ABCMdb

PMID: 15776432

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

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp The aim of this study was to determine the mechanism of dysfunction of a disease-causing mutation associated with variable phenotypes. In order to attain these objectives, we studied the effect of the p.L206W mutation on CFTR protein production and function, and we examined the genotype-phenotype correlation of [p.L206W]+[p.F508del] patients. Login to comment 4 ABCC7 p.Leu206Trp We showed that p.L206W is a processing (class II) mutation since the CFTR biosynthetic pathway was severely impaired, whereas single-channel measurements indicated ion conductance similar to the wild-type protein. Login to comment 19 ABCC7 p.Leu206Trp Individuals bearing p.L206W in Received 3 August 2004; accepted revised manuscript 30 November 2004. n Correspondence to: Pascale Fanen, INSERM U.468, Ho" pital Henri Mondor,94010 Cre¤ teil, France. Login to comment 31 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp The aim of this study was to determine the mechanism of dysfunction of a disease-causing mutation that can result in such variable phenotypes. In order to attain these objectives, we studied the effect of the p.L206W mutation on CFTR protein production and function, and we examined the genotype-phenotype correlation of [p.L206W]+[p.F508del] patients. Login to comment 32 ABCC7 p.Leu206Trp These studies showed that p.L206W is a class II but mild mutation. Login to comment 33 ABCC7 p.Leu206Trp MATERIALS AND METHODS Patients We selected the patients with p.L206W mutation for this study among patients having well documented CF (n=668) or CBAVD (n=162), who were referred to our CF diagnosis center. Login to comment 39 ABCC7 p.Leu206Trp Segregation of the p.L206W mutation was studied in the families by StyI restriction analysis. Login to comment 41 ABCC7 p.Leu206Trp Intragenic haplotypes were determined by studying three microsatellites, IVS8(CA), IVS17b (TA), and IVS17b(CA) [Morral et al., 1992], in the patients having a p.L206W allele and, when possible, in their parents. Login to comment 42 ABCC7 p.Leu206Trp Relation Between Genotype and Phenotype We extracted from the French CF registry all the patients who attended at least once in a participating care center during 1999 or 2000 and for whom the genotype was composed of the p.L206W and p.F508del mutations. Login to comment 50 ABCC7 p.Leu206Trp For statistical comparison of clinical measures, patients heterozygous [p.L206W]+[p.F508del] were compared with those homozygous for p.F508del. Login to comment 102 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp RESULTS Genotype Data Among CF PatientsWith L206W Mutation Among the 668 French CF families and 162 CBAVD patients included in our study, 12 patients were identified as heterozygotes for the p.L206W mutation. Login to comment 103 ABCC7 p.Leu206Trp Table 1 summarizes the CFTR genotype data among the 12 patients with the p.L206W mutation. Login to comment 104 ABCC7 p.Leu206Trp Because p.L206W was found to be associated with one single haplotype in four patients previously described [Desgeorges et al., 1995], we also analyzed three intragenic microsatellites IVS8(CA), IVS17b(TA), and IVS17b(CA), the variable IVS8 sequence (TG)m(T)n, and the 1540A4G (M470V) common variant. Login to comment 105 ABCC7 p.Leu206Trp All p.L206W chromosomes had the 1540A and the (TG)9(T)9 polymorphisms, six had the true [IVS8(CA)16;IVS17b(TA) 7;IVS17b(CA)17] haplotype defined by the three intragenic microsatellites, whereas six had a ''possible`` [IVS8(CA) 16;IVS17b(TA)7;IVS17b(CA)17] haplotype (see Table 1 for explanations). Login to comment 106 ABCC7 p.Leu206Trp Our data together with the study of Desgeorges et al. [1995] showed that the p.L206W mutation was associated with one haplotype using this set of polymorphic sites. Login to comment 107 ABCC7 p.Leu206Trp A more extensive study with seven diallelic and three multiallelic markers describes a second p.L206W-associated haplotype, as the result of recombination [Claustres et