Association of IFN‐γ polymorphisms with ankylosing spondylitis risk

Abstract The case‐control study was designed to investigate the genetic effects of interferon‐gamma (IFN‐γ) rs2069727 and rs1861494 polymorphisms on ankylosing spondylitis (AS) susceptibility in a Chinese Han population. Blood samples were collected from 108 AS patients and 110 healthy controls. IFN‐γ polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). Hardy‐Weinberg equilibrium (HWE) test was performed in control group. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using chi‐square test to evaluate the association between AS susceptibility and IFN‐γ polymorphisms, and the results were adjusted by logistic regressive analysis. The frequency of rs2069727 CC genotype was much higher in cases than that in controls, suggested its significant association with increased AS risk (adjusted OR = 5.899, 95% CI = 1.563‐22.261; P = .009). In addition, C allele also showed close association with increased risk of AS (adjusted OR = 2.052, 95% CI = 1.286‐1.704, P  = 0 .003). While the genotype and allele frequencies of IFN‐γ rs1861494 polymorphism were not significantly different between patients and controls (P  > 0.05 for all), IFN‐γ rs2069727 polymorphism is significantly associated with increased AS risk in a Chinese Han Population.

to the gene. 9 However, population studies have demonstrated that only 2% HLA-B27-positive patients will finally develop AS, revealing that in addition to HLA-B27 gene, some other genetic factors also take part in pathogenesis of AS. 10 Therefore, it is necessary to explore novel AS-related genes which will be of great help for prevention, diagnosis and treatment of AS in clinic.
Interferon-γ (Interferon-gamma, IFN-γ or IFNG) is an important cytokine, which is mainly secreted by natural killer cells and CD80 + T cells. 11 The human IFN-γ gene is located on chromosome 12 (12q14), containing 4 exons and 3 introns with approximately 6 kb. 12 As an immunomodulatory factors, IFN-γ plays an important role in activation of macrophage, and abnormal expression of IFN-γ has been reported to be associated with a variety of auto-inflammatory and immune diseases, such as chronic periodontitis, IgA nephropathy and multiple sclerosis. [13][14][15][16] In addition, IFN-γ could also regulate human mesenchymal stem cell (MSC) osteogenesis, and IFN-γ exposure might suppress MSC osteogenesis. 17 All the data revealed that the abnormal expression of IFN-γ might contribute to pathogenesis of AS. In AS, a study carried out by Wang H et al reported that the protein expression level of IFN-γ was significantly higher in AS patients than that in health individuals. 18 The transcription activity of IFN-γ may be regulated by its genetic mutations. 19 A related case-control study reported that IFN-γ rs2430561 polymorphism may contribute to risk of AS via influencing IFN-γ expression. 20 Rs2069727 and rs1861494 are two common polymorphisms in IFN-γ gene, and both of them have the possibility to influence production of IFN-γ protein. 19,21 According to the previous researches, we hypothesized that IFN-γ rs2069727 and rs1861494 polymorphisms might be involved in AS.
However, the related studies had been rarely reported in Chinese Han population.
In this study, we compared the genotype distributions of the IFN-γ gene rs2069727 and rs1861494 polymorphisms between AS patients and healthy controls. In addition, the genetic effects of IFNγ gene polymorphisms on AS susceptibility were also investigated among the study population.

| DNA extraction
After fasting for 10 hours, 5 mL peripheral venous blood was collected from each participant using EDTA-coated tubes. Genomic DNA was extracted from blood samples using TIANamp Genomic DNA Kit (Beijing, China) according to manufacturer's protocols and then stored at −20℃ for standby application.

| Genotyping
Genotype distributions of IFN-γ gene rs2069727 and rs1861494 polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Primer sequences for IFN-γ gene rs2069727 and rs1861494 polymorphisms were designed by Primer Premier software and showed in Table 1.
The PCR procedure was carried out according the following steps: 95℃ pre-degeneration for 5 minutes, 30 cycles of degeneration at 95℃ for 30 seconds, 57℃ annealing for 30 seconds, 72℃ extension for 30 seconds and final extension at 72℃ for 5 minutes.
Subsequently, the PCR products were digested by the corresponding restriction enzymes (HinfI for rs2069727 and MspI for rs1861494). Then, the enzyme digestion results were analysed by 2% agarose gel electrophoresis and visualized by UV light. In addition, 20% of the amplified specimens were randomly selected for direct sequencing to estimate the efficacy of digestion results.

| Statistical analysis
All the data analysis was conducted using PASW Statistics 18.0 statistical software (SPSS Inc, Chicago, IL, USA). The statistic power of our study was estimated by GPower 3.1 software. confidence intervals (CI), and the results were adjusted using logistic regression model. P value less than 0.05 was considered as statistically significant.

| Statistic power and HWE test
The statistic power of our analysis was estimated by GPower 3.

| Distributions of IFN-γ gene polymorphisms between groups
IFN-γ gene polymorphisms were genotyped by PCR-RFLP method, and the results were in line with sequencing results. The amplified fragment of rs2069727 polymorphism was digested by HinfI. As displayed in Figure 1

| D ISCUSS I ON
AS is a chronic progressive inflammatory rheumatic disease which is regulated by multiple environmental and genetic factors, especially the inflammatory system components, like cytokines. As an anti-inflammatory cytokine, IFN-γ plays a modulatory role in the immune response. In this study, we investigated the genetic asso-

IFN-γ belongs to type II class of interferons and plays crucial roles
in multiple immune and inflammatory responses. 22 Abnormalities in IFN-γ expression may result in various inflammatory and immune diseases. It has been reported that gene expression and the activity of the corresponding protein may be regulated by its single nucleotide polymorphisms (SNPs). 23 There are several polymorphisms in IFN-γ gene, and some of them have been reported to be associated with activity of IFN-γ gene, such as rs2430561. 24 The genetic variants in IFN-γ gene may be involved in human diseases via their regulatory roles in IFN-γ production and immune response. IFN-γ rs2069727 and rs1861494 are two commonly studied SNP, and their regulatory roles in transcriptional level of IFN-γ gene have also been reported in the previous relevant studies. 18,20 However, to our knowledge, the genetic association of IFN-γ rs2069727 and rs1861494 polymorphisms with AS risk has been rarely reported in Chinese Han population.
In current research, a case-control study was designed to investigate the genetic effects of rs2069727 and rs1861494 polymorphisms in IFN-γ gene on AS risk among Chinese Han population.
Our findings supported that the individuals carrying rs2069727 CC genotype showed a significant higher risk to suffer from AS, compared with those carrying TT genotype. Similarly, rs2069727 C allele also showed increased risk to develop AS. The conclusion was in line with the previous studies. The study carried out by Li et al suggested that GG genotype of IFN-γ rs2069727 polymorphism had apparently different distributions between case and control groups, and might confer increased risk of hepatocellular carcinoma. 25 Alam et al reported the significant interaction of IFNG rs2069727 with gastrointestinal GVHD. 26 In this study, TA B L E 2 Genotype and allele distributions of IFN-γ gene rs2069727 and rs1861494 polymorphisms in case and control groups A well-designed cohort study with a large sample size will be scheduled to address the above issues.
In conclusion, IFN-γ rs2069727 SNP may contribute to risk of AS in the Chinese Han population, but not rs1861494 polymorphism.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.