AutoGVP: a dockerized workflow integrating ClinVar and InterVar germline sequence variant classification.

SUMMARY: With the increasing rates of exome and whole genome sequencing, the ability to classify large sets of germline sequencing variants using up-to-date American College of Medical Genetics-Association for Molecular Pathology (ACMG-AMP) criteria is crucial. Here, we present Automated Germline Variant Pathogenicity (AutoGVP), a tool that integrates germline variant pathogenicity annotations from ClinVar and sequence variant classifications from a modified version of InterVar (PVS1 strength adjustments, removal of PP5/BP6). This tool facilitates large-scale, clinically focused classification of germline sequence variants in a research setting. AVAILABILITY AND IMPLEMENTATION: AutoGVP is an open source dockerized workflow implemented in R and freely available on GitHub at https://github.com/diskin-lab-chop/AutoGVP.

Somatic structural variation targets neurodevelopmental genes and identifies SHANK2 as a tumor suppressor in neuroblastoma.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Like many childhood cancers, neuroblastoma shows a relative paucity of somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) compared to adult cancers. Here, we assessed the contribution of somatic structural variation (SV) in neuroblastoma using a combination of whole-genome sequencing (WGS) of tumor-normal pairs (n = 135) and single-nucleotide polymorphism (SNP) genotyping of primary tumors (n = 914). Our study design allowed for orthogonal validation and replication across platforms. SV frequency, type, and localization varied significantly among high-risk tumors. MYCN nonamplified high-risk tumors harbored an increased SV burden overall, including a significant excess of tandem duplication events across the genome. Genes disrupted by SV breakpoints were enriched in neuronal lineages and associated with phenotypes such as autism spectrum disorder (ASD). The postsynaptic adapter protein-coding gene, SHANK2, located on Chromosome 11q13, was disrupted by SVs in 14% of MYCN nonamplified high-risk tumors based on WGS and 10% in the SNP array cohort. Expression of SHANK2 was low across human-derived neuroblastoma cell lines and high-risk neuroblastoma tumors. Forced expression of SHANK2 in neuroblastoma cells resulted in significant growth inhibition (P = 2.6 × 10-2 to 3.4 × 10-5) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P = 3.1 × 10-13 to 2.4 × 10-30). These data further define the complex landscape of somatic structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis in this childhood cancer.

Germline 16p11.2 Microdeletion Predisposes to Neuroblastoma.

Neuroblastoma is a cancer of the developing sympathetic nervous system. It is diagnosed in 600-700 children per year in the United States and accounts for 12% of pediatric cancer deaths. Despite recent advances in our understanding of this malignancy's complex genetic architecture, the contribution of rare germline variants remains undefined. Here, we conducted a genome-wide analysis of large (>500 kb), rare (<1%) germline copy number variants (CNVs) in two independent, multi-ethnic cohorts totaling 5,585 children with neuroblastoma and 23,505 cancer-free control children. We identified a 550-kb deletion on chromosome 16p11.2 significantly enriched in neuroblastoma cases (0.39% of cases and 0.03% of controls; p = 3.34 × 10-9). Notably, this CNV corresponds to a known microdeletion syndrome that affects approximately one in 3,000 children and confers risk for diverse developmental phenotypes including autism spectrum disorder and other neurodevelopmental disorders. The CNV had a substantial impact on neuroblastoma risk, with an odds ratio of 13.9 (95% confidence interval = 5.8-33.4). The association remained significant when we restricted our analysis to individuals of European ancestry in order to mitigate potential confounding by population stratification (0.42% of cases and 0.03% of controls; p = 4.10 × 10-8). We used whole-genome sequencing (WGS) to validate the deletion in paired germline and tumor DNA from 12 cases. Finally, WGS of four parent-child trios revealed that the deletion primarily arose de novo without maternal or paternal bias. This finding expands the clinical phenotypes associated with 16p11.2 microdeletion syndrome to include cancer, and it suggests that disruption of the 16p11.2 region may dysregulate neurodevelopmental pathways that influence both neurological phenotypes and neuroblastoma.

Neural crest-derived tumor neuroblastoma and melanoma share 1p13.2 as susceptibility locus that shows a long-range interaction with the SLC16A1 gene.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10-8). We also detected a suggestive (P < 10-7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10-4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.

