The Association Between IL-2 Gene (RS2069763( Single Nucleotide Polymorphism and Type 2 Diabetes Mellitus in Iraqi Patients

This research attempts to find the association between single nucleotide polymorphism (SNP) of IL2+166 gene (rs2069763) and type 2 diabetes mellitus (T2DM) in a sample of Iraqi patients. A total of 44 patients and 55 apparently healthy volunteers were genotyped for the SNP using polymerase chain reaction test. Three genotypes (GG, GT, and TT) corresponding to two alleles (G and T) were found to have SNP. Both study groups’ genotypes had a good agreement for the analysis of Hardy-Weinberg Equilibrium. The results revealed increased frequencies between the observed and expected GG and TT genotypes and IL2+166 SNP T allele in T2DM patients (40.9 vs. 40.0 %; OR = 1.04; 95% CI, 0.47 2.31), whereas the values in the control group were 11.4 vs. 9.1 %; OR = 1.28; 95% CI, 0.35 4.68. Nevertheless, both variations did not reach a significant level. In the Iraqi population, the IL2+166 SNP was not associated with T2DM and, therefore, no association with its etiopathogensis was found.


Introduction
Type II Diabetes Mellitus (T2DM) is an endocrinological condition resulting from increased secretion of insulin and/or irregular insulin behavior [1,2]. Reduction of insulin level results in chronic abnormally high blood sugar and resistance to glucose [3]. Currently, diabetes epidemic is related to a gene-environmental affliction. Worldwide prevalence estimates suggest that 85-95 percent of T2DM occurred in the developing countries.
In a 2010 study, the prevalence was reported to be 6.4 percent, affecting 285 million people in the age group of 20-79 years worldwide. It is expected that 438 million people will be diabetic in the developing countries by 2030, reaching 70 percent, along with a rise of 20 percent in developed countries [4]. Due to ongoing insulin resistance development and ß-cell dysfunction, the pancreas is unable to produce enough insulin to overcome insulin resistance. As a result, about 85% of the T2DM population is obese, leading to resistance to insulin [5]. Interleukin-2 (IL2) is the major growth factor for T cells and its binding to its specific receptors on T-helper cells stimulates their proliferation and production of effective cytokines from these cells [6]. This cytokine is under genetic control on chromosome locus 4q27. Expression levels of cytokine genes are often related to SNPs leading to their irregularity, which may modify the disease and thereby may lead to development of various immunological diseases, including T2DM [7]. Correlation of IL2 genetic variation with systemic lupus erythematous (SLE) [8] and Multiple sclerosis (MS) [9,10] as well as the IL 1B SNP in Rheumatoid arthritis (RA) [11] were investigated to reveal potential impact of genetic variation with the disease etiopathogensis. Therefore, this research focuses on possible association between T2DM and genetic variation of IL2 +166 G/T in a sample of Iraqi population with T2DM.

Subjects
Forty four T2DM patients (19 males and 25 females) were enrolled and their age range was 28-77 years (50.5 ± 15.5 years). The Iraqi Health Ministry Ethical Committee approved the study. During the time from February to March 2019, the patients were referred for diagnosis and treatment to the National Center for Diabetes Care and Research / Al-Mustansiriyah University. The assessment was made by the clinical consultant and was based on the updated diagnostic criteria by the American Diabetes Association (T2D ADA criteria, 2010) [12]. The study also included 55 apparently healthy control volunteers. The control subjects were matched with the patients for the race (Iraqis), gender (27 males and 28 females) and they were aged from 17 to 70 years (31.9 ± 14.9 years).

Gene polymorphism of IL2
The TonkBio Genomic DNA Extraction Kit (New Jersey, USA) was used to extract genomic DNA from EDTA blood samples. Following purity and concentration evaluation, it was subjected to specific sequence primer technique using polymerase chain reaction test (PCR-SSP). Two primers were designed (Forward: 5'-CTGGAGCATTTACTGCTGGATT-3' and Reverse: 5'-ACTCTTTACCTCAGATGAGCTGCTA -3') for the genotyping of IL2 +166 (G/T) SNP (rs2069763) using the Geneious software, version 10.1.3.
The PCR reaction was conducted at a final volume of 20 μl, including 6.5μl of the master mix (GoTaq green, Promega ® -USA), 1 μl of the forward primer (10 μM), 1 μl of the reverse primer (10 μM), 1.5 μl of DNA (50 ng), and 10 μl of nuclease free distilled water. The following steps were applied as PCR reaction conditions: an initial denaturation at 94 o C for 5 min, then 35 cycles of denaturation at 94 o C for 30 sec, followed by annealing at 63 o C for 30 sec and extension at 72 o C for 30 sec, and ended by a final extension at 72 o C for 10 min. The amplified PCR fragments were then run on agarose gel electrophoresis (1.5% agarose at 5 v/cm 2 for 45 minutes).

