Association between interleukin 2 receptor A gene polymorphisms (rs2104286 and rs12722489) with susceptibility to Multiple sclerosis in Iranian population CURRENT STATUS:

Background: Multiple sclerosis (MS) is an organ-specific autoimmune disorder with remarkable heritability. For MS disorder, interleukin 2 receptor α subunit (IL2RA) is regarded as a genetic risk factor. Results: There was a statistically significant association between alleles and genotypes of rs12722489 SNP and MS risk. The levels of mRNA expression and serum IL-2RA were higher in MS patients than in healthy controls. mRNA expression and serum concentrations of IL-2RA were higher in MS patients with CC genotype for rs12722489 compared with the rest of patients. Conclusion: Our result demonstrate that the rs12722489 SNP within IL2RA gene might be associated with MS pathogenesis through regulating the levels of IL-2RA (or CD25), which is important in the regulation of T cells.

The estimated location of the human IL2RA gene is on chromosome 10 (10p15-p14). In the recent decade, the association of two common single nucleotide polymorphisms (SNPs) in the IL2RA gene (rs12722489 and rs2104286) has been widely evaluated with several autoimmune diseases (9). The association between MS susceptibility and SNPs in the IL2RA gene has been established in many countries, including Australia, India, Japan and United States (9)(10)(11), yet it has not been assessed in Iran. In this study, the rs2104286 and rs12722489, as two SNPs in an intron of the IL2RA gene in an Iranian population were assessed for susceptibility to MS, and the impression and association of these variations in secretion of sIL-2RA, as well as its transcript level in PBMCs, were estimated.  Primers Sequences are summarized in Table 1. sIL-2RA concentration Enzyme-linked immunosorbent assay (ELISA) measurement of sIL-2RA was accomplished in accordance with the manufacturer's recommendations (BD Biosciences). Samples of serum origin were diluted 1:20 through consuming phosphate buffer saline (PBS). Reading Microtiter plates was performed utilizing a Biorad Benchmark microplate reader. Providing an outcome with a normal distribution was carried out via utilizing a log10 transformation of total sIL-2RA concentration.

Statistical analysis
To determine the association of alleles, genotypes, dominant and recessive inheritance models with the disorder risk, the chi-square test was exerted. Odds ratios (ORs) with 95% confidence intervals (95% CI) were also measured for each comparison. The Hardy-Weinberg Equilibrium (HWE) was calculated for the control group in each SNP. For all analysis, SHEsis online tool (http://shesis.bio- x.cn/SHEsis.html) and STATA software were used. The group comparisons for mRNA expression level as well as serum concentrations were performed by independent sample t-test or ANOVA. Pearson's correlation coefficient was conducted to perform correlation analysis. P values less than 0.05 were considered as statistically significant level.

Results
Allele and genotype frequencies Genotype frequencies for both rs2104286 and rs12722489 SNPs in control group did not diverge from HWE (P = 0.478 and 0.514, respectively).
Genotypic results revealed that the C allele of rs12722489 was proportionally related with a higher risk of MS (Table 2) (Table 2).
Similarly, the results of genotyping showed that the A allele of rs2104286 was proportional to an elevated risk of MS (Table 3). In the same way, the analysis suggested a notable result in the AA vs.

Gene expression
The mRNA expression level of IL-2RA gene analysis showed that IL-2RA mRNA expression was higher in MS patient compared to the control group (Figure1.A). Also, the level of expression of a series of alleles in the two genotypic groups was different in patients compared with normal subjects

Correlation analysis
According to Table 4, the expression of IL-2RA mRNA was correlated significantly with EDSS in MS patients with CC genotype for rs12722489 (r = 0.55, P = 0.0021). Moreover, a significant correlation was observed in the serum levels of IL-2RA and EDSS in MS patients with CC genotype for rs12722489 (r = 0.61, P = 0.0133).

Discussion
In spite of the notable contribution of HLA in MS disorder, there is a significant percentage of unknown genetic factors involved in MS pathogenesis. The association of two most common polymorphisms of the IL2RA gene (rs2104286 and rs12722489) with an elevated risk of MS has been evaluated in plenty of papers. However, the association of these genotypes with respect to the conflicting data is still unknown.
Being an integral membrane compound, the IL-2 receptor consists of various protein structures, such as α (CD25), β (CD122), and γ (CD132). As mentioned, the signaling of the IL-2 receptor is of paramount importance for immune cell activation, especially those involved in the pathogenesis of MS disease, such as the T cell subtypes. Consequently, there is an opinion that inhibition of IL-2 signaling can inhibit T cells (13).
The rs2104286 and rs12722489 SNPs are located inside the primary intron of the IL2RA gene.
However, these SNPs do not directly alter the IL2-RA structure, but rather can change the expression of IL2RA through influencing the utilities of the regulatory components, including messenger RNA (mRNA), and mRNA half-life (9).
In this study, it was realized that the A allele of rs2104286 was related to a higher risk of MS, which is similarly shown in previous studies including Japanese, Canadian and Indian patients (14)(15)(16).
Likewise, the C allele of rs12722489 was related with an elevated risk of MS. However, the association of this allele with MS disease in some studies has not been seen yet (17). In addition, the secretion level of this receptor was also associated with polymorphisms of rs2104286 A allele and rs12722489 C allele in MS patients compared with healthy subjects in this study. It is not clear how these polymorphisms can affect the sIL-2RA gene expression. In segregated studies, buhelt and Maier have demonstrated that the level of sCD25 is affected by the MS-associated SNP rs2104286 in the IL2RA gene (9,18).
Recently, in the clinical phase, daclizumab as a human monoclonal antibody that attaches to IL-2RA, has been tested on MS patients. Furthermore, Clinical studies have revealed that injecting daclizumab subcutaneously to patients with MS that are in the process of recovery reduces the number of lesions in the brain's MRI. Daclizumab could increase the proliferation and improve the utility of regulatory NK cells, which possess the capability to decrease activated T cells (19).
In addition, Daclizumab causes a blockade to the action of IL-2 presentation via mature dendritic cells to primed T cells, in consequence, T cells and maybe other immune cells will be significantly reduced (20). Daclizumab also prevents the meningeal lymphoid agglomeration and correlates to the immune memory responses through hindering lymphoid tissue inducer (LTi) cells (21).
By investigating the effects of daclizumab, the role of IL-2 in the pathogenesis of multiple sclerosis was clearly determined, and it is hoped that with further understanding of mutations and polymorphism of this receptor gene, a definite treatment for this autoimmune disease can be found.
In the end, some of our work restrictions need to be declared. Firstly, absence of accessibility to individual information restrained the assessment of feasible gene-gene and gene-environment interplay. Secondly, the relationship between the IL2RA polymorphisms and MS subtype such as     Figure 1 Comparison ILR-2RA gene expression between MS cases and control group Figure 2 sILR-2RA concentration in the serum of MS patient and healthy subjects