TP53 Arg72Pro polymorphism and neuroblastoma susceptibility in eastern Chinese children: a three-center case–control study

Abstract TP53 is a tumor suppressor gene that regulates cell growth, apoptosis and DNA repair. Previous studies have reported the contribution of TP53 Arg72Pro (rs1042522 C>G) polymorphism to pathogenesis of multiple tumors. Hence, we evaluated the association between this polymorphism and neuroblastoma susceptibility in eastern Chinese children. The Taqman genotyping assay was performed in 373 patients and 762 controls. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the strength of the association. No significant association was found between the TP53 gene rs1042522 C>G polymorphism and neuroblastoma susceptibility in the overall analysis (CG vs. CC: adjusted OR = 0.92, 95% CI = 0.70–1.22, P=0.567; GG vs. CC: adjusted OR = 0.99, 95% CI = 0.69–1.42, P=0.947; CG/GG vs. CC: adjusted OR = 0.94, 95% CI = 0.72–1.23, P=0.639; or GG vs. CC/CG: adjusted OR = 1.04, 95% CI = 0.75–1.43, P=0.814) and stratified analysis by age, gender, sites of origin, and clinical stages. The TP53 gene rs1042522 C>G polymorphism may not be a risk factor for neuroblastoma in eastern Chinese children. Future studies are needed to confirm this negative result and to reveal additional functional TP53 variants predisposing to neuroblastoma.


Introduction
Neuroblastoma is the most common extracranial malignant solid tumor in children, affecting 25-50 individuals per million worldwide [1], while approximately 7.7 per million in Chinese subjects [2]. Neuroblastoma accounts for 8-10% of childhood malignancies and 10% of tumor deaths [3]. It is an embryonic tumor originating from the primitive neural crest. Lesions are frequently observed in the adrenal gland, retroperitoneum, mediastinum or any spaces flanking the paraspinal sympatheti ganglia. Neuroblastoma is phenotypically hetergeneous. Patients with low-and intermediate-risk neuroblastoma, approximately 60% of cases, benefit from modern multimodality treatments and achieve greater than 95% overall survival [4]. In contrast, only 50% children with high-risk disease can attain long-time survival [4]. Neuroblastoma is also a genetic complex disease. Various genomic features including somatic genetic aberrations, chromosome copy number, transcriptomics, and epigenetics have all implicated in neuroblastoma pathogenesis [1]. However, regarding neuroblastoma risk, no defined environmental risk factors, not even maternal exposures to some harmful materials, have been established, to date. With the advances in the technology, such as genome-wide association study (GWAS), it has been unveiled that germline variations often predispose children to sporadic neuroblastoma, including single-nucleotide polymorphisms (SNPs) in CASC15, BARD1, LMO1, HACE1, LIN28B, MLF1, and CPZ genes [5][6][7][8][9].
p53 is an important tumor suppressor. Once activated, p53 initiates DNA repair or cell death depending on the degree of damage to maintain genome integrity and stability [10]. Because of its functional importance, TP53 gene mutations and functional SNPs that affect its tumor suppressing functions are detected in various types of tumors [11,12]. One well-known TP53 functional SNP is a genetic polymorphism Arg72Pro at codon 72 (rs1042522 C>G). This SNP has been well characterized, which can modify cancer susceptibility by affecting the function of p53 [13][14][15][16]. Numerous studies have indicated the association between the rs1042522 polymorphism and the risk of a variety of tumors, including breast cancer, prostate cancer, thyroid cancer, bladder cancer, hepatocellular carcinoma, cervical cancer, osteosarcoma, and Wilms tumor [17][18][19][20][21][22][23][24][25]. However, researches on the association between this SNP and neuroblastoma risk are very few. Therefore, we analyze the association in the eastern Chinese children population with 373 neuroblastoma patients and 762 healthy controls.

Study population
A total of 179 histopathologically confirmed neuroblastoma patients were enrolled from Anhui Provincial Children's Hospital, Anhui, China. All the cases were newly diagnosed between October 29, 2008 and October 31, 2019. Moreover, 264 age-and gender-matched healthy volunteers, residing in the same region, were chosen as controls (Supplementary Table S1). Beside, an additional of 158 cases and 426 controls were enrolled from Children's Hospital of Nanjing Medical University, Jiangsu, China) [26,27], and 36 cases and 72 controls enrolled from The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou area, China [28,29]. The Demographic characteristics for the 373 neuroblastoma cases and 762 controls were shown in Supplementary Table  S2. Peripheral blood samples from all participants were taken to extract DNA prior to chemotherapy or radiation therapy. The present study was approved by the Ethics Committee of each hospital, while written informed consent was obtained from all the subjects' parents or their legal guardians.

