Prediction of gastric cancer risk: association between ZBTB20 genetic variance and gastric cancer risk in Chinese Han population

Abstract Background: Gastric cancer (GC) is a complex multifactorial disease. Previous studies have revealed genetic variations associated with the risk of gastric cancer. The purpose of the present study was to determine the correlation between single-nucleotide polymorphisms (SNPs) of ZBTB20 and the risk of gastric cancer in Chinese Han population. Methods: We conducted a ‘case–control’ study involving 509 GC patients and 507 healthy individuals. We selected four SNPs of ZBTB20 (10934270 T/C, rs9288999 G/A, rs9841504 G/C and rs73230612 C/T), and used logistic regression to analyze the relationship between those SNPs and GC risk under different genetic models; multi-factor dimensionality reduction (MDR) was used to analyze the interaction of “SNP–SNP” in gastric cancer risk; ANOVA and univariate analysis were used to analyze the differences in clinical characteristics among different genotypes. Results: Our results showed that ZBTB20 rs9288999 is a protective factor for the risk of gastric cancer in multiple genetic models, of which the homozygous model is the most significant (OR = 0.48, P=0.0003); we also found that rs9288999 showed a significant correlation with reducing the risk of gastric cancer in different subgroups (BMI; age; gender; smoking or drinking status; adenocarcinoma); rs9841504 is associated with increased GC risk in the participants with BMI>24 kg/m2; rs9841504 and rs73230612 are certainly associated with clinical characteristics of platelet and carbohydrate antigen 242, respectively. Conclusion: Our results suggest that ZBTB20 rs9288999 may be important for reducing the risk of GC in the Chinese Han population.


Introduction
Gastric cancer (GC) is considered to be a common gastrointestinal tumor in the world, the incidence is second only to lung cancer, breast cancer and colorectal cancer, and the survival rate is low (1). The formation of gastric cancer is a complex process. As a multifactorial disease, gastric cancer is affected by both the environment and genetics (2). A number of studies have confirmed that genetic factors are important causes of gastric cancer: Mohammad et al.
suggested that people who are directly related to gastric cancer patients have a higher risk of gastric cancer than normal people (3); Kaurah et al. found that 30%-40% of patients with familial diffuse gastric cancer carry the CDH1 gene mutation (4). As we all know, single nucleotide polymorphisms (SNPs) are the main form of genetic differences between individuals. With the development of molecular epidemiology and the improvement and application of genetic testing techniques, genetic polymorphisms associated with GC susceptibility have been identified in different populations (5)(6)(7)(8)(9). etc. Table 1 Characteristics of patients with gastric cancer (GC) and healthy individuals.

Information about genotyping and candidate SNPs
Four candidate SNPs (rs10934270, rs9288999, rs9841504, rs73230612) on ZBTB20 were successfully genotyped. Detailed information about these four candidate SNPs was summarized in Table 2. All candidate SNPs were in HWE (p > 5%), and they are all located in the intron region.

Correlation assessment of ZBTB20 SNPs and GC risk (overall analysis)
The correlation between SNPs and GC risk under multiple genetic models was tested based on logistic regression, and the results were corrected by age and gender. The results showed that among the 4 candidate SNPs in this study, only rs9288999 had a certain correlation with gastric cancer risk. Specifically: rs9288999 of ZBTB20 is a protective factor (OR < 1) for the risk of There is no correlation between the remaining three candidate SNPs and the risk of gastric cancer.
The above results are summarized in Table 3.  Table 4.

Smoking or drinking:
The results show that rs9288999 was associated with reducing the risk of gastric cancer among participants who do not smoke or drink alcohol (OR < 1, p < 0.05) in homozygous (GG vs. AA) and recessive models (GG vs. GA-AA). In this subgroup analysis, there was no association between the remaining three candidate SNPs and risk of GC. The specific information is shown in Table 5.
In addition, we also divided the gastric cancer cases in this study according to the pathological grade (I & II vs. III & IV) and whether the lymph nodes metastasized. No association have been found between candidate SNPs and gastric cancer risk (supplemental table 2). "-" indicates Log-additive model.

