Single Nucleotide Polymorphisms in P2X7 Gene Are Associated with Serum Immunoglobulin G Responses to Mycobacterium tuberculosis in Tuberculosis Patients

Objective. Our study investigated the association between single nucleotide polymorphisms (SNPs) in P2X7 gene and serum immunoglobulin G (IgG) responses to mycobacterium tuberculosis (MTB) in TB patients. Methods. A total of 103 TB patients were enrolled as case group and 87 healthy individuals at same geographical region as control group. The SNP detection of 1513A>C and -762T>C was performed using PCR-RFLP, and the levels of serum IgG responses to MTB in all subjects were determined. Results. AC and CC of 1513A>C and TC and CC of -762T>C had higher frequencies in case group than in control group. TB patients carrying TC and CC of -762T>C had higher positive rate of IgG responses to MTB than those carrying TT. Additionally, patients carrying TC and CC of -762T>C had more MTB in sputum than those carrying TT. Conclusion. P2X7 SNPs, 1513A>C and -762T>C, may be associated with the susceptibility to tuberculosis, and -762T>C SNP may contribute to the development of MTB. The mutant genotype of -762T>C (TC and CC) may lower human capability of phagocytosis to MTB, leading to an increased morbidity of TB.


Introduction
Tuberculosis (TB) is a global public health priority and remains the second leading cause of infection-related mortality worldwide [1]. The World Health Organization (WHO) has estimated that 9 million people were diagnosed with TB and 1.5 million people died of this disease in 2013 (deaths up from 1.3 million estimated in 2012) [2]. TB is largely caused by Mycobacterium tuberculosis (MTB) which has infected around a third of the world population, but only 3∼10% of those infected progress to active disease in their lifetime, and up to 90% of infected people are asymptomatic with a latent infection [3]. Multiple factors contribute to the risk of infection and development of TB, including environmental factors such as socioeconomic conditions, acute infection and smoking, and genetic factors as well as host-pathogen interactions [4,5]. Epidemiological studies reported that the development and progression of TB in human is strongly associated with gene polymorphism; up to now, gene polymorphisms of vitamin D receptor, SLC11A1, tumor necrosis factor-alpha, Tolllike receptor 2, and monocyte chemoattractant protein-1 have been identified to be associated with the susceptibility to TB [6][7][8][9].
The P2X7 receptor is predominately expressed on hematopoietic, mesenchymal, and epithelial cells and neural lineages, playing a crucial role in immunity, inflammation, neurological function, bone homeostasis, and neoplasia [10]. Human P2X7 gene containing 13 exons encodes the P2X7 receptor and is located on chromosome position 12q24 which is a region relevant to inflammatory and psychiatric disorders [11]. P2X7 activation induces an array of downstream signaling events, in a cell specific manner, including the release of cell proliferation or death, proinflammatory mediators, and killing of intracellular pathogens [12]. Some studies reported that several single nucleotide polymorphisms (SNPs) in P2X7 gene result in the reduction or loss of receptor function, and the most common SNPs involve the 1513A>C and -762T>C [13,14]. Accumulating studies suggested that these two SNPs of P2X7 gene play an important role in TB susceptibility, while there was no significant association with -762T>C polymorphism [15,16]. However, some previous studies demonstrated no correlation of P2X7 gene SNPs with 2 Disease Markers  5 -AGACCTGCGATGGACTTCACAG-3  316  64  35  5 -GCCAGGTGGCGTAGCACCTG-3   -762  T>C   Outer primer  5 -GAAACAGGGCCCTGGGTCCTC-3  373  65  10  5 -TGGTGGGGGTGGAGGGGC-3  Inner primer  5 -GGTGTCCCTCACTGAATAGGTCAAT-3  235/186  63  30  5 -GGCAGTCCAACAAAGTTAGGTTTG-3 SNP: single nucleotide polymorphism. susceptibility to TB [17,18]. Although there were increasing studies investigating the correlation between P2X7 gene SNPs and TB, it is rare to explore whether P2X7 gene SNPs influence immunoglobulin G (IgG) responses to MTB. The detection of antibodies to MTB in a patient's serum is simple and applicable in various settings and provides extremely rapid results [19]. Several studies in humans as well as animal models have reported that anti-MTB titers rely on the state of infection and that they are associated with the degree of mycobacterial burden [20,21]. It has been demonstrated that antibody to -crystallin is a promising target for the serological diagnosis of patients with active TB patients [22]. Therefore, here we performed a case-control study to determine the association between SNPs in P2X7 gene and susceptibility to TB and further examine the role of P2X7 gene SNPs in levels of serum IgG responses to MTB in TB patients. (2) all patients were in primary pulmonary TB. Exclusion criteria were as follows: (1) individuals with similar symptoms of TB; (2) TB patients with complications of chronic obstructive pulmonary diseases (COPD), asthma, pneumonia, cancers, diabetes mellitus or hypertension, and so forth; (3) patients with heredofamilial history; (4) immunocompromised patients (with HIV infection, lipoma or long-term persistence of hormone or organ transplant, etc.). Additionally, our study also enrolled 87 healthy controls (51 male and 36 female; 35 cases of Han, 25 cases of Uygur, and 27 cases of Kazak) with a mean age of 47.0 ± 14.5 years at the same geographic region. There was no statistical difference in age, gender, and ethnic constitution between the case and control groups (all P > 0.05).

