ABCMdb

PMID: 18951463

Krasnov KV, Tzetis M, Cheng J, Guggino WB, Cutting GR
Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships.
Hum Mutat. 2008 Nov;29(11):1364-72., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with ''severe`` pancreatic insufficient cystic fibrosis (CF) and R1070W with ''mild`` pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Login to comment 4 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. Login to comment 5 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a ''severe`` phenotype. Login to comment 6 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* Reanalysis of 16 patients with the R1070Q mutation revealed that 11 carried an in cis nonsense mutation, S466X. Login to comment 7 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Ser466* All 11 patients carrying the complex allele R1070Q-S466X had severe disease, while 4 out of 5 patients with R1070Q had ''mild`` disease, thereby reconciling the apparent discrepancy between the localization studies of R1070Q and the phenotype of patients bearing this mutation. Login to comment 27 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro Patients with R1070W (c.3208C4T; p.Arg1070Trp) have pancreatic sufficient CF or congenital bilateral absence of the vas deference (CBAVD) and a normal life span, whereas patients with R1070Q (c.3209G4A; p.Arg1070Gln) and R1070P (c3209G4C; p.Arg1070Pro) have classic CF with significant clinical features of lung disease, pancreatic insufficiency, and elevated sweat chloride levels [Mickle et al., 2000]. Login to comment 28 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro Previous studies revealed that the biogenesis and function of CFTR bearing R1070W and R1070P is substantially altered, consistent with their pathologic role. Login to comment 29 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp However, CFTR with R1070Q had a less severe channel gating abnormality than R1070W and either no defect [Seibert et al., 1996; Mickle et al., 2000] or a milder defect than R1070W upon protein processing [Seibert et al., 1996]. Login to comment 30 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp These functional studies indicate that the R1070Q mutation should cause a milder phenotype than R1070W, rather than the more severe phenotype observed. Login to comment 32 ABCC7 p.Arg1070Gln We therefore hypothesized that the severe CF phenotype attributed to R1070Q was due to a localization defect in native epithelial cells. Login to comment 36 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro While CFTR bearing R1070W and R1070P displayed localization defects consistent with their associated phenotypes, R1070Q was difficult to distinguish from wild-type CFTR. Login to comment 37 ABCC7 p.Arg1070Gln These localization studies prompted a search for alternate explanations for the CF phenotype associated with the R1070Q mutation. Login to comment 85 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro RESULTS Wild-Type CFTR Is Localized toApical Membranes of MDCK Cells To determine the effect of R1070 mutations on CFTR localization, we expressed heterologous CFTR (wild-type or 1 of 3 CFTR mutants-R1070P, R1070Q, or R1070W) from an FRT integration site in MDCK type II cell lines (Fig. 1A). Login to comment 101 ABCC7 p.Arg1070Pro Proline (P) andTryptophan (W) Substitutions of Arginine at Codon 1070 Alter LocalizationWhile CFTR Bearing a Glutamine (Q) Substitution Is Found in Apical Membranes CFTR bearing R1070P, a mutation that is associated with classic CF, did not reach-or was not appreciably retained at-the cell surface, instead localizing throughout the cytoplasm. Login to comment 102 ABCC7 p.Arg1070Pro The cytoplasmic distribution seen with R1070P was similar to that of CFTR bearing the common delta-F508 mutation (aka F508 del; data not shown) that does not reach the apical membrane due to CFTR misfolding and is degraded [Cheng et al., 1990; Kartner et al., 1992; Denning et al., 1992]. Login to comment 103 ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro Immunostaining with ZO-1 (Fig. 2; R1070P, left column), Na1 /K1 ATPase (Fig. 2; R1070P, middle column), and staining of the surface apical membrane with WGA dye (Fig. 2; R1070P, right column) showed that the CFTR