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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 1  |  Page : 100-106

Association of CTLA-4 (+49A/G) gene polymorphism with type 1 diabetes mellitus in Egyptian children


Department of Pediatric, Faculty of Medicine, Menoufia University, Shebin El-Kom, Menoufia, Egypt

Date of Submission08-Jan-2015
Date of Acceptance12-Mar-2015
Date of Web Publication18-Mar-2016

Correspondence Address:
Zeinab S Abouzouna
Shebin El-Kom, 32511 Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.178996

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  Abstract 

Objective
The aim of the study was to investigate the distribution of cytotoxic T-lymphocyte antigen-4 (CTLA-4) (+49A/G) gene variants and its association with type 1 diabetes (T1D) in Egyptian children.
Background
T1D is a complex autoimmune disease characterized by T-cell-mediated destruction of the pancreatic island. Human leukocyte antigen (HLAs) account for about 60% of genetic susceptibility for the disease. About 20 non-HLA loci contributing to disease susceptibility have been identified. One among these is the CTLA-4 gene. CTLA-4 polymorphisms are associated with T1D in some but not all populations. The aim of our study was to investigate the frequency of CTLA-4 49A/G polymorphism in Egyptian children, and its susceptibility for development of T1D.
Materials and methods
A case-control study was conducted on 40 Egyptian children with T1D diagnosed according to ADA (2010). Their ages ranged from 1 to 16 years, with a mean of 8.76 ± 4.70 years; 15 were male and 25 were female. In addition, 20 age and sex-matched healthy controls were included. CTLA-4 (+49A/G) gene polymorphism typing was done by PCR amplification, followed by restriction fragment length polymorphism.
Results
The frequency of CTLA-4 polymorphisms (AA, AG, and GG) in T1D patients was 42.5, 52.5, and 5% versus 60, 40, and 0% in controls, respectively, with predominance of the AG variant in diabetic patients. Distributions of the CTLA-4 gene polymorphism on the basis of sex showed a normal pattern. The AA genotype was present in 64.7% of female diabetic patients versus 35.3% of male patients, whereas the heterozygous AG genotype was seen in 57.1% of female patients versus 42.9% of male patients, and the homozygous GG genotype was present in two female patients (5%) and was not recognized in male patients; the female predominance can be explained by steroid hormone association.
Conclusion
In this study the overall pooled prevalence of the CTLA4 (+49A/G) polymorphism was recognized but still not a risk susceptibility factor for T1D in Egyptian children.

Keywords: Cytotoxic T-lymphocyte antigen-4, Egyptian, genotyping, type 1 diabetes


How to cite this article:
Tawfik MA, Abou El-Ella SS, Abouzouna ZS. Association of CTLA-4 (+49A/G) gene polymorphism with type 1 diabetes mellitus in Egyptian children. Menoufia Med J 2016;29:100-6

How to cite this URL:
Tawfik MA, Abou El-Ella SS, Abouzouna ZS. Association of CTLA-4 (+49A/G) gene polymorphism with type 1 diabetes mellitus in Egyptian children. Menoufia Med J [serial online] 2016 [cited 2019 Sep 20];29:100-6. Available from: http://www.mmj.eg.net/text.asp?2016/29/1/100/178996


  Introduction Top


Type 1 diabetes (T1D) is a genetically complex disorder of glucose homeostasis that results from autoimmune destruction of the insulin-secreting cells of the pancreas. The development of T1D likely results from exposure to environmental factors, which interact with a number of genes that contribute to the susceptibility of the disease [1].

HLAs account for about 60% of genetic susceptibility for the disease. About 20 non-HLA loci contributing to disease susceptibility have been identified. The function of only two non-HLA loci is known: the insulin gene and the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene [2].

The CTLA-4 receptor is found on the surface of T cells. The T-cell attack can be turned off by stimulating the CTLA-4 receptor, which acts as an 'off' switch. In humans, the CTLA-4 protein is encoded by the CTLA-4 gene [3].

Polymorphisms have been identified in the CTLA-4 gene and have been associated with different susceptibilities to a wide range of T-cell-mediated autoimmune disorders [4].

Thus, it is important to study the distribution of CTLA-4 (+49A/G) gene variants and their association with clinical and laboratory findings in Egyptian children with diabetes.


