Supplementary Tables 1 - 12, Figures 1 - 5 from Common Genetic Variants in <i>NEFL</i> Influence Gene Expression and Neuroblastoma Risk

Supplementary Table 1. Functional prioritization of SNPs in linkage disequilibrium (r2=0.6) with significant rs4673067 at SCG2 locus. Supplementary Table 2. SNPs in LD (r2>0.60) with the SNP rs118727. Supplementary Table 3. SNPs in LD (r2>0.60) with the SNP rs196830. Supplementary Table 4. SNPs in LD (r2>0.60) with the SNP rs169061. Supplementary Table 5. SNPs in LD (r2>0.60) with the SNP rs11994014. Supplementary Table 6. SNPs in LD (r2>0.60) with the SNP rs17830286. Supplementary Table 7. Functional prioritization of SNPs in 3'UTR region of NEFL and in linkage disequilibrium (r2=0.6) with significant typed SNPs rs118727, rs196830, rs17830286, rs11994014. Supplementary Table 8. Prediction of mircoRNAs whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by mrSNP web tool. Supplementary Table 9. Prediction of transcriptional factors whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by HaploReg V2. Supplementary Table 10. List of UTR motifs analyzed for the SNPs rs3761, rs2979704, and rs1059111. Supplementary Table 11. Imputation results of SNPs at SCG2 and NEFL loci (+/- 1Kb) by using 1000 Genomes data. Supplementary Table 12. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in European American cohort. Supplementary Table 13. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in Italian cohort. Supplementary Figure 1. Linkage disequilibrium (LD) plot of NEFL gene (chr8: positions 24,845,004 to 24,887,674) by Haploview 4.2 for HapMap CEU subjects. Supplementary Figure 2. REST gene silencing in neuroblastoma cells. The efficiency of gene silencing mediated by lentiviral delivery of hairpin RNA directed against REST (shREST) in SH-SY5Y and SK-N-BE2c cell lines was assessed by western blotting (a-b). The bar graphs show integral optical density (OD) value for each band, normalized respect to β-Actin expression. The results are shown as mean of three experiments and are represented as fold respect to shCTR cells which are infected by lentivirus-mediated delivery of non-silencing hairpin RNA. Induction of NEFL and REST gene expression levels after REST silencing in SH-SY5Y (c) and SK-N-BE2c (d). *P<0.05 Supplementary Figure 3. (a) Association between NEFL SNP genotype and gene expression in 16 neuroblastoma cell lines. The gene expression measure of the NB1 cell line was excluded from this analysis as affected the normal distribution of data (data not shown). The analysis was performed using the SNPs rs12545967 and rs2976427 in complete linkage disequilibrium (r2=1) with rs11994014 and rs1059111, respectively. Conventionally, we report the allele code of rs11994014 and rs1059111 in the plot. (b) Association between NEFL SNP genotype and gene expression in 198 LCLs. The analysis for rs1059111 (missing in the dataset) was performed using the SNP rs2979704 in complete linkage disequilibrium (r2=1). Supplementary Figure 4. NEFL gene silencing in neuroblastoma cells with rs1059111 TA genotype. Evaluation of cell growth (a) and invasion (b) after NEFL silencing in SH-SY5Y and SK-N-BE2c cell lines. The efficiency of gene silencing mediated by lentiviral delivery of hairpin RNA directed against NEFL (shNEFL) in SH-SY5Y and SK-N-BE2c cell lines was assessed by western blotting (c). The bar graphs show integral optical density (OD) value for each band, normalized respect to β-Actin expression. The same results were observed for mRNA measurements (data not shown). The results are shown as mean of three experiments and are represented as fold respect to shCTR cells which are infected by lentivirus-mediated delivery of non-silencing hairpin RNA. *P<0.05 Supplementary Figure 5. Association between NEFL SNP genotype and neuronal marker genes in neuroblastoma cell lines. Eight cell lines (IMR-32, SK-N-DZ, SK-N-AS, KP-N-SI9s with rs1059111 TT genotype and SIMA, SK-N-BE(2), SH-SY5Y, SK-N-FI with rs1059111 TA protective genotype) with the extremes of NEFL mRNA expression were selected based on the analysis shows in the Supplementary Figure 2a. The analysis was performed using the SNPs rs2976427 in complete linkage disequilibrium (r2=1) with and rs1059111. Conventionally, we report the allele code of rs1059111 in the plot.