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

Heterozygous germline mutations in MSH3 and MLH3 are associated with increased colorectal cancer risk, suggesting they may be included in genetic screening for CRC.

Summary and findings

This study examined the impact of heterozygous germline mutations in MSH3 and MLH3 on colorectal cancer (CRC) risk. The analysis included nearly 12,000 CRC cases and 460,000 controls, revealing a 2.2-fold increase in CRC risk for MSH3 mutations and a 1.6-fold increase for MLH3 mutations. The findings suggest that these mutations may act as tumor suppressors, leading to increased somatic mutations.

Abstract

<h4>Background</h4> 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. <h4>Objectives</h4> 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. <h4>Design</h4> Nearly 12,000 CRC and multiple polyp cases and 460,000 controls were studied. 2,023 patients underwent cancer genome sequencing. <h4>Results</h4> 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. D eletions of ≥ 2bp were particularly increased (∼12-fold) and signature ID4 was usually present ( P< 0.0001). CRCs from heterozygotes without ‘second hits’ showed no hypermutation. <h4>Conclusion</h4> 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. <h4>Summary Box</h4> <h4>What is already known on this topic – summarise the state of scientific knowledge on this subject before you did your study and why this study needed to be done.</h4> Lynch syndrome and constitutional mismatch repair deficiency (cMMRd) are established cancer syndromes, respectively resulting from mono-allelic (heterozygous) and bi-allelic germline mismatch repair (MMR) mutations. Germline bi-allelic MSH3 mutations are known to predispose to a recessive syndrome of multiple colorectal adenomas and colorectal carcinoma (CRC). Weaker evidence, supported herein, suggests that bi-allelic MLH3 mutations cause a similar syndrome. Occasional, relatively small studies have reported CRC cases with heterozygous germline loss-of-function MSH3 or MLH3 mutations, leading to suggestions that these mutations increase CRC risk. However, these studies have essentially been anecdotal, presenting no statistical evidence of increased cancer risk or an identified causal mechanism. MSH3 and MLH3 deficiency in vitro have been linked to insertion-deletion mutations of a few bases, but evidence from human tumours is lacking. We hypothesised that MSH3 and/or MLH3 resemble Lynch and cMMRd genes, in that they predispose to CRC (and possibly other cancers) when heterozygous, as well as in the much rarer bi-allelic mutant state. <h4>What this study adds – summarise what we now know as a result of this study that we did not know before.</h4> In a large case-control analysis, we have shown that heterozygous loss-of-function (LoF) germline MSH3 mutations increase CRC risk about 2.2-fold. Like the Lynch syndrome genes, MSH3 in this context acts as a tumour suppressor (TSG), with somatic second hits in cancers. The second hits are sometimes loss-of-heterozygosity changes, and, of note, one LOH event can sometimes inactivate both MSH3 (chr5q14.1) and the major CRC driver gene APC (chr5q22.2). Heterozygous germline MLH3 mutations also appear to increase CRC risk (1.6-fold) in a similar fashion. The relative risks in heterozygotes are of a size that MSH3 and MLH3 could be said to be moderate risk CRC genes, among the first of this class to be found. Mismatch repair deficiency by loss of MSH3 or MLH3 function does not generally cause MSI, but results in relatively mild hypermutation via deletions of ≥ 2bp, 1bp deletions at oligonucleotide tracts, and mutational signature ID4. ID4 has no previously established cause in human tumours, but has been associated with Rnaseh2b;Tp53 deficiency in mouse and cell models. MSH3-deficient CRCs otherwise largely resemble MSS CRCs. <h4>How this study might affect research, practice or policy – summarise the implications of this study.</h4> Given that Lynch and cMMRd include patients with germline mutations in genes like PMS2 with weaker effects on cancer risk, we suggest that MSH3 and MLH3 could also be regarded as genes for these syndromes. The two genes would reasonably be included in panels used to investigate the causes of CRC and/or multiple polyps in clinical practice. The size of the increased risk suggests that heterozygous gene carriers could be offered modestly enhanced surveillance for CRC (e.g. by earlier or more frequent faecal immunochemical testing). Research into genetic variants in other DNA repair genes, even if not historically associated with CRC risk, may prove fruitful, especially if based on large genome sequencing studies of both germline and tumour genotypes.

Key findings

• Not reported in abstract.

Limitations

• Not reported in abstract.

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