RESUMEN
High-throughput sequencing efforts in molecular tumour diagnostics detect increasing numbers of novel variants, including variants predicted to affect splicing. In silico prediction tools can reliably predict the effect of variant disrupting canonical splice sites; however, experimental validation is required to confirm aberrant splicing. Here, we present RNA analysis performed for 13 canonical splice site variants predicted or known to result in splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Total nucleic acid was successfully isolated for 10 variants from eight formalin-fixed paraffin-embedded (FFPE) tumour tissues and two B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known variant in the B-cell line, aberrant splicing could only be detected after formalin fixation, which indicated that formalin fixation could possibly inhibit RNA degradation. Aberrant splicing was concluded in three of four predicted splice variants of uncertain significance, supporting their pathogenic effect. With this assay, somatic splice variants can be easily and rapidly analysed, enabling retrospective analysis to support the pathogenicity of variants predicted to result in splicing when only FFPE material is available.
Asunto(s)
Neoplasias de la Mama/genética , Pruebas Genéticas/métodos , Mutación , Empalme del ARN , Análisis de Secuencia de ARN/métodos , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína BRCA1/genética , Neoplasias de la Mama/patología , Proteínas de Unión al ADN/genética , Femenino , Pruebas Genéticas/normas , Humanos , Homólogo 1 de la Proteína MutL/genética , Proteína 2 Homóloga a MutS/genética , Análisis de Secuencia de ARN/normas , Adhesión del Tejido/métodos , Adhesión del Tejido/normas , Fijación del Tejido/métodos , Fijación del Tejido/normasRESUMEN
Many suspected Lynch Syndrome (sLS) patients who lack mismatch repair (MMR) germline gene variants and MLH1 or MSH2 hypermethylation are currently explained by somatic MMR gene variants or, occasionally, by germline POLE variants. To further investigate unexplained sLS patients, we analyzed leukocyte and tumor DNA of 62 sLS patients using gene panel sequencing including the POLE, POLD1 and MMR genes. Forty tumors showed either one, two or more somatic MMR variants predicted to affect function. Nine sLS tumors showed a likely ultramutated phenotype and were found to carry germline (n=2) or somatic variants (n=7) in the POLE/POLD1 exonuclease domain (EDM). Six of these POLE/POLD1-EDM mutated tumors also carried somatic MMR variants. Our findings suggest that faulty proofreading may result in loss of MMR and thereby in microsatellite instability.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación de la Incompatibilidad de ADN , ADN Polimerasa III/genética , ADN Polimerasa II/genética , Adulto , Anciano , Dominio Catalítico , Evolución Clonal , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , ADN Polimerasa II/química , ADN Polimerasa III/química , Femenino , Inestabilidad Genómica , Mutación de Línea Germinal , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL/genética , Proteína 2 Homóloga a MutS/genética , Proteínas de Unión a Poli-ADP-RibosaRESUMEN
BACKGROUND: Lynch syndrome (LS) is associated with a high risk for colorectal cancer (CRC) and extracolonic malignancies, such as endometrial carcinoma (EC). The risk is dependent of the affected mismatch repair gene. The aim of the present study was to calculate the cumulative risk of LS related cancers in proven MLH1, MSH2 and MSH6 mutation carriers. METHODS: The studypopulation consisted out of 67 proven LS families. Clinical information including mutation status and tumour diagnosis was collected. Cumulative risks were calculated and compared using Kaplan Meier survival analysis. RESULTS: MSH6 mutation carriers, both males and females had the lowest risk for developing CRC at age 70 years, 54% and 30% respectively and the age of onset was delayed by 3-5 years in males. With respect to endometrial carcinoma, female MSH6 mutation carriers had the highest risk at age 70 years (61%) compared to MLH1 (25%) and MSH2 (49%). Also, the age of EC onset was delayed by 5-10 years in comparison with MLH1 and MSH2. CONCLUSIONS: Although the cumulative lifetime risk of LS related cancer is similar, MLH1, MSH2 and MSH6 mutations seem to cause distinguishable cancer risk profiles. Female MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. As a consequence, surveillance colonoscopy starting at age 30 years instead of 20-25 years is more suitable. Also, prophylactic hysterectomy may be more indicated in female MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.
