A review of mismatch repair gene transcripts: issues for interpretation of mRNA splicing assays

Abstract

Many mismatch repair (MMR) gene disease-causing mutations identified in cancer patients result in aberrant messenger RNA (mRNA) splicing. However, mRNA assay interpretation can be complicated by the existence of naturally occurring alternative mRNA transcripts, most of which have not been formally described or fully characterized. Here, we provide a comprehensive catalogue of all MMR transcripts described to date, and a review of MMR nucleotide variants associated with an apparent upregulation of alternatively spliced transcripts. This work sets reference starting points for designing and interpreting MMR RNA analyses. Our database and literature searches retrieved 30 MLH1, 22 MSH2, 4 MSH6 and 9 PMS2 alternative transcripts, many predicted to introduce premature termination codons. Furthermore, we collected information on 66 MLH1, 24 MSH2 and 6 PMS2 nucleotide variants reported to be associated with altered expression of at least one of these alternative transcripts, and in many instances reported as splicing mutations. This review shows that there are many alternatively spliced MMR transcripts, which have potential to confound interpretation of splicing assays. These findings highlight the need to perform RNA analysis of patients systematically in parallel with control individuals, and call for the implementation of quantitative assessment of transcript levels for informed interpretation of mRNA assays.

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24MLH1andMSH2Gene Variants

“…S1 and S2) in patient samples, in the ex vivo minigene assay, or in both. These involved in all cases known alternatively spliced transcripts [Genuardi et al., ; Thompson et al., ]. Interestingly, for MSH2 c.2006‐6T>C, partial exon 13 exclusion was only detected by the minigene assay (Fig.…”

“…All RT‐PCR experiments were performed in duplicate. Since alternative splicing is commonly observed for MLH1 and MSH2 [Genuardi et al., ], to improve the detection and interpretation of splicing aberrations, eight control samples were also analyzed [Thompson et al., ].…”

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

“…Adapted minigenes should be designed for such variants (e.g., Naruse et al 2009;Grodecka et al 2014). Genomic regions where multiple exons are involved in alternative splicing, as is the case for MLH1 exon 9, 10, and 11, are also more challenging for a representative minigene design (Bianchi et al 2011;Thompson et al 2015).…”

“…2015), using the following symbols: Δ (skipping of exonic sequence), ▾ (inclusion of intronic sequence), p (acceptor-site shift), q (donor-site shift). We did not quantify the relative expression of different transcripts produced in the minigene assay by the same variant or wild-type allele.…”

“…(1) at the canonical À1, À2, +1, +2 position (nine variants) (2) in the consensus splice site regions excluding the canonical positions (eight variants) (3) exonic outside the consensus splice site regions (14 variants) (4) deep intronic (four variants) (Thompson et al 2015), using the following symbols: D (skipping of exonic sequence), ▼ (inclusion of intronic sequence), p (acceptor-site shift), q (donorsite shift). We did not quantify the relative expression of different transcripts produced in the minigene assay by the same variant or wild-type allele.…”