Validation of an in Vitro Mismatch Repair Assay Used in the Functional Characterization of Mismatch Repair Variants.

A significant proportion of DNA-mismatch repair (MMR) variants are classified as of unknown significance, precluding diagnosis. The in vitro MMR assay is used to assess their MMR capability, likely the most important function of an MMR protein. However, the robustness of the assay, crucial for its use in the clinical setting, has been rarely evaluated. The aim of the present work was to validate an in vitro MMR assay approach to the functional characterization of MMR variants, as a first step to meeting quality standards of diagnostic laboratories. The MMR assay was optimized by testing a variety of reagents and experimental conditions. Reference materials and standard operating procedures were established. To determine the intra- and interexperimental variability of the assay and its reproducibility among centers, independent transfections of six previously characterized MLH1 variants were performed in two independent laboratories. Reagents and conditions optimal for performing the in vitro MMR assay were determined. The validated assay demonstrated no significant intra- or interexperimental variability and good reproducibility between centers. We set up a robust in vitro MMR assay that can provide relevant in vitro functional evidence for MMR variant pathogenicity assessment, eventually improving the molecular diagnosis of hereditary cancer syndromes associated with MMR deficiency.

Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants.

The human DNA repair gene MUTYH, whose mutational loss causes a colorectal polyposis and cancer predisposition, contains three alternative first exons. In order to analyze alternative transcription and the effect of genetic alterations found in humans, we established a cell-based minigene experimental model supporting transcription and splicing and thoroughly verified its functionality. We identified highly conserved promoter areas and inactivated them in the minigene, and also introduced six human variants. Moreover, the potential contribution of CpG island methylation and specific transcription factors on MUTYH transcription was addressed. The findings allowed to attribute regulatory roles to three conserved motifs in the promoter: an M4 motif, a transcription factor IIB recognition element, and a GC box. Moreover, the data showed that three patient variants compromised MUTYH expression and therefore have the potential to cause pathogenic effects. We did not find evidence for a biologically relevant contribution of CpG island methylation or a direct transcriptional activation by DNA damage. Besides insight into the regulation of MUTYH transcription, the work therefore provides a functional MUTYH minigene experimental system suitable as a diagnostic tool for analyzing patient variants, and a functional map of the promotor that also can facilitate pathogenicity classifications of human variants.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.

Evaluation of MLH1 variants of unclear significance.

Inactivating mutations in the MLH1 gene cause the cancer predisposition Lynch syndrome, but for small coding genetic variants it is mostly unclear if they are inactivating or not. Nine such MLH1 variants have been identified in South American colorectal cancer (CRC) patients (p.Tyr97Asp, p.His112Gln, p.Pro141Ala, p.Arg265Pro, p.Asn338Ser, p.Ile501del, p.Arg575Lys, p.Lys618del, p.Leu676Pro), and evidence of pathogenicity or neutrality was not available for the majority of these variants. We therefore performed biochemical laboratory testing of the variant proteins and compared the results to protein in silico predictions on structure and conservation. Additionally, we collected all available clinical information of the families to come to a conclusion concerning their pathogenic potential and facilitate clinical diagnosis in the affected families. We provide evidence that four of the alterations are causative for Lynch syndrome, four are likely neutral and one shows compromised activity which can currently not be classified with respect to its pathogenic potential. The work demonstrates that biochemical testing, corroborated by congruent evolutionary and structural information, can serve to reliably classify uncertain variants when other data are insufficient.

Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1L749P and MLH1Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment.

Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis.

In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. 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 LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have 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. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis.

Functional testing strategy for coding genetic variants of unclear significance in MLH1 in Lynch syndrome diagnosis.

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.

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1.

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.

Promoter methylation of MLH1, PMS2, MSH2 and p16 is a phenomenon of advanced-stage HCCs.

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.

Refining the role of PMS2 in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants.

BACKGROUND AND AIM: The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. METHODS: From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. RESULTS: Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. CONCLUSIONS: Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.

Expression defect size among unclassified MLH1 variants determines pathogenicity in Lynch syndrome diagnosis.

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.

Comprehensive functional assessment of MLH1 variants of unknown significance.

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.

MUTYH gene expression and alternative splicing in controls and polyposis patients.

Mutational loss of the human DNA repair gene MUTYH in the germline predisposes for colorectal polyposis and cancer, a recessively heritable disease called MUTYH-associated polyposis. The MUTYH gene shows heavy alternative splicing, but the transcripts relevant for biological function and cancer prevention have not been determined. This knowledge is required to assess the consequences that germline variants of unknown functional significance may have. We therefore quantified expression and investigated patterns of alternative splicing in control individuals, tissue samples, and carriers of two frequent germline alterations. MUTYH expression differed organ dependently, correlating with proliferative activity. Alternative first exons were used tissue specifically; transcripts for mitochondrial proteins predominated in muscle tissues, while ascending colon and testes showed the highest fractions of transcripts for nuclear proteins. Colon cancer cell lines produced predominant transcripts for nuclear protein. Exon skipping was frequent and governed by splice-site quality. Five transcripts were found to encode the biologically relevant products of the MUTYH gene. Carriers of the disease-causing mutation c.1187G>A (p.Gly396Asp) showed normal transcript composition, but the frequent single-nucleotide polymorphism rs3219468:G>C largely reduced one transcript species of MUTYH. Since this alteration decreases protein production of the gene, an increased cancer risk for compound heterozygous carriers is possible.

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair.

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.

RNA processing in plant mitochondria is independent of transcription.

We analyzed the ability of plant mitochondria to process introduced RNA. Arabidopsis thaliana cox2 transcripts were synthesized in vitro. The in vitro transcribed mRNA was electroporated into maize and cauliflower mitochondria and incubated in organello. RNA was isolated and RT-PCR was carried out to analyze RNA processing. Our data indicate that cox2 transcripts introduced into isolated plant mitochondria are processed completely. This is the first report of in organello editing of introduced transcripts. We also found that none of the transcription, translation, or respiration inhibitors we used influenced RNA splicing or RNA editing of the cox2 transcript. Thus, our data also demonstrate that plant mitochondrial RNA processing may be independent of both transcription and respiratory regulation.

Plastid mRNAs are neither spliced nor edited in maize and cauliflower mitochondrial in organello systems.

The process of RNA editing in chloroplasts and higher plant mitochondria displays some similarities, raising the question of common or similar components in editing apparatus of these two organelles. To investigate the ability of plant mitochondria to edit plastid transcripts, we employed a previously established mitochondrial maize and cauliflower in organello system. Two plastid genes, Zea mays ndhB and ycf3 containing group II introns and several editing sites, were introduced into mitochondria. The genes were transcribed in organello. However, these transcripts of the plastid genes are neither spliced nor edited in plant mitochondria. A comparison of maize ndhB editing sites and maize mitochondrial editing sites reveals considerable sequence similarities between three ndhB editing sites and several mitochondrial sites. Nevertheless, these ndhB editing sites were not recognized in the mitochondria. Thus, we present for the first time direct evidence that the factors present in the plant mitochondria are not sufficient to allow editing and splicing of plastid transcripts.