ABCMdb

ABCC7 p.His1085Arg

Admin's notes:	Class II (maturation defect) Veit et al.
ClinVar:	c.3254A>G , p.His1085Arg D , Pathogenic/Likely pathogenic, not provided
CF databases:	c.3254A>G , p.His1085Arg (CFTR1) D , The mutation was found once in 87 non-[delta]F508 chromosomes screened. The patient is 20 years old, pancreatic insufficient with no cirrhosis. The other chromosome is [delta]F508 and the H1085R is on a B haplotype. A girl homozygous for H1085R was also found in Japan by Kunihiko Yoshimura.
Predicted by SNAP2:	A: D (85%), C: D (91%), D: D (91%), E: D (91%), F: D (91%), G: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), N: D (85%), P: D (95%), Q: D (85%), R: N (53%), S: D (85%), T: D (91%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: N, E: N, F: D, G: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: D, V: D, W: D, Y: D,

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

Publications
[hide] A Japanese patient homozygous for the H1085R mutat... Clin Genet. 1999 Aug;56(2):173-5. Yoshimura K, Wakazono Y, Iizuka S, Morokawa N, Tada H, Eto Y
A Japanese patient homozygous for the H1085R mutation in the CFTR gene presents with a severe form of cystic fibrosis.
Clin Genet. 1999 Aug;56(2):173-5., [PMID:10517260]

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1 All rights reser6ed Letter to the Editor A Japanese patient homozygous for the H1085R mutation in the CFTR gene presents with a severe form of cystic fibrosis To the Editor: Cystic fibrosis (CF), the most common autosomal recessive disorder in Caucasians, has long been believed to be extremely rare in Orientals (1, 2). Login to comment
4 Here we describe a 15-year-old female Japanese patient who exhibited typical manifestations of CF and proved to be a homozygote for a very rare missense mutation H1085R. Login to comment
15 Direct sequencing of the band clearly demonstrated an alteration of the nucleotide residue at 3386 from A to G in a homozygous fashion, resulting in His to Arg change at the amino acid 1085, a missense mutation H1085R. Login to comment
17 Further evaluation for three polymorphic loci proved that the H1085R mutation allele was associated with a haplotype of (GATT)6 in intron 6a (8), (TG)12T7 in the TG repeat and polythymidine tract at the splice acceptor site in intron 8 (9, 10), and M470 (1540A) in exon 10 (11, 12), respectively. Login to comment
18 The mutation H1085R, first described by Mercier et al. Login to comment
20 However, no other CF patients with H1085R have been reported since then, implying that it is an extremely rare mutation (14). Login to comment
21 Consistent with the previous report that other mutations located in exon 17b, such as R1066L and M1101R, were usually associated with pancreatic insufficiency, the case presented here and the French case had pancreatic insufficiency, suggesting that the H1085R is also a severe allele [(13), personal communication]. Login to comment
23 One important point is that the H1085R alleles of these 2 patients are most likely to be recurrent Letter to the Editor Fig. 1. Login to comment
24 Screening for the CFTR mutation H1085R in the patient and her family. Login to comment
27 The positions of the RsaI restriction site in the normal CFTR allele are indicated by hatched arrows, whereas the new RsaI site generated by H1085R mutation is by a solid arrow. Login to comment
36 However, it remains to be further elucidated whether this haplotype is restricted to the H1085R mutation or widespread in any other CFTR genotypes in CF patients in Japan. Login to comment

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

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

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571 This stringent sequence conservation, coupled with a cluster of missense mutations identified in this subdomain (among which two homozygosities, H1085R [Yoshimura et al. 1999] and R1066C [Casals et al. 1997], cause a severe CF phenotype), undoubtedly underlies its functional significance within CFTR. Login to comment
947 A Japanese patient homozygous for the H1085R mutation in the CFTR gene presents with a severe form of cystic fibrosis. Login to comment

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

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

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

[hide] Genotype-phenotype correlation in cystic fibrosis:... Am J Med Genet. 2002 Jul 22;111(1):88-95. Salvatore F, Scudiero O, Castaldo G
Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes.
Am J Med Genet. 2002 Jul 22;111(1):88-95., 2002-07-22 [PMID:12124743]

Abstract [show]
More than 1,000 mutations have been identified in the cystic fibrosis (CF) transmembrane regulator (CFTR) disease gene. The impact of these mutations on the protein and the wide spectrum of CF phenotypes prompted a series of Genotype-Phenotype correlation studies. The CFTR genotype is invariably correlated with pancreatic status-in about 85% of cases with pancreatic insufficiency and in about 15% of cases with pancreatic sufficiency. The correlations between the CFTR genotype and pulmonary, liver, and gastrointestinal expression are debatable. The heterogeneous phenotype in CF patients bearing the same genotype or homozygotes for nonsense mutations implicated environmental and/or genetic factors in the disease. However, the discordant phenotype observed in CF siblings argued against a major role of environmental factors and suggested that genes other than CFTR modulate the CF phenotype. A locus that modulates gastrointestinal expression was identified in mice and subsequently in humans. By analyzing nine CF patients discordant for meconium ileus we were able to show that this locus had a dominant effect. Moreover, in a collaborative study we found a higher rate of polymorphisms in beta-defensin genes 1 and 2 in CF patients and in controls. In another multicenter study mutations in alpha-1 antitrypsin (A1AT) and mannose binding lectin genes were found to be independent risk factors for liver disease in CF patients. The body of evidence available suggests that the variegated CF phenotype results from complex interactions between numerous gene products.

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46 A series of mutations usually associated with pancreatic sufficiency have been identified and defined as ''mild`` with reference to pancreatic status [Kerem et al., 1989c]: G85E, G91R, R117H, E193K, P205S, R334W, T338I, R347H, R347L, R347P, R352Q, A455E, S492F, S549N, P574H, D579G, 711 þ 5 G > A, C866Y, F1052V, H1054D, R1066H, R1068H, H1085R, D1152H, S1159P, S1251N, F1286S, G1349D, 2789 þ 5 G > A, and 3849 þ 10kb C > T [Dean et al., 1990; Cutting et al., 1990a; Cremonesi et al., 1992; Highsmith et al., 1994]. Login to comment

[hide] A finger sweat chloride test for the detection of ... Pancreas. 2004 Apr;28(3):e80-5. Naruse S, Ishiguro H, Suzuki Y, Fujiki K, Ko SB, Mizuno N, Takemura T, Yamamoto A, Yoshikawa T, Jin C, Suzuki R, Kitagawa M, Tsuda T, Kondo T, Hayakawa T
A finger sweat chloride test for the detection of a high-risk group of chronic pancreatitis.
Pancreas. 2004 Apr;28(3):e80-5., [PMID:15084988]

Abstract [show]
OBJECTIVES: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with chronic pancreatitis in Caucasians. We developed a simple method for measuring finger sweat chloride concentration to test whether CFTR dysfunction underlies chronic pancreatitis in Japan where cystic fibrosis (CF) is rare. METHODS: We studied 25 patients with chronic (21 alcoholic and 4 idiopathic) pancreatitis and 25 healthy volunteers. Sweat chloride concentrations were measured by a finger sweat chloride test. We analyzed DNA for 20 common CFTR mutations in Europeans, 9 CF-causing mutations in Japanese, and 2 polymorphic loci, a poly-T tract and (TG) repeats, at intron 8. RESULTS: Thirteen patients (52%) had sweat chloride levels >60 mmol/L, a level consistent with CF, while only 4 (16%) healthy subjects exceeded this level. The 29 CF mutations and the 5T allele were detected in neither the patients nor controls. The (TG) 12 allele was common in both the patients (58%) and controls (48%). The (TG) 12/12 genotype was common in alcoholic pancreatitis (29%) compared with the (TG) 11/11 (10%). Patients with the (TG) 12/12 genotype had significantly higher sweat chloride concentrations than the controls. CONCLUSION: CFTR dysfunction as evidenced by a finger sweat chloride test is present in about half of Japanese patients with chronic pancreatitis, suggesting that this test may be useful for detecting the high-risk group. A higher proportion of the (TG) 12 allele may be a genetic background for elevated sweat chloride concentrations in Japanese patients.

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51 The 9 CF-causing mutations (R75X, Q98R, M152R, R347H, L441P, L571S, D979A, H1085R, and T1086I) in Japa- nese20,25-28 were screened by SNP typing with Masscode System (Shimadzu, Kyoto, Japan). Login to comment

[hide] Genetic evidence for CFTR dysfunction in Japanese:... J Med Genet. 2004 May;41(5):e55. Fujiki K, Ishiguro H, Ko SB, Mizuno N, Suzuki Y, Takemura T, Yamamoto A, Yoshikawa T, Kitagawa M, Hayakawa T, Sakai Y, Takayama T, Saito M, Kondo T, Naruse S
Genetic evidence for CFTR dysfunction in Japanese: background for chronic pancreatitis.
J Med Genet. 2004 May;41(5):e55., [PMID:15121783]

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219 The nine CF causing (R75X, Q98R, M152R, R347H, L441P, L571S, D979A, H1085R, and T1086I) and two non-CF causing (Q1352H and R1453W) mutations in Japanese6 22-24 were screened by SNP typing with a Masscode system (Shimadzu, Kyoto, Japan) and confirmed by sequence analysis in positive and equivocal cases. Login to comment

[hide] Rescuing cystic fibrosis transmembrane conductance... Proc Natl Acad Sci U S A. 2004 May 25;101(21):8221-6. Epub 2004 May 12. Cormet-Boyaka E, Jablonsky M, Naren AP, Jackson PL, Muccio DD, Kirk KL
Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation.
Proc Natl Acad Sci U S A. 2004 May 25;101(21):8221-6. Epub 2004 May 12., 2004-05-25 [PMID:15141088]

Abstract [show]
Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR-processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (DeltaF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented DeltaF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not DeltaF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR.

