Correlation of -475 IL-2 Promoter Gene Polymorphisms and the Levels of Serum IL-2 on the Risk of Multiple Sclerosis

Background: The aim of present study is to asset the IL-2 promoter gene (SNP -475) as a candidate gene for multiple sclerosis (MS) susceptibility. Methods: This study included 70 patients with relapsing – remitting multiple sclerosis (RRMS) and 50 healthy controls. Following the extraction of genomic DNA from peripheral blood, frequency of genotypes and alleles of SNP -475 was calculated using Restriction fragment length polymorphism-polymer chain reaction (RFLP-PCR) and then the results were analyzed statistically. Results: The results revealed the unusual ratio for the heterozygous (AT) was 1.6972 indicating that heterozygous patients were at higher risk of multiple sclerosis than wild homozygous (AA), and homomutant (TT). The results show protective role for - 475 IL-2 promoter among individuals with multiple sclerosis, (O.R: 0.4872; C.I. 95%: 0.1617- 1.4680) and (O.R: 0.9275; C.I. 95%: 0.2476 - 3.4745) for both AA and TT genotypes, respectively. Conclusions: Our results showed that in this population of Iraqi patients, the AT genotype / A allele of -475 IL-2 promoter gene SNP may include attributed factors for MS predisposition.


Introduction
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system affecting young adults. The result is the demyelination and dense astrogliosis of the white central nervous system (CNS) substance in multiples (1,2). It is assumed that the pathogenesis of MS may involve an aberrant immune response to myelin antigens (3). Multiple sclerosis (MS) has the potential to decrease CNS connectivity, which can cause a variety of signs and symptoms, including physical, emotional, and occasionally psychological issues (4). In the clinical epidemiology study, around 2,300,000 people were worldwide affected by MS in 2013, with approximately 20,000 deaths in MS in the same year (5). The Middle East and North Africa countries are situated in a low-to moderate risk region for MS according to MS Atlas (6). Multiple sclerosis is a complex disease arising from the combined action of several genes of susceptibility and one or more environmental factors. Interleukin-2 (IL-2) is a type of cytokine that regulates the immune system functions, it is a 15,000-kDa alpha-helical cytokine produced mainly by CD4+ and CD8+ T cells that are activated, the latter to a lesser extent. lnterleukln-2 (IL-2) is essential for T-lymphocytes to proliferate and differentiate (7). Three Single nucleotide polymorphisms (SNPs) have been identified at positions 384, 475, and 631 in the 800-bp 5' upstream region of the human IL-2 gene (8,9). In the current study we have investigated the effects of the -475 A/T SNP on serum IL-2 levels on the risk of multiple sclerosis in Iraqi patients versus healthy people.

Blood Sampling
Five ml of blood were collected by vein puncture, one ml was put into EDTA tubes for molecular analysis and stored at -20 ºC until analysis, and the other 4 ml in gel tube for serum separation.

Quantitative assessment of serum IL-2
The levels of IL-2 in the blood were assessed. The blood was kept at room temperature for 30 minutes to allow for clotting before being centrifuged for 15 minutes at 2000 g. The serum was held at -20 °C until it was analyzed. The concentrations of serum cytokines were calculated using an enzyme-linked immunosorbent assay (ELISA) as directed by the manufacturer (Human Interleukin 2 ELISA/ Bt-laboratory/ China). The serum concentrations were measured in (pg/ml).

Extraction of Genomic DNA
Genomic DNA was isolated from blood samples using gSYNC™ DNA Extraction Kit (Geneaid/ Taiwan). DNA concentration and purity were determined by spectrophotometry with NanoDrop.

Genotype of -475 IL-2 promoter Polymorphism
Two primers were selected (Forward: 5'-ATAGACATTAAGAGACTTAAAC-3') and (Reverse5'-GTAACTCAGAAAATTTTCTTTG-3') (Bioneer/Korea) (9) to amplify a fragment of 332 bp for alleles detection, aliquots of amplified DNA products were digested with MseI (New England Biolabs/England). The PCR conditions were 95 °C for 2 min, amplifications were carried out in a GTC thermal cycler (Cleaver Scientific, UK) for 35 cycles, each with 30 s denaturation at 95 °C, 30 s annealing at 50 °C and 40 s extension at 72 °C. The final elongation step at 72 °C was for 5 min. The PCR product IL-2 promoter gene was digested with restriction endonucleases in a total volume of 25 μl containing 10 units of enzyme with buffers supplied by the manufacturer's instructions. The PCR products have been checked on 2% (w/v) of agarose gel for expected size. The molecular weight of the fragments was determined using a 50-bp DNA molecular weight marker (New England Biolabs/England) (11).

