver diseases appear to primarily affect the biliary epithelium, including cystic fibrosis (CF). CF results from a defect in the chloride channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of CF patients have a genomic deletion in deltaF508, other mutations of CFTR may result in less severe clinical presentations and outcomes. Recently, CFTR has been shown to be involved in secretin-stimulated choleresis in intrahepatic bile duct epithelial cells. Cholestasis in cystic fibrosis appears to result from defective chloride transport across the biliary epithelium and is the only cholestatic disease of bile ducts for which a cellular defect has been identified. Primary sclerosing cholangitis (PSC) is a cholestatic disease with histological and cholangiographic features similar to CF. The purpose of this pilot study was to explore whether there is an increased prevalence of CFTR mutations. Two patients exhibited mutations in one allele, yielding a carrier rate of 10.6%, not statistically different from the general U.S. population carrier rate of 4%.

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33 In total, 32 mutations were evaluated, which represent 90% of the most common mutations (t4): AF508 G542X G551D W1282X 3905insT NI303K 3849+ 10kbC--~T R553X 621+ IG--*T 1717- IG--,A lt)78delT 2789+5G---~A 3849+4A--~G 711+ IG---oT R1162X 1898+IG----~A R117H 3659delC G85E 2184delA A1507 R347P Y1092X R560T A455E R334W Y122X S549R(T---~G) Q493X V520F $549N R347H Patient Selection. Login to comment

[hide] Mutation characterization of CFTR gene in 206 Nort... Hum Mutat. 1996;8(4):340-7. Hughes DJ, Hill AJ, Macek M Jr, Redmond AO, Nevin NC, Graham CA
Mutation characterization of CFTR gene in 206 Northern Irish CF families: thirty mutations, including two novel, account for approximately 94% of CF chromosomes.
Hum Mutat. 1996;8(4):340-7., [PMID:8956039]

Abstract [show]
A variety of mutation detection techniques, including restriction endonuclease digestion, allele specific oligonucleotides, and automated fluorescent sequencing, were used in the identification of 15 CFTR mutations representing 86.7% of CF chromosomes in 206 Northern Irish cystic fibrosis (CF) families. A systematic analysis of the 27 exons and intron/exon boundaries of the CFTR gene was performed using denaturing gradient gel electrophoresis (DGGE) in an attempt to characterise the 55 unknown CF mutations in 51 patients. Twenty different mutations were detected by DGGE on 30 chromosomes accounting for a further 7.3% of CF alleles. Fifteen of these mutations had not previously been found in Northern Ireland, and two are novel, M1I(G > T) and V562L. In total, 30 CFTR mutations account for 93.9% of the 412 Northern Irish CF chromosomes tested. The three major CF mutations in Northern Ireland are delta F508, G551D, and R117H with respective frequencies of 68.0%, 5.1%, and 4.1%. The efficacy of the DGGE technique was proven by the detection of 77 out of 77 control variants from all the CFTR exons. DGGE is a highly efficient and sensitive method for mutation screening especially in large genes where the mutation spectrum is known to be heterogeneous.

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45 Prior to the DGGE study a variety of methods (see Tables 1 and 2) were used to screen for CFTR mutations, including single mutation assaysfor AF508, AI507, G55lD, G542X, R553X, R117H, R560T, 621+lG>T, and 3849+10kbC>T. Login to comment
53 35%) PAGE (278) Kerem et al.. 1989AF508 G551D R117H R560T G542X 621+1G>T A1507 E60X 3659delC R553X 3120G>A 1l54insTC 2789+5G>A N1303K MlI(G>T) QW P67L 557delT 711+3A>G L206W R297Q V520F V562L Y563N Y917C R1162X 3849G>A 3849 +10kbC>T 3850-1GBA W1282X 280 21 17 12 9 9 7 3 2 1 68.0 5.1 4.1 2.9 2.2 2.2 1.7 0.7 0.5 0.24 17-32-13 (38;27%j 17-31-13(24,17%) 16-07-17 16-30-13 plus14 rare haplotypes (29) 16-07-17 23-33-13 (4) 22-31-13 (2) 21-31-13 17-07-17 (5) 16-31-13 16-35-13 17-58-13 17-35-13 16-07-17 17-07-17 23-29-13 (1) 23-31-13 (1) 16-07-17 16-31-13 16-07-17 15-29-13 16-33-13 16-07-17 17-07-17 16-07-17 16-07-17 16-30-13 16-32-17 17-31-13 16-31-14 16-46-13 16-30-14 17-07-17 DGGE(2) ' RD ASO's (11) DGGE(6) RD AR (8) DGGE (1) RD PAGE (5) DGGE (2) SEQ SEQ (2) DGGE (1) RD DGGE DGGE DGGE SEQ DGGE DGGE DGGE SEQ DGGE DGGE SEQ DGGE DGGE DGGE DGGE DGGE SEQ RD DGGE DGGE Cutting et al.. 1990 Dean et al.. 1990 Kerem et al., 1990 Kerem et al.. 1990 Zielenski et al., 1991 Kerem et al.. 1990 Malone et al., CFGAC Kerem et al., 1990 Cutting et al., 1990 Zielenski et al., CFGAC lannuzzi et al., 1991 Highsmith et al., 1990 Osborne et al., 1991 this study Savov et al., 1994 Hamosh et al., CFGAC Graham et al., 1992 Petreska et al., CFGAC Claustres et al., 1993 Graham et al., 1991 Jones et al.. 1992 this study Kerem et al.. 1990 Edkins & Creegan, CFGAC Gasparini et al., 1991 Cutting et al.. 1992 Highsmith et al., 1994 Audriizet et al., 1993 Vidaud et al., 1990 "Numbers in parentheses after the microsatellite haplotypes refer to the number of alleles haplotyped when not all of the available chromosomeswere typed. Login to comment
78 R560T, 1811+1G>C V562L, Y563N, 1898+lG>T 2143delT E827X R709X, K716X R764X E831X, W846X1,2711delT 2789+5G>A Y917C S977P. Login to comment
89 G551D (5.1%), R117H (4.1%), R560T (2.9%), HUGHESET AL. Login to comment
110 Analysis of three intragenic CFTR microsatellites demonstratedthat all R117H and R560T alleles share the same respective haplotype: 16(IVS8CA)- 30(IVS17bTA)- 13(IVSl7bCA) for R117H and 16-07-17for R560T. Login to comment
120 A commercially available reverse dotblot assay (Innogenetics) is presently used in our laboratory for routine screening of eight CF mutations, of which seven are found in this population; AF508, AI507, G551D, G542X, R553X, N1303K, and W1282X and account for 78% of CFTR defects with additional mutations R560T, 621+1G>T and Rl17H screened using individual assays. Login to comment

[hide] Fluorescent multiplex microsatellites used to defi... Hum Mutat. 1996;8(3):229-35. Hughes D, Wallace A, Taylor J, Tassabehji M, McMahon R, Hill A, Nevin N, Graham C
Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes.
Hum Mutat. 1996;8(3):229-35., [PMID:8889582]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal/ carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for delta F508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, delta F508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G > T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes.

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6 Ancient mutations, AF508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13,621+1G>T with 21-31-13,3659delC with 16-35-13). Login to comment
58 As the repeat number increasesbeyond 50, it probably becomes more disposed to the addition or deletion of dinucleotide repeats during replication as all other mutations where several alleles were tested gave the same respective 17bTA repeat number, i.e., G551D (17AT repeats = 7), R560T (7), G85E (24), R117H (30), 621+1G>T (31), 3659delC (354, 1898+lG>A (45). Login to comment
74 CF 8CA-17bTA-17bCA Mutation chromosomes % Normal Laboratoryb Reference' HaplotVpe 1)15-29-13 557delT Nl Graham et al.. 1992 21 16-07-17 MU (G>T) 3) 16-24-13 4) 16-25-13 5) 16-29-13 6) 16-30-13 7) 16-30-14 8) 16-31-13 9) 16-31-14 10) 16-32-13 12) 16-33-13 13) 16-34-13 14) 16-35-13 11)16-32-17 15)1645-13 16) 1646-13 17) 1646-14 19) 17-07-17 18)16-53-13 20)17-29-14 21) 17-31-13 22) 17-32-13 23) 17-35-13 24) 17-51-11 25) 17-55-13 27) 17-58-13 28) 21-31-13 29) 22-31-13 31)23-22-17 26) 17-56-13 30) 22-33-13 32) 23-29-13 33)23-31-13 34)23-32-13 35)23-33-13 36)23-34-13 37) 23-36-13 38)24-22-17 39) 24-31-13 182delT P67L R75X L206W 1154insTC 146linsAGAT Q493x V520F 1717-1G>A G551D R560T V562L R709X S1196X L1254X R1283M G85E 2184insA 711+lG>T 3495delA 4279insA SlOR L88S R117C R117H G178R 1717-1G>A Y563N W1098R G1123R 3850- 1G>A E6OX %%deIT 1138insG R34P 2183AA>G 2184delA R1158X 1078delT R1162X 3849G>A Q141W R347P Y917C G2iX 711+3A>G 441delA 3130de115 3659delC 1898+1G>A R709X 2711delT R1158X E92K 3849+lOkbC>T 2118delAACT 4048insCC 296+1 2 T S Q22OX R297Q A1507 2789+5G>A 3120+1G>A W128W 1811+lG>C AF508 E831X R116W AF508 W846X1 3120G>A R785X R553X R553X R553X 621+1G>T G542X G542X Y1182X N1303K AF508 G54W 3041delG 1525-1G>A N1303K G542X G542X G542X 394delTT R709X N1303K 1 1 1 2 1 1 4 2 3 4 2 26 8 1 1 1 1 1 8 1 1 1 1 1 1 1 19 1 2 1 1 1 1 7 1 1 2 1 1 2 1 1 1 1 1 1 1 1 2 1 1 7 4 1 2 1 1 2 1 1 4 Asian 1 2 1Asian 5 4 i Afro-Caribbean 5 1 42 (19%) 1 1 57 (26%) 1 2 1 1 1 2 12 2 11.4 0.4 4.9 16.3 1.1 3.8 1.9 10.6 2.3 1.5 2.3 1.5 2.7 4.5 0.4 0.8 0.8 0.4 0.8 0.4 1 2 1 7 1 1 1Asian 1 1.5 0.8 0.8 NI G NI, M M NI NI. Login to comment
83 G 23-31-13 2 NI,G 24-31-13 1 C R117H 16-30-13 19 16.3 NI.G, C 1717-1G>A 16-07-17 2 M, G 16-30-13 2 M, G R553X 17-55-13 1 M 17-56-13 1 C 17-58-13 1 0.4 NI 1898+1G>A 16-45-13 4 1.5 M E60X 0.2-0.5 16-31-13 7 3.8 NI.M G85E 16-24-13 8 M. G, C 1154insTC 16-07-17 4 NI, M, G A1507 17-07-17 5 4.5 NI R560T 16-07-17 8 N1 3659delC 16-35-13 7 2.3 NI. Login to comment
91 Other relativelycommon mutations (e.g., G551D, R560T, R117H, 3659delC, and 621+lG>T) were found associatedwith the one haplotype, suggesting that they emerged more recently. Login to comment
97 Another, 16-7-17, is highly sensitive, e.g., all G551D and R560T alleles tested were found on this framework, but has a low specificity as it is common in normals and is associated with 17 different mutations. Login to comment

[hide] Haplotype analysis of 94 cystic fibrosis mutations... Hum Mutat. 1996;8(2):149-59. Morral N, Dork T, Llevadot R, Dziadek V, Mercier B, Ferec C, Costes B, Girodon E, Zielenski J, Tsui LC, Tummler B, Estivill X
Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers.
Hum Mutat. 1996;8(2):149-59., [PMID:8844213]

Abstract [show]
We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, delta F508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272-26A-->G, R1162X, and 3849 + 10kbC-->T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF.

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106 (1992) Dork et al. (1994a) Malone et al. (personal communication) Claustreset al. (1992) Ferec et al. (1992) Fanen et al. (1992) lvaschenko et al. (1991) T. Dork (personal communication) Dean et al. (1990) Dork et al. (1994a) Ferec et al. (1992) Bozon et al. (1994) Costes et al. (personal communication) Fanen et al. (1992) Audrezet et al. (personal communication) Zielenski et al. (1991a) Zielenski et al. (1991a) Granell et al. (1992) Highsmith et al. (1990) Mercier et al. (1993b) Vidaud et al. (1990) Fanen et al. (1992) Fanen et al. (1992) Dork et al. (1994b) (continued) HAPLOTYPESFOR 94 CF MUTATIONS TABLE2. CFTR HaplotvpesforDiallelic and Multiallelic DNA Markers for 94 CF Mutations"(Continued) ~~ ~ J44-GAIT- 8CA-17BTA- No. of TSU-TUB20 17BCA Mutation chromosomes % Normal Laboratory Reference 1-6-1-2 (9.1%) 1-6-2-2 (8.9%) 1-7-1-2 (3.4%) 1-7-2-2 (2.6%) 2-7-1-1 (1.2%) 2-7-2-2 (0.7%) 17-7-16 16-7-18 16-7-17 15-7-17 24-31-13 23-52-13 23-34-13 23-33-14 23-33-13 23-32-13 23-31-13 23-30-13 23-21-19 23-18-13 22-35-13 22-31-13 22-30-13 21-31-13 19-33-13 18-45-13 18-37-13 18-35-13 17-57-11 17-55-13 17-55-11 17-54-11 17-53-11 17-52-11 17-51-11 17-33-13 16-46-13 16-45-13 16-44-13 16-42-13 16-35-13 16-30-13 16-30-13 16-7-17 16-21-19 L107% L1077P 24ldelAT L719X A1507 3849+10kbC-T 2184insA 2991de132 G551D 1154insTC V520F R560T 4114ATA+lT 3667de14 435insA Q414X C225R Q39X N1303K R1162X H199Y G542X G542X w1204x R347H G542X AF50gb N1303K 2143delT 3849f 10kbC-T N1303K 681delC R347H A455E N1303K A120T 621+1 h T 574delA 1221delCT F311L R560K R553X R533X R553X Q552X R553X Q552X R116W R553X 1898+5 h T 3272-26A-G 1717-1hA 1342-2A-C A1507 2869insG 2869insG E92X 4374+1 h T 2183AA-G R117H 1609delCA I336K W1063X 1 1 1 1 6 1 3 1 1 22 17 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 4 157 7 1 2 2 1 1 2 2 1 9 1 1 1 1 1 1 6 1 1 1 2 1 3 2 1 3 1 1 1 4 2 4 1 1 - - 10.33 1.45 - - 0.48 1.45 - 0.24 1.45 0.24 - - - - 0.24 0.48 - - - - - - 0.49 0.48 - 0.24 0.24 0.24 - - - - - 0.72 0.24 0.72 - t h fP h b.fb,fP h b,fp.t t h b.fb.fp,h,t b.fb.fp,h,t t t t h b h h fP h fP fb b fP b.fb,fP,h.t fP fb b,fP,t b.fb,fp,h,t b.fb,h h h h,t t fb t b b b.fb.t fP fb fb tb h fP h h t t b h t h b b h h b,fb,h fP.h b h fP fP Bozon et al. (1994) Fanen et al. (1992) Dork et al. (1994a) Kerem et al. (1990) Dork et al. (1994~) Cutting et al. (1990) Kerem et al. (1990) lannuui et d. Login to comment

[hide] Cystic fibrosis mutation detection by hybridizatio... Hum Mutat. 1996;7(3):244-55. Cronin MT, Fucini RV, Kim SM, Masino RS, Wespi RM, Miyada CG
Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.
Hum Mutat. 1996;7(3):244-55., [PMID:8829658]

Abstract [show]
We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR.

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238 Cystic Fibrosis Mutation-Specific DNA Probe Array" Mutation Exon and column Tested Subarrayhow G85E R117H I148T 621 -+ l(G+T) 711 + 1(G+T) R334W R347H R347P 1078 delT A455E G480C Q493X A1507 F508C AF508 V520F G542X S549R(T-+ G) G551D Q552X R553X A559T R560T 1898 + l(G-,A) 2184 del A 2789 + 5(G+ A) R1066C L1077P Y1092X R1162X 3659 del C 1717-1(& A) 3272 - 26(A+ G) 3 4 4 in 4 in 5 7 7 7 7 9 10 10 10 10 10 10 in 10 11 11 11 11 11 11 11 in 12 13 in 14b in 17a 17b 17b 17b 19 19 * * * * * * * * * * * * * * * * * * * * * * * * * * * * 3849 + lOkb C-, T in 19 9,3 W1282X 20 994 3905insT 20 10.1 * N1303K 21 10,2 * * * "Row and column locations for each of the mutation specific,40 probe sets included in the specialized probe array design. Login to comment

[hide] Methods for screening in cystic fibrosis. Methods Mol Med. 1996;5:99-119. Schwarz M, Malone G
Methods for screening in cystic fibrosis.
Methods Mol Med. 1996;5:99-119., [PMID:21374513]

Abstract [show]
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Whites, with an incidence of approx 1 m 2500 live births and a carrier frequency of approx 1 in 25. Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene m 1989 (1-3), molecular genetics laboratories throughout the world have endeavored to identify the mutations present in their population of CF-bearing chromosomes. Since the entire CFTR gene and its intron-exon boundaries have been sequenced, mutation analysis in CF has become relatively simple, although time consuming. Generally, a number of different methods are applied to mutation analysis, but all involve an imtial step of amplification of part of the gene by polymerase chain reaction (PCR) (4), or a derivative of it, such as amplification refractory mutation system (ARMS) (5).

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10 Molecular D/agnoss of GenetIc Diseases Edlted by R Elles Humana Press Inc , Totowa, NJ 99 Table1 RelativeFrequenciesoftheCommonCFMutationsintheUnitedKingdom(S),NorthAmerica, andNorthernandSouthernEurope(7)" NorthNorthernSouthern UKAmericaEuropeEurope MutationExonNo.%No.%No.%No%References AF508 G551D G542X 621+l(G>T) 1717-l(G>A) sR117H =R553x 1898+l(G>A) N1303K R560T AI507 G85E 1154insTC V520F W1282X E60X 3659delC 1078delT 10738775.32690066.114,86670.28400755.033 113023.082061.973561.68370.518 111651.682342.244392.082593.569 intron4910.931541.48970.46370.51IO intron10560.57440.421600.76650.899 4450.46610.586202930.04II 11450.46960.921650.7844068 intron12450.4620.02410.191001412 21450.461301.252090.991792.4613 11410.42240.2340019009 10300.31200.19570.2750.079,14 3210.211601530014140.19II 7190.19n/an/an/an/an/an/a15 10170.17n/an/an/an/an/an/a16 20170.172452.351200.57430.5917 3160.16n/an/an/an/an/an/aMaloneb 19140.14140.133901810.019 790.0910.015302520.318 S549N1180.0850.051800920038 Q493X1070.07n/an/adan/adan/a9 R347P760.06260.25550.26240.3311 3849+10kb(C>T)intron1950.05570.55230.1180.1119 A455E930.03270.26350.17009 %/a,Datanotavadable. Login to comment
34 As an extreme example, asample may betested for R560T and R117H by ARMS duplex (primers aredetailed in Table 3,p. Login to comment
36 Becauseof the simplicity of detection of changeswhich alter restriction endonucleaserecognition sequences(Table 5, p. 107), anumber of diagnostic laboratories chooseto testfor thesemutations (e.g., R553X, R560T) at an early stage.This may be followed by further SSCPand DGGE analysis, and ultimately DNA sequencing exon by exon. Login to comment
38 Table2 Sequences,ProductSizes,andAnnealingTemperaturesofPCRPrimersUsedinCFAnalysis(seeNote1O)8 ExonPrimersequence5`to3`Name ProductAnnealing size,bptemperature,"C 3 4 7 9 10@al-Q G A10 11 12 13 14a 17b 19 CTTGGGTTAATCTCCTTGGA3i5 ATTCACCAGATTTCGTAGTC313 TCACATATGGTATGACCCTC4i5 TTGTACCAGCTCACTACCTA4i3 AGACCATGCTCAGATCTTCCAT7i5 GCAAAGTTCATTAGAACTGATC7i3 TAATGGATCATGGGCCATGT9i5 ACAGTGTTGAATGTGGTGCA9i3 GTTTTCCTGGATTATGCCTGGGCACC16B GTTGGCATGCTTTGATGACGCTTCC16D GCAGAGTACCTGAAACAGGAlOi CATTCACAGTAGCTTACCCAlOi CAACTGTGGTTAAAGCAATAGTGTlli5 GCACAGATTCTGAGTAACCATAATlli3 GTGAATCGATGTGGTGACCA1215 CTGGTTTAGCATGAGGCGGT1213 TGCTAAAATACGAGACATATTGCA13i5 ATCTGGTACTAAGGACAGCl-1M AAAAGGTATGCCACTGTTAAG14ai5 GTATACATCCCCAAACTATCT14ai3 TTCAAAGAATGGCACCAGTGT17bl5 ATAACCTATAGAATGCAGCA17bi3 GCCCGACAAATAACCAAGTGA1915 GCTAACACATTGCTTCAGGCT19i3 309 438 410 561 98 491 425 426 528 511 463 454 54 54 54 54 54 54 54 57 58 57 56 54 20 21 intron6a iniron8 intron17b intron17b mtron19 GGTCAGGATTGAAAGTGTGCA CTATGAGAAAACTGCACTGGA AATGTTCACAAGGACTCCA CAAAGTACCTGTTGCTCCA CAAGTCTTTCACTGATCTTC TGAGCAGTTGTTAATAGATAA TCTATCTCATGTTAATGCTG GTTTCTAGAGGACATGATC GACAATCTGTGTGCATCG GCTGCATTCTATAGGTTATC AAACTTACCGACAAGAGGA TGTCACCTCTTCATACTCAT AGGCTTCTCAGTGATCTGTTG GAATCATTCAGTGGGTATAAGCAG 20x547359 2Oi3 211547654 210 FC09106/l1057 6iRPT NURI22050 AT17R1.2201-30150 AT17D1.2 ACl7R214050 AC17D2 i19F43750 i19R "SequencesareprovidedcourtesyoftheCysticFibrosisGeneticAnalysisConsortium 106 Schwarz and Malone Table 3 Sequences and Product Sizes for ARMS Primers (23) Used to Detect the CF Mutations R560T and R117H in a Duplex Reactiona Mutatron Prnner Sequence 5` to 3` Product size, bp R560T RI 17H Common AAAATTTCAGCAATGTTGTTTTTGACCAAC Normal GCTTGCTAGACCAATAATTAGTTATTCAAC Mutant GCTTGCTAGACCAATAATTAGTTATTCAAG Common CACATATGGTATGACCCTCTATATAAACTC Normal CCTATGCCTAGATAAATCGCGATAGAACC Mutant CCTATGCCTAGATAAATCGCGATAGAAT 316 237 aAs for the CF(4)m test, a pan of tubes (A and B) is required for eachDNA sample. Login to comment
39 Tube A contams R560T and R117H common, R560T normal, and R117H mutant primers; tube B contams R560T and R117H common, R560T mutant and R117H normal, pnmers. Login to comment
42 Screening in Cystic Fibrosis 107 Table 5 CF Mutations Detectable by Restriction Endonuclease (RE) Digestion of PCR Products Mutation PCR primers0 RE RE digestion product sizes, bpbJ Normal Mutant G85E 621+ 1 (G`V 1154insTC R334W R347P G551D R553X R560T S549N 3849+ IOkb CC ` T) W1282X 3i5 and 313 4i5 and 4i3 Hinff MseI 105 + 204 33,35,71, 118, 181 7i5 and 7i3 MspI, RsaI 50,68,74 + 21V 715 and 7i3 MspI 192 + 218 7i5 and 7i3 CfoI 151+ 259 1li5 and 1113 Mb01 425 1115 and lli3 HzncII 186 + 239 lli5 and lli3 Mae11 425 lli5 and lli3 DdeI 13, 174 + 238 i19F and i19R HphI 88 + 349 2Oi5 and 2Oi3 Mnfl 185 + 288 309 33,35,54,71, 118, 127 50,68,76 + 21gc 410 410 182+243 425 215 + 210 13 + 412 88,127 + 222 473 'See Table 2 bThe expected digestion product sizes for both normal and mutant sequences are shown CTheseproducts may be d1stmgmshedby PAGE 1.2.3. Login to comment

[hide] The Irish cystic fibrosis database. J Med Genet. 1995 Dec;32(12):972-5. Cashman SM, Patino A, Delgado MG, Byrne L, Denham B, De Arce M
The Irish cystic fibrosis database.
J Med Genet. 1995 Dec;32(12):972-5., [PMID:8825927]

Abstract [show]
We have found records of 1014 Irish cystic fibrosis patients alive by December 1994, belonging to 883 families. Prevalence in the population is 1/3475 and incidence at birth 1/1461, with a gene frequency of 2.6%. Twenty percent of the patients are aged over 20 years, but at present survival rate falls rapidly after that age. We have identified 85% of the mutations on the CFTR gene in a sample of 29% of the families (506 CF chromosomes). Mutation delta F508 is found in 72% of Irish CF chromosomes, G551D in 6.9%, and R117H in 2%. These are the highest frequencies reported for the latter two mutations world wide. Another seven mutations are found in an additional 4% of CF families. We present new microsatellite haplotype data that could be useful for genetic counselling of CF families bearing some of the 15% of CF mutations still unidentified, and comment on possible uses of our database.

