Sequencing Of Mismatch Repair Genes Research Articles

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in MLH1, MSH2, and MSH6, including 12 missense variants that were also assessed using a cell-free in vitro MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants.

Sanger sequencing of MMR genes in a one-plate system

Abstract Both incidence and mortality of colorectal cancer (CRC) in Romania have shown a continuous increase during the last decades. Hereditary Non-Polyposic Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to mismatch repair (MMR) genes MSH2, MSH6, and MLH1. Individuals carrying germ-line mutations of these genes present high lifetime risk of colorectal and other cancers, compared to non-carriers. Oncogenetics is developed worldwide nowadays, for identifying hereditary predisposition to cancer and offering appropriate clinical follow-up to patients and mutation carriers in Lynch families. Molecular oncogenetic diagnosis in Lynch syndrome is based on complete Sanger sequencing of entire MMR genes, which is time and resources consuming, therefore needing an appropriate and adapted optimization. Conventional sequencing requires a sufficient number of available samples to be processed simultaneously, which increases the waiting time for diagnostic results. Complete analysis for only one patient meets difficult technical problems due to the complex co-amplification of all gene regions of interest within the same conditions, therefore increasing the costs and reducing the cost-effectiveness of the test. Here we present an original and robust technical protocol for sequencing the entire MSH2, MSH6, and MLH1 coding sequence for one patient in a single PCR plate. Our optimized and verified system overcomes all technical problems and offers a quick, robust, and cost-effective possibility to personalize molecular oncogenetic diagnosis in Lynch syndrome.

Importance of PCR-based Tumor Testing in the Evaluation of Lynch Syndrome-associated Endometrial Cancer.

Lynch syndrome (LS) is a hereditary cancer syndrome caused by a germline mutation in a DNA mismatch repair gene, usually MLH1, MSH2, MSH6, or PMS2. The most common cancers associated with LS are colorectal adenocarcinoma and endometrial carcinoma. Identification of women with LS-associated endometrial cancer is important, as these women and their affected siblings and children are at-risk of developing these same cancers. Germline testing of all endometrial cancer patients is not cost effective, and screening using young age of cancer diagnosis and/or presence of family history of syndrome-associated is underutilized and ineffective. Therefore, most groups now advocate for tumor tissue testing to screen for LS, with germline testing targeted to women with abnormal tissue testing results. Immunohistochemistry for MLH1, MSH2, MSH6, and PMS2 is used in many clinical laboratories for this tumor screening step, as immunohistochemistry is relatively inexpensive and is technically more accessible for smaller clinical labs. PCR-based tissue testing, whereas technically more challenging, does play an important role in the identification of these patients. MLH1 methylation analysis identifies women with tumor MLH1 loss who likely have sporadic endometrial cancer and do not need heightened cancer prevention surveillance. High levels of microsatellite instability have been identified in tumors with retained positive expression of mismatch repair proteins. Somatic sequencing of mismatch repair genes from tumor DNA, whereas not currently available in most clinical laboratories, is helpful in resolution of cases in which germline sequencing fails to identify a mutation in a mismatch repair gene. The tumor tissue testing approach can help to identify most women at-risk for germline mutations in a LS gene, but not all patients will be captured using this approach. Clinical suspicion can still play a pivotal role in accurately identifying a subset of these patients.

Report of a PMS2 Germline Mutation Patient Presenting with Endometrial and Parotid Cancer

Colorectal cancer (CRC) is the second leading cause of cancer among men and women and represents the leading cause of cancer death in Puerto Rico. Familial CRC accounts for 10-15% of the total CRC cases, with Lynch syndrome (LS) implicated in 2-4% of cases. Limited information is available on the prevalence, clinical manifestations, and genetic mutations of hereditary CRC in USA Hispanics. In this paper we report a PMS2 mutation in a Puerto Rican Hispanic patient with LS recruited through the Puerto Rico Familial Colorectal Cancer Registry. At the age of 35 years our proband was diagnosed with endometrial cancer with parotid cancer established the following year. A diagnosis of Lynch Syndrome was established through analysis of protein expression by immunohistochemistry and genetic sequencing of mismatch repair genes. Mutation in the PMS2 gene is rarely linked with LS. This case report adds Parotid Carcinoma to the spectrum of malignant conditions associated to LS. We emphasize on the importance of genetic testing in at-risk patients for hereditary CRC from various racial backgrounds, and underscore the need for genetic counselling of patients and their family members. Recognition of LS carriers will allow early detection of malignant condition and the implementation of effective therapy, which will ultimately improve prognosis.