Genotyping
The genotyping of TP53 rs1042522 C>G was performed by Taqman real-time PCR method using a 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Taqman probes for allelic discrimination were purchased from ABI company [30][31][32].

Statistical analysis
Statistical analysis was performed using the SAS software (version 9.1; SAS Institute, Cary, NC). The deviation from Hardy-Weinberg equilibrium was tested for the TP53 rs1042522 C>G polymorphism in control subjects by a goodness-of-fit χ 2 test. The association between TP53 rs1042522 C>G and neuroblastoma susceptibility was estimated by odd ratios (ORs) and 95% confidence intervals (CIs) using an unconditional logistic regression adjusted by age and gender. All tests were two-sided, and P value less than 0.05 were considered significant.

Clinical characteristics
The distributions of demographic characteristics of all participants are presented in Supplementary Table S2   Abbreviations: CI, confidence interval; OR, odds ratio. a Adjusted for age and gender, omitting the corresponding stratification factor.

Stratification analysis
Stratification analysis by age, gender, and tumor sites were further performed to explore the association between rs1042522 C>G polymorphism and susceptibility to neuroblastoma. As shown in Table 2, no significant association was found.

Discussion
TP53 is an important tumor suppressor gene, which is located on the short arm of chromosome 17; p53 protein plays a critical role in regulating cell growth, differentiation, and apoptosis [15]. The disruption or abnormally low transcription of TP53 gene can impair the tumor suppressor function of the p53 signaling pathway, thereby promoting tumorigenesis and malignant progression [33]. The polymorphic locus rs1042522 C>G is located in exon 4 of the TP53 gene, which leads to the conversion of arginine (Arg) to proline (Pro) at the 72nd codon [13]. Several lines of evidence has shown that this SNP is able to affect the function of p53, thereby modifying cancer susceptibility [13][14][15][16]. p53 is a transcription factor. It was revealed that the p53Arg and p53Pro displayed differential ability to transcriptionally activate target genes to trigger apoptosis, and to inhibit malignant transformation of cells [15]. Although the TP53 gene rs1042522 C>G has been broadly studied for its role in cancer susceptibility [17][18][19][20][21][22][23], it should be noted that there are very few publications on the association between this SNP and neuroblastoma in childhood. In the present study, we aimed to explore the association between the TP53 gene rs1042522 C>G and neuroblastoma in an eastern Chinese population. We genotyped this SNP in 373 neuroblastoma cases and 762 controls, and found that rs1042522 C>G might be not significantly associated with neuroblastoma susceptibility. In addition, stratification analysis by age, gender, and tumor sites suggested that CC genotype appears to have pathogenic effects in subjects not older than 18 months, but these results did not reach statistical significance.
He et al. previously investigated the association between the TP53 gene rs1042522 C>G polymorphism and susceptibility to neuroblastoma in southern Chinese children [34]. With 256 patients and 531 controls, they reported no significant findings in main analysis, but significant association were detected in stratified analysis [34]. In Europe, Cattelani et al. [35] found that TP53 gene rs1042522 C>G has no significant effect on the risk of neuroblastoma with 286 neuroblastoma patients and 288 healthy controls, but may be an indicator of poor prognosis. Taken together, it suggested that the rs1042522 C>G polymorphism may have a low-penetrant effect on the neuroblastoma risk. Large, or multi-center studies because of low incidence of the disease, are encouraged to dissect the effect of this SNP on neuroblastoma risk.
Several limitations should be noted. First, neuroblastoma is a multifactorial disease caused by the interaction between environmental factors and genetic background. Therefore, environmental exposures should have been also considered if data were available. Second, in the present study, only rs1042522 C>G in the TP53 gene was investigated. Many other cancer susceptibility SNPs in the TP53, such as rs78378222 and rs35850753 [36], should be also investigated in the neuroblastoma.
By analyzing the association between TP53 gene rs1042522 C>G and the risk of neuroblastoma in eastern Chinese children, we found that TP53 gene rs1042522 C>G has no significant effect on susceptibility to neuroblastoma. However, due to the limitations of the sample size and observational indicator in the present study, multi-center investigations and analysis of other functional SNPs by whole-genome sequencing are required in the future to further validate our research result.