Abstract
AbstractOver 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient’s cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.
Funder
Fundació La Marató de TV3
Institut National Du Cancer
Association Nationale de la Recherche et de la Technologie
EC | European Regional Development Fund
Direction Générale de l’Offre des Soins (DGOS) Groupement des Entreprises Françaises dans la Lutte contre le Cancer (Gefluc) OpenHealth Institute
Deutsche Krebshilfe
Wilhelm Sander-Stiftung
Publisher
Springer Science and Business Media LLC
Subject
Genetics (clinical),Genetics
Reference41 articles.
1. Lynch HT, de la Chapelle A. Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet. 1999;36:801–18.
2. Müller-Koch Y, Kopp R, Lohse P, Baretton G, Stoetzer A, Aust D, et al. Sixteen rare sequence variants of the hMLH1 and hMSH2 genes found in a cohort of 254 suspected HNPCC (hereditary non-polyposis colorectal cancer) patients: mutations or polymorphisms? Eur J Med Res. 2001;6:473–82.
3. Peltomäki P, Vasen H. Mutations associated with HNPCC predisposition—update of ICG-HNPCC/INSiGHT mutation database. Dis Markers. 2004;20:269–76.
4. Auclair J, Busine MP, Navarro C, Ruano E, Montmain G, Desseigne F, et al. Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing. Hum Mutat. 2006;27:145–54.
5. Lastella P, Surdo NC, Resta N, Guanti G, Stella A. In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects. BMC Genom. 2006;7:243.
Cited by
7 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献