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

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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.