人重组谷氨酸脱羧酶65基因的克隆表达及其在1型糖尿病诊断中的初步应用

目的 构建人重组谷氨酸脱羧酶65(GAD65)基因不同区段原核表达载体,诱导表达获得重组蛋白,并初步验证GAD65不同抗原区段在1型糖尿病GAD自身抗体检测中的价值.方法 应用巢式逆转录聚合酶链式反应(RT-PCR)技术调取目的 基因,构建相应的原核表达质粒,转化大肠杆菌E.coli HB101,诱导表达获得纯化重组蛋白,用重组蛋白作为包被抗原,初步建立检测GAD自身抗体的酶联免疫吸附测定法(ELISA)方法,评价各片段在1型糖尿病诊断中的价值.结果 获得了4种可被1型糖尿病患者血清识别的重组人GAD抗原区段,其中GAD65(180-585)抗原区段具有很好的特异性,检出率为55.3%,是首选的抗原区段.结论 所选重组人GAD65(180-585)抗原区段具有良好的抗原性,可作为1型糖尿病患者辅助诊断试剂的候选抗原.     

谷氨酸脱羧酶自身抗体对胰岛素分泌的影响

目的　探讨１ 型糖尿病胰岛素缺乏机理及 Ｇ Ａ Ｄ Ａｂ 对胰岛细胞结构及功能的影响。方法　以改良的放射配体法筛选１ 型糖尿病患者 Ｇ Ａ Ｄ Ａｂ 阳性血清，以亲和层析法从该血清中分离纯化 Ｇ Ａ Ｄ Ａｂ ，以免疫荧光法观察 Ｇ Ａ Ｄ Ａｂ 与β细胞的结合；将纯化的 Ｇ Ａ Ｄ Ａｂ 及／ 或补体加入人胎胰岛细胞培养体系。结果　 Ｇ Ａ Ｄ Ａｂ 能够与β细胞结合，并使培养胰岛细胞的胰岛素释放量减少；透射电镜观察，加 Ｇ Ａ Ｄ Ａｂ 组β细胞胞浆颗粒减少；同时加入补体组β细胞的细胞膜被破坏。结论　１ 型糖尿病患者血清 Ｇ Ａ Ｄ Ａｂ 在体外能够抑制培养的人胎胰岛β细胞的胰岛素合成和释放，并且有补体依赖性细胞毒作用。     

Clinical heterogeneity of adult Japanese diabetes depending on titers of glutamic acid decarboxylase autoantibodies

Aims/Introduction: We examined whether levels of glutamic acid decarboxylase autoantibodies (GADAb) might show the clinical heterogeneity of adult Japanese diabetes. Materials and Methods: In this cross‐sectional study, the serum levels of GADAb were measured in a total of 1857 consecutive adult diabetic patients aged 20 years or older. The patients with positive GADAb, arbitrarily defined as ≥1.5 U/mL, were divided into quartiles according to the number of patients. The age‐ and sex‐matched diabetic patients without GADAb were selected as a control group. Results: A total of 103 (5.5%) of the diabetic patients had GADAb, and showed higher HbA_1c and serum high‐density lipoprotein (HDL) cholesterol levels, lower body mass index (BMI), urinary C ‐ peptide immunoreactivity (CPR), serum triglycerides (TG) and uric acid (UA) levels, and lower prevalence of metabolic syndrome than the control group (P < 0.05). Quartiles 3 and 4 (i.e. GADAb ≥4.6 U/mL) showed a higher HbA_1c level, lower BMI, urinary CPR, serum TG and UA levels, quartile 2 (2.5 ≤ GADAb < 4.6 U/mL) showed a lower BMI level than the control group (P < 0.05). Among the clinical parameters, we observed significant upward trends for both HbA_1c and serum HDL cholesterol levels, and significant downward trends for BMI, serum TG and UA, urinary CPR levels, and prevalence of metabolic syndrome across GADAb quartiles (P < 0.05 for trend). Conclusions: These results show that the clinical phenotype of adult Japanese diabetes correlates with GADAb levels, and that patients with GADAb (≥2.5 U/mL) show different characteristics from those without GADAb, although further longitudinal studies are required. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00190.x, 2011)     

