Identification of predictive marker for type 1 diabetes development using GAD antibody epitope analysis and its application to disease prevention

虽然大多数1型糖尿病患者年轻，瘦，容易酮症，但有许多1型糖尿病患者最初被诊断为2型糖尿病，称为LADA（成人潜伏性自身免疫性糖尿病）。这些LADA患者通常在诊断后几年内进展为胰岛素缺乏状态。虽然高滴度的GAD自身抗体是LADA患者胰岛素依赖的预测标志物，但仍有一定数量的高滴度GAD自身抗体患者多年未进展为胰岛素依赖。在我们对具有GAD自身抗体的非胰岛素依赖型糖尿病患者的临床评价的横断面研究中，大约三分之二的LADA患者进展为胰岛素缺乏（阳性预测值67.1%）。因此，为了建立LADA患者中胰岛素缺乏的更好的预测标志物，进行了GAD 65自身抗体表位识别的精细定位。嵌合分子是通过用GAD 67的同源区域取代人GAD 65的区域而产生的，GAD 67是GAD的同种型，不是1型糖尿病中的主要自身抗原。我们已经创建了六个GAD 65/67嵌合构建体，命名为GAD 65-N/GAD 67-MC、GAD 65-NM/GAD 67-C、GAD 65-N/GAD 67-M/GAD 65-C、GAD 67-N/GAD 65-MC、GAD 67-NM/GAD 65-C、GAD 67-N/GAD 65-M/GAD 67-C。通过放射性配体结合试验以96孔试验形式测定血清对一系列GAD构建体的反应性。通过重组GAD 67蛋白的竞争，在来自1型糖尿病和LADA患者的血清中发现了GAD 65特异性自身抗体识别的两个主要表位，命名为表位1（E1，aa 245-360）和表位2（E2，aa 443-585）。目前正在进行全国范围的前瞻性研究，以确定GAD 65自身抗体表位，用于预测LADA患者的胰岛素依赖性。

Although the majority of patients with type 1 diabetes are young, lean, and ketosis-prone, there are number of patients with type 1 diabetes initially diagnosed as having type 2 diabetes at disease onset called LADA(Latent Autoimmune Diabetes in Adults). These patients with LADA often progress toward insulin-deficient state within several years after diagnosis. Although the high titer of GAD autoantibodies is a predictive marker of insulin dependency in LADA patients, there is a certain number of patients with high titer of GAD autoantibodies who do not progressed to insulin dependency for many years. In our cross-sectional study on the clinical evaluation of non-insulin dependent diabetic patients with GAD autoantibodies, approximately two-thirds of LADA patients progress toward insulin deficiency (positive predictive value 67.1 %). Therefore, to establish the better predictive marker for insulin deficiency in LADA patients, the fine mapping of GAD65 autoantibody epitope recognition was conducted. Chimeric molecules were generated by substitution of regions of human GAD65 with homologous regions of GAD67,an isoform of GAD that is not a major autoantigen in type 1 diabetes. We have created the six GAD65/67 chimeric constructs designated GAD65-N/GAD67-MC、GAD65-NM/GAD67-C、GAD65-N/GAD67-M/GAD65-C、GAD67-N/GAD65-MC、GAD67-NM/GAD65-C、GAD67-N/GAD65-M/GAD67-C. Serum reactivity to a series of GAD constructs were determined by radioligand binding assays in a 96-well assay format. With competition by recombinant GAD67 protein, two major epitopes recognized by GAD65-specific autoantibodies were found, designated Epitope 1(E1,aa 245-360) and Epitope 2(E2,aa 443-585), in sera from patients with type 1 diabetes and LADA. Now the nation-wide prospective study is underway to identify the GAD65 autoantibody epitope(s) for prediction of insulin dependency in LADA patients.