ABCMdb

PMID: 7525964

Miedzybrodzka ZH, Yin Z, Kelly KF, Haites NE
Evaluation of laboratory methods for cystic fibrosis carrier screening: reliability, sensitivity, specificity, and costs.
J Med Genet. 1994 Jul;31(7):545-50., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Gly542* Standard methods of CF mutation analysis include deletion detection using polyacrylamide gel electrophoresis (PAGE) of PCR product (for example, AF508 and AI507), restriction enzyme digestion ofPCR product (for example, G551D, R553X, 621 + 1(G-.T)), and probing of PCR product with labelled allele specific oligonucleotides (ASOs) (for example, G542X). Login to comment 12 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Methods The Cellmark ARMS system tests simultaneously for the mutations AF508, G551D, G542X, and 621 + 1(G--T).5 PCR is performed in two tubes, one tube containing mutation specific primers for G551D, G542X, and normal site specific primers for AF508 and 621 + 1(G-+T), the second containing mutation specific primers for AF508 and 621 + 1(G-4T) and normal site specific primers for G551D and G542X. Login to comment 19 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* We chose to evaluate dot blotting by using the Inno-LiPA CF2 kit as an example (Innogenetics).9 This kit detects the mutations AF508, AI507, G551D, G542X, N1303K, W1282Xm 1717-1,G-A, and R553X.4 12"1 Mouthwash DNA preparation (5 p1) and Taq polymerase are added to the PCR reagents provided, with biotin dUTP incorporated into PCR product as a label. Login to comment 26 ABCC7 p.Gly551Asp ABCC7 p.Gly542* 2A + B normal sample: lane A indicates normal sequence at AF508 and 621 + 1, G-T mutation sites, lane B indicates normal sequence at G542X and G551D mutation sites. Login to comment 29 ABCC7 p.Gly551Asp 7A + B G551D/AF508 compound heterozygote. Login to comment 30 ABCC7 p.Gly542* 8A + B G542X/621 + 1, G-T compound heterozygote. Login to comment 32 ABCC7 p.Gly542* 12A + B G542X carrier. Login to comment 37 ABCC7 p.Gly551Asp ABCC7 p.Arg553* Strip 7 G551D carrier, strip 10 R553X carrier. Login to comment 39 ABCC7 p.Trp1282* ABCC7 p.Gly542* Strip 12 is AF508/G542X compound heterozygote, 14 is AF508IG551D compound heterozygote, 15 is 1717-1,G--A carrier, 16 is A1507 carrier, 17 is W1282X carrier. Login to comment 40 ABCC7 p.Asn1303Lys Strip 18 represents a AF508/N1303K compound heterozygote, 19 is no DNA control, 20 is wild type control. Login to comment 42 ABCC7 p.Gly551Asp ABCC7 p.Arg553* The third method we evaluated combines deletion analysis with a restriction enzyme digest, allowing simultaneous detection of the mutations AF508, AI507, G551D, and R553X (deletion/digest/PAGE method). Login to comment 45 ABCC7 p.Gly551Asp ABCC7 p.Arg553* The mutations G551D and R553X destroy a HincII restriction site, AF508 and AI507 cause a smaller band (fig 3). Login to comment 52 ABCC7 p.Gly551Asp Lanes 4, 9, and 11 G551D carriers. Login to comment 54 ABCC7 p.Gly551Asp Lane 6 AF508/G551D compound heterozygote. Login to comment 62 ABCC7 p.Gly551Asp ABCC7 p.Arg553* DELETION/DIGEST/PAGE METHOD This method was used to test DNA from 59 carriers, 58 partners (previously tested negative), and 64 persons affected by CF, for AF508, G551D, AI507, and R553X. Login to comment 84 ABCC7 p.Gly542* The G542X mutation specific band appeared, although it was fainter than its control. Login to comment 85 ABCC7 p.Gly542* When the test was repeated on two occasions, no mutation specific band was present and direct sequencing confirmed that DNA tested had the wild type sequence at the G542X site. Login to comment 97 ABCC7 p.Gly551Asp ABCC7 p.Gly542* About half of the reported failures occurred in the first two tests in a batch, with faint extra bands in the mutant G551D and G542X positions. Login to comment 107 ABCC7 p.Gly551Asp Two out of nine G551D carriers might have been undetected had we relied upon this system alone (two false negatives) and, therefore, although we know that the system works well in other hands, we did not persist with the technique. Login to comment