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The cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene A/G was found to be associated with type 1 diabetes in many ethnic studies. Beta c ell secretory dysfunction is a key element in type 2 diabetes pathophysiology. Recent data suggests that apoptosis mechanisms may explain insulin deficiency. [1] Because CTLA-4 mediates antigen -specific apoptosis,[2] it may be a cand idate gene to confer susceptibility to type 2 diabetes. This study was undertak en to clarify the role of the CTLA-4 gene 49 A/G polymorphism in Han Chinese su bjects with type 1 or type 2 diabetes.
METHODS
Subjects
CTLA-4 exon 1 position 49 A/G (codon 17 Thr/Ala) polymorphism was studied in 31 patients with type 1 diabetes, 31 patients with type 2 diabetes and 36 controls . All subjects were Han Chinese from northwestern China. The classification an d diagnostic criteria from diabetes were based on the criteria proposed by ADA i n 1997, derived from clinical and laboratory data. Healthy controls were random ly selected from Chinese blood donors with no family history of diabetes or othe r autoimmune disorders.
Methods
DNA was prepared from 2 ml whole blood, using a Genomic DNA Minipreps Kit (Sango n, China). The CTLA-4 exon 1 position 49 polymorphism was defined, employing p olymerase chain reaction (PCR) with the following oligonucleotides: forward 5'- GCTCTACTTCCTGAAGACCT-3' and reverse 5'-AGTCTCACTCACCTTTGCAG-3'. These were d esigned according to the published human CTLA-4 complementary DNA sequence.[3] PCR was performed using 0.2 μg genomic DNA, 1U Taq polymerase (Canada). A t otal of 20 pmol of each primer and 5 nmol deoxy-NTPs under the following condit ions: initial denaturation for 4 minutes at 94℃, annealing for 45 seconds at 58 ℃, extension for 45 seconds at 72℃, denaturation for 45 seconds at 94℃ (30 cy cles), and a final extension for 4 minutes at 72℃ in a GeneAmp PCR system 2400 (Perkin Elmer).
Restriction fragment length polymorphism analysis of CTLA-4
The restriction enzyme, BbvI, cut the sequence if a G was present at position 49 , resulting in 88/74-bp fragments; if an A was present at position 49, no diges tion of the 162-bp PCR fragment occurred. Restriction fragment length polymorp hisms (RFLP) was performed on 8 μl PCR product, digested in a final volume of 1 0 μl under appropriate buffer conditions with 1U BbvI enzyme at 65℃ over 3 h. The resulting digestion products were then visualized on 3% agrose gels stained with ethidium bromide.
Statistical analysis
Analysis was performed using the χ[2 test with 95% confidence limits. P <0 .05 was considered significant. Odds ratios (OR) were calculated according to Woolf's formula. All data were analyzed by SPSS 8.0.
RESULTS
A highly significant increase in the frequency of the G allele was seen in Han C hinese patients with type 1 diabetes compared with control subjects, which infle cts an increase in the GG genotype in patients and a significant decrease in the AA genotype. The allele frequencies of A and G in Han Chinese patients with ty pe 2 diabetes were not significantly different from the control subjects. The di stribution of genotype differed significantly, reflecting an increase in the AG genotype and a decrease in the AA genotype. CTLA-4 exon 1 position 49 polymorphism in patients with type 1 and type 2 diabe tes and in controls are shown in Table. (Fig.)
