Identification of hepatoblastoma susceptibility loci in the TRMT6 gene from a seven‐center case–control study

Abstract Hepatoblastoma, the most frequently diagnosed primary paediatric liver tumour, bears the lowest somatic mutation burden among paediatric neoplasms. Therefore, it is essential to identify pathogenic germline genetic variants, especially those in oncogenic genes, for this disease. The tRNA methyltransferase 6 noncatalytic subunit (TRMT6) forms a tRNA methyltransferase complex with TRMT61A to catalyse adenosine methylation at position N1 of RNAs. TRMT6 has displayed tumour‐promoting functions in several cancer types. However, the contribution of its genetic variants to hepatoblastoma remains unclear. In this study, we investigated the association between four TRMT6 polymorphisms (rs236170 A > G, rs451571 T > C, rs236188 G > A and rs236110 C > A) and the risk of hepatoblastoma in a cohort of 313 cases and 1446 healthy controls. Germline DNA was subjected to polymorphism genotyping via the TaqMan qPCR method. Odds ratio (OR) and 95% confidence interval (CI) were used to determine hepatoblastoma susceptibility variants. The rs236170 A > G, rs236188 G > A and rs236110 C > A polymorphisms were significantly associated with hepatoblastoma risk. Combination analysis of the four polymorphisms revealed that children bearing 1–4 risk genotypes were at significantly enhanced hepatoblastoma risk compared to those without risk genotype (adjusted OR = 1.52, 95% CI = 1.19–1.95, p = 0.0008). We also conducted stratification analyses by age, sex and clinical stage. Ultimately, we found that the rs236110 C > A was significantly associated with the downregulation of MCM8, a neighbouring gene of TRMT6. In conclusion, we identified three susceptibility loci in the TRMT6 gene for hepatoblastoma. Our findings warrant further validation by extensive case–control studies across different ethnicities.

N1-methyladenosine (m 1 A), one of the chemical modifications, is located on the first nitrogen atom of adenosine in RNA.m 1 A is present ubiquitously in mRNA, rRNA, lncRNA and tRNA but is most enriched in tRNA.By affecting RNA base pairing, m 1 A profoundly impacts RNA's structure, stability, translation and function.For instance, m 1 A at position 58 of tRNA is critical for maintaining the proper structure of tRNA and stability and starting the translational process; lack of m 1 A modification in this site was reported to induce tRNA-derived small RNAs (tDRs), facilitating ribosome assembly and leading to malignant transformations. 1 Several methyltransferases that catalyse RNA m 1 A modification have been identified, including the tRNA methyltransferase 6 (TRMT6), TRMT61A, TRMT61B, TRMT10C and NML. 2 TRMT6 and TRMT61A form a functional heterotetramer complex to deposit N 1 -methylation in target RNA.TRMT6, the noncatalytic subunit of the methyltransferase complex, is responsible for tRNA binding, while TRMT61A, carrying a methyl donor binding pouch, acts as the catalytic subunit. 2 Recently, increasing evidence indicates that TRMT6 is preferentially expressed in cancerous tissues and plays an oncogenic role in various types of cancer, such as glioma, 3 bladder cancer 1 and hepatocellular carcinoma (HCC). 4,5patoblastoma, the most common primary paediatric liver malignancy, is extremely rare, with an annual incidence varying from 1.2 to 1.5 cases per million. 6In particular, it was estimated that the incidence rate of hepatoblastoma is about 1.4 per million Chinese children yearly. 7Hepatoblastoma is an embryonal tumour arising from hepatoblasts, which often exhibits mixed histological patterns representing different developmental stages of the liver.Several factors seem to increase the risk of hepatoblastoma, including inferior birth status (premature birth and very low birth), some treatments (Oxygen therapy, furosemide, total parenteral nutrition [TPN] and radiation) and toxins (e.g.plasticizers).Besides, hereditary predispositions also contribute to the development of hepatoblastoma.For instance, several constitutional genetic syndromes have shown associations with increased hepatoblastoma risk, such as Trisomy 18/Edward's syndrome, Beckwith-Wiedemann syndrome (BWS) and familial adenomatous polyposis (FAP).Unlike adult tumours with high somatic mutation prevalence, germline variants in cancer susceptibility genes are often reported in paediatric cancers, which may contribute to 8%-10% of paediatric tumours.Additionally, previous findings indicate that hepatoblastoma harbours the fewest somatic mutations out of all solid childhood tumours, underlying the importance of genetic variants in the pathogenesis of hepatoblastoma. 8[11][12][13][14][15][16][17][18] Although different research teams confirmed the contributing role of TRMT6 in the carcinogenesis of HCC, 4,5 its impacts on hepatoblastoma are unknown.Besides, no studies have reported the associations between genetic polymorphisms of the TRMT6 gene and the risk of hepatoblastoma.Our research aims to identify pathogenic genetic polymorphisms for hepatoblastoma in Chinese children with a cohort of 313 cases and 1446 healthy controls.

