Frequency analysis of the g.7081T>G/A and g.10872T>G polymorphisms in the FCGR3A gene (CD16A) using nested PCR and their functional specific effects.

Abstract

Polymorphic variants p.66L>R/H (g.7081T>G/A; rs10127939) and p.176F>V (g.10872T>G; rs396991) in FCGR3A (CD16A) have been associated with defects in cytotoxic function of natural killer (NK) cells in humans. Genotyping of these variants in genomic DNA has been ambiguous because of high degree of homology between FCGR3A and FCGR3B. We designed a strategy to genotype these polymorphisms and to evaluate their effects on NK cells' cytotoxic activity. One hundred and fifteen individuals from different geographical regions of Colombia were included. Specific primers were designed to amplify FCGR3A exons 4 and 5 encompassing g.7081T>G/A and g.10872T>G by long-range and nested polymerase chain reaction and sequencing. The binding of different monoclonal antibodies to CD16A and NK antibody-dependent cellular cytotoxicity (ADCC) were evaluated. We demonstrate that amplifying and sequencing FCGR3A allows genotyping of g.7081T>G/A and g.10872T>G without interference from FCGR3B. Allele frequencies in our population were as follows: 7081T = 0.895, 7081G = 0.065, 7081 A = 0.039, 10872T = 0.673, and 10872G = 0.326. We also observed linkage disequilibrium between variants 7081T and 10872G. Interestingly, 176FF variant affected the reactivity of MEM154 monoclonal antibody against CD16A, but it did not affect ADCC. Our studies aimed to determine whether clinical association exists between these polymorphisms and NK cell function defects in patients with compatible phenotypes.