  Materials and methods Top


This study was conducted on 40 diabetic children who attended our pediatric genetic and endocrinology unit and clinic in Menoufia University Hospital from October 2012 to January 2014. Their ages ranged from 1 to 16 years, with a mean of 8.76 ± 4.70 years. The patients included 15 boys (37.5%) and 25 girls (62.5%). The control group consisted of 20 healthy children (10 boys and 10 girls; mean age 7.0 ± 5.07 years) from our pediatric general ward in Menoufia University Hospital, matched in age and sex to the diabetic patients.

The diagnostic criteria for the diabetic children were as follows [5]:

  1. Fasting blood glucose of at least 7.0 mmol/l (≥126 mg/dl).
  2. Two-hour postprandial glucose of at least 11.1 mmol/l (≥200 mg/dl).
  3. Glycosylated hemoglobin (HbA1c) of at least 6.5.


Patients were subdivided according to the level of HbA1c (control <8% and poor control ≥8%) [6].


  Materials and methods Top


All patients and controls were subjected to the following after taking informed consent from their families

  1. Detailed history evaluation.
  2. Complete general examination including anthropometric measurements.
  3. Laboratory investigations including:
    1. Estimation of fasting blood glucose levels.
    2. Estimation of postprandial blood glucose levels.
    3. Estimation of HbA1c.
    4. Evaluation of thyroid functions by determining FT3, FT4, and TSH to exclude autoimmune diseases.
  4. Molecular study of the CTLA-4 gene:

    This was carried out in the genetic lab of our genetic and endocrinology unit and included:

    1. DNA extraction.
    2. PCR.
    3. Gel electrophoresis.
  5. Family counseling, which important in controlling the disease, and regular follow-up.
  6. Data management and statistical analysis.


Results were statistically analyzed using statistical package SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA). Two types of statistics were ascertained. P-value less than 0.05 were considered statistically significant.

  1. Descriptive statistics: presented as percentage (%), median, mean, and SD.
  2. Analytic statistics: presented using Student's t-test, the Mann-Whitney U-test, One-way analysis of variance (F test), the Kruskal-Wallis test, the ν2 -test, odds ratio (OR), post-hoc test, and Z-test; P-values less than 0.05 were considered statistically significant [7].



  Results Top


At the time of the study, the ages of patients in the diabetic group ranged from 1 to 16 years, with a mean of 8.76 ± 4.70 years, whereas the ages in the control group ranged from 3 to 15 years, with a mean of 7.0 ± 5.068 years (P = 0.004). There were 25 girls (62.5%) and 15 boys (37.5%) among the patients, whereas the control group comprised 10 boys (50%) and 10 girls (50%) (P = 0.355). The percentage of consanguinity in diabetic patients was 20 versus 80% with nonconsanguinity, and this was found to be statistically nonsignificant (P = 0.519). The percentage of positive family history in diabetic patients was 32.5%, whereas the percentage of negative family history was 67.5%, versus 100% negative family history in the control group. This was found to be significant statistically (P = 0.003) [Table 1].
Table 1: Demographic data of the studied groups

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The frequency of the A allele was 55 (68.7%) in the diabetic group (equal to 34 alleles from 17 AA variants and 21 alleles from 21 AG variants), versus 32 (80%) in controls, whereas the frequency of the G allele was 25 (31.3%) in the diabetic group (equal to four alleles from two GG variants and 21 alleles from 21 AG variants, as each variant provides two alleles), versus 20% in controls, showing statistically nonsignificant difference (P = 0.193) (OR 1.82 at 95% confidence interval in patients) [Table 2].
Table 2: Distribution of cytotoxic T-lymphocyte antigen-4 (+49A/G) alleles and genotypes in the studied groups

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The prevalence of the CTLA-4 polymorphisms AA, AG, and GG in type 1 diabetes mellitus patients was 42.5, 52.5, and 5%, respectively, versus 60, 40, and 0% in controls, showing statistically nonsignificant difference (P = 0.320) (OR 1.85 at 95% confidence interval in patients) [Table 2].

The distribution of CTLA-4 polymorphisms AA, AG, and GG on the basis of age was 23.5, 28.5, and 0.0% below the age of 5 years versus 35.5, 28.5, and 50% from 5 to 10 years, and 41.2, 42.9, and 50% after the age of 10 years, with statistically nonsignificant relation between any age group or any type of gene variant [Figure 1],[Figure 2] and [Figure 3]. In contrast, distribution of CTLA-4 polymorphisms between male and female patients was 35.3 and 64.7 for the AA genotype, 42.9 and 57.1 for the AG genotype, and 0 and 2 for the GG genotype, with apparently female predominance [Table 3].
Figure 1: Distribution of the CTLA-4+49A/G polymorphism in diabetic patients and control s by ECO911 RFLP. From left: lane 1: DNA ladder 100 bp. Band A at 162 bp, both fragments of band G at 88 and 74 bp appearing as one band because of being very close to each other. Lane 3: heterozygous (AG) genotype. Lane 7: homozygous (GG) genotype. Lane 2, 4, 5, 6, 8: homozygous (AA) genotype. CTLA-4, cytotoxic T-lymphocyte antige n-4.

