A novel exonuclease (TaqMan) assay for rapid haptoglobin genotyping.

BACKGROUND: Haptoglobin is an acute-phase binding protein that scavenges free hemoglobin. The human haptoglobin gene (HP) is polymorphic with two main alleles, haptoglobin allele 1 (Hp1) and haptoglobin allele 2 (Hp2). The smaller Hp1 allele features no duplication and consists of four exons, whereas the larger Hp2 allele, containing a 1.7 kb duplication, consists of six exons, with the fifth and sixth being highly homologous to exons 3 and 4 of Hp1. METHODS: We designed an exonuclease (TaqMan) assay targeting single nucleotide differences between the homologous regions of Hp1 and Hp2. The assay contained one probe specifically binding to a site in intron 4 of Hp2, and another probe binding equally to intron 4 of Hp1 and intron 6 of Hp2. RESULTS: Measurement of post-PCR fluorescence allowed unambiguous discrimination of HP genotypes. Comparison with genotypes obtained by a method based upon allele-specific primers yielded fully corresponding results. CONCLUSIONS: The new HP genotyping method is fast, reliable, does not require real-time instruments and may be especially useful for high-throughput genotyping.

The fibrinogen gamma (FGG) 10034C>T polymorphism is associated with venous thrombosis.

INTRODUCTION: Thrombin-induced conversion of fibrinogen to fibrin plays an essential role in hemostasis and results in the stabilization of thrombi. Elevated plasma fibrinogen levels have been associated with both increased plasma viscosity and platelet aggregability. Recently, a haplotype-tagging single nucleotide polymorphism characterized by a C to T substitution at nucleotide 10034 of the fibrinogen gamma gene (FGG 10034C>T, rs2066865), has been proposed as a novel risk factor for deep venous thrombosis (DVT). Aim of the present study was to provide further data on the role of the FGG 10034C>T polymorphism for DVT. MATERIALS AND METHODS: FGG genotypes were determined by 5'-exonuclease assay (TaqMan) in 358 patients with documented DVT and a total of 783 control subjects. RESULTS: In a multivariate analysis adjusting for age, sex, presence of factor V Leiden and carriage of prothrombin 20210A, homozygosity for the FGG 10034 TT genotype yielded an odds ratio of 2.01 (95% CI 1.23-3.31; p=0.006) for DVT. CONCLUSIONS: Our data confirm the primary finding that the FGG 10034C>T polymorphism is associated with DVT risk.