Different contribution of class II HLA in fulminant and typical autoimmune type 1 diabetes mellitus

Aims/hypothesis Fulminant type 1 diabetes, which is characterised by a markedly acute onset of diabetes and an absence of islet-related autoantibodies, accounts for 20% of type 1 diabetes in Japan. We aimed to clarify the contribution of the HLA subtype to fulminant type 1 diabetes in Japanese. Methods We determined the serological subtypes of HLA-A, -DR and -DQ in 115 patients with fulminant type 1 diabetes, 98 patients with typical type 1A diabetes and 190 normal control subjects. Results The frequency of HLA-DR4, but not DR9, was significantly higher in fulminant type 1 diabetes, while those of HLA-DR1, DR2, DR5 and DR8 were significantly lower than those in controls. In contrast, DR9 but not DR4 was more frequent and DR2 was extremely rare in typical type 1A diabetes. Haplotype analysis revealed that DR4-DQ4 was significantly more frequent, and both DR2-DQ1 and DR8-DQ1 were less frequent in fulminant diabetes. In type 1A diabetes, DR2-DQ1 was extremely rare while DR9-DQ3 was significantly more frequent. In the combination analysis, the homozygotes of DR4-DQ4 in fulminant type 1 diabetes and DR9-DQ3 in typical type 1A diabetes indicated high odds ratios (13.3 and 13.3, respectively). Conclusions/interpretation Our results suggest that class II HLA contributes to the development of fulminant type 1 diabetes. Susceptibility and resistance of the HLA subtype to type 1 diabetes are distinct between fulminant and typical autoimmune type 1 diabetes. An erratum to this article is available at .     

暴发性1型糖尿病1例病例报道暨文献复习

暴发性1型糖尿病以急骤起病,代谢紊乱严重,胰酶升高而胰腺超声检查无异常为特点.属于特发性1型糖尿病,患者自身抗体多为阴性,推测病因可能与病毒感染和自身免疫有关.     

谷氨酸脱羧酶抗体诊断成人隐匿性自身免疫性糖尿病探讨

采用放射配体法检测谷氨酸脱羧酶（ＧＡＤ６５）抗体，对１９５例≥３５岁非酮症Ⅱ型糖尿病起病半年以上者和４５例正常对照者的观察表明：本组Ⅱ型糖尿病者ＧＡＤ６５抗体阳性率为１４．８％，高于正常对照者的２．２％；发病年龄〈４０岁、有酮症史、体重指数（ＢＭＩ）〈２１ｋｇ／ｍ＾２、空腹血清Ｃ肽〈０．３ｎｍｏｌ／Ｌ和（或）胰升糖素刺激后６分钟血清Ｃ肽〈０．６ｎｍｏｌ／Ｌ者，ＧＡＤ６５抗体阳性率均高于相应对照组（     

Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with Type 1 (insulin-dependent) diabetes mellitus

Summary Antibodies to glutamic acid decarboxylase, previously known as the 64 kD antigen, appear to be more predictive of Type 1 (insulin-dependent) diabetes mellitus in Caucasoids than other autoantibodies to islet cell antigens. However, seropositivity to glutamic acid decarboxylase is not universal at the onset of Type 1 diabetes and the prevalence in Asians is low compared to Caucasoid patients. This suggests the involvement of multiple pancreatic autoantigens in the Type 1 diabetes autoimmune process or, genetic differences within and between ethnic groups that contribute to the heterogeneous autoimmune response to glutamic acid decarboxylase or both. Alternatively some cases of Type 1 diabetes could have an aetiology unrelated to autoimmunity. This study examined the differential response to glutamic acid decarboxylase according to HLA-DR and -DQ genotypes, as determined by RFLP, in 49 white Australian and 44 Asian patients with Type 1 diabetes. Among Australians heterozygous for HLA-DR3, DR4, 85% were positive for antibodies to glutamic acid decarboxylase, significantly different (p = 0.039) from the prevalence of 48% in patients with at least one HLA-DR antigen other than DR3 or DR4. Also, among Australians, the presence of low risk HLA-DQ antigens, namely DQw5, DQw6 or DQw7, reduced the prevalence of antibodies to glutamic acid decarboxylase by 40% (p = 0.064). Among Asians with Type 1 diabetes and with antibodies to glutamic acid decarboxylase, HLA-DR9 was significantly (p = 0.037) increased in frequency, at 63% compared with 22% in those without glutamic acid decarboxylase antibodies, and the presence of a low risk HLA-DQ allele reduced the antibody rates by 87% (p = 0.003). These observations may reflect differential genetic/environmental interactions in Type 1 diabetes or differential persistence of glutamic acid decarboxylase antibodies in those with different genetic backgrounds.     

