Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.
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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