R1070P protein does not localize to the apical membrane. Login to comment 104 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp CFTR bearing R1070W, a mutation associated with mild disease, localized to the apical surface of MDCK cells (see overlap with apical surface marker in Fig. 2; R1070W, left column) but was also present in other locations in the cell. Login to comment 105 ABCC7 p.Arg1070Trp The absence of colocalization with Na1 /K1 ATPase basolateral marker is consistent with presence of CFTR R1070W in the cytoplasm. Login to comment 106 ABCC7 p.Arg1070Gln On the other hand, CFTR bearing R1070Q localized primarily to the apical membrane of MDCK cells. Login to comment 107 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln Staining R1070Q cells with the apical surface dye, WGA, revealed yellow signal (Fig. 2; R1070Q, right column), indicating overlap of green GFP-CFTR and red WGA signals at the apical membrane. Login to comment 108 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln Green signal was observed in other locations in the cell (Fig. 2; R1070Q, left column) and colocalization of this signal with Na1 /K1 ATPase (Fig. 2; R1070Q, right column) suggests that a minor fraction of R1070Q is present in the basolateral membranes. Login to comment 109 ABCC7 p.Arg1070Gln Overall, CFTR R1070Q is preferentially located at the apical membrane, a pattern very similar to that observed for wild-type CFTR. Login to comment 115 ABCC7 p.Arg1070Gln CFTR bearing R1070Q is also processed to mature band C protein (Fig. 3A; lane 2) that is accessible to biotinylation at the apical membrane (Fig. 3B; lane 3). Login to comment 116 ABCC7 p.Arg1070Gln Thus, wild-type and R1070Q CFTR are processed to fully mature protein that is inserted into the apical membrane, consistent with the localization studies above. Login to comment 117 ABCC7 p.Arg1070Trp CFTR with R1070W was present in both mature (C band) and immature (B band) forms (Fig. 3A; lane 3), although the ratio of C band to B band and the ratio of C band to actin control (Fig. 3A; lane 3, lower panel) was lower than seen for wild-type CFTR. Login to comment 118 ABCC7 p.Arg1070Trp Biotinylation revealed that CFTR R1070W is inserted into the apical membrane, albeit at very low levels when compared to wild-type (Fig. 3B; lane 5). Login to comment 119 ABCC7 p.Arg1070Trp These results indicate that R1070W is able to process into mature apically-localized protein but with decreased efficiency compared to wild-type. Login to comment 120 ABCC7 p.Arg1070Trp As shown by confocal microscopy, these biochemical assays are consistent with only a fraction of R1070W at the apical membrane. Login to comment 121 ABCC7 p.Arg1070Trp There was a small amount of B band in the apical biotinylated fraction of R1070W, implying that some immature CFTR protein bearing this mutation may be inserted into the apical membrane. Login to comment 123 ABCC7 p.Arg1070Pro Finally, Western blotting revealed that R1070P is present only as partially glycosylated immature B band protein (Fig. 3A; lane 4) that is not detectable in the biotinylated apical membrane fraction. Login to comment 124 ABCC7 p.Arg1070Pro These results show that the R1070P mutation causes CFTR to be improperly processed such that it remains within the cell and does not reach the apical membrane, consistent with FIGURE 2. Login to comment 125 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Pro CFTR R1070P and R1070Wshow di¡erent cellular distribution patterns in polarized epithelial cells, whereas CFTR R1070Q shows an apical localization pattern similar to wild-type CFTR. Login to comment 127 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro Each panel shows MDCK-FRT cells expressing either wild-type, R1070P, R1070W, or R1070Q CFTR. Login to comment 132 ABCC7 p.Arg1070Pro confocal microscopy studies showing R1070P only in the cytoplasm. Login to comment 133 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro Altered Function of CFTR Bearing R1070W and R1070P Is ConsistentWith Phenotype of Patients CarryingThese Mutations A worldwide survey identified 29 patients who carried the R1070W mutation (24 of whom had detailed clinical information); 26 patients with R1070Q (16 of whom had detailed data), and 2 patients with