Case-control Study on p73 rs1801173 C > T Gene Polymorphism and Susceptibility to Gastric Cancer in a Chinese Han Population

Objective: This study aimed to investigate the association between p73 C14T (rs1801173) polymorphism and the risk of GC in a Chinese Han population. Methods: A hospital-based case-control study was conducted. A total of 577 GC cases and 678 normal controls were recruited. Their genotypes were determined using the SnapShot method. Results: The genotype frequency distribution of the case group and the control group were consistent with the Hardy–Weinberg equilibrium. No signicant difference was found in the distribution of gender, age, and drinking history between the case group and the control group. A correlation was observed between smoking and the incidence of GC (P = 0.006). Three genotypes of CC, CT, and TT were found in the rs1801173 locus of p73. The distribution of the dominant model/recessive model did not signicantly differ (P = 0.688; 0.937). No statistical difference was found even after adjustment was performed via logistic regression analysis (P = 0.703; 0.990). The frequency distribution between the two groups also did not signicantly differ (P = 0.763). Conclusion: Smoking is related to the occurrence and development of GC. No association was found between p73 rs1801173 C > T SNP and the risk of GC in a Chinese Han population. However, additional larger studies and tissue-specic biological characterization are required to conrm these ndings. studies are required. Third, the statistical power was limited because of the moderate sample size and the absence of a validation cohort. In addition, considering that gene–gene interaction and gene–environment interaction play an important role in the pathogenesis of many diseases, especially chronic diseases, p73 gene polymorphism may interact with other gene polymorphisms or environmental factors, thereby affecting the incidence of GC in the population. H. pylori infections, lifestyle, and dietary regime were not investigated. Other limitations included small sample size and sampling of individuals of the same geographical region and race. Therefore, further studies considering different geographical locations and races and a larger number of participants are necessary to conrm the results of the present study. These results failed to indicate an association between p73 rs1801173 polymorphism and risk of GC. Tissue-specic biological characterization and replication studies with larger populations are also required to conrm these ndings.


Introduction
Gastric cancer (GC) is one of the most common malignant tumors, ranking third in the number of cancer deaths worldwide [1] . The latest statistics from the Chinese National Cancer Center from a report in February 2018 showed that although the overall incidence of GC is declining, it still remains second in terms of incidence among all malignancies in China, just below lung cancer [2] . Despite remarkable progress, the current treatments for GC remain ine cacious, with overall 5-year survival rates < 30%. At the time of diagnosis, most patients have been diagnosed with advanced cancer and metastasis [3] . GC invasion and metastasis are complex processes involving multiple factors. Increasing evidence has proven that environmental and genetic factors contribute to the occurrence and development of GC. In addition to environmental risk factors, such as diet and HP infection, single nucleotide polymorphism (SNP) may play an important role in GC cancerization as a genetic factor. SNPs are polymorphisms in DNA sequences caused by mutations in the genome's single nucleotides. They are the most common form of human genetic variation, accounting for > 90% of all known morphologies [4] . Understanding the underlying mechanisms of GC initiation and progression may promote biomarker development for early detection of cancer. Previous epidemiological studies found increasing numbers of genetic variations that in uence GC susceptibility [5] . However, the association of the polymorphisms in the genes and GC susceptibility remains largely unknown. Hua et al. found that genetic variations in LIG3 may play a weak role in modifying the risk of GC [6] . He et al. systematically analyzed the associations between nine polymorphisms in four key genes (XPA, ERCC1, ERCC2, and ERCC4) in the nucleotide excision repair pathway and the risk of GC in a Chinese population. They concluded that the ERCC1 polymorphisms may affect the risk of GC in the Chinese Han population [7] . They also studied the genetic variations of mTORC1 and risk of GC in an Eastern Chinese population and found that the functional polymorphisms of mTOR may contribute to the risk of GC [8] . In 2004, the authors of the present study found that p73 rs1801173 C > T polymorphisms are associated with an increased risk of esophageal cancer in a Chinese population [9] .
Tumor protein p73 (TP73), also known as p53-like transcription factor, is a pivotal member of the p53 (TP53) family, which affects cell proliferation, apoptosis, and cell-cycle regulation. p73 is located at 1p36.33, mapping to a region that is often deleted in cancers [10] . p73 activates the transcription of p53-responsive genes, which participate in cell-cycle control, DNA repair, and apoptosis and inhibits cell growth in a p53-like manner by inducing apoptosis or G1 cell-cycle arrest [11] . Thus, p73 has tumor-suppressor functions. p73 has some signi cant differences from p53. In contrast to p53-de cient mice, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis [12] .
At least 19 polymorphisms have been recognized in p73 at present. Ten polymorphisms are in the exon region, eight are in the intron region, and one is in the region of gene promoter. However, the functionality of most of these polymorphisms has not been recognized yet [13] . The 1p36.3 region, where p73 is coded, is deleted in different malignancies via loss of heterogeneity. Although p73 mutation is rare and occurs in less than 2% of all cancers, the lack of heterogeneity in locus p73 is common in a variable rate in different cancers [14] . As a result, the genetic changes in p73 are critical in malignancies [15] . Two related SNPs in regions 4 (G > A) and 14 (C > T) of exon 2 in p73 resulted in the formation of a stem-loop structure that could affect the gene function via changes in its expression. Molecular epidemiology studies have indicated an association between this polymorphism and the risk of cancers in human; however, the results were inconsistent [16] .
Previous research demonstrated that p73 gene polymorphisms are related to the occurrence of various cancers, including esophageal cancer [17] , rectal cancer [18] , breast cancer [19] , lung cancer [20] , and cervical cancer [21] . However, no research could be found on p73 gene polymorphism and GC susceptibility. This study aimed to investigate the relation between C14T polymorphism in p73 and the risk of GC in a Chinese Han population.

