Impact of YTHDF1 gene polymorphisms on Wilms tumor susceptibility: A five‐center case‐control study

Abstract Background Wilms tumor is the most frequent renal malignancy in children. YTHDF1 is associated with the development of several kinds of cancers, yet whether common variants of the YTHDF1 gene influence Wilms tumor risk is unknown. We present, here, a hospital‐based case‐control study specifically designed to investigate the role of YTHDF1 genetic variants on Wilms tumor. Methods We successfully genotyped samples of 408 Wilms tumor cases and 1198 controls which were collected from five hospitals across China. The unconditional logistic regression was adopted to analyze the contributions of YTHDF1 gene single nucleotide polymorphisms (SNPs) to the risk of Wilms tumor. The odds ratio (OR) and 95% confidence interval (CI) were generated to evaluate the conferring risk of YTHDF1 gene SNPs (rs6011668 C>T, rs6090311 A>G). Results Neither of the two SNPs could contribute to the risk of Wilms tumor. A negative association was also detected in the combined effects of protective genotypes on Wilms tumor risk. The stratification analysis revealed that compared with those with CC genotype, rs6011668 CT/TT genotype was associated with increased Wilms tumor risk in those ≤18 months (OR = 1.54, 95% CI = 1.02–2.30, p = 0.038), and with decreased Wilms tumor risk in those >18 months (OR = 0.70, 95% CI = 0.50–0.97, p = 0.034). Conclusion Our present work sheds some light on the potential role of YTHDF1 gene polymorphisms on Wilms tumor risk.

and 95% confidence interval (CI) were generated to evaluate the conferring risk of YTHDF1 gene SNPs (rs6011668 C>T, rs6090311 A>G).
Results: Neither of the two SNPs could contribute to the risk of Wilms tumor. A negative association was also detected in the combined effects of protective genotypes on Wilms tumor risk. The stratification analysis revealed that compared with those with CC genotype, rs6011668 CT/TT genotype was associated with increased Wilms tumor risk in those ≤18 months (OR = 1.54, 95% CI = 1.02-2.30, p = 0.038), and with decreased Wilms tumor risk in those >18 months (OR = 0.70, 95% CI = 0.50-0.97, p = 0.034).

| INTRODUC TI ON
Wilms tumor (nephroblastoma) is a solid kidney tumor that mainly affects children. 1 It is a tumor of embryonic origin that originates from abnormal differentiation in primitive cells during fetal nephrogenesis. 2 The incidence of Wilms tumor is higher in the United States compared to that of Wilms tumor in China, there being about 7 new cases per million children compared to 3.3 per million. 3,4 Nearly 80% of cases are diagnosed before the age of five. 5 With the advancement of medical treatment, the 5-year survival of favorable histology Wilms tumor has exceeded over 90%. 6 For patients with unfavorable Wilms tumors, the prognosis is still disappointing. 6 Wilms tumor is a complex disease characterized by the broad heterogeneity of molecular genetic alterations. 7,8 The WT1 gene was discovered as the first identified Wilms tumor mutated gene in 1990. 9 Subsequently, mutations in the genes CTNNB1, AMER1, and DROSHA were demonstrated as Wilms tumor risk factors. 7,[10][11][12] Genome-wide analysis, as well as case-control studies also identified additional Wilms tumor risk loci. [13][14][15][16] However, all the identified gene mutations only explain a small portion of Wilms tumor origin.
Characterization of more variants will further clarify the etiology of Wilms tumor.
N6-methyladenosine (m 6 A) is one of the most seen internal modifications in mRNAs. [17][18][19][20] The m 6 A is a dynamic and reversible process where three groups of proteins take part in, including methyltransferases, demethylases, and m 6 A-specific binding proteins. 21 The binding proteins mainly include YTH-family proteins YTHDF1-3, YTHDC1-2, eukaryotic initiation factor eIF3, and insulinlike growth factor 2 mRNA-binding proteins IGF2BP1-3. 22 In the cytosol, YTHDF1 interacts with translation initiation factors eIF3 and eIF4A3 to promote the translation process of m 6 A-modified mRNAs. 23 YTHDF1 has been implicated in several types of cancers. 24

