Several neuroblastoma (NB) susceptibility loci have been identified within LINC00340, BARD1, LMO1, DUSP12, HSD17B12, DDX4, IL31RA, HACE1 and LIN28B by genome-wide association (GWA) studies including European American individuals. To validate and comprehensively evaluate the impact of the identified NB variants on disease risk and phenotype, we analyzed 16 single nucleotide polymorphisms (SNPs) in an Italian population (370 cases and 809 controls). We assessed their regulatory activity on gene expression in lymphoblastoid (LCLs) and NB cell lines. We evaluated the cumulative effect of the independent loci on NB risk and high-risk phenotype development in Italian and European American (1627 cases and 2575 controls) populations. All NB susceptibility genes replicated in the Italian dataset except for DDX4 and IL31RA, and the most significant SNP was rs6435862 in BARD1 (P = 8.4 × 10(-15)). BARD1 showed an additional and independent SNP association (rs7585356). This variant influenced BARD1 mRNA expression in LCLs and NB cell lines. No evidence of epistasis among the NB-associated variants was detected, whereas a cumulative effect of risk variants on NB risk (European Americans: P (trend) = 6.9 × 10(-30), Italians: P (trend) = 8.55 × 10(13)) and development of high-risk phenotype (European Americans: P (trend) = 6.9 × 10(-13), Italians: P (trend) = 2.2 × 10(-1)) was observed in a dose-dependent manner. These results provide further evidence that the risk loci identified in GWA studies contribute to NB susceptibility in distinct populations and strengthen the role of BARD1 as major genetic contributor to NB risk. This study shows that even in the absence of interaction the combination of several low-penetrance alleles has potential to distinguish subgroups of patients at different risks of developing NB.

The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single nucleotide polymorphisms (SNP) associated with neuroblastoma at the LINC00340, BARD1, LMO1, DUSP12, HSD17B12, HACE1 and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated 8 additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNP at these candidate genes were tested for association with disease susceptibility in 2101 cases and 4202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing and cellular differentiation assays. The neurofilament gene NEFL harbored three SNP associated with neuroblastoma (rs11994014; Pcombined=0.0050; OR=0.88, rs2979704; Pcombined=0.0072; OR=0.87, rs105911; Pcombined=0.0049; OR=0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biological investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens was associated with better overall survival (P=0.03; HR=0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression.

Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. In this paper we describe 12 IRIDA patients belonging to 7 unrelated families and identify 10 (9 novel) TMPRSS6 mutations spread along the gene sequence: 5 missense, 1 non sense and 4 frameshift. The frameshift and non sense mutations are predict to result in truncated protein lacking the catalytic domain. The causal role of missense mutations (Y141C, I212T, R271Q, S304L and C510S) is demonstrated by in silico analysis, their absence in 100 control chromosomes and the high conservation of the involved residues. The C510S mutation in the LDLRA domain in silico model causes an intra-molecular structural imbalance that impairs matriptase-2 activation. We also assessed the in vitro effect on hepcidin promoter and the proteolytic activity of I212T and R271Q variants demonstrating a reduced inhibitory effect for the former mutation, but surprisingly a normal function for R271Q which appears a silent mutation in vitro. Based on mRNA expression studies I212T could also decrease the total amount of protein produced, likely interfering with mRNA stability. Collectively, our results extend the pattern of TMPRSS6 mutations associated with IRIDA and propose a model of causality for some of the novel missense mutation. ©2010Wiley-Liss, Inc.

BackgroundCommon variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (−174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB.ResultTo evaluate the impact of IL-6 SNP rs1800795 on disease risk and phenotype, we analyzed 326 Italian NB patients and 511 controls. Moreover, we performed in silico and quantitative Real Time (qRT)-PCR analyses to evaluate the influence of the SNP on gene expression in 198 lymphoblastoid cell lines (LCLs) and in 31 NB tumors, respectively. Kaplan-Meier analysis was used to verify the association between IL-6 gene expression and patient survival. We found that IL-6 SNP is not involved in susceptibility to NB development. However, our results show that a low frequency of genotype CC is significantly associated with a low overall survival, advanced stage, and high-risk phenotype. The in silico (p = 2.61×10−5) and qRT-PCR (p = 0.03) analyses showed similar trend indicating that the CC genotype is correlated with increased level of IL-6 expression. In report gene assay, we showed that the −174 C variant had a significantly increased transcriptional activity compared with G allele (p = 0.0006). Moreover, Kaplan-Meier analysis demonstrated that high levels of IL-6 are associated with poor outcome in children with NB in two independent gene expression array datasets.ConclusionsThe biological effect of SNP IL-6–174 G>C in relation to promotion of cancer progression is consistent with the observed decreased survival time. The present study suggests that SNP IL-6–174 G>C may be a useful marker for NB prognosis.