Association of GWAS‐supported noncoding area loci rs404860, rs3117098, and rs7775228 with asthma in Chinese Zhuang population

Abstract Background Asthma is a complicated and polygenic inheritance disease, and its prevalence increases worldwide. Recent genome‐wide association studies (GWASs) identified a significant association of single nucleotide polymorphism with asthma in the Japanese population. This study aimed to examine the association of GWAS‐supported noncoding area loci, namely rs404860, rs3117098, and rs7775228, with asthma in Chinese Zhuang population. Methods A case‐control study involving 223 individuals, comprising 123 patients with asthma and 100 healthy controls, was conducted. Genotypes were determined by polymerase chain reaction (PCR)/ligase detection reaction assay. The association between gene polymorphisms and asthma risk was calculated by logistic regression analysis using different genetic models through comparisons of alleles (A vs a), homozygote genotypes (AA vs aa), heterozygote genotypes (Aa vs aa), dominant models (AA+Aa vs aa), and recessive models (AA vs. Aa+aa). Results The distribution of the genotype frequency of rs3117098 was statistically different between the case and control groups. For rs3117098, significant associations were observed through comparisons of alleles (OR: 1.832, 95% CI: 1.048‐3.204, P = .034) and dominant models (OR: 2.065, 95% CI: 1.001‐4.260, P = .050). The statistical analysis showed no significant difference for loci rs404860 and rs7775228 between patients with asthma and controls. Conclusion rs3117098 may be the risk factor for asthma in Chinese Zhuang population.


| INTRODUC TI ON
Asthma is a disease characterized by chronic airway inflammation and recurring respiratory symptoms, such as cough, chest tightness, shortness of breath, wheezing, and variable expiratory airflow limitation. Symptoms and airflow limitation characteristically vary over time in terms of intensity. Asthma also affects 1%-18% of population in different countries, and its prevalence increases in many countries. 1 Hospitalizations and deaths from asthma have declined in various areas, but the cost of asthma treatment remains a burden.
The etiology of asthma remains unclear. Asthma is a complicated and polygenic inheritance disease; familial aggregation and genetic predisposition are significant peculiarities of asthma. [2][3][4][5] Thus, the use of molecular genetic methods to explore the etiology and pathogenesis of asthma has become a research hotspot worldwide. Given the development of human genome haplotype plan data, the reduction in the costs of genome-wide single nucleotide polymorphism (SNP) typing chip, and the emergence of new statistical methods and software, genome-wide association study (GWAS) opens a new chapter in determining the genetic factors of complex diseases. GWAS, including advanced computational methods and rigorous replication, is a new platform for identifying genetic variations in complex polygenic disorders. 6 Hirota et al 7 conducted GWAS and replication study consisting of 7171 subjects with adult asthma and 27 912 controls in Japanese population; their results identified several candidate loci that are associated with susceptibility to adult asthma. Three GWAS-positive mutations are located in noncoding areas, namely rs404860 (risk allele A), rs3117098 (risk allele G), and rs7775228 (risk allele A). SNP rs404860 is located in the NOTCH4 gene in the major histocompatibility complex region, rs3117098 is located in the BTNL2 gene (a member of the immunoglobulin superfamily), and rs7775228 is located in the HLA-DQA2 gene; these genes are inflammation-related genes that may be involved in asthma development. Noncoding regions also regulate the expression of structural genes, which may be processed into various interfering RNAs to regulate the expression of other genes during transcription; these changes result in the corresponding genes being turned on, off, active, or inactive, thereby affecting the body's shape, development, and disease susceptibility. 8 Therefore, noncoding region SNPs that are located in inflammation-related genes should be studied in relation to incidence of asthma in Guangxi, China.
Several GWAS-supported SNPs have been replicated in different independent studies, but the results are inconsistent. The asthma susceptible gene in Chinese minority has also been poorly studied.
Guangxi is part of the Zhuang autonomous region and has the largest minority population in China. The Zhuang population in Guangxi also maintains ancient traditions in terms of language, ethnic culture, and lifestyle. The living environment and climate differ from the habitats of other ethnic groups. Considering the genetic backgrounds and the varying interaction between genes and environment in different races, conducting genetic research in the Guangxi Zhuang population is important. A case-control study that includes patients with asthma and healthy controls in Guangxi Zhuang population was conducted to evaluate the association between GWAS-supported noncoding area loci, namely rs404860, rs3117098, and rs7775228, and asthma.