Ethical approval of the study protoco
This study complied with the World Medical Association Declaration of Helsinki in terms of ethical conduct of research involving human subjects and/or animals and received approval from the Review Board of Jiangsu University (Zhenjiang, China). Written informed consent was provided by all the subjects in this study.

Materials And Methods
Between May 2013 and June 2017, 577 subjects with GC were recruited consecutively at the A liated People's Hospital of Jiangsu University (Zhenjiang, China). Their average age of the case group was 61.34 ± 11.097 years, with 394 males and 183 females. All cases were pathologically diagnosed as GC. The exclusion criteria were as follows: patients who previously had cancer and those with any metastasized cancer and undergoing radiotherapy or chemotherapy.
As controls, 678 patients without cancer were matched to the cases in terms of age (± 5 years) and sex. Their average age was 62.31 ± 7.549 years, with 456 males and 222 females. The controls were recruited from the abovementioned hospital during the same time period. Both groups are Chinese Han people, and they are not related by blood. Moreover, We had access to information that could identify individual participants during or after data collection.
Using a pre-tested questionnaire, trained interviewers questioned each subject personally and obtained demographic data information (e.g., age and sex) and related risk factors (such as tobacco smoking and alcohol consumption). Venous blood samples (2 mL) were collected after each interview. The de nition of "smokers" was smoking one cigarette per day for > 1 year. The de nition of "alcohol drinkers" was consumption of ≥ three alcoholic drinks a week for > 6 months.
Blood samples were collected from patients by using vacutainers and transferred to tubes lined with ethylenediamine tetra-acetic acid. Genomic DNA was isolated from whole blood by using a QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany). Sample DNA was ampli ed via PCR in accordance with the manufacturer's recommendations. The samples were genotyped using the SnapShot method, with technical support from Shanghai Biowing Applied Biotechnology Co. as previously described. For quality control, repeated analyses were performed on 160 (12.17%) randomly selected samples with high DNA quality.