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5 This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented ⌬F508-CFTR but not H1085R (a carboxy- processing mutant), whereas a carboxy fragment complemented H1085R but not ⌬F508-CFTR. Login to comment
146 All results are representative of 3-10 experiments. by comparing the transcomplementation of the ⌬F508-CFTR mutant to that of a severe processing mutant (H1085R-CFTR) for which the mutation resides in the carboxy-terminal half of the protein (27). Login to comment
147 Unlike for ⌬F508-CFTR, the processing of H1085R-CFTR could not be rescued by coexpression with the 1-633 amino fragment (Figs. 4 D and F). Login to comment
148 Importantly, however, H1085R-CFTR could be transcomplemented by a carboxy-terminal fragment (837-1,480), which had no effect on the processing of ⌬F508-CFTR or the N-tail processing mutants (Figs. 4 D, E, and F). Login to comment
170 (D) ⌬F508-CFTR was not transcomplemented by a carboxy fragment (837-1,480), whereas H1085R-CFTR, a cytosolic-loop-4-processing mutant that associates with severe disease, was complemented by fragment 837-1,480 but not by fragment 1-633. Login to comment
193 We thank M. J. Welsh for the H1085R-CFTR construct. Login to comment

[hide] A novel missense mutation A1081P in the cystic fib... J Trop Pediatr. 2004 Aug;50(4):239-40. Ngukam A, Jacquemont ML, Souville I, Viel M, Beldjord C, Hubert D, Hughes JN, Bienvenu T
A novel missense mutation A1081P in the cystic fibrosis transmembrane conductance regulator (CFTR) gene identified in a Laotian patient with congenital bilateral absence of the vas deferens.
J Trop Pediatr. 2004 Aug;50(4):239-40., [PMID:15357566]

Abstract [show]
Cystic fibrosis is the most common autosomal disorder in the Caucasion population. However, the disease is rare in Asia and little is known about the spectrum of CF transmembrane conductance regulator, CFTR, mutations in this population. We studied a 39-year-old Loatian patient with congenital bilateral absence of the vas deferens and identified a novel missense mutation in exon 17b (3373G>C). Identification of novel mutations in this Asian population is of particular interest when designing a genetic testing strategy in Asian countries and also in other countries where immigration from Asia is common.

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4 Only a few CFTR mutations have been identified in that population (L88X, M152R, K166E, F508del, 1742delAC, 1525-18G>A, 1540del10, L568X, 1898ϩ1G>T, 1898ϩ5G>T, G970D, 451-458del8, 3121-2A>G, H1085R).1-6 We report here a novel missense mutation in a Laotian patient with congenital bilateral absence of the vas deferens (CBAVD). Login to comment

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

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

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

[hide] The chemical chaperone CFcor-325 repairs folding d... Biochem J. 2006 May 1;395(3):537-42. Loo TW, Bartlett MC, Wang Y, Clarke DM
The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants.
Biochem J. 2006 May 1;395(3):537-42., 2006-05-01 [PMID:16417523]

Abstract [show]
Most patients with CF (cystic fibrosis) express a CFTR [CF TM (transmembrane) conductance regulator] processing mutant that is not trafficked to the cell surface because it is retained in the endoplasmic reticulum due to altered packing of the TM segments. CL4 (cytoplasmic loop 4) connecting TMs 10 and 11 is a 'hot-spot' for CFTR processing mutations. The chemical chaperone CFcor-325 (4-cyclohexyloxy-2-{1-[4-(4-methoxy-benezenesulphonyl)piperazin-1-yl]-ethy l}-quinazoline) rescued most CL4 mutants. To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. These results show that CFcor-325 rescued CFTR mutants by repairing the folding defects in the TMDs.

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4 To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Login to comment
5 Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. Login to comment
6 The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. Login to comment
21 EXPERIMENTAL Construction and expression of mutants The cDNAs of wild-type and CL4 mutants (H1054D, G1061R, L1065P, R1066H, Q1071P, L1077P, H1085R and W1098R) were inserted into pcDNA3 vector (Invitrogen, Oakville, ON, Canada) as described previously [2]. Login to comment
23 Site-directed mutagenesis was used to introduce the Q1071P or H1085R mutations into the double cysteine mutants as described previously [11]. Login to comment
31 Disulphide cross-linking analysis The cDNAs of double cysteine mutants M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) constructed in wild-type, mutant Q1071P or mutant H1085R backgrounds were expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325. Login to comment
37 Briefly, cells expressing wild-type, mutant Q1071P or H1085R CFTR were grown with or without 3 µM CFcor-325. Login to comment
51 Accordingly, HEK-293 cells were transfected with mutants H1054D, G1061R, L1065P, R1066H, Q1071P, L1077P, H1085R or W1098R cDNAs. Login to comment
57 Expression of mutants H1054D, G1061R, R1066H, Q1071P, L1077P, H1085R and W1098R in the presence of 3 µM CFcor-325, however, induced expression of the 190 kDa mature protein. Login to comment
59 The chemical chaperone most efficiently rescued mutants R1066H, Q1071P, H1085R and W1098R as the amount of mature CFTR was approx. Login to comment
73 Two CL4 mutants, Q1071P and H1085R, were selected for cross-linking analysis because they were efficiently rescued with CFcor-325. Login to comment
74 Expression of mutants Q1071P or H1085R in the presence of CFcor-325 increased the amount of mature CFTR from less than 5% (in the absence of corrector) of total to more than 30% (Figures 2A and 2B). Login to comment
77 Mature 190 kDa CFTR, however, contains complex carbohydrate groups that are resistant to endo H but not to PNGase F. Accordingly, cells expressing wild-type, mutant Q1071P or mutant H1085R CFTR and grown in the presence or absence of CFcor-325 were extracted with SDS and then treated with endoglycosidases. Login to comment
78 Figure 3 shows that the 170 kDa immature CFTRs but not the 190 kDa mature CFTRs of wild-type, mutant Q1071P or mutant H1085R were sensitive to digestion with endo H. Login to comment
79 Both the 190 kDa mature and 170 kDa immature CFTRs were sensitive to digestion with PNGase F. Therefore expression of mutant Q1071P and H1085R in the presence of CFcor-325 promoted maturation and trafficking of the proteins from the ER to the Golgi where complex carbohydrates are added to the protein. Login to comment
81 Cells expressing wild-type, mutant Q1071P or mutant H1085R CFTRs were grown in the presence or absence of 3 µM CFcor-325. Login to comment
82 The cells Figure 3 Endoglycosidase digestion Whole cell SDS extracts of HEK-293 cells expressing wild-type, mutant Q1071P or mutant H1085R in the absence (-) or presence (+) of 3 µM CFcor-325 were treated with endo H (H), PNGase F (F) or no endoglycosidase (-). Login to comment
85 Figure 4 Cell surface labelling of CFTR (A) BHK cells (Control) or BHK cells stably expressing wild-type (Wild-type), mutant Q1071P or mutant H1085R CFTRs were grown in the presence (+) or absence (-) of 3 µM CFcor-325. Login to comment
93 Figure 4(A) Figure 5 Measurement of cAMP-stimulated iodide efflux activities Iodide efflux assays were performed on BHK cells stably expressing wild-type, mutant Q1071P or mutant H1085R CFTRs that were grown in the presence (+CFcor-325) or absence (untreated) of CFcor-325 as described in the Experimental section. Login to comment
94 The activity of mutant H1085R grown without CFcor-325 was similar to that of mutant Q1071P (untreated) and is omitted for clarity. Login to comment
97 Similarly, labelled mature 190 kDa CFTR proteins were detected in mutants Q1071P and H1085R only after rescue with CFcor-325. Login to comment
98 Figure 4(B) shows that there was good correlation between the amount of biotinylated CFTR proteins in mutants Q1071P and H1085R with the amount of mature CFTRs detected in Figure 2(B) (63 and 50% of wild-type respectively). Login to comment
99 Next, we tested whether mutant Q1071P or H1085R was active at the cell surface after rescue with CFcor-325. Login to comment
100 Mutants Q1071P and H1085R CFTRs were stably expressed in BHK cells because adherent cells are essential for the iodide efflux assays. Login to comment
102 The BHK cells stably expressing mutants Q1071P or H1085R were treated for 48 h with or without 3 µM CFcor-325 and then used in iodide efflux assays. Login to comment
106 In contrast, BHK cells stably expressing CFTR mutants Q1071P or H1085R demonstrated iodide efflux activity only when grown in the presence of CFcor-325 (Figure 5). Login to comment
109 Accordingly, M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were introduced into a Q1071P or H1085R CFTR background. Login to comment
110 Mutants Q1071P or H1085R containing M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were then transiently expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325 for 48 h and then treated with the homobifunctional cross-linkers M5M, M8M or M17M. Login to comment
115 Mature mutant Q1071P/ M348C(TM6)/T1142C(TM12) protein was cross-linked with Figure 6 Disulphide cross-linking analysis of CFTR processing mutants HEK-293 cells expressing mutants Q1071P/M348C(TM6)/T1142C(TM12), Q1071P/T351C- (TM6)/T1142C(TM12) and Q1071P/W356C(TM6)/W1145C(TM12) (A), mutants H1085R/ M348C(TM6)/T1142C(TM12), H1085R/T351C(TM6)/T1142C(TM12) and H1085R/W356C- (TM6)/W1145C(TM12) (B) or wild-type, mutant Q1071P or mutant H1085R (C) were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Login to comment
120 Similar results were observed with the H1085R double cysteine mutants (Figure 6B). Login to comment
122 In contrast, wild-type, mature mutant Q1071P or H1085R CFTRs lacking the introduced cysteine residues was not cross-linked by M5M, M8M or M17M (Figure 6C). Login to comment
129 Similar results were observed with mutant H1085R/M348C(TM6)/T1142(TM12) (results not shown). Login to comment