Common variants upstream of MLF1 at 3q25 and within CPZ at 4p16 associated with neuroblastoma.

Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly presents in young children and accounts for approximately 12% of pediatric oncology deaths. Here, we report on a genome-wide association study (GWAS) in a discovery cohort or 2,101 cases and 4,202 controls of European ancestry. We identify two new association signals at 3q25 and 4p16 that replicated robustly in multiple independent cohorts comprising 1,163 cases and 4,396 controls (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16-1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21-1.40). The 4p16 signal maps within the carboxypeptidase Z (CPZ) gene. The 3q25 signal resides within the arginine/serine-rich coiled-coil 1 (RSRC1) gene and upstream of the myeloid leukemia factor 1 (MLF1) gene. Increased expression of MLF1 was observed in neuroblastoma cells homozygous for the rs6441201 risk allele (P = 0.02), and significant growth inhibition was observed upon depletion of MLF1 (P < 0.0001) in neuroblastoma cells. Taken together, we show that common DNA variants within CPZ at 4p16 and upstream of MLF1 at 3q25 influence neuroblastoma susceptibility and MLF1 likely plays an important role in neuroblastoma tumorigenesis.

Fine mapping of 2q35 high-risk neuroblastoma locus reveals independent functional risk variants and suggests full-length BARD1 as tumor-suppressor.

A previous genome-wide association study (GWAS) identified common variation at the BARD1 locus as being highly associated with susceptibility to high-risk neuroblastoma, but the mechanisms underlying this association have been not extensively investigated. Here, we performed a fine mapping analysis of BARD1 locus (2q35) using GWAS data from 556 high-risk neuroblastoma patients and 2,575 controls of European-American ancestry, and identified two independent genome-wide neuroblastoma-associated loci. Functional single-nucleotide polymorphism (SNP) prioritization identified two causative variants that independently contributed to neuroblastoma risk, and each replicated robustly in multiple independent cohorts comprising 445 high-risk cases and 3,170 controls (rs17489363: combined p = 1.07 × 10-31 , OR:1.79, 95% CI:1.62-1.98 and rs1048108: combined p = 7.27 × 10-14 , OR:0.65, 95% CI:0.58-0.73). Particularly, the T risk allele of rs17489363 in the canonical promoter region of full-length BARD1 altered binding site of the transcription factor HSF1 and correlated with low expression of full-length BARD1 mRNA and protein. Low-level expression of full-length BARD1 associated with advanced neuroblastoma. In human neuroblastoma cells, attenuating full-length BARD1 increased proliferation and invasion capacity. In conclusion, we have identified two potentially causative SNPs at the BARD1 locus associated with predisposition to high-risk neuroblastoma, and have shown that full-length BARD1 may act as tumor suppressor.

A family-based study of gene variants and maternal folate and choline in neuroblastoma: a report from the Children's Oncology Group.

PURPOSE: Neuroblastoma is a childhood cancer of the sympathetic nervous system with embryonic origins. Previous epidemiologic studies suggest maternal vitamin supplementation during pregnancy reduces the risk of neuroblastoma. We hypothesized offspring and maternal genetic variants in folate-related and choline-related genes are associated with neuroblastoma and modify the effects of maternal intake of folate, choline, and folic acid. METHODS: The Neuroblastoma Epidemiology in North America (NENA) study recruited 563 affected children and their parents through the Children's Oncology Group's Childhood Cancer Research Network. We used questionnaires to ascertain pre-pregnancy supplementation and estimate usual maternal dietary intake of folate, choline, and folic acid. We genotyped 955 genetic variants related to folate or choline using DNA extracted from saliva samples and used a log-linear model to estimate both child and maternal risk ratios and stratum-specific risk ratios for gene-environment interactions. RESULTS: Overall, no maternal or offspring genotypic results met criteria for a false discovery rate (FDR) Q-value <0.2. Associations were also null for gene-environment interaction with pre-pregnancy vitamin supplementation, dietary folic acid, and folate. FDR-significant gene-choline interactions were found for offspring SNPs rs10489810 and rs9966612 located in MTHFD1L and TYMS, respectively, with maternal choline dietary intake dichotomized at the first quartile. CONCLUSION: These results suggest that variants related to one-carbon metabolism are not strongly associated with neuroblastoma. Choline-related variants may play a role; however, the functional consequences of the interacting variants are unknown and require independent replication.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

