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Saudi Journal of Kidney Diseases and Transplantation
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Table of Contents   
ORIGINAL ARTICLE  
Year : 2011  |  Volume : 22  |  Issue : 2  |  Page : 273-281
Association of HLA class II alleles and CTLA-4 polymorphism with type 1 diabetes


1 Department of Biological & Environmental Sciences, Faculty of Science, Beirut Arab University, Beirut, Lebanon
2 Department of Physiology, Faculty of Pharmacy, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
3 Department of Biochemistry, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia

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Date of Web Publication18-Mar-2011
 

   Abstract 

Type-1 diabetes mellitus (T1DM) is a progressive complex autoimmune disease in which combinations of environmental as well as genetic factors contribute to T-cell mediated destruction of insulin-secreting β-cells of the pancreas. HLA class II alleles on chromosome 6p21 [insulin dependent diabetes mellitus 1 (IDDM1)], especially DR and DQ, show strong association with T1DM. In addition, several studies have suggested that polymorphisms in the CTLA-4 gene (IDDM12) on chromosome 2q33 form part of the genetic susceptibility for type 1 diabetes. The aim of this study was to analyze HLA alleles of the DQB1 and DRB1 genes using polymerase chain reaction using sequence specific primers (PCR-SSP) technique and to investigate the asso­ciation of the A49G CTLA-4 polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in Lebanese T1DM patients. The study was conduc­ted on 39 Lebanese T1DM patients. Results of HLA typing showed an increased frequency of the HLA-DQB1*0201, HLA-DQB1*0302, HLA-DRB1*0301 and HLA-DRB1*0401 alleles, sugges­ting risk association and thus can be considered as susceptibility alleles. On the other hand, strong protection against the disease was conferred by the HLA-DRB1*110101, HLA-DQB1*0301 and HLADQB1*0601 alleles. RFLP analysis of the A49G polymorphism showed a significant increase in the G allele and GG genotype frequencies in patients, suggesting that CTLA-4 may be considered as a susceptibility gene for the development of T1DM in the Lebanese population. Analysis of the two polymorphisms showed no detectable association between the two genes. However, a significant negative association of the G allele with the DQB1*0201 allele was ob­served. This might indicate that the two genetic risk factors, namely HLA and CTLA-4, act independently of each other with no additive effect.

How to cite this article:
EI Wafai RJ, Chmaisse HN, Makki RF, Fakhoury H. Association of HLA class II alleles and CTLA-4 polymorphism with type 1 diabetes. Saudi J Kidney Dis Transpl 2011;22:273-81

How to cite this URL:
EI Wafai RJ, Chmaisse HN, Makki RF, Fakhoury H. Association of HLA class II alleles and CTLA-4 polymorphism with type 1 diabetes. Saudi J Kidney Dis Transpl [serial online] 2011 [cited 2014 Nov 26];22:273-81. Available from: http://www.sjkdt.org/text.asp?2011/22/2/273/77603

   Introduction Top


Type 1 diabetes mellitus (T1DM) or insulin dependent diabetes mellitus (IDDM) is a com­mon multifactorial autoimmune disease that arises from specific destruction of insulin sec­reting beta cells of the  Islets of Langerhans More Details by autoreactive T lymphocytes. [1] The pathogenesis of T1DM is complex and multifactorial in­volving interaction between both genetic and environmental factors. [2] T1DM is a genetically polygenic disease caused by multiple suscepti­bility and protective alleles interacting with each other. [3]

The study of candidate genes led to the iden­tification of two major susceptibility genes for IDDM: IDDM1 encoded in the HLA region of the Major Histocompatibility complex (MHC) locus on chromosome 6p21 and IDDM2 enco­ded by the insulin gene region mapped to the Variable Number of Tandem Repeats (VNTR) region 5' of the insulin gene on chromosome 11p15. [4] The study of candidate genes also revealed the association of a third locus: IDDM12 region on chromosome 2q33 which contains several possible candidate genes such as the cytotoxic T lymphocyte associated antigen 4 (CTLA-4) gene. [5]

The IDDM1 susceptibility locus encompasses the HLA genes, located within the MHC. The MHC spans a 3.5 Mb region of chromosome 6p21 and consists of over 200 genes arranged as three subregions: class I, class II, and class III. [6] HLA class II genes, DRB1 and DQB1, are the major determinants of IDDM1 encoded sus­ceptibility to type 1A diabetes.

