A rapid detection method for PAI-1 promoter insertion/deletion polymorphism (4G/5G)

METHODOLOGY

A rapid detection method for PAI-1 promoter insertion/deletion polymorphism (4G/5G)

Joyce M. Annichino-Bizzacchi, Luciana Pugliese and Valder R. Arruda

Centro de Hematologia-Hemoterapia, Universidade Estadual de Campinas, Caixa Postal 6198, 13081-970 Campinas, SP, Brasil. Fax: [phone]. Send correspondence to J.M.A.-B.

ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is an important inhibitor of fibrinolysis, and increased levels of PAI-1 are associated with atheroma and myocardial infarction. A common 4G/5G insertion/deletion polymorphism located in the promoter region of PAI-1 gene has been described associated with PAI-1 activity in plasma levels. Genotyping of this polymorphism is commonly conducted with an allele-specific oligonucleotide melting technique. In the present study, we describe a quick, easy method for genotyping 4G/5G polymorphism in the promoter region of the PAI-1 gene.

INTRODUCTION

Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of fibrinolysis, and increased levels of PAI-1 are associated with coronary atheroma and myocardial infarction (Sprengers and Kluft, 1987). Prospective studies (Meade et al., 1993; Ridker et al., 1994; Hamsten et al., 1987) described a relationship between fibrinolysis and development of vascular diseases. Regulation of PAI-1 expression in vivo is complex. There are intra-individual and circadian variations in PAI-1 levels, which behaves as an acute phase reactant, increasing plasma levels after inflammation and trauma, as well as after myocardial infarction. An important issue is whether increased PAI-1 levels verified in these patients are a consequence of coronary disease or a pre-existing risk factor. The influence of genetic factors on PAI-1 levels cannot be excluded. This molecular characterization is interesting, since PAI-1 genotype could be useful in recognizing high-risk individuals before a cardiovascular event.

A common 4G/5G single nucleotide insertion/deletion polymorphism in the PAI-1 promoter region has been identified 675-bp upstream from the start of transcription (Dawson et al., 1993), and was associated with plasma PAI-1 activity (Dawson et al., 1991). Individuals with the 4G allele have higher plasma levels of PAI-1 than those with 5G allele.

Genotyping for this polymorphism is commonly conducted with an allele-specific oligonucleotide melting technique. Here we describe a quick and easy method for genotyping 4G/5G polymorphism in the promoter region of the PAI-1 gene.

MATERIAL AND METHODS

This new method was based on PCR technique followed by non-radioactive single strand conformation polymorphism (SSCP) analysis.

PCR was performed on DNA extracted by standard method, using primers previously described (Hamsten et al., 1987). An 890-bp amplified fragment was submitted to non-radioactive SSCP. In the present study, SSCP analysis was performed with PhastSystem apparatus (Pharmacia, Uppsala, Sweden). The PCR product was diluted 1:8 with loading buffer (95% formamide containing 10 mmol/l EDTA, 0.1% bromophenol blue), heated at 94°C for 5 min and electrophoresed on a premade 20% homogeneous polyacrylamide gel with strips of agarose as buffer. Running conditions were 400 V for 3 h at 15°C. DNA bands were visualized by automated silver staining (Figure 1) involving consecutive washes in 20% trichloroacetic acid (5 min at 20°C), 5% glutaraldehyde (5 min at 50°C), water (2 washes of 2 min at 50°C), 0.4% AgNO₃ (8 min at 40°C), water (2 washes of 30 s each at 30°C), 2.5% Na₂CO₃ 0.013 formaldehyde (4 min at 30°C), and 5% acetic acid (2 min at 50°C).

Figure 1
- SSCP pattern of 4G/5G polymorphism performed by PhastSystem.

Three patterns were visualized, and the PCR products of each one were sequenced to identify the 4G and 5G alleles (Figure 2).

Figure 2
- Direct sequencing autoradiograph of 4G and 5G alleles.

RESULTS AND DISCUSSION

This method allowed us to determine the genotype of all samples tested, with no doubt about whether the individual was heterozygous or homozygous. This is a reproducible, reliable method that can be used in laboratories with PhastSystem equipment, since it is very easy to perform. Therefore, we believe that this method is more simple than that previously described (Hamsten et al., 1987), and facilitates determination of 4G/5G polymorphism, principally if a large number of samples have to be analyzed, like in population studies. This analysis could become more important if the PAI-1 genotype is confirmed to be a risk factor for myocardial infarction.

ACKNOWLEDGMENTS

Research and publication supported by FAPESP.

RESUMO

O inibidor do ativador do plasminogênio-1 (PAI-1) é o inibidor principal da fibrinólise. Estudos populacionais prospectivos e retrospectivos demonstraram que níveis plasmáticos elevados de atividade do PAI-1 são um fator de risco para doença coronariana isquêmica. Recentemente descreveu-se um polimorfismo de inserção/deleção (4G/5G), localizado na região promotora do gene do PAI-1, relacionado aos níveis plasmáticos do PAI-1. A presença do alelo 4G está associada a níveis elevados de atividade do PAI-1. A metodologia comumente empregada para a determinação desse polimorfismo é a amplificação do DNA pela PCR, utilizando-se oligonucleotídeos alelo-específicos. Esta técnica demanda tempo e em análises populacionais, que geralmente avaliam um elevado número de amostras, técnicas otimizadas seriam de grande interesse. Neste artigo descrevemos a determinação deste polimorfismo pela técnica de PCR e SSCP, utilizando o aparelho PhastSystem. Este método mostrou-se uma alternativa ao método anterior, de fácil realização e com resultados reprodutíveis.

(Received October 20, 1997)

References

• Dawson, S., Hamsten, A., Wiman, B., Henney, A. and Humphries, S. (1991). Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasma plasminogen activator inhibitor-1 activity. Arterioscler. Thromb. 11: 183-190.
• Dawson, S., Wiman, B., Hamsten, A., Green, F., Humphries, S. and Henney, A. (1993). The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in hepG2 cells. J. Biol. Chem. 268: 10739-10743.
• Hamsten, A., de Faire, U., Waldius, G., Dahlén, G., Szamosi, A., Landou, C., Blomback, M. and Wiman, B. (1987). Plasminogen activator inhibitor in plasma: risk for recurrent myocardial infarction. Lancet 2: 3-9.
• Meade, T.W., Ruddock, V., Stirling, Y., Chakrabarti, R. and Miller, G.J. (1993). Fibrinolytic activity, clotting factors and long-term incidence of ischaemic heart disease in the Northwick Park Heart Study. Lancet 342: 1076-1079.
• Ridker, P.M., Hennekens, C.H., Stampfer, M.J., Manson, J. and Vaughan, D.E. (1994). Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet 343: 940-943.
• Sprengers, E.D. and Kluft, C. (1987). Plasminogen activator inhibitors. Blood 69: 381-387.

Publication Dates

• Publication in this collection
  23 Feb 1999
• Date of issue
  Sept 1998

History

• Received
  20 Oct 1997