Statistical analyses
The distributions of demographic characteristics, selected variables, and genotypes of the p73 variant differences between the cases and controls were evaluated using the χ2 test. Logistic regression analyses was used to estimate the associations between the SNPs and the risk of GC for crude and adjusted ORs when adjusting for age, sex, smoking, and drinking status. The Bonferroni correction procedure was also applied because of the number of comparisons.
The Hardy-Weinberg equilibrium (HWE) was tested using the goodness-of-t χ 2 test to compare the observed genotype frequencies to the expected ones among the controls. All statistical analyses were performed on SPSS 20.0 (SPSS Inc., Chicago, Illinois, USA). Table 1 shows that the rs1801173 of p73 was located in the rst chromosome. Their category was protein cod. The minor allele frequency of rs1801173 in the controls was 0.267. The result of HWE tests for the controls was 0.229 (P > 0.05), indicating that the sample population was representative. The snapshot method was used for genotyping, and the percentage of successful tests was 98.96%.    Table 3 The frequency distribution and logistic regression analysis of the p73 gene rs1801173 polymorphism in gastric cancer and control group showed that with reference to wild-type CC, the frequency distribution of TC heterozygous mutations in GC cases was 60.67% higher than 59.55 in controls group, but there was not statistically signi cant between the two groups (P = 0. 657) and there was no statistical difference in gender, age, smoking, and alcohol consumption after logistic regression adjustment (P = 0.691); the frequency distribution of TT homozygous mutants was 5.52% higher than 5.41 in controls group, but was also not statistically signi cant (P = 1.000), and there was no statistical difference after logistic regression adjustment (P = 0.979). In the dominant model, the frequency distribution of TC + TT mutations was 39.33% lower than 40.45% in controls group, but there was not statistically signi cant in the case-control group (P = 0.688), and the difference was not statistically signi cant after regression adjustment (P = 0.703). In the recessive model, the frequency distribution was 5.52% higher than 5.41 in controls group, but there was not statistically different (P = 0.937). According to gender, age, smoking, and drinking, after logistic regression analysis, there was still no statistical difference between the two groups (P = 0.990).The results of rs1801173 allele frequency distribution of p73 gene in the two groups showed that the allelic frequency distribution was not statistically signi cant in the case-control group (P = 0.763).  Table 4 shows that regardless of the gender, the distribution frequency of the four modes, namely, mutant, wild-type, recessive model, and dominant model, had no difference in the distribution of the two groups, suggesting that gender and p73. No statistical correlation was found between the four genetic models and the occurrence of GC. Age, smoking, drinking, and temporarily also showed no statistical association. However, a notable detail that in Table 1, a statistically signi cant difference was found in the association between smoking and the occurrence of GC. In the hierarchical analysis, the genetic model of the four genes did not show a statistically signi cant association, which is worthy of follow-up study to investigate and determine whether smoking factors are related to the rs1801173 polymorphisms of p73.

