ABCMdb

PMID: 9499426

Mickle JE, Macek M Jr, Fulmer-Smentek SB, Egan MM, Schwiebert E, Guggino W, Moss R, Cutting GR
A mutation in the cystic fibrosis transmembrane conductance regulator gene associated with elevated sweat chloride concentrations in the absence of cystic fibrosis.
Hum Mol Genet. 1998 Apr;7(4):729-35., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Ser1455* We report the identification of a 6.8 kb deletion (del14a) and a nonsense mutation (S1455X) in the CFTR genes of a mother and her youngest daughter with isolated elevated sweat chloride concentrations. Login to comment 2 ABCC7 p.Ser1455* We report the identification of a 6.8 kb deletion (del14a) and a nonsense mutation (S1455X) in the CFTR genes of a mother and her youngest daughter with isolated elevated sweat chloride concentrations. Login to comment 4 ABCC7 p.Ser1455* CFTR mRNA transcripts bearing the S1455X mutation were stable in vivo, implying that this allele encoded a truncated version of CFTR missing the last 26 amino acids. Login to comment 5 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. Login to comment 6 ABCC7 p.Ser1455* Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. Login to comment 7 ABCC7 p.Ser1455* Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. Login to comment 8 ABCC7 p.Ser1455* Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. Login to comment 28 ABCC7 p.Ser1455* Shading indicates CFTR alleles: del14a alleles are solid, S1455X alleles are hatched and the wild-type allele is unshaded. Login to comment 29 ABCC7 p.Ser1455* Shading indicates CFTR alleles: del14a alleles are solid, S1455X alleles are hatched and the wild-type allele is unshaded. Login to comment 54 ABCC7 p.Ser1455* Identification of nonsense mutation S1455X in mother and daughter with elevated sweat chloride concentrations Screening for 16 CF mutations common among Caucasians by reverse dot-blot assay did not detect a mutation in any family member. Login to comment 55 ABCC7 p.Ser1455* Identification of nonsense mutation S1455X in mother and daughter with elevated sweat chloride concentrations Screening for 16 CF mutations common among Caucasians by reverse dot-blot assay did not detect a mutation in any family member. Login to comment 71 ABCC7 p.Ser1455* ABCC7 p.Ser1455* This novel mutation is predicted to change the codon for serine at residue 1455 to an opal termination codon, and is designated S1455X.Both theproband`s mother and sister are compound heterozygotes for deletion 14a and S1455X (Fig. 1). Login to comment 72 ABCC7 p.Ser1455* ABCC7 p.Ser1455* This novel mutation is predicted to change the codon for serine at residue 1455 to an opal termination codon, and is designated S1455X.Both theproband`s mother and sister are compound heterozygotes for deletion 14a and S1455X (Fig. 1). Login to comment 73 ABCC7 p.Ser1455* Since the del14a mutation caused the CF phenotype in the homozygous state, we predicted that the S1455X mutation was the cause of the isolated elevated sweat chloride concentrations in the sister and mother. Login to comment 74 ABCC7 p.Ser1455* ABCC7 p.Ser1455* ABCC7 p.Ser1455* CFTR mRNA transcript bearing the S1455X mutation is stable RT-PCR was used to determine the consequences of the del14a and S1455X mutations upon transcript splicing and stability. Login to comment 75 ABCC7 p.Ser1455* ABCC7 p.Ser1455* CFTR mRNA transcript bearing the S1455X mutation is stable RT-PCR was used to determine the consequences of the del14a and S1455X mutations upon transcript splicing and stability. Login to comment 78 ABCC7 p.Ser1455* Since nonsense mutations frequently cause severe reduction in mRNA levels (28), the stability of mRNA transcripts bearing the nonsense mutation S1455X was assessed as follows. Login to comment 79 ABCC7 p.Ser1455* Since nonsense mutations frequently cause severe reduction in mRNA levels (28), the stability of mRNA transcripts bearing the nonsense mutation S1455X was assessed as follows. Login to comment 81 ABCC7 p.Ser1455* Immunoprecipitation of wild-type and S1455X CFTR