[hide] Specific rescue of cystic fibrosis transmembrane c... Mol Pharmacol. 2006 Jul;70(1):297-302. Epub 2006 Apr 19. Wang Y, Bartlett MC, Loo TW, Clarke DM
Specific rescue of cystic fibrosis transmembrane conductance regulator processing mutants using pharmacological chaperones.
Mol Pharmacol. 2006 Jul;70(1):297-302. Epub 2006 Apr 19., [PMID:16624886]

Abstract [show]
Most mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) that cause severe symptoms of cystic fibrosis do not reach the cell surface because they are defective in folding. Many CFTR folding mutants, however, including the DeltaF508 mutant found in more than 90% of cystic fibrosis patients, are potentially functional at the cell surface if they can be induced to fold correctly. In a previous study (Mol Pharm 2:407-413, 2005), we reported that a quinazoline derivative (CFcor-325) could rescue CFTR processing mutants. The corrector was not specific however, as it could also rescue a processing mutant of CFTR's sister protein, the multidrug resistance P-glycoprotein. The goal of this study was to test whether it was possible to specifically rescue CFTR processing mutants using a pharmacological chaperone. In this article, we report that two compounds, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol (CFpot-532) and 2-phenylamino-4-(4-ethylene-phenyl)-thiazole (corr-2b) could rescue CFTR processing mutants such as DeltaF508 CFTR but not a P-glycoprotein processing mutant. The compound CFpot-532 also acts as a potentiator of DeltaF508 CFTR channel activity. Therefore, the results suggest that the mechanism whereby CFpot-532 and corr-2b promote folding of CFTR processing mutants is through direct interaction with the CFTR mutant proteins. The compound CFpot-532 could be a particularly useful lead compound for treatment of cystic fibrosis because it is both a CFTR channel potentiator as well as a specific pharmacological chaperone.

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30 Wild-type, ⌬F508, and H1085R CFTR cDNAs were inserted into the pcDNA3 vector (Invitrogen Canada Inc., Burlington, ON, Canada) as described previously (Loo et al., 2006). Login to comment
138 It has been reported that it was possible to specifically rescue CFTR processing mutants containing a processing mutation in the front half of the protein (⌬F508) by coexpression with a CFTR NH2-half molecule whereas processing mutations located in the back half of the protein (H1085R) could be rescued by coexpression with a CFTR COOH-half molecule (Cormet-Boyaka et al., 2004). Login to comment
140 Therefore, we tested whether a specific CFTR pharmacological chaperone such as CFpot-532 could rescue a CFTR processing mutant with a mutation in the COOH half of the molecule (H1085R). Login to comment
141 HEK 293 cells transiently expressing CFTR processing mutant H1085R were incubated in the presence or absence of 10 ␮M CFpot-532 for 48 h. Whole-cell extracts were subjected to immunoblot analysis. Login to comment
142 Figure 7 shows that expression of CFTR H1085R in the presence of 10 ␮M CFpot-532 promoted maturation of the protein. Login to comment
170 HEK 293 cells were transiently transfected with CFTR wild-type or H1085R cDNAs and then expressed in the absence (-) or presence (ϩ) of 10 ␮M CFpot-532 for 48 h. Whole-cell extracts were subjected to immunoblot analysis with a CFTR polyclonal antibody. Login to comment

[hide] Additive effect of multiple pharmacological chaper... Biochem J. 2007 Sep 1;406(2):257-63. Wang Y, Loo TW, Bartlett MC, Clarke DM
Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants.
Biochem J. 2007 Sep 1;406(2):257-63., 2007-09-01 [PMID:17535157]

Abstract [show]
The most common cause of CF (cystic fibrosis) is the deletion of Phe(508) (DeltaF508) in the CFTR [CF TM (transmembrane) conductance regulator] chloride channel. One major problem with DeltaF508 CFTR is that the protein is defective in folding so that little mature protein is delivered to the cell surface. Expression of DeltaF508 CFTR in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein. Unfortunately, the efficiency of corrector-induced maturation of DeltaF508 CFTR is probably too low to have therapeutic value and approaches are needed to increase maturation efficiency. We postulated that expression of DeltaF508 CFTR in the presence of multiple correctors that bound to different sites may have an additive effect on maturation. In support of this mechanism, we found that expression of P-glycoprotein (CFTR's sister protein) processing mutants in the presence of two compounds that bind to different sites (rhodamine B and Hoechst 33342) had an additive effect on maturation. Therefore we tested whether expression of DeltaF508 CFTR in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. It was found that the combination of the quinazoline VRT-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of DeltaF508 CFTR (approx. 40% of total CFTR was mature protein) compared with expression in the presence of a single compound. The additive effect of the correctors on DeltaF508 CFTR maturation suggests that they directly interact at different sites of the protein.

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33 MATERIALS AND METHODS Construction and expression of mutants The construction of F508 and H1085R CFTR cDNAs and insertion into the pcDNA3 vector (Invitrogen Canada, Burlington, ON, Canada) was described previously [23]. Login to comment
34 The CFTR cDNAs coding for NBD2 (where NBD2 is nucleotide-binding domain 2), H1085R CFTR (residues 1-1196) as well as P-gp cDNAs for mutant P709G P-gp and P-gp truncation mutants NBD2 P-gp (residues 1-1023), TMD1+2 {TMD1 [N-terminal TMD (TM domain) containing TM segments 1-6] and TMD2 (C-terminal TMD containing TM segments 7-12)}, P-gp (residues 1-379 plus 681-1025) and TMD1 P-gp (residues 1-379) were modified to contain the epitope for monoclonal antibody A52 at the C-terminal ends and subcloned into the mammalian expression vector pMT21 as described previously [21]. Login to comment
103 Figure 4 Effect of correctors on maturation of CFTR processing mutant H1085R CFTR processing mutant H1085R was expressed for 24 h in the absence (-) or presence (+) of 3 µM VRT-325, 10 µM corr-2b, 10 µM corr-4a, combination of 3 µM VRT-325 and 10 µM corr-2b, combination of 3 µM VRT-325 and 10 µM corr-4a, or combination of 10 µM corr-2b and 10 µM corr-4a respectively. Login to comment
112 First, we tested the correctors on a CFTR processing mutant (H1085R) with a mutation in the fourth cytoplasmic loop (CL4) connecting TM segments 10 and 11 in the CO2H half of the molecule, where a relatively large number of clinically relevant processing mutations are found [35]. Login to comment
113 The H1085R CFTR mutant was selected for use in our initial studies because expression of its cDNA in HEK-293 cells yields a small amount of mature protein (approx. Login to comment
115 Therefore the use of mutant H1085R in the presence of combinations of correctors would enable us to determine if the compounds increase or decrease the efficiency of maturation. Login to comment
116 CFTR processing mutant H1085R was transiently expressed in HEK-293 cells and incubated in the presence or absence of 3 µM VRT-325, 10 µM corr-2b and 10 µM corr-4a, combination of VRT-325 and corr-2b, combination of VRT-325 and corr-4a, or combination of corr-2b and corr-4a for 24 h. Whole cell extracts were subjected to immunoblot analysis. Login to comment
119 Expression of mutant H1085R CFTR in the presence of a combination of VRT-325 and corr-2b caused the largest increase in maturation efficiency as 48% of total CFTR was present as mature protein. Login to comment
122 It has been postulated that correctors promote maturation of CFTR processing mutants by promoting interactions between the Figure 5 Effect of correctors on maturation of CFTR truncation mutant NBD2 H1085R HEK-293 cells were transfected with NBD2 H1085R followed by a 24 h incubation in the absence (-) or presence (+) of 3 µM VRT-325, 10 µM corr-2b, 10 µM corr-4a, combination of 3 µM VRT-325 and 10 µM corr-2b, or combination of 3 µM VRT-325 and 10 µM corr-4a respectively. Login to comment
126 To determine if interactions between the NBDs were required for the enhanced maturation of mutant H1085R in the presence of VRT-325 together with corr-2b or corr-4a, we utilized an H1085R mutant that lacked NBD2 ( NDB2 H1085R CFTR) (Figure 1B). Login to comment
127 The NDB2 H1085R CFTR mutant was transfected into HEK-293 cells and expressed in the presence of VRT-325, corr-2b, corr-4a, VRT-325 plus corr-2b, VRT-325 plus corr-4a or no correctors for 24 h. Whole cell extracts were then subjected to immunoblot analysis. Login to comment

[hide] Pharmacological chaperoning: two 'hits' are better... Biochem J. 2007 Sep 1;406(2):e1-2. Skach WR
Pharmacological chaperoning: two 'hits' are better than one.
Biochem J. 2007 Sep 1;406(2):e1-2., 2007-09-01 [PMID:17680777]

Abstract [show]
Protein folding disorders comprise a rapidly growing group of diseases that involve virtually every organ system and affect individuals of all ages. Their principal pathology is the inability of a protein to acquire or maintain its physiological three-dimensional structure. In cells, this generally results in one of three outcomes: accumulation of misfolded protein aggregates, cell death, or recognition by cellular quality control machinery and rapid degradation. Large-scale screening efforts to identify and design small molecules that either repair the folding defect or enable the protein to escape degradation have been encouraging. However, most compounds identified to date restore only a small fraction of molecules to the normal folding pathway, and hence are relatively poor therapeutic candidates. Results published by Wang et al. in this issue of the Biochemical Journal show that, for mutant forms of two ABC (ATP-Binding-Cassette) transporters, P-glycoprotein and CFTR (cystic fibrosis transmembrane conductance regulator), modest correction of trafficking by single agents can be additive when multiple compounds are used in combination. These findings raise the intriguing possibility that corrector molecules acting at different steps along the folding pathway might provide a multidrug approach to human protein folding disorders.

