Abstract 1112: Splicing regulation and BRCA1/2-associated breast cancer risk

Background and Hypotheses: Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are associated with high but variable lifetime risks of breast cancer. The true contribution of BRCA1/2-associated mutations to inherited breast cancer may therefore be underestimated. Several potential categories of deleterious mutations seen in other disease-associated genes might be missed in BRCA1/2 genes using conventional screening methods. In this study we explore whether previously undetected BRCA1 or BRCA2 null mutations may be identified by analysis of allele-specific mRNA accumulation. We also investigate the possibilities that allele- and tissue-specific splice variants of BRCA2 may contribute to its multiple functions and complex mutant phenotypes. Methods: Screening for BRCA1/2 mRNA-null phenotypes was performed in 324 patients from the University of Chicago Cancer Risk Clinic after obtaining IRB-approved consent. Genomic DNA and mRNA were prepared from lymphoblastoid cells, and tested for the presence of heterozygous exonic SNPs. We developed a panel of 20 exonic BRCA1/2 SNPs, at least one of which is heterozygous in 94% of samples tested. Genomic DNA and RT-PCR products were sequenced in the region surrounding the heterozygous SNPs to determine if both alleles were present in the mRNA pool. Splice variants of BRCA2 were identified using full coverage exon-flanking RT-PCR primers. Products were resolved by gel electrophoresis, excised and sequenced. Analysis was performed in breast cell lines and lymphoblastoid cell lines from unaffected individuals or BRCA2 mutation-carriers. Cells were alternately grown in the presence of puromycin or cyclohexamide at empirically determined IC50 levels. Results and conclusions: 1) Analysis of allele-specific mRNA accumulation is a robust method for detecting phenotypic molecular null alleles without prior knowledge of mutation location or even gene function. This method can be applied to any gene. 2) Previously unexamined potential categories of BRCA1/2 mutations have been assayed collectively and are unlikely to contribute to breast cancer risk. Of the 62 samples we have analyzed thus far (average BRCAPRO score 11.3), no allele-specific mRNA-null phenotypes were detected. 3) Three splice variants were detected in BRCA2 (delta exon 3, delta exon 6+7+2 bases, and delta exon 12). Splicing patterns were identical in the normal and cancer cell lines, as well as the lymphoblastoid cell lines from unaffected individuals and BRCA2 mutation carriers. Thus, we observed no tissue- or disease-specific splicing patterns, nor is there evidence of nonsense-associated altered splicing. 4) Finally, the splicing patterns were identical in the presence of protein synthesis inhibitors, suggesting BRCA2 splice patterns are not maintained by aggressive NMD of mis-spliced mRNA, but rather high levels of splicing fidelity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1112.