Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome

Somatic epimutations in the MLH1 promoter mimic the phenotype of Lynch syndrome. To date, no somatic hypermethylation of the MLH1 promoter in the carrier of a pathogenic MLH1 germline mutation has been identified, prompting the recommendation that a germline mutation in MLH1 should only be sought in the absence of tumour tissue methylation. We aimed to determine whether methylation of the MLH1 promoter may coexist in carriers of a pathogenic germline mutation in MLH1. We examined the methylation status of the MLH1 promoter in 123 tumour tissue samples, demonstrating high microsatellite instability and loss of expression of a mismatch repair protein (60 cases with MLH1 germline mutation, 25 cases without mutation, 38 cases with MSH2 mutations), using combined bisulphite restriction analysis (COBRA) and SNaPshot analysis. Methylation of the MLH1 promoter was found in two patients with pathogenic germline mutations, one a carrier of a MLH1 mutation and the other a carrier of a MSH2 mutation. Our results demonstrate that methylation of the MLH1 promoter region does not exclude the presence of a germline mutation in a mismatch repair (MMR) gene. Hypermethylation of the MLH1 promoter may be present in most cases of sporadic colorectal cancers, but this does not exclude a diagnosis of Lynch syndrome.     

Understanding the Pathogenicity of Noncoding Mismatch Repair Gene Promoter Variants in Lynch Syndrome

Lynch syndrome is the most common familial cancer condition that mainly predisposes to tumors of the colon and endometrium. Cancer susceptibility is caused by the autosomal dominant inheritance of a loss‐of‐function mutation or epimutation in one of the DNA mismatch repair (MMR) genes. Cancer risk assessment is often possible with nonsynonymous coding region mutations, but in many cases patients present with DNA sequence changes within noncoding regions, including the promoters, of MMR genes. The pathogenic role of promoter variants, and hence clinical significance, is unclear and this hinders the clinical management of carriers. In this review, we provide an overview of the classification of MMR gene variants, outline the laboratory assays and online resources that can be used to assess the causality of promoter variants in Lynch syndrome, and highlight some of the practical challenges of demonstrating the pathogenicity of these variants. In conclusion, we propose a guide that could be integrated into the current InSiGHT classification scheme to help determine if a MMR gene promoter variant is pathogenic.     

Accurate classification of MLH1/MSH2 missense variants with multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR)

Lynch syndrome, also known as hereditary nonpolyposis colon cancer (HNPCC), is the most common known genetic syndrome for colorectal cancer (CRC). MLH1/MSH2 mutations underlie approximately 90% of Lynch syndrome families. A total of 24% of these mutations are missense. Interpreting missense variation is extremely challenging. We have therefore developed multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR), a bioinformatic algorithm that effectively classifies MLH1/MSH2 deleterious and neutral missense variants. We compiled a large database (n>300) of MLH1/MSH2 missense variants with associated clinical and molecular characteristics. We divided this database into nonoverlapping training and validation sets and tested MAPP-MMR. MAPP-MMR significantly outperformed other missense variant classification algorithms (sensitivity, 94%; specificity, 96%; positive predictive value [PPV] 98%; negative predictive value [NPV], 89%), such as SIFT and PolyPhen. MAPP-MMR is an effective bioinformatic tool for missense variant interpretation that accurately distinguishes MLH1/MSH2 deleterious variants from neutral variants.     

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.244G>A (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. Hum Mutat 33:1576–1588, 2012. © 2012 Wiley Periodicals, Inc.     

Germ line MLH1 and MSH2 mutations in Taiwanese Lynch syndrome families: characterization of a founder genomic mutation in the MLH1 gene

This multicenter study evaluated the mutation spectrum and frequencies of the MLH1 and MSH2 genes and determined the occurrence of large genomic deletions in 93 unrelated Taiwanese families that fulfilled the Amsterdam criteria II by denaturing high-performance liquid chromatography analysis, DNA sequencing for aberrant chromatograms, and multiplex ligation-dependent probe amplification analysis. In total, 38 pathogenic mutations (10 large deletions and 28 point mutations or small deletion/insertions) in the MSH2 or MLH1 gene were identified in 61 of the 93 families (66%). Three of the 10 large deletions and 14 of the 28 point mutations or small insertions/deletions have not been reported elsewhere. Three mutations in the MLH1 gene, the MLH1 c.1846_1848delAAG (5 families), deletion exons 11–15 (4 unrelated families), and MLH1 c.793C>T (13 unrelated families), accounted for 35% of all cases with pathogenic mutations. Haplotype analysis indicated that mutant c.793C>T alleles were derived from two distinct common founders that might be inherited from a single ancestor of presumably Chinese origin. As a mutation detection strategy for Taiwanese Lynch syndrome patients, we recommend that diagnosis starts with screening for large genomic deletions and continues by screening for common mutations in exons 10 and 16 of the MLH1 gene prior to searching for small mutations in the remaining exons.     