| Study participants
A total of 123 patients with asthma and 100 healthy controls were nonconsecutively recruited from Guangxi Zhuang autonomous region. All the participants were natives of Guangxi Province and unrelated to one another. These participants belong to the Zhuang ethnic minority and permanently reside in Guangxi (three generations or more). These participants were diagnosed with asthma by at least two respiratory physicians in accordance with the guidelines of the Global Initiative for Asthma. 9 A self-report questionnaire including general conditions, medical history, and family history was

| DNA isolation and SNP genotyping
Genome was extracted from 2 mL of peripheral venous blood by using a DNA extraction kit (Tiangen, Shanghai, China) in strict accordance with laboratory procedures. The presence of the three noncoding SNPs (rs404860, rs3117098, and rs7775228) for asthma was analyzed. Primer design (Primer 3 Online), synthesis, and genotype test were performed by Shanghai BioWing Applied Biotechnology Company (http://www.biowi ng.com.cn/). The primer sequences of the three SNPs are presented in Table 1. All the three SNPs were genotyped using the PCR/ligase detection reaction assay.

TA B L E 1 Primers of target genes used in the PCR
The target DNA sequences were amplified using a multiplex PCR method. The reaction course included initial denaturation for 2 minutes at 95°C, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 53°C for 90 seconds, extension at 65°C for 30 seconds, and a final extension at 65°C for 10 minutes. After the reaction, 2 µL of each product was run in a 3.0% agarose gel to determine whether the reaction was successful. Approximately 5% of the DNA samples were selected randomly and retested under blind conditions to assess the quality of genotypic data. The concordance rate was 100%.

| Statistical analysis
Measurement and enumeration data were analyzed using SPSS 16.0.
Chi-square (χ 2 ) test was used to determine whether the genotype frequency distribution of the control was in the Hardy-Weinberg equilibrium (HWE) and evaluate differences in genotypic distribution between the two groups. The association between the three polymorphisms and risk of asthma was calculated by logistic regres-

| HWE test
Three SNPs, namely rs404860, rs3117098, and rs7775228, were detected in the Guangxi Zhuang population. The results showed that the genotypes were found in all SNPs. Genotyping analyses revealed that rs404860 and rs3117098 were distributed in HWE (P > .05) in the control group. However, the distribution of rs7775228 was not in HWE (P < .05) in the control group.

| Polymorphism of the three SNPs in Zhuang population
The distribution of the genotype frequency of rs3117098 was statistically different between the case and control groups, as shown by χ 2 test (P < .05, Table 3).

| Association of the three SNPs in Guangxi Zhuang population with asthma
Logistic regression was used to assess the association between SNPs and asthma in Guangxi Zhuang, and the effects of gender, age, height, weight, exposure to tobacco smoke, occupation, and exposure to allergens were adjusted. The results of the three SNPs in different genetic models are shown in Table 4.

| D ISCUSS I ON
To our knowledge, this study is the first to report the association of the polymorphisms of GWAS-supported noncoding area loci, namely rs404860, rs3117098, and rs7775228, with asthma in the Chinese Zhuang population. The results indicated that rs3117098 was significantly associated with asthma susceptibility in the Chinese Zhuang population.
SNP rs3117098 is located within the BTNL2 intron. BTNL2 is a member of the immunoglobulin superfamily, and butyrophilin proteins share the same sequence homology with B7 molecules. 10 The B7 family is the second signal of T-cell activation, that is, this family is a costimulatory molecule that regulates T-cell activation and tolerance. Thus, BTNL2 is the first member of the butyrophilin family that regulates T-cell activation, which has implications in immune diseases and immunotherapy. According to the homology of B7-1, BTNL2 is a costimulatory molecule that is involved in T-cell activation. 11 The occurrence of asthma is related to T-cell imbalance. Therefore, the occurrence of asthma may be related to BTNL2.
Konno et al 12   In summary, our case-control study failed to replicate the GWASidentified association of rs404860 and rs7775228 with asthma.
However, significant findings showed that rs3117098 polymorphism was associated with asthma.

CO N FLI C T S O F I NTE R E S T
All authors report no conflicts of interest.