R1070P (1 of whom had detailed data). Login to comment 136 ABCC7 p.Arg1070Trp Out of 24 patients who carry the R1070W mutation, 16 had F508del (c.1521_1523delCTT; p.Phe508del) on the other allele and all were diagnosed with either pancreatic-sufficient CF or CBAVD (Table 1). Login to comment 137 ABCC7 p.Arg1070Trp Pancreatic status was unavailable for 6 out of the 16 patients with the R1070W and F508del genotype. Login to comment 140 ABCC7 p.Gly542* ABCC7 p.Lys710* ABCC7 p.Arg668Cys ABCC7 p.Arg1070Trp As for the remaining R1070W patients (8/24 with detailed clinical information), five individuals carried a CFTR mutation associated with pancreatic insufficiency (2869insG (c.2737insG, p.Tyr913fs); G542X (c.1624G4 T, p.Gly542X); R668C-K710X (c. Login to comment 142 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg ABCC7 p.Arg668Cys [Arg668Cys,Lys710X]); N1303 K (c.3909C4G, p.Asn1303Lys); and S1235R (c.3705T4G, p.Ser1235Arg). Login to comment 144 ABCC7 p.Lys710* ABCC7 p.Arg668Cys ABCC7 p.Arg1070Trp The one remaining individual with the R1070W/R668C-K710X genotype had pancreatic-insufficient CF. Login to comment 145 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp Overall, the R1070W mutation was associated with either pancreatic-sufficient CF or CBAVD when in trans with a CFTR mutation that confers pancreatic insufficiency, indicating that the R1070W mutation generally conferred mild disease. Login to comment 146 ABCC7 p.Arg1070Pro Two patients were found to carry the R1070P mutation and only one of them has detailed clinical information; therefore, only limited conclusions can be drawn. Login to comment 149 ABCC7 p.Arg1070Pro Based on the latter case, the R1070P mutation is considered to be associated with severe disease. Login to comment 150 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Ser466* CFTR R1070Q With an in cis Nonsense Mutation, S466X (c.1397C4G; p.Ser466X), Is Associated With Severe CF Previous studies have reported that patients with R1070Q have classic CF; however, studies shown here indicate that CFTR FIGURE 3. Login to comment 152 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro A: Western blotting of MDCK cell lysates expressing wild-type, R1070Q, R1070W, or R1070P CFTR. Login to comment 154 ABCC7 p.Arg1070Gln CFTR R1070Q (lane 2) has a pattern similar to wild-type, primarily C band with some B band visible. Login to comment 155 ABCC7 p.Arg1070Gln The quantity of CFTR R1070Q mutant protein is slightly lower than wild-type CFTR (note actin control loading in lower panel). Login to comment 156 ABCC7 p.Arg1070Trp CFTR R1070W has a lower amount of stable protein expressed and is readily visible as both C and B bands (lane 3). Login to comment 157 ABCC7 p.Arg1070Pro CFTR R1070P has minimal stable protein and is present only as B band (lane 4). Login to comment 159 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro B:Western blotting of the apical biotinylated fraction (odd-numbered lanes) or total lysates (even-numbered lanes) of MDCK cells expressing wild-type, R1070Q, R1070W, or R1070P CFTR.Wild-type CFTR (lanes 1 and 2) and CFTR R1070Q (lanes 3 and 4) have a 205-kDa band in the biotinylated fraction that is consistent with the presence of mature protein in the apical membrane.The fraction of R1070Q inserted into the apical membrane is similar to wild-type.CFTR R1070W (lanes 5 and 6) has a faint 205-kDa band and a very faint 175-kDa band, suggesting that mature and some immature protein is inserted in the apical membrane.CFTR R1070P (lanes 7 and 8) has no bands visible in the biotinylated fraction and only a 175-kDa band is seen in the total lysate, indicating that this mutant protein is not present in the apical membrane.The lower panel is an immunoblot of each apical fraction and each total lysate after incubation with an actin antibody. Login to comment 162 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro ABCC7 p.Ser466* Summarized Clinical Information on R1070 Patients Patient mutations R1070W R1070P R1070Q R1070Q in cis S466X Number of patientsa 24 2 5 11 Second mutaiton dF508 16 1 0 7 other 8 1 5 4 Disease diagnosis CBACD (infertility) 15 0 3 0 Nonclassic CF 9 1 1 0 Classic CF 1 1 1 11 Pancreatic status Su/cient 9 0 1 0 Insu/cient 4a 1 1 10 Not reported 11b 