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Ankylosing spondylitis is an inflammatory rheumatic disease that predominantly affects the spine and may be associated with peripheral arthritis. ERAP1 gene has the highest association with AS susceptibility after HLA-B27. The purpose of this study was to determine the association of 8 important SNPs of ERAP1 with the susceptibility of the disease, besides evaluation of the effects that two of selected SNPs (rs30187 and rs28096) might have on the expression of ERAP1 and disease severity.
Methods. In present study 399 patients suffering from AS and 322 healthy controls were recruited. All 8 SNPs of ERAP1 were genotyped using allelic discrimination method. Expression of ERAP1 gene having homozygote genotypes of rs30187 and rs28096 was performed using Real Time PCR TaqMan gene Expression.
Results. The rs27044, rs10050860, rs30187, rs28096 and rs26653 were significantly associated with AS. In patients with rs28096 GG genotype, ERAP1 gene expression was 1.5 times higher than patients with rs28096 AA genotype (P=0.007). This polymorphism also had a highly significant association with BASMI index of the disease.
Conclusion. This study confirmed association of ERAP1 with ankylosing spondylitis in Iranian patients. Various SNPs of ERAP1 not only were associated with the onset of the disease, but also they were associated with the disease severity and the rate of ERAP1 gene expression. Based on our results, other studies need to be performed on other SNPs of ERAP1 and determine their correlation with disease severity in Iranian population.
Key Words: Ankylosing Spondylitis, ERAP1, and BASMI
Ankylosing Spondylitis (AS) is a chronic inflammatory disease that primarily involving the sacroiliac joints and the axial skeleton. In fact, AS is the prototypic disease of a group of chronic inflammatory rheumatic diseases known as spondyloarthropathies (SPAs) (1). AS is characterized by inflammation of the spine and sacroiliac joints, causing pain, stiffness, initial bone and joint erosion, and ultimately ankylosis and fibrosis (2, 3). Inflammation may also involve extra-articular sites such as the uveal tract, tendon insertions, proximal aorta and, rarely, lungs and kidneys (3).
The etiology of AS is influenced by both environmental and genetic factors (4). Genetic predisposition to this disease became evident in early 1970s, with the discovery of association of HLA-B27 with the disease. However, despite the fact that 90% of patients with AS are HLA-B27 positive, only < 5% of those individuals in the general population develop the disease (4-7). Apart from the well-known HLA-B27 association, recent genome wide association studies demonstrated that ERAP1 (Endoplasmic Reticulum Aminopeptidase-1), IL23R (IL-23 Receptor), ANTXR2 (Anthrax Toxin Receptor-2) and IL1R2 (IL-1 Receptor, type II) genes, are associated with AS. These studies have also identified two intergenic regions at chr2p15 and chr21q22 as the loci associated with AS (4, 8, 9). Several replication studies have confirmed that ERAP1 and IL23R are strongest candidate genes responsible for AS (4, 10-14).
ERAP1 was the first non-MHC gene with definitive association with AS (1). The discovery of the ERAP1 association has brought new insights into the field of AS research. ERAP1 has two known functions in the immune system, both of which provide important clues to the pathogenesis of AS. First, it participates in the shedding of the membrane-bound cytokine receptors; TNFRI, IL-1R II, and IL-6RÎ±. Second, it contributes to trimming of peptides down to 8-9 amino acids in length before presentation by HLA Class I molecules. These functions are being further analysis to better identify the exact role of ERAP1 in AS (1, 15-17).
In this study, we genotyped 8 SNPs in ERAP1 gene. Our aim was to identify the possible links between ERAP1 single nucleotide polymorphisms (SNPs) and haplotypes, with the susceptibility, and disease severity of AS in Iranian population. We evaluated the probable role of these SNPs in altering the expression level of ERAP1 gene and Bath Ankylosing Spondylitis Metrology Index (BASMI) that indicate the long-term effects of AS disease on each patient.
PATIENTS AND METHODS
The study group consists of 399 unrelated AS patients and 322 ethnically matched healthy controls. All patients and healthy controls have Iranian ancestry. Diagnosis of AS patients was based on 1984 modified New York criteria (18). All patients were recruited from Rheumatology Research Center of Tehran University of Medical Sciences, Shariati hospital and Iranian Association of AS. The patients group included 325 men and 74 women, with mean (SD) age of 38.07 (±10.0) years and disease duration of 15.53 (±9.4) years. Most of the patients (73.4%) were HLA-B27 positive. The healthy controls group included 231 men and 91women, with mean age of 34.1 (±10.6) years. Control subjects and their family didn't have any autoimmune and rheumatic diseases. Only 3.4% of healthy controls group was HLA-B27 positive. Ethics approval for this study was obtained from Tehran University of Medical Sciences, and all individuals in both groups provided their informed consent. A qualified rheumatologist, to obtain the BASMI, performed metrology Index.