BACKGROUND: High-risk neuroblastoma is a complex genetic disease that is lethal in more than 50% of patients despite intense multimodal therapy. Through genome-wide association studies (GWAS) and next-generation sequencing, we have identified common single nucleotide polymorphisms and rare, pathogenic or likely pathogenic germline loss-of-function variants in BARD1 enriched in neuroblastoma patients. The functional implications of these findings remain poorly understood. METHODS: We correlated BARD1 genotype with expression in normal tissues and neuroblastomas, along with the burden of DNA damage in tumors. To validate the functional consequences of germline pathogenic or likely pathogenic BARD1 variants, we used CRISPR-Cas9 to generate isogenic neuroblastoma (IMR-5) and control (RPE1) cellular models harboring heterozygous BARD1 loss-of-function variants (R112*, R150*, E287fs, and Q564*) and quantified genomic instability in these cells via next-generation sequencing and with functional assays measuring the efficiency of DNA repair. RESULTS: Both common and rare neuroblastoma-associated BARD1 germline variants were associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using isogenic heterozygous BARD1 loss-of-function variant cellular models, we functionally validated this association with inefficient DNA repair. BARD1 loss-of-function variant isogenic cells exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA double-strand break sites, and enhanced sensitivity to cisplatin and poly (ADP-ribose) polymerase (PARP) inhibition both in vitro and in vivo. CONCLUSIONS: Taken together, 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.

Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation.

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

Germline pathogenic variants in neuroblastoma patients are enriched in BARD1 and predict worse survival.

BACKGROUND: Neuroblastoma is an embryonal cancer of the developing sympathetic nervous system. The genetic contribution of rare pathogenic or likely pathogenic germline variants in patients without a family history remains unclear. METHODS: Germline DNA sequencing was performed on 786 neuroblastoma patients. The frequency of rare cancer predisposition gene pathogenic or likely pathogenic variants in patients was compared with 2 cancer-free control cohorts. Matched tumor DNA sequencing was evaluated for second hits, and germline DNA array data from 5585 neuroblastoma patients and 23 505 cancer-free control children were analyzed to identify rare germline copy number variants. Patients with germline pathogenic or likely pathogenic variants were compared with those without to test for association with clinical characteristics, tumor features, and survival. RESULTS: We observed 116 pathogenic or likely pathogenic variants involving 13.9% (109 of 786) of neuroblastoma patients, representing a statistically significant excess burden compared with cancer-free participants (odds ratio [OR] = 1.60, 95% confidence interval [CI] = 1.27 to 2.00). BARD1 harbored the most statistically significant enrichment of pathogenic or likely pathogenic variants (OR = 32.30, 95% CI = 6.44 to 310.35). Rare germline copy number variants disrupting BARD1 were identified in patients but absent in cancer-free participants (OR = 29.47, 95% CI = 1.52 to 570.70). Patients harboring a germline pathogenic or likely pathogenic variant had a worse overall survival compared with those without (P = 8.6 x 10-3). CONCLUSIONS: BARD1 is an important neuroblastoma predisposition gene harboring both common and rare germline pathogenic or likely pathogenic variations. The presence of any germline pathogenic or likely pathogenic variant in a cancer predisposition gene was independently predictive of worse overall survival. As centers move toward paired tumor-normal sequencing at diagnosis, efforts should be made to centralize data and provide an infrastructure to support cooperative longitudinal prospective studies of germline pathogenic variation.

The mechanistic basis of Myxococcus xanthus rippling behavior and its physiological role during predation.

Myxococcus xanthus cells self-organize into periodic bands of traveling waves, termed ripples, during multicellular fruiting body development and predation on other bacteria. To investigate the mechanistic basis of rippling behavior and its physiological role during predation by this Gram-negative soil bacterium, we have used an approach that combines mathematical modeling with experimental observations. Specifically, we developed an agent-based model (ABM) to simulate rippling behavior that employs a new signaling mechanism to trigger cellular reversals. The ABM has demonstrated that three ingredients are sufficient to generate rippling behavior: (i) side-to-side signaling between two cells that causes one of the cells to reverse, (ii) a minimal refractory time period after each reversal during which cells cannot reverse again, and (iii) physical interactions that cause the cells to locally align. To explain why rippling behavior appears as a consequence of the presence of prey, we postulate that prey-associated macromolecules indirectly induce ripples by stimulating side-to-side contact-mediated signaling. In parallel to the simulations, M. xanthus predatory rippling behavior was experimentally observed and analyzed using time-lapse microscopy. A formalized relationship between the wavelength, reversal time, and cell velocity has been predicted by the simulations and confirmed by the experimental data. Furthermore, the results suggest that the physiological role of rippling behavior during M. xanthus predation is to increase the rate of spreading over prey cells due to increased side-to-side contact-mediated signaling and to allow predatory cells to remain on the prey longer as a result of more periodic cell motility.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

