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

静脉血栓形成（VT）是导致死亡率和发病率的主要原因之一，在美国每年导致约30万人住院和5万人死亡，发病率为每10万非洲裔美国人141例（每1000人1.4例），每10万高加索人104例（每1000人1例），西班牙裔55例（每1000人0.6例），亚洲/太平洋岛民21例（每1000人0.2例）。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11项基因检测可将静脉血栓检测的阳性预测值提高至少30倍。我们设计了一种方法（Method Evolved for Recognition of Thrombophilia MERT，专利申请中），通过快速、同步筛选所有已知的145种静脉血栓形成相关的复发突变和多态性，预测和准确评估几个民族人群的遗传性血栓形成，包括抗凝血酶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）和纤溶酶原激活物抑制剂-1 （PAI-1）基因。MERT将帮助我们制定适应风险的分层预防方案。我们设计了291个寡核苷酸25mer探针，将其定位到微阵列上，并通过多重PCR试验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方法的分析有效性。

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.