Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 3  |  Page : 973-978

Association of cytotoxic T lymphocyte 4 gene with auto-antibodies in children with type 1 diabetes


1 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pediatrics, Damnhor Medical Institute, Damnhor, Elbehira, Egypt

Date of Submission03-Nov-2019
Date of Decision27-Nov-2019
Date of Acceptance01-Dec-2019
Date of Web Publication18-Oct-2021

Correspondence Address:
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_344_19

Rights and Permissions
  Abstract 


Objective
The aim of the study was to detect the association of CTLA4 gene with anti-insulin antibodies in type 1 diabetes in children.
Background
The objective was to evaluate the diagnostic workup done for diabetic children in its relation to clinical findings and diabetic control, and to study association of CTLA4 gene and anti-insulin antibodies in type 1 diabetes in children.
Materials and methods
The results showed no significant difference between male and females regarding serum anti-insulin antibodies and CTLA4 gene. There was a significant positive correlation between serum anti-insulin antibody and duration of disease of type 1 diabetes, but there was an insignificant association between CTLA4 gene and anti-insulin antibodies and type 1 diabetes.
Results
The study was carried out in the Pediatric Endocrinology and Genetic Units in Menoufia University Hospital and Damnhour Medical Institute. One hundred children with type 1 diabetes were enrolled (44 males and 56 females). Their age ranged from 2 to 17 years, with a mean of 9.3 ± 13.6 years. Data were collected including detailed history and thorough clinical examination, including anthropometric measures (body weight, standing height, BMI, and laboratory data). Assessments of serum anti-insulin antibodies and CTLA4 gene were done for all cases and for 100 apparently healthy control children (51 males and 49 females), whose age ranged from 3 to 16, with mean of 8.45 ± 3.48 years.
Conclusion
This study concluded that serum anti-insulin antibodies can be used as a marker in diagnosis of new cases of type 1 diabetes in children.

Keywords: anti-insulin, autoantibodies, children, cytotoxic T lymphocyte 4 gene, type I diabetes


How to cite this article:
Tawfik MA, El-Ella SS, Barseem NF, Zeariban NH, Moaty GS. Association of cytotoxic T lymphocyte 4 gene with auto-antibodies in children with type 1 diabetes. Menoufia Med J 2021;34:973-8

How to cite this URL:
Tawfik MA, El-Ella SS, Barseem NF, Zeariban NH, Moaty GS. Association of cytotoxic T lymphocyte 4 gene with auto-antibodies in children with type 1 diabetes. Menoufia Med J [serial online] 2021 [cited 2024 Mar 29];34:973-8. Available from: http://www.mmj.eg.net/text.asp?2021/34/3/973/328333




  Introduction Top


Diabetes mellitus is a group of metabolic disorders characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism, resulting from defects in insulin secretion, insulin action, or both [1]. Most of the children who develop type 1 diabetes have genetic predisposition for the disease. The HLA gene complex is responsible for ∼50% of the genetic risk for type 1 diabetes, and the remaining genetic susceptibility is conferred by a large number of loci, where most of them have minor effects. Other genes known to have effect on the risk of type 1 diabetes include insulin (INS), cytotoxic T lymphocyte antigen-4 (CTLA-4), and protein tyrosine phosphatase N22 (PTPN22) [2]. The appearance of autoantibodies against islet cell antigens is the first easily detectable sign of a possible beta cell autoimmunity. In young children, the insulin autoantibodies are often the first ones to appear, in contrast to adult diabetics who usually display GAD65 or IA-2 autoantibody positivity when diagnosed [3]. Preclinical diabetes refers to the months or years preceding the clinical presentation of type 1diabetes, when antibodies can be detected as markers of β-cell autoimmunity such as Islet cell autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), IA-2 (also known as Islet cell cytoplasmic antibodies (ICA 512) or tyrosine phosphatase auto antibodies), and insulin autoantibodies (IAA) [4]. Testing positive for multiple autoantibodies is often indicative of an increased risk of progression to clinical type 1 diabetes [5].

The aim of the present research was to study the association of anti-insulin autoantibodies and CTLA gene in type 1 diabetic child and it is relation to clinical and laboratory findings.


