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Lynch syndrome is caused by inactivating mutations in the MLH1 gene, but genetic variants of unclear significance frequently preclude diagnosis. Functional testing can reveal variant-conferred defects in gene or protein function. Based on functional defect frequencies and clinical applicability of test systems, we developed a functional testing strategy aimed at efficiently detecting pathogenic defects in coding MLH1 variants. In this strategy, tests of repair activity and expression are prioritized over analyses of subcellular protein localization and messenger RNA (mRNA) formation. This strategy was used for four unclear coding MLH1 variants (p.Asp41His, p.Leu507Phe, p.Gln689Arg, p.Glu605del + p.Val716Met). Expression was analyzed using a transfection system, mismatch repair (MMR) activity by complementation in vitro, mRNA formation by reverse transcriptase-PCR in carrier lymphocyte mRNA, and subcellular localization with dye-labeled fusion constructs. All tests included clinically meaningful controls. The strategy enabled efficient identification of defects in two unclear variants: the p.Asp41His variant showed loss of MMR activity, whereas the compound variant p.Glu605del + p.Val716Met had a defect of expression. This expression defect was significantly stronger than the pathogenic expression reference variant analyzed in parallel, therefore the defect of the compound variant is also pathogenic. Interestingly, the expression defect was caused additively by both of the compound variants, at least one of which is non-pathogenic when occurring by itself. Tests were neutral for p.Leu507Phe and p.Gln689Arg, and the results were consistent with available clinical data. We finally discuss the improved sensitivity and efficiency of the applied strategy and its limitations in analyzing unclear coding MLH1 variants., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Introduction: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated., Methods: Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively., Results: MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability., Conclusions: These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.

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., (©2013 AACR.)

Lynch syndrome is associated with germline mutations in DNA mismatch repair (MMR) genes. Up to 30% of DNA changes found are variants of unknown significance (VUS). Our aim was to assess the pathogenicity of eight MLH1 VUS identified in patients suspected of Lynch syndrome. All of them are novel or not previously characterized. For their classification, we followed a strategy that integrates family history, tumor pathology, and control frequency data with a variety of in silico and in vitro analyses at RNA and protein level, such as MMR assay, MLH1 and PMS2 expression, and subcellular localization. Five MLH1 VUS were classified as pathogenic: c.[248G>T(;)306G>C], c.[780C>G;788A>C], and c.791-7T>A affected mRNA processing, whereas c.218T>C (p.L73P) and c.244A>G [corrected] (p.T82A) impaired MMR activity. Two other VUS were considered likely neutral: the silent c.702G>A variant did not affect mRNA processing or stability, and c.974G>A (p.R325Q) did not influence MMR function. In contrast, variant c.25C>T (p.R9W) could not be classified, as it associated with intermediate levels of MMR activity. Comprehensive functional assessment of MLH1 variants was useful in their classification and became relevant in the diagnosis and genetic counseling of carrier families., (© 2012 Wiley Periodicals, Inc.)

Lynch syndrome is characterized by germline mutations of the DNA mismatch repair genes MLH1 and MSH2. The tumor spectrum includes early onset colorectal, urogenital and other cancers. Soft tissue sarcomas have been anecdotally reported in patients with Lynch syndrome, but coincidental manifestation could not be excluded. In this report, we screened a cohort of Lynch syndrome families for tumors outside the established tumor spectrum. We identified two patients with Lynch syndrome and a malignant fibrous histiocytoma (MFH). In both families a causative MSH2 germline mutation (MSH2 c.2038C ≥ T or MSH2 c.942 ± 3A ≥ T) could be detected. Archival tumor material from both resected MFH was analyzed for microsatellite instability expression of MLH1 and MSH2. A mutator phenotype was detected in both MFH with loss of MSH2 protein expression. Subsequently, the causative MSH2 germline mutation was confirmed in both patients. Of note, both tumors were diagnosed at a local advanced stage but could be curatively resected 21 and 11 year ago, respectively. Both patients are alive without local or distant recurrence. In conclusion, our data further support that patients with Lynch syndrome are at increased risk for rare tumors such as MFH. However, the prognosis compared to sporadic MFH seems to be favorable.

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.

