Microsatellite instability in mismatch repair proficient colorectal cancer: clinical features and underlying molecular mechanisms.

BACKGROUND: Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. METHODS: From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. FINDINGS: In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. INTERPRETATION: This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. FUNDING: This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).

A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

High-throughput sequencing and in-silico analysis confirm pathogenicity of novel MSH3 variants in African American colorectal cancer.

The maintenance of DNA sequence integrity is critical to avoid accumulation of cancer-causing mutations. Inactivation of DNA Mismatch Repair (MMR) genes (e.g., MLH1 and MSH2) is common among many cancers, including colorectal cancer (CRC) and is the driver of classic microsatellite instability (MSI) in tumors. Somatic MSH3 alterations have been linked to a specific form of MSI called elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that is associated with patient poor prognosis and elevated among African American (AA) rectal cancer patients. Genetic variants of MSH3 and their pathogenicity vary among different populations, such as among AA, which are not well-represented in publicly available databases. Targeted exome sequencing of MSH3 among AA CRC samples followed by computational bioinformatic pipeline and molecular dynamic simulation analysis approach confirmed six identified MSH3 variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) that corresponded to MSH3 amino-acid changes (p.E413K; p.S466N; p.S920F; p.E976K; p.H1010Y; p.E1081K). All identified MSH3 variants were non-synonymous, novel, pathogenic, and show loss or gain of hydrogen bonding, ionic bonding, hydrophobic bonding, and disulfide bonding and have a deleterious effect on the structure of MSH3 protein. Some variants were located within the ATPase site of MSH3, affecting ATP hydrolysis that is critical for MSH3's function. Other variants were in the MSH3-MSH2 interacting domain, important for MSH3's binding to MSH2. Overall, our data suggest that these variants among AA CRC patients affect the function of MSH3 making them pathogenic and likely contributing to the development or advancement of CRC among AA. Further clarifying functional studies will be necessary to fully understand the impact of these variants on MSH3 function and CRC development in AA patients.

MSH3-related adenomatous polyposis in a patient with the negative family history of colorectal polyps.

Recently, biallelic MSH3 germline pathogenic/likely pathogenic variants have been recognized as a rare cause of adenomatous polyposis. We present a 49-year-old woman who was admitted to our high-risk colorectal cancer clinic after incidental detection of a biallelic MSH3 (likely) pathogenic variant when tested for the germline (likely) pathogenic variants in hereditary breast and ovarian cancer related genes. The focus of this case report is to describe the genotype and phenotype of our patient with MSH3-related adenomatous polyposis. More than half of the polyps (13/19) were located in the right colon. In addition, benign and malignant extraintestinal lesions may be common as our patient had simple liver and kidney cysts and two basal cell skin carcinomas.

Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo.

The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate­resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double­strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)­resistant HT29 cells [DM­ and homogeneously staining region (HSR)­containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM­containing cells. No corresponding changes were found in HSR­containing cells. The present study further verified the involvement of MSH3­regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3­depleted DM­containing cells. Finally, changes in MSH3 expression could affect DHFR amplification­derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA­based drug resistance in cancer.

MSH3: a confirmed predisposing gene for adenomatous polyposis.

BACKGROUND: The MSH3 gene is part of the DNA mismatch repair system, but has never been shown to be involved in Lynch syndrome. A first report of four patients from two families, bearing biallelic MSH3 germline variants, with a phenotype of attenuated colorectal adenomatous polyposis raised the question of its involvement in hereditary cancer predisposition. The patients' tumours exhibited elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a hallmark of MSH3 deficiency. METHODS: We report five new unrelated patients with MSH3-associated polyposis. We describe their personal and familial history and study the EMAST phenotype in various normal and tumour samples, which are relevant findings based on the rarity of this polyposis subtype so far. RESULTS: All patients had attenuated colorectal adenomatous polyposis, with duodenal polyposis in two cases. Both women had breast carcinomas. EMAST phenotype was present at various levels in different samples of the five patients, confirming the MSH3 deficiency, with a gradient of instability in polyps depending on their degree of dysplasia. The negative EMAST phenotype ruled out the diagnosis of germline MSH3 deficiency for two patients: one homozygous for a benign variant and one with a monoallelic large deletion. CONCLUSION: This report lends further credence to biallelic MSH3 germline pathogenic variants being involved in colorectal and duodenal adenomatous polyposis. Large-scale studies may help clarify the tumour spectrum and associated risks. Ascertainment of EMAST may help with the interpretation of variants of unknown significance. We recommend adding MSH3 to dedicated diagnostic gene panels.

Identification of disulfidptosis related subtypes, characterization of tumor microenvironment infiltration, and development of DRG prognostic prediction model in RCC, in which MSH3 is a key gene during disulfidptosis.