al., 1996]. Login to comment 108 ABCC7 p.Leu206Trp The p.L206W mutation was found to account for 0.6% of the CF genes identified in our specialized center, and 0.9% in CBAVD patients. Login to comment 110 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Relation Between Genotype and Phenotype To investigate the conditions associated with the p.L206W mutation, we first described the clinical data of 12 patients with the p.L206W mutation identified in our center, compared with 24 cases for which clinical data are available in the literature. Login to comment 111 ABCC7 p.Leu206Trp Table 2 summarizes the clinical features and CFTR genotype in p.L206W patients. Login to comment 112 ABCC7 p.Gly542* ABCC7 p.Leu206Trp ABCC7 p.Glu60* ABCC7 p.Arg851* ABCC7 p.Trp216* For most patients (30/36), p.L206W was combined with a severe mutation (p.F508del, p.I507del, p.G542X, p.W216X, p.R851X, and p.E60X) on the other CFTR allele. Login to comment 114 ABCC7 p.Leu206Trp The patient conditions varied from isolated CBAVD to severe CF, even within the same genotype [p.L206W]+[p.F508del]. Login to comment 115 ABCC7 p.Leu206Trp Interestingly, one newly identified CBAVD patient had 5(T) adjacent to 12 (TG) repeats at the IVS8(TG)m(T)n locus in trans with p.L206W, consistent with the recent demonstration that the (TG)12(T)5 allele is associated with disease penetrance [Groman et al., 2004]. Login to comment 117 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp All patients presenting with pulmonary outcomes had the [p.L206W]+[p.F508del] or [p.L206W]+[p.I507del] genotype, except one [p.L206W]+[?] Login to comment 119 ABCC7 p.Leu206Trp The phenotypes of the 12 patients identified in the present study did not differ from those previously described in the literature and suggest that the p.L206W mutation is mostly associated with a mild CF or CBAVD phenotype. Login to comment 120 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp To examine the relationship between morbidity and the p.L206W mutation further, we performed a multicenter cross-sectional study, in which every CF patient with the [p.L206W]+[p.F508del] genotype was matched by age and sex to a p.F508del homozygote from the same center in France. Login to comment 123 ABCC7 p.Leu206Trp Patients with the [p.L206W]+[p.F508del] genotype had a significantly better nutritional status and absence of diarrhea or meconium ileus at diagnosis. Login to comment 124 ABCC7 p.Leu206Trp Pancreatic insufficiency occurred very rarely (1/12) and the lower frequency of Pseudomonas aeruginosa lung colonization explains the absence of i.v. antibiotic therapy in the compound heterozygous group for p.L206W. Login to comment 126 ABCC7 p.Leu206Trp The mean age at diagnosis was borderline significant (P=0.057) when diagnosis based on neonatal screening was excluded: 13.7 years for [p.L206W]+[p.F508del] patients and 3.2 years for p.F508del homozygotes. Login to comment 127 ABCC7 p.Leu206Trp Altogether, these results indicate milder CF disease in patients carrying the [p.L206W]+[p.F508del] genotype. Login to comment 128 ABCC7 p.Leu206Trp Based on the pancreatic sufficient phenotype (>90% of patients), the p.L206W mutation should be considered as a mild mutation. Login to comment 129 ABCC7 p.Leu206Trp Processing of CFTR Mutants To determine why patients with the p.L206W-CFTR allele suffered from mild CF, we first studied the maturation of CFTR in HeLa cells transiently transfected with cDNA encoding the wild-type and mutated CFTR proteins. Login to comment 134 ABCC7 p.Leu206Trp Immunoprecipitation experiments at steady-state show that both wild-type and CF-associated p.L206W CFTR produced mature, fully glycosylated protein (band C), while none of the mock-transfected cells produced CFTR (Fig. 1A). Login to comment 139 ABCC7 p.Leu206Trp By using a classical 15-minute radioactive metabolic labeling of proteins, the kinetics of wild-type and p.L206W core-glycosylated forms of CFTR were identical, whereas the mature band C of the mutant was not detected at any time of a 4-hr chase (Fig. 2A and B) and a even 24-hr chase (data