Sample Collection.
Peripheral blood (10 mL) was collected from all subjects in the morning after fasting for 10 to 12 h and placed into two tubes (5 mL/tube). The first 5 mL was added with ethylenediaminetetraacetic acid (EDTA) for anticoagulation. After naturally cooling to room temperature for one hour later, the mixture was centrifuged at 3000 rpm for 10 min to separate peripheral blood mononuclear cell. DNA was extracted from peripheral white blood cells using a DNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd., China, DP318-03). Then the other 5 mL peripheral blood was centrifuged at 3000 rpm for 10 min at 4 ∘ C to collect supernatant. Subsequently, the supernatant was centrifuged again at 12,000 rpm for 5 min at 4 ∘ C to extract serum. The serum was subpackaged into 200 L tubes and then stored at −80 ∘ C in refrigerator for further usage.

SNP Detection.
Two SNPs (1513A>C and -762T>C) in P2X7 gene were selected for research targets. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for analyzing 1513A>C and PCR for -762T>C SNPs. PCR primers were designed by Premier 5.0 software (http://www.premierbiosoft.com/) and synthetized by Shanghai biological engineering company. The SNPamplified, primer sequences, fragment length, annealing temperature, and cycle number were listed in Table 1 included initial denaturation at 95 ∘ C for 3 min, denaturation at 94 ∘ C for 50 s, annealing at 64 ∘ C for 45 s, and extension at 72 ∘ C for 30 s, with a total of 35 cycles, followed by the final extension at 72 ∘ C for 10 min. The amplified fragment length of 316 bp was obtained. The PCR product was digested at 37 ∘ C for 3 h with Hae II (Promega, America) and the product underwent electrophoresis in a 1.5% agarose gel to determine the genotype. The PCR amplification for -762T>C was as follows: initial prenaturation at 95 ∘ C for 5 min, denaturation at 94 ∘ C for 20 s, annealing at 65 ∘ C for 30 s, and extension at 72 ∘ C for 30 s, with a total of 10 cycles, followed by the final extension at 72 ∘ C for 10 min. The amplification products were visualized by 1.5% agarose gel electrophoresis to determine the genotype. The electropherograms of 1513 and -762 sites in P2X7 gene after enzyme digestion and PCR were presented in Figure 1. As shown in Figure 1(a), when 1513 site was AA, there was no restriction enzyme site and the fragment length was 316 bp after restriction enzyme digestion by Hae II. When 1513 site was CC, 199 bp and 120 bp fragments were obtained. When 1513 site was AC, 316 bp, 199 bp, and 120 bp fragments were exhibited. As shown in Figure 1(b), when -762 site was CC, the PCR products were 373 bp and 235 bp. When -762 site was TT, the PCR products were 373 bp and 186 bp. When -762 site was TC, the PCR products were 373 bp, 235 bp, and 186 bp.

Detection of Serum IgG Responses to MTB Antigens.
Protein chip kit for detection of IgG responses to MTB (Nanjing Potomac Bio-Technology Co., Ltd., China, S20050089) was adopted, using three MTB complex specific antigens (16 kDa, 38 kDa, and lipoarabinomannan (LAM)) to determinate IgG responses to MTB in serum. The result was considered positive if one of the three antibodies was detected positive; otherwise the result was considered negative. Sputum from patients was mounted on slide. After acid-fast staining, the slide was examined under light microscope. The number and distribution of cells were observed under low magnification (400x), and even distribution and great morphology were observed again under oil-immersion lens (1000x) for acidfast bacillus. Continuous observation of no piece (100x) twice was considered as acid-fast bacilli negative and continuous observation of 3∼9 pieces (100x) twice as acid-fast bacilli positive.
2.6. Statistical Analysis. All data were analyzed by SPSS 18.0 software (IBM Corporation, Somers, NY, USA). Continuous data were expressed as mean ± standard deviation ( ± SD), and t-test or variance analysis was performed to detect intergroup comparisons. Categorical data were expressed as percentage, which were compared by 2 test and Fisher's exact test. Hardy-Weinberg equilibrium test was applied to detect whether the two groups were representative. In addition, SHEsis software was used to analyze whether these two SNPs were in linkage disequilibrium. Genotype frequencies comparisons between groups were presented as odds ratio (OR) and 95% confidence interval (CI). All P values were twosided, and the level of significance was set at P < 0.05.