1 3b 1 Sweat chloride levels Normal or low 12 0 1 0 Elevated460 mmol/L 4 1 1 10 Not reported 8b 1 2b 1 a One patient has classic CF; the other three have normal sweat chloride levels and high FVC values. Login to comment 165 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln R1070Q functions and localizes like wild-type CFTR, suggesting that an alternative mechanism exists to create the severe phenotype associated with the R1070Q mutation. Login to comment 166 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Ser466* A literature review of patients carrying R1070Q uncovered a report that briefly mentioned two Serbian patients with classic CF who carried the R1070Q mutation in cis with S466X, both having the F508del mutation on the other CFTR gene [Radivojevic et al., 2004]. Login to comment 167 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* We found 14 R1070Q patients with detailed clinical information (16 patients in total when including those of the Radivojevic et al. [2004] group) and sequencing of CFTR exon 10 showed that 11 out of 16 patients carried an in cis S466X mutation (Table 1). Login to comment 168 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln ABCC7 p.Ser466* ABCC7 p.Ser466* Seven of these 11 R1070Q-S466X patients had F508del as the other allele and the remaining four had a variety of CF alleles in trans, one each of N1303 K, 62111G4T (c.48911G4T), 7111 3A4G (c.57913A4G), and R1070Q-S466X (Supplementary Table S1). Login to comment 169 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* Regardless of the CF mutation on the other CFTR gene, all 11 R1070Q-S466X patients had pancreatic insufficient CF. Login to comment 170 ABCC7 p.Arg1070Gln Of the five patients with R1070Q only, three had CBAVD and two were diagnosed as CF. Login to comment 171 ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Asp1152His ABCC7 p.Phe1337Val Of those with CBAVD, one patient carried a mutation known to cause pancreatic insufficient CF (S549N [c.1646G4A; p.Ser549Asn]), a second patient carried a mutation associated with CBAVD (D1152 H [c.3454G4C; p.Asp1152His]), and the third carried a mutation of unknown disease association (F1337 V [c.4009T4G; Phe1337Val]). Login to comment 172 ABCC7 p.Glu822* Of the two patients diagnosed with CF, a female patient with the E822X (c.2464G4T; p.Glu822X) mutation in her other CFTR gene had pancreatic insufficient CF and a patient carrying 278915G4A (c.265715G4A) had pancreatic-sufficient CF. Login to comment 173 ABCC7 p.Glu822* The E822X mutation has been associated with pancreatic-insufficient CF while 278915G4T is a pancreatic-sufficient mutation (www.genet.sickkids.on.ca/cftr). Login to comment 174 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* In summary, R1070Q alone appears to be able to confer mild disease (i.e., CBAVD) in some cases when paired with a known ''severe`` CF mutation, while the presence of the in cis S466X mutation was consistently associated with pancreatic-insufficient CF. Login to comment 178 ABCC7 p.Arg1070Gln Prior studies had evaluated the effect of R1070Q in nonpolarized cells [Cotten et al., 1996; Seibert et al., 1996; Mickle et al., 2000]. Login to comment 180 ABCC7 p.Arg1070Gln We had hypothesized that the severe CF phenotype associated with the R1070Q mutation was caused by abnormal trafficking of CFTR to apical membranes. Login to comment 181 ABCC7 p.Arg1070Gln However, CFTR bearing R1070Q localizes to apical membranes in a manner that is qualitatively similar to wild-type CFTR. Login to comment 182 ABCC7 p.Arg1070Gln This result led to new theories explaining the phenotype associated with R1070Q that ranged from underestimation of the chloride transport defect to lack of interaction with the macromolecular complex at the apical membrane. Login to comment 183 ABCC7 p.Arg1070Gln Before embarking on more detailed analysis of R1070Q CFTR properties, the clinical features of patients bearing this mutation were revisited to confirm its propensity to cause disease. Login to comment 184 ABCC7 p.Arg1070Gln Additionally, other deleterious variants that may exist in the CFTR gene bearing R1070Q were considered. Login to comment 185 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* A literature review uncovered a report that