Accurate detection of constitutional mismatch repair deficiency by a simple, sequencing-based microsatellite instability assay

Constitutional mismatch repair deficiency (CMMRD) is a rare but highly penetrant, cancer-predisposition syndrome, which manifests in early childhood through to adolescence. Patients benefit from clinical surveillance and altered treatment. CMMRD is caused by biallelic, germline mutations of MMR genes, but genetic diagnosis can be confounded by variants of unknown significance and pseudogenes. Assays have been developed to detect low-level microsatellite instability (MSI) in non-neoplastic tissues, a hallmark of CMMRD, but are either laborious or of limited sensitivity. We aimed to develop a simple and scalable, sequencing-based MSI assay for CMMRD diagnostics. 24 short (7-12bp) and monomorphic microsatellites were amplified and sequenced from peripheral blood leukocyte DNAs of 94 anonymised controls, and 32 genetically confirmed CMMRD patients. The CMMRD patients include approximately 15% of the known, global population and mutations in each of the MMR genes implicated in the condition. By analysing low frequency microsatellite length variation, the assay was able to distinguish CMMRD from control patients with 100% accuracy and provides an inexpensive tool for rapid CMMRD diagnosis and screening.