| Patient and study design
Patients were diagnosed with hepatoblastoma as manifested by evidence from clinical examinations, laboratory testing, pathological examination and imaging.Sufficient peripheral whole blood samples were obtained from participants for analysis.Cases (n = 313) were all Han Chinese descendants younger than 14 years of age.They were diagnosed in seven independent hospitals in Guangzhou, Kunming, Changsha, Taiyuan, Xi'an, Zhengzhou and Shenyang.Children who underwent health check-ups in those hospitals during a similar period were recruited as healthy controls (n = 1446) to minimize selection bias.Patients and controls were matched concerning age and sex (Table S1). 12We staged the patients according to the PRETEXT classification. 19All patients offered informed consent for molecular research before being recruited.The study was conducted with a protocol (No: 202016601) authorized by the institutional review board of Guangzhou Women and Children's Medical Center.Participants' epidemiological and clinical characteristics were described previously. 12

| Genotyping and selecting polymorphisms
We arbitrarily chose the candidate single nucleotide polymorphisms (SNPs) for this study from the dbSNP database compared to those without risk genotype (adjusted OR = 1.52, 95% CI = 1.19-1.95,p = 0.0008).We also conducted stratification analyses by age, sex and clinical stage.
Ultimately, we found that the rs236110 C > A was significantly associated with the downregulation of MCM8, a neighbouring gene of TRMT6.In conclusion, we identified three susceptibility loci in the TRMT6 gene for hepatoblastoma.Our findings warrant further validation by extensive case-control studies across different ethnicities.(http:// www.ncbi.nlm.nih.1][22] Only SNPs having potential biological functions, as suggested by SNPinfo (https:// snpin fo.niehs.nih.gov), were qualified for the study.Moreover, we only chose SNPs with low linkage disequilibrium (LD) (R 2 < 0.8) (https:// ldlink.nih.gov/?tab= ldmatrix).The four TRMT6 polymorphisms (rs236170 A > G, rs451571 T > C, rs236188 G > A and rs236110 C > A) showed low LD with each other, with R 2 varying from 0.066 to 0.732.Regarding potential biological functions, the rs236110 C > A is located in an exonic splicing enhancer or exonic splicing silencer, to which a specialized protein binds to elevate or decrease the efficiency of exon inclusion.The rs236170 A > G in the miRNA binding site of the TRMT6 gene may impact the stability of its transcripts.The rs236188 G > A in the transcription factor binding site may potentially alter the affinity between certain transcription factors and the promoters of the TRMT6 gene.Finally, the rs451571 T > C and rs236110 C > A are missense variants in the coding sequence and may lead to changes in amino acids during translation.Genomic DNA was extracted from participants' peripheral blood samples donated before treatment using the Tiangen Blood DNA Extraction kits (Tiangen Biotechnology).Genotyping assays were performed using Taqman qPCR on a TaqMan platform (Applied Biosystems).

| Statistical analysis
We first performed a goodness-of-fit chi-square test to check these SNPs' Hardy-Weinberg equilibrium (HWE) in the controls.Next, we assessed the SNPs' associations with hepatoblastoma susceptibility using the logistic regression analysis after adjustment for age, sex and clinical stage.The resulting odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the significance of the associations.In the multivariate analyses, age, sex and the clinical stage were used as adjusted covariates.The following genetic models were employed to evaluate the association between the four SNPs and hepatoblastoma susceptibility: homozygous (WW vs. VV), heterozygous (WW vs. WV), dominant (WW vs. WV/VV) and recessive (WW/WV vs. VV) models.W and V depicted wild type and variant alleles of an SNP, respectively.The stratified analyses by age, sex and clinical stage were also carried out.In the last, we investigated the association between the above SNPs and expression levels of relevant genes, that is, expression quantitative trait locus (eQTL), using a web tool based on Genotype-Tissue Expression (GTEx) project. 23All analyses were two-sided using SAS v9.1 software (SAS Institute Inc., Cary, NC), and a significance level of 0.05 was adopted.

| Association study
Overall, 310 cases and 1444 healthy controls were genotyped successfully among the 313 cases and 1446 controls.Four potential functional TRMT6 polymorphisms (rs236170 A > G, rs451571 T > C, rs236188 G > A and rs236110 C > A) were successfully genotyped and analysed for their contributions in hepatoblastoma susceptibility.The results are summarized in Table 1.We first performed a single locus analysis after confirming that the genotype distributions of these SNPs were not divergent from the Hardy-Weinberg equilibrium.Multivariate regression analysis demonstrated that three TRMT6 polymorphisms (rs236170 A > G, rs236188 G > A and rs236110 C > A) were significantly associated with susceptibility to hepatoblastoma (Table 1).

| Stratification analysis
We also stratified the association study by age, sex and clinical stage (Table 2).Under the dominant genetic model, the association between rs236170 and hepatoblastoma susceptibility remained significant in the subgroup of I + II stages (adjusted OR = 0.66, 95% CI = 0.48-0.92,p = 0.015).Regarding the recessive genetic model, the rs236110 was significantly associated with the risk of hepatoblastoma, regardless of age, sex and clinical stage.Finally, 1-4 risk genotypes significantly contributed to hepatoblastoma predisposition among both age groups, boys and subgroups of clinical I + II stages (Table 2).

