AUTHORS: T. Awata, S. Kurihara, M. Iitaka, S. Takei, I. Inoue, C. Ishii, K. Negishi Taro Izumida, Y. Yoshida, R. Hagura, N. Kuzuya, Y. Kanazawa, S. Katayama

Both IDDM and autoimmune thyroid disease (AITD; Graves’ disease or Hashimoto’s thyroiditis) are believed to develop through a T-cellmediated autoimmune process, and genetic factors clearly play an important role in this process. To date, studies have shown that the development of IDDM is controlled by multiple susceptibility genes, including HLA class II genes (IDDM1) and a variable number of tandem repeats (VNTR) near the insulin gene (IDDM2) (1). Less is known about AITD susceptibility genes (2). Although several studies have shown that AITD is associated with HLA class II genes, the observed association appeared to be weak. Recently, associations of CTLA-4 gene polymorphisms with both Graves’ disease and IDDM have been reported in white populations (35). The CTLA-4linked IDDM susceptibility locus is currently designated IDDM12. CTLA-4 antigen, expressed on activated T-cells, is considered to be important in T-cell regulation (6). In the present study, as a follow-up to our previous studies of HLA class II (DR, DQ) and insulin VNTR genes (7,8), we analyzed CTLA-4 gene polymorphism in Japanese patients with IDDM or AITD.

We studied 173 Japanese IDDM patients, 112 Graves’ disease patients, and 88 Hashimoto’s thyroiditis patients. The diagnosis of IDDM was based on both clinical features and laboratory data, including islet cell antibody (ICA) and the postprandial serum C-peptide value. The diagnosis of AITD was based on clinical findings, thyroid function (thyroid hormones and thyroid-stimulating hormone [TSH]), serum levels of thyroidal antibodies, ultrasonic examination, and thyroidal 123I uptake. The mean age at IDDM onset was 24.5 years (range: 169), that of Graves’ disease was 36.9 years (1272) and that of Hashimoto’s thyroiditis was 43.1 years (1273). In addition, 425 healthy Japanese subjects (age 1871 years) were randomly selected as control subjects. The A-G transition polymorphism at position 49 (exon 1, codon 17) of the CTLA-4 gene was determined by polymerase chain reaction restriction-fragment length polymorphism (PCR-RFLP) method using the PCR primers as described (4). The PCR products were then digested by Fnu4HI, followed by 3% agarose gel electrophoresis and ethidium bromide staining. The G allele sequence was digested by Fnu4HI, while the A allele sequence was not.

As shown in Table 1, the frequency of the G allele was significantly higher in AITD patients than in control subjects (71 vs. 63%, P = 0.006); a similar high frequency of the G allele was observed in the Graves’ disease and Hashimoto’s thyroiditis groups. In contrast, the G allele was not significantly associated with IDDM or early-onset (less than 15 years) IDDM. However, the frequency of the G allele was significantly higher in patients with both acute onset (defined by insulin requirement less than 1 month after onset) and insulin depletion (defined by postprandial serum C-peptide less than 0.033 nmol/l) than in control subjects (80 vs. 63%, P = 0.012). All acute-onset and insulin-depleted patients carried at least one G allele.

Yanagawa et al. (3) first reported an association between Graves’ disease in the U.S. white population and the 106-bp microsatellite allele in the 3′-untranslated region of the CTLA-4 gene, which shows a strong linkage disequilibrium with the G allele of position 49. Subsequent studies have reported associations between the G allele and Graves’ disease in German and Canadian whites (5) and in Hong Kong Chinese (4). Our present data are very similar to the data in the Chinese population (G allele frequency: 73% in patients and 62% in control subjects) and support the susceptibility effect of the G allele to Graves’ disease. A significant increase in allele frequency was even more evident in male Graves’ patients (n = 20, 80%; P = 0.020), which is similar to previous reports (3,5). These results suggest that the CTLA-4 locus is a susceptibility factor for Graves’ disease. Furthermore, we found that the G allele was also associated with Hashimoto’s thyroiditis, another type of autoimmune thyroid disease. It may be conceivable that the CTLA-4 region is a shared susceptibility factor between Graves’ disease and Hashimoto’s thyroiditis in view of their etiological similarities and familial co-occurrence. However, the association with Hashimoto’s thyroiditis should be further evaluated in other ethnic populations, and family-based studies will be necessary to confirm the association of the CTLA-4 gene polymorphism with AITD.

The G allele of CTLA-4 gene polymorphism was also associated with IDDM in whites in two independent case-control studies (4,5). Furthermore, the association was confirmed by family-based association studies in several but not all populations (4). In a case-control study (5), the increased frequency of the G allele in IDDM was less pronounced compared to Graves’ disease. In the present study, although the frequency of the G allele was not significantly higher in Japanese IDDM patients, the increase was significant in acute-onset and insulin-depleted IDDM, i.e., so-called “typical” IDDM. There are a considerable number of “atypical” IDDM patients in the Japanese population who demonstrate clinical features of slow progression and residual ß-cell function (9). The underlying genetic basis to discriminate between typical and atypical IDDM is unclear at present. However, one study reported that a particular HLA class I allele may be related to complete ß-cell destruction (10). The present data appear to indicate that CTLA-4 polymorphism contributes to the rate and degree of disease progression. Given the association between CTLA-4 polymorphism and defective CTLA-4 expression or function, this observation may be conceivable because CTLA-4 plays a critical role in the negative regulation of the T-cell activation process. However, further studies are clearly necessary to confirm our present findings.

In conclusion, the present results in combination with those of previous studies in white populations suggest that CTLA-4 gene polymorphism contributes to a common autoimmune process in IDDM and autoimmune thyroid diseases.