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

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.

DNA Methylation in the Diagnosis of Monogenic Diseases

“…In addition, about 5%-15% of colorectal carcinomas (CRCs) display reduced MLH1 immunostaining and MMR deficiency due to promoter hypermethylation. In most cases, epigenetic inactivation is a somatic event, but constitutional MLH1 hypermethylation has been reported in several patients [125][126][127][128]. Overall, this mechanism explains 1.5%-10.5% of CRCs associated with abnormal expression of MLH1, and might account for up to 3% of LS [128].…”

“…In most cases, epigenetic inactivation is a somatic event, but constitutional MLH1 hypermethylation has been reported in several patients [125][126][127][128]. Overall, this mechanism explains 1.5%-10.5% of CRCs associated with abnormal expression of MLH1, and might account for up to 3% of LS [128]. In a few cases, the epigenetic abnormality is accompanied by deletions or single nucleotide substitutions, namely a specific MLH1 haplotype bearing the variants c.27C>A and c.85G>T (secondary epimutation), and inheritance of these genetic variations and associated epimutation has been observed in some families [129].…”

“…However, more frequently, the aberrant methylation pattern is not accompanied by DNA sequence alterations (primary epimutation) and is usually reverted across generations, although occasional inheritance has been observed [130]. MLH1 epimutations tend to present as sporadic cases affected with multiple primary tumors, with an earlier average age at cancer diagnosis compared to the general population [128]. A handful of mosaic cases have been reported, including a case showing levels of methylated MLH1 alleles as low as 1% in blood and other tissues, which implies an opportunity to use very sensitive techniques [131].…”

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation

“…Our MS-MCA approach allowed the robust and highly sensitive detection of MLH1 methylation in blood, whereas no evidence of methylation was detected in controls. In contrast, pyrosequencing and MS-MLPA, widely used in clinical diagnostics for MLH1 methylation detection [12,14,23,24], display a lower analytical sensitivity (5-10%) than MS-MCA (1%) [19], potentially overlooking low-level epigenetic mosaicism. Moreover, the background signal observed in pyrosequencing analyses could account for the high proportion (78%) of low-level (< 10%) methylation levels previously reported in healthy controls [17].…”

“…Ninety-seven index cases with a constitutional MLH1 epimutation have been reported so far [8][9][10][11][12]. Most are considered primary, arising apparently de novo and reversible between generations, whereas secondary epimutations are associated with a genetic variant in cis.…”