ABCMdb

PMID: 10439967

Liechti-Gallati S, Schneider V, Neeser D, Kraemer R
Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease.
Eur J Hum Genet. 1999 Jul;7(5):590-8., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Arg560Ser ABCC7 p.Ala613Thr ABCC7 p.Thr1299Ile Using simultaneously SSCP and heteroduplex (HD) analysis, a total of 80 known CF mutations (28 missense, 22 frameshift, 17 nonsense, 13 splicesite) and 20 polymorphisms was analysed resulting in a detection rate of 97.5% including the 24 most common mutations worldwide. The ability of this technique to detect mutations independent of their nature, frequency, and population specificity was confirmed by the identification of five novel mutations (420del9, 1199delG, R560S, A613T, T1299I) in Swiss CF patients, as well as by the detection of 41 different mutations in 198 patients experimentally analysed. Login to comment 20 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Glu585* ABCC7 p.Gly85Glu ABCC7 p.Arg560Thr ABCC7 p.Thr338Ile ABCC7 p.Arg560Ser ABCC7 p.Met1101Lys ABCC7 p.Arg1070Gln ABCC7 p.Ser1235Arg ABCC7 p.Arg792* ABCC7 p.Glu1371* ABCC7 p.Arg1066Cys ABCC7 p.Ala120Thr ABCC7 p.Asn1303His ABCC7 p.Gln220* ABCC7 p.Tyr1092* ABCC7 p.Ile1005Arg ABCC7 p.Gln525* ABCC7 p.His199Tyr ABCC7 p.Ser1255* ABCC7 p.Ser977Pro ABCC7 p.Ser945Leu ABCC7 p.Gln39* ABCC7 p.Gln552* ABCC7 p.Asp1152His ABCC7 p.Trp401* ABCC7 p.Gln30* ABCC7 p.Arg1283Met ABCC7 p.Lys710* ABCC7 p.Gly27Glu The distribution of analysed known mutations is similar to that of the total number of mutations in the entire CFTR gene: missense mutations account for 35% (G27E, G85E, R117H, A120T, I148T, H199Y, R334W, T338I, R347P, R347H, A455E, M718K, S5449N, S5449I, G551D, R560T, R560S, S945L, S977P, I1005R, R1066C, R1070Q, M1101K, D1152H, S1235R, R1283M, N1303K, N1303H), followed by 28% of frameshift mutations (175delC, 394delTT, 457TAT- > G, 905delG, 1078delT, I507, F508, 1609delCA, 1677delTA, 2143delT, 2176insC, 218delA, 2184insA, 2869insG, 3659delC, 3732delA, 3821delT, 3905insT, 4016insT, 4172delGC, 4382delA), 21% of nonsense mutations (Q30X, Q39X, Q220X, W401X, Q525X, G542X, Q552X, R553X, V569X, E585X, K710X, R792X, Y1092X, R1162X, S1255X, W1282X, E1371X), and 16% of splice site mutations (621 + 1G- > T, 711 + 1G- > T, 711 + 5G- > A, 1717-1G- > A, 1898 + 1G- > A, 1898 + 5G- > T, 2789 + 5G- > A, 3271 + 1G- > A, 3272-26A- > G, 3601-17T- > C, 3849 + 4A- > G, 3849 + 10kbC- > T, 4374 + 1G- > T). Login to comment 22 ABCC7 p.Asn1303Lys Polymerase Chain Reaction Amplification The 27 exons (including exon/intron boundaries) as well as intron 19 of the CFTR gene were amplified by the polymerase chain reaction (PCR)13 using approximately 200 ng of genomic DNA, 10 mM dNTPs (PCR Nucleotide Mix, Boehringer/ Roche Diagnostics Rotkreuz, ZG, Switzerland), 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 2.5 unit Taq polymerase (Boehringer/Roche Diagnostics Rotkreuz, ZG, Switzerland) and 20 pmol of each primer in a total volume of 50 µl. Twenty-eight cycles of PCR with denaturation at 94°C for 15 s, annealing at either 61°C (exons 1, 2, 5-12, 13CD, 