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47 Steady-state levels (determined by immunoblotting) of the CFTR mutant H1085R revealed that combinations of corr-2b/VRT325 or corr-4a/ corr-2b rescued more CFTR than each compound independently. Login to comment
48 Similar additive effects were observed for combinations of corr-2b/VRT325 or corr-4a/corr-2b for the CFTR mutant H1085R, in which the second nucleotide-binding domain was removed. 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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90 In some additional examples, a number of mutations found in the fourth intracellular loop (H1054D, G1061R, L1065P, R1066C/H/L, Q1071P, L1077P, H1085R, W1098R, M1101K/ R) also affect the biosynthetic processing of CFTR (although function was not tested) (73); some intracellular loop 4 mutants (F1052V, K1060T, A1067T, G1069R, R1070Q/W) can process CFTR to the complex-glycosylated ("Band C") form but have altered channel activity compared to wild type. Login to comment

[hide] A truncated CFTR protein rescues endogenous DeltaF... FASEB J. 2009 Nov;23(11):3743-51. Epub 2009 Jul 20. Cormet-Boyaka E, Hong JS, Berdiev BK, Fortenberry JA, Rennolds J, Clancy JP, Benos DJ, Boyaka PN, Sorscher EJ
A truncated CFTR protein rescues endogenous DeltaF508-CFTR and corrects chloride transport in mice.
FASEB J. 2009 Nov;23(11):3743-51. Epub 2009 Jul 20., [PMID:19620404]

Abstract [show]
Cystic fibrosis (CF) is most frequently associated with deletion of phenylalanine at position 508 (DeltaF508) in the CF transmembrane conductance regulator (CFTR) protein. The DeltaF508-CFTR mutant protein exhibits a folding defect that affects its processing and impairs chloride-channel function. This study aimed to determine whether CFTR fragments approximately half the size of wild-type CFTR and complementary to the portion of CFTR bearing the mutation can specifically rescue the processing of endogenous DeltaF508-CFTR in vivo. cDNA encoding CFTR fragments were delivered to human airway epithelial cells and mice harboring endogenous DeltaF508-CFTR. Delivery of small CFTR fragments, which do not act as chloride channels by themselves, rescue DeltaF508-CFTR. Therefore, we can speculate that the presence of the CFTR fragment, which does not harbor a mutation, might facilitate intermolecular interactions. The rescue of CFTR was evident by the restoration of chloride transport in human CFBE41o- bronchial epithelial cells expressing DeltaF508-CFTR in vitro. More important, nasal administration of an adenovirus expressing a complementary CFTR fragment restored some degree of CFTR activity in the nasal airways of DeltaF508 homozygous mice in vivo. These findings identify complementary protein fragments as a viable in vivo approach for correcting disease-causing misfolding of plasma membrane proteins.

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215 As previously reported, the rescue was specific to mutations located in the amino-terminal part of CFTR (N-tail and NBD1), as the CFTR fragment 1-633 did not rescue a CFTR mutant harboring a mutation located close to the carboxyl tail of CFTR, H1085R-CFTR (14). Login to comment

[hide] Unique mutations of the cystic fibrosis transmembr... Intern Med. 2009;48(15):1327-31. Epub 2009 Aug 3. Izumikawa K, Tomiyama Y, Ishimoto H, Sakamoto N, Imamura Y, Seki M, Sawai T, Kakeya H, Yamamoto Y, Yanagihara K, Mukae H, Yoshimura K, Kohno S
Unique mutations of the cystic fibrosis transmembrane conductance regulator gene of three cases of cystic fibrosis in Nagasaki, Japan.
Intern Med. 2009;48(15):1327-31. Epub 2009 Aug 3., [PMID:19652440]

Abstract [show]
Cystic fibrosis (CF), the most common lethal hereditary disorder in Caucasians, is quite rare in Southeast Asia including Japan. Here, we report three CF cases encountered in Nagasaki, Japan. Case 1; a 24-year-old man with dyspnea and cough was diagnosed as CF with a missense mutation Q98R in exon 4 and a polymorphic 125C in exon 1 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Case 2; a 13-year-old woman born of consanguineous parents was diagnosed as CF with homozygous Q98R mutations in exon 4. Case 3; a 29-year-old woman complaining of cough and sputum was diagnosed as CF with a heterozygous R347H mutation in exon 7 and a polymorphic 125C in exon 1. These mutations have been previously reported in Caucasian patients, but are considered very rare. Although the numbers of individuals with CF are very limited, the profiles of CFTR mutations in those patients are likely diverse in Japan.

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16 The other novel or rare mutations such as R347H, D 979A, 1724delAG, H1085R, M152R and 1540del10 have The Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Department of Respiratory Medicine, National Hospital Organization Minami-Kyushu National Hospital, Kagoshima, Department of Respiratory Medicine, National Hospital Organization Ureshino Medical Center, Ureshino, Department of Laboratory Medicine, Nagasaki University School of Medicine, Nagasaki and Department of Respiratory Medicine, Respiratory Center, Toranomon Hospital, Tokyo Received for publication January 20, 2009; Accepted for publication April 14, 2009 Correspondence to Dr. Koichi Izumikawa, koizumik@nagasaki-u.ac.jp Figure1. Login to comment

[hide] Identification of a novel water-soluble activator ... Eur Respir J. 2010 Aug;36(2):311-22. Epub 2010 Jan 28. Bertrand J, Boucherle B, Billet A, Melin-Heschel P, Dannhoffer L, Vandebrouck C, Jayle C, Routaboul C, Molina MC, Decout JL, Becq F, Norez C
Identification of a novel water-soluble activator of wild-type and F508del CFTR: GPact-11a.
Eur Respir J. 2010 Aug;36(2):311-22. Epub 2010 Jan 28., [PMID:20110398]

Abstract [show]
One of the major therapeutic strategy in cystic fibrosis aims at developing modulators of cystic fibrosis transmembrane conductance regulator (CFTR) channels. We recently discovered methylglyoxal alpha-aminoazaheterocycle adducts, as a new family of CFTR inhibitors. In a structure-activity relationship study, we have now identified GPact-11a, a compound able not to inhibit but to activate CFTR. Here, we present the effect of GPact-11a on CFTR activity using in vitro (iodide efflux, fluorescence imaging and patch-clamp recordings), ex vivo (short-circuit current measurements) and in vivo (salivary secretion) experiments. We report that GPact-11a: 1) is an activator of CFTR in several airway epithelial cell lines; 2) activates rescued F508del-CFTR in nasal, tracheal, bronchial, pancreatic cell lines and in human CF ciliated epithelial cells, freshly dissociated from lung samples; 3) stimulates ex vivo the colonic chloride secretion and increases in vivo the salivary secretion in cftr(+/+) but not cftr(-/-) mice; and 4) is selective for CFTR because its effect is inhibited by CFTR(inh)-172, GlyH-101, glibenclamide and GPinh-5a. To conclude, this work identifies a selective activator of wild-type and rescued F508del-CFTR. This nontoxic and water-soluble agent represents a good candidate, alone or in combination with a F508del-CFTR corrector, for the development of a CFTR modulator in cystic fibrosis.

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45 Human lung tissues were obtained from seven patients: three non-CF males with a mean age of 61 yrs, a non-CF female aged 65 yrs, a F508del/F508del-CFTR male aged 37 yrs, a F508del/R1066C female aged 26 yrs and a F508del/ H1085R-CFTR female aged 23 yrs. Login to comment
118 The mutation H1085R is a severe and rare missense mutation identified by MERCIER et al. [32]. Login to comment
119 H1085R-CFTR protein presents a trafficking defect which can be corrected by F508del-CFTR corrector [33]. Login to comment
155 Structural determinants for CFTR activation In the chemical structure of GPact-11a (fig. 1b), three main elements can be distinguished: 1) the purine aromatic heterocycles composed of fused pyrimidine and imidazole rings; -15 ΔFx 35 25 15 5 -5 45 CF-KM4 F508del-CFTR HEK-293 CFPACCuFi-1CF15 -5 ΔFx 15 10 5 0 20 F508del/ H1085R F508del/ F508del F508del/ R1066C 0 Activation% 80 60 40 20 100 -2-3-4-5 Log [GPact-11a] M -6-7 Fx 40 20 0 -20 60a) b) c) d) 2015105 Time min GPact-11a CFTRInh-172 0 ● ● ● ● ● ● ● ● ●●●●●●●●●●●●● ●● ●●● ●●●● ● ● ● ● ● ● ●● ● ● ● ● ● FIGURE 5. Login to comment
161 d) Effect of GPact-11a on human bronchial ciliated epithelial cells cells freshly isolated from lungs with different cystic fibrosis genotypes (F508del/F508del-CFTR (n54), F508del/R1066C (n57) and F508del/H1085R-CFTR (n59)). Login to comment

[hide] Benzbromarone stabilizes DeltaF508 CFTR at the cel... Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3. Loo TW, Bartlett MC, Clarke DM
Benzbromarone stabilizes DeltaF508 CFTR at the cell surface.
Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3., 2011-05-31 [PMID:21520952]

Abstract [show]
Deletion of Phe508 from the first nucleotide-binding domain of the CFTR chloride channel causes cystic fibrosis because it inhibits protein folding. Indirect approaches such as incubation at low temperatures can partially rescue DeltaF508 CFTR, but the protein is unstable at the cell surface. Here, we show that direct binding of benzbromarone to the transmembrane domains promoted maturation and stabilized DeltaF508 CFTR because its half-life at the cell surface was ~10-fold longer than that for low-temperature rescue. Therefore, a search for small molecules that can rescue and stabilize DeltaF508 CFTR could lead to the development of an effective therapy for cystic fibrosis.