Distributions of P2X7 SNPs.
Genotype frequency distributions of 1513A>C and -762T>C in two groups were consistent with Hardy-Weinberg equilibrium, which provided evidence of group representativeness for these two SNPs (all P > 0.05). These two SNPs were in linkage disequilibrium with 1.000 of and 0.726 of 2 . Table 2 presented the comparison  Table 2 suggested that 1513A>C and -762T>C SNPs may correlate to the susceptibility to TB. The positive rates of IgG responses to MTB in different SNPs of case group were identified, which was shown in Table 3. TB patients carrying TC and CC of -762T>C had higher positive rate of IgG responses to MTB than those carrying TT (TC: P = 0.008; CC: P = 0.030). However, there was no significant difference about the positive rate of IgG responses to MTB antigens between patients carrying AC and CC of 1513A>C and those carrying AA (all P > 0.05). Table 3, -762T>C SNP in P2X7 gene was associated with the level of serum IgG responses to MTB. Examination under the light microscope was visualized in Figure 2, from which we found that positive rates of acid-fast bacilli were 19.0% in sputum of TT carriers, 48.9% of TC carriers, and 57.1% of CC carriers, and TC (Figure 2(b)) and CC (Figure 2(c)) carriers of -762T>C had greater MTB in sputum than those TT carriers (Table 4).

Discussion
In the present study, we evaluated whether P2X7 polymorphism confers susceptibility to TB and investigated the association of SNPs in P2X7 gene with levels of serum IgG responses to MTB antigen in TB patients. Our results suggested that 1513A>C and -762T>C SNPs may be a risk factor for the development and progression of TB. The mutant genotype of -762T>C (TC and CC) may lower human capability of phagocytosis to MTB, leading to an increased morbidity of TB. One of our findings showed that an increased frequency of AC and CC of 1513A>C and TC and CC of -762T>C was observed in TB patients, suggesting that SNPs in P2X7 gene were associated with TB. Development and progression of TB is a result of complicated host-pathogen interactions involving many elements in the innate together with adaptive immune systems [24]. Macrophages are the primary Disease Markers 5   host cells for intracellular replication of mycobacteria and participate in controlling human infection, which serve as antigen presenting cells in reactivation of lymphocytes at the infection sites [25]. Extracellular adenosine triphosphate (ATP) mediates the germicidal activity of macrophages by activation of the P2X7 receptor resulting in apoptosis of the macrophage, which functions as a crucial host defense mechanism against MTB infection [26]. The purinergic P2X7 receptor is an ATP-gated cation channel expressed in immune cells, which affects the release of proinflammatory cytokines from monocytes and macrophages [27]. Collectively, the P2X7 receptor is highly expressed on the macrophage cell surface and activities of infected cells via extracellular ATP have been demonstrated to kill intracellular bacteria and parasites [28]. The P2X7 gene is highly polymorphic and a variety of nonsynonymous SNPs have been reported which were involved in alternation of receptor function or expression, especially 1513A>C and -762T>C SNPs [13]. A previous study published by Gartland and his colleagues reported 50% reduction in monocyte P2X7 receptor function in 1513C compound heterozygotes as well as complete loss of function for 1513C homozygote [29]. Fernando et al. also suggested that the 1513A>C SNP enhances susceptibility to TB, and this defect is correlated to the reduction in the capacity of macrophages to kill MTB [30]. In addition, -762T>C transformation may downregulate function of the binding site of some transcription factor in sequence, including CCAAT enhancer binding protein and GATA binding factor, inducing the decline of P2X7 receptor function [25]. In our study, patients carrying TC and CC of -762T>C had greater MTB in sputum than those carrying TT. Therefore, P2X7 gene SNPs cause the reduction of P2X7 receptor function, setting a barrier in the bonding of P2X7 receptor with ATP, thereby reducing the capacity of macrophages to kill MTB, which was associated with the pathogenesis of TB.
Another important finding of our study was that TB patients carrying TC and CC of -762T>C had higher positive rate of IgG responses to MTB than those carrying TT. In our study, we used three MTB-specific antigens (16 kDa, 38 kDa, and LAM) to determinate antibody (anti-MTB) in serum. The 38 kDa antigen is a secretory, MTB antigen can reach a specificity of as high as 90% for TB diagnosis, and the 16 kDa antigen is an immunodominant antigen and contains 6 Disease Markers B-cell epitopes specific for the MTB complex [31]. LAM, as an inherent constituent of the MTB cell wall, can induce the human body to engender anti-LAM antibodies [32]. Millington et al. have described that antigens which elicit strong T-cell immunity are specific for MTB infection and also are crucial for immunodiagnosis [33]. In our study, an elevated positive rate of IgG responses to MTB was detected in -762T>C SNP. Hur et al. also demonstrated that significantly higher IgG responses to 16 kDa and 38 kDa were observed, which was consistent with our study suggesting that -762T>C SNPs were correlated with the development of MTB [34].
In conclusion, our study demonstrated that 1513A>C and -762T>C SNPs in P2X7 gene may be associated with the susceptibility to TB, and -762T>C SNP may contribute to the development of MTB. The mutant genotype of -762T>C (TC and CC) may lower human capability of phagocytosis to MTB, leading to an increased morbidity of TB. Functional research including large sample size will need to confirm the role of P2X7 gene SNPs in the development and progression of TB and the growth of MTB.

Disclosure
Jiangdong Wu and Lijun Lu are considered co-first author.

Conflict of Interests
No competing financial interests exist.