The rs878081 polymorphism of AIRE gene increases the risk of rheumatoid arthritis in a Chinese Han population: a case-control study

The autoimmune regulator (AIRE), a transcriptional regulator expressed in medullary thymic epithelial cells, plays an important role in thymocyte education and negative selection. Several citations studying the association between the rs878081 exon polymorphism of the AIRE gene and the risk of rheumatoid arthritis (RA) in different populations have yielded conflicting findings. Thus, this case-control study involving 300 RA cases and 300 controls was aimed to identify whether such association existed in a Chinese Han population from East China. The rs878081 polymorphism of the AIRE gene was genotyped. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using the chi-squared test, genetic model analysis, and stratification analysis. Genetic model analysis showed significant correlations between the TT genotype and the risk of RA (OR: 1.89, 95%CI: 1.03-3.47 in TT vs CC; OR: 1.84, 95%CI: 1.02-3.31 in TT vs CC+TC). Stratification analyses of sex, age, smoking, and alcoholism suggested that the rs878081 polymorphism of the AIRE gene increased RA risk among non-smokers. In conclusion, rs878081 polymorphism of AIRE gene increases the risk of RA in a Chinese Han population.


Introduction
Rheumatoid arthritis (RA), an autoimmune disease affected by multiple factors (1), could cause critical functional impairment and work-related disability despite its low prevalence (B1%) (1). No significant reduction in mortality in different RA populations worldwide has been demonstrated (2,3). It has also been reported that the improvement in survival lags behind the recent advances in RA management (2). In addition, the etiology and pathogenesis of RA are still unknown (4). The combination of environmental and genetic factors plays a major regulating role in the development and progression of RA.
The autoimmune regulator (AIRE) is a transcriptional regulator expressed in thymic medullary cells (5). Experimental studies show that single nucleotide polymorphisms (SNPs) alter transcription of the AIRE gene, which is located in the 21q22.3 region, B12.5 kb long, encoding a 545 amino acid protein of 58 kDa by 14 exonial sequences (6). SNPs thereby provide a less efficient negative selection, and improve the survival of autoimmune T-cells and the susceptibility to autoimmune diseases (7)(8)(9). A genomewide association study in a Japanese population identified two SNPs (rs2075876 and rs760426) in the AIRE gene significantly associated with RA risk (10). Moreover, several citations (11)(12)(13) studying the association between the AIRE gene rs878081 polymorphism and RA risk in different populations brought conflicting findings. According to dbSNP database, rs878081 is located in the exon region of the AIRE gene. The minor allele frequency (MAF) of T allele in rs878081 polymorphism was 0.133 according to the 1000 Genomes Browser (https://www.ncbi.nlm.nih.gov/ variation/tools/1000genomes/). The MAFs of T allele in rs878081 polymorphism in the cases were 0.196, 0.074, and 0.290 in a Spanish population (11), a Chinese population from Hebei province (12), and a Chinese population from Shanxi province (13), respectively. To date, there is no association study of this SNP among the Chinese Han population in East China. Thus, this case-control study aimed to investigate whether rs878081 polymorphism of the AIRE gene was associated with the risk and development of RA in a Chinese Han population from Jiangsu province (East China).

Patients and methods
In this hospital-based case-control design, 300 hospitalized RA patients (79 males and 221 females) were recruited from the Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University or the Second Affiliated Hospital of Jiaxing University between January 2014 and October 2017. They were diagnosed following the criteria of the American College of Rheumatology/European League against Rheumatism Collaborative Initiative for RA (14). All patients were clinically evaluated by two rheumatologists. The demographic and RA data were obtained from patient data sheets. All the recruited people were Chinese Han.
Patients with other nationalities, consanguinity, other major systemic disease, collagen vascular disease, other autoimmune diseases, hepatitis C virus infection, or family history of autoimmune diseases were excluded. Also, 300 unrelated healthy controls (128 males and 172 females) were randomly recruited from the subjects receiving regular health examinations at either of the two hospitals during the same period.
A detailed questionnaire about smoking and alcoholism habits was completed for each participant by a trained interviewer. Informed consent was obtained from all participants prior to their participation. Ethical approval, which was in line with the Declaration of Helsinki standards, for the study protocol was obtained from the Ethics Committees of the two Hospitals.

