Analysis of molecular and cellular issues of Lynch Syndrome MLH1 Mutations

Das Lynch-Syndrom ist eine autosomal dominante Prädisposition für Krebs und verantwortlich für 2 – 5 % der Kolonkarzinome. Es wird durch eine heterozygot vorliegende Keimbahn-Mutation in einem der Mismatch-Reparatur-Gene MSH2, MSH6, MLH1 und PMS2 ausgelöst. Die Mismatch-Reparatur (MMR) beseitigt Basenfehlpaarungen, die während der Replikation durch Fehler der Polymerasen entstehen. Die meisten Lynch-Syndrom-Mutationen treten in MLH1 auf und ein Drittel dieser Veränderungen sind Missense-Mutationen. In der vorliegenden Arbeit wurde ein Screening von 58 Missense-Varianten durchgeführt. Aus dem Screening wurden Varianten für die Untersuchung der Dimerisierung von MLH1 und PMS2 ausgewählt, sowie Varianten mit unterschiedlich reduzierter MLH1-Expression. Diese Varianten wurden verwendet, um die nötige MLH1-Menge zu validieren, die eine ausreichende Reparatur gewährleistet und damit zur Apathogenität führt.The lynch syndrome is an autosomal-dominant predisposition for cancer and accounts for 2 – 5 % of all colon tumors. It is caused by heterozygous mutations in one of the mismatch repair genes MSH2, MSH6, MLH1 and PMS2. The mismatch repair (MMR) system removes base mismatches occurring during replication due to errors of the polymerase. Most mutations occur in MLH1 and approximately one third of them are missense mutations. In this work, a screening von 58 Variants was performed, including the analysis of MLH1 expression and repair activity. Variants from the screening were selected to analyze the dimerization of MLH1 and PMS2 and to validate the MLH1 amount required to ensure the repair

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

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.Barretos Cancer Hospital was partially funded by FINEP‐CT‐INFRA, Grant Number: 02/2010, Radium Hospital Foundation (Oslo, Norway), Helse Sør‐Øst (Norway); Deutsche Forschungsgemeinschaft, Grant Number: PL688/2‐1info:eu-repo/semantics/publishedVersio

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

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. (C) 2013 AACR

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

Influence of fluorescent labeling of MutLα on protein expression levels.

<p>To analyze the effect of fluorescent dyes on protein expression levels, HEK293T cells were transiently cotransfected with different MutLα constructs (see below) and (A) Western blot analysis was carried out after 48 h using anti-MLH1 or anti-PMS2, respectively, controlled by β-actin detection. (B) Amounts of expressed proteins were assessed by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: negative control (untransfected). 2: MLH1/PMS2 unlabeled; 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone-0031863-g001" target="_blank">Figure 1</a>.</p

Dye tags influence single expression of MLH1 and PMS2.

<p>To determine the influence of fluorescent tags on single expressed MLH1 or PMS2 variants, HEK293T cells were transfected with different (A) MLH1 or (B) PMS2 constructs. Amounts of expressed proteins were assessed after Western blotting by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: MLH1 unlabeled; 2: MLH1-GFP-N; 3: MLH1-GFP-C; 4: MLH1-Red-N; 5: MLH1-Red-C; 6: PMS2 unlabeled; 7: PMS2-GFP-N; 8: PMS2-GFP-C; 9: PMS2-Red-N; 10: PMS2-Red-C.</p

Influence of fluorescent tags on MMR activity of MutLα.

<p>HEK293T cells were transiently cotransfected with various labeled or unlabeld MutLα constructs and 48 h post transfection MMR activity of different MutLαs were assessed in vitro in parallel with unlabeled MutLα by quantifying the 3′-nick-directed correction of a G-T mismatch in a restriction site of a plasmid substrate as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#s4" target="_blank">Materials and Methods</a>” and as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone.0031863-Brieger3" target="_blank">[10]</a>. <i>In vitro</i> repair was scored on a 2-kb circular DNA substrate that contains an <i>EcoRV</i> site which is destroyed by a G-T mismatch. Upon repair of the G-T mismatch to an A-T base pair the intact <i>EcoRV</i> site together with an <i>AseI</i> site gives rise to a 0.8- and a 1.2-kb fragment, whereas unrepaired DNA is only linearized by <i>AseI</i> to a 2-kb fragment. Repair efficiency was assessed by measuring the signal intensities of linearized and digested vector with Bio-Rad Quantity One software using the “rolling ball” baseline correction. The signal intensity of the repair bands was divided by the intensity of all three bands. Repair efficiency of unlabeled MutLα was set at 100 percent and repair of fluorescent tagged MutLα was determined in relation to the wild-type sample that was expressed, processed and tested in parallel. Average repair values and standard deviations (±) were determined from four independent experiments. Single PMS2 tagged MutLαs, single MLH1-GFP-N tagged MutLαs as well as MLH1-GFP-N coexpressed with PMS2-Red-N or MLH1-Red-N coexpressed with PMS2-GFP-N were MMR proficient while all other tagged variants showed MMR deficiency. 1: mock control (untransfected). 2: MLH1/PMS2 unlabeled (positive control); 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone-0031863-g001" target="_blank">Figure 1</a>.</p

Putative three-dimensional structure models of fluorescent labeled MutLα.

<p>Using PyMol (Warren DeLano, <a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>), GFP or Red fluorescent proteins were attached to (A) N-termini of MLH1 and PMS2 or (B) C-termini of MutLα. C-terminal tags seem to hide the C-terminal region of MutLα and consequently might avoid DNA interaction. (C) Corresponding amino acid sequences of linker regions are shown. The dashed line between PMS2 and the N-terminal fluorescent tag illustrates a putative α-helix (unknown structure) of the first thirty amino acids of PMS2.</p