MLH1 methylation screening is effective in identifying epimutation carriers

• Published in: European journal of human genetics : EJHG 2012-12, Vol.20 (12), p.1256-1264
• Main Authors: PINEDA, Marta, MUR, Pilar, BRUNET, Joan, NAVARRO, Matilde, BLANCO, Ignacio, CAPELLA, Gabriel, INIESTA, Maria Dolores, BORRAS, Ester, CAMPOS, Olga, VARGAS, Gardenia, IGLESIAS, Silvia, FERNANDEZ, Anna, GRUBER, Stephen B, LAZARO, Conxi
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adult; Age; Aged; Aged, 80 and over; Alleles; Biological and medical sciences; Bisulfite; Carrier Proteins - genetics; Carrier Proteins - metabolism; Colorectal cancer; Colorectal Neoplasms, Hereditary Nonpolyposis - diagnosis; Colorectal Neoplasms, Hereditary Nonpolyposis - genetics; Deoxyribonucleic acid; Disease transmission; DNA; DNA Methylation; Epigenesis, Genetic; Epigenetics; Female; Fundamental and applied biological sciences. Psychology; Gastroenterology. Liver. Pancreas. Abdomen; Gene expression; Gene Silencing; General aspects. Genetic counseling; Genetic Carrier Screening; Genetic testing; Genetics; Genetics of eukaryotes. Biological and molecular evolution; Genomics; Germ-Line Mutation; Haplotypes; Heritability; Heterozygote; Humans; Lymphocytes; Male; Medical genetics; Medical sciences; Melting; Melting curve; Methylation; Middle Aged; Mismatch repair; MLH1 protein; Molecular and cellular biology; Mutation; MutL Protein Homolog 1; Nuclear Proteins - genetics; Oncology; Organ Specificity; Pedigree; Polymorphism, Genetic; Probes; Protein expression; Public health; Sequence Analysis, DNA; Sperm; Stomach. Duodenum. Small intestine. Colon. Rectum. Anus; Transcription, Genetic; Tumors
• ISSN: 1018-4813; 1476-5438
• DOI: 10.1038/ejhg.2012.136

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.