Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes

Abstract

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 <40% as unclear. We defined isoforms up to 10% and exon-specific exceptions as alternative splicing, set the cut-off for SSD in cDNA + P to 30-50%, and regard >10% and <30% as unclear. FLT cDNA analyses designated 16% of all putative missense variants and 12% of VUS as SSD, detected MMR-defects in 19% of the unsolved patients, and re-classified >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.

Fig. 1

Alternative splicing (AS) of a complete exon, a combination of two exons (with bracket), or a partial exon (black triangle) is depicted for the four MMR transcripts, as we detected it by LR-PCR in ten MMR-proficient control-pairs of cDNA-P/+P. The maximum relative intensity of AS in cDNA+P is given in % below the exon number, the frequency how often AS was found in the 20 samples is indicated by +/light gray for 1–3 samples, ++/medium gray for recurrence in 4–6 samples, and +++/dark gray for frequent AS detected in seven or more samples, and the predicted effect of AS on the reading frame is given: if = in frame, oof = out-of frame

Fig. 2

Schematic presentation of splicing intensities in cDNA+P found for variants in the four MMR transcripts MLH1, MSH2, MSH6, and PMS2 sorted by exon numbers. Exon skipping in normal controls (gray dots) was defined as alternative splicing, with their frequency correlated to the thickness of the line, the height giving the range of splicing intensity in %. Alternative splicing usually ranged from 0% to 10%, but showed a few exon-specific exceptions. Based on this, the lower, light gray zone comprises splice-neutral variants. In samples with a designated splice site defect aberrant splicing was expected and found in a range of 30–50%. The gray zone above was defined to designate splicing defects. After definition of the cut-off values, we investigated the splicing of the variant-bearing exon in samples with a PTC, pathogenic missense variant, or VUS including potential SSD, predicted missense or synonymous variant