Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15

Abstract

A relevant fraction of BRCA2 variants is associated with splicing alterations and with an increased risk of hereditary breast and ovarian cancer (HBOC). In this work, we have carried out a thorough study of variants from BRCA2 exons 14 and 15 reported at mutation databases. A total of 294 variants from exons 14 and 15 and flanking intronic sequences were analyzed with the online splicing tools NNSplice and Human Splicing Finder. Fifty-three out of these 294 variants were selected as candidate splicing variants. All variants but one, were introduced into the minigene MGBR2_ex14-20 (with exons 14–20) by site-directed mutagenesis and assayed in MCF-7 cells. Twelve of the remaining 52 variants (23.1%) impaired splicing at different degrees, yielding from 5 to 100% of aberrant transcripts. Nine variants affected the natural acceptor or donor sites of both exons and three affected putative enhancers or silencers. Fluorescent capillary electrophoresis revealed at least 10 different anomalous transcripts: (E14q5), Δ (E14p10), Δ(E14p246), Δ(E14q256), Δ(E14), Δ(E15p12), Δ(E15p13), Δ(E15p83), Δ(E15) and a 942-nt fragment of unknown structure. All transcripts, except for Δ(E14q256) and Δ(E15p12), are expected to truncate the BRCA2 protein. Nine variants induced severe splicing aberrations with more than 90% of abnormal transcripts. Thus, according to the guidelines of the American College of Medical Genetics and Genomics, eight variants should be classified as pathogenic (c.7008-2A > T, c.7008-1G > A, c.7435+1G > C, c.7436-2A > T, c.7436-2A > G, c.7617+1G > A, c.7617+1G > T, and c.7617+2T > G), one as likely pathogenic (c.7008-3C > G) and three remain as variants of uncertain clinical significance or VUS (c.7177A > G, c.7447A > G and c.7501C > T). In conclusion, functional assays by minigenes constitute a valuable strategy to primarily check the splicing impact of DNA variants and their clinical interpretation. While bioinformatics predictions of splice site variants were accurate, those of enhancer or silencer variants were poor (only 3/23 spliceogenic variants) which showed weak impacts on splicing (∼5–16% of aberrant isoforms). So, the Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively) prediction algorithms require further improvement.

Variant effect on splicing regulatory elements, branchpoint usage, and pseudoexonization: Strategies to enhance bioinformatic prediction using hereditary cancer genes as exemplars

“…Three previously characterized ESE variants, c.8378G>A (exon 19), c.8969G>A (exon 23), and c.9006A>T (exon 23) (Acedo et al, 2012), lie within microdeletions shown to impact mRNA splicing, thereby demonstrating the utility of this strategy to locate putative ESE variants (Acedo et al, 2015). Fraile‐Bethencourt et al adapted this systematic minigene assay approach to map active ESEs in BRCA2 exons 2–9 and 14–18 (Fraile‐Bethencourt et al, 2017; Fraile‐Bethencourt, Valenzuela‐Palomo, Díez‐Gómez, Acedo, & Velasco, 2018; Fraile‐Bethencourt, Valenzuela‐Palomo, Díez‐Gómez, Caloca, et al, 2019; Fraile‐Bethencourt, Valenzuela‐Palomo, Díez‐Gómez, Goina, et al, 2019). Selection of variants within the microdeletion‐mapped ESEs improved the specificity of bioinformatic predictions (Fraile‐Bethencourt, Valenzuela‐Palomo, Díez‐Gómez, Goina, et al, 2019).…”

Alternative mRNA splicing can attenuate the pathogenicity of presumed loss-of-function variants in BRCA2

“…For many BRCA1 and BRCA2 variants (both intronic and exonic) an effect on mRNA splicing has been reported using either patient RNA or minigene analysis. [12][13][14][15][16][17][18][19][20][21] The analysis of patient RNA is however often hampered by the inability to determine allele-specific transcript expression. It then remains unclear if and to what extent wild type (WT) mRNA is still produced from the variant allele.…”

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

“…Moreover, this construct was fully functional and induced a full-length transcript of the expected size and sequence (Figures 2B,C). Likewise, the mutant constructs (c.864+5G>T and c.996+2_996+5del) yielded clean splicing patterns (Figure 2C), where three aberrant transcripts [ (E1q141), (E2), and (E2q135)] were identified with high resolution and sensitivity by fluorescent fragment analysis (Fraile-Bethencourt et al, 2019a). Thus, c.864+5G>T induced a deletion of 141 nt of exon 1, similar to previously reported variant c.864+1G>C (Gantla et al, 1998), but also a significant proportion of the full-length transcript (29.6%) (Figures 2B-E).…”

“…Variants and transcripts were described according to the Human Genome Variation Society (HGVS) guidelines on the basis of the UGT1A1 GenBank sequence NM_00463.2. For simplification, transcripts were annotated with a shortened code, as described previously (Fraile-Bethencourt et al, 2019a;Lopez-Perolio et al, 2019):…”