Abstract 3804: Rare DNA variants are enriched at the BARD1 locus and likely influence neuroblastoma susceptibility.
Authors: Wood Andrew C. , Pugh Trevor J. , Morozova Olena , Koster Jan , Molenaar Jan J. , Pineros Vanessa , Bosse Kristopher R. , Perin Juan C. , Diskin Sharon , Diamond Maura A. , Marra Marco , Meyerson Matthew , Versteeg Rogier , Maris John M. 
Published: August 2014

Abstract
BACKGROUND: A genome-wide association study (GWAS) in neuroblastoma identified 8 regions where common polymorphisms are associated with neuroblastoma susceptibility. However, the biological mechanisms and causal SNPs are largely unknown. Rare variants, potentially in linkage with common polymorphisms but with a higher effect size, may contribute to cancer susceptibility at GWAS-defined loci and provide biological insight into mechanism.
METHODS: To identify rare variants at neuroblastoma GWAS loci we performed ultradeep targeted resequencing of BARD1 and LMO1 on peripheral blood DNA using RainDance microdroplet RDT1000 PCR enrichment, and HiSeq2000 single end 100 bp reads. We aggregated existing whole-genome and exome sequence data from constitutional DNA of neuroblastoma cases (Molenaar et al, Nature 2012; NCI TARGET) on all replicated neuroblastoma GWAS regions.
RESULTS: Exon sequence data were available for 517 cases for BARD1, and 330 cases for LMO1, LINC00340, LIN28B-HACE1, LMO1, DUSP12, DDX4-IL3RA, and HSB17B12 (all genes with replicated associations to NB). The highest frequency of rare potentially damaging variants occurred in BARD1 with 7/517 cases (1.4%) containing novel (n=4) or rare (n=3) variants as defined by ESP6500, 1000 genomes project, and dbSNP135. Rare variants at BARD1 were associated with homozygosity or heterozygosity for the protective alleles. Using SIFT, PolyPhen-2 or MutationTaster, 6/7 variants were predicted to be deleterious, including a nonsense mutation in the domain mediating BARD1-BRCA1 heterodimerization and a nonsense mutation in a BRCT domain implicated in homology directed repair. Sanger sequencing of tumor DNA and RNA confirmed both nonsense mutations were present in their respective primary tumors. HEK293 cells transfected with BARD1 nonsense variants showed decreased stabilization of BRCA1.
CONCLUSION: Novel and potentially damaging mutations in BARD1 exons are present in the constitutional DNA of ∼1% of patients with neuroblastoma and may contribute to disease susceptibility. However, BARD1 rare variants were not tagged by the common risk alleles providing an alternative mechanism to prime neuroblasts towards transformation. Further functional work, expansion of the targeted resequencing discovery cohort as a prelude to a larger case-control comparison, and incorporation of ENCODE data into analysis of non-coding SNPs may provide critical insights into neuroblastoma pathogenesis.
Citation Format: Andrew C. Wood, Trevor J. Pugh, Olena Morozova, Jan Koster, Jan J. Molenaar, Vanessa Pineros, Kristopher R. Bosse, Juan C. Perin, Sharon Diskin, Maura A. Diamond, Marco Marra, Matthew Meyerson, Rogier Versteeg, John M. Maris. Rare DNA variants are enriched at the BARD1 locus and likely influence neuroblastoma susceptibility. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2013-3804