Appliance of the DNA-Micro Array Technique for the Identification of Patients with Thrombophilic Diathesis. I-Comparison to the Classic PCR Analysis.

Introduction: DNA Micro array have the ability to test a large number of samples for a large number of mutations on the same time, something practically impossible with the traditional hybridization methods. The goal of this study is to compare the ability of DNA micro array application to recognize genetic predisposition to thrombosis, with the one of RLF-PCR.

Material: DNA samples from 37 subjects (24 male with mean age 41.9 and 13 female with mean age 38.8) were studied both with micro arrays and PCR analysis. All of these subjects had a history of either recurrent thrombosis or thrombosis at unusual sites (e.g. retina’s central vein thrombosis).

Method: The thrombo-check microarray contains the following mutations and polymorphisms: Factor V Leiden (FV G1691A), FII G20210A (prothrombin mutation), FV Cambridge (FV G1091C), MTHFR C677T, MTHFR A1298C, CBS 844ins68, Plasminogen Activator Inhibitor (PAI 1 4G/5G). The thrombo-check microarray can distinguish between the normal sequence, as well as heterozygosity and homozygosity for the mutated sequence. To ensure the result is correct specific probes are included on the microarray to control for the RLF-PCR reaction, the hybridization reaction and the silver staining procedure. In addition, the correct functioning of the entire reaction can be checked using an external control DNA.

Results: As long as FVL and FII are concerned, in 35 out of 37 patients (pts) both methods gave similar results. However, the results of DNA analysis were different for the other two patients. The first one was found to be, using the micro array DNA method, a double heterozygous carrier of both FVL and FII mutations, while RLF-PCR did not relieve any such genetic defects. The other patient was found to be, using the RLF-PCR a homozygous carrier for the prothrombin mutation, while micro array also revealed heterozygous mutation of FV Leiden. Moreover, the micro array method detected totally 1 patient with FVC, 4 pts homozygous for MTHFR1, 6 pts homozygous for PAI and 4 pts homozygous for MTHFR2 mutation. Further more, micro array detected the co-existence of FVL and homozygocity in a) FII mutation (2 pts), b) PAI mutation (1patient) and c) MTHFR2 (2pts). Finally, 1 patient with FII was found to be also homozygous for MTHFR1 mutation.