ABCMdb

ABCC7 p.Gly622Asp

ClinVar:	c.1865G>A , p.Gly622Asp D , Likely pathogenic
CF databases:	c.1865G>A , p.Gly622Asp (CFTR1) ? , G622D was found in a patient with oligospermia. This putative mutation creates a new MboII site.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (71%), E: D (95%), F: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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No. Sentence Comment
296 G622D and R792G have reduced intrinsic chloride channel activities whereas H620Q and A800G resulted in increased intrinsic chloride transport properties [160]. Login to comment

[hide] Complete mutational screening of the cystic fibros... Hum Reprod. 1999 Dec;14(12):3035-40. Pallares-Ruiz N, Carles S, Des Georges M, Guittard C, Arnal F, Humeau C, Claustres M
Complete mutational screening of the cystic fibrosis transmembrane conductance regulator gene: cystic fibrosis mutations are not involved in healthy men with reduced sperm quality.
Hum Reprod. 1999 Dec;14(12):3035-40., [PMID:10601093]

Abstract [show]
Based on the analysis of the most frequent mutations responsible for cystic fibrosis (CF), a higher than expected frequency of CF mutations was recently reported in men with infertility due to reduced sperm quality. To further document whether this condition is associated with severe or mild abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) functions, we carried out a complete scanning of CFTR sequences using a strategy that detects almost all 850 mutations and 150 polymorphisms reported to date in the CFTR gene. We have investigated a cohort of 56 patients with severe oligoasthenoteratozoospermia (OAT) and 50 controls from southern France for CFTR gene mutations and variations. The frequencies of CF-causing mutations and CFTR variations identified in this OAT sample did not differ significantly from the frequencies found in the normal population. However, we observed a 1.7-fold increase in the proportion of homozygotes for a specific CFTR haplotype (TG11-T7-G1540) in the OAT group (P = 0.025). Our results do not confirm a link between CF mutations and reduced sperm quality. Further studies are needed to substantiate the hypothesis that a combination of variants affecting expression and function of the CFTR protein is associated with male infertility.

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37 G622D has been reported previously (Zielenski approved by the Clinical Research Committee of the University et al., 1996) in a patient with oligozoospermia, and V562L Hospital Montpellier. Login to comment
56 Four OAT men had a missense mutation on one chromosome, 1540 (M470V) in exon 10 was significantly different between Table I. Characterization of CFTR genotypes in 56 patients with oligoasthenoteratozoospermia (OAT) and in 50 controls Mutations IVS8(T)n 1540A/G Other variations IVS8(TG)n OAT 1 I1230T 7/7 A/G 1655T/G 12/12 1 D1152H 7/7 G/G - 11/11 1 F1052V 7/7 A/A 875ϩ40A/G 10/10 1 M952I 7/7 G/G 4404C/T 11/11 1 - 7/7 G/G 4404C/T 11/11 2 - 7/7 A/G 875ϩ40A/G 10/11 2 - 7/7 A/G 125G/C 11/12 1 - 7/7 A/A 125G/C 12/12 1 - 7/9 A/A 1716G/A, 3041-71G/C ϩ 4002A/Ga 11/10 1 - 7/7 G/G 356G/A, 405ϩ46G/T, 4374ϩ13A/G 11/11 1 - 7/7 A/A 875ϩ40A/G, 3499ϩ37G/A 10/10 1 - 7/9 A/A 1859G/C ϩ 2134C/Ta 10/10 1 - 7/7 A/G 4002A/G 12/12 1 - 7/9 A/G 4002A/G 11/10 1 - 7/7 G/G 2377C/T 11/11 1 - 7/7 A/A 875ϩ40A/G, 1716G/A 10/10 1 - 7/7 G/G 3417A/T 11/11 1 - 7/7 A/G 3417A/T 11/12 24 - 7/7 G/G - 11/11 2 - 7/9 A/G - 10/12 3 - 7/9 A/G - 11/10 2 - 7/9 A/A - 10/10 1 - 9/9 A/A - 10/10 2 - 7/7 A/G - 10/11 1 - 7/7 A/A - 10/10 1 - 7/7 G/G - 8/11 Controls 1 ∆F508 7/9 A/G - 10/12 1 ∆F508 7/9 A/A 875ϩ40A/G 10/11 1 V562L 7/7 A/A 223C/T 10/10 1 G622D 7/9 A/G 3041-71G/C ϩ 4002A/Ga 10/11 1 - 7/7 A/A 3419T/G 10/11 1 - 7/7 G/G 4002A/G 11/11 3 - 7/7 A/G 125G/C 11/11 1 - 7/7 G/G 125G/C 11/11 1 - 7/7 A/A 125G/C 10/11 1 - 7/7 A/A 125G/C 11/12 2 - 7/5 A/G 875ϩ40A/G 11/11 1 - 7/7 A/G 875ϩ40A/G 10/10 1 - 7/7 A/A 875ϩ40A/G, 125G/C 10/11 1 - 7/7 G/G 356G/A 11/11 1 - 7/7 A/G 356G/A 10/10 1 - 9/9 A/A 3041-71G/C ϩ 4002A/Ga 10/10 1 - 7/7 G/G 406-6T/C 11/11 1 - 7/7 A/G 3417A/T 10/11 1 - 7/7 G/G 4404C/T 11/11 1 - 7/7 A/G 1859G/Cϩ2134C/Ta 10/11 11 - 7/7 G/G - 11/11 6 - 7/7 A/G - 10/10 2 - 7/7 A/G - 11/11 1 - 7/7 A/G - 10/11 5 - 7/9 A/G - 10/12 2 - 7/5 G/G - 10/11 aDouble mutant alleles, In bold: mutations or variations previously undescribed. Login to comment

[hide] A new approach for identifying non-pathogenic muta... Hum Genet. 2000 Feb;106(2):172-8. Bombieri C, Giorgi S, Carles S, de Cid R, Belpinati F, Tandoi C, Pallares-Ruiz N, Lazaro C, Ciminelli BM, Romey MC, Casals T, Pompei F, Gandini G, Claustres M, Estivill X, Pignatti PF, Modiano G
A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.
Hum Genet. 2000 Feb;106(2):172-8., [PMID:10746558]

Abstract [show]
Given q as the global frequency of the alleles causing a disease, any allele with a frequency higher than q minus the cumulative frequency of the previously known disease-causing mutations (threshold) cannot be the cause of that disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whether they are the cause of cystic fibrosis. A total of 191 DNA samples from random individuals from Italy, France, and Spain were investigated by DGGE (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE were identified by sequencing. The sample size was sufficient to select essentially all mutations with a frequency of at least 0.01. A total of 46 mutations was detected, 20 of which were missense mutations. Four new mutations were identified: 1341+28 C/T, 2082 C/T, L1096R, and I11131V. Thirteen mutations (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2694 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classified as non-CF-causing alleles on the basis of their frequency. The remaining mutations have a cumulative frequency far exceeding q; therefore, most of them cannot be CF-causing mutations. This is the first random survey capable of detecting all the polymorphisms of the coding sequence of a gene.

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No. Sentence Comment
80 Many (13 out of 20) of the missense mutations change highly conserved (5/5 species analyzed) amino acid residues (R75Q, G85E, I148T, I506V, R668C, G622D, L997F, I1027T, F1052V, L1096R, I1131V, R1162L, N1303K); others affect amino acid residues conserved in 4/5 species (K68 E, R170H, M470V, V562L, S1235R), or in 3/5 species (R31C and G576A; Tucker et al. 1992). Login to comment
96 Moreover, 1525-61 A/G (i 9) and 3601-65 C/A (i 18) were detected by SSCA performed in the Spanish sample only (14/82 and 12/80, respectively); these mutations were not identifiable by DGGE as used in the present work The totals are: a378; b362; c380; d356 genes eCertainly a CF-causing mutations fThe most common allele at this site is (TTGA)7 gThe most common allele at this site is T7 hThe frequency shown is that of the M allele Mutation Position North-Central Southern Spain Total East Italy Italy France 82 genes 100 genes 100 genes 100 genes 382 genes % 125 G/C 5`UTR 1 2 7 3 13 3.4 R31C 2 1 1 1 0 3 0.8 K68E 3 1 0 0 0 1 0.3 R75Q 3 1 1 2 0 4 1.0 G85Ee 3 0 1 0 0 1 0.3 406-6 T/C i 3 0 0 1 0 1 0.3 I148T 4 1 0 0 0 1 0.3 621+3 A/G i 4 0 1 0 0 1 0.3 R170H 5 1 0 0 0 1 0.3 875+40 A/G i 6a 11 5 5 2 23 6.0 (TTGA)6 f i 6a 17 11 7 13 48 12.6 1341+28 C/T i 8 1 0 0 0 1 0.3 IVS8-6g T5 i 8 8 2 4 3/78 17a 4.5 IVS8-6g T9 i 8 10 7 10 11/78 38a 10.0 M470Vh 10 42 30 39 27 138 36.1 I506V 10 1 0 0 0 1 0.3 ∆F508e 10 1 0 2 0 3 0.8 1716 G/A 10 2 1 0 5 8 2.1 V562L 12 0 0 1 0 1 0.3 G576A 12 1 0/80 1 0 2b 0.6 G622D 13 0 0/80 1 0 1b 0.3 R668C 13 1 0/80 1 0 2b 0.6 2082 C/T 13 1 0/80 0 0 1b 0.3 2377 C/T 13 0 0/80 0 1 1b 0.3 2694 T/G i 14a 33 23 33 14/80 103c 27.1 2752-15 C/G i 14b 0 3 0 0 3 0.8 3041-71 G/C i 15 0 1 2 0 3 0.8 L997F 17a 0 2 0 0 2 0.5 I1027T 17a 1 0 0 0 1 0.3 F1052V 17b 1 0 0 0 1 0.3 L1096R 17b 0 0 1 0 1 0.3 3417 A/T 17b 1 0 1 0 2 0.5 I1131V 18 0 1 0 0 1 0.3 R1162L 19 0 1 0 0 1 0.3 3690 A/G 19 0 0 0 1/80 1c 0.3 S1235R 19 1 0 0 0 1 0.3 4002 A/G 20 2 3 3 3/80 11c 2.9 4005+28insA i 20 0 1 0 0 0.3 4029 A/G 21 1 0 0 0 1 0.3 N1303Ke 21 1 0 0 0 1 0.3 4404 C/T 24 1 0 1 0 2 0.5 4521 G/A 24 21 16 14/80 15/76 66d 18.5 Total 165 113 137 98 513 encountered in the present survey are possible. Login to comment