A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific glutamic acid decarboxylase antibodies in childhood IDDM

Summary Insulin-dependent diabetes mellitus (IDDM) is associated with autoreactivity against GAD but the diagnostic sensitivity (positivity in disease) and specificity (negativity in health) of isoform-specific GAD antibodies have yet to be defined in assay systems suitable for screening large number of samples. One set of IDDM patient (n=10) and control (n=50) standard sera were used to develop quantitative antibody assays with in vitro synthesized recombinant ³⁵S-methionine-labelled GAD65 and GAD67, respectively, and protein A-Sepharose to separate free from antibody-bound ligand. Binding levels were not normally distributed (p<0.0001) and therefore, the diagnostic accuracy of GAD antibodies was analysed by the ROC plots in population-based, consecutively-diagnosed, recent onset, 0–14 year-old patients (n=105), and matched, healthy control subjects (n=157). The ROC plots showed that the diagnostic sensitivity of GAD65 antibodies was 77% and the specificity 92% compared with 8% and 98%, respectively for GAD67 antibodies. In the IDDM sera, GAD65 and GAD67 antibodies were concordant in 7% (6 of 81) and GAD65 antibodies and ICA in 89% (72 of 81) without a correlation between the autoantibody levels. Autoantibodies to recombinant human islet GAD65 are specific and sensitive markers for childhood IDDM in this immunoassay with in vitro synthesized ³⁵S-methioninelabelled recombinant GAD.Abbreviations IDDM insulin-dependent diabetes mellitus - GAD glutamic acid decarboxylase - ROC receiver-operating characteristic - ICA islet cell antibodies - JDF Juvenile Diabetes Foundation     

Insulin VNTR I/III genotype is associated with autoantibodies against glutamic acid decarboxylase in newly diagnosed type 1 diabetes

BACKGROUND: In type 1 diabetes (T1D), the influence of age at diagnosis and of the IDDM1 and IDDM2 genetic susceptibility loci on the profile of beta-cell autoantibodies has been demonstrated. We studied these associations in a group of 92 patients (children, adolescents and adults, aged 2-62 years) with newly diagnosed T1D. METHODS: The prevalence of the HLA-DQB1*02 and *0302 alleles and of the classes of variable number of tandem repeats (VNTR) of the insulin gene (INS), and of beta-cell autoantibodies (GADA, IA-2A, ICA and IAA) was determined. Statistical analysis was performed using linear and logistic regression models. RESULTS: The presence of IAA, IA-2A and ICA, but not of GADA, was negatively associated with age at diagnosis. Younger patients were more likely to have multiple autoantibodies. There was a tendency of a higher prevalence of IAA in patients with the HLA-DQB1*02/0302 genotype or with the DQB1*0302 allele compared to patients lacking these markers. As a novel observation, the INS VNTR I/III genotype was significantly associated with the presence of GADA (OR = 4.79; p = 0.018). CONCLUSION: The association between the INS VNTR I/III genotype and GADA may suggest that in patients with T1D lacking the INS VNTR I/I genotype, the effect of other susceptibility factors prevails, which promotes the development of autoimmunity to beta-cell antigens other than insulin.     