| Polymorphism selection and genotyping
We chose two SNPs of YTHDF1 gene, rs6011668 C>T and rs6090311 A>G, to genotype. The reasons for choosing these two SNPs were described in our previous study. 29 To be specific, the following criteria were adopted to choose potentially functional polymorphisms: (1) the minor allele frequency (MAF) reported in HapMap was >5% for Chinese Han subjects; (2) putative functional potentials SNPs located in the 5'-flanking region, exon, 5'-untranslated region (5' UTR), and 3' UTR, which might affect transcription activity or binding capacity of the microRNA binding site; (3) SNPs in low linkage disequilibrium with each other (R 2 < 0.8). Both the two SNPs (rs6011668 C>T and rs6090311 A>G) are located in the transcription factor binding sites (TFBS). There is no significant linkage disequilibrium (LD) (R 2 < 0.8) between rs6011668 C>T and rs6090311 A>G in YTHDF1 gene (R 2 = 0.094). Genomic DNA was isolated from peripheral blood according to the standard protocol. Genotyping was performed by TaqMan SNP Genotyping Assay, by means of an ABI 7900HT (Applied Biosystems). 30 In each genotyping plate, we inserted negative control samples (water) to ensure the quality of genotyping. 10% of randomly selected replicates from the study sample were re-genotyped. Concordance rates for the original and replicate samples were 100%.

| Statistical analysis
For the analyzed SNPs, a goodness-of-fit χ 2 test was used to test for deviations from Hardy-Weinberg equilibrium (HWE). To test the Conclusion: Our present work sheds some light on the potential role of YTHDF1 gene polymorphisms on Wilms tumor risk.

K E Y W O R D S
case-control study, polymorphism, risk, Wilms tumor, YTHDF1 difference in the distribution of demographic variables between cases and controls, a Chi-square test for categorical variables and a Student t-test for continuous variables were conducted. The association between the SNPs and Wilms tumor risk was determined using unconditional logistic regression computing odds ratios (ORs) and 95% confidence intervals (CIs). Stratified analyses were carried out across the strata of the following factors: age, sex, and clinical stages. In all analyses, a two-tailed p value <0.05 was considered statistically significant. Statistical calculations were done with the SAS statistical software package version 9.1 (SAS Institute Inc.). 31 Table S1 gives information on the baseline characteristics of the in-  Table 1.

| Effect of YTHDF1 gene SNPs on Wilms tumor risk
As expected, the genotype distributions of rs6011668 C>T (P for HWE = 0.490) and rs6090311 A>G (P for HWE = 0.378) polymorphism in controls did not deviate from HWE. We evaluated the association of rs6011668 C>T and rs6090311 A>G with Wilms tumor risk but did not find statistical significance in all genotype models.
Non-significant association results remained unchanged after adjusting by age and sex. We then allocated rs6011668 CT/TT and rs6090311 AG/GG genotypes as protective genotypes. Compared to 0 protective genotype, 1, 2, and 1-2 protective genotypes could not decrease Wilms tumor risk.

| Stratification analysis
We next determined the association between YTHDF1 gene polymorphisms and susceptibility to Wilms tumor in subgroups separated by age, sex, and clinical stages ( We admit that our study has its weakness. First, the sample size, although enrolled from several hospitals, may be too small to explain the effects of an entire population. Second, we had no access to other environmental factors, which could have biased Wilms tumor risk assessment without adequate adjustment for these covariates in the risk evaluation model. Third, all the participants were Chinese, and the applicability of the findings to other populations requires confirmation.
In conclusion, our study did not find strong evidence that YTHDF1 gene variants influence Wilms tumor risk. Our results require independent replication in larger studies, preferably with more detailed information on environmental effect analysis, functional experiments, and across other populations.

ACK N OWLED G M ENTS
This study was supported by grants from National Natural

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used in the current study are available from the corresponding author upon reasonable request.