SLAMF6 is associated with the susceptibility and severity of rheumatoid arthritis in the Chinese population

Objectives A recently published genome-wide association study identified six novel loci associated with rheumatoid arthritis (RA) in Korean population. We aimed to investigate whether these newly reported RA-risk loci are associated with RA in the Chinese population and to further characterize the functional role of the susceptible gene. Methods The susceptible variants of RA were genotyped in 600 RA patients and 800 healthy controls, including rs148363003 of SLAMF6, rs117605225 of CXCL13, rs360136 of SWAP70, rs111597524 of NFKBIA, rs194757 of ZFP36L1 and rs1547233 of LINC00158. Synovial tissues were collected from the knee joint of 50 RA patients and 40 controls without osteoarthritis for the gene expression analysis. Inter-group comparisons were performed with the Chi-square test for genotyping data or with Student's t-test for gene expression analysis. Result For rs148363003 of SLAMF6, RA patients were observed to have a significantly lower frequency of genotype CC (4.5% vs. 0.9%, p = 0.004) as compared with the controls. The frequency of allele C was remarkably higher in the patients than in the controls (11.5% vs. 8.0%, p = 0.002), with an odds ratio of 1.49 (95% CI = 1.16–1.92). There was no significant difference between the patients and the controls regarding genotype or allele frequency of the other 5 variants. The mRNA expression of SLAMF6 was 1.6 folds higher in the RA patients than in the controls. Moreover, SLAMF6 expression was 1.5 folds higher in patients with genotype CC than in the patients with genotype TT. Conclusions SLAMF6 was associated with both the susceptibility and severity of RA in the Chinese population. Moreover, rs148363003 could be a functional variant regulating the tissue expression of SLAMF6 in RA patients. It is advisable to conduct further functional analysis for a comprehensive knowledge on the contribution of this variant to the development of RA.


Introduction
Rheumatoid arthritis (RA) is a complex autoimmune disorder characterized by destruction of cartilage and joint caused by inflammatory synovitis [1,2]. RA usually develops between 40 and 50 years of age. The prevalence of the disease was estimated to be 0.5-1% among the world population and was more frequently developed in women than in men [3][4][5]. Although the etiology of RA is yet to be uncovered, earlier studies demonstrated that both environmental and genetic risk factors might play a major role in the onset and progression of the disease [6][7][8]. Previous familial studies indicated the risk of RA was remarkably higher in subjects with a familial history of RA, which was likely to be attributed to both inherited and environmental factors [9,10]. The overall heritability of RA has been estimated to be about 50-65% by twin studies [11,12].
Apparently, a combination between genetic background and environmental triggers confers an increased risk for RA.
To date, numerous efforts have been undertaken to identify specific genes and pathways implicated in the development of RA. Genome-wide association studies (GWAS) have discovered more than 100 RA-associated genetic variants in multiple ancestries [13][14][15][16][17]. Julia et al. [18] analyzed 317,503 SNPs in 400 RA patients and 400 controls and identified KLF12 as a new susceptibility gene for RA in the Spanish population. Saxena et al. [19] tested > 7 million SNPs in a total of 511 RA cases and 352 healthy controls and reported 2 novel associations specific to Arab populations at the 5q13 and 17p13 loci. According to the most recent studies, previously reported loci were able to explain only about 15% of the total risk in susceptibility to RA [15]. Considering substantial missing fraction of heritability, continuous effort in identifying additional RA variants is thus necessary to better understand the disease etiology.
In a recently published meta-analysis of GWAS for 4068 RA cases and 36,487 healthy controls, Kwon et al. [15] identified six novel RA-risk loci that reached the genome-wide significance in Korean population. It is now well established that for GWAS findings in complex disease genetics, one of the pivotal tasks is to further replicate genetic changes in different populations and link these susceptible variants to potential gene functions. In this study, we aimed to investigate whether the newly reported RA-risk loci were associated with RA in the Chinese population and to further characterize the functional role of the susceptible gene.

Subjects
Under the approval of the Ethics Committee of our institutions, a cohort of 600 RA patients who visited our center for treatment between June 2014 and October 2020 were enrolled in this study. All the patients were from Chinese Han population, who were diagnosed as RA according to the 2010 American College of Rheumatology criteria for RA classification [20]. The 28-Joint Count Disease Activity Score (DAS28) was used to assess the disease activity for each patient [21,22]. Patients with DAS28 > 3.2 were classified as active RA, and patients with DAS28 ≤ 3.2 were classified as inactive RA. Eight hundred age-matched healthy subjects were recruited as normal controls. The baseline characteristics of RA patients were collected from the medical records, including age, gender, body mass index (BMI), the serum level of rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP).

