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


METHODS
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.


RESULTS
The developed method has excellent analytical characteristics (linear and efficient amplification, precision), has wide DeltaT(m) between the two alleles H and R (11.53 degrees 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.


CONCLUSIONS
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, chi(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.
Kroupis
Christos
C
Department of Clinical Biochemistry, Attikon University Hospital, University of Athens Medical School, 1 Rimini Street, Haidari, Greece. ckroupis@med.uoa.gr
Theodorou
Maria
M
Kounavi
Maria
M
Oliveira
Samantha C
SC
Iliopoulou
Eugenia
E
Mavri-Vavayanni
Mary
M
Melissari
Efthimia N
EN
Degiannis
Dimitrios
D
D001483:Base Sequence; D004247:DNA; D005838:Genotype; D006493:Heparin / Q000009:adverse effects*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D017452:Receptors, IgG / Q000235:genetics*; D013921:Thrombocytopenia / Q000139:chemically induced* / Q000235:genetics
C445323:FCGR2A protein, human; D017452:Receptors, IgG; D006493:Heparin; D004247:DNA