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39 Care Table 2 Haplotypes associated with the most common CF mutations in Ireland, as well as with Irish CF chromosomes with unidentified mutations and non-CF chromosomes Mutation NmlNcf % X/Kt Mt Microsatellite§ No chrsll AF508 367/506 72-5 A 1 See table 3 2 B 2 116 C - 0 D 2 2 G551D 35/506 6-9 B 2 16-7-17 20 R117H 10/506 2-0 C 1 16-30-13 5 C 1 16-31-13 1 G542X 5/506 1-0 B 2 17-32-13 2 621 + 1G-T 4/506 0-8 B 2 21-7-17 2 B 2 21-31-13 2 R560T 4/506 0-8 D 2 16-7-17 1 D 2 16-31-17 1 1717-1G-A 3/506 0-6 C 1 16-32-13 2 A 2 17-32-13 1 N1303K 2/506 0-4 B 2 23-29-13 2 3659delC 2/506 0-4 C 1 16-35-13 2 AI507 2/506 0-4 ND ND R352Q 1/506 0-2 C 1 16-31-13 1 R553X 0/506 0 0 ND ND 1078delT 0/506 0.0 ND ND Total identified 435/506 85-6 Unidentified 71/506 14-4 A 1 See table 3 3 A 2 5 B 2 17 C 1 5 D 2 8 Normal A 1 See table 3 16 A 2 1 B 2 3 C 1 13 C 2 1 D 2 2 * Nm = number of chromosomes bearing the mutation. Login to comment
69 For instance, table 2 shows that mutations G542X, 621 + 1G-T, N1303K, 3659delC, R560T, and Ri 17H are associated with the same haplotypes, or with haplotypes probably derived from these by slippage of the DNA polymerase, as has been seen in patients from other European populations.7'0 Since those haplotypes are relatively rare in normal chromosomes (table 3), the constancy of the association has been explained as suggesting identity by descent of all chromosomes bearing the same mutations.9 Haplotype 16-7-17 is found in CF non-AF508 chromosomes more frequently than would be expected from its frequency in normal chromosomes (p<0 01, table 3), suggesting perhaps that another fairly common CF mutation may be associated with this haplotype in Ireland. Login to comment
38 Care Table 2 Haplotypes associated with the most common CF mutations in Ireland, as well as with Irish CF chromosomes with unidentified mutations and non-CF chromosomes Mutation NmlNcf % X/Kt Mt Microsatellite&#a7; No chrsll AF508 367/506 72-5 A 1 See table 3 2 B 2 116 C - 0 D 2 2 G551D 35/506 6-9 B 2 16-7-17 20 R117H 10/506 2-0 C 1 16-30-13 5 C 1 16-31-13 1 G542X 5/506 1-0 B 2 17-32-13 2 621 + 1G-T 4/506 0-8 B 2 21-7-17 2 B 2 21-31-13 2 R560T 4/506 0-8 D 2 16-7-17 1 D 2 16-31-17 1 1717-1G-A 3/506 0-6 C 1 16-32-13 2 A 2 17-32-13 1 N1303K 2/506 0-4 B 2 23-29-13 2 3659delC 2/506 0-4 C 1 16-35-13 2 AI507 2/506 0-4 ND ND R352Q 1/506 0-2 C 1 16-31-13 1 R553X 0/506 0 0 ND ND 1078delT 0/506 0.0 ND ND Total identified 435/506 85-6 Unidentified 71/506 14-4 A 1 See table 3 3 A 2 5 B 2 17 C 1 5 D 2 8 Normal A 1 See table 3 16 A 2 1 B 2 3 C 1 13 C 2 1 D 2 2 * Nm = number of chromosomes bearing the mutation. Login to comment

[hide] Genetic and clinical features of patients with cys... Thorax. 1995 Dec;50(12):1301-4. Gan KH, Geus WP, Bakker W, Lamers CB, Heijerman HG
Genetic and clinical features of patients with cystic fibrosis diagnosed after the age of 16 years.
Thorax. 1995 Dec;50(12):1301-4., [PMID:8553305]

Abstract [show]
BACKGROUND: Cystic fibrosis is usually diagnosed in childhood, but a number of patients are not diagnosed until adulthood. The aim of this study was to investigate whether patients diagnosed at an older age had a different genetic constitution, manifestations of disease, and prognosis from those diagnosed at an early age. METHODS: Clinical data and results of lung function tests and DNA analysis of 143 adult patients with cystic fibrosis were entered into a computerised database. Patients diagnosed before their 16th birthday (early diagnosis, ED) were compared with those diagnosed at 16 years of age or older (late diagnosis, LD). RESULTS: Mean age of diagnosis of the ED group was 4.6 years compared with 27.7 years for the LD group. Mean (SD) percentage predicted pulmonary function was better for the LD group than for the ED group: forced expiratory volume in one second (FEV1) 72.5 (31.1)% and 52.0 (24.8)%, and forced vital capacity (FVC) 89.8 (25.7)% and 71.9 (23.0)%, respectively. Colonisation with Pseudomonas aeruginosa was present in 70% of the ED group and 24% of the LD group. In the ED group 81% had pancreatic insufficiency compared with only 12% of the LD group. None of the LD group was homozygous for delta F508 compared with 58% of the ED group. In the LD group 72% were compound AF508 heterozygotes and 28% had two non-delta F508 mutations. CONCLUSIONS: Among this group of 143 adult patients with cystic fibrosis late diagnosis is caused mainly by delayed expression and mild progression of clinical symptoms. Late diagnosis is associated with milder pulmonary disease, less pancreatic insufficiency, and different cystic fibrosis mutations. Since mortality in cystic fibrosis depends on the progression of pulmonary disease, patients with a late diagnosis have a better prognosis than those diagnosed early.

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41 DNA was analysed for the following mutations: E60X, R117H, A455E, AI507, AF508, G542X, S549N, G550X, G551D, R553X, R560T, R1162X, S1251N, W1282X, N1303K, 621 + 1G-+T, 1717-1G--+A. These mutations represent 80% ofthe expected cystic fibrosis mutations in The Netherlands. Login to comment
65 Table 3 CFTR mutations in 278 chromosomes of adult cystic fibrosis patients Early diagnosis Late diagnosis (n= 118) (n=25) n % n % AF508s 175 74-2 18 36-0 A455Em 12 5-1 14 28-0 1717-15 6 2-5 1 2-0 G542X' 4 1-7 - W1282X1 3 1-3 - R553X' 1 04 1 2-0 S1251N 2 0-8 - N1303K' 1 0 4 - E60X 1 0-4 3 6-0 Not identified 31 13-2 13 26-0 Total 236 50 Mutations not found: RI 17H, AI507, S549N, G550X, G551D, R560T, R1162X, 621+1G-+T. Login to comment

[hide] Correlation of sweat chloride concentration with c... J Pediatr. 1995 Nov;127(5):705-10. Wilschanski M, Zielenski J, Markiewicz D, Tsui LC, Corey M, Levison H, Durie PR
Correlation of sweat chloride concentration with classes of the cystic fibrosis transmembrane conductance regulator gene mutations.
J Pediatr. 1995 Nov;127(5):705-10., [PMID:7472820]

Abstract [show]
OBJECTIVE: To compare differences in epithelial chloride conductance according to class of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. METHODS: We evaluated the relationship between the functional classes of CFTR mutations and chloride conductance using the first diagnostic sweat chloride concentration in a large cystic fibrosis (CF) population. RESULTS: There was no difference in sweat chloride value value between classes of CFTR mutations that produce no protein (class I), fail to reach the apical membrane because of defective processing (class II), or produce protein that fails to respond to cyclic adenosine monophosphate (class III). Those mutations that produce a cyclic adenosine monophosphate-responsive channel with reduced conductance (class IV) were associated with a significantly lower, intermediate sweat chloride value. However, patients with the mutations that cause reduced synthesis or partially defective processing of normal CFTR (class V) had sweat chloride concentrations similar to those in classes I to III. CONCLUSION: Studies of differences in chloride conductance between functional classes of CFTR mutations provide insight into phenotypic expression of the disease.

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43 Defined mutations (each mutation cited in references 8, 23, and 24; numerals in parentheses indicate number of patients): Nonsense mutations-----class I: Frameshift mutations---class I: Splice site mutations-class I: Missense mutations---class HI: Missense mutations---class IV: Partially defective processing---class V: Alternative spficing-----classV: R1162X (3), Y1092X (3), G542X (21), Q552X (2), Q493X (2), w1282x (2), E1104X (1), R553X (6), E585X (l), (all PI) 3659delC (5), 2184delA (4), 4010de14 (1), 556delA (1), 3002delG (1) 3905insT (1), 4016insT (3), 1154insTC (l), 441delA (1), 2184insA (2), 1078delT (1), 4326delTC (3) (all PI) I717-1G--~A (4), 621+lG--*T (10), 711+IG--~T (3), 875+1G-+C (2), 3120+IG-~A (1) (18 PI, 2 PS) G551D (25), N1303K (7), R560T (8), I148T (1), G85E (3), A559T (1), L1077P (2), T1234V (1), (47 PI, 1 PS) R117H (10), R347H (3), R347P (1), D614G (1), S1251N (2), (all PS) P574H (2), A455E (2), (all PS) 3272-26A-+G (4), 3849+10KbC---~T (2), 3120G-+A (1), (all PS) analysis, we further grouped the patients according to the molecular consequences conferred by the CFTR alleles. Login to comment

[hide] CFTR gene variant for patients with congenital abs... Am J Hum Genet. 1995 Oct;57(4):958-60. Zielenski J, Patrizio P, Corey M, Handelin B, Markiewicz D, Asch R, Tsui LC
CFTR gene variant for patients with congenital absence of vas deferens.
Am J Hum Genet. 1995 Oct;57(4):958-60., [PMID:7573058]

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22 W1282X Unknown 2 R553X Unknown ............ 20.0 4173delC Unknown1 1 D614G Unknown 1 1716+12T- C Unknown 1 J Unknown Unknown ............ .15 21.4 NOTE.-The known CFTR mutations screened included AF508, G542X, GSS1D, N1303K, R553X, W1282X, AI507, 1717-1G-A, R560T, S549N, 621+1G--T, and R117H. Login to comment

[hide] Screening Young syndrome patients for CFTR mutatio... Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1353-7. Friedman KJ, Teichtahl H, De Kretser DM, Temple-Smith P, Southwick GJ, Silverman LM, Highsmith WE Jr, Boucher RC, Knowles MR
Screening Young syndrome patients for CFTR mutations.
Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1353-7., [PMID:7551394]

Abstract [show]
Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles.

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78 Of the 13 Young syndrome patients, we identified one (Patient 5) who was het- CBAVD Dl152H D1270N G576A* R75Q* P67L Rl17H 3849 + 10 KB C > T G551S Rl17H Pancreatic Sufficient, Moderate Pulmonary Symptoms, Normal Sweat Chloride Concentrations Pancreatic Sufficient, Moderate Pulmonary Symptoms R347P 2789 + 5 G > A R334W G85E R347H R347L Rl17H G91R A455E S945L Y563N Q1291H R297Q R352Q L1065P 3850-3 T > G F1286S 3849 + 10 KB C > T TABLE 1 CFTR MUTATION SCREENING PANEL Severe M508 G551D R553X N1303K W1282X G542X 1717-1 G > A ~1507 R560T 3659deiC 621 + 1 G > T S549N TABLE 2 CLINICAL FEATURES OF YOUNG SYNDROME PATIENTS Patient Age Sweat CI- FEV, Paranasal Sputum No. Login to comment

[hide] A change in gating mode leading to increased intri... EMBO J. 1995 Jun 1;14(11):2417-23. Champigny G, Imler JL, Puchelle E, Dalemans W, Gribkoff V, Hinnrasky J, Dott K, Barbry P, Pavirani A, Lazdunski M
A change in gating mode leading to increased intrinsic Cl- channel activity compensates for defective processing in a cystic fibrosis mutant corresponding to a mild form of the disease.
EMBO J. 1995 Jun 1;14(11):2417-23., [PMID:7540133]

Abstract [show]
The effects of the mild cystic fibrosis (CF) mutation P574H were analysed and compared with those of three severe ones (delta I507, delta F508 and R560T). Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H >> delta F508 >> R560T approximately 0. Patch-clamp analysis indicates that the open probability of P574H Cl- channels is almost twice as high as that of the wild type CFTR-Cl- channel. This increased intrinsic activity of individual P574H CFTR-Cl- channels compensates for the lower number of P574H CFTR-Cl- channels reaching the cell surface, and probably explains the milder form of CF associated with the P574H mutation. NS004, a recently described activator, restores near normal CFTR activity in cells expressing the P574H-CFTR channel. The P574H mutation modifies the gating mode of the channel with a large increase (approximately x 7) in the mean channel open time. Proline 574 might play an important role in the process connecting ATP hydrolysis at the nucleotide binding domain and opening and closing events of the CFTR-Cl- channel.

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1 Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H > > AF508 > > R560T - 0. Login to comment
21 The properties of this mutation are compared with those of three severe mutations A1507, AF508 and R560T, also affecting the first nucleotide binding domain. Login to comment
23 Immunodetection of wild type, AF508-, R560T- and P574H-CFTRs in Vero cells. Login to comment
24 Standard fluorescence microscopic photomicrographs (A, C, E and G) and XY and XZ confocal optical section (B, D, F and H) of Vero cells expressing wild type (A and B), P574H- (C and D), AF508- (E and F) and R560T-CFTRs (G and H). Login to comment
27 A1507, AF508, R560T and to compare them with the P574H mutation. Login to comment
32 The R560T mutation resulted in very low immunolabelling of R560T-CFTR expressing cells, which was strictly located within the cytosol. Login to comment
38 Figure 2 shows the percentage of cells that were positive for membrane immunolabelling after incubation with the 80 c ._ CD 60 m 40 E c 20 0 _ Vero M Chang I -E _m7A - WT P574H AF508 R560T Fig. 2. Login to comment
39 Analysis of the cell surface expression of wild type, AF508-, R560T- and P574H-CFTRs. Login to comment
40 The number of Vero and Chang cells used for the analysis was equal to 125 and 223 for wild type CFTR, 59 and 288 for AF508, 169 and 317 for R560T, 88 and 479 for P574H respectively. Login to comment
46 No R560T-expressing cells exhibited plasma-membrane immunolabelling. Login to comment
52 In addition, the absence of fully glycosylated forms of CFTR in cells expressing A1507, AF508, R560T and P574H mutant proteins confirms that these mutations are associated with defective processing. Login to comment
53 Functional characterization of P574H-CFTR as compared with other CFTR mutant proteins The functional activity of CFTR mutant proteins was measured in two different experimental conditions that permit activation of the CFTR-CI- channels. Login to comment
55 Western blotting analysis of the 'mock'-infected, wild type, AF508-, R560T- and P574H-CFTRs. Login to comment
66 When 1251- efflux were performed on Vero cells expressing A1507 and R560T CFTRs, no significant stimulation of the efflux was observed, even in conditions permitting a maximal activation of wild type CFTR or of AF508 mutant protein, i.e. 10 ,uM forskolin + 20 jM NS004 (Gribkoff et al., 1994). Login to comment
71 1050 Istim-Ictrl 700 (pA) 350 P574H R560T A1507 urIC) 0L It) C~ a ° Fig. 4. Login to comment
72 Functional analysis of the A1507-, R560T- and P574H-CFTRs. Login to comment
74 (B) Time-course of the activation of the R560T-CFTR mediated 1251- efflux by 10 ,uM forskolin (EO1) and 10 jM forskolin + 20 jM NS004 (A). Login to comment
75 (C) Time-course of the activation of the P574H-CFTR mediated 125p- efflux by 10 ,uM forskolin (O), 20 ,uM NS004 (M) and 10 jM forskolin + 20 ,uM NS004 (A). Login to comment
77 Results are shown as mean SD (D) Whole cell patch-clamp studies of wild type, A1507, AF508, R560T and P574H after stimulation by 10 jM forskolin. Login to comment
78 (E) Activation of the A1507, R560T and P574H -C1- currents (Istim) by 20 jM NS004 and/or forskolin 10 jiM. Login to comment
86 A Cl- current in R560T and A1507 expressing cells was stimulated by forskolin (Figure 4D), but its amplitude remained always very small in comparison with the Cl- current generated by the expression of wild type CFTR (2.6 and 3.7% of the wild type, for A1507 and R560T respectively), and the stimulating effect of NS004 remained modest (Figure 4E). Login to comment
87 The mutant protein P574H has a higher intrinsic Ct channel activity than wild type In order to understand why the P574H mutant protein displayed a high residual C1- channel activity, despite its defective cell-surface expression, the properties of the P574H-Cl- channel were compared with those of the wild type CFTR using the cell-attached patch-clamp technique. Login to comment
96 All mutations affecting the first nucleotide binding domain that have been described so far lead to a 2420 5 rstim rbas 3 B R560T 5 - on=4 A n=8 3 1 ' . Login to comment
97 .-.. 2 4 time(minutes) 6 Istim-lctd (pA) I J * NS004 1OgM U ForskliO±M O NS004 + Forsk 1 E WT P574H N-4. Login to comment
109 No significant Cl- channel activity was observed in cells expressing the two mutant proteins A1507 and R560T-CFTR corresponding to severe mutations. Login to comment
114 Since no A1507 or R560T proteins could be detected at the plasma membrane, and since their expression generated <4% of the whole-cell Cl- current generated by the wild type (Figure 4D), these mutant channels can be considered as members of class II. Login to comment
115 It may be that in addition to their severe sorting defect, A1507-CFTR and R560T-CFTR also have altered Cl- channel properties, as has been reported for the AF508 mutant protein (Dalemans et al., 1991). Login to comment
117 A different situation was observed with the P574H mutation. Login to comment
164 Light fluorescence microscopy Vero and Chang cells were grown on Lab Tek chamber slides and were infected using a recombinant vaccinia virus expression system carrying either the wild type or the mutant (AF508, R560T or P574H CFTR) cDNAs under the control of the T7 promoter. Login to comment
47 No R560T-expressing cells exhibited plasmamembrane immunolabelling. Login to comment
56 Western blotting analysis of the 'mock'-infected, wild type, AF508-, R560T- and P574H-CFTRs. Login to comment
67 When 1251efflux were performed on Vero cells expressing A1507 and R560T CFTRs, no significant stimulation of the efflux was observed, even in conditions permitting a maximal activation of wild type CFTR or of AF508 mutant protein, i.e. 10 ,uM forskolin + 20 jM NS004 (Gribkoff et al., 1994). Login to comment
73 Functional analysis of the A1507-, R560T- and P574H-CFTRs. Login to comment
79 (E) Activation of the A1507, R560T and P574H -C1- currents (Istim) by 20 jM NS004 and/or forskolin 10 jiM. Login to comment
112 No significant Cl-channel activity was observed in cells expressing the two mutant proteins A1507 and R560T-CFTR corresponding to severe mutations. Login to comment
118 It may be that in addition to their severe sorting defect, A1507-CFTR and R560T-CFTR also have altered Cl-channel properties, as has been reported for the AF508 mutant protein (Dalemans et al., 1991). Login to comment
167 Light fluorescence microscopy Vero and Chang cells were grown on Lab Tek chamber slides and were infected using a recombinant vaccinia virus expression system carrying either the wild type or the mutant (AF508, R560T or P574H CFTR) cDNAs under the control of the T7 promoter. Login to comment

[hide] Fluorescent multiplex microsatellites used to iden... Hum Genet. 1995 Apr;95(4):462-4. Hughes D, Hill A, Redmond A, Nevin N, Graham C
Fluorescent multiplex microsatellites used to identify haplotype associations with 15 CFTR mutations in 124 Northern Irish CF families.
Hum Genet. 1995 Apr;95(4):462-4., [PMID:7535745]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator gene contains three highly informative microsatellites; IVS8CA, IVS17BTA and IVS17BCA. Fluorescent multiplexes of these microsatellites were assayed in 124 CF families carrying 15 different CFTR mutations, from N. Ireland.

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19 Other common mutations (G551D, R560T, Rll7H and 621 + I G > T) were found associated with the one respective haplotype, suggesting that they emerged in the recent past. Login to comment
22 R560T and RI17H (haplotypes 16-7-17 and 16-30-13, respectively) Table 1 Association of microsatellite haplotypes with 15 CFTR Mutations from N. Ireland. Login to comment
24 All mutations have a distinct associated haplotype, except G551D/R560T, which are both on 16-7-17, and I507/R297Q on 17-7-17. Login to comment
25 Nine haplotypes having specific CF mutations are not present in normal chromosomes Mutation Alleles tested (%) AF508 41 (34.2) 32 (26.7) 22 (18.3) G551D 16 R560T 08 621 + 1G > T 08 Rll7H 05 G542X 03 02 E60X 02 M507 02 R297Q 01 R553X 01 3849 G > A 01 N1303K 01 3659delC 01 557delT 01 Q2X 01 Frequency Haplotype of mutation in % 8AC 17AT 17AC 463 % in normal chromosomes 58.0 23 31 13 - 17 32 13 01 17 31 13 - 4.0 16 07 17 03 2.5 16 07 17 03 1.7 21 31 13 - 2.1 16 30 13 16 1.7 23 33 13 01 22 31 13 - 0.6 16 31 13 03 0.8 17 07 17 08 0.2 17 07 17 08 0.2 17 58 13 - 0.2 16 31 14 - 0.4 23 31 13 - 0.2 16 35 13 03 0.2 15 29 13 - 0.2 23 34 13 01 Table 2 Frequent haplotypes generated from normal and uncharacterised CF chromosomes in N. Ireland. Login to comment

[hide] Cystic fibrosis: genotypic and phenotypic variatio... Annu Rev Genet. 1995;29:777-807. Zielenski J, Tsui LC
Cystic fibrosis: genotypic and phenotypic variations.
Annu Rev Genet. 1995;29:777-807., [PMID:8825494]

Abstract [show]
Cystic fibrosis (CF) is a common genetic disorder in the Caucasian population. The gene was identified in 1989 on the basis of its map location on chromosome 7. The encoded gene product, named cystic fibrosis transmembrane conductance regulator (CFTR), corresponds to a cAMP-regulated chloride channel found almost exclusively in the secretory epithelial cells. Although the major mutation that results in a single amino acid deletion (F508) accounts for 70% of the disease alleles, more than 550 additional mutant alleles of different forms have been detected. Many of these mutations can be divided into five general classes in terms of their demonstrated or presumed molecular consequences. In addition, a good correlation has been found between CFTR genotype and one of the clinical variables--pancreatic function status. An unexpected finding, however, is the documentation of CFTR mutations in patients with atypical CF disease presentations, including congenital absence of vas deferens and several pulmonary diseases. Thus, the implication of CFTR mutation is more profound than CF alone.

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551 FIBROSIS Table 1 Most common CFTR mutations in the world Name of Mutation �F508 0542X 0551D NI303K WI282X R553X 621 + 10 � T 1717-10 � A RI17H R1162X R347P 3849 + IOkbC � T �1507 394delTT 085E R560T A455E 1078deiT 2789 + SO � A 3659deiC R334W 1898 + 10 � T 711 + 10 --. Login to comment

[hide] Independent origins of cystic fibrosis mutations R... Am J Hum Genet. 1994 Nov;55(5):890-8. Morral N, Llevadot R, Casals T, Gasparini P, Macek M Jr, Dork T, Estivill X
Independent origins of cystic fibrosis mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T provide evidence of mutation recurrence in the CFTR gene.
Am J Hum Genet. 1994 Nov;55(5):890-8., [PMID:7526685]

Abstract [show]
Microsatellite analysis of chromosomes carrying particular cystic fibrosis mutations has shown different haplotypes in four cases: R334W, R347P, R1162X, and 3849 + 10kbC-->T. To investigate the possibility of recurrence of these mutations, analysis of intra- and extragenic markers flanking these mutations has been performed. Recurrence is the most plausible explanation, as it becomes necessary to postulate either double recombinations or single recombinations in conjunction with slippage at one or more microsatellite loci, to explain the combination of mutations and microsatellites if the mutations arose only once. Also in support of recurrence, mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T involve CpG dinucleotides, which are known to have an increased mutation rate. Although only 15.7% of point mutations in the coding sequence of CFTR have occurred at CpG dinucleotides, approximately half of these CpG sites have mutated at least once. Specific nucleotide positions of the coding region of CFTR, distinct from CpG sequences, also seem to have a higher mutation rate, and so it is possible that the mutations observed are recurrent. G-->A transitions are the most common change found in those positions involved in more than one mutational event in CFTR.

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112 CT................... 3863: G--oA .................. G-.T ................... 3980: G-jA .................. G--)T.................... 4374+1: G-A .................. G--oT.................... L88S L88X L88X G. Malone, personal communication Savov et al. 1994b Macek et al. 1992 406-1G--.C Bonizzato et al. 1992 406-1G- T T. Bienvenu, personal communication E92K Nunes et al. 1993 E92X Will et al. 1994 S549N Cutting et al. 1990 S5491 Kerem et al. 1990 R560K Ferec et al. 1992 R560T Kerem et al. 1990 Y563D A. Hamosh, personal communication Y563N Kerem et al. 1990 1898+1CG-.A Strong et al. 1992 1898+1GC-.C Cuppens et al. 1993 1898+3A-)C W. Lissens, personal communication 1898+3A--4G Cremonesi et al. 1992 G628R G628R 2183AA- G 2184delA 2184insA M1101K M1101R 3667del4 3667ins4 3791delC T12201 G1244E G1244V R1283K R1283M Fanen et al. 1992 Cuppens et al. 1993 Bozon et al. 1994 Dork et al., in press N. Kilin, personal communication Zielenski et al. 1993 Mercier et al. 1993 Chillon et al. 1994a Sangiuolo et al. 1993 M. Macek, Jr., personal communication Ghanem et al. 1994 Devoto et al. 1991 Savov et al. 1994a Chevalier et al., in press Cheadle et al. 1992 4374+1G-*A Fanen et al. 1992 4374+1G--iT Dork et al. 1993 of the most common allele. Login to comment

[hide] High-density multiplex detection of nucleic acid s... Nucleic Acids Res. 1994 Oct 25;22(21):4527-34. Grossman PD, Bloch W, Brinson E, Chang CC, Eggerding FA, Fung S, Iovannisci DM, Woo S, Winn-Deen ES
High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.
Nucleic Acids Res. 1994 Oct 25;22(21):4527-34., [PMID:7526344]

Abstract [show]
We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

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55 Probes for multiplex CF OLA assay CF Locus S549R2-WT S549R2-MUT S549R2-COM S549N WT S549N-MUT S549N-COM G542-WT G542-MUT G542X-COM R553-WT R553X-MUT R553-COM G551-WT G55ID-MUT G55ICOM W1282-WT W1282X-MUT W1282-COM R560T-WT R560T-MUT R560-COM 1717-WT 1717-MUT 1717-COM 3905-WT 39O51NST-MUT 3905-COM Sequence (5'-3') (HEO)2-TTGCTCGTTGACCTCCA (HEO)3-TTGCTCGTTGACCTCCC PO4-CTCAGTGTGATTCCACCT-FAM (HEO)4-TGCTCGTTGACCTCCAC (HEO)5-TGCTCGTTGACCTCCAT PO4-TCAGTGTGATTCCACCTTC-FAM (HEO)6-GTGATTCCACCTTCTCC (HEO)7-GTGATTCCACCTTCTCA PO4-AAGAACTATATTGTCTTTCTCT-FAM (HEO)8-TGCTAAAGAAATTCTTGCTCG (HEO)9-TTGCTAAAGAAATTCTTGCTCA PO4-TTGACCTCCACTCAGTGTGA-FAM (HEO)IO-TAAAGAAATTCTTGCTCGTTGAC (HEO)11-TAAAGAAATTCTTGCTCGTTGAT PO4-CTCCACTCAGTGTGATTCCA-FAM (HEO) 12-TATCACTCCAA AGGCTTTCCTC (HEO) 13-TATCACTCCAAAGGCTTTCCTT PO4-CACTGTTGCAAAGTTATTGAATCC-FAM (HEO)14-TAGACCAATAATTAGTTATTCACC (HEO)15-TAGACCAATAATTAGTTATTCACG PO4-TTGCTAAAGAAATTCTTGCTCG-FAM (HEO) 16-TCTGCAAACTTGG AGATGTCC (HEO) 17-TCTGCAAACTTGGAGATGTCT PO4-TATTACCAAAAATAGAAAATTAGAGA-FAM (HEO) 18-A AGAGTACTTTGTTATCAGCTTTTTT (HEO)19-AAGAGTACTnUTIATCAGCTnTnT PO4-GAGACTACTGAACACTGAAGGAG-FAM Oligonucleotide ligation assay kinetics study For the study of ligation reaction kinetics, probe concentrations were 5 nM, and oligonucleotide target concentration was 0.5 nM. Login to comment
157 Variations in OLA product yield seen in Figure 4 can more likely be attributed to the high degree of overlap of the probes used in the exon 11 mutational hotspot (G542X, S549N, S549R2, G551D, R553X, R560T). Login to comment

[hide] Mutation analysis in 600 French cystic fibrosis pa... J Med Genet. 1994 Jul;31(7):541-4. Chevalier-Porst F, Bonardot AM, Gilly R, Chazalette JP, Mathieu M, Bozon D
Mutation analysis in 600 French cystic fibrosis patients.
J Med Genet. 1994 Jul;31(7):541-4., [PMID:7525963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 600 unrelated cystic fibrosis (CF) patients living in France (excluding Brittany) was screened for 105 different mutations. This analysis resulted in the identification of 86% of the CF alleles and complete genotyping of 76% of the patients. The most frequent mutations in this population after delta F508 (69% of the CF chromosomes) are G542X (3.3%), N1303K (1.8%), W1282X (1.5%), 1717-1G-->A (1.3%), 2184delA + 2183 A-->G (0.9%), and R553X (0.8%).

