RESUMEN
INTRODUCTION: Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are hereditary cancer syndromes associated with mismatch repair (MMR) deficiency. Tumours show microsatellite instability (MSI), also reported at low levels in non-neoplastic tissues. Our aim was to evaluate the performance of high-sensitivity MSI (hs-MSI) assessment for the identification of LS and CMMRD in non-neoplastic tissues. MATERIALS AND METHODS: Blood DNA samples from 131 individuals were grouped into three cohorts: baseline (22 controls), training (11 CMMRD, 48 LS and 15 controls) and validation (18 CMMRD and 18 controls). Custom next generation sequencing panel and bioinformatics pipeline were used to detect insertions and deletions in microsatellite markers. An hs-MSI score was calculated representing the percentage of unstable markers. RESULTS: The hs-MSI score was significantly higher in CMMRD blood samples when compared with controls in the training cohort (p<0.001). This finding was confirmed in the validation set, reaching 100% specificity and sensitivity. Higher hs-MSI scores were detected in biallelic MSH2 carriers (n=5) compared with MSH6 carriers (n=15). The hs-MSI analysis did not detect a difference between LS and control blood samples (p=0.564). CONCLUSIONS: The hs-MSI approach is a valuable tool for CMMRD diagnosis, especially in suspected patients harbouring MMR variants of unknown significance or non-detected biallelic germline mutations.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Inestabilidad de Microsatélites , Proteína 2 Homóloga a MutS/genética , Síndromes Neoplásicos Hereditarios/genética , Adolescente , Adulto , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/patología , Niño , Preescolar , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/sangre , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Reparación de la Incompatibilidad de ADN/genética , Femenino , Mutación de Línea Germinal/genética , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Síndromes Neoplásicos Hereditarios/sangre , Síndromes Neoplásicos Hereditarios/patología , Adulto JovenRESUMEN
Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer.Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches.MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient's tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative.Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.