Disulfidptosis is a newly discovered mode of cell death induced by disulfide stress. However, the prognostic value of disulfidptosis-related genes (DRGs) in renal cell carcinoma (RCC) remains to be further elucidated. In this study, consistent cluster analysis was used to classify 571 RCC samples into three DRG-related subtypes based on changes in DRGs expression. Through univariate regression analysis and LASSO-Cox regression analysis of differentially expressed genes (DEGs) among three subtypes, we constructed and validated a DRG risk score to predict the prognosis of patients with RCC, while also identifying three gene subtypes. Analysis of DRG risk score, clinical characteristics, tumor microenvironment (TME), somatic cell mutations, and immunotherapy sensitivity revealed significant correlations between them. A series of studies have shown that MSH3 can be a potential biomarker of RCC, and its low expression is associated with poor prognosis in patients with RCC. Last but not least, overexpression of MSH3 promotes cell death in two RCC cell lines under glucose starvation conditions, indicating that MSH3 is a key gene in the process of cell disulfidptosis. In summary, we identify potential mechanism of RCC progression through DRGs -related tumor microenvironment remodeling. In addition, this study has successfully established a new disulfidptosis-related genes prediction model and discovered a key gene MSH3. They may be new prognostic biomarkers for RCC patients, provide new insights for the treatment of RCC patients, and may inspire new methods for the diagnosis and treatment of RCC patients.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

A large family with MSH3-related polyposis.

Biallelic MSH3 germline variants are a rare cause of adenomatous polyposis as yet reported in two small families only. We describe the phenotype of a third family, the largest thus far, with adenomatous polyposis related to compound heterozygous MSH3 pathogenic variants. The index patient was a 55-years old male diagnosed with rectal cancer and adenomatous polyposis (cumulatively 52 polyps), with a family history of colorectal polyposis with unknown cause. Next-generation sequencing and copy number variation analysis of a panel of genes associated with colorectal cancer and polyposis revealed compound heterozygous germline pathogenic variants in the MSH3 gene. Nine out of 11 siblings were genotyped. Three siblings carried the same compound heterozygous MSH3 variants. Colonoscopy screening showed predominantly right-sided adenomatous polyposis in all compound heterozygous siblings, with a cumulative number of adenomas ranging from 18 to 54 in an average of four colonoscopies, and age at first adenoma detection ranging from 46 to 59. Microsatellite analysis demonstrated alterations at selected tetranucleotide repeats (EMAST) in DNA retrieved from the rectal adenocarcinoma, colorectal adenomas as well as of normal colonic mucosa. Gastro-duodenoscopy did not reveal adenomas in any of the four patients. Extra-intestinal findings included a ductal adenocarcinoma in ectopic breast tissue in one female sibling at the age of 46, and liver cysts in three affected siblings. None of the three heterozygous or wild type siblings who previously underwent colonoscopy had adenomatous polyposis. We conclude that biallelic variants in MSH3 are a rare cause of attenuated adenomatous polyposis with an onset in middle age.

SYVN1-mediated ubiquitination and degradation of MSH3 promotes the apoptosis of lens epithelial cells.

The pathology of age-related cataract (ARC) mainly involves the misfolding and aggregation of proteins, especially oxidative damage repair proteins, in the lens, induced by ultraviolet-B (UVB). MSH3, as a key member of the mismatch repair family, primarily maintains genome stability. However, the function of MSH3 and the mechanism by which cells maintain MSH3 proteostasis during cataractogenesis remains unknown. In the present study, the protein expression levels of MSH3 were found to be attenuated in ARC specimens and SRA01/04 cells under UVB exposure. The ectopic expression of MSH3 notably impeded UVB-induced apoptosis, whereas the knockdown of MSH3 promoted apoptosis. Protein half-life assay revealed that UVB irradiation accelerated the decline of MSH3 by ubiquitination and degradation. Subsequently, we found that E3 ubiquitin ligase synoviolin (SYVN1) interacted with MSH3 and promoted its ubiquitination and degradation. Of note, the expression and function of SYVN1 were contrary to those of MSH3 and SYVN1 regulated MSH3 protein degradation via the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. Based on these findings, we propose a mechanism for ARC pathogenesis that involves SYVN1-mediated degradation of MSH3 via the ubiquitin-proteasome pathway and the autophagy-lysosome pathway, and suggest that interventions targeting SYVN1 might be a potential therapeutic strategy for ARC.

Variant profiling of colorectal adenomas from three patients of two families with MSH3-related adenomatous polyposis.

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21-42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.

FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.

The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain.

Profiling diverse sequence tandem repeats in colorectal cancer reveals co-occurrence of microsatellite and chromosomal instability involving Chromosome 8.

We developed a sensitive sequencing approach that simultaneously profiles microsatellite instability, chromosomal instability, and subclonal structure in cancer. We assessed diverse repeat motifs across 225 microsatellites on colorectal carcinomas. Our study identified elevated alterations at both selected tetranucleotide and conventional mononucleotide repeats. Many colorectal carcinomas had a mix of genomic instability states that are normally considered exclusive. An MSH3 mutation may have contributed to the mixed states. Increased copy number of chromosome arm 8q was most prevalent among tumors with microsatellite instability, including a case of translocation involving 8q. Subclonal analysis identified co-occurring driver mutations previously known to be exclusive.

