Abstract #LB-252: Splicing analysis of BRCA1/2 exonic and intronic sequence variants indicates that clinical testing is warranted for variants located outside the donor and acceptor dinucleotides
Authors: Phillip Whiley, Logan Walker, Sue Healey, kConFab, Leo Da Silva, Sunil Lakhani, Fergus Couch, Sean Tavtigian, David Goldgar, Melissa Brown, Amanda Spurdle
Journal: Cancer Research (2009)

Classification of intronic and predicted missense sequence variants in the breast cancer susceptibility genes  BRCA1  and  BRCA2  is essential for appropriate genetic counselling of individuals carrying these variants. However, it remains a significant challenge to determine whether such sequence changes alter gene function sufficiently to predispose the individual to breast cancer. Unclassified variants located in the splice site recognition sequences and intronic or exonic regulatory sequences may disrupt messenger RNA splicing and lead to an imbalance of alternately spliced transcripts, impaired protein function or complete protein loss. To date  in silico  prediction algorithms have limited capacity to accurately predict the effect of variants on splicing, in particular the use of cryptic splice sites after disruption of donor and acceptor sites, and also the creation of  de novo  splice sites. We are conducting ongoing studies assessing the clinical significance of  BRCA1/2  sequence variants identified within families from the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab), using a multifactorial likelihood modelling approach. In addition, mRNA splicing analysis by reverse transcriptase PCR and sequencing is being conducted for the subset of variant carriers with available lymphoblastoid cell lines (LCLs), using a large panel of controls to establish the reference RNA splicing pattern. Functional analyses have also been conducted for a subset of the variants that encode missense substitutions.  In vitro  mRNA analysis identified aberrant splicing patterns for 8/12 variants under current study:  BRCA1  IVS 19-12 G>A and 14+2 ins8 cause out of frame insertions.  BRCA1  IVS 23+5 G>C,  BRCA2  IVS 6+1 G>T,  BRCA2  IVS 25+3,  BRCA2  IVS 4-12_IVS 4-8 del5 and  BRCA2  8396 A>G  (D2723G) create whole or part exon deletions.  BRCA2  8216 A>T (E2663V) causes upregulation of truncating splice isoforms. However, sequencing and realtime expression analysis indicates that the wildtype isoform expressed in carriers of  BRCA2  8396 A>G  ( D2723G) and 8216 A>T (E2663V) would encode both wildtype and missense protein. Assays of protein function indicate that both of these missense proteins have abrogated function consistent with pathogenicity. These results have implications for the application of prior probabilities in multifactorial likelihood analysis. Importantly, the results reinforce the utility of splicing assays as a method for identifying clinically significant variants in high-risk cancer genes, including variants outside the conserved donor and acceptor dinucleotides which are not routinely tested in clinical laboratories.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-252.