Genomic DNA extracted from peripheral blood samples with standard phenol-chloroform-proteinase-K method. Samples were genotyped for 8 selected SNPs including: rs1065407, rs2287987, rs10050860, rs27044, rs26653, rs17481856, rs28366066, and rs28096. Real-Time PCR allelic discrimination TaqMan genotyping assays (Applied Biosystems, Foster city, USA) were applied to genotyping each study participant, using ABI 7300 Real-Time PCR system. Reactions were processed following standard protocols for Applied Biosystems. The allelic call was performed by the analysis of allelic discrimination plots, using ABI SDS V 1.4 software.
ERAP1 Gene Expression
After ERAP1 genotyping, based on homozygote genotypes of wild type and mutant rs30187 and rs28096 SNPs, we selected 40 patients and 40 controls for ERAP1 gene expression analysis. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque gradient. Total cellular RNA was extracted from PBMCs, using High Pure RNA Isolation Kit, (Roche, Germany) according to the manufacturer's protocoles. Quantity and quality of RNA was confirmed by using a spectrophotometer (NanoDrop ND-2000C Spectrophotometer, Thermo Fisher Scientific, USA). Isolated RNA was used to amplify cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). In order to analyze gene expression, cDNA samples were used as template for comparative CT Real Time PCR detection of ERAP1 using StepOne Plus Real Time PCR instrument (Applied Biosystems). Duplicate reactions of the target and housekeeping genes were performed simultaneously for each cDNA template, which analyzed with quantitative relative software using the comparative CT method (ABI StepOne V.2.1).
A test for deviation from Hardy-Weinberg equilibrium (HWE) was performed for each SNP in the control population using the genetic package of the R statistical software.
In order to investigate the association between one SNP of the ERAP1 gene and the AS disease, the chi-square test and Cochren-Armitage trend test were applied. In order to adjust for multi-colinearity when all SNPs are included, the binary or ordinal logistic regression was employed. To adjust for multiple testing the Benjamini-Hochberg method to control the false discovery rate (FDR) in the logistic regression analysis. Results with Two-sided of P- value < 0.05 (after adjustments) were considered statistically significant (19).
Haplotype blocks and LDs of the SNPs were determined using the Haploview V4.1 software. The Haplo EM command in genetic package of the R statistical software was also employed to determine the haplotype frequencies.
The one-way ANOVA with Bonferroni correction was used to study the effect of different genotypes of each studied SNP on disease severity. All statistical analyses were done in the R statistical software V2.15 (20).
Single-marker association of ERAP1 SNPs with AS
No marker deviated significantly from Hardy-Weinberg Equilibrium (HWE) (Table 1). Significant ERAP1 single-marker associations were noted for rs28096 (OR 0.69, 95% CI (0.55-0.88); P=0.0013), rs2287987 (OR 0.58, 95% CI (0.39-0.85); P=0.0013), rs10050860 (OR0.58, 95% CI (0.4-0.85); P=0.0013), rs27044 (OR1.37, 95% CI (1.07-1.72); P=0.0022), and rs26653 (OR 1.30, 95% CI (1.04-1.61); P=0.0035) (Table 1). According to our findings, the minor alleles of rs27044 and rs26653 have a predisposing effect on AS disease; however rs10050860, rs2287987, and rs28096 have a protective role in AS disease for minor alleles.
Identification of haplotypes associated with AS in Iranian population
In our previous study, rs30187, rs27434, rs469876, and rs13167972 were analyzed (21). These SNPs were used for determination of haplotypes in present study and consequently the influences of ERAP1 SNP haplotypes on AS susceptibility were examined. Haplotype blocks and linkage disequilibrium (LD) of SNPs (Figure 1) was determined using Haploview V4.1 software. Only 2 out of the 12 SNPs at ERAP1 (rs2287987 and rs10050860) were approximately in complete LD (r2 = 0.96, D'=0.99). Haplotype frequencies between patients and controls were determined, using haplotype blocks. The block 1 indicated protective (rs17481856, rs27434, rs469876, rs10050860, and rs30187/GCATC), and predisposing haplotypes (rs17481856, rs27434, rs469876, rs10050860, and rs30187/GCACT) (Table 2). The block 2 also demonstrated protective (rs27434, rs2287987, and rs26653/ATC) and predisposing haplotypes (rs27434, rs2287987, and rs26653/GCG)(Table 2).