Genotyping of target SNPs
With the informed consent obtained from the subjects, blood samples were then collected for genomic DNA extraction using the commercial kit (QIAGEN, Tokyo, Japan), which were then stored at − 20 °C. Approximately 10 ng of the DNA sample was used for genotyping of the following 6 SNPs with TaqMan SNP Genotyping Assay, including rs148363003 of SLAMF6, rs117605225 of CXCL13, rs360136 of SWAP70, rs111597524 of NFK-BIA, rs194757 of ZFP36L1 and rs1547233 of LINC00158. Amplification was performed in 20 μl reaction volumes, containing 2 μl of TaqMan genotyping assay mix, 6 μl of genomic DNA, 2 μl of distilled deionized water and 10 μl of the TaqMan genotyping master mix. The interpretation of genotyping assay results was read on ABI 7900HT sequence detection system (Applied Biosystem, Foster City, CA). Twenty percent of the tests were randomly repeated to validate the reproducibility of the genotyping results.

Gene expression analysis of SLAMF6
During total knee replacement surgery, samples of synovial tissues were collected from the knee joint of 50 RA patients. Forty patients undergoing surgery for cruciate ligament rupture or meniscus tear were recruited as the control group. The mean age of the RA patients was 44.6 ± 11.9 years and their mean disease duration was 52.1 months (range 19-129 months). The mean ages of the control group were 42.2 ± 9.5 years. The total RNA was extracted from synovial tissues using Trizol reagent (QIAGEN, Tokyo, Japan) according to the standard protocol, which was then reversely transcribed by the Pri-meScriptRT Master Mix kit (TaKaRa, Tokyo, Japan). The mRNA expression of SLAMF6 was quantified by quantitative real-time PCR (qPCR) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as internal controls. The gene-specific primers were as follows, forward 5′-GAG TCC GCA AGG AAC CTA GAG-3′, reverse 5′-TCC CTG TTT GAA TGA GTG ACTGA-3′ for POC5, and forward 5′-GAG TCA ACG GAT TTG GTC GT-3′, reverse 5′-TTG ATT TTG GAG GGA TCT CG-3′ for GAPDH that functions as endogenous control. All amplification reactions were carried out in triplicate.

Statistical analysis
For continuous descriptive data, the results were displayed as the mean ± standard deviation (SD). The Hardy-Weinberg equilibrium (HWE) method was used to examine the genotype frequency of the normal controls. The Chi-square analysis was used to compare the frequency of genotype and risk allele between the RA cases and controls, with the odds ratio (OR) and 95%

Demographic data of the subjects
The baseline clinical characteristics of the subjects are summarized in Table 1

Replication of RA-associated variants
HWE test revealed no remarkable difference regarding the genotype frequency among the controls. As shown in Table 2, for rs148363003 of SLAMF6, RA patients were observed to have a significantly higher frequency of genotype CC (4.5% vs. 0.9%, p = 0.004) as compared with the controls. The frequency of allele C was remarkably higher in the patients than in the controls (11.5% vs. 8.0%, p = 0.002), with an odds ratio of 1.49 (95% CI = 1.16-1.92). There was no significant difference between the patients the controls regarding genotype or allele frequency of the other 5 variants.

Tissue expression of the SLAMF6
A total of 50 RA patients and 40 non-RA controls were included in expression analysis. The two groups were matched in terms of age (44.6 ± 11.9 years vs. 42.2 ± 9.5 years, p = 0.29) and BMI (24.8 ± 3.6 vs. 24.1 ± 3.9, p = 0.37). As illustrated in Fig. 1A, the mRNA expression of SLAMF6 was 1.6 folds higher in the RA patients than in the controls (0.000625 ± 0.0000232 vs. 0.000390 ± 0.000132, p < 0.001). For RA patients, there were 40 cases with genotype TT of rs148363003 and 10 cases with genotype CC. As shown in Fig. 1B, the mRNA expression of SLAMF6 was 1.5 folds higher in patients with genotype CC than in the patients with genotype TT (0.000853 ± 0.000148 vs. 0.000568 ± 0.000214, p < 0.001).

Relationship between SLAMF6 and clinical phenotypes of RA
Of the 50 RA patients included in tissues analysis, 27 patients had DSA28 of more than 3.2, who were thus assigned to active RA group. Inter-group comparison   17:13 showed that patients in active RA group (n = 27) had significantly higher SLAMF6 expression than those in inactive RA group (n = 23) (0.000708 ± 0.000210 vs. 0.000527 ± 0.000222, p = 0.005) (Fig. 2a). As shown in Fig. 2b, the mRNA expression level of SLAMF6 was remarkably correlated with serum RF (r = 0.30, p = 0.03). As for ESR (r = 0.08, p = 0.60) and CRP (r = − 0.15, p = 0.29), no significant correlation with SLAMF6 expression was found.

