Minigene-based microdeletion assays have been used to map ESEs in BRCA2
Minigene constructs, containing a genomic segment from the gene of interest that includes the alternatively spliced exon(s) and flanking intronic regions, express pre-mRNAs. These constructs provide a rapid assay for SRE function, and effect of trans-acting factors on splicing regulation (Cooper, 2005).
Acedo et al. (2015) functionally mapped the ESE-rich regions inBRCA2 exons 19-27 using minigene splicing assays to improve ESE predictions and facilitate identification of ESE-disrupting spliceogenic variants. Since the density of active ESEs is highest near splice sites (∼50 nt at both exon ends) (Fairbrother, Holste, Burge, & Sharp, 2004), they mapped functional ESEs by introducing 34 30-nt microdeletions at the ends of each exon (Acedo et al., 2015). They found a microdeletion in exon 19 and another in exon 20 that clearly affected the splicing process, and six other microdeletions in exons 19, 20, 21 and 23 that had weak effects. 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), lay within microdeletions shown to impact mRNA splicing, so 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). Results from these assays have also been useful in re-classifying variants. For example, two variants in ClinVar, c.441A>G [p.(Gln147=), likely benign] and c.451G>A [p.(Val151Ile), VUS], were designated as spliceogenic variants and re-interpreted as VUS and likely pathogenic, respectively (Fraile-Bethencourt, Valenzuela-Palomo, Díez-Gómez, Goina, et al., 2019).