Quantitative determination of Fcgamma receptor genes by means of fluorescence-based real-time polymerase chain reaction.

The granulocyte antigens HNA-1a, -1b and -1c reside on the FcgammaIIIb receptor, which is exclusively expressed on neutrophils. They are involved in autoimmune and alloimmune neutropenias as well as in severe transfusion reactions. Recent family studies show the HNA-1c antigen to be inheritably linked to HNA-1a, resulting in individuals carrying three genes. Using sequence-specific primers, the HNA antigens can be discerned in a polymerase chain reaction (PCR), but not the number of coding FCGR3B genes present in each individual. Therefore a real-time kinetic PCR method using the LightCycler technique was established to ascertain the amount of FCGR3B genes in each individual. For this purpose, the FCGR3B genes of four HNA-1a,-1b,-1c (+), one HNA-1b,1c (+) and one HNA-1b (+) individual were quantified. In addition, two families, in which the mother was FCGR3B deficient, were analyzed. Quantification showed two of the four HNA-1a,-1b and-1c (+) individuals to have three genes and the other two to have only two genes. The HNA-1b,-1c (+) individual had also only two genes. As expected, the HNA-1b donor was typed for two genes. No FCGR3B genes could be detected in the FCGR3B-deficient mothers of either family; their husbands however, carried two FCGR3B genes. Accordingly, quantitative PCR showed the offspring of both families to have only one FCGR3B gene. Quantitative PCR with the Light Cycler has been revealed to be a fast and reliable method for the determination of FCGR3B genes present. FCGR3B quantification confirms the idea of the HNA-1c antigen to be inherited not only linked to HNA-1a, but also to be passed down on its own.