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21 Among the 104 other CFTR mutations tested on the 373 non-AF508 CF chromosomes, none of the following 58 mutations were found: G91R, 435 insA, 444delA, D11OH, 556delA, 557delT, R297Q, 1154insTC, R347L, R352Q, Q359K/T360K, 1221delCT, G480C, Q493R, V520F, C524X, 1706dell7, S549R (A-C), S549N, S549I, G551S, 1784delG, Q552X, L558S, A559T, R560T, R560K, Y563N, P574H, 2307insA, 2522insC, 2556insAT, E827X, Q890X, Y913C, 2991de132 (Dork et al, personal communication), L967S, 3320ins5, 3359delCT, H1085R, R1158X, 3662delA, 3667del4, 3667ins4, 3732delA, 3737delA, W1204X, 3750delAG, I 1234V, Q1238X, 3850- 3T-+G, 3860ins31, S1255X, 3898insC, D1270N, R1283M, F1286S, 4005 + I G-A. Forty-six other mutations were found on at Distribution of CFTR mutations found in our sample ofpopulation (1200 CF chromosomes) Mutations tested No of CF chromosomes Haplotypes Method with the mutation XV2C-KM19 (% of total CF alleles) Exon 3: G85E 4 (033) 3C HinfI/ASO394delTT 2 2B PAGEExon 4: R117H 1 B ASOY122X 2 2C MseI/sequenceI148T 1 B ASO621+IG-J* 1 B MseIIASOExon 5: 711+1G--T 8(07) 8A ASOExon 7: AF311 1 C PAGE/sequencelO78delT 5 (0-42) 5C PAGE/ASOR334W 5 (0-42) 2A,2C,ID MspIlASOR347P 5 (042) 5A CfoI/NcoIR347H 1 Cfol/sequenceExon 9: A455E 1 B ASOExon 10: S492F I C DdeI/sequenceQ493X 1 D ASOl609deICA 1 C PAGE/Ddel/sequenceA1507 3 (025) 3D PAGE/ASOAF508 827 (69) 794B,30D,2C,IA PAGEl677delTA 1 A PAGE/sequenceExon I11: 1717-IG--.A 16(1-3) 14B Modified primers + AvaIIG542X 40 (3-3) 29B,5D,2A Modified primers + BstNiS549R(T--*G) 2 2B ASOG551D 3 (025) 3B HincII/Sau3AR553X 10(0-8) 6A,1B,2C,ID Hincll/sequenceExon 12: 1898+IG--A 1 C ASO1898+ IG-C 2 IC ASOExon 13: l9l8deIGC 1 A PAGE/sequence1949de184 I C PAGE/sequenceG628R(G-+A) 2 2A Sequence2118de14 I c PAGE/sequence2143de1T 1 B PAGE/modified primers2184de1A+2183A--*G 11 (0-9) lIB PAGE/ASO2184de1A 1 ASOK710X 3 (025) IC XmnI2372de18 1 B PAGE/sequenceExon 15: S945L 1 C TaqlExon 17b:L1065P I MnlIL1077P 1 A ASOY1092X 3 (025) 2C,IA Rsal/ASOExon 19: RI1162X 6 (0-5) 5C,IA DdeI/ASO3659delC 3 (025) 3C ASOExon 20: G1244E 2 2A MboIIS1251N 2 2C RsaI3905insT 4 (0-33) 4C PAGE/ASOW1282X 18 (105) 15B,1D MnlI/ASOR1283K 1 C Mnll/sequenceExon 21: N1303K 22 (1-8) 18B,lA,ID Modified primers+BstNI 47 mutations 1031 (85 9) least one CF chromosome (table): 21 of them are very rare as they were found on only one CF chromosome in our population. Login to comment

[hide] Sensitivity of single-strand conformation polymorp... Hum Mol Genet. 1994 May;3(5):801-7. Ravnik-Glavac M, Glavac D, Dean M
Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene.
Hum Mol Genet. 1994 May;3(5):801-7., [PMID:7521710]

Abstract [show]
The gene responsible for cystic fibrosis (CF) contains 27 coding exons and more than 300 independent mutations have been identified. An efficient and optimized strategy is required to identify additional mutations and/or to screen patient samples for the presence of known mutations. We have tested several different conditions for performing single-stranded conformation polymorphism (SSCP) analysis in order to determine the efficiency of the method and to identify the optimum conditions for mutation detection. Each exon and corresponding exon boundaries were amplified. A panel of 134 known CF mutations were used to test the efficiency of detection of mutations. The SSCP conditions were varied by altering the percentage and cross-linking of the acrylamide, employing MDE (an acrylamide substitute), and by adding sucrose and glycerol. The presence of heteroduplexes could be detected on most gels and in some cases contributed to the ability to distinguish certain mutations. Each analysis condition detected 75-98% of the mutations, and all of the mutations could be detected by at least one condition. Therefore, an optimized SSCP analysis can be used to efficiently screen for mutations in a large gene.

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67 On the other hand, it was hard to distinguish among mutations G551D (G-A at 1784) R560T t t t t t t t t ait- < •- " •- < t t t i » t t at •a Si t- £ 5 a Figure 5. Login to comment
121 1078delT (35), L327R (Ravnik-Glavac a al., unpublished), R334W (36), D36K (31), R347L (26), R347P (14), A349V (26), R352Q (30), 1221delCT (34); Exon 8: W401X (31), 1342-1G-C (25); Exon 9: G458V (37), 1525 -1G-A (38); Exon 10: S492F (34), Q493X (39), 1609delCA (40,17), deltaI507 (39,41), deltaF5O8 (3), 1717-1G-A (39,42); Exon 11: G542X (39), S549N, G551D, R553X (43), R553Q (44), A559T (43), R560K (Fine et al., pers. comm.), R560T (39); Exon 12: Y563N (39), 1833delT (Schwartz et al., pers. comm.), P574H (39), 1898 + 1G-C (31), 1898+3A-G (Ferrari et al., pers. comm.); Exon 13: G628R(G-C) (31), Q685X (Firec et al., pers. comm.), K716X (26), L719X (Dork etal., pers. comm.), 2522insC (15), 2556insAT (45), E827X (34); Exon 14a: E831X (Ffrec et al., pers. comm.), R851X (29), 2721delll (31), C866Y (Audrezet et al., pers. comm.); Exon 14b: 2789+5G-A (Highsmith et al., pers. comm.); Exon 15: 2907denT (21), 2991del32 (Dark and TQmmler, pers. comm.), G970R (31); Exon 16: S977P, 3100insA (D6rk et al., pers. comm.); Exon 17a: I1005R (Dork and TQmmler, pers. comm.), 3272-1G-A (46); Exon 17b: H1054D (F6rec et al., pers. comm.), G1061R (Fdrec et al., pers. comm.), 332Oins5, R1066H, A1067T (34), R1066L (Fe"rec etal., pers. comm.), R1070Q (46), E1104X (Zielenski el al., pers. comm.), 3359delCT (46), L1077P (Bozon « a/., pers. comm.), H1085R (46), Y1092X (Bozon etal., pers. comm.), W1098R, M1101K (Zielenski et al., pers. comm.); Exon 18: D1152H (Highsmith et al., pers. comm.); Exon 19:R1162X (36), 3659delC (39), 3662delA (25), 3667del4 (Chillon et al., pers. comm.), 3737ddA (35), 3821ddT (15), I1234V (35), S1235R (31), Q1238X (26), 3849G-A (25), 385O-3T-G (38); Exon20:3860ins31 (Chillon etal., pers. comm.), S1255X (47), 3898insC (26), 3905insT (Malik et al., pers. comm.), D127ON (48), W1282X (49), Q1291R (Dork et al., pers. comm.), Exon 21: N1303H (35), N13O3K (50), W1316X (43); Exon 22: 11328L/4116delA (Dork and TQmmler, pers. comm.), E1371X (25); Exon 23: 4374+ 1G-T (38); Exon 24: 4382delA (Claustres et al., pers. comm.). Login to comment

[hide] Analysis of the CFTR gene confirms the high geneti... Hum Genet. 1994 Apr;93(4):447-51. Chillon M, Casals T, Gimenez J, Ramos MD, Palacio A, Morral N, Estivill X, Nunes V
Analysis of the CFTR gene confirms the high genetic heterogeneity of the Spanish population: 43 mutations account for only 78% of CF chromosomes.
Hum Genet. 1994 Apr;93(4):447-51., [PMID:7513293]

Abstract [show]
We have analysed 972 unrelated Spanish cystic fibrosis patients for 70 known mutations. Analysis was performed on exons 1, 2, 3, 4, 5, 6a, 6b, 7, 10, 11, 12, 13, 14a, 14b, 15, 16, 17b, 18, 19, 20 and 21 of the cystic fibrosis transmembrane regulator gene using single strand conformation polymorphism analysis and denaturing gradient gel electrophoresis. The major mutation delta F508 accounts for 50.6% of CF chromosomes, whereas another 42 mutations account for 27.6% of CF chromosomes, with 21.8% of Spanish CF chromosomes remaining uncharacterized. At present, we have identified 36 mutations that have frequency of less than 1% and that are spread over 15 different exons. This indicates that, in the Spanish population, with the exception of delta F508 (50.6%) and G542X (8%), the mutations are not concentrated in a few exons of the gene nor are there any predominating mutations. This high degree of genetic heterogeneity is mainly a result of the different ethnic groups that have populated Spain and of the maintenance of separated population sets (Basques, Arab-Andalusian, Mediterranean, Canarian and Gallician). The high proportion of CF chromosomes still unidentified (21.8%) together with association analysis with intragenic markers suggest that at least 100 different mutations causing CF are present in our population.

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31 At present, we have not detected any Spanish CF chromosomes bearing any of the following mutations: 394delTA, Y122X, 556delA, 852de122, R347P, $492F, 1677delTA, V520F, Q552X, R553X, L559S, R560K, R560T, Y563N, P564H, 2043delG, 3320ins5, R1066H, A1067T, H1085R, 3732delA, 3737delA, I1234V, S1255P, 3898insC, Q1291H or 4005+ 1G---~A. Login to comment

[hide] Genetic analysis of Hispanic individuals with cyst... Am J Hum Genet. 1994 Mar;54(3):443-6. Grebe TA, Seltzer WK, DeMarchi J, Silva DK, Doane WW, Gozal D, Richter SF, Bowman CM, Norman RA, Rhodes SN, et al.
Genetic analysis of Hispanic individuals with cystic fibrosis.
Am J Hum Genet. 1994 Mar;54(3):443-6., [PMID:7509564]

Abstract [show]
We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry delta F508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849 + 10kbC-->T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of delta F508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.

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45 The following CFTR gene mutations were identified by published methods: AF508 (Rommens et al. 1990); G542X (Kerem et al. 1990); GS51D and R553X (Cutting et al. 1990); R1162X (Gasparini et al. 1991); W1282X (Vidaud et al. 1990); N1303K (Osborne et al. 1991); 3849 +lOkbC- T (Highsmith et al., submitted); and R117H, Y122X, 1148T, 621+1G-*oT, 711+1G- T, G314E, 1078AT, R334W, R347P, Q493X, A1507, V520F, 1717 -1G-oA, R560T, and 3569AC (J. DeMarchi et al., submitted). Login to comment
54 COther = A1507, 621+1G- T, R117H, N1303K, 711+1G-*.T, 1717-1G-.A, R560T, Y122X, 1148T, G314E, 1078AT, R347P, Q493X, V520F, and 3659AC. Login to comment
56 The G542X mutation was found in 5.4% of Hispanic CF chromosomes, similar to the 3% frequency in the general population. Login to comment
47 The following CFTR gene mutations were identified by published methods: AF508 (Rommens et al. 1990); G542X (Kerem et al. 1990); GS51D and R553X (Cutting et al. 1990); R1162X (Gasparini et al. 1991); W1282X (Vidaud et al. 1990); N1303K (Osborne et al. 1991); 3849 +lOkbC-T (Highsmith et al., submitted); and R117H, Y122X, 1148T, 621+1G-*oT, 711+1G-T, G314E, 1078AT, R334W, R347P, Q493X, A1507, V520F, 1717 -1G-oA, R560T, and 3569AC (J. DeMarchi et al., submitted). Login to comment

[hide] Direct sequencing of the complete CFTR gene: the m... Hum Mol Genet. 1993 Oct;2(10):1551-6. Cheadle JP, Goodchild MC, Meredith AL
Direct sequencing of the complete CFTR gene: the molecular characterisation of 99.5% of CF chromosomes in Wales.
Hum Mol Genet. 1993 Oct;2(10):1551-6., [PMID:7505689]

Abstract [show]
We have performed an extensive mutation analysis on 184 CF families in Wales. In our previous study, mutations on 329/369 CF chromosomes were identified after screening for delta F508 and sixteen other mutations. To identify the mutations on the remaining 40 uncharacterized CF chromosomes, we have carried out direct DNA sequencing over the complete coding region, intron splice sites, and part of the promoter region of the CFTR gene. During this study we have designed a set of internal sequencing primers which allow clear sequencing through the aforementioned regions. Sequence analysis revealed 15 further mutations (4 of which are novel), and 10 previously described polymorphisms. In total, we have identified 29 mutations, the distribution of which provides further insight into the functional domains of the CFTR protein. We have characterised 99.5% of the CF chromosomes (365/367, one sample degraded). In order to ascertain accurate frequency data for the Welsh population, CF families with at least 3 'Welsh' grandparents were strictly regarded as 'Welsh'. Of these 91 families, delta F508 accounts for 71.6%, 621 + 1G-->T 6.6% and 1898 + 1G-->A 5.5%. The implications for CF population screening in Wales are discussed.

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64 17b 18 19 20 21 22 23 24 M1V E60X O220X | R117H G85E 621*1G>T 977msA AF508 I AI607 1898*1G>A G642X 2184delA 1078delT I I 8549N 2184insA I I 1154insTC S549R G651D I R553X I R560T I 1717-1G>A W846X1 3272-26A>G L1077P R12B3U 3669delC 4 016in aT I N1303K 3849*10kbC>T Figure 2. Login to comment
115 Further ARMS tests are in development and in principle it should be possible to use a three tube ARMS test to assay rapidly for 13 mutations (delta F508, 621 + 1G-T, G551D, G542X, R1283M, W1282X, R553X, N13O3K, delta 1507, 1898+1G-A, R117H, R560T, and 1717-1G-A) (S.Little, personal comm.). Login to comment

[hide] Cystic fibrosis mutations impair the fertilization... Hum Reprod. 1993 Aug;8(8):1259-63. Patrizio P, Ord T, Silber SJ, Asch RH
Cystic fibrosis mutations impair the fertilization rate of epididymal sperm from men with congenital absence of the vas deferens.
Hum Reprod. 1993 Aug;8(8):1259-63., [PMID:7691870]

Abstract [show]
One of the limiting factors for the successful treatment of male sterility due to congenital absence of the vas deferens (CAVD) is the low (< 20%) and extremely unpredictable rate of in-vitro fertilization of their epididymal spermatozoa. The recent demonstration that CAVD is a mild form of cystic fibrosis (CF) disease, almost exclusively involving the genital tract, has prompted us to investigate the hypothesis of whether there could be an association between particular CF genotype mutations and the in-vitro performance of epididymal sperm. In this study 63 patients with surgically confirmed diagnosis of CAVD undergoing microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF) were evaluated. The genetic screening was carried out on DNA extracted from peripheral lymphocytes and amplified by the polymerase chain reaction. A total of 12 mutations in the cystic fibrosis transmembrane regulator gene (CFTR), representing approximately 90% of the total known CF mutations, were tested. The presence or absence of mutations, as well as the type of mutation found, was correlated with the IVF and pregnancy rates. Of the 63 patients examined, 40 (64%) were positive to the CF screening and 23 were negative. In three cases no spermatozoa were found because of rete testis blockage. The difference in fertilization rates between patients testing positive and negative was highly significant (P = 0.000003), 13 versus 23%, respectively. In order to pinpoint which mutation could account for the most deleterious effect on the IVF results, we analysed these by keeping the patients separated by CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

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38 Briefly, genomic DNA extracted from peripheral lymphocytes was amplified by the polymerase chain reaction (PCR) and analysed for 12 mutations in the CFTR gene: Delta F508, G542X, G551D, R553X, W1282X, N1303K, Delta 1507, 1717G-A, R560T, S549N, R117H and 621 + 1. Login to comment

[hide] A novel mutation in exon 3 of the CFTR gene. Hum Genet. 1993 Apr;91(3):233-5. Guillermit H, Jehanne M, Quere I, Audrezet MP, Mercier B, Ferec C
A novel mutation in exon 3 of the CFTR gene.
Hum Genet. 1993 Apr;91(3):233-5., [PMID:7682984]

Abstract [show]
We have screened the 27 exons of the cystic fibrosis transmembrane conductance regulator gene in 87 non-delta F508 chromosomes of Breton origin using the combined techniques of denaturing gradient gel electrophoresis and direct sequencing. By this process, we have detected a new missense mutation, G91R, which results in an arginine for glycine at codon 91. Three affected patients with a delta F508/G91R genotype are pancreatic sufficient. Such observations could facilitate a better understanding of the functional importance of different regions of the encoded product and of the pathogenesis of the disease.

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60 Most of the missense mutations so far reported in the first NBF (G551D, V520F, R560T, etc.) result in a severe phenotype. Login to comment

[hide] Multiplex PCR amplification from the CFTR gene usi... Hum Mol Genet. 1993 Feb;2(2):159-63. Richards B, Skoletsky J, Shuber AP, Balfour R, Stern RC, Dorkin HL, Parad RB, Witt D, Klinger KW
Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs.
Hum Mol Genet. 1993 Feb;2(2):159-63., [PMID:7684637]

Abstract [show]
Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

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108 observed AF5O8 G542X G551D W1282X R553X R560T 1717-1 N1303K 621 + 1 R117H S549N 1507 280 7 16 2 4 2 2 5 6 11 1 •Includes affected individuals and known carriers. Login to comment

[hide] Efficient 12-mutation testing in the CFTR gene: a ... Hum Mol Genet. 1993 Feb;2(2):153-8. Shuber AP, Skoletsky J, Stern R, Handelin BL
Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis.
Hum Mol Genet. 1993 Feb;2(2):153-8., [PMID:7684636]

Abstract [show]
The identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has led to the identification of more than 225 presumed disease-causing mutations at the locus. The diagnosis of cystic fibrosis or the carrier state by direct DNA analysis is hindered by this large number. A practical assay must be able to detect enough mutations to achieve clinically significant sensitivity. The use of allele-specific oligonucleotide probes is the most promising of the available methods. However, to date this has generally involved tedious probe-by-probe hybridizations, due to variations in the oligonucleotides' denaturation temperatures caused by differences in their G-C base-pair content. We have developed a rapid, cost-effective assay that simultaneously detects 12 CFTR mutations after multiplex polymerase-chain-reaction amplification of genomic DNA. The test may be readily extended to detect additional mutations at minimal increase in the cost per test or the turnaround time. We improve specificity and avoid the need for individual hybridizations by the use of tetramethylammonium chloride to virtually eliminate the effects of G-C differences. Coupled with non-invasive sample-collection methods, this is an immediately practical assay for cystic fibrosis. More generally, it will serve as a model for the development of diagnostic tests in other genetic disorders involving complex mutation analysis.

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50 Percent GC content of allele-speciik oligonucleotides GC Content/ASO Mutation G542X G551D R553X W1282X N1303K DI507 1717-1 G - A R560T S549N R117H 621 + 1 G - T 40 53 53 47 41 30 41 53 53 47 30 HS«3X W12»2X N1J0JK £507 H117M • 21 SS4tN RSOOT Table 2. Login to comment
53 1) Number of ASOs (N = 12) 2) Mutation Frequencies # ofCF Mutation Chromosomes Frequency A508 G551D G542X W1282X N1303K R553X 621 + 1 G - T R117H 1717-1 G - A R560T AI507 S549N unknown Total 548 15 13 13 12. Login to comment
54 9 9 '3 3 2 1 1 133 764 • Minimal Number of Independent Conclusion (4 hybridizations) Filter # 1 N(A5O8) 2 A5O8 72.0% 2.0% 1.7% 2.0% 1.5% 1.1% 1.1% 0.4% 0.4% 0.3% 0.1% 0.1% 17.0% Hybridizations 3 4 G551D 621 + 1 G - T G542X R117H W1282X 1717-1 G - A N13O3K R560T R553X A507 S549N with the remaining six mutations: 621 + 1 G^T, R117H, 1717-1 G - A , R560T, AI507, and S549N. Login to comment
61 These results were scored as 1010 for the sample 2 3 4 5 6 7 8 9 10 N(A5.6) A ( + )Conlrol> 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 M C o n l r o l s ' 2 3 4 5 6 7 8 * 1 0 1 1 1 2 OS42X Pool GS51D , R553X W 1 2 6 2 X N1303K < 2 3 4 S 8 7 8 9 10 1 1 1 2 /.507 R1 17H Peel 62 1 + 1 2 S54»N R560T 1 7 1 7 - 1 5: in lane 4, rows D and E; 1001 for the sample in lane 3, rows D and E; and 1101 for the sample in lane 5, rows D and E. Login to comment
72 Example of results obtained from hybridizing four identical filters with ASOs specific for N (the normal sequence at position 508), AF508 (a 3 bp deletion at position 508), pool 1 (G542X, G551D, R553X, W1282X, and N13O3K), and pool 2 (AI507, R117H, 621 + 1 G - T , S549N, R560T, 1717-1 G-A). Login to comment
73 Row A contains positive control samples with the following genotypes: 1A (AF508/AF508); 2A (AF508/G542X); 3A (AF5O8/G551D); 4A (N/R553X); 5A (NAV1282X); 6A (AF5O8/N13O3K); 7A (N/AI5O7); 8A (N/R117H); 9A (N/621 + 1 G-T); 10A (AF508/S549N); HA (AF508/R560T); and 12A (N/1717-1 G-A). Login to comment
80 Pool 2: lane 1 (AI507); lane 2 (Rl 17H); lane 3 (621 +1 G-T); lane 4 (S549N); lane 5 (R560T); lane 6 (1717-1 G - A ) . Login to comment
86 Row A contains positive control samples: lane 2 (G542X); lane 3 (G551D); lane 4 (R553X); lane 5 (W1282X); lane 6 (N13O3K); lane 7 (AI507); lane 8 (R117H); lane 9 (621 +1 G-T)); lane 10 (S549N); lane 11 (R560T); lane 12 (1717-1 G-A)). Login to comment

[hide] Aetiology of congenital absence of vas deferens: g... Hum Reprod. 1993 Feb;8(2):215-20. Patrizio P, Asch RH, Handelin B, Silber SJ
Aetiology of congenital absence of vas deferens: genetic study of three generations.
Hum Reprod. 1993 Feb;8(2):215-20., [PMID:8473422]

Abstract [show]
Bilateral congenital absence of the vas deferens (CAVD) is a form of male sterility (found in otherwise normal men) of unknown aetiology. Because males with cystic fibrosis (CF) almost invariably have CAVD as well, we investigated the hypothesis that men with isolated CAVD might share a common genetic background with males with CF. Genetic testing for CF was carried out in three generations of subjects: 44 patients with CAVD and their wives, 24 of their parents, and 13 of their offspring generated by microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF). DNA extracted from peripheral lymphocytes was amplified by the polymerase chain reaction (PCR) and then analysed for 12 mutations in the cystic fibrosis transmembrane conductance regulatory (CFTR) gene. Among 44 patients tested with CAVD, 26 (59%) were positive for at least one CF mutation, while the carrier frequency for CF mutations in the general population is only 4%. Four patients were found to be compound heterozygotes, three with genotypes Delta F-508/R117H, one with R553X/R117H. Among 24 parents tested, 15 (seven fathers, eight mothers) had sons with CAVD who were positive for CF mutations. Of these, nine (four fathers and five mothers) were found to be carriers for CF mutations. These four fathers, although carriers of CF mutations, were obviously fertile. Of the 13 offspring tested, six (three boys and three girls) had CF positive fathers. Of these, three (two girls and one boy) were found to be carriers for CF mutations. These MESA/IVF children are the first offspring to whom men with CAVD have been able to transmit CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

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51 Exon 4,10,11,20 and 21 were amplified in a multiplex reaction followed by allele specific oligonucleotide (ASO) probe analysis for the mutations Delta F508, G542X, G551D, R553X, W1282X, N13O3K, Delta 1507, 1717G-A, R560T, S549N, R117H and 621 + 1. Login to comment

[hide] Effects of the delta F508 mutation on the structur... J Bioenerg Biomembr. 1993 Feb;25(1):11-9. Thomas PJ, Pedersen PL
Effects of the delta F508 mutation on the structure, function, and folding of the first nucleotide-binding domain of CFTR.
J Bioenerg Biomembr. 1993 Feb;25(1):11-9., [PMID:7680027]

Abstract [show]
The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (delta F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a beta-stranded structure in solution. The delta F508 mutation causes a diminution in the amount of beta-stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that delta F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of delta F508 in situ and for the rational design of new CF drugs.