[hide] The phenotypic consequences of CFTR mutations. Ann Hum Genet. 2003 Sep;67(Pt 5):471-85. Rowntree RK, Harris A
The phenotypic consequences of CFTR mutations.
Ann Hum Genet. 2003 Sep;67(Pt 5):471-85., [PMID:12940920]

Abstract [show]
Cystic fibrosis is a common autosomal recessive disorder that primarily affects the epithelial cells in the intestine, respiratory system, pancreas, gall bladder and sweat glands. Over one thousand mutations have currently been identified in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that are associated with CF disease. There have been many studies on the correlation of the CFTR genotype and CF disease phenotype; however, this relationship is still not well understood. A connection between CFTR genotype and disease manifested in the pancreas has been well described, but pulmonary disease appears to be highly variable even between individuals with the same genotype. This review describes the current classification of CFTR mutation classes and resulting CF disease phenotypes. Complex disease alleles and modifier genes are discussed along with alternative disorders, such as disseminated bronchiectasis and pancreatitis, which are also thought to result from CFTR mutations.

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No. Sentence Comment
78 Three mutant CFTR proteins, G622D, R792G and E822K, that were transiently expressed in COS cells showed lower chloride channel activities when compared to wild-type CFTR, whereas mutants H620Q and A800G showed increased activities. Login to comment

[hide] Nucleotide-binding domains of human cystic fibrosi... Cell Mol Life Sci. 2004 Jan;61(2):230-42. Callebaut I, Eudes R, Mornon JP, Lehn P
Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer.
Cell Mol Life Sci. 2004 Jan;61(2):230-42., [PMID:14745501]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.

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No. Sentence Comment
251 The G622D mutant (class IV) should be associated with modified properties of the ATP-binding pocket of the 'unconventional` site A. Login to comment

[hide] Preconception and prenatal cystic fibrosis carrier... Genet Med. 2004 May-Jun;6(3):141-4. Monaghan KG, Bluhm D, Phillips M, Feldman GL
Preconception and prenatal cystic fibrosis carrier screening of African Americans reveals unanticipated frequencies for specific mutations.
Genet Med. 2004 May-Jun;6(3):141-4., [PMID:15354332]

Abstract [show]
PURPOSE: It is recommended that cystic fibrosis (CF) carrier screening be made available to African Americans who are either pregnant or planning a pregnancy. We analyzed the carrier and mutant allele frequencies for African Americans undergoing CF carrier screening in our laboratories. METHODS: Between December 2001 and September 2003, we performed carrier screening for 2189 African Americans, testing for at least the 25 recommended mutations. RESULTS: A total of 33 CF carriers were identified. The most common mutations detected were deltaF508, G622D, R117H/7T, and G551D. The G622D allele frequency among African Americans was 0.18%. We did not detect any 3120 + 1G --> A carriers, although 4 were expected (P < 0.05). CONCLUSIONS: When considering only the 25 recommended CF mutations, 1 in 75 African Americans screened in our laboratories were carriers (within the expected range, given a 69% mutation detection rate). The addition of 2 mutations, G622D and Q98R (incidentally identified while screening for ACOG/ACMG mutations), increased the observed carrier frequency to 1 in 66, which is not significantly different from the known African American carrier frequency of 1 in 65. The frequencies of several specific mutations detected were unanticipated, as was the absence of 3120 + 1G --> A carriers. Further studies on African American patients with classic CF are needed to examine the incidence of CF mutations that are not part of the current panel, such as G622D.

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No. Sentence Comment
29 During this phase of testing, G622D, Q98R, and variants of unknown clinical significance were identified by heteroduplex analysis as abnormal band shifts and were sequenced in the forward and reverse directions using the dRhodamine dye terminator sequencing kit (Applied Biosystems). Login to comment
41 The next most common mutations detected were G622D (19%), R117H/7T (12%), which was increased compared to previous estimates of this mutant allele frequency (P Ͻ 0.001), and G551D (6%). Login to comment
50 ⌬F508 was the most common CF mutation identified followed by G622D, R117H/7T, and G551D. Login to comment
51 R117H has been previously reported at an increased frequency among individuals undergoing carrier screening compared to those with a diagnosis of cystic fibrosis.8 An unexpected result was the lack of 3120ϩ1G3A carriers, although 4 were expected given that this mutation accounts for Ϸ12% of the CF muta- Table 1 Summary of carrier screening results using various methods employed between December 2001 and September 2003 OLA v2.0, heteroduplex analysis (exons 4 and 13) and RFLP analysis (3120ϩ1G3A) OLA v3.0 INNO-LiPA Total screened 818 1274 97 No. of carriers identified 16 14 3 Observed carrier frequency 1/51 1/81 Mutations identified ⌬F508 (6), G622D (3), R117H/7T (3), I148T (3199del6 negative), Q98R, 1898ϩ1G3A, and G551Da ⌬F508 (14), R117H/7T, R553X, and G551D a In addition, 2 persons were positive for F693L (TTG) and 1 was positive for P140S (C3T at 550); both are variants of unknown clinical significance. Login to comment
57 Two mutations, G622D and Q98R, and two variants of unknown clinical significance, F693L (TTG) and P140S (C3T), which are not part of the recommended CF screening panel, were incidentally detected while using heteroduplex analysis to screen for ACOG/ACMG recommended mutations (Table 2). Login to comment
66 Functional studies have demonstrated abnormal chloride channel activities for G622D.11 However, this mutation has been only reported in one patient with oligospermia and no other identifiable CF mutation on the opposite chromosome,9 so it is not known if this mutation is associated with classic CF. Login to comment
70 A total of 4088 individuals (pan-ethnic), including 818 African Americans, have been screened for G622D. Login to comment
85 Further studies are needed on the incidence of CF mutations that are not part of the current panel, including G622D, to give an estimate of the true frequency of these alleles in African Americans and the general population in the United States. Login to comment

[hide] A large-scale study of the random variability of a... Eur J Hum Genet. 2005 Feb;13(2):184-92. Modiano G, Bombieri C, Ciminelli BM, Belpinati F, Giorgi S, Georges M, Scotet V, Pompei F, Ciccacci C, Guittard C, Audrezet MP, Begnini A, Toepfer M, Macek M, Ferec C, Claustres M, Pignatti PF
A large-scale study of the random variability of a coding sequence: a study on the CFTR gene.
Eur J Hum Genet. 2005 Feb;13(2):184-92., [PMID:15536480]

Abstract [show]
Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (n(g) approximately 100-150 genes). In the present investigation, a large random European population sample (average n(g) approximately 1500) was studied for a single gene, the CFTR (Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q > 0.005), much lower than that of the synonymous (S) substitutions, but they showed a similar rate of subpolymorphic (q < 0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic.

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No. Sentence Comment
33 In the Tajima`s test,19 the null hypothesis of neutrality is rejected if a statistically significant difference between p Common and rare nonsynonymous and synonymous cSNSs G Modiano et al European Journal of Human Genetics Table 1 List of the 61 cSNSsa encountered in the present survey The random samples of genes (and the technique utilized) cSNS variants found NE Italy (DGGE) Central Italy (DGGE) Southern France (DGGE) Northern France (DHPLC) Spain (SSCA) Czechia (DGGE) Hb Â 104 Exon Exon Length (bp) Ref. no. SNS SASc 1st 100d 2nd 500 1st 100d 2nde 1st 100d 2nd 500 1st 100 2nde 82d 72 Abs. Freq. Total sample size q Â 104 se Â 104 NSf Sf 1g 53 0 0 0 0 0/452 0 924 2 111 1 223C4T R31C 1 1 1/500 1 1 0 0/450 0 5 (11) 1 932 (2 432) 45.23 13.61 90 2 224G4T R31L 0 0 0/500 0 0 0 1/450 0 1 1 932 5.17 5.17 10 3 257C4T S42F 0 0 1/500 0 0 0 0/450 0 1 1 932 5.17 5.17 10 3 109 4 334A4G K68E 1 0 0 0/498 0 0 0 0/452 0 0 1 2 504 3.99 3.99 8 5 352C4T R74W 0 0 0 0/498 0 0 0 1/452 0 0 1 2 504 3.99 3.99 8 6 356G4A R75Q 1 7 1 7/498 2 9 2 9/452 0 2 40 (40) 2 504 (2 544) 157.23 24.66 310 7 386G4A G85E 0 0 1 1/498 0 0 0 0/452 0 0 2 2 504 7.99 5.65 16 4 216 8 482G4A R117H 0 0 0 0/292 0 2 0 1/456 0 0 3 2 302 13.03 7.52 26 9 528T4G I132M 0 0 0 0/292 0 0 0 1/456 0 0 1 2 302 4.34 4.34 8 10 575T4C I148T 1 2 0 1/292 0 0 0 1/456 0 1 6 2 302 26.06 10.63 52 5 90 11 640C4T R170C 0 0 0 0/6 0 0 1/448 0 1 1 436 6.96 6.96 14 12 641G4A R170H 1 1 0 0/6 0 0 2/448 0 4 (4) 1 436 (1 930) 20.73 10.35 41 6a 164 0 0 0/6 0 0 0/432 0 0 992 6b 126 0 0 0/6 0 0 0/454 0 942 7 247 0 0 0/6 0 0 0/796 0 1 284 8 93 13 1281G4A L383 0 0 0 0/6 0 0 1/456 0 0 1 1 516 6.60 6.60 13 9 183 14 1402G4A G424S 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 15 1459G4T D443Y 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 10 192 16 1540A4G M470Vh 42 197 30 37/96 39 199 (i) (i) 27 571(736) 1 484 (1 912) 3849.37 111.28 4 735 17 1598C4A S489X 0 0 0 0/96 0 0 0 1/796 0 1 2 374 4.21 4.21 8 18 1648A4G I506V 1 0 0 0/96 0 0 0 0/796 0 1 2 374 4.21 4.21 8 19 1655T4G F508C 0 1 0 0/96 0 0 0 1/796 0 2 2 038 8.42 5.96 17 20 1716G4A Q528 2 16 1 0/96 0 19 i I 5 43 (58) 1 478 (2 024) 286.56 37.08 557 11 95 21 1756G4T G542X 0 2 0 0/134 0 0 0/796 0 0 2 1 984 10.08 7.12 20 22 1764T4G G544 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 23 1784G4A G551D 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 12 87 24 1816G4A V562I 0 0 0 0 1 0 0/450 0 0 1 (1) 2 004 (2 504) 3.99 3.99 8 25 1816G4C V562L 0 0 0 1 0 0 1/450 0 0 2 (3) 2 004 (2 504) 11.98 6.91 24 26 1859G4C G576A 1 2 0 1 11 0 8/450 0 0 23 (27) 2 004 (2 538) 106.38 20.36 213 13 724j 449 27 1997G4A G622D 0 0 0/80 0/96 1 0 0 0/444 0 1 2 002 5.00 5.00 10 28 2082C4T F650 1 0 0/80 0/20 0 0 0 0/444 0 1 (1) 1 926 (2 412) 4.15 4.15 8 29 2134C4T R668C 1 2 0/80 0/96 1 11 0 12/444 0 27(32) 2 002 (2 558) 125.10 21.98 247 275 30 2377C4T L748 0 0 0/6 0 1 1 388 25.77 25.77 52 14a 129 31 2670G4A W846X 0 0 0/6 0 1 0/452 0/80 0 1 1 010 9.90 9.90 20 32 2694T4G T854 33 23 0/6 33 38 149/452 14/80 11 301 1 010 2980.20 143.92 4 184 33 2695G4A V855I 0 0 0/6 0 0 1/452 0/80 0 1 1 010 9.90 9.90 20 14b 38 0 0 0 0/520 0 0 0 0/446 0 2 448 15 251 34 2816G4C S895T 0 0 0/6 0 0 2/436 0 0 2 996 20.08 14.18 40 35 2831A4C N900T 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 36 2988G4C M952I 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 37 3030G4A T966 (2)k (1)k 0 6/436 0 6 (25)k 618 (1814)k 137.82 27.37 272 38 3032T4C L967S 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 16 80 0 0 0/498 0 0 0/450 0 0 1 502 17a 151 39 3123G4C L997F 0 2 2 1/494 0 7 1 4/454 0 0 17 2 502 67.95 16.42 135 40 3157G4A A1009T 0 2 0 0/494 0 0 0 0/454 0 0 2 2 502 7.99 5.65 16 41 3212T4C I1027T 1 0 0 0/494 0 0 0 0/454 0 0 1 2 502 4.00 4.00 8 17b 228 42 3286T4G F1052V 1 1 0 1/194 0 0 0 0/452 0 0 3 (3) 2 200 (2 240) 13.39 7.73 27 43 3337G4A G1069R 0 1 0 0/194 0 0 0 0/452 0 0 1 2 200 4.55 4.55 9 CommonandrarenonsynonymousandsynonymouscSNSs GModianoetal 186 EuropeanJournalofHumanGenetics 44 3345G4T Q1071H 0 0 0 0/194 0 1 0 0/452 0 0 1 2 200 4.55 4.55 9 45 3417A4T T1995 1 3 0 0/194 1 1 0 0/452 0 0 6 (8) 2 200 (2 506) 31.92 11.27 64 46 3419T4G L1096R 0 0 0 0/194 1 0 0 0/452 0 0 1 2 200 4.55 4.55 9 47 3477C4A T1115 0 0 0 0/194 0 0 0 1/452 0 0 1 2 200 4.55 4.55 9 18 101 48 3523A4G I1131V 0 0 1 0/10 0 0 0/448 0 0 1 (2) 1 512 (1 908) 10.48 7.07 21 49 3586G4C D1152H 0 0 0 0/10 0 0 1/448 0 0 1 1 512 6.61 6.61 13 19 249 50 3617G4T R1162L 0 0 1 1/494 0 0/260 0 0/454 0 0 2 2 262 8.84 6.25 18 51 3690A4G Q1186 0 0 0 0/494 0 0/260 0 0/454 1 0 1 2 262 4.42 4.42 9 52 3813A4G L1227 0 1 0 0/494 0 0/260 0 0/454 0 0 1 2 262 4.42 4.42 9 53 3837T4G S1235R 1 1 0 1/494 0 4/260 0 7/454 0 1 15 (15) 2 262 (2 310) 69.94 16.71 140 20 156 54 4002A4G P1290 2 3 0/6 3 5 18/454 3/80 2 36 1 012 357.73 58.22 690 21 90 55 4009G4A V1293I 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 56 4029A4G T1299 1 0 0/6 0 1/300 0 1/456 0 0 3 (8) 1 316 (2 330) 34.33 12.12 69 57 4041C4G N1303K 1 0 0/6 0 0/300 0 0/456 0 0 1 1 316 7.60 7.60 15 58 4085T4C V1318A 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 22 173 0 0 0/18 0 0 0/450 0 0 1 022 23 106 0 0 0 0/6 0 0 0/448 0 1 436 24l 198+3 59 4404C4T Y1424 1 0 0/6 1 2 5/420 0 2 11 (32) 980 (2 516) 127.19 22.34 251 60m 4521G4A Q1463 (21) (16) (3/32) (14/80) (30) (94/420) 15/76 (17) 15 (227) 76 (1052) 2142.86 131.07 3 367 61 4563T4C D1477 0 0 0/6 0 1 0/420 0 0 1 980 10.20 10.20 20 Totals 6 525 9 584 16 109 The bracketed figures include also the RFLP analysis data (see Materials and methods); the NE Italy, Central Italy, Southern and Northern France are each subdivided into two samples where the 1st is made up of 100 genes. 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[hide] Detection of cystic fibrosis transmembrane conduct... Hum Reprod. 2007 May;22(5):1285-91. Epub 2007 Feb 28. Ratbi I, Legendre M, Niel F, Martin J, Soufir JC, Izard V, Costes B, Costa C, Goossens M, Girodon E
Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling.
Hum Reprod. 2007 May;22(5):1285-91. Epub 2007 Feb 28., [PMID:17329263]

Abstract [show]
BACKGROUND: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene have been widely detected in infertile men with congenital bilateral absence of the vas deferens (CBAVD). Despite extensive analysis of the CFTR gene using varied screening methods, a number of cases remain unsolved and could be attributable to the presence of large gene rearrangements, as recently shown for CF patients. METHODS: We carried out a complete CFTR gene study in a group of 222 CBAVD patients with strict diagnosis criteria and without renal anomaly, and searched for rearrangements using a semi-quantitative assay in a subgroup of 61 patients. RESULTS: The overall mutation detection rate was 87.8%, and 82% of patients carried two mutations. Ten out of the 99 different mutations accounted for 74.6% of identified alleles. Four large rearrangements were found in patients who already carried a mild mutation: two known partial deletions (exons 17a to 18 and 22 to 23), a complete deletion and a new partial duplication (exons 11 to 13). The rearrangements accounted for 7% of the previously unknown alleles and 1% of all identified alleles. CONCLUSIONS: Screening for rearrangements should be part of comprehensive CFTR gene studies in CBAVD patients and may have impacts on genetic counselling for the patients and their families.

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93 1 Two CFTR mutations 15 0-15 0 [R117H] þ [(TG)13(T)5] 1 [R117H] þ [(TG)12(T)5] 1 [R117H] þ [(TG)11(T)5] 1 [R117H] þ [M952I] 1 [D1152H] þ [(TG)12(T)5] 2 [D1152H] þ [Y1032C] 1 [(TG)11(T)5;V562I] þ [L997F] 1 [(TG)11(T)5;V562I] þ [S977F] 1 [E1473X] þ [(TG)13(T)5] 1 [V232D] þ [(TG)12(T)5] 1 [R334W] þ [(TG)12(T)5] 1 [G622D] þ [(TG)12(T)5] 1 [3272-26A . Login to comment

[hide] Proteasome-dependent pharmacological rescue of cys... J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29. Norez C, Bilan F, Kitzis A, Mettey Y, Becq F
Proteasome-dependent pharmacological rescue of cystic fibrosis transmembrane conductance regulator revealed by mutation of glycine 622.
J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29., [PMID:18230692]

Abstract [show]
The most common mutation (F508del) causing cystic fibrosis (CF) results in misfolding of the CF transmembrane conductance regulator (CFTR), leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. A structure-activity relationship for F508del functional correction revealed the following profile: MPB-104-91-07-80 > 05 > 89 >> 9-hydroxyphenanthrene = phenanthrene. Coimmunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with heat shock protein (HSP)70, HSP90, or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A (BFA) but potentiated by thapsigargin (TG) and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly affected that of MPB. It is noteworthy that MPB inhibited proteasome activity in F508del-CFTR-expressing cells but did not directly affect the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent, but it also follows similar structure-activity relationship as demonstrated by functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.

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1 To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. Login to comment
2 The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. Login to comment
3 G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. Login to comment
9 With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Login to comment
11 We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation. Login to comment
26 The G622D-CFTR mutant, in which the pathophysiology is unclear, has been provisionally classified as a class 3 missense mutation after its identification in CF patients (http://www.genet.sickkids.on.ca/cftr) (Vankeerberghen et al., 1998). Login to comment
28 The G622D mutant forms a cAMP-regulated chloride channel with significantly lower Po than the wild-type channels (Vankeerberghen et al., 1998). Login to comment
36 In the present work, we performed a comparative analysis of the effect of several chemically diverse MPB and phenanthrene derivatives on two CFTR mutants, F508del and G622D. Login to comment
38 A N C Wt 620H-E-G-S-S- G622D 620H-E-D-S-S- NBD1 NBD2 R-domain N C Wt 505N-I-I-F-G- F508del 505N-I-I-G NBD1 NBD2 R-domain N+ OH Cl Cl5-butyl-10-chloro-6-hydroxybenzo[c]quinolizinium chloride MPB-91 phenanthrene OH 9-hydroxyphenanthrene MPB-104 NCl OH Cl MPB-89 NCl B N+ OH Cl6-hydroxybenzo[c]quinolizinium chloride MPB-05 N+ Cl OH Cl6-hydroxy-10-chlorobenzo[c]quinolizinium chloride MPB-07 MPB-07 N+ OH F Cl- 10-fluoro-6-hydroxybenzo[c]quinolizinium MPB80MPB-80MPB-05 N+ OH F Cl- 10-fluoro-6-hydroxybenzo[c]quinolizinium MPB80 MPB-80 MPB-91 Fig. 1. Login to comment
39 A, scheme illustrating the position of F508del and G622D mutations on a CFTR protein cartoon. Login to comment
42 For this study, we used the human nasal airway epithelial cell line JME/CF15, derived from a CF patient homozygous for the F508del mutation (Jefferson et al., 1990), and COS-7 cells stably transfected with green fluorescent protein (GFP)-tagged CFTR vectors containing either wild-type (wt)-, F508del-, or G622D-CFTR. Login to comment
59 The wt-, F508del-, and G622D-CFTR Cl-channel activities were assayed by measuring the rate of iodide (125 I) efflux from CF15 cells and COS-7 cells as described previously (Melin et al., 2004; Norez et al., 2006a). Login to comment
77 Results MPB Compounds Rescue F508del-CFTR but Not G622D-CFTR. Login to comment
78 The plasmid construction (see Materials and Methods) allowed stable expression in COS-7 cells of GFP-tagged F508del-CFTR and GFP-tagged G622D-CFTR. Login to comment
82 Figure 2A shows an example of CFTR-dependent iodide efflux in F508del-, G622D-, and wt-CFTR-expressing COS-7 cells. Login to comment
84 For G622D-CFTR-expressing cells (Fig. 2A, black triangles), Fsk ϩ Gst stimulated the iodide efflux (kpeak - kbasal ϭ 0.046 Ϯ 0.020 min-1 ) to a level Ϸ3-fold lower than that of wt-CFTR cells (Fig. 2A, black squares; kpeak - kbasal ϭ 0.155 Ϯ 0.009 min-1 ). Login to comment
86 These results suggest that, although functional (in agreement with Vankeerberghen et al., 1998), G622D-expressing COS-7 cells have a reduced transport activity compared with wt-CFTR cells. Login to comment
88 An alternative explanation would be that the trafficking of G622D is abnormal. Login to comment
92 Figure 2B presents typical iodide efflux responses, showing that in G622D-CFTR cells incubated with miglustat (black triangles), Fsk ϩ Gst increased (Ϸ2-fold) iodide efflux compared to untreated cells (Fig. 2B, open squares; p Ͻ 0.001). Login to comment
93 However, with G622D-CFTR cells incubated with MPB-104 (Fig. 2B, black circles), the Fsk ϩ Gst-stimulated iodide efflux was not different from control. Login to comment
95 Therefore, these results show a partial trafficking defect of G622D-CFTR that can be reversed by miglustat as with the F508del mutant. Login to comment
98 It is surprising to note that the incubation of cells with any of the MPB correctors did not rescue G622D-CFTR (Fig. 2C, gray bars). Login to comment
99 If G622D is a partial trafficking-deficient mutant, as our functional data suggest, then we should observe less band C forms of the mutant compared to wt-CFTR, indicating a reduced pool of mature proteins. Login to comment
100 Thus, we performed biochemical assays to compare the G622D-CFTR protein expression in the presence or absence of these compounds. Login to comment
103 Anti-GFP immunoblotting in COS-7 cells expressing G622D-CFTR (Fig. 2D, lane 2) shows a reduced amount of band C form but a much higher quantity of band B form for G622D versus wt proteins, confirming the partial trafficking defect of G622D. Login to comment
104 In COS-7 cells expressing G622D-CFTR incubated for 2 h with 100 ␮M miglustat, anti-GFP immunoblotting revealed an increased band C intensity (Fig. 2D, lane 4). Login to comment
107 However, and contrary to F508del-CFTR rescued by MPB derivatives (Dormer et al., 2001a), the abnormal processing of G622D-CFTR cannot be rescued by MPB. Login to comment
124 The efflux Band C Band B wt G622D MPB-104 Miglustat - - - - + - - + Band C Band B wt G622D MPB-104 Miglustat - - - - + - - + Band C Band B wt G622D MPB-104 Miglustat - - - - - - - - + - + - - + - + C A 0.00 0.05 0.10 0.15 0.20 F508del-CFTR G622D-CFTR ns ** ns*** Ctrl MPB-07 MPB-80 MPB-91 - - - - + - - - - + - - - - + Miglustat - - - - + - - - - - - - - - - MPB-104 - - - -- + - - - - + - - - - + - - - - + - - - - + - - - - - - - - - - - - - -- + 12 8 8 8 812 8 8 8 81624 kpeak-kbasal(min-1 ) B 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 0.25 wt-CFTR G622D-CFTR F508del-CFTR Fsk + Gst Time (min-1 ) k(min-1 ) D 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 untreated MPB-104 Miglustat Fsk + Gst Time (min) k(min-1 ) 1 2 3 4 Fig. 2. Login to comment
125 Rescue of F508del-CFTR but not of G622D-CFTR trafficking by MPB derivatives. Login to comment
126 A, examples of iodide efflux curves obtained in COS-7 cells stably transfected with wt-, F508del-, or G622D-CFTR and stimulated by Fsk/Gst as indicated by the horizontal bar above the traces. Login to comment
127 B, examples of iodide efflux curves obtained for G622D-CFTR-expressing COS-7 cells treated or not with miglustat (100 ␮M, 2 h) or MPB-104 (100 ␮M, 2 h) and stimulated by Fsk/Gst. Login to comment
128 C, bar graph showing the Fsk ϩ Gst-dependent iodide efflux in COS-7 cells stably transfected with F508del-or G622D-CFTR and treated by MPB compounds or miglustat (100 ␮M, 2 h). Login to comment
132 ‫,ءءء‬ p Ͻ 0.001, ns, nonsignificant difference. D, expression of wt-CFTR or G622D-CFTR in COS-7 cells treated as indicated above each lane. Login to comment
209 Because this effect could be attributed to a nonspecific action of MPB, we also measured the proteasome activity in mock COS-7 cells (Fig. 7A) or COS-7 cells stably expressing F508del-CFTR (Fig. 7B) or G622D-CFTR (Fig. 7C). Login to comment
215 Finally, because we showed that MPB correctors are not effective on G622D-CFTR, we asked whether this mutation could also affect the MPB-mediated inhibition of the degradation machinery. Login to comment
216 To study this theory, the proteasome activity was also measured in COS-7 cells expressing G622D proteins. Login to comment
219 Altogether, these results demonstrate that MPB correctors inhibit the cell proteasome activity in a CFTR-dependent manner and suggest that the mutation G622D prevents this inhibition. Login to comment
220 Discussion The present experiments demonstrate the following: 1) MPB derivatives are pharmacological chaperones of F508del-CFTR but not of G622D-CFTR mutants; 2) MPB corrects the F508del-CFTR-trafficking defect via a specific structure-activity relationship; 3) MPB does not prevent the interaction of F508del-CFTR with the ER resident calnexin or cytosolic HSP70 and HSP90 molecular chaperones; 4) MPB correctors inhibit the proteasome activity only in CFTR-expressing cells and have no effect on the activity of purified 20S proteasome; and 5) the mutant G622D-CFTR prevents rescue of CFTR by MPB but also abolishes the inhibition of the proteasome by MPB derivatives. Login to comment
227 ‫,ءءء‬ p Ͻ 0.001 and ‫,ء‬ p Ͻ 0.05, ns, nonsignificant difference. A B C 0 25 50 75 100 125 MG132 Lactacystine MPB-104 MPB-91 MPB-80 MPB-07 MPB-89 phenanthrene DMSO Ctrl + ns ns *** ** ns Proteasome activity in Cos-7 F508del-CFTR cells (%) 0 25 50 75 100 125 MG132 Lactacystine MPB-104 MPB-91 MPB-80 MPB-07 MPB-89 phenanthrene DMSO Ctrl + ns *** ns Proteasome activity in Cos-7 G622D-CFTR cells (%) 0 25 50 75 100 125 MG132 Lactacystine MPB-104 MPB-91 MPB-80 MPB-07 MPB-89 phenanthrene DMSO Ctrl + ns *** ns Proteasome activity in Cos-7 mock cells (%) Fig. 7. Login to comment
229 Histograms showing the effect of MPB correctors on proteasome activity in mock COS-7 cells (A) or COS-7 cells stably transfected with F508del-CFTR (B) or G622D-CFTR (C). Login to comment

[hide] Best practice guidelines for molecular genetic dia... Eur J Hum Genet. 2009 Jan;17(1):51-65. Epub 2008 Aug 6. Dequeker E, Stuhrmann M, Morris MA, Casals T, Castellani C, Claustres M, Cuppens H, des Georges M, Ferec C, Macek M, Pignatti PF, Scheffer H, Schwartz M, Witt M, Schwarz M, Girodon E
Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders--updated European recommendations.
Eur J Hum Genet. 2009 Jan;17(1):51-65. Epub 2008 Aug 6., [PMID:18685558]

Abstract [show]
The increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.

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144 A (T)5 variant can either be associated with (TG)11, (TG)12, (TG)13, and rarely (TG)15 repeats.74 When (T)5 is found in diagnostic testing, for example, for CBAVD or atypical presentation, determination of Table 4 Classification of CFTR mutations with regard to their potential for causing disease Mutation group Examples CF-causing F508del Mainly nonsense, frameshift, splicing (invariant dinucleotide): G542X, R553X, W1282X, 2183AA4G, 3659delC, 1717-1G4A, 3120+1G4A Missense that severely affects CFTR synthesis or function: G551D, N1303K, R347P 2789+5G4A, 3849+10kbC4T, 3272-26A4G, L206Wa , D1152Ha , (TG)13(T)5a CFTR-related disorders associated L206Wa , D1152Ha , (TG)13(T)5a [R117H;(T)7], (TG)12(T)5, L997F, V562I, [R668C;G576A;D443Y], [R74W;D1270N] (TG)11(T)5b , S1235Rb No clinical consequences 875+40A4G, M470V (1540A4G), I506V (1648A4G), F508C (1655T4G), 1716G4A, 2694T4G, 4002A4G, 2752-15G4C (TG)11(T)5b , S1235Rb Unproven or uncertain clinical relevance Mainly missense mutations G622D, R170H, V938G, I125T Putative splice mutations: 406-6T4C, 2752-26A4G, 3601-17T4C Only a fraction of mutations and patients have been characterized in detail and, with the exception of frequent mutations, only small numbers of patients have been available for the study of most mutations. Login to comment

[hide] Amniotic fluid digestive enzyme analysis is useful... Clin Chem. 2009 Dec;55(12):2214-7. Epub 2009 Oct 15. Oca F, Dreux S, Gerard B, Simon-Bouy B, de Becdelievre A, Ferec C, Girodon E, Muller F
Amniotic fluid digestive enzyme analysis is useful for identifying CFTR gene mutations of unclear significance.
Clin Chem. 2009 Dec;55(12):2214-7. Epub 2009 Oct 15., [PMID:19833837]

Abstract [show]
BACKGROUND: The large number of CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations and the existence of variants of unclear significance complicate the prenatal diagnosis of cystic fibrosis (CF). The aim of this study was to determine whether the pattern of amniotic fluid digestive enzymes (AF-DEs) could be correlated with the severity of CFTR mutations. METHODS: The AF-DE pattern (gamma-glutamyltranspeptidase, aminopeptidase M, and the intestinal isoform of alkaline phosphatase) was retrospectively analyzed in 43 AF samples. All fetuses presented 2 CFTR mutations, which were classified according to the severity of the disease: CF/CF (n = 38); CF/CFTR-related disorders (n = 1); and CF/unknown variant (n = 4). The relationships between clinical CF status, CFTR mutations, and AF-DE pattern were studied. RESULTS: Of 38 severely affected CF fetuses, an "obstructive" AF-DE pattern was observed in 15 of 15 samples collected before 22 weeks, irrespective of the CFTR mutation (diagnostic sensitivity, 100%; diagnostic specificity, 99.8%). In the 23 fetuses evaluated after 22 weeks, the AF-DE pattern was abnormal in 7 cases and noncontributive in 16 (diagnostic sensitivity, 30.4%; diagnostic specificity, 99.8%). Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849+10kbC>T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. The AF-DE pattern (<22 weeks) was typical in 3 cases but abnormal in the last 2 cases. CONCLUSIONS: AF-DE analysis is of value for prenatal CF diagnosis in classic forms of CF and could be helpful in nonclassic CF.

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No. Sentence Comment
10 Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849ϩ10kbCϾ T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. Login to comment
84 In the last case (F508del/G622D), we have no clear explanation for the abnormal AF-DE pattern. Login to comment
86 The G622D variant, identified in infertile patients, was found at an allelic frequency of 0.18% in a population of African Americans (17). Login to comment
89 CFTR mutation Cases, n Outcome/follow-up CF/CF mutation (n ϭ 38) F508del/F508del 21 TOPa (n ϭ 20); birth, severe CF (n ϭ 1) F508del/unidentified severe mutationb 3 TOP (n ϭ 3) F508del/G551D 2 TOP (n ϭ 2) F508del/4005ϩ1GϾA 1 TOP F508del/2711delT 1 Birth, severe CF F508del/297-3CϾT 1 TOP F508del/3120ϩ1GϾA 1 TOP F508del/405ϩ1GϾA 1 TOP F508del/711ϩ1GϾT 1 TOP F508del/Q1042X 1 TOP F508del/dele22-23 1 TOP F508del/2789ϩ5GϾA 1 Birth, severe CF dele19/dele19c 1 Birth, severe CF W1282X/dele2-6b 1 TOP 1078delT/394delTT 1 TOP CF/unknown variant (n ϭ 4) F508del/G622D 1 Birth, no clinical sign of CF F508del/N1224K 1 Birth, no clinical sign of CF F508del/L73F 1 Birth, no clinical sign of CF 3849ϩ10kbCϾT/G1127E 1 Birth, no clinical sign of CF CF/CFTR-related disorder (n ϭ 1) F508del/S1235R 1 Birth, no clinical sign of CF (del22q11 ϩ imperforate anus)d a TOP, termination of pregnancy. Login to comment
94 One hypothesis is that the G622D phenotype is clinically expressed in intestinal microvilli only during fetal life and is silent thereafter. Login to comment

[hide] C terminus of nucleotide binding domain 1 contains... J Biol Chem. 2010 Jul 16;285(29):22132-40. Epub 2010 Apr 30. Billet A, Melin P, Jollivet M, Mornon JP, Callebaut I, Becq F
C terminus of nucleotide binding domain 1 contains critical features for cystic fibrosis transmembrane conductance regulator trafficking and activation.
J Biol Chem. 2010 Jul 16;285(29):22132-40. Epub 2010 Apr 30., 2010-07-16 [PMID:20435887]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest beta strands (beta(c)5 and beta(c)6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the beta(c)5-beta(c)6 hairpin, within the beta(c)5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the beta(c)6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.

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2 We mutated CFTR amino acids located in the betac5-betac6 hairpin, within the betac5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the betac6 strand. Login to comment
3 Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. Login to comment
4 For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. Login to comment
5 In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Login to comment
6 Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Login to comment
33 We have mutated these two glycine residues in aspartic acid (G622D) and arginine (G628R) and considered other mutants in their neighborhood (H620Q, E621G, S623A, and S624A). Login to comment
87 RESULTS Expression of the NBD1 C-terminal CFTR Mutants-We have introduced EGFP-tagged CFTR proteins into HEK293 cells, wt CFTR and six CFTR mutants, i.e. H620Q, E621G, G622D, S623A, S624A, and G628R. Login to comment
91 However, no mature-glycosylated C-band form for G622D and G628R was detected (Fig. 2, lanes 6 and 7). Login to comment
102 Effect of the CFTR corrector miglustat on G622D and G628R. Login to comment
103 A, upper, Western blot showing wt-CFTR, G622D-, and G628R-CFTR expression with or without pretreatment with miglustat (100 ␮M) and detected with CFTR NBD2 C-terminal antibody. Login to comment
110 Third, the current densities for G622D and G628R, although not abolished, are both significantly reduced (p Ͻ 0.001) compared with wt (supplemental Table 2). Login to comment
115 Because we observed a pronounced reduction of the current density for the mutants G622D and G628R, we incubated transfected HEK293 and BHK-21 cells with this corrector and analyzed the corresponding CFTR activity. Login to comment
117 Similarly in BHK-21 cells the iodide efflux responses stimulated by Fsk and genistein was significantly increased for G622D and G628R after treatment with the corrector (Fig. 4B). Login to comment
118 This observation indicates that the diminution of cAMP-induced Cl- current is probably due to the diminution or to the absence of a mature form of G622D and G628R mutants. Login to comment
119 In support of this hypothesis, Western blot analysis of cells treated with miglustat shows enhanced mature C-band for G622D and G628R mutants (Fig. 4A). Login to comment
133 MPB-91 stimulated the Cl- current for each CFTR mutants studied except the glycine mutants G622D and G628R (Fig. 6 and supplemental Table 3). Login to comment
134 For them, comparison of the corresponding IV slope (Fig. 7A) did not reveal significant differences between basal condition (I/V slope of G622D, 0.032 Ϯ 0.001, n ϭ 7; G628R, 0.046 Ϯ 0.002, n ϭ 5) and in presence of 50 ␮M MPB-91 (I/V slope of G622D, 0.037 Ϯ 0.001, n ϭ 7; G628R, 0.053 Ϯ 0.003, n ϭ 5) indicating the absence of a response of the two mutated channels to that agent. Login to comment
136 Results in Fig. 8 show a potentiation of the cAMP-dependent Cl- current by MPB-91 for wt channels but neither for G622D nor G628R FIGURE 5. Login to comment
141 We also tested analogues of MPB-91 (12), but again for the mutant G622D or G628R, the Cl-channel function of CFTR was not stimulated by MPB-95 and MPB-97 (Fig. 9A). Login to comment
143 Importantly, all of these CFTR activators stimulated the Cl-channel activity of G622D and G628R CFTR, suggesting the relative specificity of the effect observed in the presence of the benzoquinolizinium drugs (Fig. 9B). Login to comment
146 Using Western blot analysis, we now observed a strong decrease of the mature-glycosylated form for the two mutants G622D and G628R. Login to comment
158 Consistently, the defect in the maturation process induced by the G622D and G628R mutations, which causes the retention of the mutated CFTR protein, can be clearly explained by the key role of these glycine residues at the structure level. Login to comment
184 In contrast, for the two mutants G622D and G628R, no activation was recorded in the presence of MPB-91 or other MPB compounds. Login to comment
187 However, this is not the case since xanthine (isobutylmethylxanthine), RP-107, or iso- flavonoide (genistein) successfully stimulated the channel activity of G622D and G628R CFTR channels. Login to comment
188 Therefore, one could hypothesize that the mechanism of activation of CFTR by MPB was itself affected by the mutations G622D and G628R and that the binding site of MPB might be located, on the folded protein, in the vicinity of the last beta hairpin of CFTR NBD1. Login to comment
189 However, the perturbation induced by the G622D and G628R mutations on the overall NBD1 structure might be felt at long range and thus influence potential binding sites, which may be distant at the three-dimensional level. Login to comment
193 A sliding of the two NBDs has been predicted to occur when CFTR evolves toward a closed form of the channel (inward-facing conformation) (29) in which, FIGURE8.KineticofactivationofwholecellCl- currentsforwt,G622D,andG628RCFTR.A,representative time course of whole cell Cl- current densities at 40 mV recorded in presence of 1 ␮M Fsk and 1 ␮M ϩ50 ␮M MPB-91. Login to comment
196 Iodide efflux of G622D and G628R CFTR-expressing cells in the presence of different MPB compounds or different activators. Login to comment

[hide] Measurement of nasal potential difference in young... Thorax. 2010 Jun;65(6):539-44. Sermet-Gaudelus I, Girodon E, Roussel D, Deneuville E, Bui S, Huet F, Guillot M, Aboutaam R, Renouil M, Munck A, des Georges M, Iron A, Thauvin-Robinet C, Fajac I, Lenoir G, Roussey M, Edelman A
Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis.
Thorax. 2010 Jun;65(6):539-44., [PMID:20522854]

Abstract [show]
BACKGROUND: A challenging problem arising from cystic fibrosis (CF) newborn screening is the significant number of infants with hypertrypsinaemia (HIRT) with sweat chloride levels in the intermediate range and only one or no identified CF-causing mutations. OBJECTIVES: To investigate the diagnostic value for CF of assessing CF transmembrane conductance regulator (CFTR) protein function by measuring nasal potential difference in children with HIRT. METHODS: A specially designed protocol was used to assess nasal potential difference (NPD) in 23 young children with HIRT (3 months-4 years) with inconclusive neonatal screening. Results were analysed with a composite score including CFTR-dependent sodium and chloride secretion. Results were correlated with genotype after extensive genetic screening and with clinical phenotype at follow-up 3 years later. RESULTS: NPD was interpretable for 21 children with HIRT: 13 had NPD composite scores in the CF range. All 13 were finally found to carry two CFTR mutations. At follow-up, nine had developed a chronic pulmonary disease consistent with a CF diagnosis. The sweat test could be repeated in nine children, and six had sweat chloride values >or=60 mmol/l. Of the eight children with normal NPD scores, only two had two CFTR mutations, both wide-spectrum mutations. None had developed a CF-like lung disease at follow-up. The sweat test could be reassessed in five of these eight children and all had sweat chloride values <60 mmol/l. CF diagnosis was ruled out in six of these eight children. CONCLUSION: Evaluation of CFTR function in the nasal epithelium of young children with inconclusive results at CF newborn screening is a useful diagnostic tool for CF.

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130 Table 3 Genotypes of the children with HIRT according to the diagnostic score cut-off in the 21 patients with reliable NPD tests; results after extensive genetic analysis CFTR genotypes Diagnosis score >0.27 (8 patients) £0.27 (13 patients) A/A 0 F508del/621+3A/G F508del/Q1291R A/AB F508del/R347H F508del/R117H;T7 W846X/R117C n¼2 F508del/R1070W 2183AA/G/L206W F508del/3272-26A/G F508del/R117H;T7; n¼4 A/D 0 F508del/R933G G551D/R352Q B/D G622D/3849+45G/A 0 A/0 F508del/0 n¼2 0 0/0 3 0 0, no identified mutation; A, CF-causing mutation; B, mutation associated with cystic CFTR-related disorders; C, mutation with no clinical consequence ; D, mutation of unknown or uncertain clinical relevance; AB, mutation that is associated with a wide phenotypic spectrum that might belong to either group A or B. CFTR, cystic fibrosis transmembrane conductance regulator; HIRT, hypertrypsinaemia; NPD, nasal potential difference. Login to comment

[hide] An update on cystic fibrosis screening. Clin Lab Med. 2010 Sep;30(3):533-43. Goetzinger KR, Cahill AG
An update on cystic fibrosis screening.
Clin Lab Med. 2010 Sep;30(3):533-43., [PMID:20638569]

Abstract [show]
Cystic fibrosis (CF) is a monogenic, autosomal recessive disorder, which ultimately leads to multisystem organ dysfunction and a subsequent decrease in life expectancy. Because of the sizeable number of disease causing mutations (>1000) and expansive ethnic and racial distribution, CF has presented a challenge for prenatal diagnosis. This article aims to review the genetics of CF, its spectrum of genotypic-phenotypic variations, current prenatal carrier screening and diagnostic recommendations, ultrasonographic markers of CF, and available reproductive options for carrier couples.

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32 Two additional mutations also not included in the current carrier screening panel, G622D and Q98R, were incidentally identified. 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] Pharmacological therapy for cystic fibrosis: from ... J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45. Becq F, Mall MA, Sheppard DN, Conese M, Zegarra-Moran O
Pharmacological therapy for cystic fibrosis: from bench to bedside.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45., [PMID:21658632]

Abstract [show]
With knowledge of the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR), its physiological role and dysfunction in cystic fibrosis (CF), therapeutic strategies are now being developed that target the root cause of CF rather than disease symptoms. Here, we review progress towards the development of rational new therapies for CF. We highlight the discovery of small molecules that rescue the cell surface expression and defective channel gating of CF mutants, termed CFTR correctors and CFTR potentiators, respectively. We draw attention to alternative approaches to restore epithelial ion transport to CF epithelia, including inhibitors of the epithelial Na(+) channel (ENaC) and activators of the Ca(2+)-activated Cl(-) channel TMEM16A. The expertise required to translate small molecules identified in the laboratory to drugs for CF patients depends on our ability to coordinate drug development at an international level and our ability to provide pertinent biological information using suitable disease models.

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116 MPB-07, MPB-91, analogues [104] human CF nasal epithelial cells, CF15 F508del, G622D Biochemistry, iodide efflux, degradation assay MPB compounds protect a proteolytic cleavage site by directly binding to the NBD1 domain of F508del-CFTR. Login to comment

[hide] Characterization of 19 disease-associated missense... Hum Mol Genet. 1998 Oct;7(11):1761-9. Vankeerberghen A, Wei L, Jaspers M, Cassiman JJ, Nilius B, Cuppens H
Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator.
Hum Mol Genet. 1998 Oct;7(11):1761-9., [PMID:9736778]

Abstract [show]
In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

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7 Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Login to comment
68 Primers used for mutagenesis Primer Sequence I601F (a1933t) 5'-CTA ACA AAA CTA GGT TTT TGG TCA CTT C-3' L610S (t1961c) 5'-CTA AAA TGG AAC ATT CAA AGA AAG CTG-3' A613T (g1969a) 5'-CAT TTA AAG AAA ACT GAC AAA ATA TTA-3' D614G (a1973g) 5'-CAT TTA AAG AAA GCT GGC AAA ATA TTA A-3' I618T (t1985c) 5'-GAC AAA ATA TTA ACT TTG CAT GAA GG-3' L619S (t1988c) 5'-GAC AAA ATA TTA ATT TCG CAT GAA GGT-3' H620P (a1991c) 5'-CAA AAT ATT AAT TTT GCC TGA AGG TAG C-3' H620Q (t1992g) 5'-AAT ATT AAT TTT GCA GGA AGG TAG CAG-3' G622D (g1997a) 5'-TTG CAT GAA GAT AGC AGC TAT TTT TAT G-3' G628R (g2014c) 5'-GCA GCT ATT TTT ATC GGA CAT TTT C-3' L633P (t2030c) 5'-CAT TTT CAG AAC CCC AAA ATC TAC AGC-3' D648V (a2075t) 5'-CTC ATG GGA TGT GTT TCT TTC GAC C-3' T665S (a2125t) 5'-CAA TCC TAA CTG AGT CCT TAC ACC G-3' F693L (t2209c) 5'-CAG ACT GGA GAG CTT GGG GAA AAA AG-3' R766M (g2429t) 5'-GCA CGA AGG ATG CAG TCT GTC CTG-3' R792G (c2506g) 5'-CAG CAT CCA CAG GAA AAG TGT CAC TG-3' A800G (c2531g) 5'-CTG GCC CCT CAG GGA AAC TTG ACT G-3' I807M (a2553g) 5'-CTG AAC TGG ATA TGT ATT CAA GAA GG-3' E822K (g2596a) 5'-GGC TTG GAA ATA AGT AAA GAA ATT AAC G-3' E826K (g2608a) 5'-GAA GAA ATT AAC AAA GAA GAC TTA AAG-3' Selection primer BstBI 5'-CTC TGG GGT CCG GAA TGA CCG AC-3' Two primers were used for each mutagenesis reaction. Login to comment
85 The remainder (G622D, D648V, F693L, R766M and I807M) did not significantly affect chloride transport ability when compared with wild-type CFTR channels. Login to comment
87 Maturation pattern of RD mutations and their associated phenotype found in patients with the indicated genotype (when the mutation is associated with CF, only the pancreas status is given) Mutation A-form B-form C-form Clinical data Genotype Phenotype Reference I601F + + - I601F/G542X PS M. Schwarz, personal communication L610S + + - Unknown Unknown A613T + + - Unknown Unknown D614G + + - D614G/unknown PI 14 I618T + + - I618T/dF508 PS G.R. Cutting, personal communication L619S + + - L619S/unknown PI B. Tümmler, personal communication H620P + + - H620P/R1158X PS M. Schwarz, personal communication H620Q + + + H620Q/dF508 PI T. Dörk, personal communication G622D + + + G622D/unknown Oligospermia J. Zielenski, personal communication G628R + + - Unknown Unknown L633P + + - L633P/3659delC M. Schwarz, personal communication D648V + + + D648V/3849+10kb C/T PI C. Ferec, personal communication T665S + + + Unknown Unknown F693L + + + F693L/W1282X Healthy C. Ferec; CF Genetic Analysis Consortium R766M + + + R766M/R792G CBAVD D. Glavac, personal communication R792G + + + R766M/R792G CBAVD D. Glavac, personal communication A800G + + + A800G/unknown CBAVD 34 I807M + + + I807M/unknown CBAVD Our observation E822K + + + E822K/unknown PI 35 E826K + + + E826K/unknown Thoracic sarcoidosis C. Bombieri, personal communication +, the protein matures up to that form; -, the protein does not reach the respective maturation step. Login to comment
97 G622D, R792G and E822K gave rise to a CFTR chloride channel with a significantly lower Po than wild-type CFTR; H620Q and A800G CFTR resulted in channels with significantly higher Po. Login to comment
123 Mutations that did not affect maturation (H620Q, G622D, D648V, T665S, F693L, R766M, R792G, A800G, I807M, E822K and E826K) were subsequently analysedat theelectrophysiologi- cal level. Login to comment
124 Three of these (G622D, R792G and E822K) gave rise to chloride channels with significantly lower Po than the wild-type channel. Login to comment

[hide] Conformational changes relevant to channel activit... J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21. Hudson RP, Chong PA, Protasevich II, Vernon R, Noy E, Bihler H, An JL, Kalid O, Sela-Culang I, Mense M, Senderowitz H, Brouillette CG, Forman-Kay JD
Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21., [PMID:22722932]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between beta-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with beta-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

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308 Mutations of residues in these C-terminal strands of NBD1, specifically G622D and G628R, have been demonstrated to perturb the pharmacological effects of dual "MPB (benzo(c)- quinolizinium)" compounds, characterized by their ability to both activate CFTR and rescue defective trafficking (56). Login to comment
306 Mutations of residues in these C-terminal strands of NBD1, specifically G622D and G628R, have been demonstrated to perturb the pharmacological effects of dual "MPB (benzo(c)- quinolizinium)" compounds, characterized by their ability to both activate CFTR and rescue defective trafficking (56). Login to comment

[hide] Novel pharmacological strategies to treat cystic f... Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006. Hanrahan JW, Sampson HM, Thomas DY
Novel pharmacological strategies to treat cystic fibrosis.
Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006., [PMID:23380248]

Abstract [show]
Cystic fibrosis (CF) is a lethal disease caused by mutations in the CFTR gene. The most frequent mutation is deletion of a phenylalanine residue (DeltaF508) that results in retention of the mutant, but otherwise functional, protein in the endoplasmic reticulum (ER). There have been recent advances in the identification of chemically diverse corrector compounds that allow DeltaF508-CFTR protein to traffic from the ER to the plasma membrane. The most studied correctors fall into two categories, pharmacological chaperones that bind to the mutant protein and circumvent its recognition by the cellular protein quality control systems and proteostasis regulators that modify the cellular pathways responsible for protein quality control and trafficking. This review focuses on recent advances in the field, strategies for the development of drugs from corrector compounds for the treatment of CF, and identification of their targets and mechanism(s) of action.

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140 VRT-325 and VRT-532 modify the activity of purified reconstituted CFTR channels [48,49], whereas MPB compounds activate CFTR channels when added acutely [45], but not the G622D mutant. 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

[hide] The p.Gly622Asp (G622D) mutation, frequently found... J Cyst Fibros. 2015 May;14(3):305-9. doi: 10.1016/j.jcf.2014.11.001. Epub 2014 Nov 28. Marion H, Natacha G, Brigitte M, Francois C, Michel R, Corinne T, Emmanuelle G, Thierry B
The p.Gly622Asp (G622D) mutation, frequently found in Reunion Island and in black populations, is associated with a wide spectrum of CF and CFTR-RD phenotypes.
J Cyst Fibros. 2015 May;14(3):305-9. doi: 10.1016/j.jcf.2014.11.001. Epub 2014 Nov 28., [PMID:25443471]

Abstract [show]
Examination of genotype-phenotype correlations along with functional evaluation of CFTR mutations may not be straightforward. The c.1865G>A, p.Gly622Asp (G622D), located at the NBD1 C terminus of the CFTR protein, was initially reported in patients with male infertility. However, the substitution of Gly622 by an aspartic acid in vitro would perturb the local structure or even affect the CFTR folding itself. In order to determine whether p.Gly622Asp affects the risk of developing a CFTR-Related disorder (CFTR-RD) or cystic fibrosis (CF), we analyzed the phenotype of subjects bearing the p.Gly622Asp mutation. We report molecular and clinical analyses in eleven unrelated patients with CF or CFTR-RD with compound heterozygosity for the p.Gly622Asp mutation. On the basis of the clinical features presented by the eleven patients, we postulate that the p.Gly622Asp might be associated with a wide spectrum of phenotypes including classical cystic fibrosis.