Biostatistical consideration
The SAS (Statistical Analysis System. Version 9.1th ed. SAS. Inst. Inc. Cary. N.C. USA. 2012) was used to investigate the differences between the study's variables. To compare percentage differences, a Chi-square test was used, at P-values of 0.05 and 0.01. The CI. 95% and odds ratio were determined.

Results
This research included 70 MS patients and 50 healthy controls (Table 1).
Mean age for the case group were (37.17±8.987) years, while the control group were (36.66±8.858) years. In the cases, there were 44 (62.8%) females and 26 (37.1%) males, while in the control groups, there were 29 (58%) females and 21 (42%). In regarding the gender distribution, there was no statistically significant difference between MS cases and healthy controls (0.0678 at p≤ 0.05).

Distributions of Genotypes and Allele Frequency of the SNP (-475) of IL-2 promoter gene Polymorphisms
The SNP (-475) of IL-2 promoter gene polymorphisms were amplified using PCR and unique primers. The results indicated that molecular size of -475 IL-2 promoter was approximately 332 bp in length. Detection of the SNP (-475) of IL-2 promoter gene polymorphism, PCR-RFLP was achieved by a specific restriction enzyme MseI for digestion of PCR product of -475 IL-2 promoter gene. Table 3 shows the distribution of the observed SNP (-475) of IL-2 promoter gene genotypes, alleles frequencies, genotype frequencies of AA, AT, and TT of SNP (-475) of IL-2 promoter gene polymorphism in the control and cases individuals. The results show that there were no significant differences of SNP (-475) IL-2 promoter genotypes cases and control groups. The odd ratio for the heterozygous (AT) was 1.6972 indicating that heterozygous patients are in higher risk of multiple sclerosis than wild homozygous (AA) and homo-mutant (TT), while the result show protective role for -475 IL-2 promoter among individuals with multiple sclerosis. The allele frequency for case group (A), T were found 0.549 and 0.449, respectively, in patients, while for control group the allele frequency of A and T were 0.51 and 0.49, respectively.

Association between genotype of -475 IL-2 promoter gene and serum IL-2
Regarding the association between genotype of SNP (-475) of IL-2 promoter gene polymorphism and serum IL-2, there were no differences among case and control groups ( Table 4). The findings of this analysis revealed that the -475 IL-2 promoter TA genotype reported the highest value serum level of IL-2 among MS patients (1069.031±393.0203 pg/ml), compared to the TA genotype (259.9776±187.4645 pg/ml) for control groups, but not significate.

Discussion
IL-2 is a cytokine which is released by TH1 cells in response to mitogenic or antigenic activation (12). Several studies have shown that the level of serum IL-2 in MS patients is higher than healthy people (13)(14)(15)(16)(17)(18).
In case of chronic progressive MS, in vitro studies revealed higher IL-2 level (20), whereas other researchers could not confirm this finding (21,22). Trotter et al. (22) found a positive and significant linkage between serum IL-2 levels and disabilities in patients with progressive MS.
One study on the Iranian population found that the frequency of IL-2(-475) polymorphisms was slightly higher in MS patients compared to healthy controls, but these variations in frequency were not significant (23). So, our findings agree with these results (24,25). Sayad et al., noticed an increase in IL-2 plasma concentration in MS patients. Thus, some polymorphisms may play a key role in MS disease pathogenesis especially in the promoter region of the IL-2 gene (26). Additionally, to study in Iranian achieved by Sayad et al., (23) they found no evidence of a connection between IL-2 polymorphisms -574 and -631 and MS disease. The SNP -475 IL-2 promoter is located in the IL-2 promoter's distal region Ward et al., showed that the distal region of the SNP -475 IL-2 promoter can act as a stable nucleation and/or initiator site (28).
The relationship of IL-2 SNP to MS conditions has been reported already by Asouri et al. (29). They found that IL-2RA rs12722489 is significantly associated with the annual attack rate adjusted by the disease duration.
The present results showed the AT genotype / A allele of -475 IL-2 promoter gene SNP may include attributed factors for MS predisposition among Iraqi patients.