Association of a Novel Point Mutation (C159G) of the CTLA4 Gene With Type 1 Diabetes in West Africans but not in Chinese

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.