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

静脉血栓形成（VT）是导致死亡和发病的主要原因之一，在美国每年约有300，000例住院治疗和50，000例死亡，非洲裔美国人的发病率为141/100，000（1.4/1000）、104/100000（1/1000）、55/100000（0.6/1000）和21/100000（0.2/1000）。然而，如果采用现有的预防性治疗，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个基因检测增加了静脉血栓形成检测的阳性预测值至少30倍。我们设计了一种方法（Method Evolved for Recognition of Thrombophilia MERT，patent pending），其将允许通过快速、同时筛选9种分子抗凝血酶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转化酶（ACE）和纤溶酶原激活物抑制剂-1（派-1）基因。MERT将帮助我们制定风险适应性预防的分层方案。我们设计了291个寡核苷酸25聚体探针点样到微阵列上，通过多重PCR方法在一次扩增反应中扩增出40个扩增子，覆盖了9个不同基因的变异序列。我们现在正在验证我们的方法的分析有效性。 我们将类似的方法应用于设计用于筛选对年龄相关性黄斑变性发展的易感性的寡核苷酸阵列（用于识别和测试年龄相关性黄斑变性的方法演进-MERT-ARMD，专利申请中）。视网膜相关性黄斑变性（ARMD）是美国和发达国家65岁及以上人群中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。我们以前的报告描述了筛查一个或多个与ARMD相关的多态性。一般来说，这些检测方法的使用是有限的，因为它们具有较低的预测价值，并且仅检测高加索人群中流行的突变。我们设计了一种MERT-ARMD，使用基于杂交的高密度寡核苷酸阵列技术，同时筛查16种分子（CFH、LOC 387715、BF、C2、ABCR、Fibulin 5、VMD 2、TLR 4、CX 3CR 1、CST 3、MnSOD、MEHE、对氧磷酶、APOE、APOVL 4和hemicentin-1基因）中105种已知的年龄相关性黄斑变性相关突变和多态性。

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.