MLH1 germline epimutations in selected patients with early-onset non-polyposis colorectal cancer

Recently, the germline epigenetic inactivation of MLH1 has been reported in a number of patients with early-onset colorectal cancer among other characteristics. The aim of the present study is to evaluate the presence of MLH1 germline epimutations in selected colorectal cancer patients suspected of hereditary non-polyposis colorectal cancer (HNPCC) in order to determine in which patients the MLH1 epigenetic test should be performed. From a total of 109 microsatellite instability (MSI)-positive HNPCC-suspected patients, 11 showed a lack of MLH1 expression in tumor tissue and no germline mutations in the mismatch repair (MMR) genes. In nine of these cases and in three additional patients with multiple tumors, the study of the germline MLH1 promoter hypermethylation was performed by means of methylation-specific PCR and combined bisulfite-restriction analysis techniques. One of the selected patients resulted positive for the MLH1 epimutation, which was confirmed in the DNA extracted from buccal lavage. The patient with the epimutation had developed an epidermoid lip carcinoma and an early-onset colorectal tumor with MSI, no MLH1 expression, and loss of heterozygosity of the gene. Parents and siblings did not carry the epigenetic alteration, suggesting a de novo mechanism. Although germline MLH1 epimutations seem to be mostly uncommon, when the cases are well selected, the probability of finding them increases. Thus, taking into account ours and previous reports, we propose that screening for MLH1 epimutations in blood DNA could be performed in early-onset colorectal cancer patients with MSI, lack of MLH1 expression in the tumor, and no germline mutations in the MMR genes.     

Evidence of constitutional MLH1 epimutation associated to transgenerational inheritance of cancer susceptibility

Constitutional epimutations of DNA mismatch repair (MMR) genes have been recently reported as a possible cause of Lynch syndrome. However, little is known about their prevalence, the risk of transmission through the germline and the risk for carriers to develop cancers. In this study, we evaluated the contribution of constitutional epimutations of MMR genes in Lynch syndrome. A cohort of 134 unrelated Lynch syndrome-suspected patients without MMR germline mutation was screened for constitutional epimutations of MLH1 and MSH2 by quantitative bisulfite pyrosequencing. Patients were also screened for the presence of EPCAM deletions, a possible cause of MSH2 methylation. Tumors from patients with constitutional epimutations were extensively analyzed. We identified a constitutional MLH1 epimutation in two proband patients. For one of them, we report for the first time evidence of transmission to two children who also developed early colonic tumors, indicating that constitutional MLH1 epimutations are associated to a real risk of transgenerational inheritance of cancer susceptibility. Moreover, a somatic BRAF mutation was detected in one affected child, indicating that tumors from patients carrying constitutional MLH1 epimutation can mimic MSI-high sporadic tumors. These findings may have important implications for future diagnostic strategies and genetic counseling.     

Cancer Risks for MLH1 and MSH2 Mutation Carriers

We studied 17,576 members of 166 MLH1 and 224 MSH2 mutation‐carrying families from the Colon Cancer Family Registry. Average cumulative risks of colorectal cancer (CRC), endometrial cancer (EC), and other cancers for carriers were estimated using modified segregation analysis conditioned on ascertainment criteria. Heterogeneity in risks was investigated using a polygenic risk modifier. Average CRC cumulative risks at the age of 70 years (95% confidence intervals) for MLH1 and MSH2 mutation carriers, respectively, were estimated to be 34% (25%–50%) and 47% (36%–60%) for male carriers and 36% (25%–51%) and 37% (27%–50%) for female carriers. Corresponding EC risks were 18% (9.1%–34%) and 30% (18%–45%). A high level of CRC risk heterogeneity was observed (P < 0.001), with cumulative risks at the age of 70 years estimated to follow U‐shaped distributions. For example, 17% of male MSH2 mutation carriers have estimated lifetime risks of 0%–10% and 18% have risks of 90%–100%. Therefore, average risks are similar for the two genes but there is so much individual variation about the average that large proportions of carriers have either very low or very high lifetime cancer risks. Our estimates of CRC and EC cumulative risks for MLH1 and MSH2 mutation carriers are the most precise currently available.     