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22 The H1085R mutation is located in TMD2 within the intracellular loop (ICL) connecting TM10 and TM11.24 Mature protein was observed when H1085R CFTR was expressed in the presence of 38À100 μM benzbromarone (Figure 1A). Login to comment
26 To test if the CFTR mutants were active after benzbromarone rescue, we performed iodide efflux assays on BHK cells stably expressing ΔF508, H1085R, or wild-type CFTR proteins. Login to comment
27 It was found that both ΔF508 and H1085R exhibited forskolin-activated iodide efflux after rescue with benzbromarone (Figure 1B). Login to comment
47 (A) Immunoblot analysis of cells expressing CFTR mutant ΔF508 or H1085R or the P-gp G251V processing mutant after treatment with the indicated concentrations of benzbromarone (Benz) for 40 h. Login to comment
48 (B) Iodide efflux assays performed on BHK cells stably expressing wild-type, ΔF508, or H1085R CFTR. Login to comment

[hide] Repair of CFTR folding defects with correctors tha... Methods Mol Biol. 2011;741:23-37. Loo TW, Clarke DM
Repair of CFTR folding defects with correctors that function as pharmacological chaperones.
Methods Mol Biol. 2011;741:23-37., [PMID:21594776]

Abstract [show]
The major cause of cystic fibrosis is the presence of processing mutations in CFTR (such as deletion of Phe-508 (F508del-CFTR)) that disrupt folding of the protein and trafficking to the cell surface. Processing mutations appear to inhibit folding of CFTR so that it accumulates in the endoplasmic reticulum as a partially folded protein. Expressing the proteins in the presence of small molecules called correctors can repair CFTR folding defects. Some correctors appear to function as pharmacological chaperones that specifically bind to the CFTR processing mutants and induce them to complete the folding process. In this chapter, we describe techniques to examine the effects of correctors on folding of CFTR processing mutants.

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147 HEK 293 cells expressing the CFTR processing mutants, F508del, Q1071P, or H1085R, were incubated with media containing no corrector (-) or 0.05 mM VX-325 plus 0.015 mM corr-4a (+). 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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52 Mutation 0.46-0.35 9 c.1078delT #, p.R347P # 8 p.G85V, c.621 + 1G > T #, p.S549R (T > G) #, p.R553X #, c.3849 + 10kbC > T # 7 p.R347H #, c.1812-1G > A, p.R709X 0.30-0.10 6 p.H199Y, p.P205S, 5 p.R117H #, p.G551D #, p.W1089X, p.Y1092X, CFTR50kbdel 4 c.296 + 3insT, c.1717-1G > A #, c.1949del84, c.3849 + 1G > A 3 p.E92K, c.936delTA, c.1717-8G > A, c.1341G > A, p.A561E, c.2603delT, p.G1244E, [p.D1270N; p.R74W] 2 p.Q2X, p.P5L, CFTRdele2,3, p.S50P, p.E60K, c.405 + 1G > A, c.1677delTA, p.L558S, p.G673X, p.R851X, p.Y1014C, p.Q1100P, p.M1101K, p.D1152H, CFTRdele19, p.G1244V, p.Q1281X, p.Y1381X <0,1 1 c.124del23bp, p.Q30X, p.W57X, c.406-1G > A, p.Q98R, p.E115del, c.519delT, p.L159S, c.711 + 3A > T, p.W202X, c.875 + 1G > A, p.E278del, p.W361R, c.1215delG, p.L365P, p.A399D, c.1548delG, p.K536X, p.R560G, c.1782delA, p.L571S, [p.G576A; p.R668C], p.T582R, p.E585X, c.1898 + 1G > A, c.1898 + 3A > G, c.2051delTT, p.E692X, p.R851L, c.2711delT, c.2751 + 3A > G, c.2752-26A > G, p.D924N, p.S945L, c.3121-1G > A, p.V1008D, p.L1065R, [p.R1070W; p.R668C], [p.F1074L; 5T], p.H1085R, p.R1158X, c.3659delC #, c.3667del4, c.3737delA, c.3860ins31, c.3905insT #, c.4005 + 1G > A, p.T1299I, p.E1308X, p.Q1313X, c.4095 + 2T > A, rearrangements study (n = 4) Mutations identified in CF families with mixed European origin: c.182delT, p.L1254X, c.4010del4. 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
67 To test the effect of P-gp on rescue of CFTR, cells were cotransfected with the H1085R CFTR processing mutant plus wild-type P-gp or an inactive P-gp mutant containing mutations to the catalytic carboxylate residues (E556Q/E1201Q) [26]. 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
185 Fig. 8. Effect of wild-type P-gp expression (+P-gp) on rescue of CFTR mutant H1085R with corrector KM11060. 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
210 3.4. Effect of P-gp expression on rescue of CFTR mutant H1085R We predicted that the efficiency of CFTR rescue with correctors would be reduced if the correctors were also substrates of drug pumps such as P-gp. Login to comment
215 To test if P-gp expression would alter CFTR rescue with KM11060, mutant H1085R was expressed in the presence of various concentrations of KM11060 in the presence of an inactive P-gp containing mutations to the catalytic carboxylate residues (E556Q/E1201Q) in the nucleotide binding domains [26] or in the presence of wild-type P-gp. Login to comment
216 CFTR mutant H1085R was tested because ithas a higher efficiency of rescue with correctors compared to DF508 CFTR [16] and the mature protein is more stable (Fig. 7). Login to comment
219 The R50 for KM11060 when H1085R CFTR was expressed with noexogenous P-gp was similar to that obtained with inactive P-gp (data not shown) suggesting that expression of P-gp did not adversely affect the cellular folding machinery. Login to comment

[hide] Multiplex ligation-dependent probe amplification i... J Mol Diagn. 2008 Jul;10(4):368-75. Epub 2008 Jun 13. Schrijver I, Rappahahn K, Pique L, Kharrazi M, Wong LJ
Multiplex ligation-dependent probe amplification identification of whole exon and single nucleotide deletions in the CFTR gene of Hispanic individuals with cystic fibrosis.
J Mol Diagn. 2008 Jul;10(4):368-75. Epub 2008 Jun 13., [PMID:18556774]

Abstract [show]
A disparity between Caucasian and Hispanic mutation detection for cystic fibrosis continues to exist, although the carrier frequency is only moderately lower in Hispanics. We aimed to identify exonic rearrangements that remained undetected by conventional methods. In seven of 32 cystic fibrosis-affected self-identified Hispanics for whom only one or no mutations were identified by extensive molecular testing, exon deletions appeared to be present with a multiplex ligation-dependent probe amplification (MLPA) assay. Two recurrent deletions (of exons 2-3 and exons 22-23) were identified in one and three patients, respectively (12.5%, 11.1% of unidentified alleles). Two apparently novel deletions (exons 6b and 20) were identified in three additional patients. Subsequent sequencing to characterize deletion breakpoints, however, identified single nucleotide deletions at the probe binding sites close to the ligation point. All resulted in false positive MLPA deletion signals. Interestingly, these mutations were not common in Caucasians, and one (935delA) was common in U.S. Hispanics. On examination of all probe binding sites, we identified a total of 76 reported mutations and five silent variants that immediately surrounded the MLPA ligation sites, with 22 occurring in non-Caucasians. These mutations are not all rare. Thus, apparent exon deletions by MLPA may indicate the presence of both large deletions and point mutations, with important implications for pan-ethnic MLPA testing in cystic fibrosis and other genetic conditions.

