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

Abstract

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.

Cytoprotective Activity of Polyamines Is Associated with the Alternative Splicing of RAD51A Pre-mRNA in Normal Human CD4+ T Lymphocytes

“…Dominant negative mutations in RAD51 protein variants are suggested to be associated with the Fanconi anemia subtype [51]. Pre-mRNAs of different members of RAD51 are subjected to alternative splicing and are believed to have impacts on cancer progression [52][53][54]. Our results correspond to the inability of truncated RAD51A splice variants to promote homological recombination and reduce DNA damage because Put induced the predominance of the FL variant and prevented the death of normal CD4 + T cells.…”

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C

“…As already justified in previous studies by our group [ 19 , 20 , 25 ], once experimental splicing data were available, splicing predictive codes PVS1 and PP3 did not contribute to our final classification. Similarly, in HBOPC_ATMv1 specifications, functional splicing codes replace rather than combine with predictive splicing codes.…”

“…MCF-7 and MDA-MB-231 cell culture, transfection, and inhibition of the nonsense-mediated decay were performed as previously described [ 19 , 25 ].…”

Splicing predictions, minigene analyses, and ACMG‐AMP clinical classification of 42 germline PALB2 splice‐site variants

“…Functional assays of VUS provide critical information for their clinical interpretation. Aberrant splicing is a known frequent mechanism of gene inactivation associated with germline variants in BC genes [20,21,24,39]. Herein, we have focused on 82 PALB2 variants located at the splice-site boundaries detected in the BRIDGES cohort, so that we have accomplished the largest splicing functional study of PALB2 by minigenes to date.…”

“…We had previously shown that spliceogenic variants make a significant contribution to the overall pool of germ‐line loss of function alleles in the BC/OC genes RAD51C [ 20 ], RAD51D [ 21 ], and BRCA2 [ 22 , 23 , 24 ]. The present work was carried out in the context of the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project ( https://bridges-research.eu/ ), where 34 known or suspected BC/OC genes were sequenced in 60,466 BC cases and 53,461 controls [ 8 ].…”