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.

Keywords: BRCA2; DNA variants; breast cancer; hybrid minigenes; splicing.

FIGURE 1

Minigene MGBR2_14-20. (A) Schematic representation of MGBR2_14-20. BRCA2 exons 14–20 are represented in blue boxes. Red lines represented the shortened introns. (B) Expected MGBR2_14-20 transcript (1,806 nt) and the amplified RT-PCR product. Specific amplification primers are shown as red arrows. (C) Capillary electrophoresis result of the functional assay of the wild type MGBR2_14-20 in MCF-7 cells. The full-length transcript is shown as a blue peak. The Genescan Liz-1200 size standard is shown as orange/faint peaks. Fragment sizes (nt) and relative fluorescent units are indicated on the x- and y-axes, respectively.

FIGURE 2

Exon 15 ESE mapping: functional assay of c.7463_7492del positive microdeletion. (A) Schematic representation of BRCA2 exons 14 and 15 and the eight microdeletions: 1, c.7010_7039del; 2, c.7035_7064del; 3, c.7378_7407del; 4, c.7402_7432del; 5, c.7438_7467del; 6, c.7463_7492del; 7, c.7561_7590del; 8, c.7586_7615del. (B) Capillary electrophoresis and sequence results of functional assays of microdeletion c.7463_7492del in MCF-7 cells.

FIGURE 3

Functional assays of the BRCA2 exons 14 and 15 variants using the MGBR2_14-minigene. (A) Exon 14 variants. (B) Exon 15 variants. On the left, capillary electropherograms are shown. Transcripts were amplified using pMAD_607FW-FAM and RTBR2_Ex17RV2. Labeled transcripts are shown as blue peaks, LIZ1200 was used as size standard (orange peaks). The expected size of the full length transcript is 1028 nt (1018–1019 nt according to Peak Scanner). On the right, splicing patterns are represented. While blue boxes are natural exons, red boxes represent aberrant exons; dashed black and red lines show canonical and aberrant splicing events, respectively.