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107 For example, the G458V (Cuppens et al., 1990) and G1244E (Devoto et al., 1991) mutations which change the first glycine residue in the Gx 4GKT sequence of the A homology region and the R560T (Kerem et al., 1990) mutation in the Rx6.sh4D sequence of the B homology region are known to cause CF. Login to comment

[hide] Mutation analysis of 184 cystic fibrosis families ... J Med Genet. 1992 Sep;29(9):642-6. Cheadle J, Myring J, al-Jader L, Meredith L
Mutation analysis of 184 cystic fibrosis families in Wales.
J Med Genet. 1992 Sep;29(9):642-6., [PMID:1357180]

Abstract [show]
We describe a molecular analysis of 184 cystic fibrosis (CF) families in Wales. To determine accurate frequency data for the CF mutations in the Welsh population, families with at least three Welsh grandparents were strictly regarded as Welsh. Of these 74 families, we have identified approximately 90% of mutations causing CF, with delta F508 accounting for 71.8% and 621 + 1G greater than T 6.7%. We observed a significant difference between the Welsh and Scottish frequencies of 621 + 1G greater than T. To allow the rapid and efficient screening for the more common mutations we modified a multiplex used by Watson et al enabling the detection of delta F508, G551D, and R553X simultaneously with 621 + 1G greater than T. In parallel to this system we ran the Cellmark Diagnostics ARMS multiplex kit, which detects delta F508, 621 + 1G greater than T, G551D, and G542X. RFLP analysis of the 184 families shows that the delta F508 chromosomes are almost exclusively found on the B haplotype (XV2c 1, KM19 2); the other CF mutations have more heterogeneous backgrounds. Strong haplotype correlations exist between the markers XV2c, KM19, D9, and G2 and the other CF mutations. Haplotype data suggest that there are at least seven mutations that remain to be identified in these families.

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61 Welsh Mixed Undefined Total Mutation No % No % No % No % AF508 107/149 71-8 92/126 73 0 69/94 73 4 268/369 72-6 621 + 1G>T 10/42* 6-7 5/34* 4-0 4/25* 4-3 19/101* 51 G551D 2/42* 1-3 6/34* 4-8 3/25* 3-2 11/101* 3 0 G542X 4/42* 2-7 4/34* 3-2 1/25* 1.1 9/101* 2-4 G85E 0/41* 0-0 2/34* 1 6 3/24* 3*4 5/99* 1-4 R553X 2/42* 1-3 2/34* 16 0/25* 00 4/101* 1-1 R1283M 3/42* 2.0 0/34* 0.0 0/25* 0.0 3/101* 0-8 N1303K 1/42* 0 7 1/34* 0-8 0/24* 0.0 2/100* 0-6 AI507 2/149 1-3 0/126 0.0 0/94 0.0 2/369 0-5 R117H 1/42* 0 7 1/34* 0-8 0/25* 0.0 2/101* 0-5 1717- 1G>A 2/42* 1-3 0/34* 0 0 0/25* 0 0 2/101* 0-5 R560T 0/42* 00 0/34* 00 1/25* 1 1 1/101* 03 1154InsTC 0/40* 0 0 1/33* 0 9 0/24* 0.0 1/97* 0-3 V520F 0/42* 0 0 0/34* 0 0 0/25* 0.0 0/101* 0 0 W1282X 0/42* 0 0 0/34* 0.0 0/25* 0.0 0/101* 0 0 R347P 0/42* 0 0 0/34* 0 0 0/24* 0.0 0/100* 0 0 Q493X 0/42* 0 0 0/34* 0 0 0/24* 0 0 0/100* 00 Total (%) 89-8 90 7 86-5 891 * Non-AF508 chromosomes. Login to comment
97 We wish to thank Professor P S Harper, Dr D Shaw, and Dr A Clarke for their useful comments on this manuscript, Dr L Sandkuijl for his help with the statistical analyses, Dr M 0 621 + 1G> T Total G551D Total G542X Total G85E Total R553X Total R1283M Total N1303K Total AI507 Total RI 17H Total 1717-1 Total R560T Total 1154Ins TC Total 0 1 1 o o o 0 o 0 0 2 0 o o 0 1 0 0 I 1 1 1 I I I Al-Jader, Meredith Table S Distribution of haplotypes in the uncharacterised CF chromosomes. Login to comment

[hide] Development, multiplexing, and application of ARMS... Am J Hum Genet. 1992 Aug;51(2):251-62. Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G, Little S
Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.
Am J Hum Genet. 1992 Aug;51(2):251-62., [PMID:1379414]

Abstract [show]
The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.

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65 ARMS tests were developed for the following mutations: AF508 (Riordan et al. 1989), A1507 (Schwarz et al. 1991), R117H (Dean et al. 1990), 621 + 1G-T (Zielenski et al. 1991), 1717G--oA (Guillermit et al. 1990), W1282X (Vidaud et al. 1990), GSS1D and R553X (Cutting et al. 1990), G542X and R560T (Kerem et al. 1990), and N1303K (Osborne et al. 1991). Login to comment
115 The mutations for which ARMS tests were developed were R560T, R553X, GS5iD, G542X, 1717-1G--A, 621 + 1G--T, N1303K, and W1282X. Login to comment
136 Exon 11: TAAAATTTCAGCAATGTTGTTTTTGACC ............ R560T GCTTGCTAGACCAATAATTAGTTATTCATC. Login to comment
139 CCTATGCCTAGATAAATCGCGATAGAAC ............. CCTATGCCTAGATAAATCGCGATAGAAT ............ N1303K:b CTCAATTTCTTTATTCTAAAGACATTGG ............ GATCACTCCACTGTTCATAGGGATCCAAG .......... GATCACTCCACTGTTCATAGGGATCCAAC ........... 621 + 1G>T: TCACATATGGTATGACCCTCTATATAAACT. Login to comment
155 Shown are the results a n m n m R560T 317bp c n m n m 0551 D 286bp obtained using primers RH-d-N and RH-d-M at 0.86 pM (a), at reduced primer concentration 0.10 ItM (b), at 0.86 FM with the inclusion of AAT1 /2 control primers (c), and using primers RHh-N and RH-h-M (which are identical to RH-d-N/M, except for C/C mismatch at position - 2). Login to comment
66 ARMS tests were developed for the following mutations: AF508 (Riordan et al. 1989), A1507 (Schwarz et al. 1991), R117H (Dean et al. 1990), 621 + 1G-T (Zielenski et al. 1991), 1717G--oA (Guillermit et al. 1990), W1282X (Vidaud et al. 1990), GSS1D and R553X (Cutting et al. 1990), G542X and R560T (Kerem et al. 1990), and N1303K (Osborne et al. 1991). Login to comment
116 The mutations for which ARMS tests were developed were R560T, R553X, GS5iD, G542X, 1717-1G--A, 621 + 1G--T, N1303K, and W1282X. Login to comment
158 Shown are the results a n m n m R560T 317bp c n m n m 0551 D 286bp obtained using primers RH-d-N and RH-d-M at 0.86 pM (a), at reduced primer concentration 0.10 ItM (b), at 0.86 FM with the inclusion of AAT1 /2 control primers (c), and using primers RHh-N and RH-h-M (which are identical to RH-d-N/M, except for C/C mismatch at position - 2). Login to comment

[hide] Incidence and expression of the N1303K mutation of... Hum Genet. 1992 Aug;89(6):653-8. Osborne L, Santis G, Schwarz M, Klinger K, Dork T, McIntosh I, Schwartz M, Nunes V, Macek M Jr, Reiss J, et al.
Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene.
Hum Genet. 1992 Aug;89(6):653-8., [PMID:1380943]

Abstract [show]
The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15,000 CF chromosomes tested, accounts for 1.5% of all CF chromosomes. The frequency of the N1303K allele varies significantly between countries and ethnic groups, being more common in Southern than in Northern Europe. This variation is independent of the delta F508 allele. It was not found on UK Asian, American Black or Australian chromosomes. N1303K is associated with four different linked marker haplotypes for the polymorphic markers XV-2c, KM.19 and pMP6d-9. Ten patients are homozygous for this mutation, whereas 106 of the remainder carry one of 12 known CF mutations in the other CF allele. We classify N1303K as a "severe" mutation with respect to the pancreas, but can find no correlation between this mutation, in either the homozygous or heterozygous state, and the severity of lung disease.

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75 N/A data not available Genotype Number Meana (range) age Male/ Pancreatic status Meconium Sputum female ileusb colonisation ofof patients Current At diagnosis PS PI P.aeruginosa Yes No Mean (range) of FEVj percentage of that predicted AF508 79 10 0.7 • 0.6 16 / 20 0 55 10/58 20 8 N1303K (0.5-36) (0-2.5) N1303K 10 7 1 • 1 3/3 0 6 1/6 3 1 N1303K (3-12) (0.1-1.5) G551D 6 19 9.7 • 2.2 1/ 2 0 4 0/4 3 1 N1303K (17-22) (8-12) G542X 8 7 0 • 0 2/3 0 4 4/5 0 2 N1303K (0.1-12) 621+lG---~T 1 22 18 1/0 0 1 0/1 1 0 N1303K W1282X 4 13.5 0.7 • 0.6 3 / 1 0 4 0/4 0 1 N1303K (5-23) (0.25-1.7) R560T 1 5 41 1 / 0 0 1 0/1 0 1 N1303K R553X 1 N/A N/A N/A 0 1 0/1 N/A N/A N1303K R334W 1 19 N/A 1/0 0 1 0/1 N/A N/A N1303K R1162X 1 N/A N/A N/A N/A N/A N/A N/A N/A N1303K 1717-1G---~A 2 7 N/A 0/1 N/A N/A N/A N/A N/A N1303K 3659delC 1 21 74 0/1 0 1 0/1 1 0 N1303K 1078delT 1 N/A N/A N/A N/A N/A N/A N/A N/A N1303K Other 90 12 4.3 • 5.6 9/9 5 19 2/17 4 10 N1303K (2-24) (0.1-15) 63 (32-101) 65 (46-84) 80 (66-93) N/A 55 70 N/A N/A N/A N/A N/A 26 N/A 66 a Mean +_SD b Shown as a fraction of the patients for whom data was available the percentage of FEV1 between age-matched N1303K homozygotes and AF508/N1303K heterozygotes (65% vs 75%, P> 0.1). Login to comment

[hide] Analysis of four diverse population groups indicat... Am J Hum Genet. 1992 Jun;50(6):1185-94. Cutting GR, Curristin SM, Nash E, Rosenstein BJ, Lerer I, Abeliovich D, Hill A, Graham C
Analysis of four diverse population groups indicates that a subset of cystic fibrosis mutations occur in common among Caucasians.
Am J Hum Genet. 1992 Jun;50(6):1185-94., [PMID:1376017]

Abstract [show]
To determine the nature and frequency of non-delta F508 cystic fibrosis (CF) mutations among diverse populations, we have sequenced exons 9-12 and 19-23 of the CF transmembrane conductance regulator (CFTR) gene from 128 CF chromosomes (39 U.S. Caucasian, 27 African-American, 42 Northern Irish, and 20 Israeli chromosomes). These regions were chosen because they encode the two putative ATP-binding folds of CFTR, domains which appear to have functional significance. In addition, CFTR exons 1 and 2 were analyzed in the American patients. Mutations were found on 49 of the 128 CF chromosomes. Nineteen different mutations were observed; six were novel, while the remaining 13 had been reported previously by our group or by other investigators. Six of nine different mutations found in African-American patients were unique to that population. However, the vast majority of the mutations found in U.S. Caucasians (eight of nine), Northern Irish (four of five), and Israelis (three of three) also occurred in other Caucasian groups. The preponderance of previously reported mutations in these three groups suggested that a subset of the non-delta F508 mutations occur in common among Caucasians. A survey of mutation frequencies in other Caucasian groups confirmed this observation. Unfortunately, this subset accounts for less than half of non-delta F508 CF mutations in most groups. These data suggest that screening for delta F508 and this select group of mutations will efficiently and economically maximize the number of CF mutations identified in Caucasian groups. However, it will be difficult to detect more than 90% of mutant CFTR alleles except in ethnically and geographically discrete populations where CF is the result of founder effect.

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104 Another exon 11 mutation, R560T, originally reported on a single chromosome (Kerem et al. 1990) was found five times in the Northern Irish. Login to comment
150 The frequent observation of the R560T mutation in Northern Irish may be an example of drift. Login to comment

[hide] Genetic determination of exocrine pancreatic funct... Am J Hum Genet. 1992 Jun;50(6):1178-84. Kristidis P, Bozon D, Corey M, Markiewicz D, Rommens J, Tsui LC, Durie P
Genetic determination of exocrine pancreatic function in cystic fibrosis.
Am J Hum Genet. 1992 Jun;50(6):1178-84., [PMID:1376016]

Abstract [show]
We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.

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10 This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as AF508, A1507, Q493X, G542X, R553X, W1282X, 621 + 1G-PT, 1717-1G--'A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T. Login to comment
58 Intron 10: 1717-1G-'A Exon 11: G542X .......... S549R ........... G551D .......... R553X .......... R560T .......... Exon 12: Y563N .......... P574H .......... Exon 19: 3659delC ....... Exon 20: W1282X ....... Exon 21: N1303K ..... G460-C A deletion G482-'A A deletion T575-C 621 + 1G-T C1132-T C1172- G C1496-A G1505-'T G1570-T C1609-T 3-bp deletion 3-bp deletion G1690-T G1717-1-A G1756-T T1779-G G1784- A C1789-T G1811-C T1819- A C1853- A C deletion G3978-A C4041-G Asp 110-His Frameshift Arg 117-His Frameshift Ile 148-Thr Splice mutation Arg 334-Trp Arg 347-Pro Ala 455- Glu Gly 458-'Val Gly480-Cys Gln 493- stop del of Ile 507 del of Phe 508 Val 520-Phe Splice mutation Gly 542- stop Ser 549-'Arg Gly 551-WAsp Arg 553- stop Arg 560- Thr Tyr 563- Asn Pro 574-His Frameshift Trp 1282-stop Asn 1303-Lys Dean et al. 1990 White et al. 1991 Dean et al. 1990 Zielenski et al. 1991a F. Rininsland, D. Bozon, and L.-C. Tsui, unpublished data Zielenski et al. 1991a Gasparini et al. 1991 Dean et al. 1990 Kerem et al. 1990b Cuppens et al. 1990 Strong et al. 1991 Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1989b Jones et al. 1991 Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1990b Cutting et al. 1990 Cutting et al. 1990 Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1990b Vidaud et al. 1990 Osborne et al. 1990 PI or PS, but not with both. Login to comment
66 As shown in table 3, meconium ileus Table 2 1181 Table 3 Frequency of 25 CF Mutations in Chromosomes of the Toronto Study Population Mutation AF508 ...... G551D...... G542X...... 621 +1G-'T N1303K..... W1282X..... R1 17H...... 1717-1G-~A R560T...... A1507 ...... R553X...... V52OF ...... R334W ..... A455E...... I148T ...... Q493X...... P574H...... R347P ...... SS6delA ..... 3659delC .... G480C...... 444delA ..... D110H...... G458V...... S549R ...... Y563N...... Login to comment
73 Complete CF Genotypes for 394 Patients No. OF PATIENTS GENOTYPE WITH Allele 1 Allele 2 pla P AF508 ...... AF508 277 (49) 2 G551D 21 (1) 0 G542X 18 (9)c 0 621+1G-~T 11 (1) 0 AI507 7 (1) 0 N1303K 6 (1) 0 R560T 5 0 1717-lG-A 5 (1) 0 556delA 3 0 Q493X 3 0 R553X 3 (1) 0 W1282X 3 0 3659delC 2 0 1148T 1 0 R117H 0 9 A445E 0 2 P574H 0 2 R347P 0 1 G551D ..... 1717-lG-~A 2 0 621+1G-~T 1 0 G480C 1 0 G551D 1 0 V520F 1 (1) 0 G542X ..... V520F 1 0 1148T ...... W1282X 1 (1) 0 W1282X .... W1282X 1 0 N1303K .... R553X 1 (1) 0 R117H ..... R117H 0 1 G542X 0 1 R334W ..... R334W 0 1 a1 Numbers in parentheses are number of patients with neonatal meconium ileus. Login to comment
85 Accordingly, the mutations R117H, R334W, R347P, A455E, and P574H may be regarded as mild, whereas AF508, AI507, Q493X, G542X, R553X, W1282X, 621 + 1G-T, 1717-1G--A, 556delA, 3659delC, 1148T, G480C, V520F, GSS1D, and R560T are severe. Login to comment

[hide] A new missense mutation (R1283M) in exon 20 of the... Hum Mol Genet. 1992 May;1(2):123-5. Cheadle JP, Meredith AL, al-Jader LN
A new missense mutation (R1283M) in exon 20 of the cystic fibrosis transmembrane conductance regulator gene.
Hum Mol Genet. 1992 May;1(2):123-5., [PMID:1284468]

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5 From a sample of 373 CF chromosomes, 318 proved to have one of the following mutations: delta F508, delta 1507, G551D, R553X, G542X, R117H, 1717-1G>A, R560T and 621 + 1G>T. Login to comment

[hide] The gene defect in cystic fibrosis and clinical ap... J R Soc Med. 1992;85 Suppl 19:6-8. Super M
The gene defect in cystic fibrosis and clinical applications of the knowledge.
J R Soc Med. 1992;85 Suppl 19:6-8., [PMID:1375961]

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51 Factors outside the gene seem to be associated with differing 1008 818 81.15% levels ofseverity and, ofcourse, one must remember G551D 215* 34 2.986 differing environmental influences such as time of G542X 203* 11 1.02% diagnosis, vigour oftreatment and social class which R560T 113* 6 1.00% may play a role. Login to comment

[hide] Effects of cystic fibrosis and congenital bilatera... Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4. Mickle JE, Milewski MI, Macek M Jr, Cutting GR
Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels.
Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4., [PMID:10762539]

Abstract [show]
The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.

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47 DNA was assayed for 16 common CFTR mutations (R117H, 62111GrT, R334W, R349P, A455E, DI507, DF508, 1717-1GrA, G542X, S549N, G551D, R553X, R560T, 3849110 Kb CrT, W1282X, and N1303K), by reverse dot-blot hybridization (Mickle et al. 1998). Login to comment

[hide] Pancreatic function and extended mutation analysis... J Pediatr. 2000 Aug;137(2):214-20. Massie RJ, Wilcken B, Van Asperen P, Dorney S, Gruca M, Wiley V, Gaskin K
Pancreatic function and extended mutation analysis in DeltaF508 heterozygous infants with an elevated immunoreactive trypsinogen but normal sweat electrolyte levels.
J Pediatr. 2000 Aug;137(2):214-20., [PMID:10931414]

Abstract [show]
BACKGROUND: Newborn screening for cystic fibrosis (CF) with immunoreactive trypsinogen (IRT) and DeltaF508 analysis followed by sweat testing misses some infants with CF and detects more DeltaF508 carriers than expected. Some of the apparent DeltaF508 carriers may be DeltaF508 compound heterozygotes with normal sweat electrolyte levels. METHODS: Infants identified by newborn screening with an elevated IRT level, one DeltaF508 allele, and a sweat chloride level <60 mmol/L underwent CF mutation analysis, pancreatic stimulation testing, and repeat IRT analysis followed by clinical review and repeat sweat test at 12 months. RESULTS: Over a 24-month period we identified 122 DeltaF508 heterozygotes and recruited 57; 4 had borderline sweat chloride levels (40 to 60 mmol/L), 5 (8.8%, 95% CI 1.4, 16.2) had a second CF mutation (R117H), and 11 (20%, 95% CI 10, 30) had the intron 8 5T allele. Three had clinical CF at 12 months (initial sweat chloride levels: 53, 51, and 32 mmol/L). Pancreatic electrolyte secretion in the subjects with a borderline sweat chloride level was similar to that in patients with known CF. CONCLUSION: The excess of DeltaF508 heterozygotes detected by IRT/DNA screening is associated with the presence of a second mutation or the 5T allele in some infants. Screened infants with borderline sweat chloride levels almost certainly have CF, but long-term follow-up of the infants with the genotype DeltaF508/R117H and DeltaF508/5T is required to determine their outcome. In the meantime, newborn screening should be confined to severe mutations associated with classic CF.

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26 Measurement of Cl levels was done by colorimetry, and measurement of sodium levels was done by flame cytometry.16 Gene Mutation Analysis Blood was taken and DNA extract- ed17 for an extended cystic fibrosis transmembrane conductance regulator protein gene mutation analysis as described previously.18 The following mutations were included: ࢞F508, ࢞I507, R117H, G551D, A455E, G542X, N1303K, W1282X, 1717-1G࢐A, R560T, R347P, R334W, R553X, R1162X, S549N, 3849+10C࢐T, and 621+1G࢐T. Login to comment

[hide] Analysis of 31 CFTR mutations in 55 families from ... Early Hum Dev. 2001 Nov;65 Suppl:S161-4. Gomez-Llorente MA, Suarez A, Gomez-Llorente C, Munoz A, Arauzo M, Antunez A, Navarro M, Gil A, Gomez-Capilla JA
Analysis of 31 CFTR mutations in 55 families from the South of Spain.
Early Hum Dev. 2001 Nov;65 Suppl:S161-4., [PMID:11755047]

Abstract [show]
We carried out a molecular analysis of 350 chromosomes from 55 families originating from the South of Spain (Andalucia) who were diagnosed with cystic fibrosis (CF). We used polymerase chain reaction, followed by an oligonucleotide ligation assay (OLA) and sequence-coded separation using capillary electrophoresis. A frequency of 43.5% for DeltaF508 was found, making it the most common CF mutation in our sample. Seven more mutations (G542X, R334W, R1162X, 2789+5G-->A, R117H, DeltaI507 and W1282X) were detected and accounted for 24.7% of the total. The remaining mutations (31.8%) were undetectable with the methodology used in this study.

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30 T I.5 R553X E.11 1078delT E.7 R560T E.11 R347P E.7 S549R E.11 R347H E.7 S549N E.11 R334W E.7 3849 + 10kbC ! Login to comment

[hide] Effect of genotype on phenotype and mortality in c... Lancet. 2003 May 17;361(9370):1671-6. McKone EF, Emerson SS, Edwards KL, Aitken ML
Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study.
Lancet. 2003 May 17;361(9370):1671-6., [PMID:12767731]

Abstract [show]
BACKGROUND: Over 1000 mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) that cause cystic fibrosis have been identified. We examined the effect of CFTR genotype on mortality and disease phenotype. METHODS: Using the US Cystic Fibrosis Foundation National Registry, we did a retrospective cohort study to compare standardised mortality rates for the 11 most common genotypes heterozygous for DeltaF508 with those homozygous for DeltaF508. Of the 28455 patients enrolled in the registry at the time of our analysis, 17853 (63%) were genotyped. We also compared the clinical phenotype, including lung function, age at diagnosis, and nutritional measures, of 22 DeltaF508 heterozygous genotypes. Mortality rates and clinical phenotype were also compared between genotypes classified into six classes on the basis of their functional effect on CFTR production. FINDINGS: Between 1991 and 1999, genetic and clinical data were available for 17853 patients with cystic fibrosis, which was 63% of the total cohort. There were 1547 deaths during the 9 years of follow-up. In the analysis of the 11 most common genotypes, DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/3849+10kbC-->T, and DeltaF508/2789+5G-->A had a significantly lower mortality rate (4.7, 8.0, 11.9, and 4.4, respectively) than the genotype homozygous for DeltaF508 (21.8, p=0.0060). DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/ 3849+10 kbC-->T, DeltaF508/2789+5G-->A, and DeltaF508/A455E have a milder clinical phenotype. Outcomes for all functional classes were compared with that of class II (containing DeltaF508 homozygotes) and classes IV and V had a significantly lower mortality rate and milder clinical phenotype. INTERPRETATION: Patients with cystic fibrosis have distinct genetic subgroups that are associated with mild clinical manifestations and low mortality. These differences in phenotype are also related to the functional classification of CFTR genotype.

