Insulin-dependent diabetes mellitus(IDDM) is caused by a chronic, clinically silent autoimmune process that gradually destroys the insulin-producing beta-cells. In the early pre-clinical phase of IDDM, antibodies to islet cell antigens are detected. Indeed, some IDDM-associated antibodies, which are present months to years before the onset of that metabolic disease, identify individuals who are high risk for IDDM. The ability to identify such individuals reliably may allow therapeutic intervention with immunoregulatory agents to prevent IDDM^1,2).

The most practical markers of beta-cell autoimmunity currently available are circulating antibodies against islet antigens and they are islet cell cytoplasmic antibody(ICA)³⁾, islet cell surface antibody(ICSA)⁴⁾, insulin autoantibody(IAA)⁵⁾, and Mr-64000(64kD) autoantibody etc. Among these autoantibodies, an antibody to Mr-64kD antigen, which is a component of pancreatic beta-cells, was detected about 10 years ago⁶⁾. Mr-64kD protein recently appears to be the gamma-aminobutyric acid(GABA)-synthesizing enzyme glutamic acid decarboxylase(GAD) and is regarded as a major target antigen in Type 1 diabetes⁷⁾. 64k autoantibodies are detected at a high frequency in new onset Type 1 diabetic patients and may precede clinical onset by several years and they have also been detected in pre-diabetic period in the first degree relatives of Type 1 diabetic patients who later develop diabetes^8–10). 64k autoantibodies may be detected when there are no residual pancreatic beta-cell function¹¹⁾ but not associated with pancreatic β-cell function¹⁰⁾. The prevalence of 64k autoantibody may be influenced by genetic background including HLA-DR, DQ types^8,12).

There is an unanswered question: why are there marked differences in the prevalence of GAD autoantibodies in Caucasoid Type 1 diabetic patients(63%) compared with various Asian populations(5–31%)(P. Zimmet, unpublished observations). In this study, we report the analysis of the relationships between the prevalence of the 64k autoantibodies, residual pancreatic β-cell function, HLA-DR antigens and HLA-DQ genes in Korean patients with IDDM.

The mean age of the 35 IDDM patients was 14 years(range 4–49years) and the mean duration of diabetes was 1.7years(range 0.02–17years). The 64k autoantibody was detected in 12 of 35(35%) patients with IDDM and in none of 10(0%) normal controls(Table 1).

The 64k autoantibody assay, developed by Baekkeskov et al⁶⁾ in 1982, involves the immunoprecipitation of radiolabled islet membrane fractions by patient sera and now revealed GAD⁷⁾. There are two GAD isoforms,. Mr-65,300 (GAD₆₅) and Mr-66,660(GAD₆₇), which are highly expressed in the brains of higher vertebrates and rat pancreatic islet cells¹⁵⁾, but also expressed in testes, ovaries, adrenal medulla etc. There are no known biochemical differences in GAD between brain and pancreatic islets and isolated human pancreatic islets demonstrated an exclusive expression of the GAD₆₄ encoding transcription in contrast to rat pancreatic islets, and brains of vertebrates expressed both GAD isoforms^16,17).

The role and significance of islet GAD and GABA are unclear, these are expressed only in beta-cells of the pancreas¹⁸⁾, regardless of insulin secretory granules, which reside in cytoplasm^19,20). The GABA produced by GAD appears to be involved in the regulation of glucagon secretion in the islets and may have other physiological roles as well²¹⁾. In isolated pancreatic islet-cell cultures, synthesis of GAD was increased according to glucose concentration^22,23) and GAD-associated antibodies have been described as having an inhibitory effect on islet insulin secretion²⁴⁾. These findings emphasize the potential role of GAD in the pathogenesis of IDDM and make it important to characterize its biological function in the islets and to elucidate its potential pathogenic involvement in beta-cell destruction. Although 64k autoantibody may have greater sensitivity, specificity and predictive value for human IDDM, than other established markers¹⁰,¹¹,^25–27), the 64k autoantibody assays have not been widely used because islet-based immunoprecipitation assays are expensive and laborious, preventing completion of large studies^28–32).

Among the autoantibodies of IDDM, the best studied is ICA, and several groups have reported that ICA is a good predictive marker in relatives of patients with IDDM^33–36). High and persistent titers for ICA, especially in the pediatric age-group, are reliable predictors³⁶⁾. It was known that ICA was detected about 80% sera of patients with recent-onset IDDM, but usually appears after onset of diabetes and a rapid drop in serum titer during disease progression, compared to 64k autoantibody^37,38). The presence of ICA may indicate the prognostic marker of IDDM, rather than a pathogenic role to develop diabetes. The Mr 64000(64k) islet cell protein, now recognized an glutamic acid decarboxylase (GAD), is a major target antigen in Type 1 diabetes. Immunoprecipitating antibodies to this protein have been found in 80% of newly diagnosed cases^1,37), and they have also been detected in the pre-diabetic period in first degree relatives of Type 1 diabetic patients who later develop diabetes^38,39). 64k autoantibody may persist, even when there are no residual beta-cell functions, but is rarely detected in the normal population^16,37,40). Zimmer et al¹²⁾ reported that there were marked differences in the prevalence of GAD antibodies in Caucasoid Type 1 diabetic patients (63%) compared with various Asian populations (5–31%).