  Materials and methods Top


The study protocol was approved by the Local Ethics Committee of the Menoufia University, and written consent was obtained from the parents. This case–control study was conducted in the Endocrinology and Genetic Units of Menoufia University Hospital and Damnhour Medical Institute. One hundred children (44 males and 56 females) were diagnosed as type 1 diabetic according to criteria of diagnosis of ADA [6]; moreover, 100 apparently healthy children (51 males and 49 females) as controls were included in this study. Inclusion criteria were newly diagnosed children up to 1-year duration. All the included patients were subjected to the following: full history taking, complete clinical examination, anthropometric measurements (weight, height, and BMI). Routine laboratory investigations and specific laboratory examinations were done such as anti-insulin antibodies and molecular genetic study for CTLA4 (+49A/G) gene polymorphism, which was done in the Genetic Laboratory of Genetic Unit of Pediatric Department of Menoufia University Hospitals, including DNA extraction, PCR, and gel electrophoresis.

Statistical analysis

The collected data were tabulated and analyzed by using an IBM personal computer with Statistical Package for the Social Sciences, version 20, for Windows (SPSS; SPSS Inc., Chicago, Illinois, USA). The results were expressed by applying ranges, means ± SD, χ2-test, Mann–Whitney test, t-test, and Kruskal–Wallis test. Pearson's correlation was used for normally distributed quantitative variables, whereas Spearman's correlation was used for quantitative variables that were not normally distributed or when one of the variables is qualitative. P value less than 0.05 was considered to be significant.


  Results Top


In this study, there was no significant difference between cases and controls regarding age, sex, BMI, and residence, as P values equal 0.11, 0.39, 0.32, and 0.06, respectively. However, there is a significant difference between them in consanguinity and a highly significant difference with family history of diabetes, as P values equal 0.04 and 0.001, respectively. Regarding fasting blood sugar, HbA1C, and C peptide, there is a significant difference between diabetic and controls, but no significant difference regarding thyroid stimulating hormone. Anti-insulin autoantibodies were positive in 17 (17%) children, with highly statistically significant value of less than 0.001 in children with type 1 diabetes. Anti-insulin autoantibodies in type IDM ranged from 0 to 130 mIU, with mean value 10 ± 19.59, and in control groups ranged from 0 to 7.0 mIU, with mean value 2.08 ± 2.36. Distribution of the two studied groups regarding CTLA4 gene showed no statistically significant relation in type one diabetes [Table 1], [Table 2], [Table 3]. There was a statistically significant relation between positive and negative cases of anti-insulin autoantibodies regarding cholesterol and low-density lipoprotein and no statistically significant relation between positive and negative cases of anti-insulin autoantibodies regarding triglyceride and other parameters such as age, sex family history, consanguinity, and presentation of diabetes (P > 0.05). There was a statistically significant difference regarding duration of disease between positive and negative anti-insulin autoantibodies (P < 0.05) [Table 4]. Gel electrophoresis of CTLA4 gene A/G49 polymorphism is shown in [Figure 1].
Table 1: Data of studied groups

Click here to view
Table 2: Laboratory investigations among studied groups

Click here to view
Table 3: Genotype results of cases and control group regarding CTLA 4 gene and CTLA 4 alleles

Click here to view
Table 4: Relationship between anti-insulin autoantibodies and some data of diabetic children

Click here to view
Figure 1: Gel electrophoresis for CTLA4 gene polymorphism.

Click here to view



  Discussion Top


Type one diabetes mellitus is caused by several pathogenic processes such as an autoimmune or idiopathic process. These include destruction of the beta cells of the pancreas, with the occurrence of decreased or absence of insulin secretion [2].

In our study, the results of demographic data showed that the patient group and control group were matched, without significant difference regarding age, sex, and residence. These results are considered an important point to eliminate the effect of age, sex, and residence on the net results and eliminate the demographic risk factors in comparison of the final results. Ages of our patients ranged from 2 to 17 years (mean age of 9.31 ± 3.6 years). There were 56 (56%) females and 44 (44%) males. This female predominance result was in agreement with Tawfik et al. [7], where there were 53 (60.3%) females vs 35 (39.7%) males; they suggested that female predominance is encountered in regions with low incidence, mainly populations of non-European origin. There was no statistically significant difference between the patient and control groups (P > 0.05) regarding weight, height, and BMI. Median age at the onset of diabetes was 7 (1–16) years, with a mean duration of diabetes being 7.09 ± 4.78 months, and ranged from 1 to 12 months.

The percentage of consanguinity in diabetic patients was 39%. Moreover, the percentage of family history in diabetic patients was 21%, with a statistically significant difference between patient and control (P < 0.04). This is in agreement with Tawfik et al. [7], who reported that the risk to develop T1D increases to 1 in 20 in population who have first-degree relative with T1DM, whereas the risk among general population is 1 in 300.

Regarding laboratory data of the studied groups, type one diabetic patients showed higher levels of fasting blood sugar, which ranged from 69 to 330 mg/dl, with a mean 162 ± 64.21, and postprandial blood sugar ranged from 78 to 550 mg/dl, with a mean 264.9 ± 114.7. This is in agreement with diagnostic criteria of type one diabetes mellitus according to American Diabetic Association [1].

