A novel G/A and the 4G/5G polymorphism within the promoter of the plasminogen activator inhibitor-1 gene in patients with deep vein thrombosis.

Plasma plasminogen activator inhibitor-1 (PAI-1) level was observed to be associated with sequence variations at the PAI-1 locus. Therefore, PAI-1 gene promoter was screened for possibly new polymorphisms and to investigate the contribution of these sequence variations to PAI-1 levels in patients with deep vein thrombosis (DVT). DNA was isolated from blood of 83 consecutive unrelated patients (42 +/- 11 years old) and from 50 apparently healthy subjects of similar age and gender distribution. Six fragments covering DNA sequence- 1523 base pairs (bp) upstream from the start of PAI-1 gene transcription to +90 bp in the first exon, were amplified by polymerase chain reaction and analyzed by single-strand conformation polymorphisms. Two polymorphisms were found: a previously described 4G/5G deletion/insertion polymorphism -675bp upstream from the start of transcription and a novel G/A single base substitution polymorphism further upstream at -844 bp. The two polymorphisms were in strong linkage disequilibrium. Significant differences between patients and controls were observed neither for the frequencies of the 4G/5G alleles (0.60/0.40 and 0.59/0.41, respectively) nor for the frequencies of the G/A alleles (0.33/0.67 and 0.41/0.59, respectively). The distribution of both polymorphisms was similar in idiopathic and secondary DVT as well as in first and recurrent DVT. In patients association between the 4G/5G genotypes and PAI activity was observed, with the highest values in the 4G/4G genotype (13.3 U/mL), median values in the 4G/5G genotype (9.8 U/mL) and the lowest values in the 5G/5G genotype (2.0 U/mL). Despite the lack of association between the G/A genotypes and plasma PAI-1 levels, electrophoretic mobility shift assay showed specific binding of a nuclear protein from human vascular endothelial cells extracts to both the G and the A variant, suggesting functional importance of this novel G/A polymorphism in regulating the expression of PAI-1 gene.