the nonsense mutation S466X had been found in cis with R1070Q in two patients [Radivojevic et al., 2004]. Login to comment 186 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* ABCC7 p.Ser466* Demonstration that S466X correlated with the CF phenotype in 11 patients combined with the known severe functional consequences of nonsense mutations (nonsense-mediated RNA decay or, less commonly, protein truncation) indicated that S466X, rather than R1070Q, was responsible for the observed severe phenotype. Login to comment 187 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Pro Thus, functional studies were informative for all three R1070 mutations, whereby R1070P and R1070W revealed processing defects that are consistent with their role in disease, while the properties of CFTR bearing R1070Q provoked a reevaluation of the established genotype-phenotype relationship that led to a more plausible explanation for the pathology observed in patients with a glutamine substitution at codon 1070. Login to comment 189 ABCC7 p.Arg1070Pro Observed in only two patients to date, this mutation causes classic CF and, although it was partly functional in nonpolarized cells, R1070P did not localize to the cell surface in polarized MDCK cells. Login to comment 192 ABCC7 p.Arg1070Pro ABCC7 p.Arg1070Pro Since R1070P was not detectable on the apical membrane of MDCK-FRT cells by biotinylation studies but was present on the membrane of nonpolarized HEK-293 cells [Mickle et al., 2000], the localization machinery for R1070P is apparently different in polarized and nonpolarized cells. Login to comment 194 ABCC7 p.Arg1070Trp Although CFTR R1070W is primarily associated with male infertility (CBAVD), previously published reports and the current study showed that the tryptophan mutation consistently has decreased protein expression levels and incomplete apical localization. Login to comment 195 ABCC7 p.Arg1070Trp CFTR R1070W transiently expressed in nonpolarized HEK-293 cells [Mickle et al., 2000], COS-1 cells [Seibert et al., 1996], and the stable expression in isogenic MDCK cell lines shown here all demonstrated lower levels than wild-type CFTR. Login to comment 197 ABCC7 p.Arg1070Trp ABCC7 p.Asn287Tyr Partial mislocalization of CFTR R1070W may be due to alteration in the rate of membrane recycling, as has previously been observed with N287Y, a CFTR cytoplasmic loop 2 mutant [Silvis et al., 2003]. Login to comment 199 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* The presence of nonsense mutation S466X in CFTR genes bearing R1070Q provides a parsimonious explanation for the CF phenotype in patients with this combination. Login to comment 200 ABCC7 p.Arg1070Gln However, the five patients carrying R1070Q alone manifested phenotypes ranging from male infertility (mild) to pancreatic insufficient CF (severe). Login to comment 201 ABCC7 p.Arg1070Gln While it is possible that the severe phenotype was due to other environmental factors, genetic modifiers, or erroneous information, the abnormal clinical features in the remaining four patients indicate that R1070Q has some deleterious effect upon CFTR function. Login to comment 202 ABCC7 p.Arg1070Gln This conclusion is consistent with the observation that CFTR R1070Q has a reduced steady-state protein level compared to CFTR wild type in MDCK cells. Login to comment 203 ABCC7 p.Arg1070Gln ABCC7 p.Arg1070Gln On the other hand, studies in nonpolarized COS-1 cells showed wild-type-like levels of R1070Q protein yet generation of only one-half the chloride efflux of wild-type cells; additionally, R1070Q had normal levels of conductance in CHO cells but a decreased open probability [Seibert et al., 1996]. Login to comment 204 ABCC7 p.Arg1070Gln One or more of these moderate dysfunctions caused by R1070Q may explain why it is associated with disease of varying severity when appearing alone. Login to comment 205 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* The combination of R1070Q and S466X adds to the growing list of complex alleles reported in CFTR [Claustres et al., 2000]. Login to comment 208 ABCC7 p.Arg1070Gln ABCC7 p.Ser466* Similarly, interpretation of the clinical spectrum associated with R1070Q also requires determination of the presence or absence of S466X. Login to comment