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.
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Acknowledgements
We thank the patients who participated in this study, Gemma Aiza for technical assistance, Javier Carmona for his assistance with pyrosequencing and Dr Juana Fernández for her assistance in skin fibroblast isolation and culture. This work was supported by grants from Ministerio de Ciencia e Innovación (SAF 06-06084; 09-07319), Fundació Gastroenterologia Dr Francisco Vilardell (F05-01), Ministerio de Educación y Ciencia Spanish Networks RTICCC (RD06/0020/1050, 1051), Acción en Cáncer (Instituto de Salud Carlos III), Fundación Científica AECC and NCI U19 CA 148107-02.
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Pineda, M., Mur, P., Iniesta, M. et al. MLH1 methylation screening is effective in identifying epimutation carriers. Eur J Hum Genet 20, 1256–1264 (2012). https://doi.org/10.1038/ejhg.2012.136
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DOI: https://doi.org/10.1038/ejhg.2012.136
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