| Expression quantitative trait loci (eQTL) analysis
We finally interrogated whether the significant SNPs affected the expression of the TRMT6 gene or its nearby genes.The data from the GTEx website unveiled that the rs236110 C > A polymorphism was related to the altered expression of the minichromosome maintenance 8 (MCM8) gene, located near the TRMT6 gene.Liver samples carrying minor alleles of rs236110 C > A (CA and AA) have significantly lower expression levels of the MCM8 gene than those with CC alleles (Figure 1), suggesting the potential impacts of the SNP on the expression of crucial genes.
5][26][27] Unlike adult cancers, the few recurrent somatic mutations are insufficient to interpret paediatric tumours' initiation and clinical heterogeneity and to facilitate precision therapies.Instead, several studies suggest that specific pathogenic germline variants are promising in directing clinical management and risk stratification for solid tumours in children. 28,29Therefore, discovering disease-predisposing germline variants in paediatric neoplasms is indispensable for clinical decision-making, disease surveillance and risk evaluation for patients, parents and siblings.
This study aimed to interrogate whether genetic variants in the TRMT6 gene predispose to hepatoblastoma.Our results demonstrated that three TRMT6 polymorphisms (rs236170 A > G, rs236188 G > A and rs236110 C > A) were able to modify hepatoblastoma risk in Chinese children individually.Moreover, the four studied SNPs collaboratively affected susceptibility to hepatoblastoma.We previously reported several hepatoblastoma-predisposing genes that regulate different types of RNA methylation, including YTHDF1, 17  Abbreviations: CI, confidence interval; HWE, Hardy-Weinberg equilibrium; OR, odds ratio.a Chi-square test for genotype distributions between hepatoblastoma patients and cancer-free controls.
b Adjusted for age and sex.
c Risk genotypes were rs236170 AA, rs451571 CC, rs236188 AA and rs236110 AA.WTAP, 12 WDR4 10 and METTL1. 9For instance, the rs7766006 in the WTAP gene, encoding a 'writer' that facilitates N6-methyladenosine (m 6 A) methylation of RNAs, decreased the risk of hepatoblastoma. 12YTHDF1 can recognize the m 6 A modification in the RNAs and regulate their stability.The YTHDF1 rs6090311 G allele protects carriers from developing hepatoblastoma risk, 17 and eQTL analysis elucidated the correlation between the YTHDF1 rs6090311 polymorphism and downregulated expression of its surrounding genes. 17 TRMT6 interacts with TRMT61A and assists the latter in installing m 1 A modification in RNA by receiving and binding to target tRNA.
Elevated expression levels of TRMT6 have been observed in several cancers and often predict inferior prognosis.and wound healing assays. 3Wang and colleagues demonstrated that TRMT6/TRMT61A accelerated liver tumorigenesis by mediating m 1 A methylation of PPARδ translation-related tRNAs. 5The increased PPARδ protein products upregulated cholesterol synthesis, further stimulating hedgehog signalling and fueling liver CSCs' self-renewal. 5Interestingly, the TRMT6/61A complex also assists the base methylation of tRNA-derived fragments.F I G U R E 1 GTEx analysis for the association between TRMT6 rs236110 C > A polymorphism and MCM8 gene expression in liver tissue.
the degree of mitochondrial RNA modification, verified across various tissue types. 30They reported that MRPP3 rs11156876 was significantly associated with increased methylation level of tRNA P9. 30 TRMT61B rs11684695 TT genotype displays the highest methylation levels of RNR2 RNA among GG, TG and TT genotypes. 30They also demonstrated that genetic variants associated with altered RNA modification levels were disease-causing among several disorders, such as abnormal blood pressure, breast cancer and psoriasis. 30Therefore, it is biologically reasonable that potential functional SNPs that affect the expression and function of TRMT6 may influence disease susceptibility.
Despite the exciting findings of the study, limitations are unavoidable.First, this study enrolled only participants of Han Chinese ethnicity.Therefore, our results may not be directly extrapolated to different ethnic groups.Second, the sample size was relatively moderate, especially the number of cases.Third, we did not explore the effects of these SNPs on clinical outcomes because we failed to obtain relevant information.Finally, function analyses should be conducted for the gene and significant SNPs.
In conclusion, we identified three hepatoblastoma susceptibility SNPs of the TRMT6 gene.These findings may facilitate the de-

CO N FLI C T O F I NTER E S T S TATEM ENT
None declared.
1,3-5 Wang et al. reported that the knockdown of TRMT6 impaired glioma cells' proliferation, migration and invasion, as revealed by CCK8, colony formation, Edu, transwell Stratification analysis of the association between TRMT6 genotypes and hepatoblastoma susceptibility.
4These studies indicate that TRMT6 is closely implicated in cancer.Ali et al. unveiled associations between genetic alterations and TA B L E 2 Abbreviations: AOR, adjusted odds ratio; CI, confidence interval.a Adjusted for age and sex, omitting the corresponding stratification factor.