Development of a real-time PCR detection method for a FCGR2A polymorphism in the LightCycler and application in the heparin-induced thrombocytopenia syndrome

• Published in: Clinical biochemistry 2009-11, Vol.42 (16), p.1685-1693
• Main Authors: Kroupis, Christos, Theodorou, Maria, Kounavi, Maria, Oliveira, Samantha C., Iliopoulou, Eugenia, Mavri-Vavayanni, Mary, Melissari, Efthimia N., Degiannis, Dimitrios
• Format: Article
• Language: English
• Subjects: Base Sequence; CD62P-selectin; DNA; FCGR2A; Flow cytometry; Genotype; Heparin; Heparin - adverse effects; HIT; Humans; LightCycler; Melting curve analysis; Molecular Sequence Data; Platelet microparticles; Polymerase Chain Reaction - methods; Polymorphism; Polymorphism, Genetic; Real-time PCR; Receptors, IgG - genetics; Thrombocytopenia - chemically induced; Thrombocytopenia - genetics
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2009.07.026

The FcγRIIa receptor is responsible for the activation of platelets by antibodies in heparin-induced thrombocytopenia (HIT). The c.497G > A polymorphism in the corresponding FCG2RA gene (H131R) has been implicated in the HIT syndrome and we aimed at its rapid and reliable determination. We designed a novel asymmetric real-time PCR method in the LightCycler that uses two hybridization probes and is followed by melting curve analysis. Seventy-one post-cardiac-surgery HIT Greek patients well ascertained by clinical data, immunological and functional tests (PAT, CD62P-selectin and microparticle flow cytometric detection) were studied, along with a clinically relevant group of 49 thrombocytopenic control patients and 119 healthy subjects. The developed method has excellent analytical characteristics (linear and efficient amplification, precision), has wide Δ T m between the two alleles H and R (11.53 °C), and is in 100% concordance with validated controls and another commonly used screening method. The RR percentage increased from 10% in the control populations to 24% in the HIT patient group. The described method is technically simple, robust, fast, and accurate. A statistically significant difference was found in the comparison between the groups of HIT patients and healthy subjects [RR vs. RH+ HH, χ 2 test, p = 0.01, OR (95% C.I.) 2.81 (1.21–4.68)]. The RR frequency in the Greek population was found to be the lowest among Caucasians.