Click here to view
Figure 2: Distribution of the CTLA-4+49A/G polymorphism in diabetic patients and controls by ECO911 RFLP. From left: Lane 1: DNA ladder 100 bp. Band A at 162 bp. Both fragments of band G at 88 and 74 bp appearing as one band because of being very close to each other. Lanes 2, 4, 6, 8: heterozygous (AG) genotype. Lanes 3, 5, 7: homozygous (AA)
genotype. CTLA-4, cytotoxic T-lymphocyte antige n-4.


Click here to view
Figure 3: Distribution of the CTLA-4+49A/G polymorphism in diabetic patients and controls. Genotyping of the CTLA-4+49A/G polymorphism by ECO911 RFLP. From left: lane 1: DNA ladder 100 bp. Band A at 162 bp. Both fragments of band G at 88 and 74 bp appearing as one band because of being very close to each other. Lanes 2, 3, 4, 5, 7, 8: heterozygous (AG) genotype. Lane6: homozygous (GG) genotype. CTLA-4, cytotoxic T-lymphocyte antige n-4.

Click here to view
Table 3: Distribution of cytotoxic T-lymphocyte antigen-4 (+49A/G) genotypes in relation to clinical data of type 1 diabetic patients

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The distributions of CTLA-4 gene polymorphisms AA, AG, and GG on the basis of consanguinity and family history showed a statistically nonsignificant relation. Also, the distributions of CTLA-4 gene polymorphisms on the basis of age of onset and duration of illness showed statistically nonsignificant relation (P > 0.05) [Table 3].

The distribution of the CTLA-4 gene polymorphisms AA, AG, and GG on the basis of thyroid function tests related to TSH (P = 0.978), FT3 (P = 0.931), and FT4 (P = 0.799) revealed a statistically nonsignificant difference. There was no statistically significant difference in the distribution of the CTLA-4 gene polymorphisms on the basis of HbA1c, both patient controlled and uncontrolled [Table 4].
Table 4: Distribution of the cytotoxic T-lymphocyte antigen-4 (+49A/G) genotype in relation to laboratory data of type 1 diabetic patients

Click here to view


Family counseling is a simple process by which patients, their families, and those at risk for an inherited disorder are advised about the nature of the disease and its consequences. It included education of patients and their families about the pathogenesis of diabetes, chronicity of the disease, and complications, how to inject insulin by pen, its dose, and precautions to be taken during injection. Counseling was also given on manifestation of hyperglycemia and hypoglycemia, how to deal with them, importance of diet control, exercise, and the importance of regular follow-up by HbA1C to detect the degree of diabetic control [8].


  Discussion Top


Susceptibility to type 1 diabetes mellitus is determined by complex interactions between several genetic loci and environmental factors.

Approximately 20 non-HLA loci contributing to disease susceptibility have been identified. The function of only two non-HLA loci is known: the insulin gene and the CTLA-4 [9].

The CTLA-4 protein receptor is encoded by the CTLA-4 gene. The CTLA-4 receptor is found on the surface of T cells. The T-cell attack can be turned off by stimulating the CTLA-4 receptor, which acts as an 'off' switch. In humans, the CTLA-4 protein is encoded by the CTLA-4 gene [4].

A single nucleotide polymorphism present in exon 1, leading to a substitution of an alanine to a threonine amino acid in position 49 (49A/G), has been associated with predisposition to autoimmune diseases [10].

Association of CTLA-4+49A/G gene variants with T1D has been examined in more than one ethnic group, with contradictory results even in the same population [11].

In our study, the frequency of the G allele was 31.3% in the diabetic group versus 20% in controls, without statistical significance (P = 0.193); the heterozygous AG genotype was present in 21 diabetic patients (54.5%) versus eight controls (40%). The homozygous GG genotype was found in two diabetic patients (5%) but it was absent in the control group (0%). The AA genotype was present in 17 diabetic patients (42.5%) versus 12 controls (60%).

