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[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.