In around 30% of families with colorectal adenomatous polyposis, no germline mutation in the previously-implicated genes APC, MUTYH, POLE, POLD1, or NTHL1 can be identified, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis was performed. We identified two unrelated individuals with differing compound-heterozygous loss-of-function germline mutations in the mismatch repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1g>a, c.2760delC, c.3001-2a>c) was indicated on RNA and protein level. Analysis of the diseased individuals’ tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST) and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the loss-of-function effect and causal relevance of the mutations. The pedigrees, genotypes, and the frequency of MSH3 mutations in the general population are consistent with an autosomal recessive mode of inheritance. Both index persons had an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing Constitutional Mismatch Repair Deficiency Syndrome (CMMRD). Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study we describe biallelic germline mutations of MSH3 in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis.

Purpose: Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can be applied clinically.Experimental Design: We assessed the expression, stability, and mismatch repair activity of 38 MLH1 missense variants and determined the pathogenicity status of recurrent variants using clinical data.Results: Four recurrent variants were classified as neutral (K618A, H718Y, E578G, V716M) and three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair but showed defects in expression. Quantitative PCR, pulse-chase, and thermal stability experiments confirmed decreases in protein stability, which were stronger in the pathogenic variants. The minimal cellular MLH1 concentration for mismatch repair was determined, which corroborated that strongly destabilized variants can cause repair deficiency. Loss of MLH1 tumor immunostaining is consistently reported in carriers of the pathogenic variants, showing the impact of this protein instability on these tumors.Conclusions: Expression defects are frequent among MLH1 missense variants, but only severe defects cause Lynch syndrome. The data obtained here enabled us to establish a threshold for distinguishing tolerable (clinically neutral) from pathogenic expression defects. This threshold allows the translation of laboratory results for uncertain MLH1 variants into pathogenicity statements for diagnosis, thereby improving the targeting of cancer prevention measures in affected families.

Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome that predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C-terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1-PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1-CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased coexpression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations. Hum Mutat 31:975-982,