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 cha in reaction (PCR) method to determine a polymorphism located in the pr omoter region of the plasminogen activator inhibitor-1 (PAI-1) gene (D awson et al, J Biol Chem 1993; 268: 10739-10745), This polymorphism, l ocated 675 basepair (bp) upstream of the transcriptional start site, c onsists of a single guanosine insertion/deletion variation, resulting in two alleles containing either 4 or 5 guanosines in a row, The indiv iduals homozygous for the 4G allele have significantly higher PAI-1 co ncentrations in plasma. The method utilizes two allele specific primer s in combination with a third downstream primer and a fourth primer lo cated upstream of the polymorphic region to give a positive control in the PCR reaction, On some subjects, either heterozygotes or homozygou s for the 4G or the 5G allele, a comparison with allele specific oligo nucleotide hybridizations or DNA sequencing have been performed with c omplete agreement between the different methods. The newly developed P CR method is very useful in screening for this polymorphism in larger population studies, Using this method we have genotyped 308 healthy bl ood 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 4 G/5G (48.1%) and 73 with 5G/5G (23.7%), If the individuals were divide d 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 signi ficant difference was observed between sexes.