Association study of IL2RA and CTLA4 Gene Variants with Type I Diabetes Mellitus in children in the northwest of Iran

Introduction

Type 1 diabetes mellitus (T1D) (OMIM 222100) is an organ-specific and autoimmune disease that primarily affects children and adolescents. It is caused by destruction of the beta cells of Langerhans of the pancreas that secrete insulin, which occurs through the autoimmune processes. Therefore, the affected patients have to use insulin throughout their lives.¹ The clinical symptoms of this disease include frequent urination (polyuria), excessive thirst (polydipsia), increased hunger (polyphagia), and weight loss.^2-5 T1D comprises 5%–10% of the total number of diabetes cases, and the risk for each individual in a specified population is estimated to be 4%. This risk would be further increased by more than 1%, if the mother suffers from T1D, and by more than 3%, if the father is also affected.⁶ In recent decades, the prevalence of T1D has been increasing at a rapid rate, with an increase by about 40% from 1998 till 2010.⁷ T1D is a heterogeneous and complex disease, and genetic and environmental factors are involved in increasing the risk of its incidence. In this regard, several studies have been conducted on family cases and monozygotic and dizygotic twins, which have reported the genetic factor as one of the important factors in this disease.^8,9 Diabetes is an epidemic disease and a major health risk in the world, and the situation is rapidly worsening. In the last three decades, numerous studies have been conducted on T1D cases to assess complex multi-factors and identify susceptibility genes. These studies have resulted in the identification of more than 40 different genetic loci associated with this disease.^10,11 Several loci are more commonly associated with T1D, including human leukocyte antigen (HLA) region on chromosome 6p21, the PTPN22 on 1p13, IL2RA on 10p15, the insulin gene (INS-VNTR) locus on 11p15, and the cytotoxic T-lymphocyte associated protein 4 (CTLA4) locus on 2q33.¹² Although numerous genes have been associated with T1D, the frequency of each of these genes associated with this disease in various populations is very different.^7,9

In 1996, the CTLA4 gene was reported as one of the important susceptibility genes in T1D.¹³ CTLA4 is one of the members of the immunoglobulin family that has four exons and hence plays an important role in the pathogenesis of autoimmune diseases such as T1D.¹⁴ This gene encodes a cell surface receptor that is expressed on activated T cells and functions as a negative regulatory factor.¹⁵ A common polymorphism in the CTLA4 gene coding region is +49 A>G, which is a conversion of alanine to threonine in exon one.¹⁶

In addition, it was reported in 2005 for the first time that interleukin 2 receptor subunit alpha (IL2RA gene or CD25) is strongly associated with T1D.¹⁷ IL2RA gene has eight exons and encodes the α-chain of the interleukin-2 receptor complex. IL2 is a strong growth factor for lymphocytes. It is known that the expression of IL2RA in CD4+CD25+ regulatory T-cells is essential to control the immune response of T cells and to prevent autoimmune diseases.¹⁸ The ss52580101 polymorphism located in intron 1 in this gene is associated with T1D.^19,20 Studies on different populations have reported different results regarding the association between CTLA4 and IL2RA variants. Some of these studies have shown a significant correlation between CTLA4 and IL2RA polymorphisms and T1D^16,21,22 while some studies reported no such relationship.^23-25 Therefore, these gene polymorphisms can be a new strategy for the prognosis of T1D and can offer improved treatment strategies.^2,6 Based on this evidence and because there is no information on the association of CTLA4 and IL2RA variants with T1D in the northwest of Iran, the present study was conducted to confirm the association of these variants in children in the northwest of Iran.

Materials and methods

Results

As described in methods, a total of 100 subjects (50 T1D patients and 50 healthy subjects) were selected in this study. The demographic data and clinical characteristics of the subjects are presented in Table 2.

At first, the aim of this study was to estimate the prevalence of common polymorphisms of the genes CTLA4 (+49 A > G) and IL2RA (ss52580101 C > A) via Sanger sequencing, then two new variants chr2:203868145 A>C and chr10: 6072892 A>G were detected for CTLA4 and IL2RA genes, respectively.

Discussion

Diabetes is a relatively common chronic disease in the world that affects various races and populations. The prevalence of this disease in several communities especially in developing countries is increasing. Studies of the entire human genome with the aim of screening loci involved in the development of diabetes in families with more than one individual with T1D found different gene polymorphisms linked to T1D. The most important genes include HLA, PTPN22, CTLA4, INF, IL2RA, and TGFB1.^9,28 In recent years, several case-control studies have been performed to assess the relationship between polymorphisms of these genes with T1D.¹⁶ As a result, it is reasonable to conduct a separate associative study on any specific ethnic population.^7,12 In this regard, in the northwest of Iran, studies on the relationship between HLA²⁹ and PTPN22 gene polymorphisms³⁰ had been conducted on children with T1D, with no significant relationship observed between these genes and T1D.

