Correlation between IL‐7R gene variants and breast cancer susceptibility in Chinese Han women

IL-7R is involved in the occurrence and development of breast cancer by binding to its ligand IL-7. This study aimed to explore the potential relationships of IL-7R polymorphisms with breast cancer susceptibility in Chinese Han women. Methods Five polymorphisms (rs969129, rs10213865, rs10053847, rs118137916, and rs6451231) of IL-7R genewere genotyped in 553 patients and 550 healthy healthy individuals from the in Chinese Han women using the Agena MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated used to evaluate the relationship. Results IL-7R rs10213865, rs969129 and rs6451231 was correlated with an increased the risk of breast cancer in multiple genetic models. Age stratified analysis revealed that rs6451231 was correlated with an increased breast cancer risk at age > 52 years. Additionally, rs10213865 was correlated with tumor site and ER expression, rs969129 was related to tumor size and Ki67 expresses status, and rs6451231was related to tumor size. Haplotype CGAG (rs969129, rs10213865, rs10053847 and rs118137916) were observed to a decrease breast cancer risk. IL-7R were


Introduction
IL-7R is involved in the occurrence and development of breast cancer by binding to its ligand IL-7. This study aimed to explore the potential relationships of IL-7R polymorphisms with breast cancer susceptibility in Chinese Han women.

Methods
Five polymorphisms (rs969129, rs10213865, rs10053847, rs118137916, and rs6451231) of IL-7R genewere genotyped in 553 patients and 550 healthy healthy individuals from the in Chinese Han women using the Agena MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated used to evaluate the relationship.

Results
IL-7R rs10213865, rs969129 and rs6451231 was correlated with an increased the risk of breast cancer in multiple genetic models. Age stratified analysis revealed that rs6451231 was correlated with an increased breast cancer risk at age > 52 years. Additionally, rs10213865 was correlated with tumor site and ER expression, rs969129 was related to Introduction Breast cancer, with about 2.1 million new cases and 0.6 million deaths in GLOBOCAN 2018 data, has been recognized as the most common type of cancer and a main cause of cancer death among women worldwide [1]. The data from China's urban cancer registration institutions showed that the incidence of breast cancer has increased by 20-30%, and the annual growth rate was 35% in the past 30 years. It is estimated that by 2021, the number of breast cancer cases in Chinese women aged 35 and 49 will reach 2.2 million [2].
Although the current development of medical technology has contributed to reduce the death rate of breast cancer, the specific cause of breast cancer has not been clarified. In addition to environmental factors, including radiation exposure and lifestyle, genetic factors are also crucial to the development of breast cancer. These genetic factors include single nucleotide polymorphism (SNPs) in the genome, and genetic variations in susceptible genes resulting in about 5-10% breast cancer [3,4].
Interleukin 7 receptor (IL-7R) is one of the Ⅰ type cytokines receptors family members secreted by stromal cells, and its coding protein is interleukin-7 (IL-7) receptor [5].
Previous studies have shown that IL-7 binds to IL-7R can was essential for inflammatory or the immune response [6,7]. Recently, increasing studies have shown that IL-7R influences the occurrence of various tumors, such as breast cancer, by forming signal complexes with its ligand IL-7 [8]. Binding IL-7R to IL-7 triggers a cascade of phosphorylation induced by signaling molecules that also participated in cell behavior such as cell division, cell adhesion, and cell differentiation [9,10]. Rawi et al. pointed out the abnormal expression of IL-7R and IL-7 in breast cancer, and also found that IL-7 could accelerated the growth of breast cancer cells through wortmannin-sensitive pathway [11,12]. These studies suggested that IL-7R is closely related to the occurrence of breast cancer. Moreover, a large amount of evidence has been explored that single nucleotide polymorphisms (SNPs) affect an individual's risk of breast cancer [13,14]. However, the relationship between IL-7R polymorphisms and breast cancer risk remains unclear.
Therefore, we conducted an association study based on Chinese Han population to clarify the correlation between IL-7R SNPs and the risk of breast cancer, which is of great significance for the early molecular diagnosis of breast cancer.

Ethics statement
Our research was approved by the First Affiliated Hospital of Xi'an Jiaotong University and conducted in accordance with the ethical standards of the Helsinki declaration and national and international norms. All subjects signed informed consent forms after full understanding of the purpose of this research, agreeing to provide personal and relevant clinical information.

Participates statement
The study finally included 681 cases and 756 controls recruited from the the First Affiliated Hospital of Xi'an Jiaotong University. All subjects of cases were confirmed as breast cancer by pathology, excluding those who had received radiotherapy, chemotherapy, and other types of cancers. Data on the patient's clinicopathological characteristics (such as tumor location, tumor size, progesterone receptor (PR) status, estrogen receptor (ER) status, and Ki-67 status etc) were obtained from the patient's medical records. The control groups were healthy women with no disease and no history of any disease who were recruited from the same hospital for physical examination at the same time. All the participants were Chinese Han population and were not related to each other.

