In pathogenicity assessment, RNA-based analyses are important for the correct classification of variants, and require gene-specific cut-offs for allelic representation and alternative/aberrant splicing. Beside this, the diagnostic yield of RNA-based techniques capable to detect aberrant splicing or allelic loss due to intronic/regulatory variants has to be elaborated. We established a cDNA analysis for full-length transcripts (FLT) of the four DNA mismatch repair (MMR) genes to investigate the splicing pattern and transcript integrity with active/inhibited nonsense-mediated mRNA-decay (NMD). Validation was based on results from normal controls, samples with premature termination codons (PTC), samples with splice-site defects (SSD), and samples with pathogenic putative missense variants. The method was applied to patients with variants of uncertain significance (VUS) or unexplained immunohistochemical MMR deficiency. We categorized the allelic representation into biallelic (50 ± 10%) or allelic loss (≤10%), and >10% and 10% and 30% of VUS. Our method allows a standardized, systematic cDNA analysis of the MMR FLTs to assess the pathogenicity mechanism of VUS on RNA level, which will gain relevance for precision medicine and gene therapy. Diagnostic accuracy will be enhanced by detecting MMR defects in hitherto unsolved patients. The data generated will help to calibrate a high-throughput NGS-based mRNA-analysis and optimize prediction programs.

Over 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.