[The application of DNA sequencing in studying haplotypes of PAI-1 gene in patients with coronary artery disease].

OBJECTIVE: Using simple and efficient DNA sequencing method to determine the haplotypes derived from 4G/5G and G/A polymorphisms in promoter of plasminogen activator inhibitor-1 (PAI-1) gene and trying to associate those haplotypes with coronary artery disease(CAD).
METHODS: The frequencies and genotype distribution of the two PAI-1 gene variants in 93 CAD patients and 145 control subjects were determined via denaturing high-performance liquid chromatography (DHPLC) method. The heterozygotes for both polymorphism sites were directly sequenced to define the haplotypes based on the principle that heterozygotes of 4G/5G genotype were one guanine deletion and therefore one base shift from this site in sequencing graph results in particular wave pattern on -844G/A site.
RESULTS: Only A-4G and G-5G haplotypes were detected in carriers heterozygous for both -6754G/5G and -844G/A polymorphisms sites though there are G-4G and A-5G haplotypes in carriers homozygous for both sites. No significant differences in haplotypes distribution were seen between CAD patients and control subjects.
CONCLUSION: The DNA sequencing is objective and correct in determining haplotypes derived from two polymorphisms with distance less than about 400 bp apart and with one insertion/deletion polymorphism. And the haplotypes derived from -675 4G/5G and -844G/A polymorphisms are not associated with pathogenosis of CAD.