Here, we report on the detection of a novel point mutation of the CTLA4 gene at nucleotide position 159 (C→G) leading to amino acid substitution at position 53 (I→M), as well as its association with type 1 diabetes in two ethnically distinct populations. Subjects included 182 unrelated type 1 diabetes children and 201 control subjects from Ghana, West Africa. The Chinese study population consisted of 350 type 1 diabetic children and 420 healthy control subjects from central China. Polymerase chain reaction–single-strand conformation polymorphism and sequence analysis were used to screen for polymorphisms in the CTLA4 gene. CTLA4 49 (A→G) mutation conferred a risk of type 1 diabetes in the Chinese children (odds ratio 1.78, 95% CI 1.58–2.0), but not in the West African children (1.17, 0.84–1.64). On the other hand, the novel CTLA4 159 (C→G) mutation conferred a risk of type 1 diabetes in the West African children (2.1, 1.54–2.86), but not in the Chinese type 1 diabetic children. The novel CTLA4 gene polymorphism at nucleotide position 159 significantly associated with type 1 diabetes in West Africans, but not in Chinese. On the other hand, the CTLA4 gene polymorphism at nucleotide position 49 significantly associated with type 1 diabetes in Chinese, but not in West Africans.

Type 1 diabetes is an organ-specific chronic autoimmune disease with a long prediabetes phase that often precedes its rapid onset. Type 1 diabetes has a weak genetic link; its association with HLA antigens is well established. The environmental trigger in type 1 diabetes may be a viral infection; however, there has been a gradual increase in the amount of literature suggesting a genetic linkage for this disorder. The disorder is characterized by lymphocytic infiltration into pancreatic islets and the presence of pancreas-specific autoantibodies in the serum, leading to selective destruction of the pancreatic β-cells that synthesize and produce insulin. The interaction of a large number of susceptibility genes and environmental factors are thought to influence the development of the disease. Type 1 diabetes is thus a multifactorial disorder that can be influenced by the age of onset, family history, extra-pancreatic autoimmune disease, the extent of hyperglycemia before insulin dependency, and the occurrence of diabetic complications (1,2,3). The CTLA4 (cytotoxic T-cell–associated antigen-4) gene has been known to encode the T-cell receptor responsible for T-cell proliferation and apoptosis. This gene has thus been reported to be a strong candidate for autoimmune disorders that are mediated by T-cell (1,2,3). Previous studies have reported on a polymorphism at position 49 (A-G) in exon 1 of the CTLA4 gene; this polymorphism is purported to be associated with type 1 diabetes in Caucasians but not in the Chinese. However, Lee et al. (1) reported that the polymorphism is associated with type 1 diabetes in their Chinese subjects. Type 1 diabetes is a complex genetic disease known to be linked to a number of genes (2,3). Two genomic regions have been reported to be associated with type 1 diabetes, namely, IDDM1 and IDDM2, identified as HLA complex and a variable number of tandem repeats in the 5′ region of the insulin gene (VNTR-INS), respectively (4,5,6). Mapping by linkage disequilibrium has suggested that a type 1 diabetes locus is located close to the microsatellite marker locus D2S152 (IDDM7) on chromosome 2q31 (7). CTLA4 and CD28 are encoded by genes that are located on chromosome 2q33. It has also been established that CTLA4 and CD28, glycoprotein receptors that are expressed in reactive T-cells, play a role in the initial T-cell activation by antigen-presenting cells (APCs) and the resulting regulation of cellular immunity (1). Two published studies have shown a significant association of the CTLA4 gene polymorphism (IDDM17) with type 1 diabetes (2,7,8).

Until now, there has been no comparative study on the genetic basis of type 1 diabetes and its related risk factors in West Africans and Chinese. Therefore, the present study reports on the detection of a novel point mutation of the CTLA4 gene at nucleotide position 159 (C→G) that results in amino acid substitution at position 53 (I→M), as well as its association with type 1 diabetes in two ethnically distinct populations.

Three different polymorphic patterns were detected for the previously determined mutation (CTLA4 A49G) and for the novel mutation CTLA4 C159G. Polymerase chain reaction (PCR)–single-strand conformation polymorphism (SSCP) and sequence analysis revealed all three variants of the CTLA4 (A49G) gene polymorphism in the two populations. The genotype and allele frequencies of the CTLA4 49 gene polymorphism in Chinese type 1 diabetic children differed significantly from control subjects (allele frequencies: χ2 = 30.46, P < 0.0001). However, the difference between genotype and allele frequencies with respect to West African type 1 diabetic children and control subjects was not significant (gene frequencies: χ2 = 0.83, P = 0.36) (Table 1). Genotype distribution and gene frequencies of the novel CTLA4 polymorphism (C159G) at nucleotide position 159 in the West African type 1 diabetic children differed significantly from the control subjects (allele frequencies: χ2 = 16.8, P < 0.0001) (Table 1). Analysis by PCR–direct sequencing of each of the products revealed three genotypic variants of two alleles corresponding to a transition of C to G at nucleotide position 159. Our study is the first to report on the existence of this mutation and on its association with type 1 diabetes in West African children, but not in Chinese. The CTLA4 A49G mutation is designated as AA, AG, and GG. The novel mutation CTLA4 C159G is designated as CC, CG, and GG, resulting in the substitution of isoleucine to methionine (I→M) at codon position 53. The CTLA4 49 (A→G) mutation conferred a risk of type 1 diabetes in the Chinese children (odds ratio [OR] 1.78; 95% CI 1.58–2.0). However, no significant association was observed in the West African children (1.17, 0.84–1.64) (Table 1). On the other hand, the novel CTLA4 159 (C→G) mutation conferred a risk of type 1 diabetes in the West African children (2.1, 1.54–2.86) but not in the Chinese type 1 diabetic children (Table 1). Overall, no significant association with type 1 diabetes was observed in subjects exhibiting both polymorphisms (data not shown).

