Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18

Abstract

Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ex14-20 produced a stable transcript of the expected size (1,806 nucleotides) and structure (V1-[BRCA2_exons_14–20]–V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3’ end of exon 17 (c.7944-7973) and the 5’ end of exon 18 (c.7979-7988, c.7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c.7806-2A>G, c.7806-1G>A, c.7806-1G>T, c.7806-1_7806-2dup, c.7976+1G>A, c.7977-3_7978del, c.7977-2A>T, c.7977-1G>T, c.7977-1G>C, c.8009C>A, c.8331+1G>T and c.8331+2T>C) or likely pathogenic (c.7806-9T>G, c.7976G>C, c.7976G>A, c.7977-7C>G, c.7985C>G, c.8023A>G, c.8035G>T and c.8331G>A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17–18 at the BRCA Share database. The remaining 8 variants (c.7975A>G, c.7977-6T>G, c.7988A>T, c.7992T>A, c.8007A>G, c.8009C>T, c.8009C>G, and c.8072C>T) induced partial splicing anomalies with important ratios of the full-length transcript (≥70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA.

Splicing Characterization of CLCNKB Variants in Four Patients With Type III Bartter Syndrome

“…In fact, exon 3 is alternatively spliced in physiological conditions. This similar alternative splicing was also reported in several large multiexon minigenes of the BRCA2 gene (Acedo et al, 2015;Fraile-Bethencourt et al, 2017). The CLCNKB gene has a natural transcript (NM_001165945, ENST00000375667.7) in which exon 3 is excluded, producing a smaller isoform (517aa).…”

Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing:BRCA2c.7976+5G > T as a case study

“…Normalized CE data from full‐length transcript (FL) showed a two‐fold reduction in carriers compared to controls, suggesting that the variant allele is not producing FL (Figure b). To test this, since allele‐specific assays could not be performed due to the lack of heterozygous informative loci in patient sample, the mutant allele (c.7976 + 5G > T) was artificially interrogated using a pSAD‐derived minigene with BRCA2 exons 14 to 20, constructed and functionally validated as previously described in Fraile‐Bethencourt et al., . A wild‐type (wt) construct and a variant construct BRCA2 c.7976 + 1G > A were used as negative and positive controls, respectively.…”

“…From these, variants Trp2626Cys, Ile2627Phe, Leu2653Pro, and Arg2659Lys had been previously evaluated by multifactorial analysis and classified as pathogenic (Class 5) (Easton et al, 2007). Other variants causing exon 17 skipping (c.7976G > A, c.7976 + 1G > A and c.7976 + 3_7976 + 4del) have been identified in HBOC patients and reported as deleterious (Brandão, Roozendaal, Tserpelis, García, & Blok, 2011;Fraile-Bethencourt et al, 2017;Hofmann, Horn, Hüttner, Classen, & Scherneck, 2003;Thirthagiri et al, 2008;Wu et al, 2005). Moreover, allele-specific assessment was performed in patient RNA for variant c.7976 + 3_7976 + 4del, and only detected transcript lacking exon 17 (Brandão et al, 2011).…”

“…Current ENIGMA guidelines do not consider construct-based mRNA assays alone as a sufficiently robust approach to be used as evidence for variant classification. However, in this study, we used a validated minigene (MGBR2_ex14-20) that confers high reproducibility of splicing patterns as previously described in Fraile-Bethencourt et al (2017). More specifically, authors analyzed variants involving exon 17 (c.7806-9T > G, c.7975A > G, c.7976G > A, and c.7976G > C) using the MGBR2_ex14-20 minigene and compared the splicing patterns with patient RNA results published in previous works, and identified the same splicing results for both approaches in all cases.…”

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

“…Resultantly, splicing reporter minigenes have emerged as an alternative approach to test a variant from the splicing perspective (Tammaro et al, 2014). Remarkably, minigenes of the splicing vector pSAD have been successfully used to assay more than 300 variants of the breast cancer gene BRCA2 (Acedo et al, 2015;Fraile-Bethencourt et al, 2017, 2019b.…”