Targeted CRT sequencing of 1000 genes in KPD patients

描述（由申请人提供）：糖尿病（DM）是一种复杂的疾病，遗传和环境因素共同影响其发病、进展和严重程度。尽管缺乏自身免疫性1型糖尿病的典型特征，但一部分糖尿病患者仍容易发生酮症酸中毒。这些酮症易感性糖尿病（KPD）患者的表型强烈暗示胰腺2细胞功能障碍作为病因的新机制。最近，我们的研究小组在400多名患者中发现了四种不同的KPD表型，并进行了临床纵向研究。分配基于A2分类系统，该系统对KPD患者的定义具有较高的准确性和对长期临床结果的良好预测。4个KPD亚群分别为A+2-、A-2-、A+2+和A-2+，频率分布分别为20%、20%、10%和50%。四组表型差异背后的基因序列变异的作用在很大程度上是未知的，推测涉及2细胞发育、功能、扩展和再生的遗传因素，以及涉及2细胞线粒体和脂质代谢的遗传因素。考虑到KPD综合征潜在病因的复杂性，每个KPD患者中多达1000个基因可能需要重新测序以系统地确定病因。目前的Sanger方法在这种规模上对候选基因进行重测序过于昂贵、劳动密集和耗时。本R21提案的目标是通过在高密度寡核苷酸芯片上靶向1,000个候选基因来评估我们下一代循环可逆终止（CRT）测序方法的可行性。选择候选基因的基本原理是基于那些已知与2细胞发育和功能有关的基因，那些与1型或2型糖尿病有关的基因，以及那些与生酮有关的基因。最近，我们描述了一种新的可逆终止体（RT）化学范式，它具有超越电流读取长度的传统RT的内在优势。我们利用固定在标准显微镜载玻片上的合成模板开发并表征了四个荧光标记的3'无阻断RTs，以证明靶向重测序的可行性。在这里，我们建议开发一种1000个基因芯片，利用我们的CRT方法对50名KPD患者的基因组DNA进行重测序。这些数据将通过使用传统的Sanger测序取样~10%的发现变异来验证，以评估CRT方法的准确性。这项研究的结果将导致对CRT技术的全面评估，同时确定KPD患者中假定的基因序列变异，所有这些都可能导致扩大的R01研究，以评估病例对照和病例比较的两层方法（例如，对于2-组：a -与a +，相反，对于a -组，2-与2+）。

DESCRIPTION (provided by applicant): Diabetes mellitus (DM) is a complex disease with a combination of genetic and environmental factors contributing to its onset, progression, and severity. A subset of DM patients is prone to developing ketoacidosis despite lacking the typical features of autoimmune type 1 diabetes. The phenotypes of these patients with ketosis-prone diabetes (KPD), strongly implicate novel mechanisms of pancreatic 2-cell dysfunction as etiologic factors. Recently, our group has identified four distinct phenotypes of KPD in more than 400 patients, followed in a longitudinal research clinic. The assignment is based on the A2 classification system that defines KPD patients with high accuracy and good prediction for long-term clinical outcome. The four KPD subgroups are A+2-, A-2-, A+2+, and A-2+, with a frequency distribution of 20%, 20%, 10%, and 50%, respectively. The role of sequence variation in genes underlying the phenotypic differences of the four groups is largely unknown and presumed to involve genetic factors in 2-cell development, function, expansion and regeneration, as well as those involved in 2-cell mitochondrial and lipid metabolism. Given the complexity of potential etiologic factors underlying the KPD syndromes, as many as 1,000 genes in each KPD patient may require resequencing to systematically pinpoint causative factors. Current Sanger methods for resequencing candidate genes at this scale are too expensive, labor-intensive, and time consuming. The goal of this R21 proposal is to evaluate the feasibility of our next-generation, cyclic reversible termination (CRT) sequencing approach by targeting 1,000 candidate genes on high-density oligonucleotide chips. The rationale for selecting the candidate genes is based on those known to be involved in 2-cell development and function, those associated with type 1 or type 2 DM, and those involved in ketogenesis. Recently, we have described a novel paradigm in reversible terminator (RT) chemistry, which has intrinsic advantages over conventional RT's with potential of exceeding current read lengths. We have developed and characterized four, fluorescently-labeled, 3'-unblocked RTs using synthetic templates fastened on standard microscope slides to demonstrate the feasibility of targeted resequencing. Here, we propose development of a 1,000 gene chip utilizing our CRT approach to resequencing genomic DNA from 50 KPD patients. These data will be validated by sampling ~10% of the discovered variants using traditional Sanger sequencing to assess accuracy of the CRT method. The outcome of this research will result in a comprehensive assessment of the CRT technology, while identifying putative gene sequence variations in KPD patients, all of which could lead to an expanded R01 study to evaluate a two-tiered approach of case- control and case-case comparisons (e.g., for the 2- group: A- versus A+, and conversely for the A- group, 2- versus 2+).