Abstract BRCA2 is one of the two principal tumor suppressor genes responsible for Hereditary Breast and Ovarian Cancer (HBOC) Syndrome. Most deleterious mutations in BRCA2 are nonsense, frame-shifting insertions/deletions, or splicing mutations resulting in premature mRNA stop codons. However, numerous sequence variants of uncertain significance (VUS) are routinely seen during clinical testing. Some of these could potentially affect splicing fidelity by altering splice site choices or affecting the ratios of normally occurring splice variants, which have yet to be defined for BRCA2. To characterize the normally occurring splice variants and possible alternate protein products of BRCA2, we have performed a complete cDNA exon scan of BRCA2 cDNA in MCF7 (a breast cancer cell line), 184A1 (a non-cancerous breast cell line), and two lymphoblastoid cell lines from unaffected wild type. In all cell lines we identified three BRCA2 splice variants: delta exon 3, delta exon 12, and delta exons 6&7+2bp. While the first two variants retain the BRCA2 translational reading frame and could theoretically result in alternate BRCA2 protein products, the third is an out-of-frame variant predicted to result in a premature stop codon. We have further developed a set of splice variant-specific detection assays that revealed all three splice variants were present in 14 breast tumor/adjacent normal tissue pairs. Surprisingly, all three BRCA2 splice variants were comparatively more abundant in the tumor cells than their normal adjacent tissues. To characterize the regulators of BRCA2 splicing in tumor cells, we repeated the exon scan in the presence of cyclohexamide or puromycin to inhibit nonsense-mediated mRNA decay (NMD). We saw no evidence of accumulated mis-spliced BRCA2 mRNA products in the presence of the inhibitors, indicating NMD does not play a major role in regulating the accumulation of normally occurring splice variants. To determine whether nonsense-associated alternate splicing (NAS) promotes skipping of exons containing frame shifting or nonsense mutations, we repeated the BRCA2 exon scan in four lymphoblastoid cell lines containing BRCA2 mutations 5578delAA, 852delC, 6174delT and S1955X. In all cases, the splicing patterns were the same as breast and wild type lymphoblastoid cell line patterns, indicating NAS does not generally promote alternate BRCA2 protein products in response to truncating mutations. We conclude that, in addition to full-length BRCA2 mRNA, there are three additional splice variants that accumulate to comparatively higher levels in tumors than in adjacent normal tissue. This suggests breast tumor tissues have altered splicing regulation, altered regulation of selective mRNA degradation, or possibly both, and could reflect a level of epigenetic misregulation of BRCA2 and possibly other genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2011-2029