In our study, 64k autoantibody was detected in 12 of 35(34%) patients with IDDM but in none of 10(0%) normal controls. Among patients studied, there was a patient whose duration of diabetes was 17 years, the fasting plasma C-peptide level was less than 0.1ng/ml, was positive in 64k autoantibody. According to these results, 64k autoantibody seems not to have sensitivity but has specificity to be a diagnostic marker of IDDM in Korea, and 64k autoantibody may persist even though there is no residual pancreatic β-cell function. Hagopian et al¹¹⁾ reported that there was a tendency that the prevalence of 64k autoantibodies are increased in patients with recent-onset diabetes and younger-aged patients. In our study, 64k autoantibody was detected in 40% of patients of recent onset(duration<6months), and in 39% of patients of the younger -aged group(age<15years) in contrast to 34% of overall patients with IDDM. Several groups^38,41) reported that, at the time IDDM developed, ICA positive patients lost their pancreatic beta-cell function more rapidly than ICA negative patients. In the high risk group of diabetes, like first relatives of patients with IDDM, the presence of 64k autoantibody is a better predictive marker of a decrease in their insulin secreting functions and becoming diabetic patients than other autoantibodies^7–9). But Tuomi et al¹⁰⁾ reported that there were no differences in pancreatic beta-cell function between 64k autoantibody positive and negative groups. In our study, there were also no differences in fasting and postprandial 2 hours plasma C-peptide levels of recent-onset(duration <6momths) and overall patients between 64k autoantibody positive and negative groups. According to these results, the presence of 64k autoantibody may not be a predictive marker of residual pancreatic beta-cell function and insulin secretory capacity. But ICA positive IDDM patients have nearly the same insulin secretory function as ICA negative patients during their first 3 months of diabetes and, thereafter, they lost their secretory function more rapidly¹⁰⁾. To confirm the exact relationship between 64k autoantibody and pancreatic β-cell function, further and serial studies are warranted.

In Caucasians, 50–60% of the population have HLA-DR3 or DR4, which are known to be a genetic susceptibility factor of IDDM, and over 95% of patients with IDDM have at least one of them^25,42). But a smaller percent population of Asians, including Koreans, have less HLA-DR3 or DR4 than Caucasians and this difference may explain why there are fewer IDDM patients in Asians^12,27,42,43). Seiβler et al⁹⁾ reported that there was an increased prevalence of 64k autoantibody significantly in normal non-diabetic children who have HLA-DR4 and HLA-DQ β chain with 57th non-asparic acid, than those without any of them. In the report of Serjeantson et al¹²⁾, among Australian patients with IDDM, heterozygous for HLA-DR3 and DR4, 85% were positive for antibodies to GAD, significantly different from the prevalence of 48% in patients with, at least, one HLA-DR antigen other than DR3 or DR4, and these observations may reflect differential genetic and environmental interactions in IDDM or differential persistence of GAD antibodies in those with different genetic backgrounds. In Caucasians, HLA-DR3 and DR4 confer a particular risk alleles for susceptibility to IDDM⁴²⁾, whereas in the Chinese, the high risk are HLA-DR3 and DR9⁴⁾. In the Japanese, in whom HLA-DR3 is virtually absent, the high risk IDDM alleles are HLA-DR4 and DR9⁴⁵⁾. In our study, among patients, heterozygous for HLA-DR3 and DR4, all 3 patients were positive to 64k autoantibody and 10 of 27(37%) patients, with at least one of them, were positive, but only 1 of 7(14%) patients without DR3 nor DR4 was positive to 64k autoantibody. Accordingly, HLA-DR3 and DR4, especially HLA-DR3/DR4 heterozygote, may be associated with the presence of 64k autoantibody.

Recently, HLA-DQ gene analysis is becoming more meaningful for the predictive marker of genetic susceptibility of IDDM, rather than HLA-DR antigens¹⁾, and there were several reports that individuals with HLA-DQA1*0301 and HLA-DQB1*0302 were significantly more susceptible to IDDM than those without them^1,46,47). According to studies about Koreans, Lee et al⁴⁸⁾ reported that HLA-DQA1*0301, DQB1*0201 and DQB1*0303 genes were significantly increased in patients with IDDM than normal controls, and Hong et al⁴⁹⁾ and Hahn et al⁵⁰⁾ reported that HLA-DQA1*0301 gene was significantly increased in patients with Korean IDDM patients than the normal population. But there is no report about the relationship between HLA-DQ genes and the prevalence of 64k autoantibody in Korea. In our study, among 34 patients, the number of patients who have HLA-DQA1*0301 and DQA1*0401, *0501 are 30(88%) and 17(50%) respectively, and all 34 patients had at least one of them. All 12 patients who were positive to 64k autoantibody have DQA1*0301 in contrast to 18 of 22(82%) patients negative to 64k autoantibody. In the HLA-DQB1 gene analysis, the patients with HLA-DQB1*0201, DQB1*0301 and DQB1*0303 genes showed slightly increased prevalences, respectively, in 64k autoantibody positivity than those without them.

In our study, a small number of patients and a smaller prevalence of 64k autoantibody, compared to Caucacians, had limited statistically significant results. Further serial and large scale studies of 64k autoantibody are warranted to understand the pathogenesis of IDDM and the exact role of that autoantibody.