BARD1germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma

Abstract

AbstractImportanceHigh-risk neuroblastoma is a complex genetic disease that is lethal in 50% of patients despite intense multimodal therapy. Our genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) within theBARD1gene showing the most significant enrichment in neuroblastoma patients, and also discovered pathogenic (P) or likely pathogenic (LP) rare germline loss-of-function variants in this gene. The functional implications of these findings remain poorly understood.ObjectiveTo define the functional relevance ofBARD1germline variation in children with neuroblastoma.DesignWe correlatedBARD1genotype withBARD1expression in normal and tumor cells and the cellular burden of DNA damage in tumors. To validate the functional consequences of rare germline P-LPBARD1variants, we generated isogenic cellular models harboring heterozygousBARD1loss-of-function (LOF) variants and conducted multiple complementary assays to measure the efficiency of DNA repair.Setting(N/A)Participants(N/A)Interventions/Exposures(N/A)Main Outcomes and MeasuresBARD1expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associatedBARD1germline variants.ResultsBoth common and rare neuroblastoma associatedBARD1germline variants were significantly associated with lower levels ofBARD1mRNA and an increased burden of DNA damage. Using neuroblastoma cellular models engineered to harbor disease-associated heterozygousBARD1LOF variants, we functionally validated this association with inefficient DNA repair. TheseBARD1LOF variant isogenic models exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA doublestrand break sites, and enhanced sensitivity to cisplatin and poly-ADP ribose polymerase (PARP) inhibition.Conclusions and RelevanceConsidering that at least 1 in 10 children diagnosed with cancer carry a predicted pathogenic mutation in a cancer predisposition gene, it is critically important to understand their functional relevance. Here, we demonstrate that germlineBARD1variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications, and these findings may also extend to other cancers harboring germline variants in genes essential for DNA damage repair.Key PointsQuestionHow do neuroblastoma patient BRCA1-associated RING domain 1 (BARD1) germline variants impact DNA repair?FindingsNeuroblastoma-associated germlineBARD1variants disrupt DNA repair fidelity. Common risk variants correlate with decreasedBARD1expression and increased DNA double-strand breaks in neuroblastoma tumors and rare heterozygous loss-of-function variants induceBARD1haploinsufficiency, resulting in defective DNA repair and genomic instability in neuroblastoma cellular models.MeaningGermline variation inBARD1contributes to neuroblastoma pathogenesis via dysregulation of critical cellular DNA repair functions, with implications for neuroblastoma treatment, risk stratification, and cancer predisposition.