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、瘦弱且容易酮症，但也有一些1型糖尿病患者在发病时被诊断为2型糖尿病，称为LADA（成人潜伏自身免疫性糖尿病）。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GAD自身抗体的高滴度是LADA患者胰岛素依赖的预测指标，但也有一定数量GAD自身抗体高滴度的患者多年未发展为胰岛素依赖。在我们对非胰岛素依赖型糖尿病患者GAD自身抗体临床评估的横断面研究中，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，为了建立更好的预测LADA患者胰岛素缺乏症的标志物，我们对GAD65自身抗体表位识别进行精细定位。嵌合分子是通过用GAD67的同源区域取代人GAD65的区域产生的，GAD67是GAD的一种同型异构体，不是1型糖尿病的主要自身抗原。我们创建了六个GAD65/67嵌合结构，分别为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。血清对一系列GAD构建物的反应性通过96孔放射配体结合试验确定。通过重组GAD67蛋白的竞争，在1型糖尿病和LADA患者的血清中发现了gad65特异性自身抗体识别的两个主要表位，分别为表位1（E1,aa 245-360）和表位2（E2,aa 443-585）。目前，一项全国性的前瞻性研究正在进行中，以确定GAD65自身抗体表位，以预测LADA患者的胰岛素依赖。

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.