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47 ARTICLES 1672 THE LANCET ߦ Vol 361 ߦ May 17, 2003 ߦ www.thelancet.com Panel 1: Frequencies of CFTR mutations* CFTR Allele CFTR Allele mutation frequency (%) mutation frequency èc;F508 69&#b7;4% 2789+5G࢐A 0&#b7;3% Unknown 15&#b7;7% R1162X 0&#b7;3% G542X 2&#b7;3% G85E 0&#b7;3% G551D 2&#b7;2% R560T 0&#b7;2% èc;I507 1&#b7;6% R334W 0&#b7;2% W1282X 1&#b7;4% 3659èc;C 0&#b7;2% N1303K 1&#b7;2% A455E 0&#b7;1% R553X 0&#b7;9% 711+1G࢐T 0&#b7;1% 621+1G࢐T 0&#b7;8% 1898+1G࢐A 0&#b7;1% R117H 0&#b7;7% 2184èc;A 0&#b7;1% 3849+10 kbC࢐T 0&#b7;7% S549N 0&#b7;1% 1717-IG࢐A 0&#b7;5% 1078èc;T 0&#b7;03% R347P 0&#b7;3% *n=17 853. Login to comment
48 Panel 2: Functional classification of CFTR alleles Class Functional effect of Allele mutation I Defective protein G542X, R553X, W1282X, production R1162X, 621-1G࢐T, 1717-1G࢐A, 1078èc;T, 3659èc;C II Defective protein èc;F508, èc;I507, N1303K, processing S549N III Defective protein G551D, R560T regulation IV Defective protein R117H, R334W, G85E, conductance R347P V Reduced amounts of 3849+10KbC࢐T, functioning CFTR protein 2789+5G࢐A, A455E Unknown 711+1G࢐T, 2184DA, 1898+1G࢐A Total cohort Genotyped cohort (n=28 455) (n=17 853) Person-years at risk 152 011 96 870 Sex (% male) 53% 52% Race (% white) 96% 96% Age (years) 11&#b7;9 (11&#b7;1) 10&#b7;9 (11&#b7;2) Age at diagnosis (years) 3&#b7;5 (7&#b7;1) 3&#b7;6 (7&#b7;5) Sweat test (mmol/L) 101 (19) 100 (20) FEV1 (L) 1&#b7;72 (0&#b7;91) 1&#b7;80 (0&#b7;92) FEV1 (% predicted) 69 (29) 72 (28) FVC (L) 2&#b7;41 (1&#b7;18) 2&#b7;50 (1&#b7;21) FVC (% predicted) 81% (28) 84% (24) Height (cm) 121% (41) 117% (41) Weight (kg) 30&#b7;0 (21&#b7;3) 28&#b7;6 (21&#b7;8) Pancreatic insufficiency (%) 90% 87% P aeruginosa colonisation (%) 49% 46% Number of deaths (%) 3548 (12%) 1547 (9%) Data are mean (SD) unless otherwise stated. Login to comment
67 Table 2: Standardised and crude mortality rates (including organ transplantation) by genotype Genotype No of Age at Sweat FEV1 FVC Height Weight Pancreatic P&#b7; aeruginosa Subjects Diagnosis Chloride (% predicted)* (% predicted)* (cms)* (kg)* Insufficiency Colonization (yrs) (mmol) (%)ߤ (%)ߤ èc;F508/èc;F508 6 213 2&#b7;5 &#b1; 0&#b7;1 104 &#b1; 0&#b7;2 77 &#b1; 0&#b7;3 89 &#b1; 0&#b7;3 141 &#b1; 0&#b7;2 37&#b7;0 &#b1; 0&#b7;1 92 (91-92) 60 (59-61) èc;F508/G551D 411 3&#b7;7 &#b1; 0&#b7;3ߥ 108 &#b1; 0&#b7;9ߥ 76 &#b1; 1&#b7;2 89 &#b1; 1&#b7;2 142 &#b1; 0&#b7;7&#a7; 38&#b7;2 &#b1; 0&#b7;6&#a7; 92 (89-94) 59 (54-64) èc;F508/G542X 389 1&#b7;9 &#b1; 0&#b7;2 104 &#b1; 0&#b7;8 79 &#b1; 1&#b7;2 91 &#b1; 1&#b7;2 141 &#b1; 0&#b7;7 37&#b7;3 &#b1; 0&#b7;5 93 (89-95) 57 (52-62) èc;F508/N1303K 213 2&#b7;1 &#b1; 0&#b7;3 106 &#b1; 1&#b7;2 80 &#b1; 1&#b7;8 91 &#b1; 1&#b7;7 141 &#b1; 1&#b7;0 37&#b7;1 &#b1; 0&#b7;6 92 (87-95) 61 (55--68) èc;F508/W1282X 205 1&#b7;6 &#b1; 0&#b7;2 103 &#b1; 1&#b7;2 80 &#b1; 1&#b7;7 92 &#b1; 1&#b7;6 141 &#b1; 0&#b7;9 37&#b7;4 &#b1; 0&#b7;7 94 (90-97) 59 (52-65) èc;F508/R553X 164 2&#b7;5 &#b1; 0&#b7;4 106 &#b1; 1&#b7;4 76 &#b1; 1&#b7;8 89 &#b1; 1&#b7;6 139 &#b1; 0&#b7;9 35&#b7;4 &#b1; 0&#b7;7&#a7; 90 (85-94) 60 (53-67) èc;F508/621-1G 162 2&#b7;5 &#b1; 0&#b7;4 107 &#b1; 1&#b7;3 78 &#b1; 1&#b7;8 89 &#b1; 1&#b7;5 143 &#b1; 1&#b7;0&#a7; 38&#b7;8 &#b1; 0&#b7;8&#a7; 87 (80-91)&#a7; 57 (49-64) èc;F508/èc;I507 149 8&#b7;5 &#b1; 1&#b7;1ߥ 95 &#b1; 1&#b7;9ߥ 86 &#b1; 2&#b7;1ߥ 93 &#b1; 1&#b7;8&#a7; 137 &#b1; 1&#b7;4&#a7; 37&#b7;4 &#b1; 1&#b7;25 84 (78-89)ߥ 39 (31-48)ߥ èc;F508/R117H 123 13&#b7;7 &#b1; 1&#b7;2ߥ 80 &#b1; 1&#b7;9ߥ 91 &#b1; 2&#b7;1ߥ 97 &#b1; 1&#b7;7ߥ 143 &#b1; 1&#b7;8 42&#b7;9 &#b1; 1&#b7;7ߥ 65 (55-73)ߥ 22 (16-29)ߥ èc;F508/3849+10 kB 114 11&#b7;3 &#b1; 0&#b7;9ߥ 72 &#b1; 2&#b7;5ߥ 77 &#b1; 2&#b7;1 87 &#b1; 1&#b7;9 144 &#b1; 1&#b7;4&#a7; 41&#b7;2 &#b1; 1&#b7;2ߥ 66 (57-74)ߥ 69 (59-77) èc;F508/2789+5G 63 13&#b7;4 &#b1; 1&#b7;6ߥ 102 &#b1; 2&#b7;1 88 &#b1; 2&#b7;8ߥ 97 &#b1; 2&#b7;3ߥ 140 &#b1; 2&#b7;5 41&#b7;8 &#b1; 2&#b7;2&#a7; 71 (59-81)ߥ 32 (22-44)ߥ èc;F508/1717-1G 74 1&#b7;3 &#b1; 0&#b7;3 103 &#b1; 2&#b7;0 75 &#b1; 2&#b7;7 86 &#b1; 2&#b7;4 139 &#b1; 1&#b7;5 35&#b7;7 &#b1; 0&#b7;9 96 (88-99) 59 (48-69) èc;F508/R560T 46 1&#b7;7 &#b1; 0&#b7;5 104 &#b1; 2&#b7;0 84 &#b1; 3&#b7;3ߥ 96&#b1; 2&#b7;8&#a7; 142 &#b1; 1&#b7;9 38&#b7;4 &#b1; 1&#b7;4 91 (79-97) 63 (48-75) èc;F508/R347P 44 5&#b7;9 &#b1; 1&#b7;1&#a7; 105 &#b1; 2&#b7;6 76 &#b1; 3&#b7;0 90 &#b1; 2&#b7;9 142 &#b1; 2&#b7;4 38&#b7;7 &#b1; 1&#b7;8 67 (52-79)ߥ 53 (38-68) èc;F508/G85E 43 9&#b7;2 &#b1; 1&#b7;8ߥ 99 &#b1; 2&#b7;3&#a7; 76 &#b1; 2&#b7;5 90 &#b1; 2&#b7;5 142 &#b1; 2&#b7;9 38&#b7;3 &#b1; 2&#b7;2 88 (75-95) 52 (35-68) èc;F508/3659DC 40 1&#b7;1 &#b1; 0&#b7;4 105 &#b1; 2&#b7;1 76 &#b1; 3&#b7;9 88 &#b1; 4&#b7;1 139 &#b1; 1&#b7;9 36&#b7;6 &#b1; 1&#b7;2 92 (77-97) 55 (39-69) èc;F508/A455E 29 14&#b7;3 &#b1; 2&#b7;0ߥ 89 &#b1; 3&#b7;1ߥ 98 &#b1; 4&#b7;0ߥ 104 &#b1; 3&#b7;4ߥ 138 &#b1; 3&#b7;4 42&#b7;1 &#b1; 2&#b7;5&#a7; 60 (41--76)ߥ 17 (8-32)ߥ èc;F508/R334W 28 13&#b7;2 &#b1; 3&#b7;0ߥ 104 &#b1; 3&#b7;2 86 &#b1; 3&#b7;4&#a7; 94 &#b1; 3&#b7;3 138 &#b1; 3&#b7;2 42&#b7;3 &#b1; 3&#b7;5 67 (46-82)ߥ 51 (32--70) èc;F508/R1162X 26 1&#b7;9 &#b1; 1&#b7;1 101 &#b1; 2&#b7;3 77 &#b1; 4&#b7;2 92 &#b1; 4&#b7;6 138 &#b1; 1&#b7;8 36&#b7;5 &#b1; 1&#b7;4 92 (75-98) 65 (47-80) èc;F508/1898+1G 20 1&#b7;2 &#b1; 0&#b7;3 99 &#b1; 2&#b7;8 83 &#b1; 4&#b7;1 94 &#b1; 4&#b7;4 138 &#b1; 3&#b7;3 35&#b7;1 &#b1; 2&#b7;1 85 (61--95) 63 (39-82) èc;F508/2184DA 20 2&#b7;3 &#b1; 0&#b7;9 106 &#b1; 5&#b7;3 82 &#b1; 4&#b7;3 92 &#b1; 4&#b7;4 141 &#b1; 3&#b7;0 36&#b7;5 &#b1; 1&#b7;5 94 (69-99) 60 (38-79) èc;F508/711+1G 17 1&#b7;3 &#b1; 0&#b7;5 108 &#b1; 4&#b7;6 83 &#b1; 4&#b7;2 94 &#b1; 4&#b7;4 137 &#b1; 3&#b7;4 36&#b7;7 &#b1; 2&#b7;9 100 73 (50-88) èc;F508/S549N 11 6&#b7;4 &#b1; 1&#b7;9&#a7; 109 &#b1; 5&#b7;7 67 &#b1; 6&#b7;1 77 &#b1; 7&#b7;2 140 &#b1; 3&#b7;2 36&#b7;7 &#b1; 2&#b7;6 92 (62-99) 71 (40--90) èc;F508/Other 2 262 5&#b7;8 &#b1; 0&#b7;2ߥ 99 &#b1; 0&#b7;4ߥ 80 &#b1; 0&#b7;5ߥ 91 &#b1; 0&#b7;5ߥ 141 &#b1; 0&#b7;3 38&#b7;1 &#b1; 0&#b7;3ߥ 86 (84-87)ߥ 50 (48-52)ߥ Other/Other 1 551 7&#b7;5 &#b1; 0&#b7;3ߥ 93 &#b1; 0&#b7;6ߥ 82 &#b1; 0&#b7;6ߥ 90 &#b1; 0&#b7;6&#a7; 141 &#b1; 0&#b7;4 38&#b7;3 &#b1; 0&#b7;3ߥ 81 (80-84)ߥ 40 (38-43)ߥ Data are mean (SE) unless otherwise indicated. Login to comment

[hide] Simultaneous screening for 11 mutations in the cys... Mol Cell Probes. 1992 Feb;6(1):33-9. Cuppens H, Buyse I, Baens M, Marynen P, Cassiman JJ
Simultaneous screening for 11 mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex amplification and reverse dot-blot.
Mol Cell Probes. 1992 Feb;6(1):33-9., [PMID:1372093]

Abstract [show]
An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.

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19 Frequency of 31 mutations in the CFTR gene in 194 Belgian CF chromosomes The 51255X, W1316X ;5 S549N, G551D, R553X, A559T;6 D110H, R117H, R347P;' Q493X, S5491, S549R(T-+G), R560T, Y563N, P574H ;9 W846X, Y913C;10 2556insAT;" R334W;" S549R(A-+C);'6 444delA, 3821deIT;" 621 +1G-*T18 mutations were not present in this random sample of the Belgian CF population . Login to comment

[hide] Cystic fibrosis. Am J Clin Pathol. 2003 Dec;120 Suppl:S3-13. Lewis MJ, Lewis EH 3rd, Amos JA, Tsongalis GJ
Cystic fibrosis.
Am J Clin Pathol. 2003 Dec;120 Suppl:S3-13., [PMID:15298139]

Abstract [show]
On a daily basis, pathologists examine the fundamental basis of human diseases using morphologic, immunologic, and molecular techniques. Cystic fibrosis (CF), as a clinically heterogeneous disease, exemplifies the complex challenges of genetic diseases for the pathologist who attempts to explain the mechanisms of disease and provide rationale for clinical management. This review includes an overview of CF and a discussion of pathophysiologic features and practical components of clinical and anatomic pathology, and concludes with a review of molecular diagnostics.

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95 ēa; ēa;Table 3ēa; ēa; Recommended Mutation Panel for Cystic Fibrosis Carrier Screening ࢞F508 ࢞I507 G542X G551D W1282X N1303K R553X 621+1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711+1G>T 1898+1G>A 2184delA 1078delT 3849+10kbC>T 2789+5G>A 3659delC I148T 3120+1G>A I506V* I507V* F508C* 5T/7T/9T* * Reflex tests. Login to comment

[hide] Diagnosis of cystic fibrosis in adults with diffus... J Cyst Fibros. 2004 Mar;3(1):15-22. Hubert D, Fajac I, Bienvenu T, Desmazes-Dufeu N, Ellaffi M, Dall'ava-Santucci J, Dusser D
Diagnosis of cystic fibrosis in adults with diffuse bronchiectasis.
J Cyst Fibros. 2004 Mar;3(1):15-22., [PMID:15463882]

Abstract [show]
We assessed the contribution of the sweat test, genotyping and nasal potential difference (NPD) in the diagnosis of cystic fibrosis (CF) in adults with diffuse bronchiectasis (DB). Among 601 adults referred for DB from 1992 to 2001, 46 were diagnosed with CF. The sweat test was positive in 37 patients and normal or intermediate in nine patients. Two CF mutations were identified in 18 patients (39%) by screening for 31 mutations and in 36 patients (78%) after complete genetic analysis. NPD was suggestive of CF in 71% of the patients. The combination of the sweat test and genetic analysis led to the diagnosis of CF in 45 patients. In the nine patients with normal or intermediate sweat test, the diagnosis was confirmed by screening for 31 mutations in five, by complete genetic screening in three, and by NPD in the remaining patient. Searching for CF should start with sweat test. If the sweat test is normal or intermediate, screening for 31 mutations may help to diagnose CF. A complete genetic analysis is indicated when only one mutation is detected and/or when other clinical features, such as obstructive azoospermia or pancreatic insufficiency, are suggestive of CF. NPD measurement is indicated in controversial cases.

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47 We used an oligonucleotide ligation assay using a commercially available kit (Cystic Fibrosis Assay, Applied Biosystems, Foster City, CA, USA) to seek 31 mutations in the CFTR gene (F508del, I507del, Q943X, V520F, 1717y1GࡊA, G542X, G551D, R553X, R560T, S549R, S549 N, 3849q10kbCࡊT, 3849q4AࡊG, R1162X, 3659delC, W1282X, 3905insT, 621q1GࡊT, R117H, Y122X, 711q1GࡊT, 1078delT, R347P, R347H, R334 W, A455E, N1303K, G85E, 1898q1GࡊA, 2183AAࡊG, 2789q5GࡊA) which allowed to detect 82% of the CF alleles in France. Login to comment
129 * 31 mutations: F508del, I507del, Q493X, V520F, 1717y1GࡊA, G542X, G551D, R553X, R560T, S549R, S549 N, 3849q10kbCࡊT, 3849q ** 4AࡊG, R1162X, 3659delC, W1282X, 3905insT, 621q1GࡊT, R117H, Y122X, 711q1GࡊT, 1078delT, R347P, R347H, R334 W, A455E, N1303K, G85E, 1898q1GࡊA, 2183AAࡊG, 2789q5GࡊA. that the laboratory criteria for the diagnosis of CF should be expanded to include identification of CFTR mutations and abnormal bioelectrical properties of the nasal epithelium, in addition to the sweat test w7x. Login to comment

[hide] A 96-well formatted method for exon and exon/intro... Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5. Lucarelli M, Narzi L, Piergentili R, Ferraguti G, Grandoni F, Quattrucci S, Strom R
A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene.
Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5., [PMID:16635477]

Abstract [show]
Full genotypic characterization of subjects affected by cystic fibrosis (CF) is essential for the definition of the genotype-phenotype correlation as well as for the enhancement of the diagnostic and prognostic value of the genetic investigation. High-sensitivity diagnostic methods, capable of full scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, are needed to enhance the significance of these genetic assays. A method for extensive sequencing of the CFTR gene was optimized. This method was applied to subjects clinically positive for CF and to controls from the general population of central Italy as well as to a single subject heterozygous for a mild mutation and with an uncertain diagnosis. Some points that are crucial for the optimization of the method emerged: a 96-well format, primer project and purification, and amplicon purification. The optimized method displayed a high degree of diagnostic sensitivity; we identified a subset of 13 CFTR mutations that greatly enhanced the diagnostic sensitivity of common methods of mutational analysis. A novel G1244R disease causing mutation, leading to a CF phenotype with pancreatic sufficiency but early onset of pulmonary involvement, was detected in the subject with an uncertain diagnosis. Some discrepancies between our results and previously published CFTR sequence were found.

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26 None of these subjects showed any clinical manifestations of CF, nor were any positive for CFTR mutations when analyzed by means of the PCR/OLA/SCS method (Celera Diagnostics) [21], which searches for the most common worldwide 31 CFTR mutations (G85E, R117H, Y122X, 621+1G->T, 711+1G->T, 1078delT, R347P, R347H, R334W, A455E, DF508, DI507, Q493X, V520F, 1717-1G->A, G542X, G551D, R553X, R560T, S549R(T->G), S549N, 1898+1G->A, 2183AA->G, 2789+5G->A, R1162X, 3659delC, 3849+10kbC->T, 3849+4A->G, W1282X, 3905insT, N1303K), including the 12 most common in Italy [1,22]. Login to comment

[hide] CFTR gene analysis in Latin American CF patients: ... J Cyst Fibros. 2007 May;6(3):194-208. Epub 2006 Sep 11. Perez MM, Luna MC, Pivetta OH, Keyeux G
CFTR gene analysis in Latin American CF patients: heterogeneous origin and distribution of mutations across the continent.
J Cyst Fibros. 2007 May;6(3):194-208. Epub 2006 Sep 11., [PMID:16963320]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is the most prevalent Mendelian disorder in European populations. Despite the fact that many Latin American countries have a predominant population of European-descent, CF has remained an unknown entity until recently. Argentina and Brazil have detected the first patients around three decades ago, but in most countries this disease has remained poorly documented. Recently, other countries started publishing their results. METHODS: We present a compilation and statistical analysis of the data obtained in 10 countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Uruguay and Venezuela), with a total of 4354 unrelated CF chromosomes studied. RESULTS: The results show a wide distribution of 89 different mutations, with a maximum coverage of 62.8% of CF chromosomes/alleles in the patient's sample. Most of these mutations are frequent in Spain, Italy, and Portugal, consistent with the origin of the European settlers. A few African mutations are also present in those countries which were part of the slave trade. New mutations were also found, possibly originating in America. CONCLUSION: The profile of mutations in the CFTR gene, which reflects the heterogeneity of its inhabitants, shows the complexity of the molecular diagnosis of CF mutations in most of the Latin American countries.

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78 At least another 38 mutations have been searched for, but none of them were found in the CF patients from Latin America: p.E60X, p.Y122X, p.G178R, p.G330X, p.R347H, p.R352Q, p.S364P, p.A455E, p.Q493X, p.V520F, p.C524X, p.R560T, p.Y563D, p.P574H, p.K710X, p.Q890X, p. R1158X, p.S1196X, p.S1255X, p.D1270N, p.W1310X, p. W1316X, c.405+1G-A, c.444delA, c.556delA, c.574delA, c.1677delTA, c.2043delG, c.2307insA, c.2909delT, c.3120G-A, c.3358delAC, c.3662delA, c.3750delAG, c.3791delC, c.3821delT, c.3849+4A-G, c.3905insT. Login to comment

[hide] Newborn screening for cystic fibrosis in Alberta: ... Paediatr Child Health. 2010 Nov;15(9):590-4. Lilley M, Christian S, Hume S, Scott P, Montgomery M, Semple L, Zuberbuhler P, Tabak J, Bamforth F, Somerville MJ
Newborn screening for cystic fibrosis in Alberta: Two years of experience.
Paediatr Child Health. 2010 Nov;15(9):590-4., [PMID:22043142]

Abstract [show]
On April 1, 2007, Alberta became the first province in Canada to introduce cystic fibrosis (CF) to its newborn screening program. The Alberta protocol involves a two-tier algorithm involving an immunoreactive trypsinogen measurement followed by molecular analysis using a CF panel for 39 mutations. Positive screens are followed up with sweat chloride testing and an assessment by a CF specialist. Of the 99,408 newborns screened in Alberta during the first two years of the program, 221 had a positive CF newborn screen. The program subsequently identified and initiated treatment in 31 newborns with CF. A relatively high frequency of the R117H mutation and the M1101K mutation was noted. The M1101K mutation is common in the Hutterite population. The presence of the R117H mutation has created both counselling and management dilemmas. The ability to offer CF transmembrane regulator full sequencing may help resolve diagnostic dilemmas. Counselling and management challenges are created when mutations are mild or of unknown clinical significance.

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46 These include the following mutations: delF508, I507del, G542X, G85E, R117H, 621+1G࢐T, 711+1G࢐T, G551D, R334W, R347P, A455E, 1717-1G࢐A, R560T, R553X, N1303K, 1898+1G࢐A, 2184delA, 2789+5G࢐A, 3120+1G࢐A, R1162X, 3659delC, 3849+10kbC࢐T, W1282X, 1078delT, 394delTT, Y122X, R347H, V520F, A559T, S549N, S549R, 1898+5G࢐T, 2183AA࢐G, 2307insA, Y1092X, M1101K, S1255X, 3876delA and 3905insT. Login to comment

[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]

Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

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167 The èc;I507 mutant that also causes severe disease (36) was among those, as was the R560T missense mutation, prominent in some ethnic populations (37). Login to comment
181 B, C) HEK293 cells were transiently transfected with CFTR with misprocessing mutations located in CL1 (H139R), CL2 (R258G), and CL3 (S945L) (B), or in NBD1 (èc;I507 and R560T; C). Login to comment
197 Two other severe disease-causing mutations in NBD1 (èc;I507 and R560T) that also were not temperature sensitive were unresponsive to VX-809. Login to comment

[hide] PGD for cystic fibrosis patients and couples at ri... Reprod Biomed Online. 2013 May;26(5):420-30. doi: 10.1016/j.rbmo.2013.01.006. Epub 2013 Jan 29. Rechitsky S, Verlinsky O, Kuliev A
PGD for cystic fibrosis patients and couples at risk of an additional genetic disorder combined with 24-chromosome aneuploidy testing.
Reprod Biomed Online. 2013 May;26(5):420-30. doi: 10.1016/j.rbmo.2013.01.006. Epub 2013 Jan 29., [PMID:23523379]

Abstract [show]
Preimplantation genetic diagnosis (PGD) for inherited disorders is presently applied for more than 300 different conditions. The most frequent PGD indication is cystic fibrosis (CF), the largest series of which is reviewed here, totalling 404 PGD cycles. This involved testing for 52 different CFTR mutations with almost half of the cases (195/404 cycles) performed for DeltaF508 mutation, one-quarter (103/404 cycles) for six other frequent mutations and only a few for the remaining 45 CFTR mutations. There were 44 PGD cycles performed for 25 CF-affected homozygous or double-heterozygous CF patients (18 male and seven female partners), which involved testing simultaneously for three mutations, resulting in birth of 13 healthy CF-free children and no misdiagnosis. PGD was also performed for six couples at a combined risk of producing offspring with CF and another genetic disorder. Concomitant testing for CFTR and other mutations resulted in birth of six healthy children, free of both CF and another genetic disorder in all but one cycle. A total of 96 PGD cycles for CF were performed with simultaneous aneuploidy testing, including microarray-based 24-chromosome analysis, as a comprehensive PGD for two or more conditions in the same biopsy material.

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42 [1075C>A; 1079C>A] p.[Gln359Lys; Thr360Lys] Exon 8 1 1 1 4 1 1 R297Q c.890G>A p.Arg297Gln Exon 8 1 1 1 2 0 0 R347P c.1040G>C p.Arg347Pro Exon 8 3 5 2 4 1 1 T338I c.1013C>T p.Thr338Ile Exon 8 1 1 1 2 1 1 DF508 c.1521_1523delCTT p.Phe508del Exon 11 130 195 172 345 88 (4) 92 DI507 c.1519_1521delATC p.Ile507del Exon 11 1 5 5 11 2 1 Q493R c.1478A>G p.Gln493Arg Exon 11 5 5 2 2 2 2 1717-1G-A c.1585-1G>A - Intron 11 6 10 9 18 6 8 G542X c.1624G>T p.Gly542X Exon 12 14 17 15 34 10 10 G551S c.1651G>A p.Gly551Ser Exon 12 1 1 1 2 1 1 G551D c.1652G>A p.Gly551Asp Exon 12 12 22 19 33 7 8 I556V c.1666A>G p.Ile556Val Exon 12 1 2 2 4 1 1 R553X c.1657C>T p.Arg553X Exon 12 3 4 2 4 0 0 R560T c.1679G>C p.Arg560Thr Exon 12 1 1 1 2 1 2 1898+1G-A c.1766 &#b1; 1G>A - Intron 13 1 1 1 2 1 1 2184delA c.2052delA p.Lys684AsnfsX38 Exon 14 1 1 0 0 0 0 G622D c.1865G>A p.Gly622Asp Exon 14 1 1 1 3 0 0 N703S c.2108A>G p.Asn703Ser Exon 14 1 2 2 3 2 2 S737F c.2210C>T p.Ser737Phe Exon 14 1 1 0 0 0 0 2622+1G-A c.2490 &#b1; 1G>A - Intron 14 1 5 5 13 1 1 2752-26A-G c.2620-26A>G - Intron 15 1 2 2 4 0 0 2789+5G-A c.2657 &#b1; 5G>A - Intron 16 3 5 4 8 0 0 3120G-A c.2988G>A - Exon 18 2 2 1 2 1 0 3067-72del c.3067_3072del p.Ile1023_Val1024del Exon 19 1 1 1 1 0 0 I1027T c.3080T>C p.Ile1027Thr Exon 19 1 1 1 1 0 0 L997F c.2991G>C p.Leu997Phe Exon 19 1 2 2 4 1 (1) 0 M1028R c.3083T>G p.Met1028Arg Exon 19 1 1 1 2 1 2 F1052V c.3154T>G p.Phe1052Val Exon 20 1 1 0 0 0 0 Y1092X c.3276C>A p.Tyr1092X Exon 20 1 2 1 2 1 1 A1136T c.3406G>A p.Ala1136Thr Exon 21 1 2 1 2 1 0 D1152H c.3454G>C p.Asp1152His Exon 21 3 7 7 15 1 1 3659 del C c.3528delC p.Lys1177SerfsX15 Exon 22 2 4 3 7 3 3 R1162X c.3484C>T p.Arg1162X Exon 22 1 3 2 5 2 2 S1235R c.3705T>G p.Ser1235Arg Exon 22 2 3 3 5 2 1 3849+10kbC>T c.3717 &#b1; 12191C>T - Intron 22 2 4 4 5 0 0 W1282X c.3846G>A p.Trp1282X Exon 23 15 20 20 42 11 11 N1303K c.3909C>G p.Asn1303Lys Exon 24 9 12 11 24 4 5 Q1352H c.4056G>C p.Gln1352His Exon 25 1 1 1 1 1 1 Total 265 404 345 685 172 (6a ) 175 Values are n unless otherwise stated. Login to comment
56 (CA)n EXON 4 (GATT)n Intron 4 Poly T tract Intron 10 R117H G--A R75XH C--T A120T G--A I148T T--C A349V C--T 1259 Ins A 621+1 G--T EXON 3 EXON 7 EXON 8 Delta I 507 EXON 10 Delta F 508 EXON 11 1717-1 G--A G542X G--T G550X G--T G551D G--A R553X C--T R560T G--C EXON 19 EXON 20 EXON 21 R1162X C--T W1282X G--A N1303K C--G IVS 1 Mutations in CFTR gene (PGD PERFORMED FOR 52 MUTATIONS) IVS 6 a IVS 8 (CA)n (CA)n IVS 17b (TA)n (CA)n D7S486 D7S522 D7S633 D7S677 D7S2847 D7S655 115,89 116.07 117.01 117.13 117.19 117.20 118.6 118.81 Mb IVS8-1 IVS8-2 Figure 1 Mutations (above) and linked markers (below) in CFTR that were used in multiplex PCR. Login to comment

[hide] Clinical and genetic features in patients with cys... Iran J Pediatr. 2013 Apr;23(2):212-5. Farjadian S, Moghtaderi M, Kashef S, Alyasin S, Najib K, Saki F
Clinical and genetic features in patients with cystic fibrosis in southwestern iran.
Iran J Pediatr. 2013 Apr;23(2):212-5., [PMID:23724185]

Abstract [show]
OBJECTIVE: Cystic fibrosis (CF) is a common autosomal recessive genetic disease caused by a mutation in the CF transmembrane conductance regulatory (CFTR) gene. This study attempted to identify the most common CFTR mutations and any correlations between certain mutations and the clinical presentation of the disease in CF patients in southwestern Iran. METHODS: Twenty nine common CFTR gene mutations were examined in 45 CF patients. FINDINGS: Chronic cough, intestinal obstruction, dehydration, heat exhaustion and steatorrhea were the most common early clinical symptoms among our patients. The most common mutation was DeltaF508, with an allele frequency of 21%. The homozygous DeltaF508 mutation was observed in eight patients (18%), and three patients (7%) were DeltaF508 carriers. The 2183AA > G mutation was observed in four patients, one of whom was also a DeltaF508 carrier. The R1162X mutation was detected in two patients. The G542X, R334W and N1303K mutations were detected each in one patient, the first of whom was also a DeltaF508 carrier. CONCLUSION: Out of 45 patients, 27 (60%) had none of the CFTR gene mutations we tested for. The most frequent mutations in southwestern Iranian patients with CF should be identified by sequencing the entire CFTR gene in order to optimize the design of a diagnostic kit for common regional mutations.

