Heterozygous germline MSH3 mutations, and probably MLH3 mutations, act as classical tumour suppressors, leading to excess somatic deletion mutations, signature ID4 and increased colorectal cancer risk

Background. MSH3 and MLH3 are non-canonical DNA mismatch repair genes, involved in repairing insertion-deletion mutations. Colorectal cancer (CRC) and adenomas have been reported in patients with bi-allelic germline MSH3 mutations, and in a very few bi-allelic MLH3 mutation carriers. Objectives. We hypothesised that germline loss-of-function MSH3 and MLH3 mutations were akin to constitutional mismatch repair deficiency (cMMRd) and Lynch syndrome, such that CRC could result from either bi-allelic germline mutations, or heterozygous germline mutations after second hits. Design. Nearly 12,000 CRC and multiple polyp cases and 460,000 controls were studied. 2,023 patients underwent cancer genome sequencing. Results. One CRC/multiple polyp case had bi-allelic MSH3 mutations and another, bi-allelic MLH3 mutations. MSH3 and MLH3 germline heterozygotes had an increased risk of CRC (respectively 2.2-fold, P=6.6x10-5 and 1.6-fold, P=0.028), owing to somatic second hits that inactivated the wildtype allele. Single second hits sometimes inactivated both MSH3 and the nearby APC gene. All CRCs with MSH3 or MLH3 deficiency were microsatellite-stable, but hypermutant. Deletions of 2 or more base pairs were particularly increased (about 12-fold) and signature ID4 was usually present (P<0.0001). CRCs from heterozygotes without second hits showed no hypermutation. Conclusion. The phenotypes of bi-allelic MSH3 and MLH3 mutation carriers resemble some cMMRd patients. Heterozygous germline MSH3 and MLH3 alleles have incomplete penetrance, but increase CRC risk via hypermutation, phenotypically resembling PMS2-mutant Lynch syndrome. A causal association with specific mutations has not previously been reported for ID4 in human tumours. ID4 probably does not have a single aetiology, but can result from MSH3 or MLH3 deficiency. The findings of this manuscript are provisional and should not be used to guide clinical practice, health-related behavior, or policy.