Importance: High-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. Objective: To define the functional relevance of BARD1 germline variation in children with neuroblastoma. Design: We 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 Measures: BARD1 expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associated BARD1 germline variants. Results: Both 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 Relevance: Considering 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 Points: Question: How do neuroblastoma patient BRCA1-associated RING domain 1 ( BARD1 ) germline variants impact DNA repair? Findings: Neuroblastoma-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. Meaning: Germline 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.

AutoGVP: a dockerized workflow integrating ClinVar and InterVar germline sequence variant classification.

With the increasing rates of exome and whole genome sequencing, the ability to classify large sets of germline sequencing variants using up-to-date American College of Medical Genetics - Association for Molecular Pathology (ACMG-AMP) criteria is crucial. Here, we present Automated Germline Variant Pathogenicity (AutoGVP), a tool that integrates germline variant pathogenicity annotations from ClinVar and sequence variant classifications from a modified version of InterVar (PVS1 strength adjustments, removal of PP5/BP6). This tool facilitates large-scale, clinically-focused classification of germline sequence variants in a research setting.

Alternative lengthening of telomeres (ALT) in pediatric high-grade gliomas can occur without ATRX mutation and is enriched in patients with pathogenic germline mismatch repair (MMR) variants.

BACKGROUND: To achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. METHODS: We performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n = 40/579) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on pediatric high-grade gliomas (pHGGs) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. RESULTS: ALT is common in pHGGs (n = 24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (<3%). Somatic ATRX mutations occur in 50% of ALT+ pHGGs and in 30% of ALT- pHGGs. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. CONCLUSIONS: We demonstrate that ATRX is mutated in only a subset of ALT+ pHGGs, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with the development of ALT in patients with pHGG.

Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma.

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop opsoclonus myoclonus ataxia syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor-infiltrating T and B cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non-OMAS-associated neuroblastomas. We found greater B and T cell infiltration in OMAS-associated tumors compared to controls and showed that both were polyclonal expansions. Tertiary lymphoid structures (TLSs) were enriched in OMAS-associated tumors. We identified significant enrichment of the major histocompatibility complex (MHC) class II allele HLA-DOB∗01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal auto-reactive B lymphocytes act as antigen-presenting cells and drive TLS formation, thereby supporting both sustained polyclonal T cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

Germline pathogenic variants in 786 neuroblastoma patients.