Purpose: Germline mutations in DNA mismatch repair genes, mainly MLH1 or MSH2, have been shown to predispose with high penetrance for the development of the clinical phenotype of hereditary nonpolyposis colorectal cancer (Lynch syndrome). Here, we describe the discovery and first functional characterization of a novel germline MLH1 mutant allele., Experimental Design: A large kindred including 54 potential carriers was investigated at the molecular level by using different types of PCR experiments, gene cloning, transfection studies, Western blot experiments, and mismatch repair assays to identify and characterize a novel MLH1 mutant allele. Twenty-two of 54 putative carriers developed colon cancer or other tumors, including breast cancer., Results: The identified MLH1 mutant allele emerged from an interstitial deletion on chromosome 3p21.3, leading to an in-frame fusion of MLH1 (exons 1-11) with ITGA9 (integrin alpha 9; exons 17-28). The deleted area has a size of about 400 kb; codes for LRRFIP2 (leucine-rich repeat in flightless interaction protein 2), GOLGA4 (Golgi autoantigen, golgin subfamily a, 4), and C3orf35/APRG1 (chromosome 3 open reading frame 35/AP20 region protein 1); and partly disrupts the AP20 region implicated in major epithelial malignancies. Tumor cells lost their second MLH1 allele. The MLH1*ITGA9 fusion protein provides no capability for DNA mismatch repair. Murine fibroblasts, expressing a doxycycline-inducible MLH1*ITGA9 fusion gene, exhibit a loss-of-contact inhibition phenotype., Conclusions: This is the first description of a functional gene fusion of the human MLH1 gene, resulting in the loss of mismatch repair capabilities. The MLH1*ITGA9 fusion allele, together with deletions of the AP20 region, presumably defines a novel subclass of Lynch syndrome patients, which results in an extended tumor spectrum known from hereditary nonpolyposis colorectal cancer and Muir-Torre syndrome patients.

Background: Inflammatory bowel diseases (IBDs; ulcerative colitis, UC, and Crohn's disease, CD) show familial clustering suggestive of a genetic background. A linkage susceptibility region for these diseases (IBD9) lies on chromosome 3p and includes the DNA mismatch repair gene MLH1. Loss of MLH1 confers the characteristic microsatellite instability (MSI) phenotype which is also frequently found in the mucosa of IBD patients. A common germline alteration of MLH1 (655A>G) results in the amino acid exchange MLH1 I219V. Conflicting data exist on its effect on the function of the protein and it has recently been reported to cosegregate with refractory UC, suggesting that this alteration may impair mismatch repair activity and thereby contribute to certain forms of UC., Methods: We analyzed the MLH1 I219V alteration using in silico and biochemical analyses and assessed its appearance in 67 well-classified UC patients in comparison to 40 healthy individuals., Results: The analyses showed that I219 is a conserved, buried hydrophobic residue, and that I219V is unlikely to abolish MLH1 function but may modulate it. Quantitative biochemical evaluation showed identical stability and activity of the protein. Furthermore, the alteration occurred equally frequently in analyzed patients and healthy volunteers., Conclusions: The MLH1 I219V alteration does not directly contribute to the etiology of UC through an impairment of mismatch repair. A putative linkage disequilibrium of MLH1 I219V with the causative gene(s) of the IBD9 locus is rather distant.

For hMLH1, a key enzyme of DNA mismatch repair and frequently mutated in human cancers, several additional functions have been suggested. We now identified Thymosin beta4 (Tbeta4), an actin-binding and cell motility regulating protein, by bacterial two-hybrid screening. Interaction was confirmed by coimmunoprecipitation. Tbeta4 was weakly expressed in the hMLH1-deficient cell lines 293T and HCT-116. Reconstitution of hMLH1 resulted in strong expression of Tbeta4. Confocal laser microscopy revealed nuclear colocalization of both proteins. Reconstitution with hMLH1 mutants lacking a functional nuclear localization sequence resulted in cytoplasmatic retention of both proteins. After Tbeta4- or hMLH1-siRNA treatment, cell migration of hMLH1-proficient cells was markedly decreased. Our results show that hMLH1 interacts with Tbeta4 and regulates its expression and nuclear transport. Moreover, loss of hMLH1 causes Tbeta4 deprivation and results in reduced migratory activity in vitro. These data give insight into novel functions of hMLH1 and probably disease related dysregulated mechanisms.