ALLELE-SPECIFIC PCR FOR DETECTION OF A SEQUENCE POLYMORPHISM IN THE PROMOTER REGION OF THE PLASMINOGEN-ACTIVATOR INHIBITOR-1 (PAI-1) GENE

We have elaborated a rapid and reliable allele specific polymerase chain reaction (PCR) method to determine a polymorphism located in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene (Dawson et al, J Biol Chem 1993; 268: 10739-10745), This polymorphism, located 675 basepair (bp) upstream of the transcriptional start site, consists of a single guanosine insertion/deletion variation, resulting in two alleles containing either 4 or 5 guanosines in a row, The individuals homozygous for the 4G allele have significantly higher PAI-1 concentrations in plasma. The method utilizes two allele specific primers in combination with a third downstream primer and a fourth primer located upstream of the polymorphic region to give a positive control in the PCR reaction, On some subjects, either heterozygotes or homozygous for the 4G or the 5G allele, a comparison with allele specific oligonucleotide hybridizations or DNA sequencing have been performed with complete agreement between the different methods. The newly developed PCR method is very useful in screening for this polymorphism in larger population studies, Using this method we have genotyped 308 healthy blood donors of different age and sex, The distribution of the genotypes in this material was determined as: 87 with 4G/4G (28.2%), 148 with 4G/5G (48.1%) and 73 with 5G/5G (23.7%), If the individuals were divided in two halves according to age, the younger half (19-51 years of age, n = 150) had a significantly higher frequency of the 4G/4G genotype as compared to the older group (52-71 years of age, n = 158). No significant difference was observed between sexes.