To identify sequence variants that are associated with susceptibility to develop neuroblastoma, we compared single marker allele and genotype frequencies in our discovery case series using the χ2
and Cochran-Armitage trend test statistics. The top three SNPs showing significant association to neuroblastoma were in tight linkage disequilibrium (LD) at chromosome 6p22 (rs6939340, rs4712653, rs9295536) yielding P
-values of 1.71 × 10-9
- 7.01 × 10-10
(allelic odds ratio 1.39-1.40; , , Table S2
). Two additional SNPs at chromosome 20p11 (rs3790171 and rs7272481) showed genome-wide significant single marker P
-values, and many others were very close to the genome-wide significance threshold (). However, only the chromosome 6 association results retained genome-wide significance after further correction for population substructure using principal components analyses as implemented in Eigenstrat20,21
(). The chromosome 6 signal falls within a 94.2 Kb LD block containing the predicted overlapping genes FLJ22536
Summary of the neuroblastoma GWAS results in the discovery set
Neuroblastoma genome-wide association study results for discovery case and control subjects.
Pairwise linkage disequilibrium diagram of the 6p22 locus and association results for regional SNPs
We next sought to replicate the chromosome 6p22 and 20p11 association signals in three separate pairings of neuroblastoma patients with controls. As shown in , the chromosome 6 risk alleles identified in the discovery phase were also significantly over-represented in all three sets of neuroblastoma cases compared to their controls, yielding a combined P
-value and allelic odds ratio for the most strongly associated SNP rs6939340 of 9.33×10-15
and 1.37 (95% CI 1.27-1.49). There was no support for the chromosome 20 alleles identified in the discovery cohort truly being associated with predisposition to neuroblastoma in the three replication case series (Supplementary Table S3
Replication effort of chromosome 6 association results.
To determine if the risk alleles at the chromosome 6p22 locus were differentially associated with patient subsets and outcome, we analyzed the associated SNPs frequency distribution from the discovery phase COG case series with respect to prognostically relevant clinical and biological covariates present at diagnosis and survival rates. For all three SNPs, there was a significantly different allele frequency distribution indicating that the risk alleles were more likely to be present in patients with a more malignant clinical presentation and disease course (). Accordingly, neuroblastoma patients homozygous for the at-risk alleles were more likely to develop tumors that were metastatic at diagnosis (P
=0.0060, 0.0084, and 0.0245 for rs4712653, rs9295536, and rs6939340 respectively), to have somatically acquired amplification of the MYCN
oncogene in tumor cells (P
=0.0018, 0.0028, and 0.0061 respectively), and to have a high-risk classification for the purposes of treatment stratification (P
=0.0018, 0.0004 and 0.0107 respectively), compared to neuroblastoma patients homozygous for the non-risk alleles (Supplemental Table S4
). In addition, patients homozygous for the at-risk alleles had a significantly decreased event-free survival probability (P
=0.0158 for SNP rs6939340; Supplemental Figures S2
). This association with more malignant disease may explain why we were able to show replication of results in the relatively small series of 59 cases from CCG that consisted solely of high-risk patients ().
Chromosome 6p22 risk alleles are associated with a more malignant neuroblastoma phenotype