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. 2009 May;30(5):757-70.
doi: 10.1002/humu.20936.

Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation, and tumor characteristics

Sven Arnold  1 , Daniel D Buchanan, Melissa Barker, Lesley Jaskowski, Michael D Walsh, Genevieve Birney, Michael O Woods, John L Hopper, Mark A Jenkins, Melissa A Brown, Sean V Tavtigian, David E Goldgar, Joanne P Young, Amanda B Spurdle
Affiliations

Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation, and tumor characteristics

Sven Arnold et al. Hum Mutat. 2009 May.

Abstract

Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice-site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data.

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Figures

Figure 1
Figure 1
PCR amplification of LCL-derived cDNA from variant carriers a. The target amplicon incorporates c.-28 (5'UTR) to c.1426 of MLH1. Lanes A (variant MSH2 c.913G>A) and D (variant MLH1 c.1668-1G>A) display normal wildtype product, with two additional fainter products representing native alternately-spliced mRNA (exon 9 exclusion, exon 9 and 10 exclusion). Lane B (variant MLH1 c.116+5G>C) shows the inclusion of 227bp (uppermost product, Arrow 1) and a concatenated wildtype/variant product (above wildtype product). Lane C (variant MLH1 c.208-3C>G) shows the exclusion of exon 3 (Arrow 2) overlying the native exon 9 exclusion product. b. The target amplicon incorporates c.441 to c.1054 of MLH1. Lanes A (variant MSH2 c.571_573delCTC), B (variant MSH2 c.942+3A>T) and D (variant MSH2 c.913G>A) display normal wildtype product, with fainter product (Arrow 1) comprising a concatenation of wildtype and native alternately-spliced mRNA excluding exon 9. Lane C (variant MLH1 c.790+2_+3insT) shows increased concatenation of wildtype and exon 9 exclusion (Arrow 1, Lane C). Genotyping for the presence of an in cis 655A>G polymorphism in exon 8 confirmed that no wildtype mRNA expressed by the variant allele, and that all normal length mRNA in both upper bands (Arrows 1 and 2, Lane C) was derived from the wildtype allele. The product indicated by Arrow 3 comprises variant-derived product exclusive of exon 9, and that indicated by Arrow 4 comprises a variant-derived product exclusive of both exons 9 and 10. c. The target amplicon incorporates c.1392 to c.1912 of MLH1. Lanes A (variant MSH2 c.1906G>A), C (variant MSH2 c.942+3A>T) and D (variant MSH2 c.913G>A) display normal wildtype band weights. In lane B (variant MLH1 c.1668-1G>A), the band indicated by Arrow 1 comprises a concatenation of wildtype and exon 15 exclusion products, while the product indicated by Arrow 2 comprises exon 15 exclusion only. d. The target amplicon incorporates c.1392 to c.2278 of MLH1. Lanes A (variant MSH2 c.913G>A), B (variant MSH2 c.1865C>T) and D (variant MSH2 c.1906G>C) display normal wildtype product. In lane B (variant MLH1 c.1732-1G>A), the band indicated by Arrow 1 comprises a concatenation of wildtype and exon 16 exclusion products, while the band indicated by Arrow 2 comprises a exon 16 exclusion product only. e. The target amplicon incorporates c.1781 to c.2278 of MLH1. Lanes A (variant MSH2 c.1865C>T), B (variant MSH2 c.1906G>C) and D (variant MSH2 c.571_573delCTC) display normal wildtype products. In lane C (variant MLH1 c.1990-1 g>A) the band indicated by Arrow 1 comprises a concatenation of wildtype and exon 18 exclusion product, while Arrow 2 comprises the exon 18 exclusion product only. f. The target amplicon incorporates c.627 to c.1296 of MSH2. Lanes A (variant MLH1 c.1732-1G>A), B (variant MLH1 c.208-3C>G) and D (variant MLH1 c.116+5G>C) display normal wildtype products. In lane C (variant MSH2 c.942+3A>T), the band indicated by Arrow 1 comprises a concatenation of wildtype and exon 5 exclusion product, while the band indicated by Arrow 2 comprises the exon 5 exclusion product only.
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References

    1. Aretz S, Uhlhaas S, Sun Y, Pagenstecher C, Mangold E, Caspari R, Moslein G, Schulmann K, Propping P, Friedl W. Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene. Hum Mutat. 2004;24(5):370–80. - PubMed
    1. Auclair J, Busine MP, Navarro C, Ruano E, Montmain G, Desseigne F, Saurin JC, Lasset C, Bonadona V, Giraud S, et al. Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing. Hum Mutat. 2006;27(2):145–54. - PubMed
    1. Caldes T, Godino J, Sanchez A, Corbacho C, De la Hoya M, Lopez Asenjo J, Saez C, Sanz J, Benito M, Ramon YCS, et al. Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2 and MSH6 mutation carriers in hereditary non-polyposis colorectal cancer. Oncol Rep. 2004;12(3):621–9. - PubMed
    1. Cartegni L, Krainer AR. Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet. 2002;30(4):377–84. - PubMed
    1. Casey G, Lindor NM, Papadopoulos N, Thibodeau SN, Moskow J, Steelman S, Buzin CH, Sommer SS, Collins CE, Butz M, et al. Conversion analysis for mutation detection in MLH1 and MSH2 in patients with colorectal cancer. Jama. 2005;293(7):799–809. - PMC - PubMed

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