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92 on.ca/cftr/) and is considered a specific African-American mutation.24 Finally, mutation H1085R has been described repeatedly (http://www.genet.sickkids.on.ca/cftr/) and was identified in one homozygous Japanese patient who was born to consanguineous parents.28 Thus, of the 22 mutations described in non-Caucasian populations, at least six (27.3%) are recurrent and may be specific to non-Caucasian populations. Login to comment
112 Mutations under MLPA Ligation Sites Exon Probe length (nt) Ligation site sequence Mutations in area of ligation site sequence* 1,5Ј UTR 154 5Ј-GAGCAAAT-TTGGGGCC-3Ј N/A 1,5Ј UTR 238 5Ј-AAAGGGTT-GAGCGGCA-3Ј 2 198 5Ј-TTGGTATA-TGTCTGAC-3Ј (5) 3 136 5Ј-CTGCTAGT-GTTGCCAA-3Ј (3) 3 220 5Ј-TTCAAAGA-AAAATCCT-3Ј 4 247 5Ј-AGAATCAT-AGCTTCCT-3Ј 444delA, African; 451del8, Chinese; (6) 5 346 5Ј-AAATAAGT-ATTGGACA-3Ј Q179K, Hispanic (7) 6a 274 5Ј-GAGTTGTT-ACAGGCGT-3Ј L218X, Pakistani (4) 6b 301 5Ј-ATTTTCAA-TCATTTCT-3Ј 935delA, Hispanic; 936delTA, Hispanic (3) 7 337 5Ј-ACTTCAAT-AGCTCAGC-3Ј S307N, Turkish (9) 8, IVS 8 364 5Ј-TTTCTAGA-TTAAGAAG-3Ј N/A 9, IVS 8 391 5Ј-TCCATCAC-ACTGGTAG-3Ј N/A 10 463 5Ј-TCCACTGT-GCTTAATT-3Ј H484Y, Hispanic; S485C, Chinese-Caucasian (5) 11 418 5Ј-CAGAGAAA-GACAATAT-3Ј K536X, Iranian; 1742delAC, Japanese (5) 12, IVS 12 292 5Ј-TGCATTTT-ACCTCTTG-3Ј N/A 13 142 5Ј-CAGATTCT-GAGCAGGG-3Ј (1) 14a 160 5Ј-GTATGTGT-TCCATGTA-3Ј (3) 14b 178 5Ј-CTGCTTCT-TTGGTTGT-3Ј 2766del8, Tunisian (1) 15 204 5Ј-GCTTGCTA-TGGGATTC-3Ј (1) 16, IVS 16 229 5Ј-GATGTAAT-AGCTGTCT-3Ј N/A 17a 256 5Ј-TGCAACAA-AGATGTAG-3Ј 3171delC, Hispanic; 3173delAC, Turkish; F1016S, Hispanic (5) 17b 283 5Ј-CAGTATGT-AAATTCAG-3Ј H1085R, Japanese (4) 18 310 5Ј-CCATGAAT-ATCATGAG-3Ј M1137R, Hispanic (6) 19 353 5Ј-TCTGTGTA-TTTTGCTG-3Ј 3791delC, African-American (2) 20 382 5Ј-CTTGGGAT-TCAATAAC-3Ј 3960delA, Hispanic (2) 21 409 5Ј-TGCAACTT-TCCATATT-3Ј W1316X, African-American (2) 22 436 5Ј-GAACAGTT-TCCTGGGA-3Ј No mutations 23 148 5Ј-CCAGCATT-GCTTCTAT-3Ј M1407T, Turkish; E1409K, Hispanic (2) 24 190 5Ј-ATCCAGAA-ACTGCTGA-3Ј No mutations 24 172 5Ј-CTCCTCTT-TCAGAGCA-3Ј UTR, untranslated region; IVS, intervening sequence; N/A, not applicable, probes not in coding region; No mutations, no reported mutations are present in the area of the ligation site sequence, regardless of ethnicity. Login to comment
128 In all, at least six (444delA, 935delA, 936delTA, 2766del8, 3791delC, and H1085R) of 22 mutations identified in non-Caucasians (27.3%) are recurrent and may be specific to non-Caucasian populations. 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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107 f 306insA, W79X, R117C, P205S, L227R, I336K, 1248+1G>A, 1609delCA, 1717-8G>A, S549R(T>G), S549N, 1812-1G>A, P574H, 2176insC, R709X, E827X, D836Y, 3007delG, L1065P, L1077P, H1085R, M1101K, 4021insT. Login to comment

[hide] Effect of cystic fibrosis-associated mutations in ... J Biol Chem. 1996 Aug 30;271(35):21279-84. Cotten JF, Ostedgaard LS, Carson MR, Welsh MJ
Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1996 Aug 30;271(35):21279-84., [PMID:8702904]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.

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81 For example, levels of mature F1052V were similar to wild-type, whereas L1065P, R1070Q, and H1085R were similar to ⌬F508 in that they produced little mature protein (Fig. 2, A and B). Login to comment
222 For example, H1085R is misprocessed like ⌬F508, yet it is reported to occur in a patient with a pancreatic sufficient phenotype. Login to comment
80 For example, levels of mature F1052V were similar to wild-type, whereas L1065P, R1070Q, and H1085R were similar to DF508 in that they produced little mature protein (Fig. 2, A and B). Login to comment
221 For example, H1085R is misprocessed like DF508, yet it is reported to occur in a patient with a pancreatic sufficient phenotype. Login to comment

[hide] Disease-associated mutations in the fourth cytopla... J Biol Chem. 1996 Jun 21;271(25):15139-45. Seibert FS, Linsdell P, Loo TW, Hanrahan JW, Clarke DM, Riordan JR
Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity.
J Biol Chem. 1996 Jun 21;271(25):15139-45., [PMID:8662892]

Abstract [show]
A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.

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130 D: छ, WT; E, Q1071P; छϩ, W1098R; Ⅺ, H1085R; Ç, M1101K; µ, M1101R; Q, control; É, L1077P. Login to comment
136 D: L, WT; E, Q1071P; L 1, W1098R; M, H1085R; &#c7;, M1101K; &#b5;, M1101R; Q, control; &#c9;, L1077P. Login to comment

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

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

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105 CFTR Haplotypes for Diallelic and Multiallelic DNA Markers for 94 CF Mutations" J44-GATT- 8CA-17BTA- No. of T854-TUB20 17BCA Mutation chromosomes % Normal Laboratory Reference 2-7-1-2 17-47-13 (55.4%) 17-46-13 17-45-13 17-34-13 17-32-13 17-31-14 17-31-13 17-29-14 17-28-13 16-48-13 16-46-14 16-46-13 16-45-13 16-44-13 16-35-13 16-33-13 16-32-13 16-31-14 16-31-13 16-30-13 16-29-13 16-26-13 16-25-13 16-24-13 14-31-13 1-7-2-1 17-7-17 (16.8%) R334W R334W 3860ins31 G1244E R1162X R1162X R1162X G91R MllOlK R347P R334W R117C E92K 3849+lOkbC+T 3293delA 1811+1.6kb A-tG 1811+1.6kb A-tG 2184insA P205S 3659delC G673X 11005R I336K W58S R347P W846X 405+1-A G178R 3905insT R1162X R347H 3100insA E60X 1078delT 4005+1-A K710X 1677delTA H199Y 3601-2AjG 3850-3T+G 3272-26A-tG 3850-1-A 1812-1-A R117H L1059X S492F Y1092X Y569H 3732delA C866Y 711+1G+T 711+1-T G85E 1949del84 2789+5-A H1085R W1282X R1066C 2043delG V456F 2 1 1 1 2 1 6 2 2 1 2 1 1 2 1 1 4 1 1 1 3 2 1 1 1 1 1 1 2 7 1 1 1 1 2 1 1 3 19 3 3 1 1 2 1 1 5 1 1 1 1 3 6 3 5 1 13 2 1 1 - 0.48 0.48 - - - 0.24 - - - 2.65 2.40 1.93 2.65 1.68 2.65 0.72 13.94 13.46 1.93 - 0.72 0.24 3.37 - b b fP fP fP t b,fb.fP h fb t h t h h fP fP b.h b h h b h h h h h fb fb,fP.t fP fP fP9t fP b t fPh b h fb b.fb,h fb*fP b,fP h h t h fb fb,fp,h.t fP fP fb t b.fP,t b,fb,h,t b f b h h fb b,fb.fP,h fP h h Gasparini et al. (1991b) Chilldn et al. (1993a) Devoto et al. (1991) Gasparini et al. (1991b) Dork et al. (1993a) Guillermit et al. (1993) Zielenski et al. (1993) Dean et al. (1990) Dork et al. (1994a) Nunes et al. (1993) Highsmith et al. (1994) Ghanem et al. (1994) Chilldn et al. (1995) Dork et al. (1994a) Dork et al. (1993a) Chilldn et al. (1993b) Kerem et al. (1990) Dork et al. (1994a) Dork et al. (1994a) Cuppenset al. (1993) Fanen et al. (1992) Maggio et al. (personal communication) Audrezet et al. (1993) Vidaud et al. (1990) Dork et al. (1993b) Zielenski et al. (1991a) Chilldn et al. (1994b) Malik et al. (personal communication) Cremonesi et at. Login to comment

[hide] Increased incidence of cystic fibrosis gene mutati... Hum Mol Genet. 1995 Apr;4(4):635-9. Pignatti PF, Bombieri C, Marigo C, Benetazzo M, Luisetti M
Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis.
Hum Mol Genet. 1995 Apr;4(4):635-9., [PMID:7543317]

Abstract [show]
In order to identify a possible hereditary predisposition to the development of obstructive pulmonary disease of unknown origin, we have looked for the presence of Cystic Fibrosis Transmembrane Regulator (CFTR) gene mutations in unrelated patients with no signs of Cystic Fibrosis (CF). We screened for 70 common mutations, and also for rare mutations by denaturing gradient gel electrophoresis analysis. In this search, different CFTR gene mutations (R75Q, delta F508, R1066C, M1137V and 3667ins4) were found in five out of 16 adult Italian patients with disseminated bronchiectasis, a significant increase over the expected frequency of carriers. Moreover, three rare CFTR gene DNA polymorphisms (G576A, R668C, and 2736 A-->G), not deemed to be the cause of CF, were found in two patients, one of which was a compound heterozygote with R1066C. These results indicate that CFTR gene mutations, and perhaps also DNA polymorphisms, may be involved in the etiopathogenesis of at least some cases of bronchiectasis.

