4.6 Article

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

期刊

CANCERS
卷 13, 期 11, 页码 -

出版社

MDPI
DOI: 10.3390/cancers13112845

关键词

breast cancer; ovarian cancer; susceptibility genes; RAD51D; ESE; ESS; aberrant splicing; VUS; minigene; clinical interpretation

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资金

  1. European Union [634935]
  2. Spanish Ministry of Science and Innovation, Plan Nacional de I + D + I 2013-2016, ISCIII [PI17/00227, PI20/00225, PI15/00059, PI20/00110]
  3. FEDER from Regional Development European Funds (European Union)
  4. Consejeria de Educacion, Junta de Castilla y Leon [CSI242P18]
  5. AECC-Scientific Foundation, Sede Provincial de Valladolid
  6. Consejeria de Educacion, Junta de Castilla y Leon
  7. Programa Estrategico Instituto de Biologia y Genetica Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y Leon [CLU-2019-02]

向作者/读者索取更多资源

RAD51D loss-of-function variants have been shown to increase lifetime risk of breast and ovarian cancer. Splicing disruption is a common pathogenic mechanism in susceptibility genes. Through experiments on splice-site and ESE variants in a cohort of approximately 113,000 women, 41 RAD51D spliceogenic variants were identified, with minigene-based mapping of ESEs proving to be a powerful approach for identifying ESE hotspots and ESE-disrupting variants.
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 similar to 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.

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