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome

Monoallelic PMS2 germline mutations cause 5%–15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA‐ and RNA‐based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety‐one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.     

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. Hum Mutat 31:975–982, 2010. © 2010 Wiley‐Liss, Inc.     

Functional characterization of MLH1 missense variants identified in lynch syndrome patients

Germline mutations in the human DNA mismatch repair (MMR) genes MSH2 and MLH1 are associated with the inherited cancer disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer or HNPCC. A proportion of MSH2 and MLH1 mutations found in suspected LS patients give rise to single amino acid substitutions. The functional consequences in regard to pathogenicity of many of these variants are unclear. We have examined the functionality of a panel of MLH1 missense mutations found in LS families, by testing the variant proteins in functional assays, addressing subcellular localization, and protein–protein interaction with the dimer partner PMS2 and the MMR‐associated exonuclease 1. We show that a significant proportion of examined variant proteins have functional defects in either subcellular localization or protein–protein interactions, which is suspected to lead to the cancer phenotype observed in patients. Moreover, the obtained results correlate well with reported MMR activity and with in silico analysis for a majority of the variants. Hum Mutat 33:1647–1655, 2012. © 2012 Wiley Periodicals, Inc.     

A cell‐free assay for the functional analysis of variants of the mismatch repair protein MLH1

The hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ‐line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction of the gene alterations detected in suspected Lynch Syndrome patients is comprised of amino acid substitutions. The relevance for cancer risk of these variants is difficult to assess, as currently no time‐ and cost‐effective, validated, and widely applicable functional assays for the measurement of MMR activity are available. Here we describe a rapid, cell‐free, and easily quantifiable MMR activity assay for the diagnostic assessment of variants of the MLH1 MMR protein. This assay allows the parallel generation and functional analysis of a series of variants of the MLH1 protein in vitro using readily available, or preprepared, reagents. Using this assay we have tested 26 MLH1 variants and of these, 15 had lost activity. These results are in concordance with those obtained from first‐generation assays and with in silico and pathology data. After its multifocal technical and clinical validation this assay could have great impact for the diagnosis and counseling of carriers of an MLH1 variant and their relatives. Hum Mutat 30:1–7, 2010. © 2010 Wiley‐Liss, Inc.     

A novel POLD1 pathogenic variant identified in two families with a cancer spectrum mimicking Lynch syndrome

The POLD1 gene is involved in DNA proofreading to ensure accurate DNA replication. Some germline alterations in its exonuclease domain are associated with predisposition to cancers and colonic polyps. Only a few pathogenic variants have been clearly identified so far. Here we report a novel variant: c.1458G>T p.(Lys486Asn) that we classified as pathogenic, detected in two putatively unrelated families. The cancer spectrum was very similar to Lynch syndrome, implying an overlapped tissue susceptibility. The common presence of colonic polyps in carriers and the MMR proficient phenotype in tumors were distinctive features suggesting POLD1 implication. Some clinical characteristics observed in the carriers of this variant differed from those reported previously, suggesting a potential genotype/phenotype correlation, and very likely in relation to the functional importance of affected residues. Our findings provide further insight into understanding the role of POLD1 in cancer-related risk.     

Molecular assessment of colorectal cancer through Lynch syndrome screening

Since 2017, the National Institute for Health and Care Excellence (NICE) has recommended molecular testing of all patients with newly diagnosed colorectal cancer (CRC) to identify those with suspected Lynch syndrome who should be referred to clinical genetics for germline testing. The pathway involves firstly determining the mismatch repair (MMR) expression status by immunohistochemistry (IHC) or performing microsatellite instability testing. This may be followed by BRAF V600E mutation testing and then MLH1 promoter hypermethylation analysis depending on the result. This approach identifies patients that are most likely to have underlying germline mutations in the MMR genes as opposed to somatic causes of deficient MMR. Here we demonstrate a case with loss of MLH1 protein expression and discuss the subsequent testing strategy according to NICE guidance.     