DQB1 alleles coding for an amino acid other than aspartic acid in position 57 (non-Asp-57), in particular DQB1*0302 and DQB1*0201 alleles, have been associated with T1DM in all ethnic groups. Other non-Asp-57 alleles such as DQB1*0501, *0502, *0604 and *0605 are neutral. [7]

Two DRB1 alleles, DRB1*03 and DRB1*04 (which encode the DR3 and DR4 molecules, respectively), are also associated with an in­creased risk of disease. DRB1*03 is in linkage disequilibrium with the DQA1*0501-DQB1* 0201 allelic combination (forming the DR3.DQ2 haplotype), whereas DRB1*04 is in linkage disequilibrium with DQA1*0301-DQB1*0302 (forming the DR4.DQ8 haplotype). [7]

The CTLA-4 gene encodes a receptor ex­pressed by activated T cells. [8] This receptor functions as a key negative regulator of T-cell activation. In normal immune response, antigen recognition by Th cells is mediated through interaction between CD28, which is expressed on virtually all T cells, and B7 proteins on the surface of antigen presenting cells. This bin­ding between CD28 and B7 is essential for ini­tiating the responses of naïve T cells. In some cases, T cells that encounter self-antigens may begin to express CTLA-4 molecules as a pro­tective mechanism, which have high affinity for B7 molecules and deliver inhibitory signals to the T cells. [9]

Mutations and polymorphisms leading to al­tered activity of CTLA-4 are believed to play an important role in the risk of developing autoimmunity. [10],[11],[12],[13] The aim of this study was to investigate the effect of the HLA DQB1 and DRB1 alleles on the risk of developing T1DM in the Lebanese population and determine which allele represents high susceptibility to or protection from the disease. We also investi­gated whether the A49G polymorphism of the CTLA-4 gene confers susceptibility to T1DM in Lebanese patients and whether an associa­tion exists between the two loci.


   Subjects and Methods Top


Subjects

The population recruited for this study inclu­ded 39 Lebanese IDDM patients attending the outpatient clinics of the American University of Beirut. IDDM was diagnosed according to the classification of the American Diabetes Association. All patients were nonobese and <26 years of age at the time of enrolment in the study. The overall male:female ratio was 0.51:0.49. The mean age at the onset for the patients was just under 9 years (8.92 for males and 8.98 for females). The mean age of the patients was 14 years (14.57 years for females and 13.55 years for males). Control population constituted of 46 healthy students at Beirut Arab University with no clinical evidence or family history of IDDM or other autoimmune disease.

HLA DRB1 and DQB1 Typing

Five milliliters of blood was collected from both patients and controls in ethylenediamine­tetraacetic acid (EDTA). Total genomic DNA was extracted from blood samples using the glass fiber matrix (GFx) column extraction kit from Amersham Pharmacia, Bjorkgatan, Swe­den. DNA typing for HLA class II DRB1* and DQB1* alleles was analyzed by polymerase chain reaction using sequence specific primers (PCR-SSP) using the SSP2L Micro SSP DNA typing trays according to the manufacturer's manual (One Lambda Inc., Canoga Park, CA, USA). The PCR products were analyzed on 2.5% agarose gel and visualized by staining with ethidium bromide and exposure to UV light.

Detection of the CTLA-4 A49G Polymorphism by PCR-RFLP

Genomic DNA was extracted from blood samples using the GFx column. Amplification of DNA in exon 1 of CTLA-4, where an A\G polymorphism exists at position 49, was car­ried out by PCR using the primer pair:

Forward:

5'--GTCAAGGGACCATTAGAAG-3' Reverse:

5'--GTTTGCAGNAGACAGGGATGAA-3'


PCR amplification was performed in 200 ng genomic DNA in 25 μL reaction volume con­taining 200 μmole/L dNTPs and 1 μmol/L mag­nesium chloride. The amplified product was 685 bp in length and was analyzed using 2% agarose gel.