not shown). Login to comment 142 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Arg851* CFTR Genotype of CF PatientsWith p.L206W Patient Genotype 15404Ga IVS8(TG)m(T)n a Haplotypeb 2296 [p.L206W]+[p.R851X] [1540A]+[1540G] [(TG)9(T)9]+[(TG)11(T)7] [16;7;17]+[16;31;14] 1929 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;31;13]c 1749 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[? Login to comment 143 ABCC7 p.Gly542* ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Glu60* ]c 179 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;31;13] 422 [p.L206W]+[p.G542X] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;33;13] 1720 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[17;32;13]c 1878 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[17;31;13]c 626 [p.L206W]+[p.E60X] [1540A]+[1540G] [(TG)9(T)9]+[(TG)11(T)7] [16;7;17]+[16;31;13] 1455 [p.L206W]+[1342-6(T)5]] [1540A]+[1540G] [(TG)9(T)9]+[(TG)12(T)5] [16;7;17]+[16;31;14]c 2104 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;31;13]c 652 [p.L206W]+[p.E216X] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;32;13] 2345 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[17;32;13] a The DNA and mutation numbering follows the CFTR mutation database, (www.genet.sickkids.on.ca/cftr), the A of the ATG translation start codon being numbered +133 (GeneBank NM_000492.2).We followed the approved nomenclature format in mutation names at the protein level and in genotype writing. Login to comment 145 ABCC7 p.Leu206Trp c The segregation has not been studied in the family; the suggested genotype indicates a possible [IVS8(CA)16;IVS17b(TA)7;IVS17b(CA)17] haplotype linked to p.L206W, the other haplotype having been described linked to the second mutation [Morral et al.,1993]. Login to comment 146 ABCC7 p.Leu206Trp TABLE2.ClinicalDataAmongPatientsWithp.L206W GenotypeClassa Ageat diagnosis(yr) Current age(yr)Sex Genital statusb Pancreatic status Pulmonary outcomes SweatCl(mEq/liter) mean7sd(n)Reference [p.L206W]+[p.F508del]II/II523FPSMild6171(2)Desgeorgesetal. Login to comment 147 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II1525FOUPSAsthma190710(2)Desgeorgesetal. Login to comment 148 ABCC7 p.Gly542* ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.G542X]II/I2237MCBAVDPS^c 65711(3)Desgeorgesetal. Login to comment 149 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.1507del]II/II3448MCBAVDPSInfection60710(3)Desgeorgesetal. Login to comment 150 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II^^^^PSYes460Bernardinoetal. Login to comment 151 ABCC7 p.Leu206Trp [2000] [p.L206W]+[?]II/-^^^^PSYes460Bernardinoetal. Login to comment 152 ABCC7 p.Leu206Trp [2000] [p.L206W]+[p.F508del]II/II0.312F^PSMildNegativeRozenetal. Login to comment 153 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II0.515F^PSMildNegativeRozenetal. Login to comment 154 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II0.616M^PSMildNegativeRozenetal. Login to comment 155 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II^38MCBAVD^^8773(2)Rozenetal. Login to comment 156 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II^56MCBAVD^^7773(2)Rozenetal. Login to comment 157 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II^55F^^Mild^Rozenetal. Login to comment 158 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del]II/II^45F^^Mild^Rozenetal. Login to comment 159 ABCC7 p.Leu206Trp [1995] [p.L206W]+[p.F508del](n=6)II/II^^MCBAVDPSNo^Casalsetal. Login to comment 160 ABCC7 p.Leu206Trp [2000] [p.L206W]+[?]II/-^^MCBAVDPSNo^Casalsetal. Login to comment 161 ABCC7 p.Leu206Trp [2000] [p.L206W]+[3121-1G4A]II/I^^MCBAVDPSNo^Casalsetal. Login to comment 162 ABCC7 p.Leu206Trp [2000] [p.L206W]+[1949del84]II/?