Lack of correlation between MSH3 immunohistochemistry and microsatellite analysis for the detection of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in colorectal cancers.

Immunohistochemical evaluation of mismatch repair protein (MMR) expression is an important screening tool in diagnostic pathology, where it is routinely used to identify subsets of colorectal cancers (CRCs) with either inherited or sporadic forms of microsatellite instability (MSI). MSH3 is not included in current MMR panels, although aberrant MSH3 expression is reported to occur in 40-60% of CRCs and is associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and a worse prognosis. In this study, we applied MSH3 immunohistochemistry and tetranucleotide MSI analysis to a cohort of 250 unselected CRCs to evaluate the potential use of the methods in routine practice. Partial, complete, and focal loss of nuclear MSH3 and its cytoplasmic mislocalization were evident in 67% of tumors, whereas MSI was evident in two to six of a panel of six tetranucleotide repeats in 46% of cases. However, concordance between MSH3 immunohistochemistry and tetranucleotide MSI results was only 61%, indicating the unsuitability of this combination of tests in routine pathology practice. MSH3 immunostaining was compromised in areas of tissue crush and autolysis, which are common in biopsy and surgical samples, potentially mitigating against its routine use. Although tetranucleotide MSI is clearly evident in a subset of CRCs, further development of validated sets of tetranucleotide repeats and either MSH3 or other immunohistochemical markers will be required to include EMAST testing in the routine evaluation of CRCs in clinical practice.

FAN1 controls mismatch repair complex assembly via MLH1 retention to stabilize CAG repeat expansion in Huntington's disease.

CAG repeat expansion in the HTT gene drives Huntington's disease (HD) pathogenesis and is modulated by DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion.

Prevalence and Characterization of Biallelic and Monoallelic NTHL1 and MSH3 Variant Carriers From a Pan-Cancer Patient Population.

NTHL1 and MSH3 have been implicated as autosomal recessive cancer predisposition genes. Although individuals with biallelic NTHL1 and MSH3 pathogenic variants (PVs) have increased cancer and polyposis risk, risks for monoallelic carriers are uncertain. We sought to assess the prevalence and characterize NTHL1 and MSH3 from a large pan-cancer patient population. MATERIALS AND METHODS: Patients with pan-cancer (n = 11,081) underwent matched tumor-normal sequencing with consent for germline analysis. Medical records and tumors were reviewed and analyzed. Prevalence of PVs was compared with reference controls (Genome Aggregation Database). RESULTS: NTHL1-PVs were identified in 40 patients including 39 monoallelic carriers (39/11,081 = 0.35%) and one with biallelic variants (1/11,081 = 0.009%) and a diagnosis of isolated early-onset breast cancer. NTHL1-associated mutational signature 30 was identified in the tumors of the biallelic patient and two carriers. Colonic polyposis was not identified in any NTHL1 patient. MSH3-PVs were identified in 13 patients, including 12 monoallelic carriers (12/11,081 = 0.11%) and one with biallelic MSH3 variants (1/11,081 = 0.009%) and diagnoses of later-onset cancers, attenuated polyposis, and abnormal MSH3-protein expression. Of the 12 MSH3 carriers, two had early-onset cancer diagnoses with tumor loss of heterozygosity of the wild-type MSH3 allele. Ancestry-specific burden tests demonstrated that NTHL1 and MSH3 prevalence was not significantly different in this pan-cancer population versus controls. CONCLUSION: NTHL1 and MSH3 germline alterations were not enriched in this pan-cancer patient population. However, tumor-specific findings, such as mutational signature 30 and loss of heterozygosity of the wild-type allele, suggest the potential contribution of monoallelic variants to tumorigenesis in a subset of patients.

Coordinated roles of SLX4 and MutSß in DNA repair and the maintenance of genome stability.

SLX4 provides a molecular scaffold for the assembly of multiple protein complexes required for the maintenance of genome stability. It is involved in the repair of DNA crosslinks, the resolution of recombination intermediates, the response to replication stress and the maintenance of telomere length. To carry out these diverse functions, SLX4 interacts with three structure-selective endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1, as well as the telomere binding proteins TRF2, RTEL1 and SLX4IP. Recently, SLX4 was shown to interact with MutSß, a heterodimeric protein involved in DNA mismatch repair, trinucleotide repeat instability, crosslink repair and recombination. Importantly, MutSß promotes the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease. The colocalization and specific interaction of MutSß with SLX4, together with their apparently overlapping functions, are suggestive of a common role in reactions that promote DNA maintenance and genome stability. This review will focus on the role of SLX4 in DNA repair, the interplay between MutSß and SLX4, and detail how they cooperate to promote recombinational repair and DNA crosslink repair. Furthermore, we speculate that MutSß and SLX4 may provide an alternative cellular mechanism that modulates trinucleotide instability.