transiently expressed in HEK 293 cells. Login to comment 82 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Wild-type CFTR and S1455X CFTR proteins immunoprecipitated with the R-domain-specific monoclonal antibody are indicated by the arrow.Both migrateat ~175kDa.TheS1455XCFTRtruncated protein was not detected by immunoprecipitation with the C-terminus-specific antibody. Login to comment 83 ABCC7 p.Ser1455* Wild-type CFTR and S1455X CFTR proteins immunoprecipitated with the R-domain-specific monoclonal antibody are indicated by the arrow.Both migrateat ~175kDa.TheS1455XCFTRtruncated protein was not detected by immunoprecipitation with the C-terminus-specific antibody. Login to comment 85 ABCC7 p.Ser1455* the larger amplicon carrying the S1455X mutation was at similar levels to the smaller amplicon missing exon 14a (Fig. 3). Login to comment 86 ABCC7 p.Ser1455* ABCC7 p.Ser1455* ABCC7 p.Ser1455* Thus, normal CFTR mRNA transcripts and transcripts either bearing S1455X or missing exon 14a were present at similar levels, indicating that the S1455X transcripts were stable. Login to comment 87 ABCC7 p.Ser1455* ABCC7 p.Ser1455* ABCC7 p.Ser1455* To verify this result, we compared the levels of CFTR transcripts bearing S1455X with those missing exon 14a by RT-PCR amplification of exon 24 followed by hybridization with oligonucleotides specific for each transcript. Login to comment 88 ABCC7 p.Ser1455* To verify this result, we compared the levels of CFTR transcripts bearing S1455X with those missing exon 14a by RT-PCR amplification of exon 24 followed by hybridization with oligonucleotides specific for each transcript. Login to comment 89 ABCC7 p.Ser1455* Quantitation of the hybridization signals by phosphoimaging indicated that S1455X CFTR transcripts were stable, although less abundant (37.9 ± 1.99% SEM, n = 7) than del14a CFTR (62.1 ± 1.17% SEM). Login to comment 90 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Since the S1455X CFTR mRNA was stable, we predicted that CFTR missing the last 26 amino acids was produced in vivo. Login to comment 91 ABCC7 p.Ser1455* ABCC7 p.Ser1455* ABCC7 p.Ser1455* The S1455X CFTR truncated protein is processed to a mature state and functions similarly to wild-type CFTR Processing of CFTR bearing S1455X was evaluated by immunoprecipitation using commercially available monoclonal antibodies directed against the R-domain or the last four residues at the C-terminus (Genzyme). Login to comment 92 ABCC7 p.Ser1455* ABCC7 p.Ser1455* The S1455X CFTR truncated protein is processed to a mature state and functions similarly to wild-type CFTR Processing of CFTR bearing S1455X was evaluated by immunoprecipitation using commercially available monoclonal antibodies directed against the R-domain or the last four residues at the C-terminus (Genzyme). Login to comment 93 ABCC7 p.Ser1455* Both S1455X CFTR and wild-type CFTR products immunoprecipitated with the R-domain antibody have an apparent Mr of 175 kDa (Fig. 4). Login to comment 94 ABCC7 p.Ser1455* ABCC7 p.Ser1455* As expected, the S1455X CFTR mutant truncated at the C-terminus was not immunoprecipitated by the C-terminus antibody (Fig. 4). Login to comment 95 ABCC7 p.Ser1455* As expected, the S1455X CFTR mutant truncated at the C-terminus was not immunoprecipitated by the C-terminus antibody (Fig. 4). Login to comment 96 ABCC7 p.Ser1455* Since the S1455X mutant appears to undergo post-translational modifications similar to wild-type CFTR, we expected that the mutant protein would be properly folded and inserted into the cell membrane. Login to comment 97 ABCC7 p.Ser1455* ABCC7 p.Ser1455* To assay function, wild-type and S1455X CFTRcDNAs were transiently transfected into CF airway epithelial cells (IB3-1) devoid of functional CFTR. Login to comment 98 ABCC7 p.Ser1455* To assay function, wild-type and S1455X CFTRcDNAs were transiently transfected into CF airway epithelial cells (IB3-1) devoid of functional CFTR. Login to comment 99 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Whole-cell chloride current activated