^^MCBAVDPSNo^Casalsetal. Login to comment 163 ABCC7 p.Leu206Trp [2000] [p.L206W]+[p.F508del]II/II^^MCBAVDPS^^Maketal. Login to comment 164 ABCC7 p.Leu206Trp [1999] [p.L206W]+[?]II/-^^MCBAVDPS^^Maketal. Login to comment 165 ABCC7 p.Gly542* ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Glu60* ABCC7 p.Trp216* [1999] [p.L206W]+[p.W216X]II/I0.116F^PSNo75(1)Thisstudy [p.L206W]+[p.F508del]II/II0.2d 2F^PSe BronchialhyperreactivityPositiveThisstudy [p.L206W]+[p.F508del]II/II216F^PSNo54714(6)Thisstudy [p.L206W]+[p.F508del]II/II24M^PSe Bronchitis65(1)Thisstudy [p.L206W]+[p.F508del]II/II23M^PSe Bronchitis9672(2)Thisstudy [p.L206W]+[p.F508del]II/II47F^PIBronchitis5478(2)Thisstudy [p.L206W]+[p.F508del]II/I56F^PSAsthma7576(2)Thisstudy [p.L206W]+[1342-6(T)5]II/-2833MCBAVDPSBronchitis^Thisstudy [p.L206W]+[p.G542X]II/I3243MCBAVDPSNo^Thisstudy [p.L206W]+[p.F508del]II/II3740MCBAVD^^^Thisstudy [p.L206W]+[p.E60X]II/I2938MCBAVDPSNo64(1)Thisstudy [p.L206W]+[p.F508del]II/II3536MCBAVDPINo93(1)Thisstudy a Theclassi'cationofmissensemutationswasbasedonfunctionalstudies[Lietal.,1993;Chengetal.,1990;Champignyetal.,1995]. Login to comment 188 ABCC7 p.Leu206Trp D: Endoglycosidase H susceptibility of wild-type, p.F508del, and p.L206W core-glycosylated immature form of the CFTR protein.The asterisk (B*) indicates the shift of band B mobility. Login to comment 191 ABCC7 p.Leu206Trp Both sets of experiments showed that the stability of the mature band C of the p.L206W mutant was nearly unaffected when compared to the wild type. Login to comment 192 ABCC7 p.Leu206Trp Altogether, this indicates that the p.L206W mutation results in decreased conversion to the fully glycosylated protein which accounts for the low amount of band C observed in the steady-state experiments. Login to comment 195 ABCC7 p.Leu206Phe ABCC7 p.Leu206Val Changing Leu to Val (p.L206V) and Phe (p.L206F)-small and large strictly hydrophobic residues-did not alter CFTR processing. Login to comment 196 ABCC7 p.Leu206Tyr ABCC7 p.Leu206Arg ABCC7 p.Leu206Glu In contrast, CFTR misprocessing was induced specifically by different polar residues Glu (p.L206E), Arg (p.L206R), or Tyr (p.L206Y). Login to comment 197 ABCC7 p.Leu206Phe ABCC7 p.Leu206Tyr Notably, the substitution by Tyr (p.L206Y), which differs from Phe only by a hydroxyl group, significantly reduced the processing efficiency when compared to p.L206F. Login to comment 199 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Because polar residues can drive transmembrane helix associations through interhelical hydrogen bonds (H bond) [Choma et al., 2000; Zhou et al., 2000, 2001], aberrant side chain-side chain H bonds between p.L206W and the neighboring TM helices 2 or 4 could account for the misprocessing induced by the p.L206W mutation. Login to comment 200 ABCC7 p.Leu206Trp Inspection of the TM2 and TM4 sequences identified five polar residues that could interact with p.L206W, namely Tyr-122, Cys-128, Thr-135, Cys-225, and Gln-237. Login to comment 201 ABCC7 p.Leu206Trp ABCC7 p.Cys225Ala ABCC7 p.Cys128Ala ABCC7 p.Gln237Ala To test this possibility, these residues were replaced one at a time by Ala (p.T122A, p.C128A, p.S135A, p.C225A, and p.Q237A) together with the p.L206W mutation, and the processing at steady-state was examined. Login to comment 202 ABCC7 p.Leu206Trp None of these double-mutant proteins displayed improved processing when compared to the p.L206W mutation alone (data not shown). Login to comment 203 ABCC7 p.Leu206Trp The overall observations reported here indicated that the p.L206W mutation impaired the CFTR biosynthetic pathway and that a hydrophobic residue at location 206 is necessary for proper CFTR processing. Login to comment 204 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Immuno£uorescence Localization of p.L206W CFTR Mutant Figure 3 shows the influence of the p.L206W mutation on the cellular localization of CFTR in transiently transfected HeLa cells evaluated by indirect immunofluorescence using the mouse monoclonal antibody MAB25031, which recognizes the C-terminal portion of the CFTR protein. Login to comment 205 ABCC7 p.Leu206Trp Wild-type CFTR was detectable as a uniform staining over the entire cell surface, and also as a more intense perinuclear pattern, whereas the p.L206W mutation resulted in large decrease in cell surface staining while perinuclear staining remained intense (Fig. 3). Login to comment 206 ABCC7 p.Leu206Trp Hence the relative amount of the