We considered the most common SNP in CTLA4 and IL2RA genes that was associated with T1D in children in the northwest of Iran. We observed that the frequency of G allele and GG genotype of +49A>G of CTLA4 gene in these patients was significantly higher than that in the control group (the G allele frequency was 26% in patients and 11% in healthy subjects). Therefore, this study confirms the relationship of the polymorphism +49A>G with an increased risk of T1D in the study population. In addition, in the case of the new SNP (chr2: 203868145 A>C) that was identified for this gene in our population, it was found that the frequency of C allele was significantly different between two groups (p = 0.005). Moreover, we did not observe a significant relationship between ss52580101C>A polymorphism of IL2RA gene and T1D (p = 0.4). It is clear that the allele frequency of the new polymorphism of IL2RA gene (chr10:6072892A>G) was not significantly different between patients and healthy subjects (p = 0.11).

CTLA4 gene is a cell surface molecule and a member of immunoglobulin family. It is believed that mutations in this gene increase the activity of T cells, and therefore it is associated with several autoimmune diseases, including T1D, Graves’ disease, lupus, and celiac disease.^31-33 CTLA4 gene is known as one of the important genes involved in T1D. Studies have reported a relationship between the polymorphism +49 A>G and T1D in different populations, including the Japanese,³² Korean, Italian, Spanish, French,³³ Egyptian,³⁴ and Belgian.³⁵ Moreover, studies have been conducted in different parts of Iran in this regard. Mojtahedi et al reported that +49 A>C is linked to the risk of T1D in southern Iran. They showed that the frequencies of G allele were 45% in T1D patients and 33.4% in healthy subjects (p = 0.0026).³⁶ Ahmadi et al also reported that the frequencies of allele +49 A>G were 36.5% in T1D patients and 20.6% in healthy subjects, which confirms that this SNP in T1D patients is significantly different from that in the healthy subjects with Iranian Kurdish ethnicity.³⁷

In this study, we observed a similar relationship in the northwest of Iran. Therefore, it can be confirmed that the most common gene polymorphism (+49 A>G) in CTLA4 gene is important in increasing the risk of T1D in Iranian population. Of course, it should be noted that the results of studies in some populations, such as Turkish,²⁵ Chinese,³³ Japanese,³⁸ and Azerbaijan,³⁹ have reported the lack of such a relationship between CTLA4 and this disease.

IL2RA gene has a key role in regulatory T (Treg) cell proliferation. Studies have shown that ss52580101 polymorphism located in intron 1 of IL2RA gene is involved in T1D. Low et al in a comprehensive study considered 288 SNPs of IL2RA gene and proved that ss52580101 is the most common SNP associated with T1D.⁴⁰

Dendrou et al in a study on the population of Poland showed a significant increase in allele and genotype frequency of polymorphism of IL2RA in T1D patients compared to that in healthy people.⁴¹ Maier et al reported that the allele frequency of ss52580101 in T1D patients is significantly higher than that in control subjects in USA.¹⁹ In this study, based on a strong relationship between polymorphisms in IL2RA and T1D that has been proven in different populations, we examined the genotype of IL2RA. However, the results observed for ss52580101 were in contrast to the above mentioned studies. We found that ss52580101 is the most common SNP of IL2RA gene and has no relationship with T1D in our population. However, our results were consistent with a study by Kawasaki et al in Japanese population who also showed that ss52580101 SNP is not associated with T1D.²²

There are studies that have shown that polymorphisms of CTLA4 are associated with clinical characteristics and pathological conditions of T1D. Abe et al reported that polymorphism of CTLA4 is associated with the relationship between ICA512 antibodies and diabetic ketoacidosis.⁴² Balic et al reported that patients who have G allele polymorphisms +49A > G have increased glycemic levels.⁴³ In this study, we found a significant relationship between GG genotype +49A > G and glycemic level of T1D patients (p = 0.0067). Glycemic level of diabetic patients has an important role in their survival, and we can thus say that this SNP in diabetic individuals can be important. In the present study, it was found that among these four SNPs, only IL2RA polymorphism follows the Hardy–Weinberg equilibrium. The reasons for the imbalance in the other three SNPs in the studied population could be the heterogeneous nature of T1D and the small sample size. Therefore, it is suggested that in such case-control studies, large sample sizes be selected to report results with greater confidence.

Conclusion

The results of this study confirmed the relationship between CTLA-4 +49A>G polymorphism and the risk of T1D and increased blood glucose levels in patients in the northwest of Iran. In addition, a newly identified polymorphism of CTLA4 (chr2: 203868145A>C) was found to be associated with T1D.

Ethical approval

All the experiments were performed according to the guideline provided by the Ethical Committee at Zanjan University of Medical Sciences. Informed consent was also obtained from the parents or guardians of all subjects who participated in this study.

Competing interests

Authors declare no conflict of interests.

Acknowledgments

We thank the Research Deputy of Zanjan University of Medical Sciences (ZUMS) for financial support of this project (Grant No. A-12-34919, A-12-379-20).

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