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1.  A Functional Screen Identifies miR-34a as a Candidate Neuroblastoma Tumor Suppressor Gene 
Molecular cancer research : MCR  2008;6(5):735-742.
MicroRNAs are small noncoding RNAs that have critical roles in regulating a number of cellular functions through transcriptional silencing. They have been implicated as oncogenes and tumor suppressor genes (oncomirs) in several human neoplasms. We used an integrated genomics and functional screening strategy to identify potential oncomirs in the pediatric neoplasm neuroblastoma. We first identified microRNAs that map within chromosomal regions that we and others have defined as frequently deleted (1p36, 3p22, and 11q23-24) or gained (17q23) in high-risk neuroblastoma. We then transiently transfected microRNA precursor mimics or inhibitors into a panel of six neuroblastoma cell lines that we characterized for these genomic aberrations. The majority of transfections showed no phenotypic effect, but the miR-34a (1p36) and miR-34c (11q23) mimics showed dramatic growth inhibition in cell lines with 1p36 hemizygous deletion. In contrast, there was no growth inhibition by these mimics in cell lines without 1p36 deletions. Quantitative reverse transcription-PCR showed a perfect correlation of absent miR-34a expression in cell lines with a 1p36 aberration and phenotypic effect after mimetic add-back. Expression of miR-34a was also decreased in primary tumors (n = 54) with 1p36 deletion (P = 0.009), but no mutations were discovered in resequencing of the miR-34a locus in 30 neuroblastoma cell lines. Flow cytometric time series analyses showed that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoptosis. BCL2 and MYCN were identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect. These data support miR-34a as a tumor suppressor gene in human neuroblastoma.
doi:10.1158/1541-7786.MCR-07-2102
PMCID: PMC3760152  PMID: 18505919
2.  Common Variation at BARD1 Results in the Expression of an Oncogenic Isoform that Influences Neuroblastoma Susceptibility and Oncogenicity 
Cancer Research  2012;72(8):2068-2078.
The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherent, anchorage-independent, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.
doi:10.1158/0008-5472.CAN-11-3703
PMCID: PMC3328617  PMID: 22350409
genome-wide association; neuroblastoma; BARD1; cancer susceptibility genes; functional genomics; oncogenes; genotype-phenotype correlations
3.  Common variations in BARD1 influence susceptibility to high-risk neuroblastoma 
Nature genetics  2009;41(6):718-723.
We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma1. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma2, we now restricted our analysis to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 gene locus (Pallelic = 2.35×10−9 − 2.25×10−8). Each SNP association was confirmed in a second series of 189 high-risk cases and 1,178 controls (Pallelic = 7.90×10−7 − 2.77×10−4). The two most significant SNPs (rs6435862, rs3768716) were also tested in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds-ratios of 1.68 each (P = 8.65×10−18 and 2.74×10−16, respectively). Significant association was also found with known BARD1 nsSNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.
doi:10.1038/ng.374
PMCID: PMC2753610  PMID: 19412175
4.  Cortical Processing of Respiratory Occlusion Stimuli in Children with Central Hypoventilation Syndrome 
Rationale: The ability of patients with central hypoventilation syndrome (CHS) to produce and process mechanoreceptor signals is unknown.
Objectives: Children with CHS hypoventilate during sleep, although they generally breathe adequately during wakefulness. Previous studies suggest that they have compromised central integration of afferent stimuli, rather than abnormal sensors or receptors. Cortical integration of afferent mechanical stimuli caused by respiratory loading or upper airway occlusion can be tested by measuring respiratory-related evoked potentials (RREPs). We hypothesized that patients with CHS would have blunted RREP during both wakefulness and sleep.
Methods: RREPs were produced with multiple upper airway occlusions and were obtained during wakefulness, stage 2, slow-wave, and REM sleep. Ten patients with CHS and 20 control subjects participated in the study, which took place at the Children's Hospital of Philadelphia. Each patient was age- and sex-matched to two control subjects. Wakefulness data were collected from 9 patients and 18 control subjects.
Measurements and Main Results: During wakefulness, patients demonstrated reduced Nf and P300 responses compared with control subjects. During non-REM sleep, patients demonstrated a reduced N350 response. In REM sleep, patients had a later P2 response.
Conclusions: CHS patients are able to produce cortical responses to mechanical load stimulation during both wakefulness and sleep; however, central integration of the afferent signal is disrupted during wakefulness, and responses during non-REM are damped relative to control subjects. The finding of differences between patients and control subjects during REM may be due to increased intrinsic excitatory inputs to the respiratory system in this state.
doi:10.1164/rccm.200804-606OC
PMCID: PMC2556457  PMID: 18658113
central hypoventilation syndrome; respiratory-related evoked potentials; wakefulness; sleep
5.  Copy number variation at 1q21.1 associated with neuroblastoma 
Nature  2009;459(7249):987-991.
Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in cancer, we performed a genome-wide association study (GWAS) of CNVs in the childhood cancer neuroblastoma, a disease where SNP variations are known to influence susceptibility1,2. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Caucasian controls at 550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. We identified a common CNV at 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets (Pcombined = 2.97 × 10−17; OR = 2.49, 95% CI: 2.02 to 3.05). This CNV was validated by quantitative PCR, fluorescent in situ hybridization, and analysis of matched tumor specimens, and was shown to be heritable in an independent set of 713 cancer-free trios. We identified a novel transcript within the CNV which showed high sequence similarity to several “Neuroblastoma breakpoint family” (NBPF) genes3,4 and represents a new member of this gene family (NBPFX). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a novel NBPF gene in early tumorigenesis of this childhood cancer.
doi:10.1038/nature08035
PMCID: PMC2755253  PMID: 19536264
6.  A genome-wide association study identifies a susceptibility locus to clinically aggressive neuroblastoma at 6p22 
The New England journal of medicine  2008;358(24):2585-2593.
Background
Neuroblastoma is a malignancy of the developing sympathetic nervous system that most commonly affects young children and is often lethal. The etiology of this embryonal cancer is not known.
Methods
We performed a genome-wide association study by first genotyping 1,032 neuroblastoma patients and 2,043 controls of European descent using the Illumina HumanHap550 BeadChip. Three independent groups of neuroblastoma cases (N=720) and controls (N=2128) were then genotyped to replicate significant associations.
Results
We observed highly significant association between neuroblastoma and the common minor alleles of three single nucleotide polymorphisms (SNPs) within a 94.2 kilobase (Kb) linkage disequilibrium block at chromosome band 6p22 containing the predicted genes FLJ22536 and FLJ44180 (P-value range = 1.71×10-9-7.01×10-10; allelic odds ratio range 1.39-1.40). Homozygosity for the at-risk G allele of the most significantly associated SNP, rs6939340, resulted in an increased likelihood of developing neuroblastoma of 1.97 (95% CI 1.58-2.44). Subsequent genotyping of these 6p22 SNPs in the three independent case series confirmed our observation of association (P=9.33×10-15 at rs6939340 for joint analysis). Furthermore, neuroblastoma patients homozygous for the risk alleles at 6p22 were more likely to develop metastatic (Stage 4) disease (P=0.02), show amplification of the MYCN oncogene in the tumor cells (P=0.006), and to have disease relapse (P=0.01).
Conclusion
Common genetic variation at chromosome band 6p22 is associated with susceptibility to neuroblastoma.
doi:10.1056/NEJMoa0708698
PMCID: PMC2742373  PMID: 18463370
7.  The genetic landscape of high-risk neuroblastoma 
Nature genetics  2013;45(3):279-284.
Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%1. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 cases using a combination of whole exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per megabase (0.48 non-silent), and remarkably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, an additional 7.1% had focal deletions), MYCN (1.7%, a recurrent p.Pro44Leu alteration), and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1, and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies reliant upon frequently altered oncogenic drivers.
doi:10.1038/ng.2529
PMCID: PMC3682833  PMID: 23334666
8.  Integrative genomics identifies LMO1 as a neuroblastoma oncogene 
Nature  2010;469(7329):216-220.
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
doi:10.1038/nature09609
PMCID: PMC3320515  PMID: 21124317
9.  Phenotype Restricted Genome-Wide Association Study Using a Gene-Centric Approach Identifies Three Low-Risk Neuroblastoma Susceptibility Loci 
PLoS Genetics  2011;7(3):e1002026.
Neuroblastoma is a malignant neoplasm of the developing sympathetic nervous system that is notable for its phenotypic diversity. High-risk patients typically have widely disseminated disease at diagnosis and a poor survival probability, but low-risk patients frequently have localized tumors that are almost always cured with little or no chemotherapy. Our genome-wide association study (GWAS) has identified common variants within FLJ22536, BARD1, and LMO1 as significantly associated with neuroblastoma and more robustly associated with high-risk disease. Here we show that a GWAS focused on low-risk cases identified SNPs within DUSP12 at 1q23.3 (P = 2.07×10−6), DDX4 and IL31RA both at 5q11.2 (P = 2.94×10−6 and 6.54×10−7 respectively), and HSD17B12 at 11p11.2 (P = 4.20×10−7) as being associated with the less aggressive form of the disease. These data demonstrate the importance of robust phenotypic data in GWAS analyses and identify additional susceptibility variants for neuroblastoma.
Author Summary
Neuroblastoma is the most common solid tumor outside the central nervous system and is accountable for 10% of the mortality rate of all children's cancers. It has distinctive clinical behaviors and is categorized into different risk groups: high-risk, intermediate-risk, and low-risk. Genome-wide association studies have reported a number of genetic variations predisposing to high-risk neuroblastoma. This study focuses on the low-risk neuroblastoma group and identifies four novel genes (DUSP12, DDX4, IL31RA, and HSD17B12) at three distinct genomic positions that harbor disease-causing variants. This study also reports several gene sets that are enriched in overall neuroblastoma as well as in both high-risk and low-risk groups. Also of importance is that this study adopts a new computational method that identifies genes, instead of only one single nucleotide polymorphism, as disease-causing variants. Shown to have superior power of detection genome-wide association signals for neuroblastoma, the methodology presented in this study has great potential applications in case-control association studies in other diseases.
doi:10.1371/journal.pgen.1002026
PMCID: PMC3060064  PMID: 21436895

Results 1-9 (9)