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31 List of CFTR gene mutations and DNA polymorphisms screened Mutations R75Q/X/L, G85E, 394deITT 457TAT->G, R117H 621 + 1G->T 711 + 5G->A L206W 875 + 40 A->G 936 del TA 1001 + 11C->T R334W, R347 P/H/L, 1154insTC A455E, V456F DF5O8 1717-IG->A, 1717-8G->A G542X, G551D, Q552X, R553X P574H 1898 + 3A->G 2183 AA->G, 2184delA, R709X D836Y, 2694 T/G 2752-22 A/G 2789 + 5 G->A, 2790-2 A-»G Q890X 3041-71 G/C 3132delTG 3271 + 18 C-»T, 3272-26 A->G H1054D, G1061R, R1066C/H, A1067T, H1085R, Y1092X, 3320 ins5 D1152H R1162X, 3667ins4, 3737delA, 11234V 3849 + 10 kb C-»T, 3850-1 G-»A SI25IN, S1255P, 3905insT, 3898insC, D127ON, W1282X, R1283M, 4002 A/G 4005 + 1 G-»A N1303 K/H, 4029 A/G D1377H Q1411 X 4404 C/T, 4521 G/A Location e 3 e 4 i 4 i 5 e 6a i 6a e 6b i 6b e 7 e 9 e 10 i 10 e 11 e 12 i 12 e 13 e 14a i 14a i 14b e 15 i 15 e 17a i 17a e 17b e 18 e 19 i 19 e 20 i 20 e2l e 22 e 23 e24 Listing is in order of location along the CFTR gene, e = exon; i = intron. Login to comment

[hide] A cluster of cystic fibrosis mutations in exon 17b... J Med Genet. 1994 Sep;31(9):731-4. Mercier B, Lissens W, Novelli G, Kalaydjieva L, de Arce M, Kapranov N, Canki Klain N, Estivill X, Palacio A, Cashman S, et al.
A cluster of cystic fibrosis mutations in exon 17b of the CFTR gene: a site for rare mutations.
J Med Genet. 1994 Sep;31(9):731-4., [PMID:7529319]

Abstract [show]
Intensive screening has improved our understanding of the profile of mutations in the CFTR gene in which more than 400 mutations have been detected to date. In collaboration with several European laboratories we are involved in such analysis. We have identified 14 new mutations in exon 17b of CFTR, having analysed 780 CF chromosomes, and have compared the frequency of mutations in this exon with that of other regions of the CFTR gene. The results obtained indicate an accumulation of mutations, not only in regions encoding the two nucleotide binding folds, but also in those encoding transmembrane domains of the CFTR gene, in particular exon 17b.

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19 Most of these are missense mutations and as no functional test has been 732 Table 1 Mutations identified in exon 17b of the CFTR gene Mutation Nucleotide Modificationl Ethnic Rcferencesposition ongini (No) 3271-1 G--A 3272-1 G-A Belgian (1) 11F1052V 3286 T-G Belgian (1) 11HI054D 3292 CG French (1) 13G1061R 3313 G-C French (1) 113320 Dup 3320 Duplication of Breton (1) 6 CTATG R1066C 3328 CT French (1) 14 R1066L R1066H A1067T G1069R R1070Q 3359 del CT L1077P H1085R W1089X Y1092X M1IOIR 3329 3329 3331 3337 3341 G-T G-+A G-A G,A G--A 3359 3362 3386 3398 3408 3434 del CT T--C A-.G G-+A C +A T--G Spanish (5) French (1) Breton (1) Breton (1) Bulgarian (1) Bulgarian (3) Rumanian (1) Albanian (1) French (1) Italian (1) French (1) Spanish (1) French (4) Turkish (1I) * Bozon et al, personal communication. Login to comment

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

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

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

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

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

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

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

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

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

[hide] 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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64 Arginine point mutations on the predicted aqueous face of TM6 (positions 338, 341, 344, 345, 348, 351) were introduced into processing mutants DF508, G232D, and H1085R. Login to comment
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
183 This result shows that the V510D and H1085R mutants could indeed be directly rescued by a suppressor mutation. 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] Effect of ivacaftor on CFTR forms with missense mu... J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23. Van Goor F, Yu H, Burton B, Hoffman BJ
Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23., [PMID:23891399]

