ABCMdb

PMID: 1384321

Grebe TA, Doane WW, Richter SF, Clericuzio C, Norman RA, Seltzer WK, Rhodes SN, Goldberg BE, Hernried LS, McClure M, et al.
Mutation analysis of the cystic fibrosis transmembrane regulator gene in Native American populations of the southwest.
Am J Hum Genet. 1992 Oct;51(4):736-40., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Direct mutation analysis of six mutations of the CFTR gene-namely, AF508, G542X, G551D, R553X, N1303K, and W1282X-was performed on PCR-amplified genomic DNA extracted from blood samples. Login to comment 7 ABCC7 p.Gly542* Of the 12 affected individuals studied, no AF508 mutation was detected; only one G542X mutation was found. Login to comment 9 ABCC7 p.Gly542* All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Login to comment 24 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* These mutations are G551D (Cutting et al. 1990), G542X (Kerem et al. 1990), and R553X (Cutting et al. 1990), which are all in exon 11, and N1303K (Osborne et al. 1991), in exon 21. Login to comment 25 ABCC7 p.Trp1282* A mutation in exon 20, W1282X, is present in approximately 60% of CF chromosomes in the AshkenaziJewish population (Vidaud et al. 1990). Login to comment 49 ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys The reaction mix for AF508 contained 200 gM each dNTP, 0.6 gM each primer, 1 jg DNA, 2.5 U Promega Taq polymerase, and 5 jl Promega 10 X reaction buffer (500 mM KCl, 100 mM Tris-HCl pH 8.8, 15 mM MgCl2, 1% Triton X-100) in a total volume of 50 pl. Reaction mixes for the other mutations and linked markers were similar, with the following modifications: for exon 11, 150 jM each dNTP and 0.4 jM each primer; for N1303K, 200 jM each dNTP and 1 jM each primer; for XV2c, 100 jM each dNTP and 0.3 jM each primer; for KM19, 200 jM each dNTP and 0.4 jM each primer; and, for W1282X, 200 jiM each dNTP and 0.6 jM each primer. Login to comment 50 ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys Reaction mixes for the other mutations and linked markers were similar, with the following modifications: for exon 11, 150 jM each dNTP and 0.4 jM each primer; for N1303K, 200 jM each dNTP and 1 jM each primer; for XV2c, 100 jM each dNTP and 0.3 jM each primer; for KM19, 200 jM each dNTP and 0.4 jM each primer; and, for W1282X, 200 jiM each dNTP and 0.6 jM each primer. Login to comment 55 ABCC7 p.Trp1282* ABCC7 p.Trp1282* C. Restriction Digestion of Amplified Samples For exon 1 1, 12 gI PCR product were digested with 30 U HincII and/or 30 U MboI in a 30-gl reaction mix containing the recommended reaction buffer. Electrophoresis was in a 1.8% agarose gel. For KM19, 12 gI PCR product were digested with 30 U PstI in a 30-il reaction mix containing the recommended buffer. Electrophoresis was in a 1.2% agarose gel. For XV2C, 20 jl PCR product were digested with 30 U TaqI in a 30-pl reaction mix containing the recommended buffer. Electrophoresis was in a 1.8% gel. For W1282X, 20 jgl PCR product were digested with 8 U MnlI in a 30-glreaction mix containing the recommended buffer. Electrophoresis was in a 2.0% gel. Login to comment 62 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* After hybridization, the filters were washed for 5 min at room temperature in 5 x SSC and then for 30 min in 2 x SSC at the following temperatures: 49°C for the AF508 probe and 43°C for its normal homologue, 430C for G542X and its normal sequence, and 51 'C for N1303K and its normal sequence. Login to comment 78 ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys Direct mutation analysis of the CFTR gene in the patients revealed no copies of AF508, GSS1D, R553X, N1303K, or W1282X. Login to comment 79 ABCC7 p.Gly542* One patient (008-1), however, carries a single copy of G542X. Login to comment