Relationship between ERAP1 SNPs, and Bath Ankylosing Spondylitis Metrology Index (BASMI)
We also analyzed the association of ERAP1 SNPs with disease severity. Among all 12 SNPs investigated (all SNPs in our current and our previous studies), only rs28096 was associated with BASMI (Table 3).
Analysis of ERAP1 gene expression in AS patients and healthy controls
The expression level of ERAP1 (rs28096 G/G genotype compared to A/A genotype) in AS patients and healthy controls has been shown in Figure 2. The expression level of ERAP1 in patients with G/G genotype is about 1.5 times greater than the expression level of ERAP1in patients with A/A genotype (P=0.007). The expression level of ERAP1 for rs28096 mentioned genotypes remains the same among healthy controls (P=0.14). The expression level of ERAP1 (rs30187 T/T genotype compared to C/C genotype) in AS patients and healthy controls have been shown in Figure 2. The expression level of ERAP1 in patients with C/C genotype is more than the expression level of ERAP1in patients with T/T genotype but this difference was not significant (P=0.08). The expression level of ERAP1 for rs30187 mentioned genotypes were similar among healthy controls (P=0.61).
Recently many genes outside HLA region have been investigated for AS susceptibility and association with disease phenotypes. According to some genome-wide association studies, ERAP1 is one of the interesting candidate genes outside HLA, which is strongly associated with AS (13, 22-25).
In two genome-wide association studies by WTCCC and TASC, which are two of the most comprehensive GWAS in AS, thousands of SNPs were examined and revealed that ERAP1 polymorphisms are associated with AS with a high level of statistical significance(3, 26). However, further studies in several populations and ethnic groups are essential, to confirm ERAP1 as a common susceptibility locus for AS. In the present study we assessed the association of SNPs in ERAP1 gene with AS susceptibility and functional severity for the first time in Iranian population. We analyzed single-marker association of 8 SNPs with AS; and found a significant allelic association of AS with five out of the 8 genotyped SNPs. In addition, our results suggest that minor alleles of rs27044 and rs26653 serve as a predisposing effect on AS, while the minor alleles of rs10050860, rs2287987, and rs28096 have a protective role on AS.
The exact role of ERAP1 in the etiology of AS is not clear yet. According to molecular viewpoint, ERAP1 encodes an aminopeptidase in endoplasmic reticulum, which is involved in protein processing and transport(15). Thus, ERAP1 determines the antigenic repertoire of peptides presented by class I MHC molecules and consequently governs T cell responses(15). It is posible that SNPs in ERAP1 gene may influence aminopeptidase activity so that peptide trimming and presentation alter. Moreover, ERAP1 plays an important role in regulating pro-inflammatory cytokine receptors through cleavage ofpro-inflammatory cytokine receptors such as TNFR1, IL6R, and IL1R2on cell surface (27-31).
Four of the ERAP1 SNPs that we found associated with AS, were first described in a GWAS study in British and US patients with AS (the Australo-Anglo-American Spondylitis Consortium or TASC, in collaboration with the Wellcome Trust Case Control Consortium), in which the most strongly associated SNP was rs30187 (26). In our previous study rs30187 also showed the strongest association with AS among other SNPs, which is in accordance with many studies throughout the world, except one study in Hungary (3, 14, 21, 32). The rs30187 codes an amino acide which is located near the entrance of the substrate pocket (25). This strong association suggests a possible role of this SNP in the enzymatic activity of ERAP1 especially in peptide trimming. In 2006, Goto et.al. reported that the presence of T/T genotype in rs30187 plays an important role in reduction of ERAP1 aminopeptidase activity and decrease in substrate binding capacity(33). rs30187 codes Arg528 instead of Lys528 in ERAP1 molecule, which located in the proximity of the entrance of the substrate pocket. Thus, the substitution of Lys528 in wild-type protein with Arg528 may lead to a significant decrease in the enzymatic activity and substrate binding affinity (25).
We also found that two other SNPs associated with AS, rs10050860 (Asp575Asn) andrs2287987 (Met 349 Val), are in a complete linkage disequilibrium with each other (D=0.991, LOD=131.64, r2=0.96) and rs2287987 leads to rs10050860 tagging. Interestingly, in a study by WTCCC2 and TASC in 2011, it has been proved that rs10050860was in complete linkage disequilibrium with rs17482078 and rs17482078 minor allele had ~40% slower rates of substrate trimming than wild-type ERAP1 (25). Thus, it could be deduce that these three SNPs (rs2287987, rs17482078, and rs10050860) are strongly in linkage disequilibrium and lead to decrease enzyme activity.