Glycosylated hemoglobin (HbA1c) in our study ranged from 5.50 to 16.50%, with a mean of 9.66 ± 2.15 in diabetic patient vs 4–5.5%, with a mean of 4.75 ± 0.52, in control group, with a statistically highly significant difference (P < 0.001). This result supported the usage of HbA1C as diagnostic test for type one diabetes. Regarding the distribution of CTLA-4 (49A/G) polymorphisms in our study, it was revealed that there is no association of CTLA4 (+49A/G) gene polymorphism and their variants with T1DM in the studied children. Gel electrophoresis of genotyping of CTLA-4 + 49A/G polymorphism by ECO911 RFLP, showed homozygous GG genotype, with only G band being present, as seen in [Figure 1].

The genotyping results of our groups showed that control subject showed 75% AG and 25% had AA CTLA 4 genotype, whereas 72% of the patients with type 1 diabetes mellitus were type AG and 24% had AA genotyping. The homozygous GG genotype was found in four (4%) diabetic patients. None in the control group had the GG genotype. A allele was seen in 55.8% in patients vs 57.1% in controls, whereas G allele was seen in 42% in type 1 diabetic vs 42.9% in control group. All these results of CTLA-4 (+49A/G) gene polymorphism regarding G allele and the three variant pattern (AA, AG, and GG variants) showed no statistically significant difference between diabetic patients and controls, as P value was 0.12, odds ratio = 1.06, and CI 95%, and this was in agreement with Tawfik et al. [7] and Kamel et al. [8]. Other studies were reported in Egypt, such as Saleh et al. [9], and Mosaad et al. [10], where both of them supported the association of CTLA-4 (+49A/G) gene polymorphism with T1D.

Kavvoura and Ioannidis [11] suggested that according to a large meta-analysis, the distribution of the CTLA-4 exon 1 polymorphism among Asians and whites showed a clear difference. The pooled frequency of the G allele was 43.3% among control subjects (by race, frequencies were 55.4, 36.2, 33.6, 20.6, and 45.2% among controls of Asian, European, North African/Middle Eastern, Sub-Saharan African, and Pacific Asian descent, respectively). The overall pooled prevalence of G/G homozygosity was 20.4% (33.4, 12.8, 8.9, 5.7, and 22.3% in the five racial descent groups, respectively). The overall pooled prevalence of A/G heterozygosity was 44.8% (44.1, 46.8, 49.4, 31.2, and 45.7% in the five racial descent groups, respectively). Our results are concordant with the findings of this meta-analysis in a way that genotype and allele frequencies of our studied cases are in favor with those reported for Middle East populations and Sub-Saharan African. In Iranian population, and association was found only for the G allele and not for the GG genotype [12]. Moreover, CTLA-4 (+49A/G) gene polymorphism regarding G allele and variants showed no statistically significant association. This is consistent with the results from other populations, including Chinese [13], in Japanese [14], and in Turkish population [15]. On the contrary, these findings are inconsistent with results from other populations including Spanish, Italian, French, Mexican–American, Korean [13], Belgian [16], Japanese [17]), and Estonian [18], where all these studies showed association of CTLA-4 (+49A/G) gene polymorphism with T1D in their population.

In this study, there was a significant difference between anti-insulin autoantibodies and duration of illness. So in our cases, 17 (17%) were anti-insulin positive in which the mean duration of illness was 2.18. This finding is supported by the mechanism that as the disease progresses, beta cell mass decreases owing to continuous autoimmune destruction resulting in decreasing auto immune antigens and thus auto antibodies decline. This finding is in agreement with Cheng et al. [3]. The results of our study showed no association between CTLA-4 (+49A/G) gene polymorphism and anti-insulin antibodies, which can be explained by decreased number of consanguinity and family history in our patients, with no statistically significant difference, as P value was 0.12. Other parameters showed no significant relation with anti-insulin antibodies, like age, sex, residence, family history, consanguinity, and triglycerides. There was a statistically significant relation between positive and negative cases of anti-insulin autoantibodies regarding cholesterol and low-density lipoprotein, and this can be explained by poor glycemic control, as dyslipidemia has a relation with diabetes control rather than diabetes duration [3].


