| Autor: |
Sanoguera-Miralles L; Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain., Bueno-Martínez E; Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain., Valenzuela-Palomo A; Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain., Esteban-Sánchez A; Molecular Oncology Laboratory, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain., Llinares-Burguet I; Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain., Pérez-Segura P; Molecular Oncology Laboratory, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain., García-Álvarez A; Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain., de la Hoya M; Molecular Oncology Laboratory, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain., Velasco-Sampedro EA; Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain. |
| Abstrakt: |
RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1−4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants. |
