Association between METTL3 gene polymorphisms and neuroblastoma susceptibility: A nine‐centre case‐control study

Abstract Neuroblastoma ranks as the most commonly seen and deadly solid tumour in infancy. The aberrant activity of m6A‐RNA methyltransferase METTL3 is involved in human cancers. Therefore, functional genetic variants in the METTL3 gene may contribute to neuroblastoma risk. In the current nine‐centre case‐control study, we aimed to analyse the association between the METTL3 gene single nucleotide polymorphisms (SNPs) and neuroblastoma susceptibility. We genotyped four METTL3 gene SNPs (rs1061026 T>G, rs1061027 C>A, rs1139130 A>G, and rs1263801 G>C) in 968 neuroblastoma patients and 1814 controls in China. We found significant associations between these SNPs and neuroblastoma risk in neither single‐locus nor combined analyses. Interestingly, in the stratified analysis, we observed a significant risk association with rs1061027 AA in subgroups of children ≤ 18 months of age (adjusted OR = 1.87, 95% CI = 1.03‐3.41, P = .040) and females (adjusted OR = 1.86, 95% CI = 1.07‐3.24, P = .028). Overall, we identified a significant association between METTL3 gene rs1061027 C>A polymorphism and neuroblastoma risk in children ≤18 months of age and females. Our findings provide novel insights into the genetic determinants of neuroblastoma.

analyse the association between the METTL3 gene single nucleotide polymorphisms (SNPs) and neuroblastoma susceptibility. We genotyped four METTL3 gene SNPs (rs1061026 T>G, rs1061027 C>A, rs1139130 A>G, and rs1263801 G>C) in 968 neuroblastoma patients and 1814 controls in China. We found significant associations between these SNPs and neuroblastoma risk in neither single-locus nor combined analyses. Interestingly, in the stratified analysis, we observed a significant risk association with rs1061027 AA in subgroups of children ≤ 18 months of age (adjusted OR = 1.87, 95% CI = 1.03-3.41, P = .040) and females (adjusted OR = 1.86, 95% CI = 1.07-3.24, P = .028). Overall, we identified a significant association between METTL3 gene rs1061027 C>A polymorphism and neuroblastoma risk in children ≤18 months of age and females. Our findings provide novel insights into the genetic determinants of neuroblastoma.

| INTRODUC TI ON
Neuroblastoma is a solid childhood cancer arising from sympatho-adrenergic neuronal progenitors. 1 It accounts for approximately 5% of all paediatric cancers, but disproportionally causes 12% cancer mortality in children. 2 The incidence rate of neuroblastoma in America is nearly 1 out of 7000. 3 Yet, the incidence rate of neuroblastoma in China is about 1 out of 13 000. 4 Unlike other paediatric malignancies, neuroblastoma is characterized by high phenotypic heterogeneity. 5 Clinical outcomes of cases vary significantly from spontaneous recovery without treatment to therapy-resistant progression. 6,7 Survival rate could be achieved at least 95% in patients with a non-high-risk (low-and intermediate-risk) neuroblastoma. 8 Conversely, only 50% of patients with high-risk neuroblastoma achieve long-term survival. 9,10 Over the past decades, significant advances have been made towards understanding the determinants of neuroblastoma risk. 11,12 Environmental or parental exposures were reported to predispose to neuroblastoma, but warrant more validations. 13,14 Previous research has suggested that there is a strong genetic component underlying neuroblastoma susceptibility. 15 For example, most of the familial neuroblastoma harboured mutations in genes ALK 16,17 and PHOX2B. 18 A clinical trial of an inhibitor of ALK was launched soon after the initial discovery of ALK mutations.
Moreover, researchers have unceasingly found predisposing genetic polymorphisms in sporadic neuroblastoma. [19][20][21][22][23][24][25][26] To be noted, all the identified genetic variations so far only revealed a small part of the genetic landscape of this malignancy. Therefore, it would be of translational interest to determine more causal genetic risk variants for improving the prevention and prognosis of neuroblastoma.
N6-methyladenosine (m 6 A) is the most prevalent modification in RNA, especially mRNA. 27,28 The m 6 A modification mainly regulates gene expression at the post-transcriptional levels by affecting mRNA stability, mRNA translation and splicing. 23 m 6 A modifications are installed by RNA methyltransferases (METTL3, METTL14 and WTAP, known as 'writers'), removed by the demethylases (FTO and ALKBH5, known as 'erasers'), and recognized by m 6 A-binding proteins (YTHDF1/2/3 and IGF2BP1, known as 'readers'). [29][30][31][32] Emerging evidence suggests that dysregulated m 6 A modification is tightly implicated in various diseases, especially cancers. 33 To identify METTL3 genetic variations that confer susceptibility to neuroblastoma, we performed this multi-centre epidemiology study.