SNP selection and genotyping
In this study, five IL-7R SNPs (rs10213865, rs969129, rs118137916, rs10053847 and rs6451231) with minor allele frequency (MAF) value > 0.05 were selected in present study based on 1000 Genomes Project (http://www.1000genomes.org/) and dbSNP database (https: //www. ncbi.nlm.nih.gov/projects/SNP/). Participants' blood samples were stored at -80℃ in a test tube containing EDTA reagent until analyzed. Whole-blood genomic DNA extraction was performed using the GoldMag whole-blood genomic DNA purification kit (GoldMag Co. Ltd., Xi an, China), and its quality was detected by NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's s guidelines. Primer design was conducted with Agena MassARRAY Assay Design 3.0 Software (San Diego, California, USA) [15]. The corresponding primers of the selected SNPs in this study were listed in Supplementary Table S1. The SNPs genotyping was performed by two professionals using the Agena MassARRAY system (Agena, San Diego, CA, U.S.A.) software in a double-blind manner [16].

Statistical analysis
This study used SPSS 20.0 version and Microsoft Excel to analyze all relevant data, and P < 0.05 was considered statistically significant. The variation frequency of the controls was evaluated by the hardy weinberg-equilibrium (HWE) calculated by Fisher's exact test. The frequency of alleles and genotypes in cases and controls was assessed by the Pearson's χ 2 test, and the relationship between IL-7Rs SNPs and breast cancer risk was analyzed using Odds ratios (OR) and 95% confidence intervals (CI) in multiple genetic model (allele, codominant, dominant, recessive and log-additive). Linkage disequilibrium and haplotype analysis were performed using the the 4.2 version of the Haploview software package.

Results
The Basic information about the subject Basic clinical information of the cases and the control groups were summarized in Table 1. Our study containing 553 patients with an average age of 52.00 ± 9.83 years and 550 controls with an average age of 52.11 ± 9.51 years. There was no statistically significant difference in age distribution between the case group and the control group, and they were matched by age (P = 0.884).

Information about the IL7R SNPs locus
The specific information of the IL-7R polymorphisms were shown in Table 2. All SNPs in the control group were complied with Hardy-Weinberg equilibrium (HWE). In addition, we used the HaploRegv4.1 database to predict the function of IL-7R polymorphisms, and found that the SNPs sites were involved in the Proteins bound, DNAse, and motifs changed etc., suggesting that these SNPs sites had potential biological functions.  Log-additive ---------  were not related to breast cancer susceptibility (P > 0.05).

Stratification analysis of IL7R polymorphisms and breast cancer risk risk by age
To further investigate the impact of age on the potential breast cancer risk of selected IL7R polymorphisms, we stratified analysis by age under multiple genetic, we performed a stratified analysis according to age under multiple genetic models (    As shown in Table 6, we observed that individuals with rs10213865 mutational genotype  Associations between haplotype analyses and the risk of breast cancer Finally, haplotype analysis was performed to explore the relationship between interactions of the IL-7R SNPs and breast cancer risk. As shown in Fig. 1, rs10213865, rs969129, rs118137916 and rs10053847 consisted of one block in IL-7R gene. As shown in Table 7, haplotype CGAG was observed to prominently reduce the risk of breast cancer through χ 2 test analysis (OR = 0.80, 95% CI, 0.65-0.99; P = 0.043).

Discussion
Genetic variations, including single nucleotide polymorphism, were closely related to the occurrence of breast cancer. Genome-wide association studies (GWAS) have detected many SNPs were related to breast cancer susceptibility in tumor-related genes such as PD-1, FGFR2, ESR1, PTEN, and MAP3K1 [17][18][19]. The effect of IL-7R SNPs on breast cancer susceptibility in Chinese Han population is not clear at present. In this present work, we revealed that the allele and genotype frequency distribution of IL-7R SNPs (rs969129, rs10213865 and rs6451231) was significantly different between the case and control groups, and they increased the risk of breast cancer. In the age stratified analysis subgroup, IL-7R-rs6451231 significantly increased breast cancer risk among those older than 50 years. Rs10213865, rs969129 and rs6451231 were also significantly correlated with some clinical parameters, such as tumor size, ER status and Ki67 stress status. We have also observed that haplotype CGAG increases breast cancer susceptibility. These results provided some support for the impact of genetic variation of IL-7R on breast cancer rs10053847 and rs10213865 variant was observed to be correlated with a decreased breast cancer risk under multiple genetic model, and AGAA haplotypes (rs10213865, rs969129, rs118137916, and rs10053847) also increased breast cancer risk. However, our study observed that IL-7R SNPs (rs969129, rs10213865 and rs6451231) were significantly increased breast cancer risk, and haplotype CGAG (rs10213865, rs118137916, rs969129 and rs10053847) significantly increased breast cancer susceptibility. As far as we know, our study is the first to explore the relationship between IL-7R polymorphism and susceptibility to breast cancer.
There are some limitations to this study that cannot be ignored. First, this was a hospitalbased case-control study involving only Han women, and the applicability of our conclusions should be explored in other ethnic populations. Second, our sample size was relatively small, and false negative results cannot be excluded. Despite the above limitations, the results of this study provide a scientific basis for future research on IL-7R gene and breast cancer.
In conclusion, our study was the first to found that IL-7R SNPs (rs6451231, rs10213865 and rs969129) were significantly increased breast cancer risk, which provides a basis for further mechanism research of IL-7R and breast cancer.

ACKNOWLEDGMENTS
We thank all the participants in this study, including clinicians and hospital staff who provided samples for the study and those who contributed to the writing.

Consent for publication
Written informed consent was obtained from the patient for publication of this report.

Availability of data and materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.