mature CFTR band detected in steady-state experiments was consistent with the cell surface staining by indirect immunofluorescence for both the wild-type and the p.L206W CFTR mutant. Login to comment 207 ABCC7 p.Leu206Trp Chloride Channel Function of p.L206W CFTR Mutant To characterize this mutant protein functionally, the cAMP-stimulated Cl-conductance across the entire cell plasma membrane of CFTR-expressing cells was measured using a Cl- - sensitive dye fluorescent assay (Fig. 4A and B). Login to comment 209 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Cells expressing wild-type or p.L206W CFTR produced a significantly higher cAMP-dependent fluorescence change than mock-transfected cells (wild-type: 0.03170.051; L206W: 0.01270.011), indicating a CFTR-depen- FIGURE 2. Login to comment 210 ABCC7 p.Leu206Trp Pulse-chase experiments showing the turnover of the immature and mature forms of wild-type and p.L206W CFTR. Login to comment 214 ABCC7 p.Leu206Trp C: Stability of the mature wild-type and p.L206W CFTR determined upon inhibition of protein biosynthesis with cycloheximide (CHX). Login to comment 218 ABCC7 p.Leu206Trp Compared to the wild-type, cells expressing p.L206W exhibited a distribution in fluorescence change that was significantly decreased (Po0.005). Login to comment 219 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp This decrease in cAMP-dependent Cl-conductance across the entire cell plasma membrane reflected at least in part the p.L206W CFTR misprocessing, and is consistent with the CF phenotype. Login to comment 220 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Single-Channel Properties of the p.L206W Mutant Because the p.L206W mutant had altered cAMP-dependent Cl-conductance at the cell plasma membrane and produced some mature protein, single-channel properties were analyzed using excised, inside-out membrane patches. Login to comment 221 ABCC7 p.Leu206Trp None of the membrane patches of mock-transfected cells displayed a Cl- current in the presence of intracellular ATP and PKA, whereas wild-type and p.L206W mutant CFTR channels were reversibly activated (data not shown). Login to comment 222 ABCC7 p.Leu206Trp The single-channel current amplitudes of the p.L206W mutant were not different from those of the wild-type (Fig. 4C). Login to comment 223 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp By using 120 mM Cl- (symmetrically), wild-type and p.L206W mutant CFTR had a linear i-v relationship under activating conditions (Fig. 4D) and the conductance for p.L206W was 7.6270.61 pS (n=6), which was not significantly different from wild-type (7.1870.36 pS; n=6). Login to comment 225 ABCC7 p.Leu206Trp However, visual inspection suggested that the gating parameters of the p.L206W mutant were not dramatically different from those of the wild-type CFTR. Login to comment 226 ABCC7 p.Leu206Trp DISCUSSION This study provides strong clinical, molecular and functional evidence that the transmembrane CFTR p.L206W mutation should be classified as a mild class II mutation. Login to comment 227 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Based on PI incidence in the overall case reports and in the present [p.L206W]+[p.F508del] genotype-phenotype relationships study (PI occurred in o10% of patients), p.L206W should be considered as a mild CF allele. Login to comment 230 ABCC7 p.Leu206Trp Our multicenter cross-sectional study highlights the significantly better nutritional status (absence of diarrhea and meconium ileus) and confirms the presence of pulmonary outcomes in CF patients, further documenting the atypical clinical presentation of compound p.L206W patients from CBAVD to PI cystic fibrosis. Login to comment 232 ABCC7 p.Leu206Trp Of interest, p.L206W arises in a unique haplotype with IVS8 (TG)9(T)9 and the 1540A common variant. Login to comment 233 ABCC7 p.Leu206Trp This has implications concerning the phenotype associated with p.L206W. Login to comment 234 ABCC7 p.Leu206Trp First, IVS8 (TG)9 and IVS8 (T)9 alleles, which are associated with correct splicing of exon 9, result in normal levels