BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma

Abstract

ImportanceHigh-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 the BARD1 gene 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 of BARD1 germline variation in children with neuroblastoma. DesignWe correlated BARD1 genotype with BARD1 expression in normal and tumor cells and the cellular burden of DNA damage in tumors. To validate the functional consequences of rare germline P-LP BARD1 variants, we generated isogenic cellular models harboring heterozygous BARD1 loss-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 MeasuresBARD1 expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associated BARD1 germline variants. ResultsBoth common and rare neuroblastoma associated BARD1 germline variants were significantly associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using neuroblastoma cellular models engineered to harbor disease-associated heterozygous BARD1 LOF variants, we functionally validated this association with inefficient DNA repair. These BARD1 LOF 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 germline BARD1 variants 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 PointsO_ST_ABSQuestionC_ST_ABSHow do neuroblastoma patient BRCA1-associated RING domain 1 (BARD1) germline variants impact DNA repair? FindingsNeuroblastoma-associated germline BARD1 variants disrupt DNA repair fidelity. Common risk variants correlate with decreased BARD1 expression and increased DNA double-strand breaks in neuroblastoma tumors and rare heterozygous loss-of-function variants induce BARD1 haploinsufficiency, resulting in defective DNA repair and genomic instability in neuroblastoma cellular models. MeaningGermline variation in BARD1 contributes to neuroblastoma pathogenesis via dysregulation of critical cellular DNA repair functions, with implications for neuroblastoma treatment, risk stratification, and cancer predisposition.