الفهرس 
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Abstract
Diabetes mellitus (DM) is a chronic metabolic disorder caused by an absolute or relative deficiency of insulin, or loss of its action. Insulin is an anabolic hormone produced by the beta cells of the islets of Langerhans located in the pancreas, and the absence, destruction, or loss of these cells results in type 1 diabetes (insulin-dependent diabetes mellitus (IDDM). Most children with diabetes have type 1 diabetes mellitus (T1DM) and a lifetime dependence on exogenous insulinto prevent acute complications and to reduce the risk of long-term complications. The two most common forms of diabetes are type 1 diabetes (T1DM, previously known as insulin-dependent diabetes or IDDM) and type 2 diabetes (T2DM, previously known as non-insulin-dependent diabetes or NIDDM). Both are caused by a combination of genetic and environmental risk factors. Genes for Type1DM (T1DM) may provide susceptibility to, or protection from, the disease. Although many chromosomal loci associated with such activities have been located, few true genes have been identified which can divide into 2 major subdivisions: HLA genes and Non HLA genes. HLA are accounting for about 60% genetic susceptibility for the disease. The HLA genes are a cluster of genes on chromosome 6 p 21. The genes encode glycoproteins that are found on the surfaces of most cells and help the immune system to distinguish between self and nonself. About 20 non-HLA loci contributing to disease susceptibility have been identified. The function of only 2 non-HLA loci is known include: Insulin gene and the cytotoxic T-lymphocyte antigen-4 gene (CTLA-4). The Cytotoxic-T-lymphocyte antigen (CTLA-4) encoding gene is one of the most important non-HLA susceptibility genes of T1DM and other autoimmune diseases, also named as (IDDM12). IDDM12 maps to chromosome 2q33. The role of CTLA-4 in the autoimmune process has been emphasized by its association with systemic lupus erythromatosis, celiac disease, rheumatoid arthritis, and autoimmune thyroid diseases. The CTLA4 gene encodes the CTLA4 protein receptor which involved in the control of T-cell proliferation and mediates T-cell apoptosis. The CTLA4 protein is found on the surface of T-cells, which lead the cellular immune attack on antigens. The T-cell attack can be turned on by stimulating the CD28 receptor on the T-cell. The T cell attack can be turned off by stimulating the CTLA4 receptor, which acts as an ”off” switch. The human cytotoxic T lymphocyte-associated antigen-4 gene (CTLA-4) is known to contain polymorphisms in three regions: a cytosine-thymine single-base substitution in the promoter at position -318 (C-318/T-318), and adenine-guanine dimorphism in the exon 1 leader sequence at position 49 (A49/G49), and a multiallelic dinucleotide repeat in the 3’untranslated region (UTR) of exon 4. A single nucleotide polymorphism (SNP) 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. Association of CTLA-4 +49 A/G gene variants with T1D has been examined in morethan one ethnic group with contradictory results even in the same population. The aim of this work is to study the distribution of CTLA-4 (+49 A/G) gene variants and the association of these variants with the clinical and laboratory findings in Egyptian children with Diabetes. Patient and method:- The present study was carried out on forty diabetic children attended to our pediatric genetic & endocrinology unit and clinic in Menoufiya University Hospital. Twenty apparently healthy children matched with the diabetic patients in the same age and sex from our pediatric general ward in Menoufiya University Hospital as a control group. The diabetic children:- Included 15 males and 25 females, their ages ranged from one year to 16 years with mean age of (8.76±4.70) with variable duration illness and degree of diabetic control. Diagnostic criteria of diabetic children: • Fasting blood glucose ≥7.0 (≥126) mmol/l(mg/dl). • 2 hour postprandial glucose ≥11.1 (≥200) mmol/l(mg/dl). • Glycosylated Hemoglobin (HbA1c) ≥6.5. All patients and Control were subjected to the following after taking Informed consent: 1. Detailed history taking. 2. Complete general examination including anthropometric measurements. 3. Laboratory investigations including: • Estimation of fasting blood glucose levels. • Estimation of post prandial blood glucose levels. • Estimation of glycosylated hemoglobin ( HbA1c). • Evaluation of thyroid functions by determining FT3, FT4 and TSH. 4. Molecular study of CTLA-4 gene polymorphism by PCR-RFLP which was made in genetic lab of our genetic &endocrinology unit including: • DNA extraction. • Polymerase chain reaction(PCR) • Gel electrophoresis 5. Family counseling. 6. Data management and statistical analysis. This study revealed that: • The percentage of positive family history in diabetic patients was 32.5% while the percentage of negative family history was 67.5%, versus 100% negative family history in control group this was found to be significant statistically (p=0.003). Summary - 109 - • As regarding to type of presentation, the percentage of the diabetic patients presented with classic symptoms was 20% while the percentage of the diabetic patients presented with DKA (coma) and admitted to ICU was 80% . • HbA1C ranges from (9.04 ± 1.68) in diabetic group versus (5.27 ± 0.61)in control group this was found to be highly statistically significant association (p=<0.001). • Regarding to thyroid function TSH ranging from (2.30± 1.36 μIU/mL), FT3 ranging from (2.89 ± 0.86 pg/mL) and FT4 ranging from (1.57 ± 0.57 ng/dL) without statistically significant between patient and control. • The molecular study of CTLA-4 gene shows that the frequency number of the G allele was 25 (31.3%) in diabetic group (equal to 4 alleles from 2 GG variants and 21 alleles from 21 AG variants, as each variant provide two alleles), versus (20%) in controls showing insignificant statistically (P=0.193) with OR (CI95%) 1.82 (0.73-4.51) . • the frequency number of the A allele was 55 (68.7%) in diabetic group(equal to 34 alleles from 17 AA variants and 21 alleles from 21 AG variants), versus 32 (80%) in controls • As regards CTLA-4 polymorphisms (AA, AG, GG) in T1DM patients was (42.5%, 52.5%, 5%) respectively versus (60%, 40%, 0%) in controls showing insignificant statistically (p=0.320) with OR (CI95%) 1.85 (0.62-5.56). • On the analysis of the frequency of CTLA-4 alleles and genotypes in diabetic patients regarding to their ages, no statistically significant associations were found (P >0.05).• Regarding to distributions of CTLA-4 gene polymorphism with gender we found that, normal pattern AA genotype was presented in (64.7%) for female diabetic patients versus (35.3%) for male patients, whileheterozygous AG genotype in female diabetic patients (57.1%) versus (42.9%) for male patients, and the homozygous GG genotype was founded in two female diabetic patients (100%) and not recognized in male.All of these results revealed female predominance but with no statistically significant associations were found (P > 0.05). • No association was found between distributions of CTLA-4 gene polymorphisms in regarding to Consanguinity and family history. Also Statistically insignificant difference existed in the distribution of the CTLA-4 gene polymorphisms with thyroid function test; TSH (p=0.978), FT3 (p=0.931) and FT4 (p=0.799). • In our study there was statistically insignificant difference between the variables (age, presentation of diabetes, HbA1c level, and presence of other associated autoimmune diseases) and the CTLA-4 (+49A/G) genotypes and allele frequencies. On the other hand the CTLA-4 gene GG variant was found in female patients with T1D but not for male patients.