DNA extraction and genotyping
Blood samples were collected using vacutainer tubes and then transferred to EDTA tubes. Genomic DNA was isolated from whole blood using a QIAamp DNA blood mini kit (Qiagen, Germany). Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as previously described (16). SNP was genotyped by the MassARRAY system (Sequenom, USA) and by MALDI-TOF MS, which was performed without knowing patient status (case vs control) to ensure the quality of genotyping.

Genotype and gene expression correlation analysis
Genotype data of the AIRE gene rs878081 polymorphism were available online from the International HapMap Project. The mRNA expression data were available online from Genotype-Tissue Expression Portal (https://www. gtexportal.org/home/) (17).

mRNA extraction and real-time PCR
According to the manufacturer's protocol, mRNAs were extracted from blood samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc, USA). cDNA was synthesized using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., China) at 37°C for 15 min, 85°C for 5 s, and determined at 4°C, while qPCR was conducted using a SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) on a Bio-Rad iQ5 Real-Time PCR system (Bio-Rad Laboratories, Inc., USA). The following thermocycling conditions were used for the PCR: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The primer sequences for amplification were 5 0 -GAG AGTGCTGAGAAGGACA-3 0 (forward) and 5 0 -GTTTAATTTC CAGGCACATGA-3 0 (reverse). The relative expression was calculated using the 2 -DDCq method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the internal control.

Statistical analysis
Demographic characteristics and rs878081 genotypes of the AIRE gene were evaluated using a chi-squared test (w 2 ) (for categorical variables) or Student's t-test (for continuous variables). The associations between the rs878081 T/C genotypes and the risk of RA were estimated by calculating odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analysis; crude ORs and adjusted ORs were used in case of adjustment for age and sex. The Hardy-Weinberg equilibrium (HWE) was assessed by a goodness-of-fit w 2 test to compare the observed genotype frequencies with the expected frequencies in controls. This study was powered to detect the effect of rs878081 polymorphism of the AIRE gene on RA susceptibility at Po0.05 (18). All statistical analyses were performed on SAS software 9.1.3 (SAS Institute, USA).

Characteristics of the study population
The characteristics of the study population are summarized in Table 1, including DAS28 grades and morning stiffness. The case and control groups were well-matched in terms of gender (male proportion=44.67% vs 48.67%) and age (mean age=54.50 vs 53.84 years). No significant differences between groups were found in smoking or alcoholism.

Association between rs878081 polymorphism and RA risk
Genotype distributions of the rs878081 polymorphism of the AIRE gene among all subjects are reported in Table 2. The rate of genotyping was 498%, and the test polymorphism of the study population was under HWE (P40.05). Logistic regression analyses revealed the rs878081 polymorphism increased the risk of RA in two genetic models (OR: 1.89, 95%CI: 1.03-3.47 in TT vs CC, P=0.039 and OR: 1.84, 95%CI: 1.02-3.31 in TT vs CC+TC, P=0.043; Table 2). The effects of the SNP on RA risk were further evaluated according to age, gender, smoking, and drinking. The increased RA risk conferred by rs878081 was more significant in females and nonsmoking patients (Table 3). However, no significant association was found between rs878081 genotypes and clinical or biochemical characteristics except for tender joints (P=0.020, Table 4). Furthermore, no significant difference in demographic or laboratory data was found between CC+CT and TT genotypes (Table 5) or between CC and CT+TT genotypes ( Table 6).

Association between rs878081 polymorphism and RA population
According to dbSNP database and Genotype-Tissue Expression Portal (17), the TT genotype decreased the AIRE mRNA levels compared to CC genotype in the adipose-subcutaneous (Figure 1). A significant difference was found in the expression levels for rs878081 polymorphism with the expression quantitative trait loci (eQTL) analysis (Po0.01).
Similar results were observed in the patients' blood samples. A statistically significant difference in the mean levels of expression of the rs878081 alleles was found (P=0.018). The transcription of AIRE was decreased by the T allele compared with the C allele ( Figure 2).