  Conclusion and recommendations Top


This study concluded that anti-insulin antibody can be used in early diagnosis of type 1 diabetes in children regardless of their genetic background of CTLA4 gene, and also, it can be used in early detection of cases of type 1 diabetes before the appearance of symptoms in high-risk family members. It is recommended to use more than one marker of autoantibodies for TIDM to increase significance of positivity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2018; 31:S55–S60.  Back to cited text no. 1
    
2.
WHO. Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications: Report of a WHO Consultation Part 1: Diagnosis and Classifications of Diabetes Mellitus? Geneva: World Health Organization; 2017.  Back to cited text no. 2
    
3.
Cheng B, Sung F, Wang A, Hung C, Huang C, Ting W, et al. Autoantibodies against islet cell antigens in children with type 1 diabetes mellitus. Oncotarget 2018; 9:16275–16283.  Back to cited text no. 3
    
4.
Jaeger C, Winter S, Eckhard M, Hardt P, Brendel MD, Bretzel RG. Binding characteristics and crossreactivity of insulin autoantibodies and insulin antibodies directed to three different insulin molecules. Acta Diabetol 2008; 45:191–194.  Back to cited text no. 4
    
5.
Jönsson L, Hallström I, Lundqvist A. The logic of care ' parents' perceptions of the educational process when a child is newly diagnosed with type 1 diabetes. BMC Pediatr 2012; 12:165.  Back to cited text no. 5
    
6.
American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 2015; 33:S62–S69.  Back to cited text no. 6
    
7.
Tawfik MA, 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.  Back to cited text no. 7
    
8.
Kamel AM, Mira MF, Mossallam GI, Ebid GT, Radwan ER, Eldin NH, et al. Lack of association of CTLA-4+49 A/G polymorphism with predisposition to type 1 diabetes in a cohort of Egyptian families. Egypt J Med Human Genetics 2014; 15:25–30.  Back to cited text no. 8
    
9.
Saleh HM, Rohowsky N, Leski M. The CTLA4 -819 C/T and+49A/G dimorphisms are associated with Type 1 diabetes in Egyptian children. Indian J Hum Genet 2008; 14:92–98.  Back to cited text no. 9
    
10.
Mosaad YM, Elsharkawy AA, El-Deek BS. Association of CTLA-4 (+49A/G) gene polymorphism with type 1 diabetes mellitus in Egyptian children. Immunol Invest 2012; 41:28–37.  Back to cited text no. 10
    
11.
Kavvoura FK, Ioannidis JP. CTLA-4 gene polymorphisms and susceptibility to type 1 diabetes mellitus: a HuGE review and meta-analysis. Am J Epidemiol 2005; 162:3–16.  Back to cited text no. 11
    
12.
Mojtahedi Z, Omrani GR, Doroudchi M, Ghaderi A. CTLA-4+49A/G polymorphism is associated with predisposition to type 1 diabetes in Iranians. Diabetes Res Clin Pract 2005; 68:111–116.  Back to cited text no. 12
    
13.
Marron MP, Raffel LJ, Garchon HJ, Jacob CO, Serrano-Rios M, Martinez Larrad MT, et al. Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups. Human Mol Gen 1997; 6:1275–1282.  Back to cited text no. 13
    
14.
Awata T, Kurihara S, Iitaka M, Takei SI, Inoue I, Ishii C, et al. Association of CTLA-4 gene A-G polymorphism (IDDM12 locus) with acute-onset and insulin-depleted IDDM as well as autoimmune thyroid disease (Graves' disease and Hashimoto's thyroiditis) in the Japanese population. Diabetes. 1998; 47:128-9. doi: 10.2337/diab.47.1.128. PMID: 9421386.  Back to cited text no. 14
    
15.
Çelmeli F, Türkkahraman D, Özel D, Akçurin S, Yeğin O. CTLA-4 (+49A/G) polymorphism and type-1 diabetes in Turkish children. J Clin Res Pediatr Endocrinol 2013; 5:40–43.  Back to cited text no. 15
    
16.
Nisticò L, Buzzetti R, Pritchard LE, Van der Auwera B, Giovannini C, Bosi E, et al. The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Human Mol Gen 1996; 5:1075–1080.  Back to cited text no. 16
    
17.
Ide A, Kawasaki E, Abiru N, Sun F, Kobayashi M, Fukushima T, et al. Association between IL-18 gene promoter polymorphisms and CTLA-4 gene 49A/G polymorphism in Japanese patients with type 1 diabetes. J Autoimmun 2014; 22:73–78.  Back to cited text no. 17
    
18.
Haller K, Kisand K, Pisarev H. Insulin gene VNTR, CTLA-4+49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group. Tissue Antigens 2007; 69:121–127.  Back to cited text no. 18
    


    Figures

  [Figure 1]
 
 
    Tables

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



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and me...
Results
Discussion
Conclusion and r...
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed560    
    Printed22    
    Emailed0    
    PDF Downloaded60    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]