Fulminant type 1 diabetes in Korea: high prevalence among patients with adult-onset type 1 diabetes

Aims/hypothesis The aim of this study was to investigate the prevalence of fulminant type 1 diabetes and the clinical characteristics of the disease among newly diagnosed Korean patients. Methods Using data retrieved from the Seoul National University Hospital database, we identified all patients newly diagnosed with type 1 diabetes from 1 January 1999 to 31 July 2006. Information on clinical manifestations and laboratory data, including the presence of islet autoantibodies detected at diagnosis, were obtained by reviewing medical records. Results We identified 99 patients newly diagnosed with type 1 diabetes. Seven patients (7.1%) fulfilled the criteria for fulminant type 1 diabetes. Among the patients aged ≥18 years at onset, 30.4% had fulminant type 1 diabetes. Patients with this diabetes subtype tested negative for islet autoantibodies, had a higher age of onset (median 28 vs 10 years, p < 0.001) and a markedly shorter duration from onset of hyperglycaemic symptoms to first hospital visit (median 3 vs 30 days, p < 0.001) than patients with non-fulminant type 1 diabetes, and showed trends of increased serum aspartate aminotransferase and amylase levels and a decreased glucagon-stimulated serum C-peptide response. Conclusions/interpretation In Korea, the prevalence of fulminant type 1 diabetes was 7.1% among all patients newly diagnosed with type 1 diabetes and 30.4% among patients with adult-onset diabetes. The clinical and metabolic characteristics of the patients with fulminant type 1 diabetes were similar to those reported in Japanese studies. Y. M. Cho and J. T. Kim contributed equally to this work.     

国内299例暴发性1型糖尿病病例复习及其临床特点分析

目的:归纳总结国内暴发性1型糖尿病(FT1DM)的临床特征。方法:收集中文数据库中2005年1月至2018年12月公开发表的FT1DM相关文献,对其中符合纳入标准的279例病例及我院收治的20例患者资料进行汇总分析。结果:(1)自2005年起,国内对FT1DM的报道数逐年增加,南方地区报道病例数明显多于北方地区;(2)...     

Correlation of glycated albumin but not hemoglobin A1c with endogenous insulin secretion in fulminant type 1 diabetes mellitus

Aims/Introduction: Fulminant type 1 diabetes mellitus (FT1DM) develops as a result of very rapid and almost complete pancreatic β‐cell destruction. We hypothesized that in patients with FT1DM who have less endogenous insulin secretion, disease progression is more rapid, and thus glycated albumin (GA) levels are lower. This study was designed to prove this hypothesis. Materials and Methods: The present study included 42 patients with FT1DM (24 men, 18 women) in whom glycated hemoglobin (HbA_1c), GA and daily urinary C‐peptide (CPR) were measured at the time of diagnosis. Patients with complications, such as liver disease, kidney disease, anemia, or who were pregnant were excluded. Results: Urinary CPR (log transformed) was not correlated with HbA_1c (R = 0.168, P = 0.287), but was positively correlated with GA (R = 0.336, P = 0.030). It was weakly, but not significantly, correlated with GA/HbA_1c ratio (R = 0.281, P = 0.072). In patients with GA < 24.0%, urinary CPR was significantly lower than in patients with GA ≥ 24.0%. In addition, in patients with GA/HbA_1c ratio <3.8, urinary CPR was significantly lower than in patients with GA/HbA_1c ratio ≥ 3.8. Conclusions: Our findings suggest that in patients with FT1DM, GA at the time of diagnosis was correlated with endogenous insulin secretion. GA < 24.0% at the time of diagnosis is predictive for less endogenous insulin secretion in patients with FT1DM. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00050.x, 2010)     

空腹C-P、INS及胰岛细胞自身免疫抗体诊断儿童1型糖尿病的价值

为探讨儿童初发1型糖尿病（T1DM）的诊断指标，对33例T1DM患儿（观察组）分别测定其空腹血糖（FPG），糖化血红蛋白（HbA1c)，C－肽（C－P），胰岛素（INS），胰岛细胞抗体（ICA）及谷氨酸脱羧酶抗体（GAD），并与33例年龄，性别配对的健康儿童（对照组）作比较。结果显示，观察组除FPG，HbA1c显著升高外，C－P，INS，C－P／FPG，INS／FPG均显著低于对照组，以C－P／FPG最明显。提示空腹C－肽，INS，C－P／FPG，INS／FPG测定对诊断儿童初发T1DM有重要价值；胰岛细胞自免疫抗体测定对诊断T1DM有重要价值。     

暴发性1型糖尿病临床流行病学调查

目的了解住院糖尿病患者中暴发性1型糖尿病的患病状况。方法采用Hanafusa提出的标准诊断暴发性1型糖尿病。系统性回顾2001～2008年本院内分泌科住院糖尿病患者情况,分析暴发性1型糖尿病所占比例。结果8年间本院内分泌科住院糖尿病患者共8801例,其中新诊断急性起病1型糖尿病患者107例,暴发性1型糖尿病患者为11例。暴发性1型糖尿病约占本院连续住院糖尿病患者的1‰,新发1型糖尿病患者的10％。未观察到暴发性1型糖尿病发病逐年增加及月份聚集现象。结论暴发性1型糖尿病呈散发,成年人中常见,临床中应注意鉴别诊断。     

The prognostic significance of glutamic acid decarboxylase antibodies in patients with chronic pancreatitis undergoing total pancreatectomy with islet autotransplantation

AimIslet autotransplantation (IAT) is considered a ‘non-immune’ model of islet transplant, with no risk for autoimmune-mediated beta cell loss, but we have previously observed de novo type 1 diabetes in one total pancreatectomy with islet autotransplantation (TPIAT) recipient. We aimed to investigate the clinical significance of glutamic acid decarboxylase antibodies (GADA), as a sensitive marker for autoimmune diabetes mellitus (DM), in patients with chronic pancreatitis undergoing TPIAT.MethodsWe identified 9 patients undergoing TPIAT with elevated GADA pre-TPIAT (8 non-diabetic and 1 with C-peptide positive DM), otherwise demographically similar to GADA negative TPIAT recipients (n = 341). Metabolic and clinical measures related to islet cell function were recorded both before and after TPIAT.ResultsNone of the 9 TPIAT patients achieved insulin independence after surgery, vs. 33% of GADA negative patients (n = 318 with 1-yr follow-up). The two patients with the highest titters of GADA (> 250 IU/mL) both experienced islet graft failure, despite normoglycaemia pre-TPIAT and high islet mass transplanted (5276 and 9378 IEQ per kg), with elevated HbA1c levels post-TPIAT (8.3%, 9.6%). The remaining 7 seven were insulin dependent with partial graft function and HbA1c levels < 7%.ConclusionInsulin dependence was more frequent in 9 patients with elevated GADA prior to TPIAT than in GADA negative TPIAT recipients, with graft failure in 2 cases. We speculate that beta-cell autoimmunity may occur in a small subset of TPIAT recipients and that beta cell antibody testing prior to TPIAT may be warranted to identify individuals at higher risk for insulin dependence.     

暴发性1型糖尿病的患病状况及其特征

目的 探讨暴发性1型糖尿病(F1D)的患病状况和临床特征.方法 采用Hanafusa提出的诊断标准,从中南大学湘雅二医院急性酮症起病的1型糖尿病患者中筛选F1D患者,再根据胰岛自身抗体谷氨酸脱羧酶抗体(GADA)或蛋白酪氨酸磷酸酶抗体的有无将非F1D患者分为经典1型组和特发1型组,比较3组患者临床特征的差异.结果 87例急性酮症起病的1犁糖尿病患者中有8例符合F1D的诊断标准,占9.1%,在18岁以上患者中占14.0%.起病时暴发组的血糖显著高于经典1型组和特发1型组(P=0.004);暴发组血淀粉酶水平显著高于经典1型组(P=0.021).4例(50%)患者发病初期GADA阳性,其中1例柯萨奇病毒B(CVB)IgM阳性,1例人单纯疱疹病毒1(HSV1)IgM阳性.结论 F1D约占以酮症或酮症酸中毒起病的1型糖尿病患者的10%.起病时F1D患者比经典1型和特发1型糖尿病患者有更严重的代谢紊乱.病毒感染和自身免疫可能参与其发病过程.