Importance: Neuroblastoma accounts for 12% of childhood cancer deaths. The genetic contribution of rare pathogenic germline variation in patients without a family history remains unclear. Objective: To define the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in neuroblastoma patients. Design Setting and Participants: Germline DNA sequencing was performed on the peripheral blood from 786 neuroblastoma patients unselected for family history. Rare variants mapping to CPGs were evaluated for pathogenicity and the percentage of cases harboring pathogenic (P) or likely pathogenic (LP) variants was quantified. The frequency of CPG P-LP variants in neuroblastoma cases was compared to two distinct cancer-free control cohorts to assess enrichment. Matched tumor DNA sequencing was evaluated for "second hits" at CPGs and germline DNA array data from 5,585 neuroblastoma cases and 23,505 cancer-free control children was analyzed to identify rare germline copy number variants (CNVs) affecting genes with an excess burden of P-LP variants in neuroblastoma. Neuroblastoma patients with germline P-LP variants were compared to those without P-LP variants to test for association with clinical characteristics, tumor features, and patient survival. Main Outcomes and Measures: Rare variant prevalence, pathogenicity, enrichment, and association with clinical characteristics, tumor features, and patient survival. Results: We observed 116 P-LP variants in CPGs involving 13.9% (109/786) of patients, representing a significant excess burden of P-LP variants compared to controls (9.1%; P = 5.14 × 10-5, Odds Ratio: 1.60, 95% confidence interval: 1.27-2.00). BARD1 harbored the most significant burden of P-LP variants compared to controls (1.0% vs. 0.03%; P = 8.18 × 10-7; Odds Ratio: 32.30, 95% confidence interval: 6.44-310.35). Rare germline CNVs disrupting BARD1 were also identified in neuroblastoma patients (0.05%) but absent in controls (P = 7.08 × 10-3; Odds Ratio: 29.47, 95% confidence interval: 1.52 - 570.70). Overall, P-LP variants in DNA repair genes in this study were enriched in cases compared to controls (8.1% vs. 5.7%; P = 0.01; Odds Ratio: 1.45, 95% confidence interval: 1.08-1.92). Neuroblastoma patients harboring a germline P-LP variant had a worse overall survival when compared to patients without P-LP variants (P = 8.6 × 10-3), and this remained significant in a multivariate Cox proportional-hazards model (P = 0.01). Conclusions and Relevance: Neuroblastoma patients harboring germline P-LP variants in CPGs have worse overall survival and BARD1 is an important predisposition gene affected by both common and rare pathogenic variation. Germline sequencing should be performed for all neuroblastoma patients at diagnosis to inform genetic counseling and support future longitudinal and mechanistic studies. Patients with a germline P-LP variant should be closely monitored, regardless of risk group assignment.

OpenPBTA: The Open Pediatric Brain Tumor Atlas.

Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children's Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas and TP53 loss as a significant marker for poor overall survival in ependymomas and H3 K28-mutant diffuse midline gliomas. Already being actively applied to other pediatric cancers and PNOC molecular tumor board decision-making, OpenPBTA is an invaluable resource to the pediatric oncology community.

Genetic Analysis in African American Children Supports Ancestry-Specific Neuroblastoma Susceptibility.

BACKGROUND: Neuroblastoma is rarer in African American (AA) children compared with American children of European descent. AA children affected with neuroblastoma, however, more frequently develop the high-risk form of the disease. METHODS: We have genotyped an AA cohort of 629 neuroblastoma cases (254 high-risk) and 2,990 controls to investigate genetic susceptibility to neuroblastoma in AAs. RESULTS: We confirmed the known neuroblastoma susceptibility gene BARD1 at genome-wide significance in the subset of high-risk cases. We also estimated local admixture across the autosomal genome in the AA cases and controls and detected a signal at 4q31.22 where cases show an increase in European ancestry. A region at 17p13.1 showed increased African ancestry in the subgroup of high-risk cases with respect to intermediate- and low-risk cases. Using results from our published European American (EA) genome-wide association study (GWAS), we found that a polygenic score that included all independent SNPs showed a highly significant association (P value = 1.8 × 10-73) and explained 19% of disease risk variance in an independent EA cohort. In contrast, the best fit polygenic score (P value = 3.2 × 10-11) in AAs included only 22 independent SNPs with association P value < 2.75 × 10-6 in the EA GWAS, and explained 2% of neuroblastoma risk variance. The significance of the polygenic score dropped rapidly with inclusion of additional SNPs. CONCLUSIONS: These findings suggest that several common variants contribute to risk of neuroblastoma in an ancestry-specific fashion. IMPACT: This work supports the need for GWAS to be performed in populations of all races and ethnicities.

A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma.

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Joint modeling of performance and subjective reporting to assess sensitivity to drug-induced sleepiness.

In an NASA ground study, two forms of cognitive tests were evaluated in terms of their sensitivity to sleepiness induced by the drug promethazine (PMZ). Performance for the two test modes (Y(1) and Y(2)), PMZ concentration, and a self-reported sleepiness using the Karolinska Sleepiness Scale (KSS) were monitored for 12 h post dose. A problem arises when using KSS to establish an association between true sleepiness and performance because KSS scores are discrete and also because they tend to concentrate on certain values. Therefore, we define a latent sleepiness measure X as an unobserved continuous random variable describing a subject's actual state of sleepiness. Under the assumption that drug concentration affects X, which then affects Y(1), Y(2), and KSS, we use Bayesian methods to estimate joint equations that permit unbiased comparison of the performance measures' sensitivity to X. The equations incorporate subject random effects and include a negativity constraint on subject-specific slopes of performance with respect to sleepiness.