Contribution of MLH1 constitutional methylation for Lynch syndrome diagnosis in patients with tumor MLH1 downregulation.

Diana Pinto,Manuela Pinheiro,Paula Lopes,Zeremariam Yohannes,Hege Marie Vedeld,Carla Pinto,Guro Elisabeth Lind,Manuel R Teixeira,Joana Guerra,Ragnhild Lothe,Pedro Pinto,Rui Henrique,Catarina Santos,Rui Santos,Ana Peixoto

doi:10.1002/cam4.1285

Abstract

Constitutional epimutation of the two major mismatch repair genes, MLH1 and MSH2, has been identified as an alternative mechanism that predisposes to the development of Lynch syndrome. In the present work, we aimed to investigate the prevalence of MLH1 constitutional methylation in colorectal cancer (CRC) patients with abnormal expression of the MLH1 protein in their tumors. In a series of 38 patients who met clinical criteria for Lynch syndrome genetic testing, with loss of MLH1 expression in the tumor and with no germline mutations in the MLH1 gene (35/38) or with tumors presenting the BRAF p.Val600Glu mutation (3/38), we screened for constitutional methylation of the MLH1 gene promoter using methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) in various biological samples. We found four (4/38; 10.5%) patients with constitutional methylation in the MLH1 gene promoter. RNA studies demonstrated decreased MLH1 expression in the cases with constitutional methylation when compared with controls. We could infer the mosaic nature of MLH1 constitutional hypermethylation in tissues originated from different embryonic germ layers, and in one family we could show that it occurred de novo. We conclude that constitutional MLH1 methylation occurs in a significant proportion of patients who have loss of MLH1 protein expression in their tumors and no MLH1 pathogenic germline mutation. Furthermore, we provide evidence that MLH1 constitutional hypermethylation is the molecular mechanism behind about 3% of Lynch syndrome families diagnosed in our institution, especially in patients with early onset or multiple primary tumors without significant family history.

Highlights

Lynch syndrome is the most common hereditary syndrome that predisposes to colorectal cancer (CRC), corresponding to 2-5% of all CRCs [1, 2]
Analysis of the constitutional methylation status using the MS-M LPA CpG Island Methylator Phenotype (CIMP) kit in the four patients with MLH1 epimutation showed that none of the other seven genes tested (CACNA1G, CDKN2A, CRABP1, IGF2, NEUROG1, RUNX3, and SOCS1) presented hypermethylation (Table S2)
MLH1 methylation was 10.5% (4/38), signifying that MLH1 methylation may account for a non-negligible proportion of Lynch syndrome patients when analysis is restricted to those showing MLH1 abnormal expression in their tumors

Summary

Introduction

Lynch syndrome is the most common hereditary syndrome that predisposes to colorectal cancer (CRC), corresponding to 2-5% of all CRCs [1, 2] It is an autosomal dominant disease caused by germline mutations in the Mismatch Repair (MMR) genes involved in DNA repair [3, 4]. These include MLH1, MSH2, MSH6, and PMS2, about 90% of the mutations described in this syndrome occur in MLH1 or MSH2 [5–8]. MLH1 constitutional methylation and Lynch syndrome or just “epimutation” [9, 10] This phenomenon consists in constitutional transcriptional silencing of these genes by epigenetic mechanisms rather than by genetic mutations that directly affect the sequence of the gene [8–10]. MLH1 epimutations may be dichotomized into two categories: (1) those that arise spontaneously and are reversible between generations, though occasionally transmitted to the generation in a non-M endelian pattern (primary MLH1 epimutation); and (2) Mendelian epimutations that follow a classic autosomal dominant inheritance pattern due to an underlying cis-genetic cause (secondary/genetically facilitated MLH1 epimutation) [10, 13]

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