Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium

Abstract

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Δ1Aq, Δ5, Δ5q, Δ8p, Δ9, Δ(9,10), Δ9_11, Δ11q, Δ13p and Δ14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.

Naturally occurringBRCA2alternative mRNA splicing events in clinically relevant samples

“…Our data (up to 24 splicing events, including 4 predominant) suggest that BRCA2 is probably in the upper limit of an average locus for the number of alternate-splicing events. Yet, this is in contrast with BRCA1 , a genomic locus similar to BRCA2 in size (≈81 kb vs ≈84 kb) and number of exons (23 vs 27), but with a notable higher level of alternative splicing (up to 63 splicing events, including 10 predominant), according to a recent study conducted by the ENIGMA consortium with a very similar methodology 6. This difference may reflect the well-established link between protein disorder (a structural feature of BRCA1 but not BRCA2 proteins) and high levels of alternative splicing 22 23…”

“…These results are of importance to the design and interpretation of mRNA splicing assays, construct-based or of patient material, that are commonly used to assess whether VUS leads to aberrations that are phenotypically equivalent to a molecular null (ie, a gene deletion). Recent studies have shown that detection of alternate splice events can be highly variable between laboratories and is quite sensitive to variations in cell types, cell growth conditions, mRNA preparation and RT-PCR methodology 5 6 24. Recommendations have been made to help make methodologies and reporting of alternate-splicing isoforms more uniform, including inhibition of NMD, sequence confirmation, proper primer design, presentation of data from at least 10 control samples of the same cell type to reveal potentially rare alternate splice isoforms, quantifying the variant allele contribution to full-length mRNA isoforms and quantifying the level of ‘aberrant’ transcripts relative to the full-length transcript 24.…”

“…In most cases, splicing isoforms were verified by sequence analysis at the originating institution. Samples and methods have been described in detail elsewhere 6. Details are presented in the ‘Results’.…”

“…ENIGMA collaborators have recently demonstrated the value of comprehensive approaches to catalogue previously unrecognised naturally occurring BRCA1 mRNA transcripts 6. Here, we report a complementary study conducted to provide the first systematic catalogue of human alternate BRCA2 mRNA splicing events.…”

Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing

“…Consistent with our observations, cancer and other diseases have been associated with dysregulated alternative splicing, including for several UGT genes. 12,13,15,[29][30][31] In addition, coordinated temporal control of alternative splicing has been recently documented for UGT2B7 in the kidney. 15 As extended read lengths were obtained with the use of pyrosequencing, and applied to pools of drug or hormone metabolizing tissues, our approach did not allow to quantitatively assess variant expression levels in specific tissues.…”