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.

Updates on the surface antigens of basophils: CD16 on basophils of patients with respiratory or insect venom allergy and the rejection of CD203c and CD63 externalization decoupling by bisindolylmaleimides

“…It is possible that there may be differences at the individual level as there are indices that the presence of certain CD16a polymorphisms may increase CD16a expression in NK cells. 29,30 Nevertheless, among the six patients and control subjects tested in the present study, we did not see any that would express CD16b as a dominant form of surface-bound CD16 (Table S4). We reproduced these unexpected findings at the mRNA level, where we confirmed by Sanger sequencing of nested RT-PCR products that CD16a is the dominant form of CD16 in basophils although we also detected low expression of CD16b ( Figure 5).…”

Polymorphisms in Fc Gamma Receptors and Susceptibility to Malaria in an Endemic Population

“…This observation has led to the speculation that both SNPs should lead to increased levels of susceptibility to different infections and autoimmune diseases (109,112,113). However, in a study involving 115 Colombians, neither of these SNPs showed any association with the phenomenon of antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells (111). However, the authors of this study found a linkage disequilibrium between the SNP L66R/H and the SNP V176F in the FCGR3A gene.…”

“…Another important polymorphism in the FCGR3A gene is the rs10127939 SNP. This SNP results from the substitution of a T to either a G or A, thus replacing leucine by arginine or histidine at amino acid position 66 in the extracellular domain (111,112). Some authors have shown the SNP L66R/H and the SNP V176F to be associated with a reduced cytotoxic response and IgG binding capacity in the NK cells of patients experiencing recurrent infections with Herpes simplex virus, Epstein-Barr virus or Varicella zoster (111).…”

“…This SNP results from the substitution of a T to either a G or A, thus replacing leucine by arginine or histidine at amino acid position 66 in the extracellular domain (111,112). Some authors have shown the SNP L66R/H and the SNP V176F to be associated with a reduced cytotoxic response and IgG binding capacity in the NK cells of patients experiencing recurrent infections with Herpes simplex virus, Epstein-Barr virus or Varicella zoster (111). This observation has led to the speculation that both SNPs should lead to increased levels of susceptibility to different infections and autoimmune diseases (109,112,113).…”

“…However, the authors of this study found a linkage disequilibrium between the SNP L66R/H and the SNP V176F in the FCGR3A gene. Furthermore, the 176FF variant appears to result in alterations to the epitope that would normally interact with IgG1 antibodies (111). Further studies should be undertaken to specifically investigate the effect of these SNPs in a population that is endemic for malaria, as no previous research has been attempted in such a population.…”