by cAMP in IB3-1 cells transfected with wild-type CFTR or S1455X CFTR.Current-voltage plots for wild-type CFTR (left) and S1455X CFTR (right) in the presence of CPT-cAMP (200 µM) are shown as filled symbols. Login to comment 100 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Whole-cell chloride current activated by cAMP in IB3-1 cells transfected with wild-type CFTR or S1455X CFTR.Current-voltage plots for wild-type CFTR (left) and S1455X CFTR (right) in the presence of CPT-cAMP (200 &#b5;M) are shown as filled symbols. Login to comment 103 ABCC7 p.Ser1455* Incubation with a non-hydrolyzable form of cAMP (CPT-cAMP; 200 µM) evoked chloride currents of similar magnitude in wild-type CFTR (+100 mV: 1090.9 ± 84.2 SEM pA; n = 12) and S1455X CFTR (+100 mV: 938.9± 114.4 pA; n = 20) transfected cells that were significantly higher (P < 0.05) than non-transfected cells (+100 mV: 89.3 ± 34.0 pA; n = 5). Login to comment 104 ABCC7 p.Ser1455* Incubation with a non-hydrolyzable form of cAMP (CPT-cAMP; 200 &#b5;M) evoked chloride currents of similar magnitude in wild-type CFTR (+100 mV: 1090.9 &#b1; 84.2 SEM pA; n = 12) and S1455X CFTR (+100 mV: 938.9&#b1; 114.4 pA; n = 20) transfected cells that were significantly higher (P < 0.05) than non-transfected cells (+100 mV: 89.3 &#b1; 34.0 pA; n = 5). Login to comment 106 ABCC7 p.Ser1455* Whole-cell patch-clamp analysis of IB3-1 cells transfected with S1455X CFTR displayed cAMP-activated currents that were outwardly rectified and inhibited by glybenclamide (+100 mV: 302.7 ± 127.5 pA; n = 7). Login to comment 107 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Thus, S1455X CFTR generates robust cAMP-activated chloride currents similarly to wild-type CFTR. Login to comment 108 ABCC7 p.Ser1455* Thus, S1455X CFTR generates robust cAMP-activated chloride currents similarly to wild-type CFTR. Login to comment 119 ABCC7 p.Ser1455* The good health of the mother at 46 years of age suggested a very low likelihood that the S1455X/ del14a genotype causes CF. Login to comment 120 ABCC7 p.Ser1455* The good health of the mother at 46 years of age suggested a very low likelihood that the S1455X/ del14a genotype causes CF. Login to comment 126 ABCC7 p.Ser1455* The S1455X mutation was implicated as the cause of the isolated elevated sweat chloride concentrations in the mother and younger daughter based on several lines of evidence. Login to comment 127 ABCC7 p.Ser1455* The S1455X mutation was implicated as the cause of the isolated elevated sweat chloride concentrations in the mother and younger daughter based on several lines of evidence. Login to comment 131 ABCC7 p.Ser1455* Indeed, whole-cell patch-clamp data obtained from IB3-1 bronchial epithelial cells indicate that the chloride channel of S1455X CFTR functions similarly to wild-type CFTR. Login to comment 132 ABCC7 p.Ser1455* ABCC7 p.Ser1455* This probably explains why the mother and child carrying the S1455X mutation escaped the pulmonary and pancreatic manifestations of CF. Login to comment 133 ABCC7 p.Ser1455* ABCC7 p.Ser1455* However, the remote possibility exists that another undetected mutation on the S1455X allele may contribute to the phenotype. Login to comment 134 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Moreover, since the functional data were derived from a heterologous airway epithelial expression system as patient samples were not available for patch-clamp studies, S1455X CFTR dysfunction in vivo may vary in a tissue-specific manner. Login to comment 135 ABCC7 p.Ser1455* ABCC7 p.Ser1455* The S1455X mutation is predicted to eliminate a region containing two stretches of amino acids that are highly conserved among species (36-38). Login to comment 136 ABCC7 p.Ser1455* The S1455X mutation is predicted to eliminate a region containing two stretches of amino acids that are highly conserved among species (36-38). Login to comment 137 ABCC7 p.Ser1455* The phenotype associated with the S1455X mutation indicates that the C-terminal sequences of CFTR have a functional role in the sweat