of p.L206W CFTR mRNA, and the 1540A allele is described as maintaining normal CFTR function. Login to comment 235 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Because for most CF patients the mild L206W is associated with a severe mutation on the other chromosome, the particular haplotype linked to p.L206W strengthens the mild dominant effect of the p.L206W mutation. Login to comment 236 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp Second, because p.L206W is linked to a unique CFTR genetic background, it can be deduced that these polymorphisms inherited in cis with the p.L206W mutation cannot explain the phenotypic variability observed. Login to comment 237 ABCC7 p.Leu206Trp The data reported here show that p.L206W is a disease-causing mutation by decreasing CFTR processing and subsequently Cl-channel conductance at the cell plasma membrane by approximately 60%. Login to comment 238 ABCC7 p.Leu206Trp The subcellular localization of p.L206W CFTR in transfected HeLa cells differs dramatically from that of the wild-type CFTR, the mutant being mainly located in the cytoplasm. Login to comment 241 ABCC7 p.Leu206Trp We can first confirm that Leu-206 does not line the channel [Akabas, 1998] and second, that cells expressing the p.L206W mutation generate residual cAMP-dependent Cl- currents at the plasma membrane, which is believed to be sufficient to confer a variable or mild clinical phenotype. Login to comment 243 ABCC7 p.Leu206Trp Immuno£uorescence localization of wild-type and p.L206W CFTR in transiently transfected HeLa cells.Cells grown on glass slides were 'xed in methanol, permeabilized in 0.1%Triton X-100 in PBS, and visualized by indirect immuno£uorescence. Login to comment 250 ABCC7 p.Pro205Ser ABCC7 p.Leu206Trp Interestingly, the disease-causing mutation p.P205S, flanking p.L206W in the middle of the TM3, produced nearly no detectable mature protein [Sheppard et al., 1996]. Login to comment 253 ABCC7 p.Leu206Trp Thus, the p.L206W mutation might disturb local hydrophobic constraints, preventing the ''pro-folding`` activity of Pro-205. Login to comment 256 ABCC7 p.Val232Asp This hypothetical disease-causing mechanism is based on the observation that the neutral-to-charge CF mutation p.V232D in TM4 induces an aberrant side chain-side chain H bond with the neighboring wild-type Gln-207 in TM3 by using a recombinant helix-loop-helix fragment of CFTR (Therien et al., 2001). Login to comment 257 ABCC7 p.Leu206Trp ABCC7 p.Leu206Trp This mechanism seems unlikely to explain p.L206W misprocessing because we did not identify such partners for p.L206W by testing several polar residues in the two helices adjacent to TM3. Login to comment 260 ABCC7 p.Leu206Trp ClÀ channel activity of HeLa cells transiently transfected with wild-type and p.L206W CFTR. Login to comment 274 ABCC7 p.Arg117His ABCC7 p.Arg334Trp ABCC7 p.Arg347Asp ABCC7 p.Pro99Leu p.P99L, p.R117H, p.R334W, and p.R347D/H/P form ClÀ channels with altered permeation properties but are processed normally and are therefore indexed as class IV mutants [Sheppard et al., 1993, 1996; Tabcharani et al., 1993]. Login to comment 275 ABCC7 p.Pro205Ser ABCC7 p.Leu206Trp ABCC7 p.Gly85Glu Unexpectedly, the mechanism of dysfunction of p.L206W resembles that of p.G85E and p.P205S, which are misprocessed without altering ion conductance [Sheppard et al., 1996; Xiong et al., 1997]. Login to comment 283 ABCC7 p.Leu206Trp Environmental and other genetic factors might contribute to this variability [Bronsveld et al., 2000; Garred et al., 1999], and a plausible explanation that we favor is that p.L206W and other class II mutants are associated with a variable clinical phenotype because multiple potential partners could modify the processing of the CFTR protein during its course to the cell surface. Login to comment 287 ABCC7 p.Leu206Trp p.L206W, a misprocessing mutation localized in a transmembrane segment, provides a striking example that functional analysis of CFTR mutations identified in patients is necessary to assign the correct class to each particular mutation. Login to comment