Discussion
The association between the rs878081 polymorphism of the AIRE gene and the risk of RA was investigated in a Chinese population, which showed that the polymorphism conferred susceptibility to RA.     AIRE plays a functional role in thymocyte education and negative selection by controlling the thymus expression of peripheral antigens (5,19). Dysfunction of the AIRE gene could lead to autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (20,21). The association between several SNPs (rs2075876, rs760426, rs1800250, rs2776377, rs1055311, rs933150, rs1003854, rs2256817, rs374696, rs1078480) of the AIRE gene and RA risk was studied before, and a recent meta-analysis (22) proved that rs2075876 and rs760426 are significantly associated with an increased risk of RA. After genotype-tissue expression analysis indicated that TT genotype of rs878081 polymorphism significantly decreased the AIRE expression, many researchers focused on the association between AIRE rs878081 polymorphism and RA risk. Garcia et al. (11) showed that the minor allele of rs878081 was significantly more frequent in RA patients than in controls from Spain, which was in line with our findings. However, two studies (12,13) from West and North China, showed no evidence of association between rs878081 and RA risk. Such discrepancies could be attributed to four reasons. First, genetic heterogeneity may exist among populations and living environments, but RA results from the intricate interactions between various susceptibility genes and   environmental factors. The effects of some genetic variants may vary across different populations and environments (1). Second, the study designs were different. The Spanish study (11) included fewer females in the RA group than the two Chinese studies (12,13). Moreover, cases in the West China study (13) were considerably younger than cases in the North China study (mean age=43.5 vs 53.9 years). Genetic factors are often stronger in younger cases, and menopause status is an influential factor (23). Third, clinical heterogeneity may exist among studies. Fourth, since the sample sizes of some studies were too small to draw a convincing conclusion, the results may be false-positives or false-negatives. Nevertheless, a meta-analysis including this SNP is warranted to resolve inconsistence and evaluate the role of this SNP in the risk of RA. Stratification analyses showed that the risk of RA conferred by the rs878081 polymorphism of the AIRE gene remained significant in the non-smoking subgroup, which was because susceptible individuals were likely to be exposed to risk factors to some extent. In addition, the genotype distribution of rs878081 polymorphism in relation to demographic, clinical, and laboratory data was analyzed. Individuals with TT genotype had more tender joints. However, given the decreased sample sizes in the stratification analyses and the limited power, the results should be interpreted with caution. Nevertheless, our findings still provide evidence for a possible interaction between the SNP and some RA risk factors.
Our results indicated that the AIRE rs878081 polymorphism increased the risk of RA in a Chinese population. According to dbSNP database, rs878081 polymorphism will cause synonymous mutation when the nucleotide changes from C to T. Then, we investigated the mRNA levels of different genotypes of rs878081 polymorphism in the Genotype-Tissue Expression Portal (17). The TT genotype decreased the AIRE mRNA levels compare to CC genotype in the adipose-subcutaneous. Considering that our clinical specimen was whole blood, we extracted mRNA from blood sample and conducted qPCR to determine the levels of AIRE. Similar results were observed in the patients' serum. Above all, the proportion of TT genotype increased in the RA group compared to the normal group. TT genotype is more likely to cause the down-regulation of AIRE compared to CC or CT genotypes. We hypothesized that AIRE rs878081 polymorphism con-ferred susceptibility to RA by altering the expression levels of AIRE.
The association between the AIRE rs878081 polymorphism and risk of other autoimmune diseases was examined before (6,(24)(25)(26). Ferrera et al. (24) reported no significant association of this SNP with risk of systemic sclerosis in an Italian population. No significant association was found between rs878081 polymorphism and Grave's disease (6), Addison's disease (26), or type 1 diabetes (25). However, some studies demonstrated that the ARIE rs878081 polymorphism conferred susceptibility to RA. The above differences among studies may be explained by the disease-dependent functionality of rs878081 polymorphism and should be confirmed by further studies.
This case-control study has several potential limitations that merit careful consideration. First, the patients and controls recruited from hospitals may not be representative of the general population. Nonetheless, the genotype distribution of the controls was in HWE. Second, because of the limited sample size, a single case-control study may be insufficient to fully uncover the relationship between the AIRE rs878081 polymorphism and susceptibility to RA. Thus, our findings should be confirmed by larger numbers of subjects. Third, we did not obtain detailed information about RA severity and response to treatment, which restricted our analyses. Fourth, the risk of RA cannot be attributed to a single AIRE gene SNP, but should be interpreted by considering other AIRE SNPs, other genes, and some environmental factors. Fifth, the underlying mechanisms of this SNP in RA were not investigated. Finally, the true significance of the association between this SNP and RA risk should be validated by further studies in different populations.
In conclusion, our study provided strong evidence that the rs878081 polymorphism of the AIRE gene may contribute to RA risk. However, this finding was obtained with a limited sample size representing a preliminary conclusion and should be confirmed by multicenter case-control studies with diverse ethnic populations.