Restriction enzyme digestion was performed using 6.5 μL of PCR product. The sample was incubated at 37ºC overnight in a final volume of 15 μL using 200 U/mL of BbvI enzyme. The digested samples were then analyzed on 2% agarose gel.


   Statistical Analysis Top


Genotype and allele frequencies in patients and control group were calculated by direct counting. The association of the G allele with IDDM was assessed by calculating the odds ratio (OR) and 95% confidence intervals (95% CI) for the G phenotype and GG genotype. The level of significance was determined by χ2 and Fisher's exact tests.


   Results Top


HLA DRB1* Allele Frequencies

The frequencies of the HLA DRB1* alleles in both the patients and the control population are summarized in [Table 1]. Statistical analysis showed that the DRB1*030101 allele fre­quency was significantly increased in patients versus controls (46.15% vs. 9.80%; P < 0.001). In addition, a marked significant increase of the DRB1*110101 allele frequency in controls versus patients was detected (30.4% vs. 5.12%, P < 0.001). Differences in the frequencies of other DRB1* alleles between patients and con­trols were insignificant.
Table 1: HLA DRB1 allele frequencies in patients and controls.

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HLA DQB1* Allele Frequencies

The frequencies of the HLA DQB1* alleles in both the patients and the control population are summarized in [Table 2]. Results showed a significant increase in the frequency of the DQB1*020101 allele in patients compared to controls (56.41% vs. 19.50%, P < 0.001). An increased frequency of the DQB1*030201 al­lele was also observed in patients but did not reach statistical significance (17.95% vs. 10.3%, P = 0.0511). A significant increase of the DQB1*030101 allele (38.40% vs. 10.26, P <0.001) and the DQB1*0601 allele (7.3% vs. 0%, P = 0.0136) was observed in the control population compared to patients. The frequency of other DQB1* alleles did not differ signifi­cantly between patients and controls.
Table 2: HLA DQB1 allele frequencies in patients and controls.

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Statistical analysis showed a significant diffe­rence in the GG genotype frequency between patients and controls (Fisher's exact test, P = 0.0034) [Table 3]. Sample 1 was homozygous, samples 2 and 3 were heterozygous and sample 4 was normal. The G allele frequency was sig­nificantly overrepresented in the patients com­pared with the controls (OR = 3.381, 95% CI = 1.75-6.534, P = 0.0002) [Table 4].
Table 3: Genotype frequencies of the CTLA-4 A49G polymorphism in Lebanese control subjects and patients with type 1A diabetes.

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Table 4: Allele frequencies of the CTLA-4 A49G polymorphism in Lebanese T1DM and control subjects.

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Association of the CTLA-4 A49G Polymor­phism with the HLA Alleles in T1DM Patients

To study whether an association exists bet­ween the CTLA-4 polymorphism and the HLA­DR and DQ risk alleles, the allele frequencies of the CTLA-4 A49G were compared in pa­tients positive for the high-risk DR and DQ alleles versus patients negative for these alleles.

Statistical analysis revealed no significant asso­ciation between the CTLA-4 A49G polymor­phism and the DRB1 alleles [Table 5]. How­ever, a higher frequency of the predisposing G allele was observed in patients lacking the high­risk DRB1*0301 (62.5%) compared to patients carrying the allele (44.5%). Eleven out of 12 patients negative for the DRB1*0301 carried the predisposing G allele, of which 7 were he­terozygous and 4 were homozygous. In con­trast, an increased frequency of the G allele was observed in patients lacking the protective DRB1*1101 allele (52.9%) compared to pa­tients carrying this protective allele (25%). Out of 35 patients lacking the protective DRB1*1101 allele, 9 were homozygous for the G allele (GC) (GG) while 19 were heterozygous (AG). How­ever, the difference failed to reach statistical significance (P = 0.1335) [Table 5].
Table 5: CTLA-4 polymorphism alleles in T1DM patients relative to the DR alleles

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On the other hand, comparison of the DQB1 alleles, DQB1*0201 and DQB1*0302, with the CTLA-4 A49G alleles revealed a significant negative association of the G allele with the DQB1*0201 allele [Table 6]. The G allele fre­quency was significantly higher in patients ne­gative for the DQB1*0201 allele (78.6%) com­pared to T1DM patients positive for this allele (42%) (P = 0.0136). Seven out of 39 patients were negative for the DQB1*0201 allele; 4 were homozygous (GG) for the mutant G allele while 3 were heterozygous (AG). None had the wild-type (AA) genotype.
Table 6: CTLA-4 polymorphism alleles in T1DM patients relative to the DQ alleles.

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   Discussion Top


The contribution of the HLA class alleles to T1DM in Lebanese patients is still unknown. This study investigated the association of the HLA DRB1 and DQB1 alleles with T1DM and and the effect of the alleles on susceptibility or resistance to the disease.

Our results showed that the DRB1*0301 allele was associated with an increased risk for T1DM. The frequency of the DRB1*0401 was also increased in the patients compared to con­trols, but this increase was not significant thus conferring a lower risk. The frequencies of the non-Asp-57 DQB1 alleles, DQB1*0201 and DQB1*0302, associated with susceptibility to T1DM were also increased in the Lebanese patients compared to controls. Highest risk was conferred by the DQB1*0201 allele. The risk associated with the DQB1*0302 allele; which was overrepresented in patients compared to controls, was lower as the difference did not reach statistical significance.

Our results also indicated strong susceptibi­lity conferred by the DRB1*030101 allele. The results designate that a strong protection against T1DM is mediated by the DRB1*1101 allele which showed a significant increase in the control population compared to patients. On the other hand, the DQB1*0301 and DQB1*0601 alleles were also negatively associated with type 1A diabetes, thereby providing a protective effect against the disease.

The results of this study on Lebanese T1DM patients are consistent with those found in other studies on Caucasians of Arabic origin such as Egypt, [14] Kuwait [15] and Bahrain. [16] Reports from other Middle Eastern populations such as Iraq, [17] indicated that the DRB1*0301 allele was more frequently found in T1DM patients.

Studies on various Caucasian ethnic groups showed that the predominant susceptibility alleles were the DRB1*0301 and DQB1*0302, which is consistent with the results of this study. Such results were obtained from studies on T1DM patients from Uruguay, [18] Romania, [19] the United Kingdom, [20] Italy [21] and Spain. [22] The notable difference between Lebanese and other Caucasian populations was the weak associa­tion of the DRB1*0401 allele with T1DM and the absence of a protective effect of the DRB1*1501 allele.

These results distinguish Lebanese diabetics from Caucasians and other populations and point out to the differential distribution of HLA class II alleles among various ethnic and racial groups, reflected by the different disease inci­dence rates in such populations.

A recent study on Hispanic and non-Hispanic White (NHW) youth diagnosed with type 1 diabetes in Colorado showed that high-risk HLA genotypes, DRB1*03-DQB1*02/DRB1*04-DQB1*03, are becoming less frequent over time in youth with T1DM, suggesting that in­creasing environmental exposure is now able to trigger type 1 diabetes in subjects who are less genetically susceptible. [23] Similar results were also obtained in a study conducted on Caucasians, revealing a rising incidence and decreasing age at diagnosis of type 1 diabetes among children with lower risk HLA class II genes, DR4 and DR3. [24]

The second gene investigated in this study was the CTLA-4 gene. CTLA-4 plays a role in limiting T-cell proliferative response. Several hypotheses have been proposed to determine the possible mechanism by which this is a­chieved. The most known hypothesis states that disruption of the balance between CD28 and CTLA-4 interactions with B7 could lead to autoimmune disease by preventing apoptosis or down regulation of activated self-reaction T-lymphocytes. [25] Gribben et al [26] have sugges­ted that this may be through antigen-specific induction of the apoptotic pathway.

The current study investigated the A49G po­lymorphism in exon 1 of CTLA-4 gene in 40 Lebanese and 46 controls from the same ethnic background. An increase in the frequency of the G allele was discovered in patients when compared to control subjects; this difference was statistically significant, despite the small sample size.

The presence of an alanine on codon 17 of CTLA-4 has been associated with susceptibi­lity to T1DM and autoimmune thyroid di­sease. [8] CTLA-4 gene polymorphisms have been associated with autoimmune diseases such as Hashimoto's thyroditis, [8] Addison's disease, [8] celiac disease, [27] and Grave's disease. [28] Ethnic heterogeneity of the effect of CTLA-4 on T1DM has been observed. The A/G polymorphism has been shown to be associated with T1DM in various ethnic groups. [29] Our results are con­sistent with the findings of these earlier studies showing positive association of the CTLA-4 gene polymorphism with T1DM.

A lack of association of CTLA-4 gene poly­morphism with T1DM was observed in certain Caucasian populations including Sardinians, [29] US Whites, [30] Germans, [31] Danish, [32] and Tur­kish. [33] The CTLA-4 gene (IDDM12) is located near two other T1DM susceptibility regions, IDDM7 (2q31), [34] IDDM13 (2q34) [35] and the genes encoding CD28 and islet tyrosine phos­phate, [36] which may be considered candidate T1DM susceptibility genes. This closeness to other susceptibility genes and regions may ex­plain the weakness or absence of association of the CTLA-4 gene with T1DM in some po­pulations. [29],[31],[32]

Despite the limited size of our sample, our re­sults, together with other population studies, show an association of CTLA-4 with T1DM in Lebanese population, which suggests that CTLA­4 on chromosome 2q33 (IDDM12) is a possible susceptibility locus.

The CTLA-4 A49G polymorphism was ana­lyzed with the HLA-DR and DQ alleles to reveal whether an interaction exists between the two genes. The analysis led to several con­clusions. For the DRB1 alleles, no significant association was detected with the CTLA-4 G allele [Table 5]. An increased frequency of the G allele in patients negative for the high-risk DRB1*0301 allele (62.5%) compared to pa­tients positive for this allele (44.5%) was detected. This increase was found to be statis­tically insignificant. Increased frequency of G allele was also detected in T1DM patients lacking the protective DRB1*1101 allele (52.9% vs. 25%) but failed to reach statistical signi­ficance.

In contrast to DRB1 alleles, a negative asso­ciation between the CTLA-4 A49G polymer­phism and the high-risk DQB1*0201 allele was detected. The G allele was significantly overrepresented in patients lacking DQB1*0201 (78.6%) compared to those positive for these alleles (42%) [Table 6]. The finding that none of the seven patients negative for the DQB1*0201 had the wild type (AA) of the CTLA-4 polymorphism is worth noting. An increased frequency of the G allele in patients negative for the protective DQB1*0301 allele (53.1%vs. 35.7%) was also seen but was statistically insignificant.

From these findings, it can be concluded that the diabetogenic effect of the CTLA-4 G allele is more pronounced in patients negative for the high-risk DQB1*0201 allele and the protective DQB1*0301 and DRB1*1101 alleles. This is further supported by the fact that all T1DM patients negative for the high-risk DQB1*0201 allele lacked the wild-type (AA) genotype of the CTLA-4 A49G polymorphism.

The study also revealed three diabetic pa­tients who lacked the high-risk DR and DQ alleles and carried the protective and neutral ones. Two of these patients were homozygote for the G allele and carried the protective DQB1*0301 allele and the neutral DQB1*0501 allele. One of them was homozygous for the DQB1*0301 allele. This finding may indi­cate that homozygosity for the predisposing G allele of the CTLA-4 A49G polymorphism may override the protective effect of the DQB1*0301 allele. However, other polymor-phisms in the CTLA-4 gene, other susceptibility genes and several environmental factors may have con­tributed to the development of T1DM in these patients.

Few studies have investigated the association or correlation of the HLA and CTLA-4 genes. A previous report by Zalloua et al [37] on Leba­nese T1DM patients revealed results different from that of the present study. A significant increase in the GG genotype (22.2%) was observed in HLA-DQB1*0201 + /0302 + patients as compared to controls (0%). These results suggest the presence of an association between CTLA-4 A49G polymorphism and T1DM in contrast to the results of this study. Further­more, an association of this polymorphism was observed for the HLA-DQB1*0201 and DQB1 *0302 high-risk alleles. [37] This difference in the results could be a consequence of the method employed in their analysis. The study by Zal­loua et al [37] compared the frequency of the CTLA-4 A49G in HLA matched controls and T1DM patients. On the other hand, in the pre­sent study, association of CTLA-4 A49G poly­morphism was investigated in relation to HLA based on comparing the frequency of the CTLA-4 polymorphism in patients positive and negative for the high-risk and protective HLA alleles and genotypes.

