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
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
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DOI：
10.1038/s41435-017-0001-0
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发表时间：
2019-01-01
影响因子：
5
通讯作者：
Milena Trujillo-Vargas, Claudia
中科院分区：
文献类型：
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作者：
Andres Perez-Romero, Camilo;Pilar Sanchez, Isaura;Milena Trujillo-Vargas, Claudia
FCGR3A(CD16A)基因的多态变异p.66L>；R/H(g.7081T>；G/A；rs10127939)和p.176F>；V(g.10872T>；G；rs396991)与人类NK细胞的细胞毒功能缺陷有关。由于FCGR3A和FCGR3B之间的高度同源性，基因组DNA中这些变异体的基因分型一直不明确。我们设计了一种策略来对这些多态进行基因分型，并评估它们对NK细胞细胞毒活性的影响。来自哥伦比亚不同地理区域的115人参加了调查。通过长程套式聚合酶链式反应和测序，设计了FCGR3A外显子4和5的特异性引物，扩增出g.7081T>；G/A和g.10872T>；G。检测不同单抗与CD16A的结合及NK抗体依赖的细胞毒作用(ADCC)。我们证明，扩增和测序FCGR3A可以在不受FCGR3B干扰的情况下对g.7081T>；G/A和g.10872T>；G进行基因分型。等位基因频率分别为：7081T=0.895,7081G=0.065,7081A=0.039,10872T=0.673,10872G=0.326。我们还观察到变异体7081T和10872G之间的连锁不平衡。有趣的是，176FF变异影响了抗CD16A的MEM154单抗的反应性，但不影响ADCC。我们的研究旨在确定在表型相容的患者中，这些基因多态与NK细胞功能缺陷之间是否存在临床关联。
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.