DISCUSSION
Donner H et al[3] first reported that an alanine at codon 17 of CTLA4 is asso ciated with genetic susceptibility to type 1 diabetes. The transmission disequi librium test (TDT), in a multi-ethnic collection of families with one child or more affected by type 1 diabetes, revealed a highly significant deviation for t ransmission of alleles A/G in the first exon of the CTLA-4 gene.[4] Subsequen t studies have shown an correlation between the G allele and type 1 diabetes in adult-onset patients from white populations[5] and in children from central Po land.[6] Recently, Lee YJ et al[7] have also demonstrated this correlation in Han Chinese children. Our findings are similar to previous reports. A highl y significant increase in the frequency of the G allele was seen in patients wit h type 1 diabetes compared with controls (66.1%, 34.7%, respectively; P <0 .0005; OR=3.670). This reflected an increase in the GG genotype in patients ( 48.4%, 22.2%, respectively; P =0.025; OR=3.281) and a significant decreas e in the AA genotype (16.1%, 52.8%, respectively; P =0.002). The study by Rau H, et al[8] showed that the distribution of alleles as well as genotypic a nd phenotypic frequencies, were similar among Caucasian patients with type 2 dia betes and controls. Our results are not completely similar to Rau H's report.[8] The allelic frequencies of A and G in patients with type 2 diabetes were not significantly different from controls (A/G, 0.500/0.500 vs 0.653/0.347, P =not significant). The distribution of genotype did, however, differ significantly. This difference reflected an increase in the AG genotype in patients (54.8% vs 25.0%, respectively; P =0.012; OR=3.643) and a decrease in the AA genotype (22.6% vs 52.8%, respectively; P =0.011). This study shows that CTLA-4 49 AA (Thr/Thr) is protective from diabetes, whereas, CTLA-4 49 G allele (both as homozygotes and as heterozygotes) confers an increased risk of diabetes mellitus.
CTLA-4 is a negative regulator of T-cell proliferation and activation, which p lays a critical role in the induction of self-tolerance and mediates antigen-s pecific apoptosis. Type 1 diabetes is a T-cell mediated autoimmune disease, so its onset is partly associated with deficient expression and function of CTLA -4. Recent findings suggest that programmed cell death may also be involved in the pathogenesis of type 2 diabetes. Furthermore, there is an evidence favorin g a convergence in signaling pathways toward common effectors of beta-cell apop tosis elicited by stimuli implicated in the pathogenesis of type 1 and type 2 di abetes.[9] If CTLA-4 were involved in this process, its association with type 2 diabetes might be conceivable. A functional role of the CTLA-4 A/G polymorphism, encoding a threonine to alanine change within the signal peptide of CTLA-4, has several possible explanations. It may be in linkage disequilibrium with the (AT) n microsatellite in the 3'-untranslated region and could, therefore, affect ribonucleic acid stability . Equally, it may be in linkage disequilibrium with other disease-causing muta tions.[10]However, it is also possible that this signal peptide polymorph ism determines a subtle alteration in the subcellular localization of mature CTL A-4 protein or affects the interaction of the nascent peptide with chaperonins, leading to a functionally important difference in the folding of the mature pro tein.[11]
In conclusion, CTLA-4 49 AA(Thr/Thr) is protective from diabetes, while CTLA- 4 49 G allele (both as homozygotes and as heterozygotes ) confers an increased risk of diabetes mellitus.
REFERENCES
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3. Donner H, Ran H, Walfish PG, et al. CTLA-4 alanine-17 confers genetic suscept ibility to Graves' disease and to type 1 diabetes mellitus. J Clin Endocrinol M etab 1997;82:143-146.
4. Marron MP, Raffel LJ, Garchon HJ, et al. Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA-4 polymorphism in multiple ethnic groups. Hum M ol Genet 1997;6:1275-1282.
5. Djilali-Saiah I, Larger E, Harfouch-Hammoud E, et al. No major role for the C TLA-4 gene in the association of autoimmune thyroid disease with IDDM. Diabete s 1998;47:125-127.
6. Krokowski M, Bodalski J, Bratek A, et al. CTLA-4 gene polymorphism is associat ed with predisposition to IDDM in a population from central Poland. Diabetes Me tab 1998;24:241-243.
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8. Rau H, Braun J, Donner H, et al. The codon 17 polymorphism of the CTLA-4 gene in type 2 diabetes mellitus. J Clin Endocrinol Metab 2001;86:653-655.
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11. Vaidya B, Imrie H, Geatch DR, et al. Association analysis of the cytotoxin T ly mphocyte antigen-4 (CTLA-4) and autoimmune regulator-1 (AIRE-1) genes in spo radic autoimmune Addison's disease. J Clin Endocrinol Metab 2000;85:688-691.
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