The results of the present study indicate an association of the CTLA4 gene polymorphism 49 (A→G) with type 1 diabetes in Chinese children. On the other hand, the polymorphism at nucleotide position 159 (C→G) associated with type 1 diabetes in the West African subjects. To our knowledge, this report is the first to 1) study the CTLA4 gene as a whole in West Africans, 2) establish the existence of a polymorphism at nucleotide position 159 of the CTLA4 gene, and 3) report on the polymorphism’s association with type 1 diabetes in people of African origin. The CTLA4 gene is known to be polymorphic in untranslated sequences in exon 3 with variant lengths of a dinucleotide (AT)n repeat (9). Another polymorphism in exon 1 at position 49 (A→G) encoding for threonine (Thr) and Alanine (Ala), respectively, has already been characterized by previous studies (10,11,12). The CTLA4 49 G (Ala) allele has been known to be associated with type 1 diabetes in Spanish and Italian families; and more recently, it was found to be associated in Chinese type 1 diabetic children (8). However, it is also possible that different polymorphisms of the CTLA4 gene associate with type 1 diabetes differently in different ethnic groups and environments. Because type 1 diabetes is an autoimmune disorder, it is likely that the gene environment could predispose to the disorder differently. It could be speculated that although these two populations are susceptible to the same condition, different antigenic and environmental factors may trigger the different mutations that predispose to type 1 diabetes. It would be interesting to know how this new polymorphism affects other ethnic groups. Also, further studies on the level of exposure to the specific antigenic factors in the different ethnic groups in relation to these polymorphisms could provide essential information on the pathophysiological importance of these mutations. In the present study, we found that the novel CTLA4 (C159G) gene polymorphism at nucleotide position 159 was significantly associated with type 1 diabetes in our West African subjects, in contrast to the CTLA4 polymorphism at nucleotide position 49. The novel polymorphism was present on at least one allele in ∼30% of the type 1 diabetic West African patients compared with 12% in the control subjects. On the other hand, the novel CTLA4 159 G allele was present in ∼6% of the Chinese type 1 diabetes children compared with 4% in control subjects. The CTLA4 159 (C→G) mutation resulted in amino acid substitution of isoleucine (I) with methionine (M) at amino acid position 53. However, how this mutation influences CTLA4 gene products and what subsequent immunological roles it might play in the pathogenesis of type 1 diabetes in the two different ethnic groups studied has yet to be determined. Although the functional roles of these polymorphisms are not clear, the CTLA4 49 (A→G) and CTLA4 159 (C→G) mutations, respectively, may confer susceptibility to type 1 diabetes in Chinese and West African children. Further studies in different ethnic groups are necessary to characterize the distribution of this polymorphism.

Written informed consent was obtained from all of the subjects by the participating institutions. The research protocol was approved by the University of Tsukuba Ethics Committee before initiation of the study. The subjects included 182 unrelated Ghanaian children clinically diagnosed with type 1 diabetes. The West African subjects consisted of type 1 diabetic patients (96 boys, 86 girls) and healthy control subjects (101 boys, 100 girls) aged 0.3–15 years (6.9 ± 4.2 years). The West African subjects were recruited from pediatric units in four participating polyclinics located in the Ashanti region of Ghana. The Chinese study population consisted of 350 type 1 diabetic patients (246 boys, 104 girls) and 420 healthy control subjects (212 boys, 208 girls) aged 0.3–15 years (7.4 ± 3.2 years). All of the Chinese subjects lived in the same region (Taiyuan City, Shanxi, China) located in the central part of China. Diagnosis of diabetes was made according to the criteria defined by the National Diabetes Data Group (13), and classification as type 1 diabetes was based on the presence of ketosis, low BMI, and the need for insulin therapy. In all subjects the diagnosis of type 1 diabetes was confirmed by the presence of at least one of the three major islet autoantibodies: islet cell antibodies (ICAs), GAD antibodies (GAD65), and tyrosine phosphate-like molecule ICA512. Islet autoantibody tests were negative for all of the healthy control subjects (14,15).

