ABCMdb

ABCC7 p.Val232Asp

Admin's notes:	Class II-III (maturation defect, gating defect) Veit et al.
ClinVar:	c.695T>A , p.Val232Asp ? , not provided
CF databases:	c.695T>A , p.Val232Asp (CFTR1) D , This mutation was was detected by DGGE and identified by direct sequencing in the CFTR gene. The defect is a T to A change at nucleotide 827 in exon 6a which would lead to a valine-to-aspartic acid replacement in the protein sequence at residue 232. This nucleotide change has been found in an infertile man with CBAVD having neither manifestation of gastrointestinal nor pulmonary disease but with a sweat teat at mmol/
Predicted by SNAP2:	A: N (53%), C: N (61%), D: D (80%), E: D (71%), F: N (61%), G: D (75%), H: D (75%), I: N (97%), K: D (80%), L: N (93%), M: N (82%), N: D (71%), P: D (75%), Q: D (71%), R: D (80%), S: D (66%), T: D (63%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, W: D, Y: N,

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II-III (maturation defect, gating 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] Molecular analysis in Brazilian cystic fibrosis pa... Genet Test. 2000;4(1):69-74. Bernardino AL, Ferri A, Passos-Bueno MR, Kim CE, Nakaie CM, Gomes CE, Damaceno N, Zatz M
Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations.
Genet Test. 2000;4(1):69-74., [PMID:10794365]

Abstract [show]
We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.

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6 Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 6211 1GRT, V232D, 1717-1GRA, 2347 delG, R851L, 27891 5GRA, and W1089X. Login to comment
53 Seven other rare mutations were also identified : 6211 1GRT (exon 4), V232D (exon 6a), 1717-1G RA (intron 11), 2347 delG (exon 13b), R851L (exon 14a), 27891 5GRA (intron 14b), and W1089X (exon 17b). Login to comment
81 In this study, 16 mutations were identified: D F508, G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 6211 1GRT, V232D, 1717-1GRA, 2347 delG, R851L, 27891 5GRA, and W1089X. Login to comment
84 GEN OTYPES, FREQUENCIES, AN D PRESENCE OF PI FRO M 160 CF PATIE NTS (320 CF CHROM OSOM ES) Number and frequency (%) Genotype Number Frequency (%) of patients with PI D F508/D F508 47 29.40 47 (100%) D F508/G542X 13 8.10 13 (100%) D F508/R1162X 6 3.80 6 (100%) D F508/R334W 5 3.10 3 (60%) D F508/N1303K 3 1.90 3 (100%) D F508/W1282X 2 1.20 2 (100%) D F508/G58E 2 1.20 1 (50%) D F508/L206W 1 0.62 0 D F508/R553X 1 0.62 1 (100%) D F508/R851L 1 0.62 0 D F508/2789 1 5g ® A 1 0.62 0 D F508/3617delGA 1 0.62 1 (100%) D F508/3171delC 1 0.62 1 (100%) D F508/2686insT 1 0.62 1 (100%) D F508/Y275X 1 0.62 1 (100%) D F508/U 22 13.80 14 (64%) G542X/G542X 3 1.90 3 (100%) G542X/N1303K 3 1.90 2 (67%) G542X/R1162X 1 0.62 1 (100%) G542X/U 5 3.10 4 (80%) N1303K/R1162X 1 0.62 1 (100%) N1303K/G58E 1 0.62 0 2347delG/2347delG 1 0.62 1 (100%) R334W/V232D 1 0.62 0 R334W/W1089X 1 0.62 1 (100%) R334W/U 1 0.62 1 (100%) W1282X/U 1 0.62 1 (100%) G58E/U 1 0.62 1 (100%) R553X/U 1 0.62 1 (100%) L206W/U 1 0.62 0 621 1 1G ® T/U 1 0.62 1 (100%) 1717-1G ® A/U 1 0.62 Not known V201M/U 1 0.62 0 U/U 27 16.90 12 (44%) Total 160 100 - U, Unknown CF mutation. Login to comment

[hide] Heterogeneity for mutations in the CFTR gene and c... Hum Reprod. 2000 Jul;15(7):1476-83. Casals T, Bassas L, Egozcue S, Ramos MD, Gimenez J, Segura A, Garcia F, Carrera M, Larriba S, Sarquella J, Estivill X
Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.
Hum Reprod. 2000 Jul;15(7):1476-83., [PMID:10875853]

Abstract [show]
Congenital absence of the vas deferens (CAVD) is a heterogeneous disorder, largely due to mutations in the cystic fibrosis (CFTR) gene. Patients with unilateral absence of the vas deferens (CUAVD) and patients with CAVD in association with renal agenesis appear to have a different aetiology to those with isolated CAVD. We have studied 134 Spanish CAVD patients [110 congenital bilateral absence of the vas deferens (CBAVD) and 24 CUAVD], 16 of whom (six CBAVD, 10 CUAVD) had additional renal anomalies. Forty-two different CFTR mutations were identified, seven of them being novel. Some 45% of the CFTR mutations were specific to CAVD, and were not found in patients with cystic fibrosis or in the general Spanish population. CFTR mutations were detected in 85% of CBAVD patients and in 38% of those with CUAVD. Among those patients with renal anomalies, 31% carried one CFTR mutation. Anomalies in seminal vesicles and ejaculatory ducts were common in patients with CAVD. The prevalence of cryptorchidism and inguinal hernia appeared to be increased in CAVD patients, as well as nasal pathology and frequent respiratory infections. This study confirms the molecular heterogeneity of CFTR mutations in CAVD, and emphasizes the importance of an extensive CFTR analysis in these patients. In contrast with previous studies, this report suggests that CFTR might have a role in urogenital anomalies.

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73 Finally, six polymorphisms were found each in V232D 2 (1) 0 2 (1) one patient: 104G/T, 296ϩ128G/C, 741C/T, 3195A/T, 3212T/3732delA 0 2 (14) 2 (1) L383S 1 1 (7) 2 (1) C and 4029A/G. Login to comment
83 We detected only three homozygous patients (one for V232D P Ͻ 0.001). Login to comment
97 Dilatation V232D/V232D 9T/9T 1 of ejaculatory ducts, often resembling utricular cysts, was S945L/R258G 7T/7T 1 demonstrable also in some men, all of whom were azoospermicG551D/F1074L 5T/7T 1 A1006E/L383S 5T/7T 1 (Figure 1). Login to comment

[hide] Adenosine triphosphate-binding cassette superfamil... Biol Reprod. 2001 Aug;65(2):394-400. Larriba S, Bassas L, Egozcue S, Gimenez J, Ramos MD, Briceno O, Estivill X, Casals T
Adenosine triphosphate-binding cassette superfamily transporter gene expression in severe male infertility.
Biol Reprod. 2001 Aug;65(2):394-400., [PMID:11466205]

Abstract [show]
Cystic fibrosis transmembrane regulator (CFTR), multidrug-resistant (MDR)1, and multidrug resistance-associated (MRP) proteins belong to the ATP-binding cassette (ABC) transporter superfamily. A compensatory regulation of MDR1 and CFTR gene expression has been observed in CFTR knockout rodent intestine and in an epithelial cell line of human colon, whereas a high homology and similar anion binding site are shared by MRP and CFTR proteins. To provide better insight into the relationship among the expression behavior in vivo of the three genes in human testis, analysis of MDR1 and MRP gene expression in testicular biopsies was performed and related to the presence of CFTR gene mutations in congenital absence of the vas deferens (CAVD: n = 20) and non-CAVD (n = 30) infertile patients with azoospermia or severe oligozoospermia. A CFTR mutation analysis performed in both groups of patients supported the involvement of CFTR gene mutations in CAVD phenotype (85%) and in defective spermatogenesis (19%). Quantitative reverse transcription-polymerase chain reaction analysis of testicular tissue showed a CFTR-independent MDR1 and MRP gene expression in human testis, suggesting that the mechanisms underlying CFTR gene regulation in testis are different from those in intestine. These findings should contribute to the understanding of patterns of in vivo expression of CFTR, MDR1, and MRP genes in CFTR-related infertility.

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87 Phenotypical and genotypical description of CAVD and non-CAVD infertile patients.a No. patient Phenotype FSH (U/L) Non-CFTR infertility-associated factors Testicular biopsy CFTR mutation M470V polymorphism CAVD infertility 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CUAVD CUAVD CUAVD CUAVD 3.1 7.3 3.1 2.4 1.9 3.5 5.7 4.3 3.6 ND 2.2 4.8 11.3 2.1 ND 7.6 5.3 6.5 3.9 21.4 None None None None None None None None None None None None None None None None None None None Yes 1 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes V232D/V232D F508del/R117H F508del/R117H G542X/2789ϩ5GϾA F508del/D1270N ϩ R74W F508del/D1270N ϩ R74W S945L/R258G F508del/5T F508del/5T L206W/5T R117H/N F508del/N Y1014C/N 5T/N N/N N/N Y1092X/R258G 621ϩ1GϾT/5T Q890R/N N/N M/M M/M M/M M/M M/V M/V M/V M/M M/V M/V M/V M/V M/V M/V M/M V/V V/V M/V V/V M/M Non-CAVD infertility 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SA) TF (SA) TF (SSO) OA OA OA OA OA OA OA OA 42.0 15.9 34.8 8.9 26.3 6.4 7.8 15.6 8.7 3.2 3.9 12.6 4.7 1.3 5.6 3.9 6.1 9.3 8.8 19.3 9.6 ND 3.3 5.9 6.6 3.6 1.9 4.2 2.0 4.4 None None None None None None None None None None None None None None None None Yes 2 Yes 2 Yes 2, 3 Yes 4 Yes 5 Yes 6 None None None None None Yes 1 Yes 7 Yes 8 Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes F508del/N R334W/N N/N N/N N/N N/N N/N N/N N/N R75Q/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N 5T/5T N/N N/N N/N N/N N/N N/N N/N M/M V/V M/V M/V M/V M/V V/V V/V V/V V/V M/V M/V M/V ND V/V M/M M/V M/M M/V M/M M/V V/V M/V M/V M/V V/V V/V M/V M/V V/V a CFTR mutations and M470V allele are also described for each patient. Login to comment
94 CFTR Analysis We have identified 14 different CFTR mutations (R117H, L206W, V232D, R258G, F508del, G542X, 621ϩ1GϾT, Q890R, S945L, Y1014C, Y1092X, D1270N, 2789ϩ5GϾA, IVS8-6[5T]) in 17 of 20 patients of the CAVD group, giving a CFTR mutation frequency of 85%. Login to comment

[hide] Interhelical packing in detergent micelles. Foldin... J Biol Chem. 2002 Feb 22;277(8):6067-72. Epub 2001 Dec 17. Therien AG, Deber CM
Interhelical packing in detergent micelles. Folding of a cystic fibrosis transmembrane conductance regulator construct.
J Biol Chem. 2002 Feb 22;277(8):6067-72. Epub 2001 Dec 17., 2002-02-22 [PMID:11748233]

Abstract [show]
Using a helix-loop-helix construct consisting of the adjacent transmembrane segments 3 and 4 of the cystic fibrosis transmembrane conductance regulator (CFTR) labeled with pyrene at both N and C termini, we describe a system for the study of intramolecular helix-helix interactions within a polytopic membrane protein. Through measurement of pyrene excimer band intensity as a determinant of helix-helix proximity, we show that the helices retain tertiary contacts in detergent micelles. Notably, the nature of the micellar detergent can alter the stability of these contacts, with perfluorooctanoate highly supportive, lysophosphatidylcholine and lysophosphatidylglycerol somewhat less tolerant, and SDS largely intolerant of such interactions. This construct is further employed to study the role of the acyl chain length of micellar detergents in modulating interhelical packing; detergents having acyl chains of 9 carbons display the greatest extent of helical packing. These results provide important information regarding the role of lipids on membrane protein folding and conformation as well as demonstrate the usefulness of a pyrene-based system in studying the forces that govern interhelical packing.

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136 dition, since TM 3-4 constructs represent a segment of the CFTR protein, experiments become feasible which test the effects of various cystic fibrosis phenotypic mutations on interhelical packing of TMs 3 and 4, as described for the mutant V232D (21). Login to comment

[hide] ATB(0)/SLC1A5 gene. Fine localisation and exclusio... Eur J Hum Genet. 2001 Nov;9(11):860-6. Larriba S, Sumoy L, Ramos MD, Gimenez J, Estivill X, Casals T, Nunes V
ATB(0)/SLC1A5 gene. Fine localisation and exclusion of association with the intestinal phenotype of cystic fibrosis.
Eur J Hum Genet. 2001 Nov;9(11):860-6., [PMID:11781704]

Abstract [show]
The Na+-dependent amino acid transporter named ATB(0) was previously found to be located in 19q13.3 by fluorescence in situ hybridisation. Genetic heterogeneity in the 19q13.2-13.4 region, syntenic to the Cystic Fibrosis Modulator Locus 1 (CFM1) in mouse, seemed to be associated to the intestinal phenotypic variation of cystic fibrosis (CF). We performed fine chromosomal mapping of ATB(0) on radiation hybrid (RH) panels G3 and TNG. Based on the most accurate location results from TNG-RH panel, mapping analysis evidenced that ATB(0) is localised between STS SHGC-13875 (D19S995) and STS SHGC-6138 in 19q13.3, that corresponds with the immediately telomeric/distal segment of the strongest linkage region within the human CFM1 (hCFM1) syntenic region. Regarding to the genomic structure and exon organisation, our results show that the ATB(0) gene is organised into eight exons. The knowledge of the genomic structure allowed us to perform an exhaustive mutational analysis of the gene. Evaluation of the possible implication of ATB(0) in the intestinal phenotype of CF was performed on the basis of the functional characteristics of the encoded protein, its apparent relevance to meconium ileus (MI) and position in relation to the hCFM1 syntenic region. We have analysed this gene in samples from CF patients with and without MI. Several sequence variations in the ATB(0) gene were identified, although none of them seemed to be related to the intestinal phenotype of CF. Even though no particular allele or haplotype in ATB(0) appears to be associated to CF-MI disease, new SNPs identified should be useful in segregation and linkage disequilibrium analyses in families affected by other disorders caused by the impairment of neutral amino acid transport.

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151 Statistical analysis showed that the higher incidence for P17A and the lower incidence for V512L observed in the general population Table 3 CFTR mutations of the CF patients under study with and without meconium ileus (MI) CF-non MI CF-MI CFTR mutations n CFTR mutations n F508del/R117H 2 F508del/F508del 7 F508del/R334W 3 F508del/L365P 1 F508del/R347P 1 F508del/G542X 1 F508del/621+1G4Ta 1 F508del/621+IG4Ta 1 F508del/M1101K 1 F508del/R1066C 1 F508del/1609delCAa 1 F508del/W1089X 1 F508del/2789+5G4Aa 3 F508del/R1162X 1 F508del/3849+10kbC4T 1 F508del/1609delCAa 1 G542X/G85E 1 F508del/Q1281X 1 G542X/V232D 1 F508del/1811+1.6kbA4G 1 G542X/1811+1.6kb A4Ga 1 F508del/2789+5G4Aa 1 G542X/2789+5G4A 1 F508del/2869insG 1 Q890X/L206W 1 F508del/unknown 1 1811+1.6kbA4G/P205S 1 I507del/I507del 1 R1162X/3272-26A4G 1 G542X/1078delT 1 N1303K/R347H 1 G542X/1811+1.6kbA4Ga 1 N1303K/A1006E+5T 1 S549R/CFTR50kbdel 1 2789+5G4A/405+1G4A 1 R1066C/R1066C 1 W1282X/712-1G4T 1 a CF patient with a sibling presenting identical CFTR genotype and discordance of intestinal phenotype. Login to comment

[hide] Polar residues in membrane domains of proteins: mo... Biochemistry. 2002 Mar 19;41(11):3647-53. Partridge AW, Melnyk RA, Deber CM
Polar residues in membrane domains of proteins: molecular basis for helix-helix association in a mutant CFTR transmembrane segment.
Biochemistry. 2002 Mar 19;41(11):3647-53., 2002-03-19 [PMID:11888281]

Abstract [show]
Polar side chains constitute over 20% of residues in the transmembrane (TM) helices of membrane proteins, where they may serve as hydrogen bond interaction sites for phenotypic polar mutations that arise in membrane protein-related diseases. To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Ala-scanning mutagenesis of the TM4 sequence indicated that ladder formation minimally required the simultaneous presence of the Cys-225, Asp-232, and Gln-237 residues. As random hydrophobic sequences containing these three residues at TM4 equivalent positions did not oligomerize, specific van der Waals packing interactions between helix side chains were also shown to play a crucial role. Overall, the results suggest that polar mutations in membrane domains, in conjunction with critically positioned polar partner residues, potentially constitute a source of aberrant helix interactions that could contribute to loss of function when they arise in protein transmembrane domains.

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1 To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Login to comment
2 Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. Login to comment
3 However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Login to comment
19 To investigate the roles of polar side chains in TM helices, we synthesized peptides corresponding to TM4 from CFTR either with or without the CF V232D mutation. Login to comment
20 We found that whereas the wild-type TM4 peptide failed to form any noncovalent helical associations, the TM4 peptide containing the V232D mutation generates a well-defined oligomeric ladder of TM4 helices mediated through a network of interhelical side chain-side chain H-bonds between D232 † This work was supported, in part, by grants to C.M.D. from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes for Health Research (CIHR), and the National Institutes of Health (NIDDK). Login to comment
65 The TM4 peptide was designed identically to TM4-wt except that it contained the V232D mutation. Login to comment
67 Effect of the V232D Mutation on Helical Packing. Login to comment
71 However, the TM4 peptide containing the V232D mutation displays additional bands consistent with those of a tetramer, hexamer, and octamer. Login to comment
122 In this context, we simulated H-bond formation in membrane-interactive peptides using segments featuring the CF-phenotypic mutation V232D from TM4 of the CFTR transmembrane domain. Login to comment
135 The V232D peptide was reduced in the lane indicated by incubating the peptide with TCEP in aqueous solution for 5 min prior to addition of Tricine sample buffer. Login to comment
163 However, due to FIGURE 6: Molecular models proposed for the oligomerization of the CFTR transmembrane helix 4 containing the V232D mutation (TM4-VD). Login to comment
178 The V232D mutation in TM4 creates a membrane-buried electrostatic locus, whether it is in the context of a TM peptide or in the full-length protein. Login to comment
182 For example, where the V232D mutation arises in full-length CFTR, the D232 residue on one TM4 helix could partner with the Q237 residue in the complementary TM4 helix in another CFTR molecule, to produce a functionally impaired H-bonded dimer. Login to comment
185 In the single-spanning TM peptides studied here, the V232D mutation results in a homophilic association of TM4 helices mediated by D232 and Q237 interhelical H-bonding. Login to comment
187 That the V232D mutation produces a non-wild-type H-bond in both systems suggests that a given mutant polar residue arising in the CFTR membrane domain could have multiple polar partners available in vivo. Login to comment

[hide] Polytopic membrane protein folding and assembly in... Mol Membr Biol. 2004 May-Jun;21(3):163-70. Booth PJ, High S
Polytopic membrane protein folding and assembly in vitro and in vivo.
Mol Membr Biol. 2004 May-Jun;21(3):163-70., [PMID:15204624]

Abstract [show]
The insertion and folding of proteins in biological membranes during protein synthesis in vivo is fundamental to membrane biogenesis. At present, however, certain molecular aspects of this process can only be understood by complementary studies in vitro. We bring together in vitro and in vivo results, highlighting how the studies inform each other and increase our knowledge of the folding and assembly of polytopic membrane proteins. A notable recent advance is the high-resolution crystal structure of the protein machinery responsible for membrane protein insertion into the endoplasmic reticulum. This provides an opportunity to combine in vitro and in vivo studies at a more sophisticated level and address mechanistic aspects of polytopic protein insertion and folding. Quality control is another important aspect of membrane biogenesis, and we give an overview of the current understanding of this process, focusing on cystic fibrosis as a well-studied paradigm. Mutations in the associated membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), can cause the quality control mechanisms to prevent the mutant protein reaching its normal site of action, the cell surface. In vitro studies of CFTR shed light on the possible origins of other clinically relevant folding mutants and highlight the potential synergy between in vitro and in vivo approaches.

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88 In the case of cystic fibrosis (see also below), detailed studies of peptides corresponding to helixÁ/ loopÁ/helix segments of the CFTR protein have shown that a hydrophobic-to-charged point mutation (V232D) in TM helix 4 induces a hydrogen bond with Gln 207 in helix 3, where this alteration is known to result in a cystic fibrosis disease phenotype [37]. Login to comment

[hide] Misprocessing of the CFTR protein leads to mild cy... Hum Mutat. 2005 Apr;25(4):360-71. Clain J, Lehmann-Che J, Dugueperoux I, Arous N, Girodon E, Legendre M, Goossens M, Edelman A, de Braekeleer M, Teulon J, Fanen P
Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype.
Hum Mutat. 2005 Apr;25(4):360-71., [PMID:15776432]

Abstract [show]
Cystic fibrosis (CF) is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine the mechanism of dysfunction of a disease-causing mutation associated with variable phenotypes. In order to attain these objectives, we studied the effect of the p.L206W mutation on CFTR protein production and function, and we examined the genotype-phenotype correlation of [p.L206W]+[p.F508del] patients. We showed that p.L206W is a processing (class II) mutation since the CFTR biosynthetic pathway was severely impaired, whereas single-channel measurements indicated ion conductance similar to the wild-type protein. These data raise the larger question of the phenotypic variability of class II mutants, including p.F508del. Since multiple potential partners could modify the processing of the CFTR protein during its course to the cell surface, environmental and other genetic factors might contribute to this variability.

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256 This hypothetical disease-causing mechanism is based on the observation that the neutral-to-charge CF mutation p.V232D in TM4 induces an aberrant side chain-side chain H bond with the neighboring wild-type Gln-207 in TM3 by using a recombinant helix-loop-helix fragment of CFTR (Therien et al., 2001). Login to comment

[hide] Comprehensive genetic analysis of the cystic fibro... Genet Med. 2006 Sep;8(9):557-62. Kammesheidt A, Kharrazi M, Graham S, Young S, Pearl M, Dunlop C, Keiles S
Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens--implications for newborn screening.
Genet Med. 2006 Sep;8(9):557-62., [PMID:16980811]

Abstract [show]
PURPOSE: In the United States, approximately 1/3,700 babies is born with cystic fibrosis each year. The >1,300 documented sequence variants pose a challenge for detection of cystic fibrosis through genetic screening. To investigate whether comprehensive characterization of the cystic fibrosis gene is feasible using dried newborn blood specimens, we modified the whole blood Ambry Test: CF and determined its sensitivity by testing DNA from individuals with cystic fibrosis who still had unknown mutations after commercial mutation panel testing. METHODS: DNA from 42 archived newborn dried blood specimens of affected Hispanic, African-American and Caucasian individuals in California was analyzed by temporal temperature gradient electrophoresis screening and targeted sequencing, and by gross deletion analysis. RESULTS: Excluding two specimens that could not be analyzed due to poor DNA quality, we report a 100% sensitivity and clinical detection rate in the remaining 40 patients. Eighty-three mutations representing 40 different variants were detected, including 8 novel mutations. CONCLUSIONS: This study demonstrates the feasibility of temporal temperature gradient electrophoresis-based full sequence analysis and targeted sequencing from DNA in newborn blood specimens. The Ambry Test: CF, as an additional step in cystic fibrosis newborn screening models, can be used to dramatically reduce the number of cystic fibrosis carrier sweat test referrals.

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98 In states with single specimenmodels,originalspecimensaretestedforthepresenceof themostcommonmutation,deltaF508,and/orotherdeleterious Table 1 Genotype data from panel testing and comprehensive Ambry TestTM : CF analysis Case Ethnicity ABI-31 Mutation 1 ABI-31 Mutation 2 Genzyme-87 Mutation 1 Genzyme-87 Mutation 2 Ambry Mutation 1 Ambry Mutation 2 Ambry Mutation 3 1 Hispanic delF508a 4382delAa 2 Hispanic delF508 N/I delF508 N/I delF508a 1248ϩ1GϾAa 3 African-American N/I N/I N/I N/I M150K CFTRdele17A,17Bb 4 Hispanic G542X N/I G542X N/I G542Xa 1288insTAa 5 African-American N/I N/I 3120ϩ1GϾA N/I 3120ϩ1GϾAa Q98Xa 3849؉72G>A 6 Hispanic delF508 N/I delF508 N/I delF508a 2289del10ins5a 7c Hispanic N/I N/I N/I N/I H199Ya 406-1GϾAa 8 Hispanic delF508 N/I delF508 N/I delF508a CFTRdele2,3(21kbb 9 Hispanic delF508 N/I delF508 N/I delF508a 2105-2117del13insAGAAAa 10 Hispanic G542X N/I G542X N/I G542X M952I Y914X 11 Hispanic N/I N/I N/I N/I 663delT L558S 12 Hispanic N/I N/I delF311 N/I delF311a 406-1GϾAa 13 Hispanic N/I N/I 2055del9insAa 2055del9insAa 14 Hispanic delF508 N/I delF508 N/I delF508 2055del9insA 15 Hispanic delF508 N/I delF508 N/I delF508 E257X 16 Hispanic N/I N/I N/I N/I V232D V232D 17 Hispanic delF508 N/I delF508 N/I delF508 H199Y 18 Hispanic delF508 N/I delF508 4160insGGGG 19 Caucasian delF508 N/I delF508 297-1GϾA 20 Hispanic 2183delAAϾG N/I 2183delAAϾG N/I 2183de1AAϾG 3500-2AϾG 21 Hispanic delF508 N/I delF508 S492F 22 Hispanic delF508 N/I delF508 N/I delF508 935delA 23 Caucasian R1162X N/I R1162X N/I R1162X 3940delG 24 Hispanic 711ϩ1GϾT N/I 711ϩ1GϾT T465N 25 Hispanic delF508 N/I delF508 N/I delF508 406-1GϾA 26 Hispanic delF508 N/I delF508 2055del9insA 27 Hispanic delF508 N/I delF508 N/I delF508 V232D 28 Hispanic delF508 N/I delF508 N/I delF508 S1235R 29 Hispanic G542X N/I G542X N/I G542X 297-1GϾA 30 Hispanic delF508 N/I delF508 N/I delF508 Q1100P 31 Hispanic delF508 N/I delF508 W216X 32 Hispanic N/I N/I N/I N/I 406-1GϾA H199Y 33 Hispanic N/I N/I N/I N/I 3272-26AϾG R75X 34 Hispanic N/I N/I Q890X N/I Q890X 2055del9insA 35 Hispanic delF508 N/I delF508 N/I delF508 W216X 36 Hispanic delF508 N/I delF508 N/I delF508 H199Y 37 Hispanic delF508 N/I delF508 N/I delF508 1288insTA I1027T 38 Hispanic G542X N/I G542X N/I G542X 663delT 39 Hispanic delF508 N/I delF508 N/I delF508 1288insTA 40 Hispanic delF508 N/I delF508 1288insTA mutations using mutation panels. Login to comment

[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] Role of the extracellular loop in the folding of a... Biochemistry. 2007 Jun 19;46(24):7099-106. Epub 2007 May 22. Wehbi H, Rath A, Glibowicka M, Deber CM
Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin.
Biochemistry. 2007 Jun 19;46(24):7099-106. Epub 2007 May 22., 2007-06-19 [PMID:17516627]

Abstract [show]
The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.

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44 Briefly, the percent change in molecular weight (MW) was first calculated from gels by comparing the apparent MW of each TM3/4 hairpin [estimated from the migration rates of Mark-12 molecular weight markers (Invitrogen)] to its expected theoretical MW using the equation: The percent change in MW of each ECL2 replacement in the TM3/4 WT or TM3/4 V232D backgrounds was then normalized to the migration of the parental hairpin as follows: ECL2 mutants with positive percent changes thus migrate less than their parental hairpin on SDS-PAGE; those with negative values migrate further. Login to comment
97 When the changes in TM3/4 WT hairpin migration were compared to changes in overall hairpin helicity, a strong correlation (R ) 0.79) was observed (Figure 5), leading us to propose that increases in non-native R-helix structure within ECL2 might Table 1: Migration Behavior on SDS-PAGE Gels of Single and Double Mutants in the Loop Region of CFTR TM3/4 Constructs % change in apparent MW on SDS-PAGE mutant vs TM3/4 WT in WT loop mutantsa vs TM3/4 V232D in V232D loop mutantsa Pb E217G 6.8 ( 0.7 E217S 11.1 ( 3.4 5.4 ( 1.4 0.056 Q220R 15.2 ( 1.1 Q220G 0.3 ( 0.4 Q220N 2.1 ( 1.3 0.5 ( 0.3 0.108 Q220K 14.1 ( 1.0 Q220W 13.1 ( 1.3 11.5 ( 0.9 0.157 Q220E -11.1 ( 1.1 -4.0 ( 0.3 <0.001 S222G 12.0 ( 2.1 1.1 ( 0.6 0.001 S222E -0.3 ( 2.4 1.3 ( 0.5 0.512 E217G/S222G 12.4 ( 1.9 E217S/S222E 26.1 ( 4.5 averagec 10.4 ( 7.3 4.0 ( 4.2 0.067 a Values are the percentage difference vs TM3/4 WT or TM3/4 V232D migration of SDS-PAGE gels. Login to comment
112 ECL2 Mutants Are Less DisruptiVe in the Context of the Membrane-Based CF-Phenotypic TM Mutant V232D. Login to comment
114 The CF-phenotypic V232D replacement in TM helix 4 has been suggested in the TM3/4 hairpin context to form a stabilizing interhelical hydrogen bond (33, 38); the TM3/4 V232D construct has a more compact fold than the TM3/4 WT hairpin and consequently migrates faster on SDS-PAGE (33). Login to comment
115 Several ECL2 replacements characterized as described above in the TM3/4 WT background were recapitulated in the TM3/4 V232D hairpin (Figure 6, Table 1). Login to comment
116 We found that the Q220E, S222G, and E217S replacements exhibited a relatively less pronounced effect on migration in the TM3/4 V232D hairpin than the TM3/4 WT background with high (p < 0.01 for Q220E and S222G) or detectable but marginal (p ) 0.056 for E217S) statistical significance. Login to comment
117 The Q220N and Q220W replacements also affected folding less in the TM3/4 V232D background than in the TM3/4 WT hairpin (Table 1). Login to comment
118 The mean change in migration of ECL2 mutants in the TM3/4 V232D hairpin was >2-fold smaller than the WT. Login to comment
139 Several sets of experi- FIGURE 6: SDS-PAGE migration of S222G and Q220W replacements in the TM3/4 WT and TM3/4 V232D backgrounds. Login to comment
140 Hairpins containing ECL2 mutants S222G and Q220W in the TM3/4 WT background and in the TM3/4 V232D background are shown. Login to comment
143 ments suggest that TM3/4 migration patterns are not simple functions of charge: (i) The double mutant Q207L/V232D migrates identically to WT, while V232D migrates significantly faster than WT (33). Login to comment
160 The TM3/4 V232D construct exhibits marginal but discernibly smaller migration changes than TM3/4 WT when ECL2 substitutions are introduced (Table 1, Figure 6). Login to comment
161 The stronger helix-helix interactions in TM3/4 V232D may therefore help to maintain a compact hairpin structure when ECL2 loop mutants are introduced that otherwise destabilize the fold. Login to comment

[hide] Misfolding of the cystic fibrosis transmembrane co... Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15. Cheung JC, Deber CM
Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.
Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15., 2008-02-12 [PMID:18193900]

Abstract [show]
Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.

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121 V232D is the CF-phenotypic mutant in TM4. Login to comment
141 We addressed the possibility experimentally that wild type Q207 in TM3 can form an interhelical side chain-side chain H-bond with CF-phenotypic mutant V232D in TM4 in an investigation of a series of helix-loop-helix ("hairpin") constructs derived from CFTR TM helices 3 and 4. Login to comment

[hide] Detergent binding explains anomalous SDS-PAGE migr... Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5. Epub 2009 Jan 30. Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM
Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.
Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5. Epub 2009 Jan 30., 2009-02-10 [PMID:19181854]

Abstract [show]
Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.

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5 The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. Login to comment
24 We find that gel shifts strongly correlate (R2 ϭ 0.8) with changes in the SDS-loading capacity of these miniature membrane proteins, indicating that altered detergent binding explains anomalous SDS-PAGE behavior. Our results reveal a distinction between two CF-phenotypic mutants studied: V232D binds significantly less SDS than the WT protein while P205S SDS binding is indistinguishable from WT, indicating that CFTR dysfunction may arise variously as a consequence of altered protein-lipid interactions or via altered intra-protein contacts. Login to comment
61 PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment
62 V232D, V232A, P205S, and Q220W) migrated as WT within statistical significance; 2 were faster (V232D and V232K); and 4 were slower (G228L, E217V, E217F, and E217S/S222E). Login to comment
70 The mutant hairpins ranged in loading levels from 3.4-10 g SDS/g, with V232D binding significantly fewer SDS molecules than WT, and the E217F and E217S/S222E mutants binding significantly more. Login to comment
97 PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment
113 Conversely, even though it migrates faster than WT on SDS-PAGE, the V232D hairpin reports a radius approximately 20% larger than WT (Table 2). Login to comment
123 However, even if SDS/ protein stoichiometry (and by extension, gel shift) remains unchanged, increases in the conformational flexibility of non-coated regions may alter the hairpin`s hydrodynamic radius (compare Fig. 5 B-E)-a potential explanation for the as-WT gel shift but increased hydrodynamic radius relative to WT of the P205A/V232D mutant. Login to comment
141 For example, V232D and P205A/V232D display larger than WT hydrodynamic radii on SEC-HPLC (ϩ19% and ϩ21%, respectively)-even though each Asp-containing mutant migrates faster or as-WT on PAGE. Login to comment
153 Our observation that the V232D mutant binds less SDS than WT TM3/4 may therefore indicate that the dysfunction in the full-length CFTR molecule caused by this mutation arises from altered protein-lipid associations. Login to comment

[hide] Mechanisms for rescue of correctable folding defec... Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22. Grove DE, Rosser MF, Ren HY, Naren AP, Cyr DM
Mechanisms for rescue of correctable folding defects in CFTRDelta F508.
Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22., [PMID:19625452]

Abstract [show]
Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF.

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10 In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Login to comment
183 Thus, we asked to what extent can disease related folding defects caused by mutations in MSD1 (G85E, G91R, and V232D), MSD2 (M1137R), and NBD2 (N1303K) be corrected relative to those caused by deletion of F508 in NBD1 (Figure 4A). Login to comment
185 The V232D mutation has been proposed to introduce an abnormal hydrogen bond within the transmembrane (TM) region of MSD1, which impedes normal TM assembly (Therien et al., 2001), but the affect of this mutation on CFTR biogenesis is unknown. Login to comment
189 The CFTR G85E and G91R point mutations are contained within TM1, whereas the V232D mutation lies within TM4 of CFTR`s MSD1 domain. Login to comment
194 Intriguingly, introduction of the V232D mutation generates an unstable CFTR biogenic mutant with decreased levels of the immature B-form and no apparent maturation product (Figure 4A). Login to comment
195 Yet, treatment with Corr-4a resulted in a dramatic increase in accumulation of both the immature and mature forms of CFTR V232D. Login to comment
196 However, folding defects in CFTR V232D were not significantly rescued by low temperature incubations (data not shown). Login to comment
197 Pulse-chase analysis indicated that Corr-4a strongly increases the folding efficiency of CFTR V232D, and does not simply increase the synthesis of this point mutant (Figure 4B). Login to comment
198 The ability of Corr-4a to dramatically enhance CFTR V232D folding suggests that this mutant may have a single folding defect in MSD assembly that is correctable by Corr-4a. Login to comment
199 In agreement with this idea, Corr-4a is able to enhance the accumulation of the V232D-containing MSD1 fragment, CFTR 370X V232D (data not shown). Login to comment
209 (B) Corr-4a increases the biosynthetic maturation of CFTR V232D. Login to comment
210 HEK293 cells transfected with CFTR V232D (1 ␮g) were preincubated with Corr-4a or DMSO for 2 h, labeled with [35 S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Login to comment
227 Next, we probed the ability of Corr-4a to enhance the proper association of CFTR 837X V232D with CFTR 837-1480. Login to comment
228 We observed that, when compared with the wild-type 837X fragment, the steady-state levels of CFTR 837X V232D were decreased (Figure 5B). Login to comment
229 Yet, the levels of CFTR 837X V232D were increased upon Corr-4a treatment. Login to comment
230 Furthermore, pulse-chase analysis demonstrated that Corr-4a stabilizes CFTR 837X V232D (Figure 5C). Login to comment
231 In addition, the levels of the C-terminal fragment, 837-1480, were increased when coexpressed with the CFTR 837X V232D mutant fragment (Figure 5D). Login to comment
233 Yet, when Corr-4a was present, a large subpopulation of the 837X V232D and 837- Figure 5. Login to comment
238 (B) HEK293 cells transfected individually with CFTR 837X (1 ␮g), CFTR 837X⌬F508 (1 ␮g), CFTR 837X V232D (1 ␮g), or CFTR 837-1480 (1 ␮g) were cultured for 18 h before addition of Corr-4a or DMSO. Login to comment
242 (C) Corr-4a increases the stability of CFTR 837X V232D and CFTR 837-1480. Login to comment
243 HEK293 cells transfected with either CFTR 837X V232D or CFTR 837-1480 were preincubated with Corr-4a or DMSO for 2 h, labeled with [35 S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Login to comment
247 (D) HEK293 cells were transfected with either CFTR 837-1480, CFTR 837X and CFTR 837-1480, CFTR 837X⌬F508 and CFTR 837-1480, or CFTR 837X V232D and CFTR 837-1480. Login to comment
254 Thus, Corr-4a is able to correct defects in MSD1 folding to the degree that it facilitates the proper assembly of CFTR 837X V232D and CFTR 837-1480. Login to comment
257 Yet, it appears that multiple folding defects limit the rescue of CFTR⌬F508, whereas a less complex mutant, such as CFTR V232D, is readily rescued. Login to comment
309 Corr-4a is also able to act on CFTR 837X V232D, and enhance its accumulation. Login to comment
318 Surprisingly, there was a dramatic enhancement of CFTR V232D folding by Corr-4a. Login to comment
319 In addition, Corr-4a was able to enhance the accumulation of CFTR 837X V232D and to increase levels of the mature, highly glycosylated form of CFTR 837-1480 with CFTR 837X V232D. Login to comment
320 The V232D mutant presents a mild form of CF (Alonso et al., 2007); however, the molecular basis for CF associated with this mutation has not been well-defined. Login to comment
321 The V232D mutation is proposed to form an aberrant hydrogen bond in MSD1 (Therien et al., 2001; Choi et al., 2004) that may hinder formation of interdomain contacts between MSD1 and MSD2 that are proposed to occur in the folded CFTR channel (Dawson and Locher, 2006; Serohijos et al., 2008). Login to comment
325 Yet, rare mutations, such as V232D, which cause more specific folding defects (Therien et al., 2001; Choi et al., 2004) are easier to correct. Login to comment

[hide] Association of cystic fibrosis genetic modifiers w... Fertil Steril. 2010 Nov;94(6):2122-7. Epub 2010 Jan 25. Havasi V, Rowe SM, Kolettis PN, Dayangac D, Sahin A, Grangeia A, Carvalho F, Barros A, Sousa M, Bassas L, Casals T, Sorscher EJ
Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens.
Fertil Steril. 2010 Nov;94(6):2122-7. Epub 2010 Jan 25., [PMID:20100616]

Abstract [show]
OBJECTIVE: To investigate whether genetic modifiers of cystic fibrosis (CF) lung disease also predispose to congenital bilateral absence of the vas deferens (CBAVD) in association with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We tested the hypothesis that polymorphisms of transforming growth factor (TGF)-beta1 (rs 1982073, rs 1800471) and endothelin receptor type A (EDNRA) (rs 5335, rs 1801708) are associated with the CBAVD phenotype. DESIGN: Genotyping of subjects with clinical CBAVD. SETTING: Outpatient and hospital-based clinical evaluation. PATIENT(S): DNA samples from 80 subjects with CBAVD and 51 healthy male controls from various regions of Europe. This is one of the largest genetic studies of this disease to date. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotype analysis. RESULT(S): For single nucleotide polymorphism (SNP) rs 5335, we found increased frequency of the CC genotype among subjects with CBAVD. The difference was significant among Turkish patients versus controls (45.2% vs. 19.4%), and between all cases versus controls (36% vs. 15.7%). No associations between CBAVD penetrance and polymorphisms rs 1982073, rs 1800471, or rs 1801708 were observed. CONCLUSION(S): Our findings indicate that endothelin receptor type A polymorphism rs 5335 may be associated with CBAVD penetrance. To our knowledge, this is the first study to investigate genetic modifiers relevant to CBAVD.

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68 Portuguese CFTR alleles Spanish CFTR alleles Turkish CFTR alleles 5T 22 F508del 11 5T 20 F508del 14 5T 9 D1152H 14 R334W 5 D443Ya 3 D110H 3 R117H 3 G576Aa 3 F508del 2 S1235R 3 R668Ca 3 3041-11del7 2 N1303K 2 G542X 2 1767del6 2 P205S 2 R117H 2 2789þ5G>A 2 D614G 2 V232D 2 CFTRdele2(ins186) 2 G542X 1 L997F 1 3120þ1G>A 1 L206W 1 H609R 1 G1130A 1 V562I 1 N1303H 1 M952I 1 I507del 1 L206W 1 365insT 1 3272-26A>G 1 3272-26A/G 1 E585X 1 2789þ5G>A 1 L15P 1 2752-15C>G 1 G576Aa 1 R347H 1 R334Q 1 R668Ca 1 2689insG 1 R347H 1 CFTRdele2,3 1 R1070W 1 E831X 1 L1227S 1 I 1027T 1 R1070W 1 E831X 1 3272-26A>G 1 L997F 1 I853F 1 A349V 1 6T 1 Note: CFTR ¼ 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] Increased folding and channel activity of a rare c... Am J Physiol Lung Cell Mol Physiol. 2011 Sep;301(3):L346-52. Epub 2011 Jun 3. Caldwell RA, Grove DE, Houck SA, Cyr DM
Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators.
Am J Physiol Lung Cell Mol Physiol. 2011 Sep;301(3):L346-52. Epub 2011 Jun 3., [PMID:21642448]

Abstract [show]
Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, DeltaF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRDeltaF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl(-) channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl(-) conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to approximately 20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTR(inh-172) and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRDeltaF508 function.

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35 The folding defect caused by the V232D mutation appears to be due to the introduction of a charged residue into a region of CFTR that is embedded in the lipid bilayer of the ER membrane (17, 24). Login to comment
36 Since the folding defect in CFTR caused by the V232D mutation is correctable to wild-type levels, CF patients with this allele may benefit from treatment with folding correctors. Login to comment
154 Yet, the mechanism by which the V232D mutation causes CF is not well documented and whether patients with this mutation can be treated with modulators of CFTR folding and channel activity is not clear. Login to comment
156 The V232D mutation is proposed to cause aberrant hydrogen bonding between TM4 and adjacent TM segments in CFTR (24). Login to comment
165 Thus, CF patients that harbor low frequency mutations, such as V232D, might be treated with small molecules that correct CFTR misfolding or potentiate CFTR channel activity. Login to comment
169 In doing so, Corr-4a may prevent the V232D mutation from causing the aberrant hydrogen bonding between TM segments in the bilayer proposed to cause premature degradation of CFTRV232D (24). Login to comment
37 The folding defect caused by the V232D mutation appears to be due to the introduction of a charged residue into a region of CFTR that is embedded in the lipid bilayer of the endoplasmic reticulum (ER) membrane (17, 24). Login to comment
38 Since the folding defect in CFTR caused by the V232D mutation is correctable to wild-type levels, CF patients with this allele may benefit from treatment with folding correctors. Login to comment
191 Yet the mechanism by which the V232D mutation causes CF is not well documented, and whether patients with this mutation can be treated with modulators of CFTR folding and channel activity is not clear. Login to comment
193 The V232D mutation is proposed to cause aberrant hydrogen bonding between TM4 and adjacent TM segments in CFTR (24). Login to comment
202 Thus CF patients that harbor low-frequency mutations, such as V232D, might be treated with small molecules that correct CFTR misfolding or potentiate CFTR channel activity. Login to comment
206 In doing so, Corr-4a may prevent the V232D mutation from causing the aberrant hydrogen bonding between TM segments in the bilayer proposed to cause premature degradation of CFTRV232D (24). Login to comment

[hide] Testicular CFTR splice variants in patients with c... Hum Mol Genet. 1998 Oct;7(11):1739-43. Larriba S, Bassas L, Gimenez J, Ramos MD, Segura A, Nunes V, Estivill X, Casals T
Testicular CFTR splice variants in patients with congenital absence of the vas deferens.
Hum Mol Genet. 1998 Oct;7(11):1739-43., [PMID:9736775]

Abstract [show]
The involvement of the five thymidine (5T) variant in intron 8 of the cystic fibrosis membrane regulator (CFTR) gene in congenital bilateral absence of the vas deferens (CBAVD) phenotype has been extensively demonstrated. This variant leads to alternative splicing of the CFTR gene which results in a wild-type transcript and one without exon 9. Little is known about expression of the CFTR gene in the testis. We analysed the level of the aberrantly spliced transcripts in testicular biopsies and correlated it with disease expression. Quantitative RT-PCR analysis in testicular biopsies from control and CBAVD patients showed a correlation between the length of the IVS8-6(T) n tract and the level of alternatively spliced transcripts. Results from histological analysis also suggest an involvement of the alternative transcript in the spermatogenic status of patients, leading to a decreased number of mature sperm forms in the tubule.

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18 RESULTS CFTR analysis Eight different mutations (R117H, L206W, V232D, ∆F508, G542X, 711+1G→T, D1270N and 2789+5G→A) were found in nine of the 12 CBAVD patients, yielding a CFTR mutation frequencyof75%.ThreepatientspresentedtwoCFTRmutations, with one of them homozygous for the V232D mutation. Login to comment
26 CFTR genotype, IVS8-6 poly(T) allele and proportion of exon 9+ (E9+) and exon 9- (E9-) CFTR transcripts in testicular and epididymal biopsies Sample Phenotype CF mutation IVS8-6(T) Testis Epididymis n E9+ (%) E9- (%) n E9+ (%) E9- (%) 1 Non-CBAVD N/N 9T/9T 5 99 ± 0 1 ± 0 2 Non-CBAVD N/N 7T/7T 2 96 ± 2 4 ± 2 3 Non-CBAVD N/N 7T/7T 3 98 ± 0 2 ± 0 4 Non-CBAVD N/N 7T/7T 3 97 ± 1.5 3 ± 1.5 5 Non-CBAVD R334W/N 7T/7T 3 94 ± 1 6 ± 1 6 Non-CBAVD N/N 7T/7T 2 95 ± 1 5 ± 1 7 CBAVD V232D/V232D 9T/9T 4 96 ± 1.5 4 ± 1.5 8 CBAVD ∆F508/N 9T/9T 2 99 ± 0 1 ± 0 9 CBAVD ∆F508/D1270N 7T/9T 2 98 ± 1 2 ± 1 10 CBAVD G542X/2789+5G→A 7T/9T 2 96 ± 1 4 ± 1 11 CBAVD N/N 7T/7T 3 96 ± 2 4 ± 2 2 90 ± 3 10 ± 3 12 CBAVD N/N 7T/7T 2 94 ± 2 6 ± 2 5 78 ± 5 22 ± 5 13 CBAVD R117H/N 7T/7T 2 99 ± 0 1 ± 0 4 95 ± 2 5 ± 2 14 CBAVD G542X/5T 5T/9T 3 30 ± 2 70 ± 2 15 CBAVD ∆F508/5T 5T/9T 2 80 ± 5 20 ± 5 16 CBAVD L206W/5T 5T/9T 2 58 ± 2 42 ± 2 17 CBAVD 711+1G→T/5T 5T/7T 3 77 ± 4 23 ± 4 18 CBAVD 5T/N 5T/7T 5 71 ± 2 29 ± 2 The mean proportion of E9+ and E9- CFTR transcripts is calculated as the mean of the proportions found for each sample. Login to comment

[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]

Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.

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312 For example, it was demonstrated that the cystic fibrosis-phenotypic mutation V232D in CFTR TM segment 4 likely inhibits folding of CFTR by forming an unnatural hydrogen bond with Gln-207 in TM3 (47). Login to comment

[hide] Spectrum of mutations in the CFTR gene in cystic f... Ann Hum Genet. 2007 Mar;71(Pt 2):194-201. Alonso MJ, Heine-Suner D, Calvo M, Rosell J, Gimenez J, Ramos MD, Telleria JJ, Palacio A, Estivill X, Casals T
Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry.
Ann Hum Genet. 2007 Mar;71(Pt 2):194-201., [PMID:17331079]

Abstract [show]
We analyzed 1,954 Spanish cystic fibrosis (CF) alleles in order to define the molecular spectrum of mutations in the CFTR gene in Spanish CF patients. Commercial panels showed a limited detection power, leading to the identification of only 76% of alleles. Two scanning techniques, denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism/hetroduplex (SSCP/HD), were carried out to detect CFTR sequence changes. In addition, intragenic markers IVS8CA, IVS8-6(T)n and IVS17bTA were also analyzed. Twelve mutations showed frequencies above 1%, p.F508del being the most frequent mutation (51%). We found that eighteen mutations need to be studied to achieve a detection level of 80%. Fifty-one mutations (42%) were observed once. In total, 121 disease-causing mutations were identified, accounting for 96% (1,877 out of 1,954) of CF alleles. Specific geographic distributions for the most common mutations, p.F508del, p.G542X, c.1811 + 1.6kbA > G and c.1609delCA, were confirmed. Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. This updated information on the spectrum of CF mutations in Spain will be useful for improving genetic testing, as well as to facilitate counselling in people of Spanish ancestry. In addition, this study contributes to defining the molecular spectrum of CF in Europe, and corroborates the high molecular mutation heterogeneity of Mediterranean populations.

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9 Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. Login to comment
45 (%) p.F508del # E.10 1009 (51.74) p.G542X # E.11 150 (7.69) p.N1303K # E.21 57 (2.92) c.1811 + 1.6kbA > G I.11 36 (1.84) p.R334W # E.7 35 (1.79) p.L206W E.6a 32 (1.64) c.711 + 1G > T # I.5 31 (1.58) p.Q890X E.15 28 (1.43) p.R1162X # E.19 25 (1.28) c.2789 + 5G > A # I.14b 24 (1.23) p.R1066C E.17b 23 (1.18) p.I507del # E.10 21 (1.07) c.1609delCA E.10 18 (0.92) c.712-1G > T I.5 18 (0.92) c.3272-26A > G I.17a 18 (0.92) c.2183AA > G # E.13 16 (0.82) p.G85E # E.3 15 (0.77) c.2869insG E.15 15 (0.77) p.W1282X # E.20 15 (0.77) p.V232D E.6a 14 (0.71) p.A1006E * E.17a 12 (0.61) c.2184insA E.13 11 (0.56) p.K710X E.13 11 (0.56) TOTAL (n = 23) 1,634 (83.72) * , the complex allele [p.A1006E; p.V562I; IVS8-6(5T)] #, CF mutations identified with the Celera Diagnosis Cystic Fibrosis v2 genotyping assay and the Inno-Lipa CFTR12, CFTR17 + Tn Samples with microsatellite haplotypes 16/45-46-47 (IVS8CA/IVS17bTA) were submitted to direct analysis of the c.1811 + 1.6kbA > G mutation, which was found mainly associated with the 16-46 haplotype. Login to comment
62 Two mutations, p.V232D and c.1341G > A, showed the same high frequency (3/122, 2.5%) in the Castilla-Leon region. Login to comment
85 In addition, three other mild mutations, c.3272-26A > G, p.V232D and p.A1006E, showed frequencies ranging from 0.9% to 0.6% (Table 1). Login to comment
93 In addition, two other relatively frequent mutations showed unequal geographic distributions, p.V232D in the Castilla-Leon region (2.5%) and c.2789 + 5G > A in the Balearic Islands (10.5%). Login to comment

[hide] Corrector-mediated rescue of misprocessed CFTR mut... Biochem Pharmacol. 2012 Feb 1;83(3):345-54. Epub 2011 Nov 28. Loo TW, Bartlett MC, Shi L, Clarke DM
Corrector-mediated rescue of misprocessed CFTR mutants can be reduced by the P-glycoprotein drug pump.
Biochem Pharmacol. 2012 Feb 1;83(3):345-54. Epub 2011 Nov 28., [PMID:22138447]

Abstract [show]
The most common cause of cystic fibrosis is deletion of Phe508 in the first nucleotide-binding domain (NBD) of the CFTR chloride channel, which inhibits protein folding. DeltaF508 CFTR can be rescued by indirect approaches such as low temperature but the protein is unstable. Here, we tested our predictions that (1) other CFTR mutants such V232D and H1085R were more stable at the cell surface than DeltaF508 CFTR after low temperature rescue and (2) the advantages of rescue with specific correctors (pharmacological chaperones) are that they may stabilize DeltaF508 CFTR and increase the effectiveness of the correctors by bypassing drug pumps such as P-glycoprotein (P-gp) (increased bioavailability). It was found that the stability of mutants V232D and H1085R after low-temperature (30 degrees C) rescue was about 10-fold higher than DeltaF508 CFTR. We show that the corrector, 4,5,7-trimethyl-N-phenylquinolin-2-amine (5a), could stabilize DeltaF508 CFTR at the cell surface. Unlike most correctors, corrector 5a showed specificity for CFTR as it did not rescue the G268V P-gp processing mutant nor stimulate the ATPase activity of wild-type P-gp. By contrast, corrector KM11060 was a P-gp substrate as it stimulated P-gp ATPase activity and rescued the G268V mutant. Expression of wild-type P-gp reduced the effectiveness of CFTR rescue by corrector KM11060 by about 5-fold. The results underlie the importance of selecting correctors that are specific for CFTR because their efficiency can be reduced by drug pumps such as P-gp.

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14 Here, we tested our predictions that (1) other CFTR mutants such V232D and H1085R were more stable at the cell surface than DF508 CFTR after low temperature rescue and (2) the advantages of rescue with specific correctors (pharmacological chaperones) are that they may stabilize DF508 CFTR and increase the effectiveness of the correctors by bypassing drug pumps such as P-glycoprotein (P-gp) (increased bioavailability). Login to comment
15 It was found that the stability of mutants V232D and H1085R after low-temperature (30 8C) rescue was about 10-fold higher than DF508 CFTR. Login to comment
37 We show that rescue of DF508 CFTR with specific correctors resulted in a more stable protein and mutations such as V232D and H1085R are different from DF508 because they do not destabilize the rescued form of mature CFTR. Login to comment
106 (B) Samples of cells expressing DF508, V232D or H1085R CFTR mutants or P-gp mutant G268V and grown in the absence (À) or presence (+) of 20 mM VX-809 for 18 h were subjected to immunoblot analysis. Login to comment
120 We also tested whether correctors would promote maturation of CFTR mutants DF508, V232D and H1085R in HEK 293 cells in parallel because it has been reported that CFTR rescue depends on the cell system used [38-41]. Login to comment
121 Mutants V232D (TMD1) and H1085R (TMD2) were included because they are processing mutations located in different domains of CFTR (Fig. 1A) and both yield active proteins after rescue [16,42,43]. Login to comment
181 Degradation of wild-type, V232D, and H1085R CFTRs was monitored over time at 37 8C. Login to comment
203 To test if other CF mutations affect stability of CFTR, mutants V232D (TMD1) and H1085R (TMD2) were selected for study as both are processing mutations that yield active proteins after rescue [16,43]. Login to comment
204 Accordingly, we examined the stability of mutants V232D and H1085R after low-temperature rescue. Login to comment
205 Cells expressing wild-type, V232D, or H1085R CFTRs were expressed at low temperature to promote maturation of the protein. Protein synthesis was stopped by addition of cycloheximide, and turnover of the protein was monitored after incubation for 0-32 h at 37 8C (Fig. 7). Login to comment
206 It was observed that the half-lives of the mature forms of V232D and H1085R were at least 10-fold longer (about 14 and 12 h, respectively) than DF508 CFTR (about 1 h, Fig. 4). Login to comment
208 The V232D TMD1 and H1085R mutations may have less effect on the stability of mature CFTR because they have more localized effects on protein folding in the TMD1 and TMD2 domains, respectively. Login to comment
245 Corr-4a also may promote interactions between TMD1 and TMD2 as it restored folding of a processing mutant containing a charged residue (V232D) within TM4 [43] that is embedded in the lipid bilayer [56]. Login to comment
246 The V232D mutation is predicted to alter packing of the TM segments [56]. 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] Sequence hydropathy dominates membrane protein res... Biochemistry. 2012 Aug 7;51(31):6228-37. Epub 2012 Jul 25. Nadeau VG, Rath A, Deber CM
Sequence hydropathy dominates membrane protein response to detergent solubilization.
Biochemistry. 2012 Aug 7;51(31):6228-37. Epub 2012 Jul 25., [PMID:22779403]

Abstract [show]
The ability to predict from amino acid sequence how membrane protein structures will respond to detergent solubilization would significantly facilitate experimental characterization of these molecules. Here we have investigated and compared the response to solubilization by the "mild" n-dodecyl-beta-d-maltoside (DDM) and "harsh" sodium dodecyl sulfate (SDS) of wild-type and point mutant "hairpin" (helix-loop-helix) membrane proteins derived from the third and fourth TM segments of the human cystic fibrosis transmembrane conductance regulator (CFTR) and the intervening extracellular loop. Circular dichroism spectroscopy, size-exclusion chromatography, and pyrene fluorescence spectroscopy were used to evaluate the secondary structures, hairpin-detergent complex excluded volumes, and hairpin compactness of the detergent-solubilized sequences. Sequence hydrophobicity is found to be the dominant factor dictating membrane protein response to detergent solubilization by DDM and SDS, with hairpin secondary structure exquisitely sensitive to mutation when DDM is used for solubilization. DDM and SDS differ principally in their ability to promote approach of TM segment ends, although hairpin compactness remains sensitive to point mutations. Our overall findings suggest that protein-protein and protein-detergent interactions are determined concomitantly, with the net hydropathy of residues exposed to detergent dominating the observed properties of the solubilized protein.

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87 The intensity of mean residue ellipticity at 222 nm, for example, ranged by ~20 000 deg cm2 dmol-1 among the hairpin sequences (compare E217V and V232D, Figure 2A, left), a result that might not initially be anticipated for a "mild" detergent. Login to comment
89 We further noted that the spectra of mutant hairpins solubilized in DDM exhibited a gradation of intensities, whereas SDS-solubilized mutant spectra could be divided into "low helicity" (WT, V232D, A204L, and P205S) or "high helicity" (E217V, ES/SE, and E217F) clusters. Login to comment
107 The WT and mutant sequences complexed with DDM exhibited variable elution volumes, with ES/SE and V232D eluting earliest and latest from the column, respectively (Figure 3B, left). Login to comment
119 However, we noted that the elution volumes of the V232D mutant (open circle, Figure 3B) placed this hairpin outside the 95% confidence interval of the line of best fit. Login to comment
120 TM3/4-V232D can therefore be considered to differ in its hydrodynamic properties following solubilization by DDM vs SDS. Login to comment
165 N = 7 for the plots; the line of best fit omits the outlier mutant V232D (open circle, data point outside the 95% confidence interval of the fit). Login to comment
167 Note that the line of best fit has a near- unity slope, indicating that the relative order of elution is the same among the hairpin-DDM and hairpin-SDS complexes when V232D is excepted. Login to comment
172 Hydropathy Dependence in Local Sequence Context: The Cases of ES/SE and V232D. Login to comment
173 The ES/SE-DDM and V232D mutants are distinguished among the groups of DDM- or SDS-solubilized hairpins in that each of these hairpin-detergent complexes exhibits an apparent excluded volume on SEC that is larger than expected based on their rankings in helicity and low E:M ratios (compare Figures 3B, 2B, and 4B). Login to comment
174 Because the excluded volume of protein-detergent complexes is not an absolute measure of binding stoichiometry or particle size when particles deviate from compact, spherical shapes, we can only conclude at present that the shapes and/or dynamics of the ES/SE-DDM complex and the V232D-SDS complex differ in some way from the hairpin-DDM and hairpin-SDS complexes of equivalent helicities. 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] A Modified Alderman-Grant Coil makes possible an e... J Magn Reson. 2009 Nov;201(1):87-92. Epub 2009 Aug 15. Grant CV, Yang Y, Glibowicka M, Wu CH, Park SH, Deber CM, Opella SJ
A Modified Alderman-Grant Coil makes possible an efficient cross-coil probe for high field solid-state NMR of lossy biological samples.
J Magn Reson. 2009 Nov;201(1):87-92. Epub 2009 Aug 15., [PMID:19733108]

Abstract [show]
The design, construction, and performance of a cross-coil double-resonance probe for solid-state NMR experiments on lossy biological samples at high magnetic fields are described. The outer coil is a Modified Alderman-Grant Coil (MAGC) tuned to the (1)H frequency. The inner coil consists of a multi-turn solenoid coil that produces a B(1) field orthogonal to that of the outer coil. This results in a compact nested cross-coil pair with the inner solenoid coil tuned to the low frequency detection channel. This design has several advantages over multiple-tuned solenoid coil probes, since RF heating from the (1)H channel is substantially reduced, it can be tuned for samples with a wide range of dielectric constants, and the simplified circuit design and high inductance inner coil provides excellent sensitivity. The utility of this probe is demonstrated on two electrically lossy samples of membrane proteins in phospholipid bilayers (bicelles) that are particularly difficult for conventional NMR probes. The 72-residue polypeptide embedding the transmembrane helices 3 and 4 of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (residues 194-241) requires a high salt concentration in order to be successfully reconstituted in phospholipid bicelles. A second application is to paramagnetic relaxation enhancement applied to the membrane-bound form of Pf1 coat protein in phospholipid bicelles where the resistance to sample heating enables high duty cycle solid-state NMR experiments to be performed.

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94 The initial samples containing the TM3/4 V232D segment of CFTR would form ordered and well-aligned bicelles only at low concentrations, near the threshold of solid-state NMR detection. Login to comment
111 (A) One-dimensional 15 N chemical shift spectrum of uniformly 15 N-labeled CFTR TM3/4 V232D. Login to comment
124 Expression and purification The 15 N labeled form of the CFTR TM3/4 V232D helical hairpin construct was expressed and purified as previously described [26-28]. Login to comment
130 Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled TM3/4 V232D. Login to comment
136 Thrombin-treated TM3/4 V232D was purified by RP-HPLC on a C4 semipreparative column (Phenomenex) using an acetonitrile gradient. Login to comment
140 18 mg of TM3/4 V232D (>95% pure) per 1 L of minimal M9 medium. Login to comment
144 Bicelle sample preparation The uniformly 15 N-labeled TM3/4 V232D segment of CFTR and Pf1 major coat protein were reconstituted into bicelles as previously described [31,32]. 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] Positional dependence of non-native polar mutation... Biochim Biophys Acta. 2008 Jan;1778(1):79-87. Epub 2007 Sep 15. Wehbi H, Gasmi-Seabrook G, Choi MY, Deber CM
Positional dependence of non-native polar mutations on folding of CFTR helical hairpins.
Biochim Biophys Acta. 2008 Jan;1778(1):79-87. Epub 2007 Sep 15., [PMID:17949679]

Abstract [show]
Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix-loop-helix constructs) which we term 'helical hairpins'; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077-8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a 15N,13C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of 15N and 1H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.

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3 In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Login to comment
4 Over 95% of the backbone resonances of a 15 N,13 C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Login to comment
5 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
13 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence ⁎ Corresponding author. Login to comment
35 Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment
43 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
46 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
52 Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled V232D-TM3/4, or 13 C glucose (purchased from Cambridge Isotope Laboratories) for expression of 15 N/13 C isotopically labeled TM3/4; and 100 μg/mL ampicillin. Login to comment
73 Triple resonance NMR experiments for 15 N/13 C isotopically enriched V232D-TM3/4 115 mM PFO were carried out at 45 °C. Login to comment
94 Here, the double mutant Q207N/V232E migrates significantly faster than wild type, indicating that the N/E pair is likely involved in side chain-side chain interactions analogously to the Q/D pair at the corresponding positions in the TM3/4-V232D single mutant [22]. Login to comment
95 In contrast, replacement of wild type Q237 in TM4 with the non-polar Leu residue (mutant Q237L) creates a slower migrating hairpin vs. wild type (Fig. 1a), suggesting that some pre-existing inter-helical interactions - conceivably attributable to a network of H-bonding interactions - have been removed. Login to comment
99 Chemical shift assignment and secondary structure analysis of V232D-TM3/4 in PFO 1 H-15 N HSQC spectrum of 15 N-enriched V232D-TM3/4 in 115 mM PFO at 45 °C is shown in Fig. 2. Login to comment
104 Analysis of the triple resonance experiments HNCACB, CBCA(CO)NH, HNCO, (H)CC(CO)NH-TOCSY, and H(CC) (CO)NH-TOCSY of the 15 N,13 C-labeled V232D-TM3/4 enabled the assignments for 95% of the residues in the TM3/4 region, despite the fact that the analysis was complicated due to crosspeak overlapping typical of native helical TM proteins in detergent micelles. Login to comment
107 Hα, Cα, Cβ, CO, and NH chemical shifts of V232D-TM3/4 residues were analyzed using TALOS [51]. Login to comment
108 The secondary structure elements of V232D-TM3/4 thus obtained agree broadly with the formation of two helices in the micelle environment, separated by a turn. Login to comment
118 1 H-15 N HSQC spectrum of 15 N-labeled TM3/4 mutant V232D with amide chemical shift assignments. Login to comment
129 However, these NOEs were not sufficient per se for calculation of a defined tertiary structure for the V232D-TM3/4 hairpin. Login to comment
131 Analysis of 1 H-15 N HSQC spectra of WT-TM3/4 and its various mutants in PFO micelles To probe for global conformational changes of TM3/4 upon introducing a polar residue in TM4, the 1 H-15 N HSQC spectrum of wild type CFTR TM3/4 was acquired in PFO micelles under the same buffer conditions as V232D-TM3/4. Login to comment
132 With the amide chemical shift assignments of V232D-TM3/4 in hand, and because these two constructs differ by only a single residue, any major differences in the 1 H-15 N HSQC spectra should be attributable to the modification of the electrostatic environment by the non-polar-to-polar residue mutation at position 232, as well as to any resulting conformational changes. Login to comment
134 Summary of the chemical shift deviation and the sequential, mid-range NOEs for V232D-TM3/4 in PFO micelles. Login to comment
141 (a) WT; (b) V232D; (c) I231D; and (d) Q207N/V232E. Login to comment
142 Spectra were recorded in 115 mM PFO at 45 °C. Gly cross-peaks (shown in (b)) were assigned from the V232D spectrum (Fig. 2); other assignments shown were made where possible by comparison. Login to comment
144 As illustrated in the 'Gly box`, 1 H-15 N HSQC spectra of WT-TM3/ 4 (Fig. 4a) and V232D-TM3/4 (Fig. 4b) do possess some similarities. Login to comment
146 However, among the four spectra shown, significant chemical shift changes are observed for several of G213, G226, G228, G239 and G241 resonances between WT and V232D-TM3/4. Login to comment
147 As assignments were available only for the V232D-TM3/4 construct, we could not assign the full complement of WT Gly resonances specifically; however, the observed shifts are suggestive of a change of the environment of these residues. Login to comment
149 In order to further investigate the conformational changes of TM3/4 upon introduction of a polar residue into TM4, we next performed a comparative analysis of amide 1 H/15 N chemical shifts in the 1 H-15 N HSQC spectra of WT-TM3/4 vs. I231D-TM3/4 which contains a polar mutation at the position preceding the CF-phenotypic mutation V232D-TM3/4. Login to comment
150 Note that this mutant exhibited a faster migration rate than both WT-TM3/4 and V232D-TM3/4 on SDS-PAGE [22] (Fig. 1b). Login to comment
151 From the analysis of the Gly cross-peaks (Fig. 4c), one observes that the N-H chemical shifts of G3, G23, and G194 remain identical to the WT, but obvious changes occurred for G213, G226, G228, G239 and G241 to the extent that we could not formally assign them by comparison To V232D-TM3/4. Login to comment
153 We then examined the double mutant Q207N/V232E-TM3/4 in which the potential polar partners are located in the sequence at the same positions as in V232D-TM3/4 (Q207 in TM3 and D232 in TM4). Login to comment
154 This mutant similarly migrates faster than V232D-TM3/4 (Fig. 1a, b). Login to comment
155 As shown in Fig. 4b and d, a striking similarity was observed between the 1 H-15 N HSQC 'Gly box` spectra of V232D- and Q207N/V232E-TM3/4, suggesting that the cross-peaks of their Gly residues can be correspondingly assigned, and that their global conformations likely correspond closely. Login to comment
160 While some mutants do contain a change in charge vs. WT, previous work from our laboratory has established that CFTR TM3/4 migration patterns are not simple functions of charge: (i) WT TM3/4 and the double mutant Q207L/V232D the same migration rates while V232D migrates significantly faster than WT [20]; (ii) Asp substitutions at 20 different positions along TM4 between residues 221 and 241 produce TM3/4 hairpins that migrate 3-12% faster than WT [22]; if introduction of a single negative charge was the dominating effect, all 20 mutants should display similar migration rates. Login to comment
161 In the present work, Q207N/V232E-TM3/4 migrates faster than TM3/4-V232D (Fig. 1b) although they each have one added negative charge vs. WT. Login to comment
174 We further observed that the turn (approximately S222 to G226) determined between TM3 and TM4 in the V232D-TM3/4 construct is shifted approximately six residues toward TM4 vs. the one predicted (W216 to Q220) in the original schematic presented for the wild type CFTR protein [6]. Login to comment
187 Similarly, G194 present at the N-terminal end of TM3, is not affected by the V232D mutation (Fig. 4b). Login to comment
189 In addition to these "Gly box" effects, chemical shift changes vs. WT in the amide proton dimension of virtually all of the residues in the V232D-TM3/4 spectrum, including A223, A225, and G226 from the turn, as well as W216, L218, L219, Q220, and A221 near the C-terminus of the TM3 helix (data not shown), confirming that the effect is not limited to immediate neighbors. Login to comment
190 These changes, in concert with results from migration rate data on SDS-PAGE, emphasize the fact that the interactions between helices differ in WT- vs. V232D-TM3/4. Login to comment
192 To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment
194 The full amide chemical shift analysis of the 1 H-15 N HSQC shows that similarly to the V232D mutant, all TM4 amides - along with residues from W216 through A221 (C-terminus of TM3) and from S222 to G226 (turn) - are highly affected by the I231D mutation (not shown), suggesting that this mutation introduces significant changes in packing relative to WT protein. Login to comment
195 In contrast, the full 1 H-15 N HSQC spectra for V232D- and Q207N/V232E-TM3/4 exhibit striking similarities (not shown, but exemplified by the 'Gly box` comparison between Fig. 4 b and d), suggesting that these two mutants adopt similar conformations in the PFO micelle environment. Login to comment
196 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
200 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
202 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100° of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
208 Conclusion Features of the secondary structure of mutant V232D-TM3/4 helical hairpins of CFTR, along with comparisons to the WT and mutants I2231D and Q207N/V232E, have been determined using high resolution NMR spectroscopy. Login to comment
209 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment
6 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
14 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence Ìe; Corresponding author. Login to comment
36 Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment
44 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
47 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
53 Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled V232D-TM3/4, or 13 C glucose (purchased from Cambridge Isotope Laboratories) for expression of 15 N/13 C isotopically labeled TM3/4; and 100 bc;g/mL ampicillin. Login to comment
74 Triple resonance NMR experiments for 15 N/13 C isotopically enriched V232D-TM3/4 115 mM PFO were carried out at 45 &#b0;C. Login to comment
100 Chemical shift assignment and secondary structure analysis of V232D-TM3/4 in PFO 1 H-15 N HSQC spectrum of 15 N-enriched V232D-TM3/4 in 115 mM PFO at 45 &#b0;C is shown in Fig. 2. Login to comment
105 Analysis of the triple resonance experiments HNCACB, CBCA(CO)NH, HNCO, (H)CC(CO)NH-TOCSY, and H(CC) (CO)NH-TOCSY of the 15 N,13 C-labeled V232D-TM3/4 enabled the assignments for 95% of the residues in the TM3/4 region, despite the fact that the analysis was complicated due to crosspeak overlapping typical of native helical TM proteins in detergent micelles. Login to comment
109 The secondary structure elements of V232D-TM3/4 thus obtained agree broadly with the formation of two helices in the micelle environment, separated by a turn. Login to comment
119 1 H-15 N HSQC spectrum of 15 N-labeled TM3/4 mutant V232D with amide chemical shift assignments. Login to comment
130 However, these NOEs were not sufficient per se for calculation of a defined tertiary structure for the V232D-TM3/4 hairpin. Login to comment
133 With the amide chemical shift assignments of V232D-TM3/4 in hand, and because these two constructs differ by only a single residue, any major differences in the 1 H-15 N HSQC spectra should be attributable to the modification of the electrostatic environment by the non-polar-to-polar residue mutation at position 232, as well as to any resulting conformational changes. Login to comment
135 Summary of the chemical shift deviation and the sequential, mid-range NOEs for V232D-TM3/4 in PFO micelles. Login to comment
143 Spectra were recorded in 115 mM PFO at 45 &#b0;C. Gly cross-peaks (shown in (b)) were assigned from the V232D spectrum (Fig. 2); other assignments shown were made where possible by comparison. Login to comment
145 As illustrated in the 'Gly box`, 1 H-15 N HSQC spectra of WT-TM3/ 4 (Fig. 4a) and V232D-TM3/4 (Fig. 4b) do possess some similarities. Login to comment
148 As assignments were available only for the V232D-TM3/4 construct, we could not assign the full complement of WT Gly resonances specifically; however, the observed shifts are suggestive of a change of the environment of these residues. Login to comment
152 From the analysis of the Gly cross-peaks (Fig. 4c), one observes that the N-H chemical shifts of G3, G23, and G194 remain identical to the WT, but obvious changes occurred for G213, G226, G228, G239 and G241 to the extent that we could not formally assign them by comparison To V232D-TM3/4. Login to comment
156 As shown in Fig. 4b and d, a striking similarity was observed between the 1 H-15 N HSQC 'Gly box` spectra of V232D- and Q207N/V232E-TM3/4, suggesting that the cross-peaks of their Gly residues can be correspondingly assigned, and that their global conformations likely correspond closely. Login to comment
162 In the present work, Q207N/V232E-TM3/4 migrates faster than TM3/4-V232D (Fig. 1b) although they each have one added negative charge vs. WT. Login to comment
175 We further observed that the turn (approximately S222 to G226) determined between TM3 and TM4 in the V232D-TM3/4 construct is shifted approximately six residues toward TM4 vs. the one predicted (W216 to Q220) in the original schematic presented for the wild type CFTR protein [6]. Login to comment
188 Similarly, G194 present at the N-terminal end of TM3, is not affected by the V232D mutation (Fig. 4b). Login to comment
191 These changes, in concert with results from migration rate data on SDS-PAGE, emphasize the fact that the interactions between helices differ in WT- vs. V232D-TM3/4. Login to comment
193 To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment
197 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
201 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
203 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100&#b0; of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
210 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment

[hide] Coarse-grained molecular dynamics simulations of m... J Struct Biol. 2007 Mar;157(3):593-605. Epub 2006 Oct 20. Bond PJ, Holyoake J, Ivetac A, Khalid S, Sansom MS
Coarse-grained molecular dynamics simulations of membrane proteins and peptides.
J Struct Biol. 2007 Mar;157(3):593-605. Epub 2006 Oct 20., [PMID:17116404]

Abstract [show]
Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.

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220 It is thought that the Q207 sidechain in M3 may form an interhelical H-bond with a CF-phenotypic mutant V232D in M4 (Therien et al., 2001). Login to comment
219 It is thought that the Q207 sidechain in M3 may form an interhelical H-bond with a CF-phenotypic mutant V232D in M4 (Therien et al., 2001). Login to comment

[hide] Diagnostic testing by CFTR gene mutation analysis ... J Mol Diagn. 2005 May;7(2):289-99. Schrijver I, Ramalingam S, Sankaran R, Swanson S, Dunlop CL, Keiles S, Moss RB, Oehlert J, Gardner P, Wassman ER, Kammesheidt A
Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
J Mol Diagn. 2005 May;7(2):289-99., [PMID:15858154]

Abstract [show]
Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.

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83 While ⌬F508 was homozygous in six subjects, seven other less common alleles (G542X, W1204X, R75X, V232D, E116K, T501A, 3272-26 AϾG) were also seen in the homozygous state. Login to comment
103 Table 1. Continued Mutations in 257 patients Allele counts of each mutation % of variant alleles (183) % of all alleles tested (514) R1070W 1 0.55 0.19 R1158X 1 0.55 0.19 R1438W 1 0.55 0.19 R334W 2 1.09 0.39 R352W 1 0.55 0.19 R553X 2 1.09 0.39 R668C 2 1.09 0.39 R74W 3 1.64 0.58 R75X 3 1.64 0.58 S1235R 2 1.09 0.39 S492F 2 1.09 0.39 S549N 1 0.55 0.19 S573CS573C 1 0.55 0.19 S945L 1 0.55 0.19 T351S 1 0.55 0.19 T501A 2 1.09 0.39 T604ST604S 1 0.55 0.19 V11I 1 0.55 0.19 V201 mol/L 1 0.55 0.19 V232D 2 1.09 0.39 V754 mol/L 1 0.55 0.19 W1089X 2 1.09 0.39 W1098C 1 0.55 0.19 W1204X 4 2.19 0.78 Y563N 1 0.55 0.19 Y913XY913X 1 0.55 0.19 85 different mutations 183 100.00 35.60 Novel variants are in boldface, mutations on the ACMG/ACOG panel are italicized. Login to comment
187 CFTR Sequence Variants Identified in Five Comprehensive CFTR Studies in US Hispanics CFTR mutations Alleles Relative mutation frequency (%) (of 317) deltaF508 123 38.80 3876delA 15 4.70 G542X 12 3.80 406 - 1GϾA 8 2.50 3849 ϩ 10kbCϾT 5 1.60 R75X 4 1.30 935delA 4 1.30 S549N 4 1.30 W1204X 4 1.30 R334W 4 1.30 2055del9ϾA 3 1 R74W 3 1 H199Y 3 1 L206W 3 1 663delT 3 1 3120 ϩ 1GϾA 3 1 L997F 3 1 I1027T 3 1 R1066C 3 1 W1089X 3 1 D1270N 3 1 2105del13insAGAAA 3 1 Q98R 2 Ͻ1 E116K 2 Ͻ1 I148T 2 Ͻ1 R668C 2 Ͻ1 P205S 2 Ͻ1 V232D 2 Ͻ1 S492F 2 Ͻ1 T501A 2 Ͻ1 1949del84 2 Ͻ1 Q890X 2 Ͻ1 3271delGG 2 Ͻ1 3272 - 26AϾG 2 Ͻ1 G1244E 2 Ͻ1 D1445N 2 Ͻ1 R553X 2 Ͻ1 E588V 2 Ͻ1 1717 - 8GϾA 2 Ͻ1 A1009T 2 Ͻ1 S1235R 2 Ͻ1 G85E 1 Ͻ1 296 ϩ 28AϾG 1 Ͻ1 406 - 6TϾC 1 Ͻ1 V11I 1 Ͻ1 Q179K 1 Ͻ1 V201 mol/L 1 Ͻ1 874insTACA 1 Ͻ1 I285F 1 Ͻ1 deltaF311 1 Ͻ1 F311L 1 Ͻ1 L320V 1 Ͻ1 T351S 1 Ͻ1 R352W 1 Ͻ1 1248 ϩ 1GϾA 1 Ͻ1 1249 - 29delAT 1 Ͻ1 1288insTA 1 Ͻ1 1341 ϩ 80GϾA 1 Ͻ1 1429del7 1 Ͻ1 1525 - 42GϾA 1 Ͻ1 P439S 1 Ͻ1 1717 - 1GϾA 1 Ͻ1 1811 ϩ 1GϾA 1 Ͻ1 deltaI507 1 Ͻ1 G551D 1 Ͻ1 A559T 1 Ͻ1 Y563N 1 Ͻ1 (Table continues) In this study, we used temporal temperature gradient gel electrophoresis (TTGE) and direct DNA sequencing to increase the sensitivity of mutation detection in U.S. Hispanics, and to determine whether additional mutations are recurrent. Login to comment
201 Comparison of Relative Frequencies of CFTR Sequence Variants in Comprehensive CFTR Studies in US and Mexican Hispanics This study % Orozco 2000 % US/ Mexican % deltaF508 28.96 54.48 43.72 G542X 3.83 8.28 5.19 406 - 1GϾA 3.28 2.07 2.38 W1204X 2.19 Ͻ1 1.08 R74W 1.64 Ͻ1 R75X 1.64 2.07 1.51 H199Y 1.64 Ͻ1 Ͻ1 L206W 1.64 Ͻ1 L997F 1.64 Ͻ1 I1027T 1.64 Ͻ1 2055del9ϾA 1.64 1.38 1.27 D1270N 1.64 Ͻ1 E116K 1.09 Ͻ1 V232D 1.09 Ͻ1 R334W 1.09 Ͻ1 S492F 1.09 Ͻ1 T501A 1.09 Ͻ1 R553X 1.09 Ͻ1 Ͻ1 E588V 1.09 Ͻ1 R668C 1.09 Ͻ1 Q890X 1.09 Ͻ1 W1089X 1.09 Ͻ1 S1235R 1.09 Ͻ1 D1445N 1.09 Ͻ1 3876delA 1.09 3.24 1717 - 8GϾA 1.09 Ͻ1 3272 - 26AϾG 1.09 Ͻ1 A1009T 1.09 Ͻ1 deltaI507 Ͻ1 3.45 1.30 S549N Ͻ1 3.45 1.95 G567A Ͻ1 Ͻ1 I148T 2.07 1.08 I506T 1.38 Ͻ1 N1303K 2.76 1.08 935delA 1.38 1.30 2183AAϾG 1.38 Ͻ1 3199del6 1.38 Ͻ1 3849 ϩ 10kbCϾT Ͻ1 1.30 ACMG/ACOG italicized. Login to comment

[hide] CFTR Cl- channel function in native human colon co... Gastroenterology. 2004 Oct;127(4):1085-95. Hirtz S, Gonska T, Seydewitz HH, Thomas J, Greiner P, Kuehr J, Brandis M, Eichler I, Rocha H, Lopes AI, Barreto C, Ramalho A, Amaral MD, Kunzelmann K, Mall M
CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis.
Gastroenterology. 2004 Oct;127(4):1085-95., [PMID:15480987]

Abstract [show]
BACKGROUND & AIMS: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl- channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. METHODS: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl- secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-DeltaF508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl- secretion with patients in whom Cl- secretion was absent. RESULTS: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset (P < 0.0001), higher frequency of PS (P < 0.0001), and less severe lung disease (P < 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of approximately 12%-54% of controls. CONCLUSIONS: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl- secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity.

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78 Relationship Between the CFTR Genotype and Cl- Channel Function in Native Rectal Epithelia CFTR genotype Number of individuals Sweat Cl-concentration (mmol/L)a cAMP-mediated response Carbachol-induced plateau response or maximal lumen-negative response Isc-cAMP (␮A/cm2) Cl- secretion (% of control) Isc-carbachol (␮A/cm2) Cl- secretion (% of control) Cl- secretion absent R1162X/Q552X 1 71 17.1 0 0.7 0 W1282X/3121-2AϾG 1 112 1.9 0 0.6 0 1898 ϩ 1G Ͼ T/1609delCA 2b 114, 118 25.4, 13.4 0, 0 0, 0.7 0, 0 ⌬F508/Q39X 2b 127, 129 2.6, 4.4 0, 0 1.7, 3.7 0, 0 ⌬F508/G542X 1 102 29.0 0 6.6 0 ⌬F508/R553X 3 112, 102, 109 13.1, 4.5, 23.8 0, 0, 0 1.5, 4.4, 1.0 0, 0, 0 ⌬F508/E585X 1 115 1.4 0 1.1 0 ⌬F508/Q637X 1 100 2.9 0 1.2 0 ⌬F508/Y1092X 1 119 0.0 0 -0.3 0 ⌬F508/120del23c 1 72 20.1 0 3.3 0 ⌬F508/182delT 1 116 10.8 0 5.2 0 ⌬F508/3905insT 2 88, 96 8.4, 5.6 0, 0 2.3, -1.1 0, 1 ⌬F508/V520F 1 68 1.2 0 1.7 0 ⌬F508/A561E 3 113, 146, 100 17.0, 17.0, 16.0 0, 0, 0 2.1, 1.5, 3.7 0, 0, 0 ⌬F508/R1066C 1 138 0.0 0 0.0 0 ⌬F508/N1303K 3 100, 117, 94 1.7, 4.1, 1.5 0, 0, 0 -0.6, 2.2, 0.8 0, 0, 0 A561E/A561E 2 101, 116 6.6, 2.0 0, 0 7.3, 3.3 0, 0 Residual Cl- secretiond G542X/I148N 1 75 -50.1 54 -22.2 12 1898 ϩ 3A Ͼ G/1898 ϩ 3A Ͼ G 1 82 -36.8 39 -12.9 7 ⌬F508/3272-26A Ͼ G 1 116 -17.8 19 -27.2 14 ⌬F508/S108F 1 118 -15.8 17 -12.3 7 ⌬F508/R117H 1 90 -35.9 38 -207.7 109 ⌬F508/Y161Cc 1 44 -35.1 37 -45.9 25 ⌬F508/P205S 1 80 -23.3 25 -10.4 5 ⌬F508/V232D 1 120 -16.9 18 -26.9 14 ⌬F508/R334W 1 92 -22.1 23 -21.1 11 ⌬F508/R334W 1 101 -24.5 26 -37.4 20 ⌬F508/T338I 1 73 -44.4 47 -79.4 42 ⌬F508/G576A 1 40 -16.9 18 -115.5 61 ⌬F508/I1234V 1 113 -13.6 15 -8.6 5 G576A/G85E 1 95 -26.1 28 -61.6 32 F1052V/M1137R 1 47 -36.7 39 -146.6 77 M1101K/M1101K 1 94 -11.1 12 -4.8 3 S1159F/S1159F 1 67 -47.9 51 -38.7 21 N1303K/R334W 1 91 -30.3 32 -47.7 25 NOTE. CFTR Cl- channel function was determined in rectal epithelia from Cl- secretory responses induced by IBMX/forskolin (Isc-cAMP) and after co-activation with carbachol (Isc-carbachol). Login to comment
101 Functional Classification and Protein Location of CFTR Mutations Mutation type Severe mutations (protein location) Mild mutations (protein location) Missense V520F, A561E (NBD1) G85E (MSD1, TM1) R1066C (MSD2, CL4) S108F, R117H (MSD1, EL1) N1303K (NBD2) I148N, Y161Ca (MSD1, CL1) P205S (MSD1, TM3) V232D (MSD1, TM4) R334W, T338I (MSD1, TM6) G576A (NBD1) I1234V (NBD2) F1052V, M1101K (MSD2, CL4) M1137R (MSD2, TM12) S1159F (pre-NBD2) Splice 1898 ϩ 1G Ͼ T (R domain) 1898 ϩ 3A Ͼ G (R domain) 3121-2A Ͼ G (MSD2, TM9) 3272-26A Ͼ G (MSD2, TM10) Single amino acid deletion ⌬F508 (NBD1) Nonsense Q39X (N-terminus) G542X, Q552X, R553X, E585X (NBD1) Q637X (R domain) Y1092X (MSD2, CL4) R1162X (pre-NBD2) W1282X (NBD2) Frameshift 120del23a 182delT (N-terminus) 1609delCA (NBD1) 3905insT (NBD2) NOTE. Severe mutation, Cl- secretion absent; mild mutation, residual cAMP-mediated Cl- secretion. Login to comment
125 According to our functional data, 3121-2AϾG, 1898ϩ1GϾT, and V520F constitute severe mutations, whereas 1898ϩ3AϾG, I148N, Y161C, V232D, T338I, I1234V, and S1159F confer residual CFTR Cl- channel function (Table 1). Login to comment

[hide] Non-native interhelical hydrogen bonds in the cyst... Biochemistry. 2004 Jun 29;43(25):8077-83. Choi MY, Cardarelli L, Therien AG, Deber CM
Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations.
Biochemistry. 2004 Jun 29;43(25):8077-83., [PMID:15209503]

Abstract [show]
Polar residues comprise about 15% of the transmembrane (TM) domains of proteins, where they can stabilize structure via native side chain-side chain interhelical hydrogen bonds between TM helices. However, non-native H-bonds may be implicated in disease states, through limiting protein dynamics during transport and/or misfolding the protein by inducing non-native rotational positions about TM helical axes. Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G(194)LALAHFVWIAPLQ(207)VALLMGLIWELLQASAFAGLGFLIV(232)LALFQ(237)AGLG(241)) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. In the present work, a library of 21 TM3/4 constructs was prepared, where Asp residues were placed individually at TM4 positions 221-241. Using gel shift assays-in which H-bond-linked hairpins (closed conformation) migrate faster than the elongated forms (open conformation)-we found that Q207 in TM3 is able to "capture" all 21 TM4 D mutations into measurable populations of interhelical H-bonds. A similar library of TM4 D mutants-but also containing Q207L-reverted to wild-type migration rates, confirming Q207 as the polar partner for TM4 D residues. In view of the broad capture range of Q207, these results emphasize the potential consequences to folding and dynamics of introducing polar mutations into the TM domains of membrane proteins in the vicinity of a native polar TM residue.

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2 Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G194LALAHFVWIAPLQ207VALLMGLIWELLQASAFAGLGFLIV232LALFQ237AGLG241) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. Login to comment
28 1 Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; TM, transmembrane; VD, V232D; wt, wild type; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; CD, circular dichroism; H-bond, hydrogen bond; RP-HPLC, reversed-phase high-performance liquid chromatography; LB, Luria-Bertani; MW, molecular weight; TFE, trifluoroethanol. Login to comment
33 With the use of this "gel-shift assay" on SDS-PAGE, we observed that the construct derived from the phenotypic mutant Val232-Aspswhich induces a mild form of CFsmigrated significantly faster than the wt construct (22), indicating that V232D adopts a more compact conformation likely imposed by a tight interaction between the mutant Asp 232 in TM helix 4 and its polar partner Gln 207 in TM helix 3 (22). Login to comment
81 Previous studies have shown that the V232D mutant migrates at a rate faster than that of the wt in NuPAGE MES gels (22). Login to comment
82 This migration pattern was shown to be associated with a more compact conformation of the V232D construct, imposed by a tight interaction between polar partners Asp 232 and Gln 207 of the V232D TM3/4 construct. Login to comment
89 Interestingly, mutants G228D through I231D, and L233D through F236D, have a more negative value of the percent of MW decrease relative to the wt than the CF-phenotypic mutant V232D (Figure 3b) (see the Discussion). Login to comment
90 The remaining Asp mutants toward either terminus of the TM4 helix migrated slower than the V232D construct on SDS-PAGE. Login to comment
97 V232D is the original CF-phenotypic mutant studied by Therien et al. (22). Login to comment
104 V232D is the CF-phenotypic mutant in TM4. Login to comment
111 Double asterisks denote values statistically different from that of the construct V232D on SDS-PAGE gel (p < 0.01). Login to comment
120 D mutants in mid-TM4, i.e., V232D/Q237L and L230D/Q237L, continued to migrate faster than the wt, but were clearly retarded vs their D counterparts. Login to comment
121 DISCUSSION In a helical hairpin construct derived from the first transmembrane domain of CFTR, the CF-phenotypic mutant V232D in TM4 was previously found to form a non-native side chain-side chain H-bond with the wild-type residue Q207 in TM3 (22) which was proposed to be responsible, in part, for the disruption of the functional CFTR protein. Login to comment
178 From the results (Figures 5 and 7), we found that double mutants where D is relatively distant from Q207 in TM3 (F224D/ Q237L, A238D/Q237L, and G239D/Q237L) returned to the wt migration rate, whereas the other double mutants (V232D/ Q237L and L230D/Q237L) migrated more slowly than the corresponding D mutants, but still significantly faster than the wt construct. Login to comment
182 The CF-phenotypic Mutant V232D Is Not the Optimal Position for Interhelix H-Bond Formation. Login to comment
183 As elaborated in Figure 3b, the CF-phenotypic mutant V232D does not form the tightest H-bond with Q207 in TM3 in comparison to the several X f D mutants on TM4. Login to comment
184 In fact, the migration rate of V232D is statistically slower vs those of D mutants at 229, 230, 231, and 233 (double asterisks, Figure 3b). Login to comment
186 As V232D is the CF-phenotypic mutant in this series, this result initially appeared counterintuitive. Login to comment
189 It is also interesting to note that several mutantssincluding S222D, G226D, V232D, and Q237Dsdisplay locally slower migration rates. Login to comment
191 One might speculate that this is the case for CF-phenotypic mutant V232D, and therefore, its capture by Q207 necessitates major reorientation of the wild-type TM4 axissand accordingly the greatest disruption of the native folded domain structure. Login to comment

[hide] Expression and purification of two hydrophobic dou... Protein Expr Purif. 2002 Jun;25(1):81-6. Therien AG, Glibowicka M, Deber CM
Expression and purification of two hydrophobic double-spanning membrane proteins derived from the cystic fibrosis transmembrane conductance regulator.
Protein Expr Purif. 2002 Jun;25(1):81-6., [PMID:12071702]

Abstract [show]
We describe a rapid method for the expression and purification of two hydrophobic protein constructs derived from the membrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein associated with cystic fibrosis. The proteins have no sequence homology but are both predicted to contain two membrane-spanning segments. The protocol involves the expression of CFTR constructs as thioredoxin fusion proteins in Escherichia coli, followed by partial purification by affinity chromatography, removal of the thioredoxin moiety by proteolytic cleavage in the presence of detergent, and final purification by reversed-phase high-performance liquid chromatography. The method yields milligram amounts of purified constructs that spontaneously insert into detergent micelles in alpha-helical conformation. We predict that this protocol will be applicable to a variety of proteins of similar size and hydrophobicity.

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91 These constructs have proven valuable in studies of the secondary and tertiary structure of wild-type and mutant CFTR, as in our recent investigation of the structural consequences of the cystic fibrosis-phenotypic mutant V232D in TM 3-4 (9). Login to comment

[hide] Spectrum of CFTR mutations in cystic fibrosis and ... Hum Mutat. 2000;16(2):143-56. Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, Girodon E, Cazeneuve C, Bienvenu T, Lalau G, Dumur V, Feldmann D, Bieth E, Blayau M, Clavel C, Creveaux I, Malinge MC, Monnier N, Malzac P, Mittre H, Chomel JC, Bonnefont JP, Iron A, Chery M, Georges MD
Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.
Hum Mutat. 2000;16(2):143-56., [PMID:10923036]

Abstract [show]
We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

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108 g D44G, 300delA, W57X, 405+1G>A, D110H, E116K, 541del4, 542del7, L137R, 621+2T>G, I175V, H199R, H199Y, C225X, V232D, Q290X, E292X, G314V, T338I, 1221delCT, W401X, Q452P, I502T, 1716+2T>C, G544S, R560S, A561E, V562I, Y569D, 1898+3A>G, 1898+5G>A, G628R(G>A), 2143delT, G673X, R851X, Q890X, S977F, 3129del4, 3154delG, 3271+1G>A, G1061R, R1066L, R1070W, 3601-17T>C, S1196X, 3732delA, G1249R, 3898insC, 4374+1G>A, del25kb. Login to comment
152 Twenty-four non F508del mutations were found associated with the 9T allele: 394delTT, L90S, D110H, R117G, 621+1G>T, V232D, A455E, G542X, R851L, T908N, 2789+5G>A, 2896insAG, H939R, 3007delG, I980K, I1027T, R1066H, A1067T, D1154G, 3737delA, R74W+D1270N, N1303I, N1303K, D1377H. Login to comment

[hide] High heterogeneity for cystic fibrosis in Spanish ... Hum Genet. 1997 Dec;101(3):365-70. Casals T, Ramos MD, Gimenez J, Larriba S, Nunes V, Estivill X
High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes.
Hum Genet. 1997 Dec;101(3):365-70., [PMID:9439669]

Abstract [show]
We have analyzed 640 Spanish cystic fibrosis (CF) families for mutations in the CFTR gene by direct mutation analysis, microsatellite haplotypes, denaturing gradient gel electrophoresis, single-strand conformation analysis and direct sequencing. Seventy-five mutations account for 90.2% of CF chromosomes. Among these we have detected seven novel CFTR mutations, including four missense (G85V, T582R, R851L and F1074L), two nonsense (E692X and Q1281X) and one splice site mutation (711+3A-->T). Three variants, two in intronic regions (406-112A/T and 3850-129T/C) and one in the coding region (741C/T) were also identified. Mutations G85V, T582R, R851L, E692X and Q1281X are severe, with lung and pancreatic involvement; 711+3A-->T could be responsible for a pancreatic sufficiency/insufficiency variable phenotype; and F1074L was associated with a mild phenotype. These data demonstrate the highest molecular heterogeneity reported so far in CF, indicating that a wide mutation screening is necessary to characterize 90% of the Spanish CF alleles.

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33 Eight mutations have frequencies 366 Table 1 Seventy-five CFTR mutations identified in 640 Spanish families with cystic fibrosis (CF) Mutation Exon/intron CF alleles % ∆F508 E.10 681 53.20 G542X E.11 108 8.43 N1303K E.21 34 2.65 1811+1.6kbA→Ga I.11 24 1.87 711+1G→T I.5 22 1.71 R1162Xa E.19 21 1.64 R334Wa E.7 21 1.64 R1066C E.17b 14 1.09 1609delCAa E.10 13 1.01 Q890X E.15 13 1.01 G85E E.3 12 0.94 712-1G→Ta I.5 11 0.86 2789+5G→A I.14b 11 0.86 ∆I507 E.10 10 0.78 W1282X E.20 10 0.78 2869insGa E.15 9 0.70 L206W E.6a 7 0.54 R709X E.13 7 0.54 621+1G→T I.4 6 0.47 3272-26A→G I.17a 6 0.47 R347H E.7 5 0.39 2183AA→G E.13 5 0.39 K710X E.13 5 0.39 2176insC E.13 5 0.39 3849+10kbC→T I.19 5 0.39 P205Sa E.6a 4 0.31 1078delT E.7 4 0.31 R553X E.11 4 0.31 G551D E.11 4 0.31 1812-1G→Aa I.11 4 0.31 CFdel#1a E.4-7/11-18 4 0.31 V232D E.6a 3 0.23 936delTAa E.6b 3 0.23 1717-8G→A I.10 3 0.23 1949del84 E.13 3 0.23 W1089X E.17b 3 0.23 R347P E.7 3 0.23 del E.3a E.3 2 0.16 R117H E.4 2 0.16 L558S E.11 2 0.16 A561E E.12 2 0.16 2603delT E.13 2 0.16 Y1092X E.17b 2 0.16 Q1100Pa E.17b 2 0.16 M1101K E.17b 2 0.16 delE.19a E.19 2 0.16 G1244E E.20 2 0.16 P5La E.1 1 0.08 Q30Xa E.2 1 0.08 G85Va E.3 1 0.08 E92Ka E.4 1 0.08 A120Ta E.4 1 0.08 I148T E.4 1 0.08 711+3A→Ta I.5 1 0.08 H199Y E.6a 1 0.08 875+1G→A I.6a 1 0.08 Table 1 (continued) Mutation Exon/intron CF alleles % 1717-1G→A I.10 1 0.08 L571S E.12 1 0.08 T582Ra E.12 1 0.08 E585X E.12 1 0.08 1898+3A→G I.12 1 0.08 G673X E.13 1 0.08 E692Xa E.13 1 0.08 R851X E.14a 1 0.08 R851La E.14a 1 0.08 A1006E E.17a 1 0.08 L1065Ra E.17b 1 0.08 F1074La E.17b 1 0.08 R1158X E.19 1 0.08 3667del4a E.19 1 0.08 3860ins31a E.20 1 0.08 3905insT E.20 1 0.08 4005+1G→A I.20 1 0.08 Q1281Xa E.20 1 0.08 Q1313X E.21 1 0.08 Known mutations (75) 1155 90.23 Unknown mutations 125 9.77 a Mutations discovered by the CF group of the Medical and Molecular Genetics Centre - IRO, Barcelona, Spain that range between 0.5% and 0.9%, representing 6.0% of the CF chromosomes. Login to comment

[hide] CFTR gene mutations and asthma in the Norwegian En... Respir Med. 2006 Dec;100(12):2121-8. Epub 2006 May 5. Munthe-Kaas MC, Lodrup Carlsen KC, Carlsen KH, Skinningsrud B, Haland G, Devulapalli CS, Pettersen M, Eiklid K
CFTR gene mutations and asthma in the Norwegian Environment and Childhood Asthma study.
Respir Med. 2006 Dec;100(12):2121-8. Epub 2006 May 5., [PMID:16678395]

Abstract [show]
BACKGROUND: Several candidate genes have been implicated in the etiology of asthma, including the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR gene result in derangements of mucociliary clearance. Homozygotes for CFTR mutations develop cystic fibrosis (CF), a disorder characterized mainly by lung and pancreas disease. OBJECTIVE: To investigate whether there was an increased frequency of CFTR mutations in asthma patients. METHODS: Seven hundred and three subjects aged 10-11 years from the environment and childhood asthma (ECA) study were included in the present study. Possible associations between asthma, reduced lung function, bronchial hyperresponsiveness (BHR), and increased or decreased nitrogen oxide (NO) levels (based on structural parental interview, spirometry, PD20 methacholine challenge test and exhaled NO measurements), and the five most common CFTR mutations in Norway (DeltaF508, R117H, R117C, 4005+2T-->C, 394delTT), the modulating polymorphisms IVS8(TG)mTn and the IVS8-5T were investigated. RESULTS: No association were found between asthma, reduced lung function, BHR or exhaled NO levels and CF heterozygosity. However, the IVS8(TG)11T7 haplotype was associated with normal lung function. CONCLUSIONS: Our results do not support the hypothesis that CFTR mutations or polymorphisms play a role in the pathogenesis of asthma in children. However, the distribution of Tn(TG)m haplotypes differed between individuals with reduced lung function and individuals with normal lung function.

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25 CFTR mutation Alleles (%) F508del 184 (62.2) R117C 12 (4.1) R117H 12 394delTT 11 (3.8) 4005+2T-C 11 G551D 6 (2.0) 3659delC 5 (1.7) E60X 4 (1.4) V232D 4 1525-2A-G 3 (1.0) N1303K 3 G542X 2 (0.7) E279X 2 R75X 2 S912X 2 E116X 1 (0.3) L295Q 1 R347L 1 Q493X 1 I506L 1 I507del 1 R553X 1 G576A 1 621-1G-T 1 2183AA-G 1 S945L 1 R1162X 1 I1234V 1 3849+10 kbC-T 1 W1282X 1 Unknown 18 (6.5) Total alleles 296 (100%) Mutations detected with OLA31 m kit-74%. 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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42 Some have concentrated in the search of specific mutations that are Table 1 Mutations found in the Latin American CF patients Exon 1 p.L6VÌe; Exon 3 p.W57X, p.R75X, p.G85E Exon 4 p.R117H Exon 6a p.H199Y, p.V201M, p.L206W, p.Q220X, p.V232D, c.846delTÌe; Exon 6b p.Y275XÌe;, c.935delA Exon 7 p.R334W, p.R347P, p.Y362XÌe;, c.1078delT, c.1215delG Exon 8 c.1323_1324insAÌe; Exon 9 c.1460_1461delATÌe;, c.1353_1354insTÌe;,# Exon 10 p.I506T, p.I507del, p.F508del Exon 11 p.G542X, p.S549N, p.S549R, p.G551D, p.G551S, p.R553X, p.L558S, p.A559T, c.1782delA Exon 12 p.S589I Exon 13 p.H609RÌe;, p.P750L, p.V754M, c.1924_1930del, c.2055_2063del, c.2183AA NG;c.2184delA, c.2184delA, c.2185_2186insC, c.2347delG, c.2566_2567insTÌe;, c.2594_2595delGTÌe; Exon 14a p.R851L, c.2686_2687insTÌe; Exon 15 c.2869_2870insG Exon 16 c.3120+1GNA Exon 17a p.I1027T, c.3171delC, c.3199_3204del Exon 17b p.G1061R, p.R1066C, p.W1069X#, p.W1089X, p.Y1092X, p.W1098CÌe; Exon 19 p.R1162X, p.W1204X, p.Q1238X, c.3617_3618delGAÌe;#, c.3659delC Exon 20 p.W1282X, p.R1283M Exon 21 p.N1303K, c.4016_4017insT Exon 22 c.4160_4161insGGGGÌe; 5' flanking c.-834GNT Intron 2 c.297-1GNAÌe;, c.297-2ANG Intron 3 c.406-1GNA Intron 4 c.621+1GNT Intron 5 c.711+1GNT Intron 8 c.IVS8-5T Intron 10 c.1716GNA, c.1717-1GNA Intron 11 c.1811+1.6KbANG, c.1812-1GNA Intron 12 c.1898+1GNA, c.1898+3ANG Intron 14 c.2789+2_2789+3insA, c.2789+5GNA Intron 17a c.3272-26ANG Intron 17b c.3500-2ANGÌe; Intron 19 c.3849+1GNA, c.3849+10KbCNT Intron 20 c.4005+1GNA, c.4005-1GNA# Mutations are listed according to their position in the gene. Login to comment
46 of chromosomes analysed p.F508del p.G542X p.W1282X p.N1303K p.R1162X p.L6VÌe; p.W57X p.R75X p.G85E p.R117H p.H199Y p.V201M p.L206W p.Q220X p.V232D p.Y275XÌe; p.R334W p.R347P p.Y362XÌe; p.I506T Argentina 98 61 440 258 18 12 12 2 1 1 3 1 5 1 310 181 20 7 5 5 7 0 5 0 222 135 15 7 5 1 26 14 2 1 1 150 88 6 6 1 2 3 Subtotal and frequency (%) 1246 100 737 59.15 61 4.90 27 2.17 28 2.25 9 0.72 1 0.08 1 0.08 13 1.04 1 0.08 13 1.04 1 0.08 Brazil 468 221 26 11 74 38 2 1 320 155 28 3 8 8 4 1 2 1 1 8 122 62 120 38 10 3 148 38 4 0 0 48 15 154 75 5 1 0 2 0 386 154 24 6 10 17 9 0 10 1 18 4 0 0 2 0 0 0 0 Subtotal and frequency (%) 1858 100 800 43.06 99 5.33 11 0.59 34 1.83 25 1.35 13 0.70 1 0.05 2 0.11 1 0.05 1 0.05 20 1.07 1 0.05 Chile 72 21 36 11 3 0 44 22 4 3 1 1 100 45 7 5 0 2 0 2 0 Subtotal and frequency (%) 252 100 99 41.28 14 5.55 8 3.17 3 1.19 3 1.19 Colombia 184 77 7 2 1 2 1 34 13 2 1 1 Subtotal and frequency (%) 218 100 90 41.28 9 4.13 3 1.38 2 0.92 2 0.92 1 0.46 Costa Rica Frequency (%) 48 100 11 22.91 12 25.00 0 0 0 0 0 Cuba Frequency (%) 144 100 49 34.03 Ecuador 32 11 1 50 16 2 2 20 5 0 0 0 Subtotal and frequency (%) 102 100 32 31.37 2 1.96 1 0.98 2 1.96 Mexico 194 79 12 4 3 1 1 1 2 80 36 4 1 Subtotal and frequency (%) 274 100 115 41.97 16 5.84 5 1.82 3 1.09 1 0.36 1 0.36 1 0.36 2 0.73 Uruguay Frequency (%) 76 100 43 56.58 6 7.89 2 2.63 3 3.95 3 3.95 2 2.63 Venezuela 54 16 2 82 41 Subtotal and frequency (%) 136 100 57 41.91 2 1.47 Total 4354 2033 221 49 72 42 1 1 3 32 1 1 1 2 1 1 1 39 1 1 2 Frequency (%) 100 46.69 5.08 1.13 1.65 0.96 0.02 0.02 0.07 0.73 0.02 0.02 0.02 0.05 0.02 0.02 0.02 0.90 0.02 0.02 0.05 The five most frequent mutations are shown on the left-hand side, followed by the rest of the mutations in 5'-3' and exon-intron order. Login to comment
98 As an example, in the case of Argentina and Uruguay, the p.F508del mutation shows the highest frequencies (59% and Table 5 Mutations with frequencies less than 0.1% Panel A Mutation Number of chromosomes % Country p.R75X 3 0.07 Mexico c.W1089X 3 0.07 Argentina, Brazil c.406-1GNA 3 0.07 Mexico c.1898+1GNA 3 0.07 Argentina, Brazil c.2686_2687insTÌe; 3 0.07 Argentina, Brazil p.L206W 2 0.05 Brazil p.I506T 2 0.05 Mexico p.S589I 2 0.05 Argentina c.711+1GNT 2 0.05 Argentina c.935delA 2 0.05 Mexico c.2055_2063del 2 0.05 Mexico c.2347delG 2 0.05 Brazil c.2566_2567insTÌe; 2 0.05 Argentina c.2789+2_2789+3insA 2 0.05 Argentina c.3199_3204del 2 0.05 Mexico c.3272-26ANG 2 0.05 Argentina c.4016_4017insT 2 0.05 Argentina Panel B Mutation N % each Country p.L6VÌe;, p.W57X, p.Q220X, p.Y362XÌe;, p.I1027T, p.G1061R, p.R1283M, c.297-2ANG, c.1353_1354insTÌe;, c.1460_1461delATÌe;, c.1782delA, c.1898+3ANG, c.2184delA, c.2594_2595delGTÌe;, c.2869_2870insG, c.4005Ìe;1GNA, c.4005-1GNA# 17 0.02 Argentina p.R117H, p.H199Y, p.G551S, p.L558S, p.P750L, p.V754M, p.W1069X#, p.W1098CÌe;, p.W1204X, c.297-1GNAÌe;, c.846delTÌe;, c.1078delT, c.1716GNA, c.1924_1930del, c.4160_4161insGGGGÌe; 15 0.02 Mexico p.V201M, p.V232D, p.Y275XÌe;, p.R347P, p.R851L, p.Q1238X, c.3171delC, c.3617_3618delGAÌe;# 8 0.02 Brazil p.A559T, p.H609RÌe;, c.1215delG, c.1323_1324insAÌe;, c.2185_2186insC, c.3500-2ANGÌe;, c.3849+1GNA, 7 0.02 Colombia c.-834GNT 1 0.02 Uruguay The upper part (Panel A) shows the mutations found in more than one patient, whereas the lower part (Panel B) of the table shows all the mutations that are present only once in each country. Login to comment

[hide] Functional Rescue of F508del-CFTR Using Small Mole... Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012. Molinski S, Eckford PD, Pasyk S, Ahmadi S, Chin S, Bear CE
Functional Rescue of F508del-CFTR Using Small Molecule Correctors.
Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012., [PMID:23055971]

Abstract [show]
High-throughput screens for small molecules that are effective in "correcting" the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

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172 Corr-4a has a mild correction effect of the F508del-CFTR (effective in the low &#b5;M range), and a nearly complete correction effect on the rare mutant V232D-CFTR (Caldwell et al., 2011). Login to comment

[hide] Corrector VX-809 stabilizes the first transmembran... Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5. Loo TW, Bartlett MC, Clarke DM
Corrector VX-809 stabilizes the first transmembrane domain of CFTR.
Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5., [PMID:23835419]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.

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175 By contrast, other CFTR mutants defective in processing such as V232D and H1085R have half-lives similar to wild-type CFTR after rescue [14]. Login to comment
176 To test if other CFTR processing mutants could be rescued by TM6 mutations, arginine mutations were introduced at positions Ile338, Ser341, Ile344, Val345, Met348, and Thr351 of the V232D and H1085R processing mutants. Login to comment
177 None of the introduced arginines were found to promote maturation of V232D or H1085R. Login to comment
178 Examples of typical results obtained with the I344R or M348R mutations introduced into V232D or H1085R are shown in Fig. 5B. Login to comment
179 It was possible that the processing mutations in the TMDs (V232D (TMD1) or H1085R (TMD2)) cannot be rescued by a direct rescue approach using suppressor mutations. Login to comment
180 To test if the V232D or H1085R mutants could be rescued by suppressor mutations in other domains, suppressor mutations in NBD1 (I539T), the NBD1-TMD2 interface (V510D), or TMD2 (R1070W) (only V232D) locations were introduced into the mutants. Login to comment
196 RDR1 differs from VX-809 however, as we find that it does not rescue processing mutations in other domains such as V232D (TMD1) or H1085R (TMD2) (unpublished observations). Login to comment
220 None of the arginines introduced into CFTR processing mutants DF508, V232D, or H1085R promoted maturation. Login to comment
236 (B) Extracts of cells expressing wild-type CFTR or mutants V232D or H1085R with or without the I344R or M348R mutations were subjected to immunoblot analysis. Login to comment
237 (C) Extracts of cells expressing processing mutants DF508, V232D, or H1085R with or without the V510D, I539T, or R1070W suppressor mutations were subjected to immunoblot analysis. Login to comment

[hide] Bithiazole correctors rescue CFTR mutants by two d... Biochemistry. 2013 Aug 6;52(31):5161-3. doi: 10.1021/bi4008758. Epub 2013 Jul 22. Loo TW, Bartlett MC, Clarke DM
Bithiazole correctors rescue CFTR mutants by two different mechanisms.
Biochemistry. 2013 Aug 6;52(31):5161-3. doi: 10.1021/bi4008758. Epub 2013 Jul 22., [PMID:23865422]

Abstract [show]
Better correctors are needed to repair cystic fibrosis transmembrane conductance regulator (CFTR) processing mutants that cause cystic fibrosis. Determining where the correctors bind to CFTR would aid in the development of new correctors. A recent study reported that the second nucleotide-binding domain (NBD2) was involved in binding of bithiazole correctors. Here, we show that bithiazole correctors could also rescue CFTR mutants that lacked NBD2. These results suggest that bithiazoles rescue CFTR mutants by two different mechanisms.

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17 VX-809 appears to rescue CFTR processing mutants through direct interactions with TMD1.12,18 Bithiazoles, however, appear to rescue CFTR processing mutants by a different mechanism because bithiazoles have an additive effect on maturation when used in combination with VX-809.15 Identification of the bithiazole rescue site in CFTR is controversial as there is evidence that these compounds bind to NBD218 or to the TMDs.9 Evidence that bithiazoles interact with the TMDs was that 4a inhibited cross-linking between cysteines introduced into TMD1 and TMD219 and that 4a appears to stabilize TMD2.20 In addition, it was found that bithiazoles enhanced core glycosylation of a truncation mutant that contained only the TMDs.19 By contrast, it was recently reported that bithiazoles must interact with NBD2 because ƊF508 CFTR missing NBD2 was not rescued with bithiazoles and in silico docking predicted the presence of a bithiazole-binding site in NBD2.18 To test whether rescue of other CFTR processing mutants with bithiazoles (see Figure 1 for structures) was mediated by the TMDs or NBD2, we tested if bithiazoles could rescue full-length or ƊNBD2 CFTR mutants that had processing mutations in the TMDs such as G126D in TM2,21 V232D in Received: July 3, 2013 Revised: July 17, 2013 Published: July 18, 2013 Rapid Report pubs.acs.org/biochemistry (c) 2013 American Chemical Society dx.doi.org/10.1021/bi4008758 | Biochemistry 2013, 52, 5161-5163 Terms of Use TM4,20 F337R in TM6,12 and S1141R in TM12 (see Figure 2A). Login to comment
18 Mutants G126D and V232D are naturally occurring CF mutants, whereas F337R and S1141R mutants were constructed to map the orientation of the TM segments.12 The mutants were expressed for 18 h with or without VX-809 or the 4a, 4d, or 15Jf bithiazole correctors. Login to comment
20 Figure 2B shows that full-length and ƊNBD2 forms of G126D, V232D, and S1141R could be efficiently rescued with all the correctors such that the mature protein was the major product (Figure 2C,D). Login to comment
25 The second bithiazole rescue mechanism appears to involve the TMDs because removal of NBD2 had little effect on bithiazole rescue of mutants G126D, V232D, and S1141R and mutation F337R in TM6 inhibited rescue of full-length CFTR with bithiazoles. Login to comment

[hide] VX-809 corrects folding defects in cystic fibrosis... Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7. Ren HY, Grove DE, De La Rosa O, Houck SA, Sopha P, Van Goor F, Hoffman BJ, Cyr DM
VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7., [PMID:23924900]

Abstract [show]
Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

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60 There are several CF-associated mutations in MSD1 that cause defects in CFTR processing and function: N-terminal tail (E56K and P67L), TM1 (E92K), TM2 (L206W), and TM4 (V232D) (Figure 4, A-E). Login to comment
62 In contrast, 5 bc;M VX-809 only partially restored folding and function to E92K and V232D (Figure 4, A and E). Login to comment
65 V232D-CFTR was the least responsive to VX-809, and higher concentrations of the compound did not restore function beyond the 25% of normal CFTR observed in the presence of 5 bc;M VX-809. Login to comment
107 V232D-CFTR biogenesis and function to normal levels (Caldwell et al., 2011), these data suggest that correctors such as VX-809 and Corr4a can act to selectively suppress folding defects in CFTR caused by different disease-related mutations in MSD1. Login to comment

[hide] The cystic fibrosis V232D mutation inhibits CFTR m... Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9. Loo TW, Clarke DM
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9., [PMID:24412276]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Repair of CFTR mutants requires an understanding of the mechanisms of misfolding caused by processing mutations. Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Here, we performed mutational analysis to test for Asp232/Gln207 interactions in full-length CFTR. The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. We report that only Val232 mutations to charged amino acids severely blocked CFTR maturation. The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Mutation of Val232 to large nonpolar residues (Leu, Phe) had little effect. The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones.

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0 The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds Tip W. Loo a,b , David M. Clarke *,a,b a Department of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8 b Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 1. Login to comment
15 It appears that processing Biochemical Pharmacology 88 (2014) 46-57 A R T I C L E I N F O Article history: Received 11 November 2013 Received in revised form 30 December 2013 Accepted 31 December 2013 Available online 9 January 2014 Keywords: CFTR V232D processing mutation Cystic fibrosis Processing mutations Packing of TM segments Correctors A B S T R A C T Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Login to comment
17 Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Login to comment
19 The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. Login to comment
21 The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Login to comment
23 The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. Login to comment
24 These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Login to comment
25 Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Login to comment
26 Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones. Login to comment
37 Although P-gp studies have demonstrated that formation of non-native hydrogen bonds between TM segments promotes folding [22,23], a study of the V232D (TM4) CF processing mutation predicted that hydrogen bonds in CFTR TM segments would have the opposite effect and inhibit folding [24]. Login to comment
38 Therien et al. [24] used helix-loop-helix fragments of the CFTR TM3/TM4 region to study the V232D (TM4) mutation and concluded that the aspartate residue formed a non-native hydrogen bond with Gln207 in TM3 to disrupt TM3/TM4 interactions. Login to comment
39 Based on the conclusion that V232D inhibited maturation by forming a non-native hydrogen bond with Gln207, it was proposed that over 75 other CF mutations in the TMDs may also inhibit maturation through non-native hydrogen bond formation between helices [24]. Login to comment
41 In this study, we used a mutational approach to test if the mechanism of the V232D processing defect was due to formation of a non-native hydrogen bond with Gln207 in full-length CFTR. Login to comment
42 The rationale was that the V232N mutation should be just as effective as V232D to inhibit CFTR maturation and mutation of Gln207 to nonpolar residues should rescue the V232D mutant if the processing defect was caused by Asp232/Gln207 hydrogen bonds. Login to comment
70 Disulfide cross-linking analysis To compare the effect of V232D on packing of the TM segments, double cysteine mutants were generated for disulfide cross-linking analysis. Login to comment
80 Results 3.1. Replacement of Gln207 in TM3 with nonpolar residues does not rescue V232D CFTR The 1480 amino acids of CFTR are organized into two TMDs, each containing six TM segments, two NBDs, and an R domain [3] (Fig. 1A). Login to comment
85 The V232D mutation is a useful model system to examine the effects of processing mutations and correctors on CFTR maturation because it severely inhibits maturation such that CFTR accumulates as immature core-glycosylated protein in the ER. Login to comment
86 The V232D mutant however, can be efficiently rescued with correctors to yield mature protein at the cell surface with activity similar to the wild-type protein [6]. Login to comment
87 A previous study [24] on TM3/TM4 helix-loop-helix fragments suggested that V232D (TM4) caused CFTR misfolding by forming a non-native hydrogen bond with Gln207 (TM3). Login to comment
88 It was reported that the V232D mutation caused a TM3/TM4 helix-loop-helix fragment to migrate with increased mobility on SDS-PAGE gels relative to a wild-type fragment unless Gln207 was replaced by an amino acid such as leucine that did not form hydrogen bonds. Login to comment
90 If V232D inhibited CFTR maturation by forming a non-native hydrogen bond with Gln207, then we predicted that V232D would no longer inhibit maturation if Gln207 were replaced with an amino acid that could not form a hydrogen bond. Login to comment
96 Accordingly, Gln207 in the V232D mutant was replaced with a small (alanine) or large (leucine) amino acid that does not form hydrogen bonds. Login to comment
97 The Q207L mutation had previously been reported to abolish hydrogen bond interactions with V232D in TM3/4 helix-loop-helix fragments [24]. Login to comment
100 The V232D, V232D/Q207A, V232D/Q207L, and V232D/Q207C mutants were then expressed in the presence or absence of corrector VX-809 to test for maturation. Login to comment
101 Corrector VX-809 was used because it efficiently rescues V232D CFTR [33]. Login to comment
102 Whole cell SDS extracts were then subjected to immunoblot analysis. Fig. 1B shows that V232D severely inhibits maturation as only the 170 kDa immature protein was detected. Login to comment
103 Mature CFTR however, was observed when V232D was expressed in the presence of VX-809. Login to comment
104 By contrast, none of the Gln207 mutations rescued V232D CFTR (Fig. 1B and C) as no mature CFTR was observed when mutants V232D/Q207A, V232D/Q207L, or V232D/Q207C were expressed in the presence or absence of VX-809 (Fig. 1B). Login to comment
105 These results suggest that the Q207X mutations likely had deleterious effects on mutant V232D, as the mutants could no longer be rescued with VX-809. Login to comment
107 Mutations to Gln207 inhibit CFTR maturation Since V232D but not mutants V232D/Q207A, V232D/Q207L, or V232D/Q207C could be rescued with VX-809 (Fig. 1B), we tested if maturation of CFTR was also sensitive to changes to Gln207 in a wild-type background. Login to comment
109 Mutants V232D/Q207A, L, or C do not mature. Login to comment
112 (B) Whole cell extracts of cells expressing V232D or V232D/Q207A, L, or C mutants in the absence () or presence (+) of VX-809 were subjected to immunoblot analysis. Login to comment
116 An asterisk indicates significant (P < 0.05) inhibition when compared to V232D CFTR expressed in the presence of VX-809. Login to comment
125 The results suggest that Gln207 mutations might have an additive or synergistic effect on the V232D mutation. Login to comment
127 Inhibition of CFTR maturation correlates with the polarity of a Val232 mutation If V232D inhibits CFTR maturation by forming a non-native hydrogen bond with Gln207, then it would be expected that V232N and V232Q mutations would also severely inhibit maturation since asparagine and glutamine are structurally similar to aspartate and their amide groups can accept or donate two hydrogen bonds. Login to comment
130 The mutants were expressed in HEK 293 cells and whole cell extracts subjected to immunoblot analysis. Fig. 3A and B shows that the expression of mutant V232E resembled that of V232D as it significantly inhibited maturation by about 40-fold (about 2% of the CFTR protein was present as the mature protein when compared to 80% for wild-type CFTR (Fig. 3)). Login to comment
131 By contrast, the amount of mature protein was about 20-fold higher (about 40%) for mutants V232N and V232Q compared to V232D or V232E (Fig. 3). Login to comment
132 These results suggested that charge rather than non-native hydrogen bonds might be responsible for the severe reduction in CFTR maturation caused by the V232D or V232E mutations. Login to comment
157 The results suggest that the V232D mutation may severely inhibit maturation because it introduces charge into a relatively hydrophobic region in the TM segments. Login to comment
159 3.4. Rescue of Val232 processing mutants with correctors The V232D CFTR could be efficiently rescued with either VX-809 [19] or bithiazoles [33]. Login to comment
169 For example, V510D promotes maturation of mutants with processing mutations in TMD1 (V232D), TMD2 (H1085R) and NBD1 (DF508) whereas other suppressors such as I539T and R1070W promote maturation of DF508 CFTR but not mutants V232D or H1085R [19]. Login to comment
178 The properties of the V232E mutant are likely to be very similar to the V232D mutant as the V510D suppressor mutation [19] could also rescue the V232D mutant. Login to comment
195 The L305R(TM5) P-gp mutant resembles CFTR mutants V232D, V232E, and V232K The characteristics of the V232D/Q207X (Fig. 1) and V232X (Fig. 3) mutants suggest that the mechanism of the V232D mutation involves disruption of a hydrophobic pocket between TM segments. Login to comment
204 It appears that the CFTR V232D, V232E, and V232K processing mutationsresemble P-gp mutantsthatcontain acharged residueata hydrophobic interface between adjacent TM segments because they can be efficiently rescued (with VX-809). Login to comment
241 We predict that the V232D CFTR mutant might be similar to L305R P-gp because it involves insertion of a charged residue into a hydrophobic pocket. Login to comment
258 Cross-linking analysis suggests that V232D causes incomplete packing of the TM segments Previous studies on processing mutations in NBD1 (DF508) or in the fourth intracellular loop connecting TM segments 10 and 11 (Q1071P or H1085R) showed that they trapped CFTR at an early folding step resulting in incomplete packing of the TM segments [17,50]. Login to comment
263 To test if V232D trapped CFTR in a conformation with incomplete packing of the TM segments, the mutation was introduced into a Cys-less CFTR containing W356C(TM6) and W1145C(TM12). Login to comment
264 Cys-less CFTR, mutants W356C/W1145C and V232D/W356C/W1145C were expressed in HEK 293 cells in the absence or presence of 5 mM VX-809. Membranes were prepared Fig. 8. Login to comment
271 No cross-linked product was detected in Cys-less CFTR or in mutant V232D/W356C/W1145C when expressed in the absence of corrector VX-809 (Fig. 10A and B). Login to comment
272 Expression of mutant V232D/W356C/W1145C in the presence of corrector however, promoted maturation of the protein such it could be efficiently cross-linked with either M8M or BMH (about 80-85% cross-linked product; Fig. 10B). Login to comment
273 These results suggest that the V232D mutation traps CFTR in a partially folded conformation with incomplete packing of the TM segments that can be corrected with VX-809. Login to comment
274 In summary, the results suggest that: (1) the mechanism of how V232D causes protein misfolding is unlikely to involve non-native hydrogen bond interactions with Gln207 because V232N yielded about 20-fold more mature CFTR than V232D; (2) it appears that the mechanism of protein misfolding by V232D mutation involves disruption of a hydrophobic pocket since the hydrophobicity of the substituted amino acid at position 232 correlated with the amount of mature product and the ability to correct the defects with correctors or the V510D suppressor mutation; (3) the V232D mutation traps CFTR as a partially folded intermediate that can be rescued by corrector VX-809 to yield a native structure. Login to comment
276 Discussion The results in this study suggest that the mechanism for inhibition of packing of TM segments caused by the V232D mutation may be different in the full-length protein than predicted by the TM3/4 helix-loop-helix model system. Login to comment
277 Therien et al. [24] studied the V232D mutation in the TM3/4 helix-loop-helix fragment and suggested that the fragment was a useful model system for studying the mechanism of the V232D processing mutation because most consecutive TM segments in membrane proteins were in contact with one another [51]. Login to comment
278 In the TM3/4 helix-loop-helix model system, it was predicted that the two TM segments would be adjacent to each other along their entire lengths to bring Gln207(TM3) in close contact with TM4 to form a non-native hydrogen bond with the V232D processing mutation. Login to comment
279 It was postulated that the V232D mutation formed a hydrogen bond with Gln207 because replacement of Gln207 with nonpolar residues reversed the abnormal migration pattern of the V232D TM3/4 fragment in SDS-PAGE gels. Login to comment
280 They showed that the migration pattern of mutant V232D/Q207L in SDS-PAGE gels resembled that of the wild-type fragment. Login to comment
281 The results of TM3/4 helix-loop-helix model system studies were used to predict that hydrogen bond formation between V232D and Gln207 could alter the normal assembly and alignment of CFTR TM segments in full-length CFTR. Login to comment
282 Our studies on the full-length CFTR protein however, suggest that V232D inhibited maturation by disrupting a hydrophobic pocket. Login to comment
284 Although mutants such as A207A, Q207L and Q207C could be rescued with corrector VX-809 (Fig. 2), the V232D mutation appeared to have an effect that was independent of that of Gln207 since mutants Q207A/V232D, Q207L/V232D and Q207C/V232D could no longer be rescued by VX-809 (Fig. 1B). Login to comment
292 The V232D mutation inhibits packing of the TM segments. Login to comment
293 (A) Membranes prepared from cells expressing mutants W356C/W1145C, V232D/W356C/W1145C or Cys-less CFTR in the absence (none) or presence of corrector VX-809 were treated without (none) or with cross-linkers (X-linkers) M8M or BMH for 10 min at 20 8C. Login to comment
301 The P-gp L305R mutation in TM5 resembles the CFTR V232D mutation because it also inhibits maturation by disrupting a TM4/5 hydrophobic interface (Fig. 7A). Login to comment
313 We predict that V232D inhibits CFTR maturation by a mechanism that is similar to that proposed for the Q1071P, H1085R [50] and DF508 [17,26] processing mutations (see Fig. 11). Login to comment
317 The V232D mutation traps CFTR at an early folding step with incomplete packing of the TM segments and incomplete domain assembly (Fig. 11B). Login to comment
321 Corrector 4a may also efficiently rescue V232D CFTR because it also appears to interact with the transmembrane domains [18,65]. Login to comment
323 In summary, mutational analysis suggests that the mechanism of protein misfolding by the V232D mutation in TM4 does not involve non-native hydrogen bond formation with Gln207 in TM3. Login to comment
325 Understanding the mechanism of protein misfolding by V232D CFTR and its rescue by correctors is important because more than 50 other CF mutations have been identified in the TM segments of CFTR that involve a change from nonpolar to polar/charged residue [66]. Login to comment
327 Model of inhibition of CFTR by V232D and rescue by corrector VX-809. Login to comment
330 (B) The presence of V232D (red) in TM4 disrupts the hydrophobic environment and inhibits proper folding to trap the mutant protein as a partially folded intermediate. Login to comment
332 (C) Studies [19,20] suggest that corrector VX-809 interacts with TMD1 to induce V232D to complete the folding process to yield a native structure in which W356C and W1145C can be cross-linked (orange line). Login to comment

[hide] CFTR and lung homeostasis. Am J Physiol Lung Cell Mol Physiol. 2014 Dec 15;307(12):L917-23. doi: 10.1152/ajplung.00326.2014. Epub 2014 Nov 7. Collawn JF, Matalon S
CFTR and lung homeostasis.
Am J Physiol Lung Cell Mol Physiol. 2014 Dec 15;307(12):L917-23. doi: 10.1152/ajplung.00326.2014. Epub 2014 Nov 7., [PMID:25381027]

Abstract [show]
CFTR is a cAMP-activated chloride and bicarbonate channel that is critical for lung homeostasis. Decreases in CFTR expression have dire consequences in cystic fibrosis (CF) and have been suggested to be a component of the lung pathology in chronic obstructive pulmonary disease. Decreases or loss of channel function often lead to mucus stasis, chronic bacterial infections, and the accompanying chronic inflammatory responses that promote progressive lung destruction, and, eventually in CF, lung failure. Here we discuss CFTR's functional role airway surface liquid hydration and pH, in regulation of other channels such as the epithelial sodium channel, and in regulating inflammatory responses in the lung.

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130 V232D is a rare folding mutation that can be corrected to near WT, maturely glycosylated protein levels with small molecule corrector Corr-4a (11). Login to comment

[hide] [CFTR gene sequencing in a group of Chilean patien... Rev Chil Pediatr. 2014 Jul;85(4):448-54. doi: 10.4067/S0370-41062014000400007. Lay-Son R G, Vasquez D M, Puga Y A, Manque M P, Repetto L G
[CFTR gene sequencing in a group of Chilean patients with cystic fibrosis].
Rev Chil Pediatr. 2014 Jul;85(4):448-54. doi: 10.4067/S0370-41062014000400007., [PMID:25697318]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations of the CFTR gene, in which over 1,900 different mutations have been identified. In Chile, the diagnosis panel with the 36 most common mutations detects approximately 50% of all alleles, while for Caucasians, it is nearly 90%. The objective of this study is to expand the capacity of mutational screening in Chilean patients and look for recurrent mutations at the national level. METHOD: The detection of unknown pathogenic alleles was assessed by CFTR gene sequencing in a selected group of patients from the National Cystic Fibrosis Foundation (NCFF). 39 patients, who met the CF diagnostic criteria and had only one allele identified according to the mutational panel, were studied. Massive sequencing was performed throughout the investigation and the main CFTR databases were used for analysis. RESULTS: The second pathogenic allele was identified in 16 of 39 patients of this study (41%), finding eleven different mutations that had not been reported in our population. We believe that the reason is that one of the variants had not been previously described. CONCLUSIONS: Mutations that had been described mainly in Hispanic and/or Mediterranean populations were identified. We found a variation that had not been previously reported, but not enough recurrent mutations that could explain the low rate of detection were found. Knowledge about mutations can provide appropriate genetic counseling and will be critical to evaluate the potential use of new targeted therapies for treating them.

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58 Mutaciones detectadas por secuenciaci&#f3;n masiva en cohorte de 39 pacientes chilenos con FQ portadores de un alelo desconocido Mutaci&#f3;n detectada (nomenclatura actual*) n de alelos Reporte en pacientes con FQ (no de alelos) Efecto Denominaci&#f3;n antigua c.1330_1331delAT 3 Argentina (1)a Prote&#ed;na truncada por generaci&#f3;n de cod&#f3;n de t&#e9;rmino 1460delAT c.314T>A 2 Francia (1)a Cambio de amino&#e1;cido Isoleucina por Asparagina I105N c.4046G>A 2 Italia (7)b,c , EEUU (1)d Cambio de amino&#e1;cido Glicina por Aspartato G1349D c.148T>C 2 Espa&#f1;a (2)e Cambio de amino&#e1;cido Serina por Prolina S50P c.695T>A 1 Espa&#f1;a (14)e,f , EEUU (hispanos) (5)g,h Francia (2)a , Brasil (1)i Cambio de amino&#e1;cido Valina por Aspartato V232D c.3266G>A 1 Espa&#f1;a (5)e , Brasil (2)i,j , EEUU (hispanos) (2)g , Argentina (1)k , Israel (1)l Prote&#ed;na truncada por generaci&#f3;n de cod&#f3;n de t&#e9;rmino W1089X c.1647T>G 1 Emiratos &#c1;rabes Unidos (> 30)m,n , Colombia (4)o , Israel (4)p , Argelia (2)p , Marruecos (2)q , Reino Unido (2)p , Portugal (1)p , Espa&#f1;a (1)p , Francia (1)p , Italia (1)p , Brasil (1)q , Argentina (1)q Cambio de amino&#e1;cido Serina por Arginina S549R(T- >G) c.308G>A 1 No descrita previamente Cambio de amino&#e1;cido Glicina por Glutamato G103E c.1680-1G>A 1 Espa&#f1;a (1)r Alteraci&#f3;n en splicing 1812-1G->A c.1679+1G>C 1 Francia (2)s Macedonia (1)s , Alteraci&#f3;n en splicing 1811+1G->C c.490-2A>G 1 Argentina (1)t Alteraci&#f3;n en splicing 622-2A->G FQ: Fibrosis qu&#ed;stica. Login to comment