gland. Login to comment 138 ABCC7 p.Ser1455* The phenotype associated with the S1455X mutation indicates that the C-terminal sequences of CFTR have a functional role in the sweat gland. Login to comment 141 ABCC7 p.Ser1455* Loss of these sequences due to the S1455X mutation prohibits CFTR trafficking to the basolateral surface, leading to reduced chloride permeability of the duct cells and elevated chloride concentrations in the sweat. Login to comment 142 ABCC7 p.Ser1455* Loss of these sequences due to the S1455X mutation prohibits CFTR trafficking to the basolateral surface, leading to reduced chloride permeability of the duct cells and elevated chloride concentrations in the sweat. Login to comment 151 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ser549Asn ABCC7 p.Arg560Thr Mutation analysis Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G→T, R334W, R349P, A455E, 1717-1G→A, ∆I507, ∆F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C→T, W1282X, N1303K) by reverse dot-blot hybridization (46). Login to comment 152 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ser549Asn ABCC7 p.Arg560Thr Mutation analysis Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G࢐T, R334W, R349P, A455E, 1717-1G࢐A, ࢞I507, ࢞F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C࢐T, W1282X, N1303K) by reverse dot-blot hybridization (46). Login to comment 164 ABCC7 p.Ser1455* Dot-blots were hybridized at 42_C for 1 h with γ-32P-labeled S1455X mutant probe (5'-CACTTGCTTCAGTT- CCGG-3') or S1455 wild-type probe (5'-ACCGGAACTCAAG- CAAGTG-3'), washed at room temperature and exposed to X-ray film for 10 min at -80_C. Login to comment 165 ABCC7 p.Ser1455* Dot-blots were hybridized at 42C for 1 h with b3;-32P-labeled S1455X mutant probe (5'-CACTTGCTTCAGTT- CCGG-3') or S1455 wild-type probe (5'-ACCGGAACTCAAG- CAAGTG-3'), washed at room temperature and exposed to X-ray film for 10 min at -80C. Login to comment 166 ABCC7 p.Ser1455* Expression analysis The mutation S1455X was created in the CFTR-containing vector pBQ4.7 (gift from J. Rommens and L.-C. Tsui; The Hospital for Sick Children, Toronto) by single-strand mutagenesis (48) and then shuttled into the Rous sarcoma virus (RSV)-driven expression vector (pRSV-CFTR) using NcoI and SalI restriction sites common to both plasmids (30). Login to comment 167 ABCC7 p.Ser1455* Expression analysis The mutation S1455X was created in the CFTR-containing vector pBQ4.7 (gift from J. Rommens and L.-C. Tsui; The Hospital for Sick Children, Toronto) by single-strand mutagenesis (48) and then shuttled into the Rous sarcoma virus (RSV)-driven expression vector (pRSV-CFTR) using NcoI and SalI restriction sites common to both plasmids (30). Login to comment 180 ABCC7 p.Ser1455* Whole-cell patch-clamp recording Whole-cell recordings were performed on IB3-1 cells transiently transfected with either pRSV-CFTR or pRSV-CFTR/S1455X using an inverted Nikon microscope (Nikon, Inc., Melville, NY), an Axopatch amplifier (Axon Instruments, Inc., Foster City, CA) and PCLAMP 6.0 software (Axon Instruments, Inc.). Login to comment 181 ABCC7 p.Ser1455* Whole-cell patch-clamp recording Whole-cell recordings were performed on IB3-1 cells transiently transfected with either pRSV-CFTR or pRSV-CFTR/S1455X using an inverted Nikon microscope (Nikon, Inc., Melville, NY), an Axopatch amplifier (Axon Instruments, Inc., Foster City, CA) and PCLAMP 6.0 software (Axon Instruments, Inc.). Login to comment 183 ABCC7 p.Ser1455* ABCC7 p.Ser1455* IB3-1 cells were transiently transfected at 30% confluency in a 35 mm dish for 6-8 h with either 3 µg of wild-type CFTR (pRSV-CFTR) or 3 µg of CFTR bearing the mutation S1455X (pRSV-CFTR/S1455X) using 15 µl of lipofectin and 1 ml of Opti-MEM. Login to comment 184 ABCC7 p.Ser1455* ABCC7 p.Ser1455* IB3-1 cells were transiently transfected at 30% confluency in a 35 mm dish for 6-8 h with either 3 &#b5;g of wild-type CFTR (pRSV-CFTR) or 3 &#b5;g of CFTR bearing the mutation S1455X (pRSV-CFTR/S1455X) using 15 &#b5;l of lipofectin and 1 ml of Opti-MEM. Login to comment