Genomic DNA was extracted from whole-blood samples collected in disodium EDTA (3 mg/ml) according to the established protocol, with a slight modification (16). PCR-SSCP analysis and sequencing were performed according to the method by Harada et al. (17). Statistical analysis for linkage disequilibrium between this novel polymorphism and the previously determined CTLA4 gene mutation at position 49 A/G was performed. A χ2 test was performed to examine the differences in the distribution of genotypes and alleles between healthy control subjects and type 1 diabetic subjects.

TABLE 1

Genotype distribution of point mutations (A49G and C159G) of CTLA4 gene in type 1 diabetic patients and healthy control subjects

Mutation typesChinese
West African
Type 1 diabetesControl subjectsType 1 diabetesControl subjects
n 350 420 182 201 
A49G mutation     
 Genotype distribution     
  A/A 110 (31.4) 201 (47.8) 106 (58.1) 129 (64.1) 
  A/G 166 (47.4) 177 (42.2) 67 (37.1) 61 (30.2) 
  G/G 74 (21.2) 42 (10) 9 (4.8) 11 (5.7) 
 Allele frequencies     
  A 386 (55.1) 578 (68.9) 278 (76.6) 318 (79.2) 
  G 314 (44.9) 262 (31.1) 86 (23.4) 84 (20.8) 
  χ2 30.46
 
0.83
 
  P <0.0001
 
0.36
 
 OR (95% CI) 1.78 (1.58–2.0)
 
1.17 (0.84–1.64)
 
C159G mutation     
 Genotype distribution     
  C/C 312 (89.1) 387 (92.2) 102 (55.9) 147 (73.2) 
  C/G 36 (10.3) 31 (7.4) 64 (34.3) 48 (23.9) 
  G/G 2 (0.6) 2 (0.4) 18 (9.8) 6 (2.9) 
 Allele frequencies     
  C 660 (94.2) 806 (95.9) 266 (73.0) 342 (85.1) 
  G 40 (5.8) 34 (4.1) 98 (27.0) 60 (14.9) 
  χ2 2.32
 
16.8
 
  P 0.13
 
<0.0001
 
 OR (95% CI) 1.44 (0.9–2.30)
 
2.1 (1.54–2.86)
 
Mutation typesChinese
West African
Type 1 diabetesControl subjectsType 1 diabetesControl subjects
n 350 420 182 201 
A49G mutation     
 Genotype distribution     
  A/A 110 (31.4) 201 (47.8) 106 (58.1) 129 (64.1) 
  A/G 166 (47.4) 177 (42.2) 67 (37.1) 61 (30.2) 
  G/G 74 (21.2) 42 (10) 9 (4.8) 11 (5.7) 
 Allele frequencies     
  A 386 (55.1) 578 (68.9) 278 (76.6) 318 (79.2) 
  G 314 (44.9) 262 (31.1) 86 (23.4) 84 (20.8) 
  χ2 30.46
 
0.83
 
  P <0.0001
 
0.36
 
 OR (95% CI) 1.78 (1.58–2.0)
 
1.17 (0.84–1.64)
 
C159G mutation     
 Genotype distribution     
  C/C 312 (89.1) 387 (92.2) 102 (55.9) 147 (73.2) 
  C/G 36 (10.3) 31 (7.4) 64 (34.3) 48 (23.9) 
  G/G 2 (0.6) 2 (0.4) 18 (9.8) 6 (2.9) 
 Allele frequencies     
  C 660 (94.2) 806 (95.9) 266 (73.0) 342 (85.1) 
  G 40 (5.8) 34 (4.1) 98 (27.0) 60 (14.9) 
  χ2 2.32
 
16.8
 
  P 0.13
 
<0.0001
 
 OR (95% CI) 1.44 (0.9–2.30)
 
2.1 (1.54–2.86)
 

Data are n (%), unless otherwise specified.

D.O.H. and H.L. were supported by the Honjo International Scholarship Foundation and by the Asia International Scholarship Foundation, respectively.

We gratefully acknowledge the assistance and cooperation of the faculty and staff at the Shanxi Medical University, Beicheng Central Hospital, and Taiyuan City Hospital, China; and the late Dr. K.O. Safo Adu and the staff at Safo Adu Hospital, Manhyia Urban Hospital, and the School of Medical Sciences, University of Science and Technology, Kumasi, Ghana.

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Address correspondence and reprint requests to Douglas Osei-Hyiaman, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, (NIH/NIAAA/DICBR/LPS), 12420 Parklawn Dr., MSC-8115, Bethesda, MD 20892-8115. E-mail: dhyiaman@mail.nih.gov.

D.O.-H. is currently affiliated with the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland. L.H. is currently affiliated with the Sidney Kimmel Cancer Center, San Diego, California.

Received for publication 2 November 2000 and accepted in revised form 8 June 2001.

APC, antigen-presenting cell; ICA, islet cell antibody; OR, odds ratio; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism.