Application of high-throughput approaches in the study of complex disorders

高通量方法在复杂疾病研究中的应用

• 批准号: 7594270
• 负责人: Owen M Rennert
• 金额: $ 24.26万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594270
• 关键词: African American; Age related macular degeneration; Age-Years; Angiotensins; Antithrombin III; Apolipoprotein E; Asians; Biological Assay; Biomedical Research; Blindness; CX3CL1 gene; Caucasians; Caucasoid Race; Complex; Developed Countries; Developing Countries; Development; Diagnosis; Disease; Enzymes; Factor V; Fibrinogen; Genes; Genetic Polymorphism; Genetic screening method; Goals; Hispanics; Hospitalization; Incidence; Individual; Inherited; Institutes; Legal patent; Methods; Methylenetetrahydrofolate reductase (NADPH); Morbidity - disease rate; Mutation; Numbers; Oligonucleotide Microarrays; Oligonucleotides; Pacific Island Americans; Pathogenesis; Plasminogen Activator Inhibitor 1; Polymerase Chain Reaction; Population; Predictive Value; Predictive Value of Tests; Predisposition; Process; Prophylactic treatment; Protein C; Prothrombin; Protocols documentation; Reaction; Recurrence; Reporting; Risk; Risk Factors; Screening procedure; Spottings; Stratification; TLR4 gene; Technology; Testing; Thrombophilia; United States; Variant; Venous Thrombosis; aryldialkylphosphatase; base; citrate carrier; density; design; fibulin; human CX3CR1 protein; mortality

Venous thrombosis (VT) is one of the leading causes of mortality and morbidity resulting in approximately 300,000 hospitalizations and 50,000 fatalities per year in the United States with an incidence of 141 per 100 000 African-Americans (1.4 per 1000), 104 per 100 000 Caucasians (1 per 1000), 55/100 000 in Hispanics (0.6per 1000) and 21 per 100 000 Asian/Pacific Islanders (0.2 per 1000). It is, however, an avoidable disease if currently available prophylactic treatment is instituted. Our calculations demonstrated that concurrent use of a panel of 11 genetic tests increases the positive predictive value of testing for venous thrombosis at least 30-fold. We have devised an approach (Method Evolved for Recognition of Thrombophilia MERT, patent pending) that will allow prediction and accurate assessment of hereditary thrombophilia in several ethnic populations by rapid, concurrent screening of an array of all known 145 venous thrombosis-associated recurrent mutations and polymorphisms in nine molecules antithrombin III (AT III), protein C, protein S, fibrinogen, factor V (FV), prothrombin (factor II), methylenetetrahydrofolate reductase (MTHFR), angiotensin 1-converting enzyme (ACE) and plasminogen activator inhibitor-1 (PAI-1) genes . MERT will help us develop stratification protocols for risk-adapted prophylaxis. We have designed 291 oligonucleotide 25mer probes to be spotted onto the microarray and achieved to amplify 40 amplicons covering the variation sequences from nine different genes in a single amplification reaction by Multiplex PCR assay. We are now in the process of verifying the analytic validity of our method. We applied a similar approach to the design of an oligo-array for screening for susceptibility to development of age-related macular degeneration (Method Evolved for Recognition and Testing of Age-Related Macular Degeneration-MERT-ARMD, patent pending). Age-related macular degeneration (ARMD) is the most common cause of severe vision loss in the United States and developed countries among people 65 years of age and older. It has been suggested that ARMD is a multifactorial disorder. Our previous reports described screening for one or more polymorphisms associated with ARMD. In general, the use of these assays is limited because they have a low predictive value and detect mutations prevalent only in Caucasian populations. We have designed a MERT-ARMD that will concurrently screen 105 known age-related macular degeneration-associated mutations and polymorphisms in 16 molecules (CFH, LOC387715, BF, C2, ABCR, Fibulin 5, VMD2, TLR4, CX3CR1, CST3, MnSOD, MEHE, paraoxonase, APOE, ELOVL4 and hemicentin-1 genes), using hybridization-based, high-density oligonucleotide array technology.

静脉血栓 (VT) 是死亡和发病的主要原因之一，在美国每年导致约 300,000 人住院治疗和 50,000 人死亡，发病率为每 100 000 名非裔美国人中 141 例（每 1000 人中 1.4 例），每 10 万人中 104 例。白种人（每 1000 人中有 1 人），西班牙裔中有 55/100 000 人（每 1000 人 0.6 人），每 10 万名亚洲/太平洋岛民中有 21 人（每 1000 人 0.2 人）。然而，如果采取目前可用的预防性治疗，这是一种可以避免的疾病。我们的计算表明，同时使用一组 11 项基因检测可将静脉血栓形成检测的阳性预测值提高至少 30 倍。我们设计了一种方法（用于识别血栓形成倾向的方法进化 MERT，正在申请专利），该方法将通过快速、同时筛查一系列所有已知的 145 种静脉血栓形成相关的复发性突变来预测和准确评估多个种族人群的遗传性血栓形成倾向。九个分子的多态性抗凝血酶 III (AT III)、蛋白 C、蛋白 S、纤维蛋白原、因子 V (FV)、凝血酶原（因子 II）、亚甲基四氢叶酸还原酶 (MTHFR)、血管紧张素 1 转换酶 (ACE) 和纤溶酶原激活剂抑制剂 1 (PAI-1) 基因。 MERT 将帮助我们制定风险适应预防的分层方案。我们设计了 291 个寡核苷酸 25mer 探针，将其点样到微阵列上，并通过多重 PCR 测定在单次扩增反应中扩增出 40 个扩增子，涵盖来自 9 个不同基因的变异序列。我们现在正在验证我们方法的分析有效性。 我们应用了类似的方法来设计寡核苷酸阵列，用于筛选对年龄相关性黄斑变性发展的易感性（年龄相关性黄斑变性的识别和测试的进化方法-MERT-ARMD，正在申请专利）。年龄相关性黄斑变性 (ARMD) 是美国和发达国家 65 岁及以上人群严重视力丧失的最常见原因。有人认为 ARMD 是一种多因素疾病。我们之前的报告描述了筛选与 ARMD 相关的一种或多种多态性。一般来说，这些测定的使用受到限制，因为它们的预测价值较低并且检测仅在白种人群体中普遍存在的突变。我们设计了一个 MERT-ARMD，它将同时筛选 16 个分子（CFH、LOC387715、BF、C2、ABCR、Fibulin 5、VMD2、TLR4、CX3CR1、CST3、MnSOD、 MEHE、对氧磷酶、APOE、ELOVL4 和 hemicentin-1 基因），使用基于杂交的高密度寡核苷酸阵列技术。