Abstract 3867: Understanding the neuroblastoma predisposition signal at chromosome 2q35: Identification of the β-BARD1 isoform as an oncogenic driver
Authors: Kristopher R. Bosse, Sharon J. Diskin, Kristina Cole, Patrick Mayes, Cynthia Winter, Maura Diamond, Marci Laudenslager, Edward F. Attiyeh, Geoffrey Norris, Yael P. Mosse, Mario Capasso, Marcella Devoto, Eric R. Rappaport, Irmgard Irminger, John M. Maris
Journal: Cancer Research (2010)

Abstract Genome-wide association studies have proven to be a powerful tool to identify susceptibility loci in cancer, however the molecular basis of a majority of these association signals remain undefined. We have previously reported nine common single nucleotide polymorphism (SNP) alleles at chromosome 2q35, within and 5′ upstream of the BRCA1-associated RING domain (BARD1) gene, to be significantly associated with high-risk neuroblastoma (Capasso, M et al., Nature Genetics 2009). Here, we focus on understanding the biological underpinnings of this genetic association by investigating how common variation at this locus correlates with changes in BARD1 expression in both the germline and in neuroblastoma samples, and further how these BARD1 alterations are involved in driving neuroblastoma tumorigenesis. We first showed that the neuroblastoma associated SNP variation at 2q35 is in linkage disequilibrium (LD) with multiple genomic areas dense with regulatory domains, including many putative binding sites for the neuronal splicing regulator, FOX2. We next demonstrated that in peripheral blood mononuclear cells, these SNPs correlate both with a trend towards increased overall BARD1 expression and with differential BARD1 exon expression. Specifically, a majority of the neuroblastoma associated SNPs were significantly associated with decreased expression of the RING domain forming BARD1 exons 2 and 3 (P = 0.0008-0.034). We have identified 16 unique BARD1 isoforms in the neuroblastoma tissue of origin, sympathetic fetal ganglia, with the β-BARD1 isoform specifically splicing out exons 2 and 3. β-BARD1 was found to be ubiquitously expressed in a panel of fetal sympathetic ganglia, neuroblastoma cell lines, and high-risk primary tumors, with its expression being significantly increased in cell lines harboring a homozygous BARD1 risk allele genotype at the most significantly associated 2q35 SNP, rs6435862 (N = 22; P = 0.04). Transient transfection of a specific siRNA targeting the alternatively spliced boundary of β-BARD1 in a panel of neuroblastoma cell lines (N = 12) showed that neuroblastoma cells harboring at least one risk allele at SNP rs6435862 and the highest β-BARD1 expression showed a significant inhibition of cell proliferation upon β-BARD1 knockdown (% growth inhibition = 45-81%, P = 0.0004-0.034), while six of seven cell lines with a homozygous major allele genotype at rs6435862 and lower β-BARD1 expression did not show significant growth inhibition. These data are consistent with β-BARD1 acting as an oncogene in neuroblastoma and we hypothesize that common genomic variation drives formation of this isoform, underlying neuroblastoma predisposition on 2q35. Ongoing work will focus on further defining the role of common variation in BARD1's RNA splicing patterns and identifying the molecular basis of β-BARD1's oncogenicity in neuroblastoma. 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 3867.