MLH1 methylation screening is effective in identifying epimutation carriers

Abstract

Recently, constitutional MLH1 epimutations have been identified in a subset of Lynch syndrome (LS) cases. The aim of this study was the identification of patients harboring constitutional MLH1 epimutations in a set of 34 patients with a clinical suspicion of LS, MLH1-methylated tumors and non-detected germline mutations in mismatch repair (MMR) genes. MLH1 promoter methylation was analyzed in lymphocyte DNA samples by MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification). Confirmation of MLH1 constitutional methylation was performed by MS-MCA (Methylation-specific melting curve analysis), bisulfite sequencing and pyrosequencing in different biological samples. Allelic expression was determined using heterozygous polymorphisms. Vertical transmission was evaluated by MS-MLPA and haplotype analyses. MS-MLPA analysis detected constitutional MLH1 methylation in 2 of the 34 individuals whose colorectal cancers showed MLH1 methylation (5.9%). These results were confirmed by bisulfite-based methods. Both epimutation carriers had developed metachronous early-onset LS tumors, with no family history of LS-associated cancers in their first-degree relatives. In one of the cases, the identified MLH1 constitutional methylation was monoallelic and results in MLH1 and EPM2AIP1 allele-specific transcriptional silencing. It was present in normal somatic tissues and absent in spermatozoa. The methylated MLH1 allele was maternally transmitted and methylation was reversed in a daughter who inherited the same allele. MLH1 methylation screening in lymphocyte DNA from patients with early-onset MLH1-methylated LS-associated tumors allows the identification of epimutation carriers. The present study adds further evidence to the emerging entity of soma-wide MLH1 epimutation and its heritability.

Prevalence ofMLH1constitutional epimutations as a cause of Lynch syndrome in unselected versus selected consecutive series of patients with colorectal cancer

“…The low frequency of constitutional epimutations, analytical limitations and uncertainty regarding the penetrance of these epimutations, their expression and their mode of inheritance may underlie such suboptimal diagnosis. Our results confirm previous reports of a prevalence of constitutional epimutations of approximately 10% in cases fulfilling the rBG with loss of MLH1 expression and no pathogenic mutations according to standard genetic testing 4 5 11 13. These findings strongly support the routine evaluation of constitutional epimutations in cases fulfilling the rBG that lack MLH1 expression and display MLH1 hypermethylation before considering them to be sporadic CRC cases.…”

The MLH1 c.-27C>A and c.85G>T variants are linked to dominantly inherited MLH1 epimutation and are borne on a European ancestral haplotype

“…11 These cases have tended to be sporadic due to the spontaneous origination of the epimutation in carriers and its subsequent eradication in the germline. [12][13][14][15] However, a handful of familial cases with an MLH1 epimutation have been reported in which transmission of the epimutation between generations has been shown to occur in both non-Mendelian 14 and autosomal dominant patterns, with the latter linked to localized cis-acting genetic anomalies. 16,17 In a Caucasian family from Western Australia (WA Family 16), dominant transmission of a mosaic MLH1 epimutation was demonstrated through three successive generations linked to a particular haplotype bearing two single-nucleotide variants (SNVs) in tandem; promoter substitution c. À27C4A and missense variant c.85G4T (p.A29S) (according to coding reference sequence NM_000249.2).…”

Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis

“…Mangold et al found germline mutations of MLH1 or MSH2 in 62% of patients who fulfilled the revised Bethesda guidelines and had MSI-H [28]. Colorectal cancer patients with suspected Lynch syndrome, who were selected for epimutation tests because of negative germline tests for MMR genes, had MSI and IHC loss of MLH1 due to epimutation at rates of 3%–10% [11,13,14,16,17,29,30,31,32,33]. The current study suggests that similar findings may occur in endometrial cancer.…”

“…Studies of mature spermatozoa from male MLH1 epimutation carriers have shown that MLH1 methylation is absent in spermatozoa, while methylation is present soma-wide [13,31,36,37]. Constitutional epimutation is the term describing the epigenetic change found in somatic tissues, but is absent or erased in the germline.…”