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

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42 For example, severe processing mutations such as F508del-, R1066H-, and H1085R-CFTR exhibited a low level of mature CFTR (b5% normal). Login to comment
44 None M1V A46D E56K P67L R74W G85E E92K D110E D110H R117C R117H E193K L206W R334W I336K T338I S341P R347H R347P R352Q A455E L467P S492F F508del V520F A559T R560S R560T A561E Y569D D579G R668C L927P S945L S977F L997F F1052V H1054D K1060T L1065P R1066C R1066H R1066M A1067T R1070Q R1070W F1074L L1077P H1085R M1101K D1152H S1235R D1270N N1303K 0 100 200 300 400 500 600 * * * CFTR Mutation mRNA (% Normal CFTR) Fig. 1. Login to comment
64 Mutant CFTR form CFTR processing Mature/total % Normal CFTR Normal 0.89 &#b1; 0.01 100.0 &#b1; 18.5 G85E -0.05 &#b1; 0.04 -1.0 &#b1; 0.9 R560S 0.00 &#b1; 0.00 0.0 &#b1; 0.0 R1066C 0.02 &#b1; 0.01 0.0 &#b1; 0.0 S492F 0.00 &#b1; 0.00 0.1 &#b1; 0.1 R560T 0.01 &#b1; 0.01 0.2 &#b1; 0.1 V520F 0.05 &#b1; 0.03 0.3 &#b1; 0.2 M1101K 0.05 &#b1; 0.03 0.3 &#b1; 0.1 A561E 0.08 &#b1; 0.04 0.5 &#b1; 0.2 R1066M 0.02 &#b1; 0.02 0.5 &#b1; 0.4 N1303K 0.02 &#b1; 0.02 0.5 &#b1; 0.3 A559T 0.16 &#b1; 0.09 0.6 &#b1; 0.2 M1V 0.06 &#b1; 0.06 0.7 &#b1; 0.6 Y569D 0.11 &#b1; 0.04 0.6 &#b1; 0.2 R1066H 0.08 &#b1; 0.02a 0.7 &#b1; 0.2a L1065P 0.05 &#b1; 0.05 1.0 &#b1; 0.8 L467P 0.10 &#b1; 0.07 1.2 &#b1; 0.8 L1077P 0.08 &#b1; 0.04 1.5 &#b1; 0.6 A46D 0.21 &#b1; 0.08 1.9 &#b1; 0.5a E92K 0.06 &#b1; 0.05 1.9 &#b1; 1.3 H1054D 0.09 &#b1; 0.04 1.9 &#b1; 0.8 F508del 0.09 &#b1; 0.02a 2.3 &#b1; 0.5a H1085R 0.06 &#b1; 0.01a 3.0 &#b1; 0.7a I336K 0.42 &#b1; 0.05a 6.5 &#b1; 0.7a L206W 0.35 &#b1; 0.10a 6.8 &#b1; 1.7a F1074L 0.52 &#b1; 0.03a 10.9 &#b1; 0.6a A455E 0.26 &#b1; 0.10a 11.5 &#b1; 2.5a E56K 0.29 &#b1; 0.04a 12.2 &#b1; 1.5a R347P 0.48 &#b1; 0.04a 14.6 &#b1; 1.8a R1070W 0.61 &#b1; 0.04a 16.3 &#b1; 0.6a P67L 0.36 &#b1; 0.04a 28.4 &#b1; 6.8a R1070Q 0.90 &#b1; 0.01a 29.5 &#b1; 1.4a S977F 0.97 &#b1; 0.01a 37.3 &#b1; 2.4a A1067T 0.78 &#b1; 0.03a 38.6 &#b1; 6.1a D579G 0.72 &#b1; 0.02a 39.3 &#b1; 3.1a D1270N 1.00 &#b1; 0.00a,c 40.7 &#b1; 1.2a S945L 0.65 &#b1; 0.04a 42.4 &#b1; 8.9a L927P 0.89 &#b1; 0.01a,b 43.5 &#b1; 2.5a,b R117C 0.87 &#b1; 0.02a,b 49.1 &#b1; 2.9a,b T338I 0.93 &#b1; 0.03a,b 54.2 &#b1; 3.7a,b L997F 0.90 &#b1; 0.04a,b 59.8 &#b1; 10.4a,b D110H 0.97 &#b1; 0.01a,b 60.6 &#b1; 1.5a,b S341P 0.79 &#b1; 0.02a 65.0 &#b1; 4.9a,b R668C 0.94 &#b1; 0.03a,b 68.5 &#b1; 1.9a,b R74W 0.78 &#b1; 0.01a 69.0 &#b1; 2.7a,b D110E 0.92 &#b1; 0.05a,b 87.5 &#b1; 9.5a,b R334W 0.91 &#b1; 0.05a,b 97.6 &#b1; 10.0a,b K1060T 0.87 &#b1; 0.02a,b 109.9 &#b1; 28.0a,b R347H 0.96 &#b1; 0.02a,c 120.7 &#b1; 2.8a,b S1235R 0.96 &#b1; 0.00a,c 139.0 &#b1; 9.0a,b E193K 0.84 &#b1; 0.02a,b 143.0 &#b1; 17.1a,b R117H 0.86 &#b1; 0.01a,b 164.5 &#b1; 34.2a,b R352Q 0.98 &#b1; 0.01a,b 179.9 &#b1; 8.0a,c F1052V 0.90 &#b1; 0.01a,b 189.9 &#b1; 33.1a,b D1152H 0.96 &#b1; 0.02a,c 312.0 &#b1; 45.5a,b Notes to Table 1: Quantification of steady-state CFTR maturation expressed as the mean (&#b1;SEM; n = 5-9) ratio of mature CFTR to total CFTR (immature plus mature) or level of mature mutant CFTR relative to mature normal-CFTR (% normal CFTR) in FRT cells individually expressing CFTR mutations. Login to comment
74 Because the level of CFTR mRNA was similar across the panel of cell lines tested, the range in baseline activity and ivacaftor response likely reflects the severity of the functional defect and/or the 0 50 100 150 200 S341P R347P L467P S492F A559T A561E Y569D L1065P R1066C R1066M L1077P M1101K N1303K R560S L927P R560T H1085R V520F E92K M1V F508del H1054D I336K A46D G85E R334W T338I R1066H R352Q R117C L206W R347H S977F S945L A455E F1074L E56K P67L R1070W D110H D579G D110E R1070Q L997F A1067T E193K R117H R74W K1060T R668C D1270N D1152H S1235R F1052V Baseline With ivacaftor * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Chloride transport (% Normal) Mutant CFTR form 0 100 200 300 400 S341P R347P L467P S492F A559T A561E Y569D L1065P R1066C R1066M L1077P M1101K N1303K R560S L927P R560T H1085R V520F E92K M1V F508del H1054D I336K A46D G85E R334W T338I R1066H R352Q R117C L206W R347H S977F S945L A455E F1074L P67L E56K R1070W D110H D579G D110E R1070Q L997F A1067T E193K R117H R74W K1060T R668C D1270N D1152H S1235R F1052V * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mature CFTR (% Normal) Mutant CFTR form A B Fig. 2. Login to comment
82 Mutation Patientsa Chloride transport (bc;A/cm2 ) Chloride transport (% normal) EC50 Baseline With ivacaftor Baseline With ivacaftor Fold increase over baselineb Normal 204.5 &#b1; 33.3 301.3 &#b1; 33.8c 100.0 &#b1; 16.3 147.3 &#b1; 16.5c 1.5 266 &#b1; 42 G551D 1282 1.5 &#b1; 0.7 113.2 &#b1; 13.0c 1.0 &#b1; 0.5 55.3 &#b1; 6.3c 55.3 312 &#b1; 73 F1052V 12 177.3 &#b1; 13.7 410.2 &#b1; 11.3c 86.7 &#b1; 6.7 200.7 &#b1; 5.6c 2.3 177 &#b1; 14 S1235R ND 160.6 &#b1; 25.7 352.1 &#b1; 43.4c 78.5 &#b1; 12.6 172.2 &#b1; 21.2c 2.2 282 &#b1; 104 D1152H 185 117.3 &#b1; 23.0 282.7 &#b1; 46.9c 57.4 &#b1; 11.2 138.2 &#b1; 22.9c 2.4 178 &#b1; 67 D1270N 32 109.5 &#b1; 20.5 209.5 &#b1; 27.4c 53.6 &#b1; 10.0 102.4 &#b1; 13.4c 1.9 254 &#b1; 56 R668C 45 99.0 &#b1; 9.4 217.6 &#b1; 11.7c 48.4 &#b1; 4.6 106.4 &#b1; 5.7c 2.2 517 &#b1; 105 K1060T ND 89.0 &#b1; 9.8 236.4 &#b1; 20.3c 43.5 &#b1; 4.8 115.6 &#b1; 9.9c 2.7 131 &#b1; 73 R74W 25 86.8 &#b1; 26.9 199.1 &#b1; 16.8c 42.5 &#b1; 13.2 97.3 &#b1; 8.2c 2.3 162 &#b1; 17 R117H 739 67.2 &#b1; 13.3 274.1 &#b1; 32.2c 32.9 &#b1; 6.5 134.0 &#b1; 15.7c 4.1 151 &#b1; 14 E193K ND 62.2 &#b1; 9.8 379.1 &#b1; 1.1c 30.4 &#b1; 4.8 185.4 &#b1; 1.0c 6.1 240 &#b1; 20 A1067T ND 55.9 &#b1; 3.2 164.0 &#b1; 9.7c 27.3 &#b1; 1.6 80.2 &#b1; 4.7c 2.9 317 &#b1; 214 L997F 27 43.7 &#b1; 3.2 145.5 &#b1; 4.0c 21.4 &#b1; 1.6 71.2 &#b1; 2.0c 3.3 162 &#b1; 12 R1070Q 15 42.0 &#b1; 0.8 67.3 &#b1; 2.9c 20.6 &#b1; 0.4 32.9 &#b1; 1.4c 1.6 164 &#b1; 20 D110E ND 23.3 &#b1; 4.7 96.4 &#b1; 15.6c 11.4 &#b1; 2.3 47.1 &#b1; 7.6c 4.1 213 &#b1; 51 D579G 21 21.5 &#b1; 4.1 192.0 &#b1; 18.5c 10.5 &#b1; 2.0 93.9 &#b1; 9.0c 8.9 239 &#b1; 48 D110H 30 18.5 &#b1; 2.2 116.7 &#b1; 11.3c 9.1 &#b1; 1.1 57.1 &#b1; 5.5c 6.2 249 &#b1; 59 R1070W 13 16.6 &#b1; 2.6 102.1 &#b1; 3.1c 8.1 &#b1; 1.3 49.9 &#b1; 1.5c 6.2 158 &#b1; 48 P67L 53 16.0 &#b1; 6.7 88.7 &#b1; 15.7c 7.8 &#b1; 3.3 43.4 &#b1; 7.7c 5.6 195 &#b1; 40 E56K ND 15.8 &#b1; 3.1 63.6 &#b1; 4.4c 7.7 &#b1; 1.5 31.1 &#b1; 2.2c 4.0 123 &#b1; 33 F1074L ND 14.0 &#b1; 3.4 43.5 &#b1; 5.4c 6.9 &#b1; 1.6 21.3 &#b1; 2.6c 3.1 141 &#b1; 19 A455E 120 12.9 &#b1; 2.6 36.4 &#b1; 2.5c 6.3 &#b1; 1.2 17.8 &#b1; 1.2c 2.8 170 &#b1; 44 S945L 63 12.3 &#b1; 3.9 154.9 &#b1; 47.6c 6.0 &#b1; 1.9 75.8 &#b1; 23.3c 12.6 181 &#b1; 36 S977F 9 11.3 &#b1; 6.2 42.5 &#b1; 19.1c 5.5 &#b1; 3.0 20.8 &#b1; 9.3c 3.8 283 &#b1; 36 R347H 65 10.9 &#b1; 3.3 106.3 &#b1; 7.6c 5.3 &#b1; 1.6 52.0 &#b1; 3.7c 9.8 280 &#b1; 35 L206W 81 10.3 &#b1; 1.7 36.4 &#b1; 2.8c 5.0 &#b1; 0.8 17.8 &#b1; 1.4c 3.6 101 &#b1; 13 R117C 61 5.8 &#b1; 1.5 33.7 &#b1; 7.8c 2.9 &#b1; 0.7 16.5 &#b1; 3.8c 5.7 380 &#b1; 136 R352Q 46 5.5 &#b1; 1.0 84.5 &#b1; 7.8c 2.7 &#b1; 0.5 41.3 &#b1; 3.8c 15.2 287 &#b1; 75 R1066H 29 3.0 &#b1; 0.3 8.0 &#b1; 0.8c 1.5 &#b1; 0.1 3.9 &#b1; 0.4c 2.6 390 &#b1; 179 T338I 54 2.9 &#b1; 0.8 16.1 &#b1; 2.4c 1.4 &#b1; 0.4 7.9 &#b1; 1.2c 5.6 334 &#b1; 38 R334W 150 2.6 &#b1; 0.5 10.0 &#b1; 1.4c 1.3 &#b1; 0.2 4.9 &#b1; 0.7c 3.8 259 &#b1; 103 G85E 262 1.6 &#b1; 1.0 1.5 &#b1; 1.2 0.8 &#b1; 0.5 0.7 &#b1; 0.6 NS NS A46D ND 2.0 &#b1; 0.6 1.1 &#b1; 1.1 1.0 &#b1; 0.3 0.5 &#b1; 0.6 NS NS I336K 29 1.8 &#b1; 0.2 7.4 &#b1; 0.1c 0.9 &#b1; 0.1 3.6 &#b1; 0.1c 4 735 &#b1; 204 H1054D ND 1.7 &#b1; 0.3 8.7 &#b1; 0.3c 0.8 &#b1; 0.1 4.2 &#b1; 0.1c 5.3 187 &#b1; 20 F508del 29,018 0.8 &#b1; 0.6 12.1 &#b1; 1.7c 0.4 &#b1; 0.3 5.9 &#b1; 0.8c 14.8 129 &#b1; 38 M1V 9 0.7 &#b1; 1.4 6.5 &#b1; 1.9c 0.4 &#b1; 0.7 3.2 &#b1; 0.9c 8.0 183 &#b1; 85 E92K 14 0.6 &#b1; 0.2 4.3 &#b1; 0.8c 0.3 &#b1; 0.1 2.1 &#b1; 0.4c 7.0 198 &#b1; 46 V520F 58 0.4 &#b1; 0.2 0.5 &#b1; 0.2 0.2 &#b1; 0.1 0.2 &#b1; 0.1 NS NS H1085R ND 0.3 &#b1; 0.2 2.1 &#b1; 0.4 0.2 &#b1; 0.1 1.0 &#b1; 0.2 NS NS R560T 180 0.3 &#b1; 0.3 0.5 &#b1; 0.5 0.1 &#b1; 0.1 0.2 &#b1; 0.2 NS NS L927P 15 0.2 &#b1; 0.1 10.7 &#b1; 1.7c 0.1 &#b1; 0.1 5.2 &#b1; 0.8c 52.0 313 &#b1; 66 R560S ND 0.0 &#b1; 0.1 -0.2 &#b1; 0.2 0.0 &#b1; 0.0 -0.1 &#b1; 0.1 NS NS N1303K 1161 0.0 &#b1; 0.0 1.7 &#b1; 0.3 0.0 &#b1; 0.0 0.8 &#b1; 0.2 NS NS M1101K 79 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS L1077P 42 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R1066M ND 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R1066C 100 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS L1065P 25 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS Y569D 9 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS A561E ND 0.0 &#b1; 0.1 0.0 &#b1; 0.1 0.0 &#b1; 0.0 0.0 &#b1; 0.1 NS NS A559T 43 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS S492F 16 0.0 &#b1; 0.0 1.7 &#b1; 1.2 0.0 &#b1; 0.0 0.8 &#b1; 0.6 NS NS L467P 16 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R347P 214 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS S341P 9 0.0 &#b1; 0.0 0.2 &#b1; 0.2 0.0 &#b1; 0.0 0.1 &#b1; 0.1 NS NS a Number of individuals with the individual mutation in the CFTR-2 database (www.CFTR2.org). Login to comment

[hide] 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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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
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
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