MDR analysis
Subsequently, MDR analysis was used to assess the interaction of 'SNP-SNP'. Figure 1 shows the interaction between the four candidate SNPs of ZBTB20. The blue line indicates that these four SNPs may have a redundant role in regulating the risk of diabetes. All experimental results have been shown in Table 8. The best unit point model for predicting the risk of gastric cancer is: rs9288999 (testing accuracy = 0.521, CVC = 10/10, p = 0.0006); the two-site model is:rs9288999, rs73230612 (testing accuracy = 0.535, CVC = 10/10, p = 0.0003); the three-site model is: rs10934270, rs9288999 and rs73230612 (testing accuracy = 0.500, CVC = 7/10, p < 0.0001); the four-site model is: rs10934270, rs9288999, rs9841504 and rs73230612 (testing accuracy = 0.517, CVC = 10/10, p < 0.0001). Figure 2 shows the interaction of 'SNP-SNP' in different combinations of sites, among them, the light gray grid represents a low risk of gastric cancer, the darker gray grid represents a higher risk of gastric cancer, and the unfilled grid represents no data. Therefore, we can conclude that the impact of these four candidate SNPs of ZBTB20 on the risk of gastric cancer may be interdependent.

Differences in clinical indicators under different genotypes
Finally, we also evaluated the correlation between the four candidate SNPs polymorphisms (rs10934270, rs9288999, rs9841504, rs73230612) and clinical indicators of gastric cancer patients.
These clinical indicators include carcinoembryonic antigen (CEA), tumor necrosis factor (TNF),    There are differences in the incidence of gastric cancer even between north and south of China (16).Wherefore , it is very necessary to conduct gastric cancer association studies in different populations. These research results are of great significance for us to understand the susceptibility mechanism of gastric cancer, to explore the pathogenesis of gastric cancer, the risk prediction and screening of high-risk groups, and to guide the individualized treatment of gastric cancer.
Some cancer-related studies have found that NF-Κb (nf-kappa b) may cause oncogenesis, Therefore, in our study, the Chinese Han population was used as the research object to conduct a 'case-control' study, then we analyzed the correlation between ZBTB20 gene polymorphisms (rs10934270, rs9288999, rs9841504, rs73230612) and gastric cancer risk. The Based on previous studies and our study, the cause of the above results may be the interaction between environmental factors and genetic polymorphisms. what's more, ZBTB20 rs9288999 may play a certain role in that.
In addition, exploring the interaction between SNP-SNP can also help us to discover potential risk factors for the incidence of gastric cancer. Therefore, MDR was used to explore the interaction between the four candidate SNPs. The results showed that for gastric cancer risk, rs10934270, rs9288999, rs9841504 and rs73230612 showed a strong interaction.
In the correlation analysis between clinical indicators and gastric cancer risk, we also found that the ZBTB20 polymorphic site rs9841504 and platelet showed a certain significant correlation (p = 0.048). Platelet involves in cancer growth and metastasis is a long-term concept (41), and This study provides data supplements for the association between the ZBTB20 gene polymorphisms and the risk of gastric cancer in the Chinese Han population, and concludes that there may have certain association between the two. However, we must face the fact that our research has certain limitations, which is not only for the confirmation of results but also for new discoveries, a large sample size is indeed necessary. Currently, the genetic regions/sites discovered are only a small part, and there are more genetic susceptible sites/regions waiting to be discovered.
With the discovery of susceptibility sites for gastric cancer in the future, we will have a more comprehensive understanding of the genetics of gastric cancer.

Conclusion
In summary, our study is the first to find that the rs9288999 of ZBTB20 has a potential association with reducing the risk of gastric cancer in the Chinese Han population. It provides new data supplement for the study of the association between ZBTB20 gene polymorphism and gastric cancer risk.   Table 1 Characteristics of patients with gastric cancer (GC) and healthy individuals. Table 2 The basic information and HWE about the selected SNPs of ZBTB20. Table 3 Analysis of the association between susceptibility of gastric cancer and single nucleotide polymorphism of ZBTB20. Table 4 The SNPs of ZBTB20 associated with susceptibility of gastric cancer in the subgroup tests (age and gender). Table 5 The SNPs of ZBTB20 associated with susceptibility of gastric cancer in the subgroup tests (smoking and drinking). Table 6 The SNPs of ZBTB20 associated with susceptibility of gastric cancer in the subgroup tests (BMI). Table 7 The SNPs of ZBTB20 associated with susceptibility of gastric cancer in the subgroup tests (adenocarcinoma). Table 8 SNP-SNP interaction models analyzed by the MDR method. Table 9 Clinical characteristics of patients based on the genotypes of selected SNPs. The tittles and legends of figures: Tittle: Figure 1 Dendrogram analysis of SNP-SNP interaction.

Figure legends
The colors in the tree diagram represent synergy (yellow) or redundancy (blue).

Figure legends
In each box, the left bar represents cases and the right bar represents controls. The light gray boxes indicate the low risk of gastric cancer and dark gray boxes indicate the high risk, the empty boxes mean no data.