Targeted CRT sequencing of 1000 genes in KPD patients

KPD 患者 1000 个基因的靶向 CRT 测序

• 批准号: 8097662
• 负责人: Michael L. Metzker
• 金额: $ 5.13万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097662
• 关键词: Autoimmune Process; Benchmarking; Candidate Disease Gene; Cells; Chemistry; Classification; Clinic; Clinical; Collection; Complex; DNA; DNA Resequencing; Data; Data Quality; Development; Diabetes Mellitus; Dideoxy Chain Termination DNA Sequencing; Disease; Environmental Risk Factor; Frequencies; Functional disorder; Gene Chips; Genes; Genetic; Genomics; Goals; Insulin-Dependent Diabetes Mellitus; Label; Lead; Length; Methods; Microscope; Mitochondria; Natural regeneration; Non-Insulin-Dependent Diabetes Mellitus; Oligonucleotides; Outcome; Outcomes Research; Pancreas; Patients; Phenotype; Reading; Research; Role; Sampling; Severities; Slide; Source; Spottings; Subgroup; Syndrome; System; Technology; Time; Variant; base; case control; density; design and construction; interest; ketogenesis; lipid metabolism; next generation; novel

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+).

描述（由申请人提供）：糖尿病（DM）是一种复杂的疾病，其遗传和环境因素的结合有助于其发作，进展和严重性。尽管缺乏自身免疫1型糖尿病的典型特征，但DM患者的一部分容易发生酮症酸中毒。这些容易发生酮症糖尿病（KPD）患者的表型强烈暗示胰腺2细胞功能障碍的新机制是病因学因素。最近，我们的小组在400多名患者中确定了四种不同的KPD表型，随后在一个纵向研究诊所中。该分配基于A2分类系统，该系统定义了具有高精度和良好预测的KPD患者，以进行长期临床结果。四个KPD亚组为A+2-，A-2-，A+2+和A-2+，频率分布分别为20％，20％，10％和50％。序列变异在四组表型差异的基因中的作用在很大程度上是未知的，并且假定涉及2细胞发育，功能，扩张和再生以及与2细胞线粒体和脂质代谢相关的遗传因素。鉴于KPD综合征的潜在病因学因素的复杂性，每个KPD患者中多达1,000个基因可能需要重新方程，以系统地确定因果因素。当前在此规模上重新陈述候选基因的Sanger方法太昂贵，劳动力且耗时。该R21提案的目的是通过针对高密度寡核苷酸芯片的1,000个候选基因来评估我们下一代环状可逆终止（CRT）测序方法的可行性。选择候选基因的理由是基于已知参与2细胞发育和功能的基因，与1型或2型DM相关的基因以及涉及酮症的基因。最近，我们描述了可逆终结剂（RT）化学中的一种新型范式，该化学具有比常规RT具有固有的优势，具有超过电流读取长度的潜力。我们已经使用在标准显微镜载玻片上固定的合成模板开发并表征了四个，荧光标记的3'-挡板RT，以证明目标重新方程的可行性。在这里，我们提出了使用我们的CRT方法来开发1,000个基因芯片，以重塑50 kpd患者的基因组DNA。这些数据将通过使用传统的Sanger测序来评估CRT方法的准确性来对发现约10％的发现变体进行验证。这项研究的结果将导致对CRT技术的全面评估，同时确定KPD患者的假定基因序列变化，所有这些都可能导致R01的扩展研究，以评估病例控制和病例 - 病例的两层方法案例比较（例如，对于2组：A-与A+，相反，对于A-组，2- vess 2+）。