Baseline features of the participants
The baseline and laboratory indexes of the study population are listed with detail in the Table 1. The male/female distribution of the study subjects in the AS and control groups was 53 (21.2%)/197 (78.8%) and 45 (18%)/205 (82%), respectively. The age of the AS and control groups was 38.50±8.80 and 37.62±7.40, respectively. The matching of two study groups for age (P > 0.05) and sex (P > 0.05) was observed. HLA-B27 was observed to be positive in 202 (80.8%) AS patients and 18 (7.2%) healthy controls (P< 0.0001). Moreover, the C-reactive protein (CRP) level in the case group (3.14 ±2.27) was significantly higher (P<0.0001) relative to the control group (1.27±0.95).
Association test of ERAP2 polymorphisms
The frequency distribution pattern of the genotypes for ERAP2 gene rs2548538 (P=0.830), rs2287988 (P=0.443), and rs17408150 (P=0.165) SNPs in the healthy control group adhered to the HWE. In the total population analysis, results revealed that all three SNPs did not associate significantly with risk of AS (Table 3). That notwithstanding, when the HLA-B27 positive AS patients and the control group were compared, rs2287988 and rs17408150 SNPs showed statistically significant association with AS risk (Table 4).
For rs2287988, there was significant association in the minor G allele and GG genotype. The G allele was represented in 45.7% of the HLA-B27 positive AS patients and in 38.4% of the healthy controls (OR= 1.35, 95%CI= 1.03-1.76, P= 0.023). The GG genotype was frequently presented in the HLA-B27 positive AS patients relative to controls (20.3% vs. 13.6%), and the difference was statistically significant (OR= 1.91, 95%CI= 1.09-3.35, P= 0.022).
In case of rs17408150, it was observed that the C allele had significant association with higher risk of AS in the HLA-B27 positive patients (OR= 1.39, 95%CI=1.06-1.81, P= 0.013). The AA genotype was represented in the HLA-B27 positive AS group (21.8%) higher than the controls (17.2%); hence the AA genotype was associated significantly with increased AS risk (OR= 2.13, 95%CI=1.19-3.82, P= 0.010). The heterozygote AT genotype had statistically significantly difference in the distribution between HLA-B27 positive AS cases versus control group (OR= 1.91, 95%CI=1.19-3.06, P= 0.007). It was observed that the dominant model of AA+AT vs. TT for rs17408150 SNP had significant association with the higher AS risk (OR= 1.96, 95%CI=1.24-3.10, P= 0.003) in the AS group with positive HLA-B27.
Frequency of the haplotype
Regarding the haplotypic analysis (rs2548538 A/T, rs2287988 A/G, and rs17408150 A/T), it was detected that four haplotypes had significant association with AS risk. While AAA (OR= 2.13, 95%CI= 1.25-4.31, P= 0.014) and AAT (OR= 2.85, 95%CI= 1.50-05.40, P= 0.0083) haplotypes were associated with increased AS risk, the AGA (OR= 0.86, 95%CI= 0.47-0.82, P= 0.005) and TAT (OR= 0.57, 95%CI= 0.31-0.75, P= 0.004) haplotypes were associated with decreased risk of AS (Table 5).
Linkage disequilibrium test
The structure of SNP pairs in the LD block according to the SNP sequence of rs2548538, rs2287988, and rs17408150 are shown in the figure 1. The Dʹ value between rs2548538 and rs2287988 SNPs was 55% and between rs2548538 and rs17408150 SNPs was 81%. Nonetheless, a partially stronger linkage was detected when only the HLA-B27 positive patients were included in the analysis (Dʹ= 56% and 87%, respectively, for rs2548538-rs2287988 and rs2548538-rs17408150). However, the r2 values indicated no remarkable LD between SNP pairs.
mRNA expression of cytokines
The mRNA transcription level of IL-17A (fold change [FC]= 2.85, P=0.0001, Figure 2.a) and IL-23 (FC=1.63, P=0.0084, Figure 2.b) had upregulated levels in the PBMCs obtained from AS patients in relation to healthy subjects. However, there was significant underexpression of the mRNA of IL-10 (FC=0.43, P=0.0024, Figure 2.c) in the PBMCs obtained from AS patients relative to the healthy subjects. The mRNA expression of TGF-β was lower non-significantly in the case group relative to the control group (FC= 0.89, P> 0.05, Figure 2.d). The mRNA expression of all four cytokines had no significant difference among AS patients with three genotypes for rs2287988 and rs17408150 SNPs (Figure 2 and Supplementary Table 1).
Transcript levels of IL-17A (FC=3.00, P=0.0001, Figure 3.a) and IL-23 (FC=1.70, P=0.0084, Figure 3.b) had upregulated levels in the PBMCs obtained from AS patients positive for HLA-B27 gene relative to the healthy subjects. Conversely, the transcript level of IL-10 (FC=0.41, P=0.001, Figure 3.c) was significantly lower in the PBMCs obtained from AS patients positive for HLA-B27 relative to healthy subjects. The mRNA expression of TGF-β had no significant difference between HLA-B27 positive AS patients and the normal controls (FC=0.82, P > 0.05, Figure 3.d). The mRNA expression of all cytokines had no significant difference among the AS patients positive for HLA-B27 carrying three different genotypes for rs2287988 and rs17408150 SNPs (Figure 3 and Supplementary Table 1).
Serum concentration of cytokines
The serum concentrations of IL-17A (46.50±13.10 vs. 19.56±6.48, P= 0.0001, Figure 4.a) and IL-23 (388.9±38.78 vs. 200.2±24.88, P= 0.0001, Figure 4.b) were increased in the AS group relative to normal control group. However, the serum concentration of IL-10 was significantly lower in the AS group compared with the control group (1.89±0.18 vs. 3.68±1.10, P= 0.0015, Figure 4.c). However, the difference in the serum concentration of TGF-β was not statistically significant between AS group and the control group (20.4±4.51 vs. 23.4±4.21, P > 0.05, Figure 4.d). The serum concentration of all cytokines had no significant difference among the AS cases positive for HLA-B27 harboring three different genotypes for rs2287988 and rs17408150 SNPs (Figure 4 and Supplementary Table 2).
The serum concentrations of IL-17A (49.11±14.21 vs. 19.56±6.48, P= 0.0001, Figure 5.a) and IL-23 (395.4±39.7 vs. 200.2±24.88, P= 0.0001, Figure 5.b) were higher in the AS patients positive for HLA-B27 relative to normal controls. In contrast, the serum concentration of IL-10 was significantly lower in the HLA-B27 positive AS group compared with the control group (1.75±0.14 vs. 3.68±1.10, P= 0.0017, Figure 5.c). However, the difference in the serum concentration of TGF-β was not statistically significant between HLA-B27 positive AS cases and the control group (19.7±4.47 vs. 23.4±4.21, P > 0.05, Figure 5.d). The serum concentration of all cytokines had no significant difference among the AS cases positive for HLA-B27 carrying three different genotypes for rs2287988 and rs17408150 SNPs (Figure 5 and Supplementary Table 2).
Association of the genotypes and clinical manifestations
The ERAP2 gene rs2548538, rs2287988, and rs17408150 polymorphisms were investigated in association with clinical features of AS patients. It was observed that none of the characteristics of the AS patients, including CRP, disease duration, BASDAI, BASFI, BASG, and ASQoL scores had significant association with ERAP2 gene polymorphisms (Table 6). Moreover, analysis of the association of the ERAP2 gene rs2548538, rs2287988, and rs17408150 SNPs with the mentioned clinical manifestations in the HLA-B27 positive AS patients, resulting in no significant association.