Gastroenterology. 2019 Apr 15. pii: S0016-5085(19)35707-5. doi: 10.1053/j.gastro.2019.03.071. [Epub ahead of print]
Methylation Tolerance-based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome.
Bouvet D1, Bodo S1, Munier A2, Guillerm E3, Bertrand R1, Colas C4, Duval A5, Coulet F3, Muleris M6.
BACKGROUND & AIMS:
Approximately 75% of patients with suspected Lynch syndrome carry variants in MLH1 or MSH2-proteins encoded by these genes are required for DNA mismatch repair (MMR). However, 30% of these are variants of unknown significance (VUS). A assay that measures cell response to the cytotoxic effects of a methylating agent can determine the effects of VUS in MMR genes and identify patients with constitutional mismatch repair-deficiency syndrome. We adapted this method to test the effects of VUS in MLH1 and MSH2 genes found in patients with suspected Lynch syndrome.
We transiently expressed MLH1 or MSH2 variants in MLH1- or MSH2-null human colorectal cancer cell lines (HCT116 or LoVo), respectively. The MMR process causes death of cells with methylation-damaged DNA bases, so we measured proportions of cells that undergo death following exposure to the methylating agent; cells that escaped its toxicity were considered to have variants that affect function of the gene product. Using this assay, we analyzed 88 variants (mainly missense variants), comprising a validation set of 40 previously classified variants (19 in MLH1 and 21 in MSH2) and a prospective set of 48 VUS (25 in MLH1 and 23 in MSH2). Prediction scores were calculated for all VUS according to the recommendations of the American College of Medical Genetics and Genomics, based on clinical, somatic, in silico, population and functional data.
The assay correctly classified 39/40 variants in the validation set. The assay identified 12 VUS that did alter function of the gene product and 28 VUS that did not; the remaining 8 VUS had intermediate effects on MMR capacity and could not be classified. Comparison of assays results with prediction scores confirmed the ability of the assay to discriminate VUS that affected the function of the gene products from those that did not.
Using an assay that measures the ability of the cells to undergo death following DNA damage induction by a methylating agent, we were able to assess whether variants in MLH1 and MSH2 cause defects in DNA MMR. This assay might be used to help assessing the pathogenicity of VUS in MLH1 and MSH2 found in patients with suspected Lynch syndrome.
Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.
CMMRD; MNNG-induced cell death; diagnosis; genetic analysis
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