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Open AccessArticle

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

1
Splicing and Genetic Susceptibility to Cancer, Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
2
Molecular Oncology Laboratory CIBERONC, Hospital Clinico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain
3
Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
4
Knight Cancer Research Building, 2720 S Moody Ave, Portland, OR 97201, USA
5
Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge CB1 8RN, UK
6
Leiden University Medical Center, Department of Human Genetics, 2300RC Leiden, The Netherlands
*
Authors to whom correspondence should be addressed.
Cancers 2020, 12(12), 3771; https://doi.org/10.3390/cancers12123771
Received: 19 October 2020 / Revised: 8 December 2020 / Accepted: 9 December 2020 / Published: 15 December 2020
(This article belongs to the Special Issue Genetic Variants Associated with Breast and Ovarian Cancer Risk)
Genetic variants in more than 10 genes are known to confer moderate to high risks to breast and/or ovarian cancers (BC/OC). In the framework of the international project BRIDGES, a panel of 34 known or suspected BC/OC genes has been sequenced in 60,466 breast cancer patients and 53,461 controls. In this work, we focus on BRIDGES variants detected in the RAD51C gene and their impact on the gene expression step known as splicing (intron removal), whose alteration is a relevant disease mechanism. For this purpose, we bioinformatically analyzed 40 RAD51C variants from the intron/exon boundaries, 20 of which were selected. Then, we developed a biotechnological tool, called splicing reporter minigene, containing RAD51C exons 2 to 8 where any variant can be introduced by site-directed mutagenesis and functionally assayed in MCF-7 cells under the splicing perspective. Nineteen variants impaired splicing, 18 of which induced severe splicing anomalies. Finally, they were clinically interpreted according to strict guidelines whereby 15 variants were classified as Pathogenic/Likely Pathogenic, so they are clinically actionable. Therefore, carrier patients and families may benefit from tailored prevention protocols and therapies.
Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T. View Full-Text
Keywords: breast cancer; ovarian cancer; susceptibility genes; RAD51C; genetic variants; splicing; aberrant splicing; VUS; functional assay; minigene; clinical interpretation breast cancer; ovarian cancer; susceptibility genes; RAD51C; genetic variants; splicing; aberrant splicing; VUS; functional assay; minigene; clinical interpretation
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MDPI and ACS Style

Sanoguera-Miralles, L.; Valenzuela-Palomo, A.; Bueno-Martínez, E.; Llovet, P.; Díez-Gómez, B.; Caloca, M.J.; Pérez-Segura, P.; Fraile-Bethencourt, E.; Colmena, M.; Carvalho, S.; Allen, J.; Easton, D.F.; Devilee, P.; Vreeswijk, M.P.G.; de la Hoya, M.; Velasco, E.A. Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene. Cancers 2020, 12, 3771. https://doi.org/10.3390/cancers12123771

AMA Style

Sanoguera-Miralles L, Valenzuela-Palomo A, Bueno-Martínez E, Llovet P, Díez-Gómez B, Caloca MJ, Pérez-Segura P, Fraile-Bethencourt E, Colmena M, Carvalho S, Allen J, Easton DF, Devilee P, Vreeswijk MPG, de la Hoya M, Velasco EA. Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene. Cancers. 2020; 12(12):3771. https://doi.org/10.3390/cancers12123771

Chicago/Turabian Style

Sanoguera-Miralles, Lara; Valenzuela-Palomo, Alberto; Bueno-Martínez, Elena; Llovet, Patricia; Díez-Gómez, Beatriz; Caloca, María J.; Pérez-Segura, Pedro; Fraile-Bethencourt, Eugenia; Colmena, Marta; Carvalho, Sara; Allen, Jamie; Easton, Douglas F.; Devilee, Peter; Vreeswijk, Maaike P.G.; de la Hoya, Miguel; Velasco, Eladio A. 2020. "Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene" Cancers 12, no. 12: 3771. https://doi.org/10.3390/cancers12123771

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