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Article

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

1
Splicing and Genetic Susceptibility to Cancer Laboratory, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
2
Molecular Oncology Laboratory CIBERONC, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), Hospital Clinico San Carlos, 28040 Madrid, Spain
3
Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge CB1 8RN, UK
4
Cancer Genetics, Unidad de Excelencia Instituto de Biología y Genética Molecular (CSIC-UVa), 47003 Valladolid, Spain
5
Hereditary Cancer Program, Catalan Institute of Oncology, IDIBELL and CIBERONC, 08908 Hospitalet de Llobregat, Spain
6
Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands
*
Authors to whom correspondence should be addressed.
These authors are joint senior authors.
Academic Editor: Ana Vega
Cancers 2021, 13(11), 2845; https://doi.org/10.3390/cancers13112845
Received: 16 April 2021 / Revised: 1 June 2021 / Accepted: 3 June 2021 / Published: 7 June 2021
(This article belongs to the Special Issue Genetic Variants Associated with Breast and Ovarian Cancer Risk)
In the BRIDGES project, the breast/ovarian cancer gene RAD51D has been sequenced in >113,000 women. In the present study, we focused on the impact that 11 pre-selected RAD51D variants at the intron/exon boundaries had on the splicing process (intron removal). For this purpose, we developed a splicing reporter minigene, containing RAD51D-exons 2–9 wherein any variant could be introduced and functionally assayed for splicing alterations. All variants impaired splicing, 10 of which caused complete splicing aberrations. Moreover, we developed a minigene-based strategy to search for non-canonical, spliceogenic variants that disrupted splicing enhancers/silencers in the non-constitutive exon 3. Twenty-six BRIDGES and 16 artificial exon 3 variants were also tested. Thirty variants impaired splicing by producing variable amounts of the FL transcript. In total, up to 9 variants were classified as Likely Pathogenic, and therefore were clinically actionable. Carriers may benefit from tailored prevention protocols and therapies.
RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation. View Full-Text
Keywords: breast cancer; ovarian cancer; susceptibility genes; RAD51D; ESE; ESS; aberrant splicing; VUS; minigene; clinical interpretation breast cancer; ovarian cancer; susceptibility genes; RAD51D; ESE; ESS; aberrant splicing; VUS; minigene; clinical interpretation
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MDPI and ACS Style

Bueno-Martínez, E.; Sanoguera-Miralles, L.; Valenzuela-Palomo, A.; Lorca, V.; Gómez-Sanz, A.; Carvalho, S.; Allen, J.; Infante, M.; Pérez-Segura, P.; Lázaro, C.; Easton, D.F.; Devilee, P.; Vreeswijk, M.P.G.; de la Hoya, M.; Velasco, E.A. RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants. Cancers 2021, 13, 2845. https://doi.org/10.3390/cancers13112845

AMA Style

Bueno-Martínez E, Sanoguera-Miralles L, Valenzuela-Palomo A, Lorca V, Gómez-Sanz A, Carvalho S, Allen J, Infante M, Pérez-Segura P, Lázaro C, Easton DF, Devilee P, Vreeswijk MPG, de la Hoya M, Velasco EA. RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants. Cancers. 2021; 13(11):2845. https://doi.org/10.3390/cancers13112845

Chicago/Turabian Style

Bueno-Martínez, Elena, Lara Sanoguera-Miralles, Alberto Valenzuela-Palomo, Víctor Lorca, Alicia Gómez-Sanz, Sara Carvalho, Jamie Allen, Mar Infante, Pedro Pérez-Segura, Conxi Lázaro, Douglas F. Easton, Peter Devilee, Maaike P. G. Vreeswijk, Miguel de la Hoya, and Eladio A. Velasco. 2021. "RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants" Cancers 13, no. 11: 2845. https://doi.org/10.3390/cancers13112845

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