Impact of colonoscopic screening in male and female Lynch syndrome carriers with an MSH2 mutation

Stuckless S, Green JS, Morgenstern M, Kennedy C, Green RC, Woods MO, Fitzgerald W, Cox J, Parfrey PS. Impact of colonoscopic screening in male and female Lynch syndrome carriers with an MSH2 Mutation. The lifetime risk of developing colorectal cancer (CRC) in Lynch syndrome (LS) carriers is very high. To determine the impact of colonoscopic screening in 54 male and 98 female MSH2 mutation carriers, outcomes were compared with 94 males and 76 females who were not screened. CRC incidence and survival in the screened group were compared to that expected, derived from the non-screened group. To correct for survivor bias, controls were matched for age at entry into screening and also for gender. In males, median age to CRC was 58 years, whereas expected was 47 years (p = 0.000), and median survival was 66 years vs 62 years (p = 0.034). In screened females, median age to CRC was 79 years compared to 57 years in the non-screened group (p = 0.000), and median survival was 80 years compared with expected of 63 years (p = 0.001). Twenty percent of males and 7% of females developed an interval CRC within 2 years of previous colonoscopy. Although colonoscopic screening was associated with decreased CRC risk and better survival, CRCs continued to occur. CRC development may be further reduced by decreasing the screening interval to 1 year and improving quality of colonoscopy.     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA

Gene dosage abnormalities account for a significant proportion of the mutations in genes tested in DNA diagnostic laboratories. Detection of these changes has proved a challenge as the methods available to date are time consuming or unreliable. The multiplex ligation-dependent probe assay (MLPA) is a new technique allowing relative quantification of up to 40 different nucleic acid sequences in a single reaction tube. We have evaluated MLPA for potential use in the diagnostic setting against the following criteria: accuracy, reagent cost, hands-on time, reliability, and retests required. A total of 215 UK patients referred for genetic testing on the basis of a family history consistent with autosomal dominant hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) were tested by MLPA. Of these, 12 cases with deletions of one or more exons were identified, six with MLH1 deletions and six with MSH2 deletions. Test failure rates were less than 5% and overall mutation detection sensitivity in this series was increased by approximately 50% by the inclusion of MLPA for an additional testing cost of about 10%. Two novel mutations in MSH2 and 10 novel point mutations in MLH1 were also identified during the course of this study. We conclude that MLPA is a cost effective and robust gene dosage method that can be readily adopted by diagnostic services. Comprehensive mutation scanning for MSH2 and MLH1 is incomplete without gene dosage analysis.     

Different molecular mechanisms underlie genomic deletions in the MLH1 Gene

In this study we examined a series of 52 patients belonging to hereditary nonpolyposis colorectal cancer (HNPCC) or HNPCC-related families, all who had previously tested negative for mismatch repair (MMR) gene point mutations. Southern blot mutational screening of MLH1 and MSH2 genes was carried out with the aim of detecting large genomic rearrangements and of identifying the molecular mechanisms underlying the inactivation of the MMR genes. Three patients had abnormal restriction patterns and were found to carry distinct MLH1 internal deletions. Long-range PCRs identified the loss of DNA tracts spanning exon 6 (about 2.4 kb in proband A-AV20 and 0.8 kb in proband A-PD5) and exon 3 (about 2.5 kb in proband R-RM2). In A-AV20 the breakpoints occurred into identical 33-bp regions in introns 5 and 6 and a mechanism of classical Alu-mediated homologous recombination was evident. Also, in patient A-PD5 the breakpoints were located in these introns, but without direct involvement of repetitive sequences. In patient R-RM2 the breakpoints were located within repetitive L1 elements with poor homology in intron 2 and 3 and the rearranged allele was characterized by a complex insertion deletion (delCCinsACATAGTA), giving rise to a palindromic CTTAACATAGTATGTTAAG sequence in proximity of the fusion site. This study confirms that genomic rearrangements are an important component of the spectrum of MMR mutations. Although Alu repeats are likely to be implicated in the majority of cases, different molecular mechanisms may also be responsible for the observed MLH1 intragenic deletions. In particular, HNPCC resulting from L1-mediated recombination has been identified as a novel mechanism for MMR inactivating mutation.