Discussion
GC is a multifactorial disorder, in which genetic and environmental interactions serve an important role in the development and progression [23] . Increasing age, gender, lifestyle, dietary regime, environmental factors, and Helicobacter pylori infections are among the known risk factors for stomach cancer [24] . While dietary regime and lifestyle are the most recognized factors, enhanced identi cation of the genetic risk factors is expected to improve the understanding of the basic molecular events involved in tumorigenesis [25] . Genetic factors, including gene expression pro le and cancer biomarkers, such as SNPs, have a crucial role in improving the early diagnosis of GC [26] .
Recent studies have demonstrated that a high number of genes and various environmental factors are the causal agents of GC, and the presence of different forms of alleles in gene polymorphisms may promote the development of cancers. In this regard, p73 has been a research focus due to its role as a major tumor suppressor gene [27] . p73 has some functions similar to or independent of p53 and plays a role, particularly in compensation for loss of p53 function, in the regulation of cell cycle, DNA repair, apoptosis, and possibly cell differentiation. In the past decades, almost 146 unique variations were reported (shown in the Biomuta database) [28] , while numerous studies probed into the relationship of G4C14-A4T14 polymorphism and cancer genomics. G4A (rs2273953) and C14T (rs1801173) polymorphisms are located at positions 4 (G to A) and 14 (C to T) of exon 25′-untranslated region, which may in uence the initiating AUG codon by constructing a stem loop [29] . In recent years, the G4C14-A4T14 polymorphism of p73 has been identi ed to be implicated in the tumorigenesis of various cancer types. However, the data from these published case-control studies were not consistent.
Yang et al. [30] and Niwa et al. [31] reported that G4C14-A4T14 polymorphism is not associated with the susceptibility of cervical cancer in Uighur and Japanese population, respectively. However, Craveiro et al. [32] and Feng et al. [33] revealed that G4C14-A4T14 polymorphism leads to an increasing risk of cervical cancer.
Hamajima et al. [34] demonstrated no signi cant differences in the genotype frequencies among the enrolled cases and controls in their study of colorectal cancer. On the contrary, Lee et al. [35] reported that GC/AT and AT/AT genotypes are signi cantly associated with the risk of colorectal cancer in Korean population. Arfaoui et al. [36] also uncovered no remarkable differences in genotype frequencies in cancers and controls, but they found that AT/AT genotype may cause poor prognosis of colorectal cancer. Hu et al. [37] indicated that AT/AT and GC/AT variants are associated with a remarkable decrease in the risk of lung cancer. Li et al. [38] suggested that the AT/AT and GC/AT genotypes are related, with a statistically signi cantly increased risk of lung cancer. However, Choi et al. [39] revealed that the G4C14-A4T14 polymorphism of p73 does not affect the susceptibility of lung cancer in Korean population. Zheng et al. [9] found that the rs1801173 C > T SNPs of p73 are associated with increased risk of ESCC. However, p73 gene polymorphism and GC susceptibility have not been reported yet. Therefore, the rs1801173 C/T polymorphism of p73 merits further functional study to elucidate the etiology of SNP and GC.
In the present study, no statistically signi cant association was found between p73 rs1801173 C > T gene polymorphism and the risk of GC in a Chinese Han Population. Although the C mutant allele frequency was higher in patients with GC than in controls, the difference was not statistically signi cant. However, the frequency of smoking factors in the case group was 34.49%, higher than that in the control group by 27.29%, and the difference was statistically signi cant. This result showed that smoking is related to the occurrence and development of GC. Li et al. [40] studied the connection between p73 G4C14-to-A4T14 polymorphism and the risk of lung cancer. They found that the increased risk associated with the combined p73 GC/AT + AT/AT genotype in younger (≤ 50 years) subjects and light (compared with heavy) smokers suggested an early onset and lower levels of exposure characteristic of genetic susceptibility. They also observed a signi cantly higher risk in men than in women, particularly among smokers. However, the interaction between smoking and p73 polymorphism was only borderline signi cant, which warrants additional investigations with larger sample sizes.
This case-control study had several limitations. First, because the patients and controls were enrolled from hospitals, inherent bias may have resulted in spurious ndings. Second, the polymorphisms may not provide a comprehensive view of p73 genetic variability. Fine-mapping studies are required. Third, the statistical power was limited because of the moderate sample size and the absence of a validation cohort. In addition, considering that gene-gene interaction and gene-environment interaction play an important role in the pathogenesis of many diseases, especially chronic diseases, p73 gene polymorphism may interact with other gene polymorphisms or environmental factors, thereby affecting the incidence of GC in the population. H. pylori infections, lifestyle, and dietary regime were not investigated. Other limitations included small sample size and sampling of individuals of the same geographical region and race. Therefore, further studies considering different geographical locations and races and a larger number of participants are necessary to con rm the results of the present study. These results failed to indicate an association between p73 rs1801173 polymorphism and risk of GC. Tissue-speci c biological characterization and replication studies with larger populations are also required to con rm these ndings.

Data Availability
The data used to support the ndings of this study are available from the corresponding authors upon request.
Ethical Approval