Discussion
As recent meta-analysis of GWAS data in Korean cohorts identified 6 novel loci of RA [15], we replicated these variants in the Chinese RA patients and confirmed that rs148363003 of SLAMF6 was associated with RA in the Chinese population. For rs148363003, allele T was a wild-type allele, and allele C was a mutant allele. Genotype CC was indicative of remarkably higher risk of RA. We observed that allele C of rs148363003 can remarkably add to the risk of RA by 1.49 folds, which was consistent with an increased RA risk of 1.62 folds in the Korea population as reported by Kwon et al. [15]. The rs148363003 is located at 11 kb downstream of SLAMF6. Through expression quantitative trait locus mapping based on public database, Kwon et al. [15] reported that rs148363003 had chromatin interactions with 13 coding genes on chromosome 1. To further clarify the biological basis underlying the association between rs148363003 and the risk of RA, we preliminarily investigated the relationship between rs148363003 and tissue expression of SLAMF6. For the first time, we analyzed the expression of SLAMF6 gene in the synovial tissues of RA patients as well as in the controls. Remarkably higher tissue expression of SLAMF6 was observed in RA patients than in the controls. Moreover, patients with genotype CC of rs148363003 were found to have remarkably increased expression of SLAMF6 than those with genotype TT.
Since the frequency of genotype CC was remarkably higher in patients than in normal controls, it was plausible that the mutant allele C added to the risk of RA via regulation of SLAMF6. Interestingly, as shown in Hap-loReg database [23], rs148363003 is predicted to alter transcription factor-binding sites of SLAMF6. A finemapping of the genomic region around rs148363003 is warranted to further elucidate its regulatory role in the tissue expression of SLAMF6.
As reported in previous studies, the susceptible genes of RA could be associated with the severity of the disease [24][25][26]. Elkhawaga et al. [24] reported that rs28362491 of NFKB1 contributed to both susceptibility and severity of RA. Zhao et al. [25] reported that rs1801278 of IRS-1 was associated with increased risk and disease activity of RA. In this study, the relationship between SLAMF6 and RA activity was also analyzed. Patients with high RA activity were found to have significantly higher SLAMF6 expression than patients with low RA activity. Moreover, there was significant correlation between SLAMF6 expression and serum RF. Collectively, SLAMF6 may act as a modifier gene which is prognostic of the progression of RA.
To date, the exact function of SLAMF6 in RA remains obscure. The product of SLAMF6 is a member of superfamily immunoglobulin (Ig) domain-containing molecules that play a role in interactions between naive lymphocytes. Previous functional studies indicated that the SLAMF6 could be involved in a series of inflammatory diseases [27][28][29]. Wang et al. [27] reported that SLAMF6 might function as an inhibitory receptor that regulates autoimmune responses. Savola et al. [29] reported accumulation of mutations in SLAMF6 in expanded T cells may have pathogenic significance for RA. Other members of SLAMF have been reported to contribute to the accumulation of antibody producing cells in patients with systemic lupus erythematosus, which therefore were considered as potential therapeutic targets for autoimmune diseases [30]. In the current study, we preliminarily observed that the tissues expression of SLAMF6 was associated with RA activity. Further investigation into the relationship between serum soluble SLAMF6 concentration and clinical characteristics of RA patients is warranted to better decipher the potential application of SLAMF6 as a clinical biomarker for RA.
In the current study, no association between the 5 SNPs and RA in the Chinese population was found in the current study. However, we still cannot entirely exclude the association of these 5 genes with RA. Several limitations of this study should be addressed here. First, due to the inherent defect of retrospective research, the lack of replication was probably attributed to the relatively small sample size of current study. Larger sample size may help yield more convincing outcome. Second, we used patients with cruciate ligament rupture or meniscus tear as control group for gene expression of SLAMF6 in synovial tissue. It was difficult to collect synovial tissue from healthy population. The third limitation of the present study lies in that no in vivo experiment was performed to clarify the regulatory role of rs148363003 in SLAMF6 expression. Further functional experiments are therefore necessary to clarify the underlying regulatory mechanism.

Conclusions
SLAMF6 was associated with both the susceptibility and severity of RA in the Chinese population. Moreover, rs148363003 could be a functional variant regulating the tissue expression of SLAMF6 in RA patients. It is advisable to conduct further functional analysis for a comprehensive knowledge on the contribution of this variant to the development of RA.