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26 Genomic DNA was extracted from 200 &#b5;L of whole blood with the QiaAmp DNA Mini Kit (Qiagen, Valencia, CA, USA) and 29 common CFTR gene mutations (D1152H, 1717-1G>A, G542X, W1282X, N1303K, ࢞F508, 3849+10kbC>T, 394delTT, 621+1G>T, S1251N, G551D, R117H, R1162X, R334W, A455E, 2183AA>G, 3659delC, 1078delT, ࢞I507, R347P, R553X, E60X, 3120+1G>A, 2789+5G>A, 1898+1G>A, 711+1G>T, G85E, 2184delA and R560T) were analyzed with the ELUCIGENE CF29 v. 2 kit using four multiplex PCR. Login to comment

[hide] A comprehensive assay for CFTR mutational analysis... Clin Chem. 2013 Oct;59(10):1481-8. doi: 10.1373/clinchem.2013.206466. Epub 2013 Jun 17. Abou Tayoun AN, Tunkey CD, Pugh TJ, Ross T, Shah M, Lee CC, Harkins TT, Wells WA, Tafe LJ, Amos CI, Tsongalis GJ
A comprehensive assay for CFTR mutational analysis using next-generation sequencing.
Clin Chem. 2013 Oct;59(10):1481-8. doi: 10.1373/clinchem.2013.206466. Epub 2013 Jun 17., [PMID:23775370]

Abstract [show]
BACKGROUND: Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS: We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine (PGM) platform. RESULTS: The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500x to 3500x, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS: With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.

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53 of cases af9;/af9; c.1521_1523delCTT; c.1521_1523delCTT èc;F508; èc;F508 CF Yes 97 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.350Gb0e;A èc;F508; R117H CF Yes 53; 50 Dartmouth 1 af9;/af9; c.350Gb0e;A; c.1477Cb0e;T R117H; Q493*b CF Yes 52; 49 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.1000Cb0e;T èc;F508; R334W CF Yes 49; 54 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.489af9;1Gb0e;T èc;F508; 621af9;1Gb0e;T CF Yes 48; 47 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.1364Cb0e;A èc;F508; A455E CF Yes 51; 46 Dartmouth 1 af9;/af9; c.489af9;1Gb0e;T; c.2988af9;1Gb0e;A 621af9;1Gb0e;T; 3120af9;1Gb0e;A CF Yes 48; 49 Coriell 1 af9;/af9; c.1521_1523delCTT; c.1657Cb0e;T èc;F508; R553* CF Yes 44; 49c Coriell 2 af9;/af9; c.1521_1523delCTT; c.3528delC èc;F508; 3659delC CF Yes 46; 46 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.579af9;1Gb0e;T 621af9;1Gb0e;T; 711af9;1Gb0e;T CF Yes 50; 51 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.254Gb0e;A 621af9;1Gb0e;T; G85E CF Yes 50; 45 Coriell 1 af9;/af9; c.1521_1523delCTT; c.1679Gb0e;C èc;F508; R560T CF Yes 44; 52 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.1364Cb0e;A 621af9;1Gb0e;T; A455E CF Yes 50; 49 Coriell 1 af9;/af9; c.3909Cb0e;G; c.4046Gb0e;A N1303K; G1349D CF Yes 47; 52 Coriell 1 af9;/af9; c.2657af9;5Gb0e;A; c.2657af9;5Gb0e;A 2789af9;5Gb0e;A; 2789af9;5Gb0e;A CF Yes 100 Coriell 1 af9;/af9; c.1040Gb0e;C; c.1652Gb0e;A R347P; G551D CF Yes 51; 49 Coriell 1 af9;/af9; c.1000Cb0e;T; c.3368-2Ab0e;T R334W; 3500-2Ab0e;G CF Yes 53; 45 Coriell 1 af9;/af9; c.254Gb0e;A; c.3454Gb0e;C G85E; D1152H CF Yes 44; 47 Coriell 1 af9;/af9; c.1521_1523delCTT; c.350Gb0e;A èc;F508; R117H CF Yes 49; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.54-5940_273af9;10250del21kb èc;F508; CFTRdel2,3 CF Yes 47; N/Ad Coriell 1 af9;/af9; c.1521_1523delCTT; c.1766af9;1Gb0e;A èc;F508; 1898af9;1Gb0e;A CF Yes 47; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.2051_2052delAAinsG èc;F508; K684Sfs CF Yes 47; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.2052del èc;F508; K684Nfs*38 CF Yes 51; 55 Coriell 1 af9;/afa; c.1521_1523delCTT èc;F508 Carrier Yes 50c Dartmouth 16 af9;/afa; c.1652Gb0e;A G551D Carrier Yes 50c Dartmouth 5 af9;/afa; c.1519_1521delATC èc;I507 Carrier Yes 46 Dartmouth 1 af9;/afa; c.3454Gb0e;C D1152H Carrier Yes 50 Dartmouth 1 af9;/afa; c.1657Cb0e;T R553* Carrier Yes 51 Dartmouth 1 af9;/afa; c.178Gb0e;T E60* Carrier Yes 51 Dartmouth 1 af9;/afa; c.3846Gb0e;A W1282* Carrier Yes 45c Dartmouth 3 af9;/afa; c.1000Cb0e;T R334W Carrier Yes 51 Dartmouth 1 af9;/afa; c.1624Gb0e;T G542* Carrier Yes 47c Dartmouth 4 af9;/afa; c.3484Cb0e;T R1162* Carrier Yes 43 Dartmouth 1 af9;/afa; c.1766af9;1Gb0e;A 1898af9;1Gb0e;A Carrier Yes 57 Dartmouth 1 af9;/afa; c.3773_3774insT 3905insT (L1258Ffs*7) Carrier Yes 37 Dartmouth 1 af9;/afa; c.350Gb0e;A R117H Carrier Yes 50c Dartmouth 3 af9;/afa; c.1645Ab0e;C S549R Ab0e;C Carrier No N/A Dartmouth 1 af9;/afa; c.1040Gb0e;A R347H Carrier Yes 47 Dartmouth 1 af9;/afa; c.3909Cb0e;G N1303K Carrier Yes 46 Dartmouth 1 af9;/afa; c.3718-2477Cb0e;T 3849af9;10kbCb0e;T Carrier Yes 51 Coriell 1 af9;/afa; c.2988af9;1Gb0e;A 3120af9;1Gb0e;A Carrier Yes 49 Coriell 1 af9;/afa; c.489af9;1Gb0e;T 621af9;1Gb0e;T Carrier Yes 50 Coriell 1 af9;/afa; c.1585-1Gb0e;A 1717-1Gb0e;A Carrier Yes 51 Coriell 1 afa;/afa;e N/Af N/A Normal N/A N/A Dartmouth 9 a af9;/af9;, 2 pathogenic mutations; af9;/afa;, carrier of a single pathogenic mutation; afa;/afa;, absence of any pathogenic mutations. Login to comment
115 Mutation cDNA position Mutation class Sensitivity (TPa ) Specificity (TN) Accuracyb G85E c.254Gb0e;A Missense Detected (2/2) 100% (77/77) 100% R117H c.350Gb0e;A Missense Detected (6/6) 100% (73/73) 100% 621af9;1Gb0e;T c.489af9;1Gb0e;T Splice site Detected (6/6) 100% (73/73) 100% 711af9;1Gb0e;T c.579af9;1Gb0e;T Splice site Detected (1/1) 100% (78/78) 100% R334W c.1000Cb0e;T Missense Detected (3/3) 100% (76/76) 100% R347P c.1040Gb0e;C Missense Detected (1/1) 100% (78/78) 100% A455E c.1364Cb0e;A Missense Detected (2/2) 100% (77/77) 100% èc;I507 c.1519_1521delATC In-frame deletion Detected (1/1) 100% (78/78) 100% èc;F508 c.1521_1523delCTT In-frame deletion Detected (30/30) 100% (49/49) 100% G542* c.1624Gb0e;T Nonsense Detected (4/4) 100% (75/75) 100% G551D c.1652Gb0e;A Missense Detected (6/6) 100% (73/73) 100% R553* c.1657Cb0e;T Nonsense Detected (3/3) 100% (76/76) 100% R560T c.1679Gb0e;C Missense Detected (1/1) 100% (78/78) 100% 1898af9;1Gb0e;A c.1766af9;1Gb0e;A Splice site Detected (2/2) 100% (77/77) 100% 2789af9;5Gb0e;A c.2657af9;5Gb0e;A Splice site Detected (1/1) 100% (78/78) 100% 3120af9;1Gb0e;A c.2988af9;1Gb0e;A Splice site Detected (2/2) 100% (77/77) 100% R1162* c.3484Cb0e;T Nonsense Detected (1/1) 100% (78/78) 100% 3659delC c.3528del Frameshift deletion Detected (1/1) 100% (78/78) 100% 3849af9;10kbCb0e;T c.3718-2477Cb0e;T Splice site Detected (1/1) 100% (78/78) 100% W1282* c.3846Gb0e;A Nonsense Detected (3/3) 100% (75/75) 100% N1303K c.3909Cb0e;G Missense Detected (1/1) 100% (78/78) 100% 2184delA c.2052del Frameshift deletion Detected (1/1) 97% (76/78) 97% 1717-1Gb0e;A c.1585-1Gb0e;A Splice site Detected (1/1) 100% (78/78) 100% a TP, true-positive rate; TN, true-negative rate. Login to comment

[hide] Genetic testing of sperm donors for cystic fibrosi... Eur J Obstet Gynecol Reprod Biol. 2013 Sep;170(1):183-7. doi: 10.1016/j.ejogrb.2013.06.022. Epub 2013 Jul 15. Landaburu I, Gonzalvo MC, Clavero A, Ramirez JP, Yoldi A, Mozas J, Zamora S, Martinez L, Castilla JA
Genetic testing of sperm donors for cystic fibrosis and spinal muscular atrophy: evaluation of clinical utility.
Eur J Obstet Gynecol Reprod Biol. 2013 Sep;170(1):183-7. doi: 10.1016/j.ejogrb.2013.06.022. Epub 2013 Jul 15., [PMID:23866907]

Abstract [show]
OBJECTIVE: To evaluate the clinical utility of genetic testing for cystic fibrosis (CF) and spinal muscular atrophy (SMA) in sperm donors. STUDY DESIGN: We studied the results of the genetic tests for CF and SMA applied to 372 sperm donor candidates. The CF carrier screening test analysed 32 mutations on the CFTR gene. Regarding SMA, the carrier test studied possible deletions of SMN1/2 by Multiplex Ligation-dependent Probe Amplification (MLPA) methodology. RESULTS: The carrier frequency obtained was greater for SMA than for CF. After adjusting the results obtained for the sensitivity of the tests, and taking into account the prevalence of female carriers in our population, the probability of transmission of the disease to the child from a donor with a negative genetic test was about five times lower in the case of SMA than in CF, although this difference was not statistically significant. The number of donors needed to screen (NNS) to avoid the occurrence of a child being affected by CF and SMA in our population was similar in both cases (1591 vs. 1536). CONCLUSIONS: This study demonstrates the need to include SMA among the diseases for which genetic screening is performed in the process of sperm donor selection. We believe that testing donors for SMA is as important and as useful as doing so for CF.

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51 The panel of mutations studied was: S549N, S549R, R553X, G551D, V520F, I507del, F508del, 3876delA, 1717-1G->A, G542X, R560T, 3120+1G->A, A455E, R117H, 394delTT, 2183AA- >G, 2184delA, 2789+5G->A, 1898+1G->A, 621+1G->T, 711+1G- >T, G85E, R347P, R347H, W1282X, R334W, 1078delT, 3849+10kbC->T, R1162X, N1303K, 3659delC, 3905insT. Login to comment

[hide] Revertants, low temperature, and correctors reveal... Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004. Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, Da Paula AC, Williams J, Hirst S, Gomes CM, Amaral MD
Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.
Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004., [PMID:23890012]

Abstract [show]
Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

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89 As a negative control, we also analyzed the effect of low temperature on CFTR bearing another class II disease-causing mutation R560T (Roxo-Rosa et al., 2006), confirming its previously described insensitivity to low temperature rescue (Figures 4A and 4B). Login to comment
231 EXPERIMENTAL PROCEDURES Cells and Culture Conditions BHK cell lines expressing F508del-4RK (R29K/R516K/R555K/R716K)-, F508del-G550E-, F508del-R1070W-, F508del-V510D-, F508del-R555K-, F508del-V510D/G550E-, F508del-G550E/R1070W-, DAA (D567A)-, 4RK- DAA-, DD/AA (D565A, D567A)-, 4RK-DD/AA-, and R560T-CFTR were produced and cultured as previously described (Roxo-Rosa et al., 2006). Login to comment

[hide] Effect of ivacaftor on CFTR forms with missense mu... J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23. Van Goor F, Yu H, Burton B, Hoffman BJ
Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23., [PMID:23891399]

Abstract [show]
BACKGROUND: Ivacaftor (KALYDECO, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function. METHODS: The effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface. RESULTS: Ivacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance. CONCLUSIONS: These in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.

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44 None M1V A46D E56K P67L R74W G85E E92K D110E D110H R117C R117H E193K L206W R334W I336K T338I S341P R347H R347P R352Q A455E L467P S492F F508del V520F A559T R560S R560T A561E Y569D D579G R668C L927P S945L S977F L997F F1052V H1054D K1060T L1065P R1066C R1066H R1066M A1067T R1070Q R1070W F1074L L1077P H1085R M1101K D1152H S1235R D1270N N1303K 0 100 200 300 400 500 600 * * * CFTR Mutation mRNA (% Normal CFTR) Fig. 1. Login to comment
64 Mutant CFTR form CFTR processing Mature/total % Normal CFTR Normal 0.89 &#b1; 0.01 100.0 &#b1; 18.5 G85E -0.05 &#b1; 0.04 -1.0 &#b1; 0.9 R560S 0.00 &#b1; 0.00 0.0 &#b1; 0.0 R1066C 0.02 &#b1; 0.01 0.0 &#b1; 0.0 S492F 0.00 &#b1; 0.00 0.1 &#b1; 0.1 R560T 0.01 &#b1; 0.01 0.2 &#b1; 0.1 V520F 0.05 &#b1; 0.03 0.3 &#b1; 0.2 M1101K 0.05 &#b1; 0.03 0.3 &#b1; 0.1 A561E 0.08 &#b1; 0.04 0.5 &#b1; 0.2 R1066M 0.02 &#b1; 0.02 0.5 &#b1; 0.4 N1303K 0.02 &#b1; 0.02 0.5 &#b1; 0.3 A559T 0.16 &#b1; 0.09 0.6 &#b1; 0.2 M1V 0.06 &#b1; 0.06 0.7 &#b1; 0.6 Y569D 0.11 &#b1; 0.04 0.6 &#b1; 0.2 R1066H 0.08 &#b1; 0.02a 0.7 &#b1; 0.2a L1065P 0.05 &#b1; 0.05 1.0 &#b1; 0.8 L467P 0.10 &#b1; 0.07 1.2 &#b1; 0.8 L1077P 0.08 &#b1; 0.04 1.5 &#b1; 0.6 A46D 0.21 &#b1; 0.08 1.9 &#b1; 0.5a E92K 0.06 &#b1; 0.05 1.9 &#b1; 1.3 H1054D 0.09 &#b1; 0.04 1.9 &#b1; 0.8 F508del 0.09 &#b1; 0.02a 2.3 &#b1; 0.5a H1085R 0.06 &#b1; 0.01a 3.0 &#b1; 0.7a I336K 0.42 &#b1; 0.05a 6.5 &#b1; 0.7a L206W 0.35 &#b1; 0.10a 6.8 &#b1; 1.7a F1074L 0.52 &#b1; 0.03a 10.9 &#b1; 0.6a A455E 0.26 &#b1; 0.10a 11.5 &#b1; 2.5a E56K 0.29 &#b1; 0.04a 12.2 &#b1; 1.5a R347P 0.48 &#b1; 0.04a 14.6 &#b1; 1.8a R1070W 0.61 &#b1; 0.04a 16.3 &#b1; 0.6a P67L 0.36 &#b1; 0.04a 28.4 &#b1; 6.8a R1070Q 0.90 &#b1; 0.01a 29.5 &#b1; 1.4a S977F 0.97 &#b1; 0.01a 37.3 &#b1; 2.4a A1067T 0.78 &#b1; 0.03a 38.6 &#b1; 6.1a D579G 0.72 &#b1; 0.02a 39.3 &#b1; 3.1a D1270N 1.00 &#b1; 0.00a,c 40.7 &#b1; 1.2a S945L 0.65 &#b1; 0.04a 42.4 &#b1; 8.9a L927P 0.89 &#b1; 0.01a,b 43.5 &#b1; 2.5a,b R117C 0.87 &#b1; 0.02a,b 49.1 &#b1; 2.9a,b T338I 0.93 &#b1; 0.03a,b 54.2 &#b1; 3.7a,b L997F 0.90 &#b1; 0.04a,b 59.8 &#b1; 10.4a,b D110H 0.97 &#b1; 0.01a,b 60.6 &#b1; 1.5a,b S341P 0.79 &#b1; 0.02a 65.0 &#b1; 4.9a,b R668C 0.94 &#b1; 0.03a,b 68.5 &#b1; 1.9a,b R74W 0.78 &#b1; 0.01a 69.0 &#b1; 2.7a,b D110E 0.92 &#b1; 0.05a,b 87.5 &#b1; 9.5a,b R334W 0.91 &#b1; 0.05a,b 97.6 &#b1; 10.0a,b K1060T 0.87 &#b1; 0.02a,b 109.9 &#b1; 28.0a,b R347H 0.96 &#b1; 0.02a,c 120.7 &#b1; 2.8a,b S1235R 0.96 &#b1; 0.00a,c 139.0 &#b1; 9.0a,b E193K 0.84 &#b1; 0.02a,b 143.0 &#b1; 17.1a,b R117H 0.86 &#b1; 0.01a,b 164.5 &#b1; 34.2a,b R352Q 0.98 &#b1; 0.01a,b 179.9 &#b1; 8.0a,c F1052V 0.90 &#b1; 0.01a,b 189.9 &#b1; 33.1a,b D1152H 0.96 &#b1; 0.02a,c 312.0 &#b1; 45.5a,b Notes to Table 1: Quantification of steady-state CFTR maturation expressed as the mean (&#b1;SEM; n = 5-9) ratio of mature CFTR to total CFTR (immature plus mature) or level of mature mutant CFTR relative to mature normal-CFTR (% normal CFTR) in FRT cells individually expressing CFTR mutations. Login to comment
74 Because the level of CFTR mRNA was similar across the panel of cell lines tested, the range in baseline activity and ivacaftor response likely reflects the severity of the functional defect and/or the 0 50 100 150 200 S341P R347P L467P S492F A559T A561E Y569D L1065P R1066C R1066M L1077P M1101K N1303K R560S L927P R560T H1085R V520F E92K M1V F508del H1054D I336K A46D G85E R334W T338I R1066H R352Q R117C L206W R347H S977F S945L A455E F1074L E56K P67L R1070W D110H D579G D110E R1070Q L997F A1067T E193K R117H R74W K1060T R668C D1270N D1152H S1235R F1052V Baseline With ivacaftor * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Chloride transport (% Normal) Mutant CFTR form 0 100 200 300 400 S341P R347P L467P S492F A559T A561E Y569D L1065P R1066C R1066M L1077P M1101K N1303K R560S L927P R560T H1085R V520F E92K M1V F508del H1054D I336K A46D G85E R334W T338I R1066H R352Q R117C L206W R347H S977F S945L A455E F1074L P67L E56K R1070W D110H D579G D110E R1070Q L997F A1067T E193K R117H R74W K1060T R668C D1270N D1152H S1235R F1052V * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mature CFTR (% Normal) Mutant CFTR form A B Fig. 2. Login to comment
82 Mutation Patientsa Chloride transport (bc;A/cm2 ) Chloride transport (% normal) EC50 Baseline With ivacaftor Baseline With ivacaftor Fold increase over baselineb Normal 204.5 &#b1; 33.3 301.3 &#b1; 33.8c 100.0 &#b1; 16.3 147.3 &#b1; 16.5c 1.5 266 &#b1; 42 G551D 1282 1.5 &#b1; 0.7 113.2 &#b1; 13.0c 1.0 &#b1; 0.5 55.3 &#b1; 6.3c 55.3 312 &#b1; 73 F1052V 12 177.3 &#b1; 13.7 410.2 &#b1; 11.3c 86.7 &#b1; 6.7 200.7 &#b1; 5.6c 2.3 177 &#b1; 14 S1235R ND 160.6 &#b1; 25.7 352.1 &#b1; 43.4c 78.5 &#b1; 12.6 172.2 &#b1; 21.2c 2.2 282 &#b1; 104 D1152H 185 117.3 &#b1; 23.0 282.7 &#b1; 46.9c 57.4 &#b1; 11.2 138.2 &#b1; 22.9c 2.4 178 &#b1; 67 D1270N 32 109.5 &#b1; 20.5 209.5 &#b1; 27.4c 53.6 &#b1; 10.0 102.4 &#b1; 13.4c 1.9 254 &#b1; 56 R668C 45 99.0 &#b1; 9.4 217.6 &#b1; 11.7c 48.4 &#b1; 4.6 106.4 &#b1; 5.7c 2.2 517 &#b1; 105 K1060T ND 89.0 &#b1; 9.8 236.4 &#b1; 20.3c 43.5 &#b1; 4.8 115.6 &#b1; 9.9c 2.7 131 &#b1; 73 R74W 25 86.8 &#b1; 26.9 199.1 &#b1; 16.8c 42.5 &#b1; 13.2 97.3 &#b1; 8.2c 2.3 162 &#b1; 17 R117H 739 67.2 &#b1; 13.3 274.1 &#b1; 32.2c 32.9 &#b1; 6.5 134.0 &#b1; 15.7c 4.1 151 &#b1; 14 E193K ND 62.2 &#b1; 9.8 379.1 &#b1; 1.1c 30.4 &#b1; 4.8 185.4 &#b1; 1.0c 6.1 240 &#b1; 20 A1067T ND 55.9 &#b1; 3.2 164.0 &#b1; 9.7c 27.3 &#b1; 1.6 80.2 &#b1; 4.7c 2.9 317 &#b1; 214 L997F 27 43.7 &#b1; 3.2 145.5 &#b1; 4.0c 21.4 &#b1; 1.6 71.2 &#b1; 2.0c 3.3 162 &#b1; 12 R1070Q 15 42.0 &#b1; 0.8 67.3 &#b1; 2.9c 20.6 &#b1; 0.4 32.9 &#b1; 1.4c 1.6 164 &#b1; 20 D110E ND 23.3 &#b1; 4.7 96.4 &#b1; 15.6c 11.4 &#b1; 2.3 47.1 &#b1; 7.6c 4.1 213 &#b1; 51 D579G 21 21.5 &#b1; 4.1 192.0 &#b1; 18.5c 10.5 &#b1; 2.0 93.9 &#b1; 9.0c 8.9 239 &#b1; 48 D110H 30 18.5 &#b1; 2.2 116.7 &#b1; 11.3c 9.1 &#b1; 1.1 57.1 &#b1; 5.5c 6.2 249 &#b1; 59 R1070W 13 16.6 &#b1; 2.6 102.1 &#b1; 3.1c 8.1 &#b1; 1.3 49.9 &#b1; 1.5c 6.2 158 &#b1; 48 P67L 53 16.0 &#b1; 6.7 88.7 &#b1; 15.7c 7.8 &#b1; 3.3 43.4 &#b1; 7.7c 5.6 195 &#b1; 40 E56K ND 15.8 &#b1; 3.1 63.6 &#b1; 4.4c 7.7 &#b1; 1.5 31.1 &#b1; 2.2c 4.0 123 &#b1; 33 F1074L ND 14.0 &#b1; 3.4 43.5 &#b1; 5.4c 6.9 &#b1; 1.6 21.3 &#b1; 2.6c 3.1 141 &#b1; 19 A455E 120 12.9 &#b1; 2.6 36.4 &#b1; 2.5c 6.3 &#b1; 1.2 17.8 &#b1; 1.2c 2.8 170 &#b1; 44 S945L 63 12.3 &#b1; 3.9 154.9 &#b1; 47.6c 6.0 &#b1; 1.9 75.8 &#b1; 23.3c 12.6 181 &#b1; 36 S977F 9 11.3 &#b1; 6.2 42.5 &#b1; 19.1c 5.5 &#b1; 3.0 20.8 &#b1; 9.3c 3.8 283 &#b1; 36 R347H 65 10.9 &#b1; 3.3 106.3 &#b1; 7.6c 5.3 &#b1; 1.6 52.0 &#b1; 3.7c 9.8 280 &#b1; 35 L206W 81 10.3 &#b1; 1.7 36.4 &#b1; 2.8c 5.0 &#b1; 0.8 17.8 &#b1; 1.4c 3.6 101 &#b1; 13 R117C 61 5.8 &#b1; 1.5 33.7 &#b1; 7.8c 2.9 &#b1; 0.7 16.5 &#b1; 3.8c 5.7 380 &#b1; 136 R352Q 46 5.5 &#b1; 1.0 84.5 &#b1; 7.8c 2.7 &#b1; 0.5 41.3 &#b1; 3.8c 15.2 287 &#b1; 75 R1066H 29 3.0 &#b1; 0.3 8.0 &#b1; 0.8c 1.5 &#b1; 0.1 3.9 &#b1; 0.4c 2.6 390 &#b1; 179 T338I 54 2.9 &#b1; 0.8 16.1 &#b1; 2.4c 1.4 &#b1; 0.4 7.9 &#b1; 1.2c 5.6 334 &#b1; 38 R334W 150 2.6 &#b1; 0.5 10.0 &#b1; 1.4c 1.3 &#b1; 0.2 4.9 &#b1; 0.7c 3.8 259 &#b1; 103 G85E 262 1.6 &#b1; 1.0 1.5 &#b1; 1.2 0.8 &#b1; 0.5 0.7 &#b1; 0.6 NS NS A46D ND 2.0 &#b1; 0.6 1.1 &#b1; 1.1 1.0 &#b1; 0.3 0.5 &#b1; 0.6 NS NS I336K 29 1.8 &#b1; 0.2 7.4 &#b1; 0.1c 0.9 &#b1; 0.1 3.6 &#b1; 0.1c 4 735 &#b1; 204 H1054D ND 1.7 &#b1; 0.3 8.7 &#b1; 0.3c 0.8 &#b1; 0.1 4.2 &#b1; 0.1c 5.3 187 &#b1; 20 F508del 29,018 0.8 &#b1; 0.6 12.1 &#b1; 1.7c 0.4 &#b1; 0.3 5.9 &#b1; 0.8c 14.8 129 &#b1; 38 M1V 9 0.7 &#b1; 1.4 6.5 &#b1; 1.9c 0.4 &#b1; 0.7 3.2 &#b1; 0.9c 8.0 183 &#b1; 85 E92K 14 0.6 &#b1; 0.2 4.3 &#b1; 0.8c 0.3 &#b1; 0.1 2.1 &#b1; 0.4c 7.0 198 &#b1; 46 V520F 58 0.4 &#b1; 0.2 0.5 &#b1; 0.2 0.2 &#b1; 0.1 0.2 &#b1; 0.1 NS NS H1085R ND 0.3 &#b1; 0.2 2.1 &#b1; 0.4 0.2 &#b1; 0.1 1.0 &#b1; 0.2 NS NS R560T 180 0.3 &#b1; 0.3 0.5 &#b1; 0.5 0.1 &#b1; 0.1 0.2 &#b1; 0.2 NS NS L927P 15 0.2 &#b1; 0.1 10.7 &#b1; 1.7c 0.1 &#b1; 0.1 5.2 &#b1; 0.8c 52.0 313 &#b1; 66 R560S ND 0.0 &#b1; 0.1 -0.2 &#b1; 0.2 0.0 &#b1; 0.0 -0.1 &#b1; 0.1 NS NS N1303K 1161 0.0 &#b1; 0.0 1.7 &#b1; 0.3 0.0 &#b1; 0.0 0.8 &#b1; 0.2 NS NS M1101K 79 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS L1077P 42 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R1066M ND 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R1066C 100 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS L1065P 25 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS Y569D 9 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS A561E ND 0.0 &#b1; 0.1 0.0 &#b1; 0.1 0.0 &#b1; 0.0 0.0 &#b1; 0.1 NS NS A559T 43 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS S492F 16 0.0 &#b1; 0.0 1.7 &#b1; 1.2 0.0 &#b1; 0.0 0.8 &#b1; 0.6 NS NS L467P 16 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R347P 214 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS S341P 9 0.0 &#b1; 0.0 0.2 &#b1; 0.2 0.0 &#b1; 0.0 0.1 &#b1; 0.1 NS NS a Number of individuals with the individual mutation in the CFTR-2 database (www.CFTR2.org). Login to comment