We showed thatrs28096 in MAF (A) is strongly associated with AS and is protective. To our knowledge, the present study is the first to report association between rs28096 and AS. As noted earlier, the SNPs we found associated with AS in our current and previous studies (namely rs30187 (Lys528Arg), rs2287987 (Met349Val), rs10050860 (Asp575Asn), and rs26653 (Arg127Pro)), are prospective candidates to alter ERAP1 enzymatic function. In the crystal structure of the soluble domain of human ERAP1 (Figure 3), which is available in the Protein Data Bank (PDB) ID code 2YD0 (http://www.rcsb.org/pdb/explore/jmol.do?structureId=2YD0&bionumber= 1), there is a wide central cavity which accommodates Zn (II) forming substrate-binding site of the enzyme. Szczypiorska et al. have proposed that, since rs2287987 (Met349Val) reside in the proximity of this catalytic center (zinc-binding motif), it could affect ERAP1 activity(34). Harvey et al. had come up with the same idea (23). Afterwards Goto et al. described the function of rs30187 (Lys528Arg)(33). According to this study, Lys528Arg amino acid exchange causes a significant decrease in the enzymatic activity of ERAP1 and Lys528 is required for the maximal activity of the enzyme(33). Molecular modelings also revealed thatLys528 is located near the entrance of the substrate pocket so that affects the substrate-binding affinity of ERAP1 (35). Arg127 is one of the constituents of metallopeptidase domain of ERAP1(36), suggesting a probable role of this moiety in proteolysis activity of ERAP1.
Previous studies had shown that ERAP1 polymorphisms associated with AS affect the levels of expression of ERAP1 gene. It has been reported that rs28096 SNP shows the greatest association with ERAP1 expression (1.6 Ã- 10-32) (23). In our study, although no differences was observed in the expression level of ERAP1 between patients and controls, the analysis showed interesting results when comparing ERAP1 expression based on the presence of wild type and mutant homozygotic genotypes of rs30187 andrs28096. Surprisingly the expression level of ERAP1 gene in rs28096 G/G genotype was 1.48 times higher than rs28096 A/A genotype which raises a new challenge in the pathogenesis of AS. Moreover, the results indicated that ERAP1 gene expression level is associated with pain score in rs28096 G/G genotype while no similar association was seen in rs28096 A/A. (data not shown)
Then we performed haplotype analysis across 12 ERAP1 SNPs, and found two haplotype blocks. Block 1 consisted 5 SNPs (rs17481856, rs27044, rs469876, rs10050860, rs30187) and block 2 consisted 3 SNPs (rs27434, rs2287987, rs26653) (Figure 1). According to frequency analysis of haplotypes in patient and control groups, we found rs17481856, rs27434, rs469876, rs10050860, rs30187/GCATC haplotype (block 1) and rs27434, rs2287987, rs26653/ATC (block2) as protective; and rs17481856, rs27434, rs469876, rs10050860, rs30187/GCACT (block1) and rs27434, rs2287987, rs26653/GCG (block2) as being predisposing to AS (Tables 3).
When we analyzed the association of ERAP1 SNPs with disease severity, a trend of higher scores was noted for BASMI in two SNPs (rs30187 and rs28096). After controlling probable confounding variables, we found that only rs28096 had a significant relationship with BASMI (Table 3). Besides, rs28096 G/G genotype confers much higher disease severity than AA genotype regarding to BASMI, as from G/G genotype to A/A, mean BASMI increases 1.25 units (p-value = 0.008). In rs30187a trend of mean BASMI increase was also seen from T/T genotype to C/C, however the trend was not statistically significant. In fact, BASMI is the only objective index among AS patients and unearthing such a relationship with AS could provide helpful clues for individualized treatment in AS patients.
In conclusion our results confirmed ERAP1 association with ankylosing spondylitis in Iranian patients. Various SNPs of ERAP1 not only were associated with the onset of the disease, but also were associated with the severity of the disease and the rate of ERAP1 gene expression. We also showed the first evidence of influence of ERAP1 SNPs in BASMI. Other studies need to be performed on other SNPs of ERAP1 and determination of their correlation with disease severity in the current population.
Finally, we conclude that although several interesting GWAS and replication studies have been published introducing important SNPs and genetic basics of AS, the practical implications are still quite limited. The authors therefore propose that it's time to switch the gears and head toward functional studies for understanding the mechanisms of the known associations in AS pathogenesis.
We thank the Rheumatology Research Center and Iranian AS association for their valuable collaborations.