15, 16, 18-22, 24) or 53°C (exons 3, 4, 13AB, 14a, 14b, 17a, 17b, 21/N1303K, 23) for 15 s and extension at 72°C for 45 s were carried out in PE 9600 and PE 2400 thermocyclers. Login to comment 24 ABCC7 p.Asn1303Lys Sequences for the primers 1-10, 11 (forward), 12-16, 17b, 18-20, 21 (forward), 22, 23, 24 (forward) have been reported by Zielenski et al;14 exon 17a was amplified using the primer sequences described by Cheadle et al;15 the primers for the amplification of intron 19 were designed by Highsmith et al;16 and the primer sequence for the detection of N1303K was reported by Friedman et al.17 The following primer sequences were derived from the present study: Exon 11 (reverse): 5' - GTGATTCTTAACCCAC- TAGCC - 3' Exon 21 (reverse): 5' - AAGTGTGTAGAATGATGT- CAGC - 3' Exon 24 (reverse): 5' - CGAGCTCCAATTCCAT- GAGG - 3' SSCP Analysis Three microliters of the amplification product were added to 2-3 µl of SSCP buffer (95% formamide, 100 mM NaOH, 0.25% bromphenol blue, 0.25% xylencyanol) and denatured at 95°C for 2 min followed by rapid cooling on ice. Three microliters of the mixture were loaded on to a 12% nondenaturing polyacrylamide gel (99% acrylamide, 1% piperazine diacrylamide (PDA)) cast on to GelBond (Bioconcept, Allschwil, BL, Switzerland). Login to comment 34 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Gly85Glu ABCC7 p.Ser549Asn ABCC7 p.Arg560Thr ABCC7 p.Asp1152His Intron 19, all 27 exons and their exon-intron boundaries, including the 24 most common mutations worldwide (G85E, R117H, 621 + 1G- > T, 711 + 1G- > T, 1078delT, R334W, R347P, A455E, I507, F508, 1717-1G- > A, G542X, S549N, G551D, R553X, R560T, 1898 + 1G- > A, 2184delA, 2789 + 5G- > A, R1162X, 3659delC, 3849 + 10kbC- > T, W1282X, N1303K) (Cystic Fibrosis Genetic Analysis Consortium 1994), and the 15 most common mutations in our population (I148T, 1078delT, R334W, R347P, F508, 1717-1G- > A, G542X, R553X, 2347delG, D1152H, R1162X, 3849 + 10kbC- > T, 3905insT, W1282X, N1303K), were considered in this study. Login to comment 43 ABCC7 p.Arg792* The R792X nonsense mutation in exon 13 and the 4374 + 1G- > T splice site mutation in exon 23 were not detectable with our screening method, whereas 78 out of 80 mutations and all 20 polymorphisms showed band shifts either in the single or in the double strands of the amplified DNA fragments. Login to comment 44 ABCC7 p.Arg347His ABCC7 p.Thr338Ile ABCC7 p.Met1101Lys ABCC7 p.Glu1371* ABCC7 p.Arg1066Cys ABCC7 p.Tyr1092* ABCC7 p.Ser1255* Three mutations (R1066C, M1101K, E1371X) could only be identified after restriction enzyme digestion of the amplification product, and five mutations (711 + 1G- > T, R347H, T338I, Y1092X, S1255X) were discovered in the uncut, but not in the digested, PCR product. Login to comment 54 ABCC7 p.Arg560Ser R560S A transversion A- > T at nucleotide position 1812 located in exon 12 leading to the exchange of the amino acid Arg by a Ser. 4. Login to comment 55 ABCC7 p.Ala613Thr A613T A transversion G- > A at nucleotide position 1969 located in exon 13 leading to the exchange of the amino acid Ala by a Thr. Login to comment 