ABCMdb

PMID: 1284468

Cheadle JP, Meredith AL, al-Jader LN
A new missense mutation (R1283M) in exon 20 of the cystic fibrosis transmembrane conductance regulator gene.
Hum Mol Genet. 1992 May;1(2):123-5., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg560Thr From a sample of 373 CF chromosomes, 318 proved to have one of the following mutations: delta F508, delta 1507, G551D, R553X, G542X, R117H, 1717-1G>A, R560T and 621 + 1G>T. Login to comment 6 ABCC7 p.Trp1282* The remaining 55 CF chromosomes were subsequently screened for W1282X by ASO (Allele specific oligonucleotide) hybridisations on dot blot preparations of the exon 20 amplified samples. Login to comment 7 ABCC7 p.Trp1282* The exon 20 W1282X site was amplified by the polymerase chain reaction using the 20i3' and 20i5' primers (7). Login to comment 13 ABCC7 p.Trp1282* ABCC7 p.Arg1283Met ABCC7 p.Arg1283Met Lane 1 corresponds to the blank, lane 2 to the control W1282X heterozygote DNA, lanes 3-5 to the three unrelated R1283M heterozygote affecteds, lane 6 to a known normal, and lanes 7/8, 9/10, and 11/12 to the parents of each of the three R1283M affecteds. Login to comment 15 ABCC7 p.Trp1282* ABCC7 p.Trp1282* This revealed that only the control W1282X heterozygote DNA was identified as positive for the W1282X mutation. Login to comment 16 ABCC7 p.Trp1282* To confirm the absence of W1282X in our population we performed an Mnll digest on 10 jtl of the PCR products (according to the manufacturers specifications). Login to comment 17 ABCC7 p.Trp1282* Normal DNA digests generate 185, 183 and 105 bp fragments, whereas the W1282X mutation destroys a restriction site thereby yielding two fragments of 290 and 183 bp. Login to comment 22 ABCC7 p.Trp1282* To determine if our new mutation was in the same Mnll site as W1282X we performed a double digest with Hinfl. Login to comment 23 ABCC7 p.Trp1282* Since the same restriction pattern was observed with our new mutants and the W1282X control DNA, it was clear that they were in the same site. Login to comment 25 ABCC7 p.Trp1282* Sequencing from a primer 87 bp from the site of the W1282X mutation (5'-T GGA TCA GGG AAG AGT ACT TTG-3'), we identified a novel G-T transversion at position 3980. Login to comment 26 ABCC7 p.Arg1283Met ABCC7 p.Arg1283Met This substitution is predicted to cause the replacement of the amino acid arginine by methionine at residue 1283, and using the established nomenclature (2) has been called R1283M. Login to comment 27 ABCC7 p.Arg1283Met To determine whether R1283M is a harmless polymorphism or a disease causing mutation, we screened 51 normal chromosomes and 85 delta F5O8 chromosomes by Mnll digests. Login to comment 28 ABCC7 p.Arg1283Met No R1283M mutations were identified. Login to comment 29 ABCC7 p.Trp1282* ABCC7 p.Arg1283Met ABCC7 p.Arg1283Met To allow the rapid and specific detection of R1283M, we designed some ASOs (Normal: 5'-AGG CTT TCC TCC ACT G-3', Mutant: 5'-CAG TGG ATG 20i5' 473 bp -- 90 bp 15 - * • Hinfl Fokl Hnll W1282X/R1283M 2Oi3' 183 bp 308 bp 105 bp 185 bp 165 bp Figure 2. Login to comment 31 ABCC7 p.Trp1282* ABCC7 p.Arg1283Met The position of the sequencing primer and the W1282X and R1283M mutations are marked. Login to comment 33 ABCC7 p.Arg1283Met The R1283M mutation also creates a unique Fokl site in exon 20, thus providing an alternative means of identification. Login to comment 35 ABCC7 p.Arg1283Met Clinically, all three of the patients with R1283M are pancreatic insufficient, which has been proposed to result from the presence of two severe alleles (3). Login to comment 36 ABCC7 p.Arg1283Met ABCC7 p.Arg1283Met Two of the patients (L.R. and L.W.) are delta F508/R1283M heterozygotes, and since delta F508 is assumed to be a severe allele, we suggest that R1283M is also a severe allele. Login to comment 42 ABCC7 p.Arg1283Met The haplotype data suggests that the chromosome in which the R1283M mutation occurred probably carried the 1,1,2,2,2,1,2 haplotype (MetD, MetH, XV2c, KM19, D9, G2, J3.ll). Login to comment 43 ABCC7 p.Arg1283Met MUTANT A T NORMAL A T O>T at 3980 Haplotype analysis of further R1283M mutants should enable us to determine the origin of the mutation. Login to comment 44 ABCC7 p.Arg1283Met R1283M is a new missense mutation occurring in the second nucleotide binding fold of the CFTR gene. Login to comment 48 ABCC7 p.Arg1283Met It is possible however, that the identified mutation could be an uncommon sequence polymorphism; only one of the 51 normal chromosomes screened carries the unusual haplotype associated with R1283M. Login to comment 50 ABCC7 p.Trp1282* ABCC7 p.Arg1283Met In screening for R1283M by an Mnll restriction digest it is important to note that one would also identify any W1282X mutants in their population; to distinguish between them the use of ASOs or Fokl digestion is required. Login to comment 51 ABCC7 p.Arg1283Met R1283M accounts for 0.8% (3/373) of CF chromosomes in our population. Login to comment 52 ABCC7 p.Arg1283Met In two of the individuals R1283M can be traced to a Welsh origin, and in the third, either an English or a Welsh descent. Login to comment 55 ABCC7 p.Arg1283Met Haplotype associations with the R1283M mutation, where 1 denotes the absence and 2 denotes the presence of a restriction site. Login to comment 59 ABCC7 p.Arg1283Met R1283M ASO hybridisation to exon 20 amplified samples. Login to comment