[hide] Different cystic fibrosis transmembrane conductanc... Urology. 2013 Oct;82(4):824-8. doi: 10.1016/j.urology.2013.06.024. Epub 2013 Aug 14. Lu S, Yang X, Cui Y, Li X, Zhang H, Liu J, Chen ZJ
Different cystic fibrosis transmembrane conductance regulator mutations in Chinese men with congenital bilateral absence of vas deferens and other acquired obstructive azoospermia.
Urology. 2013 Oct;82(4):824-8. doi: 10.1016/j.urology.2013.06.024. Epub 2013 Aug 14., [PMID:23953609]

Abstract [show]
OBJECTIVE: To investigate cystic fibrosis transmembrane conductance regulator (CFTR) gene in Chinese men with congenital bilateral absence of vas deferens (CBAVD) and other obstructive azoospermia. MATERIALS AND METHODS: Four hundred one patients with obstructive azoospermia were included. Patients were grouped as 158 with CBAVD and 243 with other acquired obstructive azoospermia. Another 200 fertile men were used as controls. Genomic deoxyribonucleic acid was isolated from peripheral blood lymphocytes for all men. The exon 10 and 11 CFTR genes were amplified and sequenced. The frequency of CFTR gene mutations was compared among 3 groups. RESULTS: Six heterozygous mutations (+/-), I556V, M469V, E527N, F508del, S485C, and I558S, were found in 30 patients, and 1 homozygous mutation (+/+), I556V, was found in 1 patient. The overall frequency of CFTR mutations was 31 of 401 (7.7%). Of these mutations, I556V was the most common type with 24 of 31 (77.4%). In CBAVD group, 20 of 158 patients were identified with 6 different heterozygous mutations (I556V, M469V, E527N, F508del, S485C, and I558S) and 1 homozygous mutation (I556V). The rate of CFTR mutations was 12.7%. In acquired obstructive group, 11 of 243 patients were identified with 2 different heterozygous mutations, I556V and M469V; the rate of mutations was 4.5%. No CFTR mutations were identified in controls. There was significant difference among 3 groups (P = .000). The frequency of CFTR mutations in CBAVD is 2-fold higher than in other acquired obstructive group. CONCLUSION: Different CFTR mutations are observed in Chinese patients with CBAVD. I556V is the major common type of CFTR mutations in Chinese patients with CBAVD.

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70 In this study, we analyzed exons 10 and 11 CFTR gene in CBAVD and other acquired obstructive azoospermia because common mutations such as F508del, D507, G551D, G542X, R560T, and R553X associated with CF in Caucasians were frequently identified in these exons. Login to comment

[hide] Cystic fibrosis carrier screening in a North Ameri... Genet Med. 2014 Jul;16(7):539-46. doi: 10.1038/gim.2013.188. Epub 2013 Dec 19. Zvereff VV, Faruki H, Edwards M, Friedman KJ
Cystic fibrosis carrier screening in a North American population.
Genet Med. 2014 Jul;16(7):539-46. doi: 10.1038/gim.2013.188. Epub 2013 Dec 19., [PMID:24357848]

Abstract [show]
PURPOSE: The aim of this study was to compare the mutation frequency distribution for a 32-mutation panel and a 69-mutation panel used for cystic fibrosis carrier screening. Further aims of the study were to examine the race-specific detection rates provided by both panels and to assess the performance of extended panels in large-scale, population-based cystic fibrosis carrier screening. Although genetic screening for the most common CFTR mutations allows detection of nearly 90% of cystic fibrosis carriers, the large number of other mutations, and their distribution within different ethnic groups, limits the utility of general population screening. METHODS: Patients referred for cystic fibrosis screening from January 2005 through December 2010 were tested using either a 32-mutation panel (n = 1,601,308 individuals) or a 69-mutation panel (n = 109,830). RESULTS: The carrier frequencies observed for the 69-mutation panel study population (1/36) and Caucasian (1/27) and African-American individuals (1/79) agree well with published cystic fibrosis carrier frequencies; however, a higher carrier frequency was observed for Hispanic-American individuals (1/48) using the 69-mutation panel as compared with the 32-mutation panel (1/69). The 69-mutation panel detected ~20% more mutations than the 32-mutation panel for both African-American and Hispanic-American individuals. CONCLUSION: Expanded panels using race-specific variants can improve cystic fibrosis carrier detection rates within specific populations. However, it is important that the pathogenicity and the relative frequency of these variants are confirmed.

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63 This threshold could not be reached Table 1ߒ CFTR allele frequency identified by the CF32 mutation panel Varianta Number of detected alleles Mutation (%) Legacy nomenclature HGVS nomenclature F508delb p.F508del 31,142 68.69 R117Hb p.R117H 5,198 11.46 G542Xb p.G542X 1,162 2.56 G551Db p.G551D 989 2.18 W1282Xb p.W1282X 824 1.82 3120ߙ+ߙ1G>Ab c.2988ߙ+ߙ1G>A 706 1.56 N1303Kb p.N1303K 648 1.43 R553Xb p.R553X 487 1.07 3849ߙ+ߙ10kbC>Tb c.3717ߙ+ߙ12191C>T 436 0.96 621ߙ+ߙ1G>Tb c.489ߙ+ߙ1G>T 410 0.90 1717-1G>Ab c.1585-1G>A 388 0.86 2789ߙ+ߙ5G>Ab c.2657ߙ+ߙ5G>A 382 0.84 I507delb p.I507del 258 0.57 R334Wb p.R334W 257 0.57 R1162Xb p.R1162X 211 0.47 G85Eb p.G85E 199 0.44 1898ߙ+ߙ1G>Ab c.1766ߙ+ߙ1G>A 170 0.37 R347Hc p.R347H 160 0.35 3659delCb c.3528delC 155 0.34 3876delAc c.3744delA 153 0.34 R560Tb p.R560T 132 0.29 S549Nc p.S549N 125 0.28 3905insTc c.3773dupT 121 0.27 R347Pb p.R347P 117 0.26 2184delAb c.2052delA 107 0.24 A455Eb p.A455E 106 0.23 711ߙ+ߙ1G>Tb c.579ߙ+ߙ1G>T 65 0.14 394delTTc c.262_263delTT 56 0.12 V520Fc p.V520F 54 0.12 1078delTc c.948delT 52 0.11 2183AA>Ga,c c.2051_2052delAAinsG 37 0.08 S549Rc p.S549R 31 0.07 Total 45,338 100 a 2183AA>G variant was added to the panel in 2010. b Variants from ACMG/ACOG CF screening panel. c Classified as a CF-causing mutation by the CFTR2 Database. ACMG, American College of Medical Genetics and Genomics; ACOG, American College of Obstetricians and Gynecologists; CF, cystic fibrosis; HGVS, Human Genome Variation Society. Table 2ߒ Continued on next page Table 2ߒ CFTR allele frequency identified by the CF69 mutation panel Varianta Allele frequency Mutation (%) Legacy nomenclature HGVS nomenclature F508delb p.F508del 1,868 60.49 R117Hb p.R117H 274 8.87 D1152Hc p.D1152H 125 4.05 G542Xb p.G542X 98 3.17 L206Wd p.L206W 73 2.36 3120ߙ+ߙ1G>Ab c.2988ߙ+ߙ1G>A 65 2.10 G551Db p.G551D 47 1.52 N1303Kb p.N1303K 42 1.36 W1282Xb p.W1282X 38 1.23 3849ߙ+ߙ10kbC>Tb c.3717ߙ+ߙ12191C>T 28 0.91 3876delAd c.3744delA 28 0.91 F311dele p.F312del 24 0.78 I507delb p.I507del 24 0.78 R553Xb p.R553X 24 0.78 R117Cd p.R117C 22 0.71 621ߙ+ߙ1G>Tb c.489ߙ+ߙ1G>T 21 0.68 1717-1G>Ab c.1585-1G>A 18 0.58 S549Nd p.S549N 18 0.58 R334Wb p.R334W 17 0.55 2789ߙ+ߙ5G>Ab c.2657ߙ+ߙ5G>A 16 0.52 G85Eb p.G85E 14 0.45 3199del6e c.3067_3072delATAGTG 12 0.39 R1066Cd p.R1066C 11 0.36 1898ߙ+ߙ1G>Ab c.1766ߙ+ߙ1G>A 10 0.32 R347Hd p.R347H 10 0.32 R1162 Xb p.R1162X 9 0.29 W1089Xd p.W1089X 9 0.29 2184delAb c.2052delA 8 0.26 2307insAd c.2175dupA 8 0.26 1078delTd c.948delT 7 0.23 R75Xd p.R75X 7 0.23 3120G>Ad c.2988 G>A 6 0.19 3659delCb c.3528delC 6 0.19 Q493Xd p.Q493X 6 0.19 R1158Xd p.R1158X 6 0.19 R560Tb p.R560T 6 0.19 1812-1G>Ad c.1680-1G>A 5 0.16 2055del9>Ad c.1923_1931del9insA 5 0.16 406-1G>Ad c.274-1G>A 5 0.16 A559Td p.A559T 5 0.16 R347Pb p.R347P 5 0.16 S1255Xd p.S1255X 5 0.16 1677delTAd c.1545_1546delTA 4 0.13 711ߙ+ߙ1G>Tb c.579ߙ+ߙ1G>T 4 0.13 E60Xd p.E60X 4 0.13 R352Qd p.R352Q 4 0.13 Y1092Xd p.Y1092X 4 0.13 2183AA>Gd c.2051_2052delAAinsG 3 0.10 3791delCd c.3659delC 3 0.10 3905insTd c.3773dupT 3 0.10 by 10 variants: the 2143delT, A455E, S549R, Y122X, and M1101K mutations, typically observed in Caucasians; 935delA, 2869insG, and Q890X in Hispanics; and 405+3A>C and G480C in the African-American population. Login to comment

[hide] The relative frequency of CFTR mutation classes in... J Cyst Fibros. 2014 Jul;13(4):403-9. doi: 10.1016/j.jcf.2013.12.003. Epub 2014 Jan 16. De Boeck K, Zolin A, Cuppens H, Olesen HV, Viviani L
The relative frequency of CFTR mutation classes in European patients with cystic fibrosis.
J Cyst Fibros. 2014 Jul;13(4):403-9. doi: 10.1016/j.jcf.2013.12.003. Epub 2014 Jan 16., [PMID:24440181]

Abstract [show]
More than 1900 different mutations in the CFTR gene have been reported. These are grouped into classes according to their effect on the synthesis and/or function of the CFTR protein. CFTR repair therapies that are mutation or mutation class specific are under development. To progress efficiently in the clinical phase of drug development, knowledge of the relative frequency of CFTR mutation classes in different populations is useful. Therefore, we describe the mutation class spectrum in 25,394 subjects with CF from 23 European countries. In 18/23 countries, 80% or more of the patients had at least one class II mutation, explained by F508del being by far the most frequent mutation. Overall 16.4% of European patients had at least one class I mutation but this varied from 3 countries with more than 30% to 4 countries with less than 10% of subjects. Overall only respectively 3.9, 3.3 and 3.0% of European subjects had at least one mutation of classes III, IV and V with again great variability: 14% of Irish patients had at least one class III mutation, 7% of Portuguese patients had at least one class IV mutation, and in 6 countries more than 5% of patients had at least one class V mutation.

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56 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations Large deletions and insertions 1078delT; 1717-1G࢐A; 3659delC; 621+1G࢐T Class II Defective protein processing G85E, F508del, I507del, R560T, N1303K Class III Defective protein regulation ('gating`) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R117H, R334W, R347P Class V Reduced amount of functioning protein 2789+5G࢐A, 3849+10KbC࢐T, A455E Unclassified All other mutations, including those unknown. Login to comment

[hide] Impact of heterozygote CFTR mutations in COPD pati... Respir Res. 2014 Feb 11;15:18. doi: 10.1186/1465-9921-15-18. Raju SV, Tate JH, Peacock SK, Fang P, Oster RA, Dransfield MT, Rowe SM
Impact of heterozygote CFTR mutations in COPD patients with chronic bronchitis.
Respir Res. 2014 Feb 11;15:18. doi: 10.1186/1465-9921-15-18., [PMID:24517344]

Abstract [show]
BACKGROUND: Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population. METHODS: Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV1/FVC < 0.70 and FEV1 < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network's Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations. RESULTS: Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS). CONCLUSIONS: The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.

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81 As expected based on genotype-phenotype correlations in the disease [33], HBE cells derived from a F508del CFTR heterozygote had slightly lower CFTR activity at baseline than wild type monolayers as measured by Table 1 List of CFTR mutations analyzed F508del R117H 1717-1G > A R117C G85E R334W 1898 + 1G > A Y122X A455E R347P 2184delA G178R I507del R553X 2789 + 5G > A G314E G542X R560T 3120 + 1G > A G330X G551D W1282X 3659delC R347H N1303K 621 + 1G > T K710X 406-1G > A R1162X 711 + 1G > T E60X G480C R1066C W1089X V520F A559T S1196X Q1238X S1251N S1255X 663delT 935delA 1161delC 1288insTA 2184insA 2307insA 2711delT 2869insG R709X R764X R1158X 574delA Q493X 1898 + 5G > T 3905insT I506T 3849 + 10kbC > T 712-1G > T Q98R Q552X S549N 1078delT H199Y 444delA S549R (T > G) 2143delT P205S 2043delG 1811 + 1.6kbA > G 3272-26A > G L206W 3791delC Y1092X (C > G) 3199del6 F508C 2108delA Y1092X (C > A) D1152H V520I 3667del4 394delTT 3876delA M1101K 1677delTA W1098X (TGA) 1812-1G > A 4016insT 1609delCA 3171delC response to forskolin stimulation (49.3 &#b1; 11.5 bc;A/cm2 in CFTR (+/+) vs. 40.5 &#b1; 5.3 bc;A/cm2 in CFTR (+/-), although this was not statistically significant (Figure 1A,B). Login to comment

[hide] CFTR mutations spectrum and the efficiency of mole... PLoS One. 2014 Feb 26;9(2):e89094. doi: 10.1371/journal.pone.0089094. eCollection 2014. Zietkiewicz E, Rutkiewicz E, Pogorzelski A, Klimek B, Voelkel K, Witt M
CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.
PLoS One. 2014 Feb 26;9(2):e89094. doi: 10.1371/journal.pone.0089094. eCollection 2014., [PMID:24586523]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

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71 Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans Pathogenic mutations 1 1 L15Ffs10X c.43delC 175delC 1 CFMDB 1717-1G.A 2 2 G27V 21.92 c.80G.T 212G.T 1 Novel F508del 2 2 S18RfsX16 c.54-5940_273 +10250del21kb exon2,3del21kb 66 IL19 various CF mutations i2 i2 IVS2_Donor c.164+1G.A 296+1G.A 3 CFMDB various CF mutations 3 3 G85E 22.61 c.254G.A 386G.A 1 IL17 unknown 3 3 E60X c.178G.T 310G.T 0 IL17 x 3 3 L88IfsX22 c.262_263delTT 394delTT 0 IL17 x 4 4 E92K 21.92 c.274G.A 406G.A 2 CFMDB c.164+1G.A; c.2051- 2AA.G 4 4 L101X c.302T.G 434T.G 1 CFMDB c.3717+12191C.T 4 4 K114IfsX5 c.341_353del13bp 473del13bp 1 Novel F508del 4 4 R117H 20.35 c.350G.A 482G.A 5 IL17 F508del; 2x unknown 4 4 R117C 22.07 c.349C.T 481C.T 2 CFMDB S1206X;1x unknown 4 4 L137_L138insT c.412_413insACT L138ins 1 CFMDB F508del 4 4 R153I 22.61 c.458G.T 590G.T 2 Novel F508del; c.3527delC i4 i4 IVS4_Donor c.489+1G.T 621+1G.T 5 IL17 F508del; c.489+1G.T 5 5 L165X c.494T.A 626T.A 1 Novel F508del i5 i5 IVS5_Donor c.579+1G.T 711+1G.T 0 IL19 x i5 i5 IVS5_Donor c.579+3A.G 711+3A.G 2 CFMDB 2,3del21kb; c.2052-3insA i5 i5 IVS5_Donor c.579+5G.A 711+5G.A 0 IL17 x 7 8 F311L 20.90 c.933C.G 965C.G 2 CFMDB 2x F508 7 8 G314R 20.58 c.940G.A 1072G.A 4 CFMDB various CF mutations 7 8 F316LfsX12 c.948delT 1078delT 1 IL17 unkown 7 8 R334W 22.41 c.1000C.T 1132C.T 6 IL17 various CF mutations 7 8 I336K 22.07 c.1007T.A 1139T.A 2 CFMDB 2,3de21kb; F508del 7 8 R347P 22.27 c.1040G.C 1172G.C 11 IL17 various CF mutations i7 i8 IVS8_Donor c.1116+2T.A 1248+2T.A 1 Novel Q1412X 9 10 A455E 22.61 c.1364C.A 1496C.A 0 IL17 x i9 i10 IVS10_Donor c.1392+1G.A 1524+1G.A 1 CFMDB c.3816-7delGT 10 11 S466X c.1397C.G 1529C.G 1 CFMDB G542X 10 11 I507del c.1519_1521delATC 1651delATC 2 IL19 F508del 10 11 F508del c.1521_1523delCTT 1654delCTT 805 IL19 various CF mutations i10 i11 IVS11_Acceptor c.1585-1G.A 1717-1G.A 27 IL19 various CF mutations 11 12 G542X c.1624G.T 1756G.T 25 IL19 various CF mutations 11 12 G551D 21.24 c.1624G.T 1756G.T 5 IL19 various CF mutations 11 12 Q552X c.1654C.T 1786C.T 0 IL19 x 11 12 R553X c.1657C.T 1789C.T 14 IL19 various CF mutations 11 12 R560T 21.92 c.1679G.C 1811G.C 0 IL19 x i12 i13 IVS13_Donor c.1766+1G.A 1898+1G.A 6 IL19 various CF mutations i12 i13 IVS13_Donor c.1766+1G.C 1898+1G.C 1 CFMDB F508del 13 14 H620P 21.73 c.1859A.C 1991A.C 1 CFMDB F508del 13 14 R668C//G576A 21.61//1.73 c.2002C.T//c.1727G.C 2134C.T// 1859G.C 5 b CFMDB// rs1800098 c.1585-1G.A; 4 unknown 13 14 L671X c.2012delT 2143delT 27 IL17 various CF mutations 13 14 K684SfsX38 c.2051_2052delAAinsG 2183AA.G 10 IL17 various CF mutations 13 14 K684NfsX38 c.2052delA 2184delA 0 IL17 x 13 14 Q685TfsX4 c.2052_2053insA 2184insA 15 CFMDB various CF mutationsc , 1 unknown Table 2. Cont. Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans 13 14 L732X c.2195T.G 2327T.G 1 CFMDB F508del 14A 15 R851X c.2551C.T 2683C.T 3 CFMDB various CF mutations 14A 15 I864SfsX28 c.2589_2599del11bp 2721del11bp 2 CFMDB F508del; 2,3del21kb i14B i16 IVS16_Donor c.2657+2_2657+3insA 2789+2insA 1 CFMDB F508del i14B i16 IVS16_Donor c.2657+5G.A 2789+5G.A 0 IL17 unkown 15 17 Y919C 21.02 c.2756A.G 2888A.G 1 CFMDB unknown 15 17 H939HfsX27 c.2817_2820delTACTC 2949delTACTC 1 Novel unkown i15 i17 IVS17_Donor c.2908+3A.C 3040+3A.C 1 Novel F508del i16 i18 IVS18_Donor c.2988+1G.A 3120+1G.A 0 IL19 x 17A 19 I1023_V1024del c.3067_3072delATAGTG 3199del6 0 IL19 x i17A i19 IVS19 c.3140-26A.G 3272-26A.G 9 IL19 various CF mutations 17B 20 L1065R 21.90 c.3194T.G 3326T.G 1 CFMDB F508del 17B 20 Y1092X c.3276C.A 3408C.A 1 CFMDB R334W i18 i21 IVS21_Donor c.3468+2_3468+3insT 3600+2insT 11 CFMDB various CF mutationsd , 1 unknown 18 21 E1126EfsX7 c.3376_3379delGAAG 3508delGAAG 1 Novel F508del 19 22 R1158X c.3472C.T 3604C.T 2 CFMDB F508del; R553X 19 22 R1162X c.3484C.T 3616C.T 1 IL17 F508del 19 22 L1177SfsX15 c.3528delC 3659delC 4 IL17 various CF mutations 19 22 S1206X c.3617C.A 3749C.A 1 CFMDB R117C i19 i22 IVS22 c.3717+12191C.T 3849+10kbC.T 58 IL17 various CF mutations 20 23 G1244R 22.62 c.3730G.C 3862G.C 1 CFMDB F508del 20 23 S1251N 22.28 c.3752G.A 3884G.A 0 IL19 x 20 23 L1258FfsX7 c.3773_3774insT 3905insT 0 IL19 x 20 23 V1272VfsX28 c.3816_3817delGT 3944delGT 1 CFMDB c.1392+1G.A 20 23 W1282X c.3846G.A 3978G.A 9 IL19 various CF mutations 21 24 N1303K 22.62 c.3909C.G 4041C.G 18 IL19 various CF mutations 22 25 V1327X c.3979delG 4111delG 1 Novel F508del 22 25 S1347PfsX13 c.4035_4038dupCCTA c.4167dupCCTA 1 CFMDB 2,3del21kb 23 26 Q1382X c.4144C.T 4276C.T 1 CFMDB F508del 23 26 Q1412X c.4234C.T 4366C.T 2 CFMDB F508del; c.1116+2T.A i23 i26 IVS26_Donor c.4242+1G.T 4374+1G.T 1 CFMDB F508del Sequence changes of uncertain pathogenic effect, tentatively counted as mutations 6A 6 E217G 0.30 c.650A.G 782A.G 1 CFMDB; rs1219109046 unknown 7 8 R352Q 20.01 c.1055G.A 1187G.A 1 CFMDB; rs121908753 F508del 7 8 Q359R 0.33 c.1076A.G 1208A.G 1 CFMDB F508del i8 i9 IVS9 c.1210-12T5_1210- 34_35 (TG)12 1332-12Tn_- 34TGm 6 CFMDB F508del; 3x unknown i8 i9 IVS9 c.1210-12T5_1210- 34_35 (TG)13 1332-12Tn_- 34TGm 2 CFMDB 2143delT; 1x unknown i8 i9 IVS9 c.1210-12T8 1332-12Tn 1 Novel unknown 10 11 I506V 20.21 c.1516A.G 1648A.G 1 CFMDB; rs1800091 unknown 12 13 V562L 0.79 c.1684G.C 1816G.C 1 CFMDB; rs1800097 unknown 13 14 G723V 0.44 c.2168G.T 2300G.T 1 CFMDB; rs200531709 unknown 15 17 D924N 0.03 c.2770G.A 2902G.A 1 CFMDB; rs201759207 unknown patient with F508del on another allele) was not supported by the SVM value (+0.35); the patient was PS and had ambiguous chloride values (45, 64 and 83 mmol/L). 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102 a Mutations detected by two INNOLiPA_CFTR tests (legacy names): IL19 (INNOLiPA_CFTR19): F508del; G542X; N1303K; W1282X; G551D; 1717-1G.A; R553X; CFTRdele2,3(21kb); I507del; 711+1G.T; 3272-26A.G; 3905insT; R560T; 1898+1G.A; S1251N; I148T; 3199del6; 3120+1G.A; Q552X. Login to comment

[hide] Genetics of cystic fibrosis: CFTR mutation classif... Int J Biochem Cell Biol. 2014 Jul;52:94-102. doi: 10.1016/j.biocel.2014.02.023. Epub 2014 Mar 12. Fanen P, Wohlhuter-Haddad A, Hinzpeter A
Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies.
Int J Biochem Cell Biol. 2014 Jul;52:94-102. doi: 10.1016/j.biocel.2014.02.023. Epub 2014 Mar 12., [PMID:24631642]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools. This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment.