57 ABCC7 p.Thr1299Ile T1299I A transversion C- > T at nucleotide position 4028 located in exon 21 leading to the exchange of the amino acid Thr by a Ile. In nine patients we found only one mutation, the second mutation being not detectable by our screening system. Login to comment 61 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Ser549Asn ABCC7 p.Arg560Thr The slots C present wild type (wt) sequences, 1-8 present amplification products from CF patients with the following genotypes: 1 = R553X/R553X; 2 = 1717-1G- > A/wt; 3 = R553X/wt; 4 = G542X/wt; 5 = G542X/1717-1G- > A; 6 = G551D/wt; 7 = R560T/wt; 8 = S549N/wt. Login to comment 79 ABCC7 p.Ser977Pro Even the largest fragment used in this screening (exon 16, 570 bp) gave rise to the identification of a base substitution (T- > C, S977P) without digestion of the amplification product before SSCP analysis. Login to comment 92 ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Glu585* ABCC7 p.Gly85Glu ABCC7 p.Arg1158* ABCC7 p.Met1101Lys ABCC7 p.Ser1235Arg ABCC7 p.Ala120Thr ABCC7 p.Gln525* ABCC7 p.Ser945Leu ABCC7 p.Gln39* ABCC7 p.Asp1152His ABCC7 p.Lys710* ABCC7 p.Leu568Phe ABCC7 p.Met348Lys ABCC7 p.Gly126Asp The technique developed demonstrates excellent single-strand separation and non-radioactive visualisation on polyacrylamide gels, and is time-saving and directly Table 2 Known mutations identified in 198 CF patients analysed investigatively Exon (E) Number of CFTR mutations intron (I) chromosomes Patient`s nationality Highest prevalence ∆F508 E10 212 miscellaneous 3905insT E20 025 Swiss Swiss, Amish, Arcadian R553X E11 020 Swiss, German German 1717-1G->A I10 017 Swiss, Italian Italian N1303K E21 011 Swiss, French, Italian Italian W1282X E20 014 Swiss, Italian, Israelit Jewish-Askhenazi G542X E11 009 Swiss, Spanish, Italian Spanish 2347delG E13 008 Swiss R1162X E19 006 Swiss, Italian, Russian Italian 3849+10kbC->T I19 005 German, French R347P E07 004 Swiss T5 I08 004 Swiss R334W E07 003 Swiss Q525X E10 003 Swiss 3732delA E19 003 Swiss S1235R E19 003 Italian, Turkish G85E E03 002 Italian, Greek I148T E04 002 Austrian, Turkish French-Canadian 621+1G->T I04 002 French French-Canadian 1078delT E07 002 Swiss E585X E12 002 Italian 2176insC E13 002 Swiss, Italian 2789+5G->A I14b 002 Italian Spanish D1152H E18 002 Swiss, French 4016insT E21 002 Turkish Q39X E02 001 Swiss 394delTT E03 001 Swiss Nordic, Finnish R117H E04 001 Swiss A120T E04 001 Swiss G126D E04 001 Swiss 711+5G->A I05 001 Russian M348K E07 001 Italian L568F E12 001 Italian 2183AA->G E13 001 Italian Italian K710X E13 001 Swiss S945L E15 001 French 3272-26A.->G I17a 001 Swiss M1101K E17b 001 Swiss Huttite 3601-17C->T I18 001 Swiss R1158X E19 001 Swiss 4005+1G-A I20 001 Italian applicable to early diagnostic testing, carrier detection and prenatal diagnosis. Login to comment 102 ABCC7 p.Arg560Ser ABCC7 p.Ala613Thr ABCC7 p.Thr1299Ile Figure 4 SSCP gel demonstrating new mutations (*) in exon 4 (420del9), exon 7 (1199delG), exon 12 (R560S), exon 13 (A613T), and exon 21 (T1299I) of the CFTR gene compared with three control patterns. Login to comment