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0 Original Article The p.Gly622Asp (G622D) mutation, frequently found in Reunion Island and in black populations, is associated with a wide spectrum of CF and CFTR-RD phenotypes Heller Marion a , Gaitch Natacha a , Martinez Brigitte a , Cartault Fran&#e7;ois b , Renouil Michel b , Theze Corinne c , Girodon Emmanuelle a , Bienvenu Thierry a,d,Ìe; a AP-HP, Laboratoire de Biochimie et G&#e9;n&#e9;tique Mol&#e9;culaire, GH Cochin-Broca-H&#f4;tel Dieu, Paris, France b Service de G&#e9;n&#e9;tique, Centre Hospitalier Saint Denis, Saint Denis, La R&#e9;union, France c Laboratoire de G&#e9;n&#e9;tique Mol&#e9;culaire, IURC, Institut Universitaire de Recherche Clinique, 34093 Montpellier Cedex 5, France d Universit&#e9; Paris Descartes Paris, Institut Cochin, INSERM U1016, Paris, France Received 2 July 2014; revised 14 October 2014; accepted 2 November 2014 Available online 28 November 2014 Abstract Examination of genotype-phenotype correlations along with functional evaluation of CFTR mutations may not be straightforward. Login to comment
1 The c.1865G N A, p.Gly622Asp (G622D), located at the NBD1 C terminus of the CFTR protein, was initially reported in patients with male infertility. Login to comment
2 However, the substitution of Gly622 by an aspartic acid in vitro would perturb the local structure or even affect the CFTR folding itself. Login to comment
3 In order to determine whether p.Gly622Asp affects the risk of developing a CFTR-Related disorder (CFTR-RD) or cystic fibrosis (CF), we analyzed the phenotype of subjects bearing the p.Gly622Asp mutation. Login to comment
4 We report molecular and clinical analyses in eleven unrelated patients with CF or CFTR-RD with compound heterozygosity for the p.Gly622Asp mutation. Login to comment
5 On the basis of the clinical features presented by the eleven patients, we postulate that the p.Gly622Asp might be associated with a wide spectrum of phenotypes including classical cystic fibrosis. Login to comment
12 Two nucleotide substitutions were reported in the CFTR gene (NM_000492.3) at position 628 giving rise to p.Gly628Arg (G628R) in CF patients, c.1882G N A and c.1882G N C, while one mutation has been described at position 622, c.1865G N A, p.Gly622Asp (G622D), reported in 1998 by Zielenski et al. in a patient with oligospermia (http:// www.genet.sickkids.on.ca/cftr/) [1]. Login to comment
13 p.Gly622Asp was further reported at a 0.18% allelic frequency among African Americans in a carrier screening program [2]. Login to comment
14 In 1998, Vankerberghen et al. evaluated the impact of this missense change on the channel behavior and found that p.Gly622Asp did not affect chloride transport ability but gave rise to a CFTR chloride channel with Ìe; Corresponding author at: Laboratoire de Biochimie et G&#e9;n&#e9;tique Mol&#e9;culaire, H&#f4;pital Cochin, 27 rue du Faubourg Saint Jacques, 75014 Paris, France. Login to comment
19 These functional data suggested p.Gly622Asp as a possible CFTR-related disorder (CFTR-RD) associated mutation. Login to comment
20 Ten years later, Norez et al. showed that p.Gly622Asp-expressing COS7 cells have a reduced transport activity compared with wild-type CFTR cells and a partial trafficking defect that can be reversed by misglustat [3]. Login to comment
21 Recently, Billet et al. expressed mutant and WT CFTR in HEK 293 cells and detected no mature-glycosylated C-band for the mutant and a significant reduced current density, suggesting that the substitution of Gly622 by an aspartic acid would perturb the local structure or even affect the folding itself [4]. Login to comment
22 To unravel discrepancies between these previous reports, and in order to determine whether or not p.Gly622Asp affects the risk of developing a CFTR-RD or CF, we analyzed the phenotype of subjects bearing the p.Gly622Asp mutation. Login to comment
34 For patients, healthy individuals and fetuses originating for Reunion Island (903 samples in total), a systematic search for c.366T N A, p.Tyr122Ter (Y122X) and c.1865G N A, p.Gly622Asp (G622D) was performed because of their particular occurrence in this population. Login to comment
44 Results We identified a total of 24 carriers of the p.Gly622Asp mutation among 903 individuals from Reunion Island. Login to comment
51 In particular, none of the four patients carried the c.3717 + 45G N A (3849 + 45G N A) variation, which was already reported in cis with p.Gly622Asp. Login to comment
52 p.Gly622Asp was also found in eight infertile males, five with azoospermia and three with oligoasthenozoospermia (from 0.2 to 7 M spermatozoa/ml). Login to comment
53 Four of them were compound heterozygous: three Table 1 Frequency of the p.Gly622Asp missense mutation in different groups of subjects from Reunion Island. Login to comment
54 Phenotype Number of individuals p.Gly622Asp carriers (number) Allele frequency (%) Cystic fibrosis (suspected or established) 56 6 5.35 Echogenic bowel 183 7 1.91 Male infertility 222 8 1.80 Idiopathic bronchiectasis 15 0 0.0 Idiopathic pancreatitis 15 0 0.0 Unaffected individuals 412 3 0.36 carried a severe CF mutation (c.1521_1523del (F508del), c.2988 + 1G N A) and one a CFTR-RD mutation (c.1210-12T [5]). Login to comment
56 p.Gly622Asp was also detected in three fetuses who were studied because of hyperechogenic bowel and were found to carry a CFTR-RD variant such as c.2851A N G, p.Lys951Gln (K951E), c.1210-12T[5] and c.1584G N A (1716G N A) (Table 3). Login to comment
60 Because the CFTR genetic background could influence the potential pathogenicity of the p.Gly622Asp mutation, we studied the haplotype background. Login to comment
61 Three intragenic CFTR microsatellites, c.53 + 10167CA(17_26) (IVS1CA), c.1210-307GT(16_24) (IVS8CA) and c.3367 + 200TA(7_56) (IVS17bCA), were typed for seven p.Gly622Asp heterozygotes. Login to comment
63 We also studied the association of p.Gly622Asp with the c.1408A N G, p.Val470 allele in exon 11 and the c.1210-12T(5_9) (Tn repeat) located in intron 9 (traditional nomenclature), and with other variants in the CFTR gene. Login to comment
64 We focused our analysis on the 11 compound heterozygotes showing Table 2 Clinical presentations of four cystic fibrosis cases with p.Gly622Asp mutation. Login to comment
67 [Tyr122Ter];[Gly622Asp] p. Login to comment
68 [Tyr122Ter];[Gly622Asp] p. Login to comment
69 [Tyr122Ter];[Gly622Asp] c. Login to comment
70 [2988 + 1G N A];[1865G N A] Phenotype CF CF CF CF Sweat Cl- (mEq/L) 78 63 80 63 Diabetes No No No No Cirrhosis No No No No Glucose intolerance No No No Yes (12 y) Nasal polyposis No No No No Intestinal disorders No No No No Pancreatic function PS PI (10 y) PI (4 y) PS FEV1(%) 85% 76% 99% 93% FVC (%) 79% 76% 86% 106% Pseudomonas aeruginosa No No Yes (3 y) No Staphylococcus aureus Yes (6 y) Yes (7 y) Yes (3 y) No Aspergillus fumigatus No Yes (6 y) No No Streptococcus pneumoniae No No No Yes (10 y) Haemophilus influenzae No Yes (4 y) Yes (4 y) Yes (10 y) Treatment PERT PERT PERT, steroid therapy PERT, steroid therapy, bronchodilatators Table 3 CFTR genotype and CFTR haplotype in subjects bearing the p.Gly622Asp mutation. Login to comment
74 Mutation 1 Mutation 2 5/7/9T allelea TG repeatb Codon 470 c.3717 + 45G N A Codon 854 5'UTR Patients with cystic fibrosis MUC821 p.Gly622Asp p.Tyr122Ter T7;T7 nd VV No c.2562T N G homo c.-8G N C MUC822 p.Gly622Asp p.Tyr122Ter T7;T7 nd VV No c.2562T N G homo c.-8G N C MUC940 p.Gly622Asp p.Tyr122Ter T7;T7 nd VV No No c.-8G N C R156C p.Gly622Asp c.2988 + 1G N A T7;T7 nd MV No c.2562T N G No Patients with male infertility MUC2815 p.Gly622Asp c.2988 + 1G N A T7;T7 nd nd No nd No MUC3913 p.Gly622Asp p.Phe508del T7;T9 nd MV No No No MUC4216 p.Gly622Asp p.Phe508del T7;T9 nd MV Yes No No MUC4811 p.Gly622Asp c.1210-12T[5] T5;T7 TG11;TG12 VV No No No Fetuses with hyperechogenic bowel MUC5131 p.Gly622Asp c.1210-12T[5] T5;T7 TG11;TG12 MV Yes c.2562T N G No MUC6775 p.Gly622Asp p.Lys951Glu T7;T7 nd MV No c.2562T N G No MUC4196 p.Gly622Asp c.1584G N A T7;T7 nd nd No nd No a c.1210-12T(5_9). Login to comment
77 We found that the p.Gly622Asp was associated with the p.Val470 allele in all informative cases (4/4) (Table 3). Login to comment
79 We found that the p.Gly622Asp was associated with the c.1210-12T[7] allele in all informative cases (6/6) (Table 3). Login to comment
81 p.Gly622Asp was associated with the c. Login to comment
83 Our findings showed no evidence of a difference in the distribution of the c.1210-12T(5_9) alleles and in the frequency of p.Met470Val variant between the different groups of subjects who carried the p.Gly622Asp variant. Login to comment
85 Discussion c.1865G N A, p.Gly622Asp appears to be the second most common mutation after c.1521_1523del, p.Phe508del, in the African American general population, found at an allelic frequency of 0.18% (3/118 healthy individuals) while its worldwide allelic frequency is much lower, 0.05% (4/4088 healthy individuals) [2]. Login to comment
86 Observations of p.Gly622Asp in patients are scarce. Login to comment
89 Although several other studies have examined CF mutations in African Americans and African patients with a clinical diagnosis of CF, no individual bearing p.Gly622Asp was identified [5-8]. Login to comment
92 Interestingly, we identified a total of 24 carriers of the p.Gly622Asp mutation among 903 individuals from Reunion Island. Login to comment
96 Although p.Gly622Asp may be associated in cis with the c.3717 + 45G N A variant, which is considered as neutral, we could not identify a specific haplotype assigned to a CF-causing mutation or a CFTR-RD mutation. Login to comment
97 Although the existence of an undetected mutation in cis with p.Gly622Asp, possibly deep-intronic or located in regulatory regions, cannot be ruled out, it is cautious to consider p.Gly622Asp as a CF mutation. Login to comment
102 However, it seems that the CF phenotype observed in patients carrying p.Gly622Asp may be more severe, thus making genetic counseling difficult. Login to comment
103 Other authors suggested that, taking account the ethnic/ racial background of individuals, p.Gly622Asp should be included in the American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics (ACMG) CF mutation panel [2], especially with the development of specific CF assays using the next-generating sequencing technology. Login to comment
104 As long as no clear explanation to such extreme phenotypes associated with p.Gly622Asp is found, our results support the recommendation of considering a CF allele whenever p.Gy622Asp is identified and of including it in future CF mutation screening panels, possibly targeted to individuals of African origin. Login to comment