491PD - Lynch-Like Syndrome: Characterization and Comparison with Epcam Deletion Carriers

ABSTRACT Introduction: Colorectal cancers (CRCs) with MSI+ but without detectable germline mutation or hypermethylation in DNA mismatch repair (MMR) genes can be classified as Lynch-like Syndrome (LLS). The underlying mechanism and clinical significances of LLS are largely unknown. Methods: We measured MSI and MMR protein expression in 4,765 consecutive CRC cases. Among these, MSI+ cases were further classified based on clinical parameters, sequencing of MMR genes or polymerase e (POLE) and d (POLD1) and promoter methylation analysis of MLH1 and MSH2. Results: We found that MSI+ and MMR protein deficient CRCs comprised 6.3% (N = 302) of this cohort. Based on germline sequencing of 124 cases, we identified 54 LS with MMR germline mutation (LS-MMR), 15 LS with EPCAM deletions (LS-EPCAM) and 55 LLS patients. Of the 55 LLS patients, six (10.9%) had variants of unknown significance in the genes tested, and one patient had a novel somatic mutation (p.S459P) in POLE. In patients with biallelic deletions of EPCAM, all tumors and their matched normal mucosa showed promoter hypermethylation of MSH2. Conclusion: We found that patients with LLS and LS-EPCAM shared clinical features that differed from LS-MMR patients, including lower frequency of fulfillment of the revised Bethesda guidelines (83.6% and 86.7% vs. 98.1% for LS-MMR) and older mean age at CRC diagnosis (52.6 and 52.7 years vs. 43.9 years for LS-MMR). The similarity between LLS and LS-EPCAM suggests LLS as a subset of familial MSI+ CRC. Disclosure: All authors have declared no conflicts of interest.

Lynch-like syndrome: characterization and comparison with EPCAM deletion carriers.

Colorectal cancers (CRCs) with microsatellite instability-high (MSI+) but without detectable germline mutation or hypermethylation in DNA mismatch repair (MMR) genes can be classified as Lynch-like syndrome (LLS). The underlying mechanism and clinical significances of LLS are largely unknown. We measured MSI and MMR protein expression in 4,765 consecutive CRC cases. Among these, MSI+ cases were further classified based on clinical parameters, germline sequencing of MMR genes or polymerase ε (POLE) and δ (POLD1) and promoter methylation analysis of MLH1 and MSH2. We found that MSI+ and MMR protein-deficient CRCs comprised 6.3% (N = 302) of this cohort. On the basis of germline sequencing of 124 cases, we identified 54 LS with MMR germline mutation (LS-MMR), 15 LS with EPCAM deletions (LS-EPCAM) and 55 LLS patients. Of the 55 LLS patients, six (10.9%) had variants of unknown significance in the genes tested, and one patient had a novel somatic mutation (p.S459P) in POLE. In patients with biallelic deletions of EPCAM, all tumors and their matched normal mucosa showed promoter hypermethylation of MSH2. Finally, we found that patients with LLS and LS-EPCAM shared clinical features that differed from LS-MMR patients, including lower frequency of fulfillment of the revised Bethesda guidelines (83.6 and 86.7% vs. 98.1% for LS-MMR) and older mean age at CRC diagnosis (52.6 and 52.7 years vs. 43.9 years for LS-MMR). We identified somatic mutation in POLE as a rare underlying cause for MMR deficiency in LLS. The similarity between LLS and LS-EPCAM suggests LLS as a subset of familial MSI+ CRC.

International analysis of unique DNA mismatch repair gene sequence variants redefines the contribution of MSH2 and MLH1 to colorectal cancer (CRC) incidence

International analysis of unique DNA mismatch repair gene sequence variants redefines the contribution of MSH2 and MLH1 to colorectal cancer (CRC) incidence

Colorectal tumour BRAF V600E and MLH1 promoter methylation status in the assessment of mismatch repair gene sequence variants of unknown clinical significance

Lynch Syndrome is the most common hereditary cause of colorectal cancer, caused by pathogenic mutations in the mismatch repair (MMR) genes that result in functional defects in the DNA mismatch repair complex. Individuals identified with a pathogenic mutation are at high-risk of early onset colorectal and/or endometrial tumours. However, up to 50% of MMR sequence variants are reported to be of unknown clinical significance, creating a problem for clinicians and patients. We are developing a multifactorial model to assess the pathogenicity of MMR gene sequence variants of unknown clinical significance. Currently this model utilizes data on tumour microsatellite instability (MSI) and family segregation analysis to assess the likelihood that a variant carrier demonstrates features expected for a carrier of a pathogenic mutation. Tumour BRAF V600E mutation and MLH1 promoter methylation are reported to be associated with MSI-H, sporadic colorectal cancer and may thus provide a strong prediction that a tumour with MSI-H status is not from a MMR gene mutation carrier. A literature review of BRAF V600E and MLH1 promoter methylation in colorectal cancer patients was performed. Frequency of these tumour features in each study was assessed, stratified by MSI status and reported/likely MMR gene mutation status. Frequency of these characteristics was also analysed in the Colon CFR dataset, and used to estimate a likelihood ratio (LR) of pathogenicity for use in multifactorial modelling. As expected, the literature review of 4655 individuals from 33 studies revealed a high frequency of BRAF mutations in MSI-H tumours without a MMR mutation, which increased for the subset of cases with lack of MLH1 tumour protein expression. However, BRAF V600E mutation was identified in one patient with a PMS2 mutation, challenging previous assumptions that BRAF mutation status excludes positive MMR gene mutation status. MLH1 promoter methylation data generated using a variety of experimental designs were available for only 2984 individuals from 40 studies, with inconsistencies in frequency between studies. Information was reported on methylation status of 204 known MLH1 gene mutation carriers. Analysis of the Colon CFR dataset showed that BRAF tumour mutation status predicted MMR gene mutation status when combined with tumour MSI-H status. However, MSI-L and MSS with BRAF did not provide any additional predictive power over MSI-L or MSS status alone. The likelihood of a tumour with MSI-H and BRAF V600E positive status to be from a non-carrier was 11.55 and 9.85 for population-based and clinic-based patients, respectively. However, 4/98 (4.08%) of MSI-H BRAF positive tumours were from MMR mutation carriers. In conclusion, positive BRAF tumour status is not a conclusive test to exclude MMR gene mutation status for patients, but BRAF-MSI tumour status is a powerful addition to multifactorial models evaluating clinical significance of MMR gene variants.