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74 23 ACMG recommended panel of classic CF-causing mutations G85E R117H R334W R347P A455E I507del F508del G542X G551D R553X R560T R1162X W1282X N1303K 621 + 1G > T 711 + 1G > T 1717 - 1G > A 1898 + 1G > A 2184delA 2789 + 5G > A 3120 + 1G > A 3659delC 3849 + 10kbC > T Additional or alternative mutations present at significant frequencies in an ethnic population served by a newborn screening program may be assessed. Login to comment
116 Other class III mutations such as the frequent mutations p.Arg560Thr or p.Gly970Arg are listed in Fig. 3 (Seibert et al., 1996). Login to comment

[hide] New pharmacological approaches for cystic fibrosis... Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14. Bell SC, De Boeck K, Amaral MD
New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls.
Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14., [PMID:24932877]

Abstract [show]
With the discovery of the CFTR gene in 1989, the search for therapies to improve the basic defects of cystic fibrosis (CF) commenced. Pharmacological manipulation provides the opportunity to enhance CF transmembrane conductance regulator (CFTR) protein synthesis and/or function. CFTR modulators include potentiators to improve channel gating (class III mutations), correctors to improve abnormal CFTR protein folding and trafficking (class II mutations) and stop codon mutation read-through drugs relevant for patients with premature stop codons (most class I mutations). After several successful clinical trials the potentiator, ivacaftor, is now licenced for use in adults and children (>six years), with CF bearing the class III G551D mutation and FDA licence was recently expanded to include 8 additional class III mutations. Alternative approaches for class I and class II mutations are currently being studied. Combination drug treatment with correctors and potentiators appears to be required to restore CFTR function of F508del, the most common CFTR mutation. Alternative therapies such as gene therapy and pharmacological modulation of other ion channels may be advantageous because they are mutation-class independent, however progress is less well advanced. Clinical trials for CFTR modulators have been enthusiastically embraced by patients with CF and health care providers. Whilst novel trial end-points are being evaluated allowing CFTR modulators to be efficiently tested, many challenges related to the complexity of CFTR and the biology of the epithelium still need to be overcome.

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493 Class II mutations which besides F508del, include R560T (Roxo-Rosa et al., 2006), A561E (Mendes et al., 2003), R1066C (Seibert et al., 1996) and N1303K (Gregory et al., 1991) amongst others, affect CFTR protein processing due to misfolding which is recognized by endoplasmic reticulum (ER) quality control retention and which targets proteins with abnormal conformations to degradation (Amaral, 2004). Login to comment
547 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations: G542X, R1162X, RW1282X Deletions and insertions: CFTRdele2,3; 1078delT; 1717-1G ࢐ A; 3659delC; 621+1G N T Class II Defective protein processing G85E, F508del, I507del, R560T, A561E, R1066C, N1303K Class III Defective protein regulation (gating) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R334W, R347P, R117H Class V Reduced amount of functioning protein 2789+5G ࢐ A, 3272-26ANG, 3849+10KbC ࢐ T, A455E Class VI Reduced cell surface stability Rescued F508del, c.120del23 Unclassified All other mutations, including those unknown a F508del-CFTR pocket (at NBD1:ICL4 interface) (Farinha et al., 2013). Login to comment

[hide] Mechanisms of CFTR functional variants that impair... PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul. LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, Romagnuolo J, Baillie J, Alkaade S, Cote G, Gardner TB, Amann ST, Slivka A, Sandhu B, Aloe A, Kienholz ML, Yadav D, Barmada MM, Bahar I, Lee MG, Whitcomb DC
Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul., [PMID:25033378]

Abstract [show]
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

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269 67 SNPs (125GtoC, 1716G.A, 1717-1G.A, 1898+1G.A, 2183AA.G, 2184delA, 2789+5G.A, 3120+1G.A, 3659delC, 3849+10kbC.T, 621+ 1G.T, 711+5G.A, A455E, D110H, D1152H, D1270N, D443Y, D579G, F1052V, F1074L, F508C, F508del, G1069R, G1244E, G1349D, G178R, G542X, G551D, G551S, I1131L/V, I148T, I336K/T, I507del, I807M, IVS8T5, K1180T, L1065P, L967S, L997F, M1V, M470V, M952I, M952T, N1303K, P67L, Q1463Q, R1070Q, R1162X, R117C, R117H, R170H, R258G, R297Q, R31C, R352Q, R553X, R668C, R74W, R75Q, S1235R, S1255P, S485R, S977F, T338I, T854T, V201M, W1282X) were multiplexed into 6 wells; 14 SNPs (S492F, S945L, R74Q, R560T, R1162L, G85E, I1027T, R334W, R347P, G576A, 711+1G.T, 1001+11C.T, P1290P, 3199del6) were ascertained separately via TaqMan Gene Expression Assays, with repeat confirmation of all positive results. Login to comment

[hide] Molecular genetic testing for cystic fibrosis: lab... Genet Med. 2015 Mar;17(3):219-25. doi: 10.1038/gim.2014.93. Epub 2014 Jul 31. Lyon E, Schrijver I, Weck KE, Ferreira-Gonzalez A, Richards CS, Palomaki GE
Molecular genetic testing for cystic fibrosis: laboratory performance on the College of American Pathologists external proficiency surveys.
Genet Med. 2015 Mar;17(3):219-25. doi: 10.1038/gim.2014.93. Epub 2014 Jul 31., [PMID:25077647]

Abstract [show]
BACKGROUND: Molecular testing for cystic fibrosis mutations is widespread and routine in reproductive decision making and diagnosis. Our objective was to assess the level of performance of laboratories for this test. METHODS: The College of American Pathologists administers external proficiency testing with multiple DNA samples distributed biannually. RESULTS are analyzed, reviewed, and graded by the joint College of American Pathologists/American College of Medical Genetics and Genomics Biochemical and Molecular Genetics Committee. Assessment is based on genotype and associated clinical interpretation. RESULTS: Overall, 357 clinical laboratories participated in the proficiency testing survey between 2003 and 2013 (322 in the United States and 35 international). In 2013, US participants reported performing nearly 120,000 tests monthly. Analytical sensitivity and specificity of US laboratories were 98.8% (95% confidence interval: 98.4-99.1%) and 99.6% (95% confidence interval: 99.4-99.7%), respectively. Analytical sensitivity improved between 2003 and 2008 (from 97.9 to 99.3%; P = 0.007) and remained steady thereafter. Clinical interpretation matched the intended response for 98.8, 86.0, and 91.0% of challenges with no, one, or two mutations, respectively. International laboratories performed similarly. DISCUSSION: Laboratory testing for cystic fibrosis in the United States has improved since 2003, and these data demonstrate a high level of quality. Neither the number of samples tested nor test methodology affected performance.

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87 These included 621+1, F508del, A455A, 1717-1, R117H, I507del, 3659delC, G85E, G542X, G551D, R553X, R347P, W1282X, N1303K, and R560T. Login to comment

[hide] Full-open and closed CFTR channels, with lateral t... Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7. Mornon JP, Hoffmann B, Jonic S, Lehn P, Callebaut I
Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics.
Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7., [PMID:25287046]

Abstract [show]
In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.

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357 Moreover, a large ''hot spot`` region for natural CFTR mutations is located at the NBD1:ICL4 interface, involving (1) six ICL4 positions (H1054D, G1061R, L1065P, R1066H/R1066C, F1074L, and L1077P), which line the path followed by F508 during the MD1 conformational transition from its initial to its final position, and (2) seven positions in NBD1 (S492F, I507del, F508del, V520F, A559T, R560K/R560T, and A561E) (Fig. 7c). Login to comment

[hide] Improving newborn screening for cystic fibrosis us... Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209. Baker MW, Atkins AE, Cordovado SK, Hendrix M, Earley MC, Farrell PM
Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.
Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209., [PMID:25674778]

Abstract [show]
Purpose:Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology.Methods:An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation.Results:The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified.Conclusion:The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.Genet Med advance online publication 12 February 2015Genetics in Medicine (2015); doi:10.1038/gim.2014.209.

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15 Correspondence: Mei W. Baker (mwbaker@wisc.edu) Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study Mei W. Baker, MD1,2 , Anne E. Atkins, MPH2 , Suzanne K. Cordovado, PhD3 , Miyono Hendrix, MS3 , Marie C. Earley, PhD3 and Philip M. Farrell, MD, PhD1,4 Table 1ߒ CF-causing or varying consequences mutations in the MiSeqDx IUO Cystic Fibrosis System c.1521_1523delCTT (F508del) c.2875delG (3007delG) c.54-5940_273ߙ+ߙ10250del21kb (CFTRdele2,3) c.3909C>G (N1303K) c.3752G>A (S1251N) Mutations that cause CF when combined with another CF-causing mutation c.1624G>T (G542X) c.2988ߙ+ߙ1G>A (3120ߙ+ߙ1G->A) c.3964-78_4242ߙ+ߙ577del (CFTRdele22,23) c.613C>T (P205S) c.1021T>C (S341P) c.948delT (1078delT) c.2988G>A (3120G->A) c.328G>C (D110H) c.200C>T (P67L) c.1397C>A (S466X(C>A)) c.1022_1023insTC (1154insTC) c.2989-1G>A (3121-1G->A) c.3310G>T (E1104X) c.3937C>T (Q1313X) c.1397C>G (S466X(C>G)) c.1081delT (1213delT) c.3140-26A>G (3272-26A->G) c.1753G>T (E585X) c.658C>T (Q220X) c.1466C>A (S489X) c.1116ߙ+ߙ1G>A (1248ߙ+ߙ1G->A) c.3528delC (3659delC) c.178G>T (E60X) c.115C>T (Q39X) c.1475C>T (S492F) c.1127_1128insA (1259insA) c.3659delC (3791delC) c.2464G>T (E822X) c.1477C>T (Q493X) c.1646G>A (S549N) c.1209ߙ+ߙ1G>A (1341ߙ+ߙ1G->A) c.3717ߙ+ߙ12191C>T (3849ߙ+ߙ10kbC->T) c.2491G>T (E831X) c.1573C>T (Q525X) c.1645A>C (S549R) c.1329_1330insAGAT (1461ins4) c.3744delA (3876delA) c.274G>A (E92K) c.1654C>T (Q552X) c.1647T>G (S549R) c.1393-1G>A (1525-1G->A) c.3773_3774insT (3905insT) c.274G>T (E92X) c.2668C>T (Q890X) c.2834C>T (S945L) c.1418delG (1548delG) c.262_263delTT (394delTT) c.3731G>A (G1244E) c.292C>T (Q98X) c.1013C>T (T338I) c.1545_1546delTA (1677delTA) c.3873ߙ+ߙ1G>A (4005ߙ+ߙ1G->A) c.532G>A (G178R) c.3196C>T (R1066C) c.1558G>T (V520F) c.1585-1G>A (1717-1G->A) c.3884_3885insT (4016insT) c.988G>T (G330X) c.3197G>A (R1066H) c.3266G>A (W1089X) c.1585-8G>A (1717-8G->A) c.273ߙ+ߙ1G>A (405ߙ+ߙ1G->A) c.1652G>A (G551D) c.3472C>T (R1158X) c.3611G>A (W1204X) c.1679ߙ+ߙ1.6kbA>G (1811ߙ+ߙ1.6kbA->G) c.274-1G>A (406-1G->A) c.254G>A (G85E) c.3484C>T (R1162X) c.3612G>A (W1204X) c.1680-1G>A (1812-1G->A) c.4077_4080delTGTTinsAA (4209TGTT->AA) c.2908G>C (G970R) c.349C>T (R117C) c.3846G>A (W1282X) c.1766ߙ+ߙ1G>A (1898ߙ+ߙ1G->A) c.4251delA (4382delA) c.595C>T (H199Y) c.1000C>T (R334W) c.1202G>A (W401X) c.1766ߙ+ߙ3A>G (1898ߙ+ߙ 3A->G) c.325_327delTATinsG (457TAT->G) c.1007T>A (I336K) c.1040G>A (R347H) c.1203G>A (W401X) c.2012delT (2143delT) c.442delA (574delA) c.1519_1521delATC (I507del) c.1040G>C (R347P) c.2537G>A (W846X) c.2051_2052delAAinsG (2183AA->G) c.489ߙ+ߙ1G>T (621ߙ+ߙ 1G->T) c.2128A>T (K710X) c.1055G>A (R352Q) c.3276C>A (Y1092X (C>A)) c.2052delA (2184delA) c.531delT (663delT) c.3194T>C (L1065P) c.1657C>T (R553X) c.3276C>G (Y1092X (C>G)) c.2052_2053insA (2184insA) c.579ߙ+ߙ1G>T (711ߙ+ߙ 1G->T) c.3230T>C (L1077P) c.1679G>A (R560K) c.366T>A (Y122X) c.2175_2176insA (2307insA) c.579ߙ+ߙ3A>G (711ߙ+ߙ 3A->G) c.617T>G (L206W) c.1679G>C (R560T) - c.2215delG (2347delG) c.579ߙ+ߙ5G>A (711ߙ+ߙ 5G->A) c.1400T>C (L467P) c.2125C>T (R709X) - c.2453delT (2585delT) c.580-1G>T (712-1G->T) c.2195T>G (L732X) c.223C>T (R75X) - c.2490ߙ+ߙ1G>A (2622ߙ+ߙ1G->A) c.720_741delAGGGAG AATGATGATGAAGTAC (852del22) c.2780T>C (L927P) c.2290C>T (R764X) - c.2583delT (2711delT) c.1364C>A (A455E) c.3302T>A (M1101K) c.2551C>T (R851X) - c.2657ߙ+ߙ5G>A (2789ߙ+ߙ5G->A) c.1675G>A (A559T) c.1A>G (M1V) c.3587C>G (S1196X) - Mutations/variants that were validated in this study are in bold. CF, cystic fibrosis. Table 1ߒ Continued on next page reduce carrier detection and potentially improve the positive predictive value (PPV), the NBS goals of equity and the highest possible sensitivity become more difficult to achieve. Login to comment

[hide] Clinical diagnostic Next-Generation sequencing: th... Scand J Clin Lab Invest. 2015 Sep;75(5):374-81. doi: 10.3109/00365513.2015.1031689. Epub 2015 Apr 15. Loukas YL, Thodi G, Molou E, Georgiou V, Dotsikas Y, Schulpis KH
Clinical diagnostic Next-Generation sequencing: the case of CFTR carrier screening.
Scand J Clin Lab Invest. 2015 Sep;75(5):374-81. doi: 10.3109/00365513.2015.1031689. Epub 2015 Apr 15., [PMID:25874479]

Abstract [show]
A 23-mutation panel for CFTR carrier screening is recommended to women of reproductive age by the American College of Obstetricians and Gynecologists. In the present study the optimized efficiency regarding the carrier rate of Next-Generation sequencing (NGS) technology is compared to the one of limited mutation detection panels. A total of 824 consequent cases were subjected to the commercial Cystic Fibrosis Genotyping Assay. Some 188 negative samples randomly selected from the initial group of probands were further subjected to an extended mutation panel characterized by 92% detection rate, as well as to massive parallel sequencing. Twenty-two probands subjected to the commercial assay proved to carry one mutation included in the ACOG panel (carrier rate 0.0267). The latter panels revealed the presence of mutations not included in the ACOG panel in four probands, resulting to an increase of carrier rate of 0.0106 in the case of in-house panel and an increase of rate of 0.0213 if NGS was used. The above data seem to support the implementation of NGS in the routine CFTR carrier screening.

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36 Sample code Source Genotype NA18668*2 Coriell Cell Repositories* CFTR, CFdelex2,3/p.F508del NA07830 Coriell Cell Repositories* CFTR, F508del/556delA NA11275 Coriell Cell Repositories* CFTR, 3659delC/F508del NA11277 Coriell Cell Repositories* CFTR, I507del/wt NA11860 Coriell Cell Repositories* CFTR, 3849af9;10kb,Cb0e;T/3849af9;10kb,Cb0e;T 40C2 CDC** CFTR, F508del/R334W 10C4 CDC** CFTR, 2184delA/394delTT CDC2 CDC** CFTR, F508del/Exon 17&#aa;-17b-18del 212C4 CDC** CFTR, F508del/3659delC 412C2 CDC** CFTR, F508del/R334W 213C4 CDC** CFTR, W1282X/W1282X 21C2 CDC** CFTR, 1717-1Gb0e;A/1154insTC 412C5 CDC** CFTR, F508del/2183AAb0e;G 412C1 CDC** CFTR, 2184delA/394delTT 212C5 CDC** CFTR, F508del/3849af9;10KbCb0e;T 38C4 CDC** CFTR, R553X/wt 48C1 CDC** CFTR, F508del/G542X 48C3 CDC** CFTR, F508del/G551D 19C4 CDC** CFTR, F508del/R560T 19C5 CDC** CFTR, G551D/G551D 29C3 CDC** CFTR, 621af9;1Gb0e;T/N1303K 29C5 CDC** CFTR, F508del/2789af9;5Gb0e;A 49C1 CDC** CFTR, 3120af9;1Gb0e;A/L467P# 49C3 CDC** CFTR, 621af9;1Gb0e;T/R1162X 40C5 CDC** CFTR, 711af9;1Gb0e;T/wt 21C1 CDC** CFTR, A455E/F508del 112C2 CDC** CFTR, 1898af9;1Gb0e;A/F508del 214C5 CDC** CFTR, F508del/3140-26Ab0e;G *http://ccr.coriell.org/; **CDC, Center for Disease Control & Prevention, http://www.cdc.gov/; #According to CDC report, its clinical significance is unknown. Login to comment
94 Mutation cDNA Coverage Score Reference allele (F/R strand) Mutant allele (F/R strand) Genotype F508del* c.1521_1523delCTT 2080 26.5 491/557 523/504 HET 556delA c.424delA 2168 26.7 524/557 547/536 HET 3659delC* c.3528delC 2359 27.0 573/605 566/609 HET I507del c.1519_1521delATC 2246 26.8 508/612 619/501 HET 3849af9;10kb,Cb0e;T c.3717af9;12191Cb0e;T 3596 28.4 - 1834/1756 HOM 3849af9;10kb,Cb0e;T c.3717af9;12191Cb0e;T 4169 29.0 1023/1059 1116/967 HET R334W* c.1000Cb0e;T 2473 27.1 636/599 626/609 HET 2184delA* c.2052delA 3069 27.9 734/801 792/738 HET 394delTT* c.262_263delTT 3176 28.0 775/811 819/766 HET W1282X c.3846Gb0e;A 4268 29.0 - 2168/2096 HOM 1717-1Gb0e;A c.1585-1Gb0e;A 3863 28.7 922/1007 985/944 HET 1154insTC c.1022_1023insTC 4021 28.8 1058/1039 979/941 HET 2183AAb0e;G c.2051_2052delAAinsG 3927 28.8 1023/996 974/926 HET R553X c.1657Cb0e;T 6027 30.2 1532/1480 1476/1534 HET G542X c.1624Gb0e;T 3862 28.7 933/996 925/1002 HET G551D c.1652Gb0e;A 5225 29.7 1257/1351 1341/1268 HET G551D c.1652Gb0e;A 4862 29.5 - 2487/2369 HOM R560T c.1679Gb0e;C 3542 28.4 861/908 915/853 HET 621af9;1Gb0e;T* c.489af9;1Gb0e;T 2256 26.8 534/592 606/519 HET N1303K c.3909Cb0e;G 2126 26.6 534/528 492/568 HET 2789af9;5Gb0e;A c.2657af9;5Gb0e;A 3453 28.3 824/901 895/828 HET 3120af9;1Gb0e;A c.2988af9;1Gb0e;A 3021 27.8 721/787 802/707 HET L467P c.1400Cb0e;T 3848 28.7 928/993 1003/920 HET R1162X c.3484Cb0e;T 4180 29.0 1021/1065 1112/976 HET 711af9;1Gb0e;T c.579af9;1Gb0e;T 4222 29.0 1036/1072 1001/1108 HET A455E c.1364Cb0e;A 5621 30.0 1365/1443 1438/1370 HET 1898af9;1Gb0e;A c.1766af9;1Gb0e;A 2934 27.7 683/782 702/762 HET 3272-26Ab0e;G 3140-26Ab0e;G 3755 28.6 902/973 1008/867 HET of the majority of CFTR mutations carriers in our region. Login to comment

[hide] Translating the genetics of cystic fibrosis to per... Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008. Corvol H, Thompson KE, Tabary O, le Rouzic P, Guillot L
Translating the genetics of cystic fibrosis to personalized medicine.
Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008., [PMID:25940043]

Abstract [show]
Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review.

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55 C p.Arg560Thr (R560T) 0.28% c.1040G . Login to comment

[hide] The improvement of the best practice guidelines fo... Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99. Girardet A, Viart V, Plaza S, Daina G, De Rycke M, Des Georges M, Fiorentino F, Harton G, Ishmukhametova A, Navarro J, Raynal C, Renwick P, Saguet F, Schwarz M, SenGupta S, Tzetis M, Roux AF, Claustres M
The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.
Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99., [PMID:26014425]

Abstract [show]
Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.European Journal of Human Genetics advance online publication, 27 May 2015; doi:10.1038/ejhg.2015.99.

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78 Table 1 Examples of common CF-causing, indetermined, and non CF-causing variants (modified from5,8,17) HGVS nomenclature Legacy name cDNA nucleotide name Protein name CF-causing variantsa F508del c.1521_1523delCTT p.Phe508del G542X c.1624G4T p.Gly542* G551D c.1652G4A p.Gly551Asp N1303K c.3909C4G p.Asn1303Lys W1282X c.3846G4A p.Trp1282* 621+1G4T c.489+1G4T CFTRdele2,3 c.54-5940_273 +10250del21080 p.Ser18Argfs*16 E60X c.178G4T p.Glu60* G85E c.254G4A p.Gly85Glu 394delTT c.262_263delTT p.Leu88Ilefs*22 711+1G4T c.579+1G4T R347P c.1040G4C p.Arg347Pro A455E c.1364C4A p.Ala455Glu Q493X c.1477C4T p.Gln493* I507del c.1519_1521delATC p.Ile507del R553X c.1657C4T p.Arg553* R560T c.1679G4C p.Arg560Thr 1898+1G4A c.1766+1G4A 2183AA4G c.2051_2052delAAinsG p.Lys684Serfs*38 2789+5G4A c.2657+5G4A 3120+1G4A c.2988+1G4A M1101K c.3302 T4A p.Met1101Lys R1162X c.3484C4T p.Arg1162* 3659delC c.3528delC p.Lys1177Serfs*15 M1V c.1 A4G p.? Login to comment

[hide] [Male infertility caused by bilateral agenesis of ... Rev Med Interne. 1997;18(2):114-8. Durieu I, Bey-Omar F, Rollet J, Boggio D, Bellon G, Morel Y, Vital Durand D
[Male infertility caused by bilateral agenesis of the vas deferens: a new clinical form of cystic fibrosis?].
Rev Med Interne. 1997;18(2):114-8., [PMID:9092029]

Abstract [show]
Congenital bilateral absence of vas deferens causes male excretory infertility and represents 1 to 2% of male infertility. Because of a genotypic similarity with cystic fibrosis, the possible in vitro fertilization with epididymal sperm requires careful genetic counselling. We studied genotype, sweat chloride concentration, respiratory function tests, sinus abnormalities, pancreatic and hepatic functions in 22 subjects with congenital bilateral absence of vas deferens. Among them, four were compound heterozygotus, all of them with the R117H mutation. Ten had a positive sweat test, one of them also being compound heterozygotus. Congenital bilateral absence of vas deferens and double mutation or positive sweat test led to high probable cystic fibrosis diagnosis in 13 subjects. Six subjects were heterozygotus for one cystic fibrosis mutation, criterium which is not sufficient for cystic fibrosis diagnosis; five of them had sinus abnormalities, present in 11 of the 22 subjects. Only three patients had no mutation nor sweat chloride abnormalities. This work confirms the high frequency of cystic fibrosis mutations in males with congenital bilateral absence of vas deferens, with a higher frequency of positive sweat test than in other publications, and a high frequency of sinus abnormalities. This monosymptomatic phenotype of cystic fibrosis suggests new hypotheses for a relationship between genotype and phenotype.

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46 Vingt-deux mutations du gene CFTR ont Cte recher- chtes : les cinq plus frequentes (AF508, G542X, N1303K, 1717-G--A, G85E) et les 17 suivantes : R117H, 556delA, R334W, R347H, R347P, S549N, S5491, S549R, G551D, R553X,R560T,G1244E3,S1255X,W1282X,R1283K,3898 ins C, D1270N. Login to comment