A Japanese study, following the same metho­dology of analysis done in this study, demons­trated that a distinct association exists between the G allele of the CTLA-4 gene and the ab­sence of HLA-DRB1*0405 which is a suscep­tible T1DM allele among the Japanese popu­lation. [38] Therefore, the CTLA-4 mediated dia­betogenic effect is significant in the DRB1*0405-negative population but is not significant in the DRB1*0405-positive type 1A diabetic patients. [38] These results are in agreement with ours where we found a negative association between the G allele and DQB1*0201 which is a susceptibility allele among the Lebanese po­pulation.

Contradictory data have been reported among Caucasian population. Some reports are in agreement with the results reported in this study. Djilali-Saiah et al [39] showed that in the patients without DRB1*0301, the G allele of CTLA-4 has a stronger diabetogenic effect than DR3-positive subjects. In contrast to these results, previous analysis of the correlation between the polymorphism of CTLA-4 and HLA-DRB1 by Van der Auwera et al [40] revealed that the frequency of the G allele is signifi­cantly higher in patients with DRB1*0401 (DR4) than in the control subjects.

Future studies should be conducted on larger sample size and on study populations obtained from different geographic regions within Le­banon to evaluate gene-gene interaction of the HLA and CTLA-4 genes. In addition, the re­sults of such studies should be compared to those obtained from studies conducted in other Middle East countries. Other types of CTLA-4 polymorphisms such as the C-651T and C­159G should be investigated in the Lebanese population to obtain a solid evidence of their association.


   Acknowledgment Top


We appreciate Dr. Nuha Nuwayri Salti (Prof. of Histology in the medical school at American University of Beirut) for her assistance in pro­viding the blood samples for our study.

 
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28.Nakkash-Chmaisse H, Makki RF, Abdelhamid E, Fakhoury H, Salti NN, Salti I. CTLA-4 gene polymorphism and its association with Graves' disease in the Lebanese population. Eur J Immunogenet 2004;31:141-3.  Back to cited text no. 28
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29.Marron MP, Raffel LJ, Garchon HJ, et al. In­sulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in mul­tiple ethnic groups. Hum Mol Genet 1997;6: 1275-82.  Back to cited text no. 29
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30.Owerbach D, Naya FJ, Tsai MJ, Allander SV, Powell DR, Gabbay KH. Analysis of candidate genes for susceptibility to type I diabetes: a case­control and family-association study of genes on chromosome 2q31-35. Diabetes 1997; 46:1069-74.  Back to cited text no. 30
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31.Badenhoop K, Donner H, Pani M, Rau H, Sieg­mund T, Braun J. Genetic susceptibility to type 1 diabetes: clinical and molecular hetero-geneity of IDDM1 and IDDM12 in a german population. Exp Clin Endocrinol Diabetes 1999;107(Suppl3): S89-92.  Back to cited text no. 31
    
32.Larsen ZM, Kristiansen OP, Mato E, et al. IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34 in genetic susceptibility to type 1 dia-betes (insulin-dependent). Autoimmunity 1999; 31:35­-42.  Back to cited text no. 32
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40.Van der Auwera BJ, Vandewalle CL, Schuit FC, et al. CTLA-4 gene polymorphism confers sus­ceptibility to insulin-dependent diabetes mellitus (IDDM) independently from age and from other genetic or immune disease markers. The Belgian Diabetes Registry. Clin Exp Immunol 1997;110: 98-103.  Back to cited text no. 40
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Correspondence Address:
Hana Fakhoury
Department of Biochemistry, King Saud bin Abdulaziz University for Health Sciences, P.O. Box 22490, MC 3130 Riyadh 11426
Saudi Arabia
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PMID: 21422625

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    Abstract
    Introduction
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