The cost-effectiveness of genetic testing strategies for Lynch syndrome among newly diagnosed patients with colorectal cancer

PurposeTo estimate the cost-effectiveness of genetic testing strategies to identify Lynch syndrome among newly diagnosed patients with colorectal cancer and to offer targeted testing to relatives of patients with Lynch syndrome. MethodsWe calculated incremental costs per life-year saved for universal testing relative to no testing and age-targeted testing for strategies that use preliminary genetic tests (immunohistochemistry or microsatellite instability) of tumors followed by sequencing of mismatch repair genes. We also calculated incremental cost-effectiveness ratios for pairs of testing strategies. ResultsStrategies to test for Lynch syndrome in newly diagnosed colorectal tumors using preliminary tests before gene sequencing have incremental cost-effectiveness ratios of ≤$45,000 per life-year saved compared with no testing and ≤$75,000 per life-year saved compared with testing restricted to patients younger than 50 years. The lowest cost testing strategies, using immunohistochemistry as a preliminary test, cost ≤$25,000 per life-year saved relative to no testing and ≤$40,000 per life-year saved relative to testing only patients younger than 50 years. Other testing strategies have incremental cost-effectiveness ratios ≥$700,000 per life-year saved relative to the lowest cost strategies. Increasing the number of relatives tested would improve cost-effectiveness. ConclusionLaboratory-based strategies using preliminary tests seem cost-effective from the US health care system perspective. Universal testing detects nearly twice as many cases of Lynch syndrome as targeting younger patients and has an incremental cost-effectiveness ratio comparable with other preventive services. This finding provides support for a recent US recommendation to offer testing for Lynch syndrome to all newly diagnosed patients with colorectal cancer.

Microsatellite instability in the evaluation of hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) accounts for most hereditary colorectal cancers. The detection of families with HNPCC enables disease surveillance and clinical management, which significantly reduce morbidity and mortality. Mutations in DNA mismatch repair (MMR) genes underlie HNPCC and cause microsatellite instability (MSI). Although screening for pathogenic mutations in DNA MMR genes is time consuming and costly, MSI-based molecular diagnosis improves HNPCC diagnosis, which was once based on clinical characteristics and family history alone. Patients selected for MSI testing usually fulfill the Bethesda criteria. Tumor MSI status with tissue immunohistochemistry staining directs further genetic evaluation, including sequencing of MMR genes or methylation studies. This review outlines the importance of MSI status in disease diagnosis, prognosis, and treatment.

Malignant melanoma in patients with hereditary nonpolyposis colorectal cancer

Malignant melanoma (MM) is the most aggressive skin cancer. Most MMs are sporadic, and in this setting an association with mismatch repair (MMR) gene mutations, typical of hereditary nonpolyposis colorectal cancer (HNPCC) tumours, has been proposed. To characterize clinically and/or by molecular biology the patients with MM belonging to a cohort of 60 kindreds with HNPCC. Methods Patients with HNPCC with a diagnosis of MM were studied by immunohistochemistry (IHC) on tumour tissue using antibodies to MLH1, MSH2, p16, beta-catenin and E-cadherin, and by direct sequencing of MMR genes on germline DNA, and BRAF and NRAS on somatic DNA extracted from MM. Nine cutaneous MMs were detected in the tumour spectrum of eight families with HNPCC. The median age at diagnosis was 46 years. In one HNPCC family the diagnosis of MM was made in two first-degree relatives fitting the clinical definition of familial melanoma. IHC and sequencing analysis showed an MSH2 mutation in one patient with MM. Dermatological surveillance should be recommended to families in which MM is diagnosed in at least one member, especially at a young age. The combination of MMR gene mutations and abnormalities of p16 or other molecular pathways is needed to induce melanocytic carcinogenesis in a familial setting as well as in sporadic MM.