RNA sequencing uncovers clinically actionable germline intronic MSH2 variants in previously unresolved Lynch syndrome families

Background

Tumour sequencing has improved the ability to resolve a Lynch syndrome diagnosis in patients with mismatch repair-deficient (MMRd) colorectal and endometrial cancers. Laboratory and population-based cohorts have reported double somatic mutations in 24.7%– 32.8% of MMRd colorectal cancers and 13.2%–21.8% of MMRd endometrial cancers.1–3 Despite these advances in testing, approximately 25% of such cases remain unexplained.2 Recent studies show that, when added to germline DNA testing, RNA sequencing has the potential to increase diagnostic yield (number of clinically actionable variants) by reducing variants of uncertain significance (VUS) and detecting cryptic pathogenic germline variants in many patients, including cases where there is high suspicion for a hereditary cancer syndrome.4–7 We describe the detection of likely pathogenic variants (VLP) in the MSH2 gene in two individuals who proceeded with RNA sequencing and the subsequent interventions and outcomes.

Case presentation

The proband of Family A was diagnosed with endometrioid type endometrial carcinoma exhibiting loss of expression of the MSH2 protein in her early 40s. The family history met Amsterdam II criteria (figure 1). Paired tumour-germline DNA testing for Lynch syndrome revealed only a single somatic pathogenic variant in MSH2. Concurrent DNA and RNA analysis, which included high-throughput DNA and massively parallel RNA sequencing as previously described,8 identified the MSH2 c.2459-12A>G VLP in this patient. MSH2 c.2459-12A>G creates a novel splice acceptor site resulting in an abnormal transcript subject to nonsense-mediated mRNA decay (NMD) due to an insertion of 11 nucleotides from intron 14 in exon 15 (r.2458_2459ins2459-11_2459–1). This intronic variant had previously been classified as a VUS, and its reclassification to VLP following ACMG/AMP guidelines9 was based on RNA sequencing data from this patient (PS3), including the absence of r.2458_2459ins2459-11_2459–1 expression in all 345 healthy, ancestrally diverse controls used for RNA sequencing validation at this laboratory. Additional lines of evidence include previously observed phenotypic evidence where MSH2 c.2459–12A>G has been detected in individuals and families meeting Amsterdam I or II criteria for Lynch syndrome and/or with tumours exhibiting loss of MSH2 and MSH6 nuclear expression by immunohistochemistry10 11 (PP4), deleterious in silico predictions (PP3), and the variant’s rarity in the Genome Aggregation Database (gnomAD) population database (PM2) which uses whole genome sequencing of individuals to provide coverage of both exonic and intronic regions.

The proband’s affected brother, who was diagnosed with an MSH2-deficient and MSH6-deficient rectal cancer in his late 20s, also underwent paired tumour-germline DNA testing for Lynch syndrome which revealed no actionable germline findings and single somatic mutations in the MSH2 and MSH6 genes. He subsequently tested positive for the MSH2 c.2459–12A>G VLP, as did the proband’s sister who had a 10 mm MSH2-deficient colon polyp detected in her mid-30s. RNA sequencing on the proband’s sister also confirmed the presence of the same abnormal transcript as the proband.

The proband of Family B was diagnosed with endometrioid type endometrial carcinoma exhibiting loss of expression of the MSH2 and MSH6 proteins in her late 50s. The family history nearly met Amsterdam II criteria (figure 2). Paired tumour-germline DNA testing for Lynch syndrome revealed only copy-neutral loss of heterozygosity of the MSH2 and MSH6 genes. Following this result, the proband’s sister, who was diagnosed with triple negative breast cancer and colon cancer in her mid-30s, underwent concurrent DNA and RNA analysis, and the MSH2 c.2458+976A>G VLP was identified. MSH2 c.2458+976A>G, which was subsequently confirmed in this family’s proband, creates a novel splice donor site and along with an upstream cryptic acceptor site resulting in an abnormal transcript subject to NMD with insertion of 188 nucleotides from intron 14 (r.2458_2459ins2458+788_2458+975). This abnormal transcript was not identified in healthy controls used for RNA sequencing validation at this laboratory. Furthermore, this deep intronic variant was previously out of reporting range, but RNA sequencing allowed for its subsequent detection via Sanger sequencing. Utilizing RNA sequencing data in conjunction with available phenotypic evidence and this variant’s rarity in the gnomAD population database, MSH2 c.2458+976A>G was classified as likely pathogenic following ACMG/AMP guidelines,9 using the following lines of evidence: PS3, PS4, PP4 and PP3.

Investigations

We performed germline RNA sequencing for two individuals with unresolved MMRd endometrial cancer from families suspicious for Lynch syndrome. These individuals previously underwent paired germline/tumour DNA sequencing of the MMR genes (MLH1, MSH2, MSH6 and PMS2) and had only a single somatic Lynch syndrome gene mutation or copy-neutral loss of heterozygosity identified, concordant with the pattern of protein loss detected via immunohistochemistry.

Outcome and follow-up

Since the detection of MSH2 c.2459-12A>G, the proband of Family A has undergone colonoscopy and removal of a single adenomatous polyp. Upper gastrointestinal endoscopy revealed one fundic gland polyp in the gastric body. This individual also had a CT scan of the chest, abdomen and pelvis (normal) and urine cytology (normal). Both siblings in Family A who also carry the familial MSH2 VLP are undergoing intensive surveillance as recommended for families with Lynch syndrome, including annual colonoscopy, that has not identified any additional cancers to date.

Since the detection of MSH2 c.2458+976A>G, the proband of Family B has undergone colonoscopy and has increased frequency from every 3 years to annually.