| Sample selection
The current study is a hospital-based case-control study of neuroblastoma with participants recruited from nine hospitals in China

| Polymorphism selection and genotyping
Potentially functional SNPs in the METTL3 gene were screened out from the dbSNP database and SNPinfo software. 38,39 In brief, we searched for potentially functional candidate SNPs located in the 5′-flanking region, 5′-untranslated region, 3′-untranslated region and exon of METTL3. Moreover, the included SNPs should conform to: (a) the minor allele frequency >5% for Chinese Han subjects; (b) putative functional potential SNPs, which might affect transcription activity or binding capacity of the microRNA-binding site; and (c) SNPs in low linkage disequilibrium with each other (R 2 > .8). Following these criteria, four SNPs (rs1061026 T>G, rs1061027 C>A, rs1139130 A>G, and rs1263801 G>C) in the METTL3 gene were selected for the Chinese sample genotyping. As shown in Figure S1, there was no significant LD (R 2 < .8) among these four SNPs of METTL3 (R 2 = .036 between rs1061026 and rs1061027, R 2 = .009 between rs1061026 and rs1139130, R 2 = .248 between rs1061026 and rs1263801, R 2 = .453 between rs1061027 and rs1139130, R 2 = .459 between rs1061027 and rs1263801, R 2 = .387 between rs1139130 and rs1263801).
Blood samples were stored at −80°C. DNA was extracted according to standard procedure, followed by genotyping using TaqMan methodology. [40][41][42] Laboratory personnel were blinded to case/control status. We also repeatedly genotyped 10% randomly selected sample to assess the genotyping error rate and obtained concordance rates of 100%.

K E Y W O R D S
case-control study, METTL3, neuroblastoma, polymorphism, risk

| Statistical analysis
Compliance of alleles at individual loci with the Hardy-Weinberg equilibrium (HWE) was measured in controls using a chi-square test.
Differences in selected demographic variables between cases and controls were assessed by the chi-square test. Crude or adjusted (for age and gender) odds ratios (ORs) with respective 95% confidence intervals (CIs) were obtained from logistic regression analyses for the analysis of associations between polymorphisms and neuroblastoma risk. Logistic regression analyses were adopted to obtain haplotype frequencies and distinct haplotypes, with the adjustment for gender and age. 43,44 The P value level of significance was .05. We did the analyses using SAS 9.1 (SAS Institute).

| Associations between METTL3 SNPs and neuroblastoma susceptibility
The clinical characteristics of the eligible participants (968 cases and 1814 controls) were depicted in Table S1. No significant differences between cases and controls were observed with respect to age (P = .536) and gender (P = .231). The associations between the four METTL3 SNPs and neuroblastoma risk were shown in Table 1.
The P values of HWE for all SNPs were >.05 in the controls, indicating none of them departing from HWE. In the single-locus analysis, all the selected variants in the METTL3 gene showed no significant association with neuroblastoma susceptibility. Then, we analysed the combined effect of risk genotypes but still failed to detect any significant association.

| Haplotype analysis
We further examined whether the haplotypes of the four METTL3 gene SNPs are correlated to neuroblastoma risk in an order of rs1061026, rs1061027, rs1139130 and rs1263801. As shown in Table 3, the TCGG haplotype was defined as the reference group.
We failed to detect a significant relationship between neuroblastoma risk and subjects with all the haplotypes.

ACK N OWLED G EM ENTS
This study was supported by grants from the Natural Science  The haplotype order was rs1061026, rs1061027, rs1139130 and rs1263801. b Obtained in logistic regression models with adjustment for age and gender.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data were available upon request.