All of these results of the CTLA-4 (+49A/G) gene polymorphism regarding the G allele and the three variant patterns AA, AG, and GG showed no statistically significant difference (P>0.05) between diabetic patients and controls.

This result is consistent with those from other populations including the Chilean [12], the Chinese [13], the Japanese [14], the Egyptian [15], and the Turkish [16]. All of them reported that the CTLA-4 (+49A/G) gene polymorphism was not associated with T1D in their population.

In the Iranian population, association was found only for the G allele and not for the GG genotype [17].

These findings are inconsistent with the results from other populations, including Spanish, Italian, French, Mexican-American, Korean [13], Belgian [18], Japanese [19], Croatian [11], and Estonian [20]. All of those studies showed an association between the CTLA-4 (+49A/G) gene polymorphism and T1D, which can be explained by genetic heterogeneity and different environmental factors.

A noteworthy observation is that in Egypt two case-control studies [21,22] support an association between the CTLA-4 (+49A/G) gene polymorphism and T1D, whereas another cohort study in Egypt [15] suggests that the CTLA-4+49A/G polymorphism is not recognized as a risk susceptibility factor for T1D in Egyptians.

These contradictory results can be explained by the limited size of the sample (40 patients and 20 controls), genetic heterogeneity in the studied populations, the different environmental factors involved in the pathogenesis of T1D, limitations of the studies, and other methodological issues.

An analysis of the frequency of CTLA-4 alleles and genotypes in diabetic patients with respect to their ages showed no statistically significant association (P > 0.05). This result is in agreement with those from Turkey [16] and Egypt [15], where there was no relation between the distributions of the CTLA-4 gene polymorphism and age of patients. However, this result is inconsistent with the observation of Mosaad et al. [22], who suggested that the CTLA-4 (GG) genotype is associated with younger age (P = 0.027).

With regard to distributions of the CTLA-4 gene polymorphism in relation to sex, we found that a normal pattern AA genotype was present in 64.7% of female diabetic patients versus 35.3% of male patients, whereas the heterozygous AG genotype was present in 57.1% of female diabetic patients versus 42.9% of male patients, and the homozygous GG genotype was found in two female diabetic patients (5%) but was not recognized in males.

All of these results reveal female predominance. This female predominance is in agreement with the result of Saleh et al. [21], who suggested a strong association between the CTLA-4 (+49A/G) gene polymorphism and female patients, which is explained by steroid hormones, which are considered the most likely factors that trigger the onset of female sex-stratified, genetically based autoimmune diseases. This idea is reinforced by the increased prevalence of autoimmune diseases in women, the sexual dimorphism of the immune response, and the in-vitro modulatory effects of sex steroids on immune functions. These modifiers could directly or indirectly target steroid receptors that act as transcription factors for the susceptibility genes associated with T1D.

A statistically nonsignificant difference existed in the distribution of the CTLA-4 gene polymorphisms with thyroid function tests for TSH (P = 0.978), FT3 (P = 0.931), and FT4 (P = 0.799). These findings are concordant with those of Ηelmeli et al. [16], who showed no association between CTLA-4 (+49A/G) polymorphisms and autoimmune disease.

Also there was a statistically nonsignificant difference in the distribution of the CTLA-4 gene polymorphisms on the basis of HbA1c, both patient controlled and uncontrolled, which is in agreement with a Portuguese study [23] and an Egyptian study [22]. Both suggested no statistical difference between patients with diabetic control and poor control as regards CTLA-4 gene variant.

Balic et al. [10] showed that CTLA-4+49A/G could confer genetic risk for T1D, particularly with the G allele in younger individuals. Also, their data suggested that the association of CTLA-4 with T1D is concentrated in a subset of patient carriers of the G allele of +49A/G polymorphisms, with higher events of ketoacidosis and higher glycemia at diagnosis. This finding was not documented in our study, as there was no statistical difference between patients with controlled diabetes and those with poor control as regards the CTLA-4 gene variant.

In our study there was no difference between the variables age, presentation of diabetes, HbA1c level, and presence of other associated autoimmune diseases in relation to CTLA-4 (+49A/G) genotypes and allele frequencies. Nevertheless, the CTLA-4 gene GG variant was found to be predominant in female patients with T1D.


  Conclusion Top


Family counseling has an important role in the management of diabetes (by changing lifestyle, exercising, following diet control, and follow-up). The CTLA-4 (+49A/G) gene AG variant is the most prevalent in diabetic patients; nevertheless, the CTLA-4 (+49A/G) gene polymorphism is not associated with risk susceptibility for T1D in Egyptian children.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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