Abstract 4020: Characterization of novel BARD1 splice variants in colon cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BARD1 is a BRCA1 binding partner with putative tumor suppressive properties. Aberrant splice variants of BARD1 have been detected in various cancers, and it has been postulated that the presence of splice variants is cancer-specific. BARD1 expression patterns and their correlation with clinical outcome have not been assessed in colon cancer. In this study, we determined the occurrence and role of BARD1 splice variants in colon cancer. We characterized novel BARD1 splice variants and quantified the mRNA expression levels of BARD1 splice variants and full-length BARD1 in primary colon cancers and their corresponding normal tissue. Further, full-length BARD1 protein expression was correlated with clinical outcome in primary colon cancer samples. Methods: We performed PCR on cDNA from 15 colon cancer samples and matched normal colon tissues using primer targets in the first and last exon of BARD1 mRNA. The PCR produced several bands, that were submitted for direct sequencing. In a second approach the bands were subcloned and subsequently sequenced. The sequences were analyzed and splice variants were characterized. Splice variant specific qPCR primers were designed and expression levels were quantified using optimized SYBR Green qPCR assays. Control assays were performed to assure good RNA/cDNA quality and optimal reaction conditions. In addition, mRNA expression analysis was performed on a cDNA array consisting of pooled cDNA from 16 different human tissues (prostate, heart, brain, testis, placenta, leukocytes, thymus, lung, colon, pancreas, skeletal muscle, liver, kidney, spleen, small intestine, ovary). Full-length BARD1 protein expression was assessed via immunohistochemistry in a separate cohort of 82 primary colon cancer samples. The stained slides were scanned and visualized using the Aperio ImageScope system. Expression levels were assessed and correlated with clinical outcome data. Results: In addition to the full-length BARD1 mRNA, we now find 19 distinct BARD1 splice variants in colon cancer, the majority of which have not been previously described. 18 of the 19 splice variants lack one or more complete exons, thus creating new and specific exon-exon boundaries. Contrary to previous assumptions, these splice variants can also be found in the adjacent normal colon tissue as well as in various other normal human tissues. While BARD1 splice variants account for the majority of BARD1 mRNA, distinct variants show a cancer-specific regulation pattern. Further, loss of full-length BARD1 protein is associated with a poor prognosis (p=0.02). Conclusion: BARD1 splice variants commonly occur in colon cancer and normal colon tissue, with distinct variants showing a cancer-specific expression pattern. Loss of the full-length BARD1 protein correlates with a poor prognosis in colon cancer, consistent with a tumor suppressive function. Taken together, we suggest that splice variant regulation may affect BARD1 function. 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 4020. doi:10.1158/1538-7445.AM2011-4020