Statistical analysis
The age was expressed as mean ± SD, and the normality, randomization and homogeneity were calculated for the age by using IBM SPSS computer program V25.0. Frequencies of allele and genotype were given as percentage. The genotype frequencies were first tested for agreement with Hardy-Weinberg equilibrium (HWE), then Pearson's Chi-square analysis was performed to evaluate the significance of differences between the observed and expected genotype frequencies (https://www.easycalculation.com/health/hardy-weinberg-equilibrium-calculator.php). The relation Al-Malkey.
Iraqi Journal of Science, 2020, Vol. 61, No. 5, pp: 993-998 995 between IL2 SNP and T2DM was addressed in terms of odds ratio (OR) and the exact probability of two-tailed Fisher was evaluated as a significant difference [13]. WINPEPI version 11.65 software for epidemiologists was used (www.brixtonhealth.com) to achieve the latter calculations.

IL-2 +166 gene polymorphism
Genetic variations of IL2 +166 (G/T) (rs2069763) SNP investigated the change of G allele to T allele at the nucleotide position 15 of the forward DNA strand. This change occurred at chromosome 4:122456327 positions. They were genotyped by PCR with allele specific primer (PCR-ASP) technique [14,15]. The IL2 gene PCR amplified products revealed a single band of 301bp on agarose gel electrophoresis (Figure-1). Analysis of HWE indicated that there was significantly no difference between frequencies of IL2 +166 (G/T) (rs2069763) SNP in T2DM patients for both the observed and expected genotype. Therefore; these genotypes revealed a good equilibrium agreement with the HWE analysis in patients and control groups (Table-1).  Type 2 diabetes mellitus involves multiple metabolic dysfunctions resulting in hyperglycemia from decreased insulin production and increased insulin resistance, in addition to irregular glucagon metabolism and signaling pathways of insulin and beta-cells [16]. It is proposed that several interleukins contribute to T2DM's pathology and have impacts on the signaling pathways of insulin and the function of beta-cell [17]. In the current study, there is a lack of association between IL2+166 G/T SNP and T2DM in the investigated Iraqi population. A study by Howson et al., (2011) in Ulm and the surrounding area, southwest Germany, revealed an increased risk of genetic variation of IL2 +116 SNPs (rs2096763 and rs2096762). An association between IL2 (rs2069763) among 786 T2DM cases and 1,484 controls and its predisposition to adult-onset autoimmune diabetes was indicated [18], which contradicts our findings due to variations in sample size and ethnicity. A relevance association between IL2 (rs2096763) SNP and type 1 diabetes (T1DM) among white European ancestry was investigated by Howson et al., (2012) and revealed that the major allele G confers protection from T1DM, with an average of GG alleles at age of nine years old . IL2 gene expression is correlated to T1DM pathogenesis, added to that two IL2 receptor genes (IL2 RA and IL2 RB ) are both associated with type 1 diabetes [19]. It is suggested that IL2 gene variation has a strong correlation to the exacerbation of autoimmune types of diabetes mellitus with increasing age to older than 17 years [19], which was also confirmed by another two previous studies [20,21].
To the best of our knowledge, IL2 +166 (rs2069763) genetic variations and its predisposition to T2DM as a risk factor in Iraqi population have not been previously demonstrated. Meanwhile, a study by Hamid and Shani (2018) investigated the association between IL-10 (-592A/C) gene polymorphism with the progression of T2DM in Basrah Province and revealed significant association between CA genotype with the risk of T2DM and that IL-10 (-592A/C) SNP contributes to the development of T2DM [22].

Conclusions
This study results showed that GG genotype / T allele of IL2 +166 SNP is not an attributed factor for T2DM in this sample of Iraqi patients. In order to increase the statistical power for further investigations, the sample size is needed to be increased and different clinical courses for the disease could be implemented. Added to that, cytokine gene expression studies will be required with different diabetes mellitus clinical courses and associations to therapy response.