Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells

Abstract

Natural Killer cells (NK cells) are one of the main effector immune cells involved in Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). Upon recognition of cell bound IgG antibodies, which occurs through Fc gamma receptors (FCGRs) expressed on the cell surface of NK cells, NK cells become activated and lyse target tumor or infected cells. The FCGRs, FCGR3A and FCGR2C, expressed on the surface of NK cells have single nucleotide polymorphisms (SNPs) that result in differential activity of NK cells. In addition to SNP genetic variation within each of these genes, the FCGRs are subject to copy number variation (CNV), which leads to variable protein expression levels on the cell surface. Studies have found that FCGR genotype for FCGR3A and FCGR2C is associated with variation in the response to immunotherapy. Due to high sequence homology within FCGR3 and FCGR2 families, there are difficulties associated with genotyping these specific receptors related to cross-amplification of non-targeted FCGRs. To improve specificity for both FCGR3A and FCGR2C, Rnase-H primers (RH primers) were designed to amplify specifically FCGR3A (while not co-amplifying FCGR3B) and FCGR2C (while not co-amplifying FCGR2B). In addition, fluorescently labeled Locked Nucleic Acid (LNA) probes provide additional precision for determination of the SNPs within both FCGR3A and FCGR2C. For CNV determination, separate fluorescently labeled probes for FCGR3A, and for FCGR2C, can be used with the same RH primers for each gene. These probes can be combined in the same well with control primers/probe for a known diploid gene and used to calculate the copy number of both FCGR3A and FCGR2C. Here we provide new detailed methodology that allows for the specific amplification of these FCGRs in a single PCR reaction, allowing for genotyping of both the SNPs and CNVs using Real Time PCR.

An Engineered Human Fc variant With Exquisite Selectivity for FcγRIIIaV158 Reveals That Ligation of FcγRIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages

“…As previously described (46, 47), FCGR3A genes were specifically amplified using the following primers, 5′-TCC AAA AGC CAC ACT CAA AGA CAG CGC-3′ and 5′-GAT GGT GAT GTT CAC AGT CTC T-3′ and the sequence of amplified FCGR3A was analyzed. FCGR3A SNP was also confirmed by nested PCR using following primer sets; 5′-TCC AAA AGC CAC ACT CAA AGA CAG CGC-3′ and 5′-CTC TGA AGA CAC ATT TTT ACT CCC AAA-3′ for FCGR3A F158, and 5′-TCC AAA AGC CAC ACT CAA AGA CAG CGC-3′ and CTC TGA AGA CAC ATT TTT ACT CCC AAC for FCGR3A V158.…”

FCGR Polymorphisms Influence Response to IL2 in Metastatic Renal Cell Carcinoma

“…For genotyping the FCGR3A SNP, Rnase H primers were developed to specifically amplify FCGR3A while not co-amplifying FCGR3B . These primers were paired with specific probes to determine the SNP (33). For genotyping the FCGR2C -C/T SNP, Rnase H primers were developed to specifically amplify this gene while not co-amplifying FCGR2B .…”

“…Primers and probes for both FCGR3A and FCGR2C were designed through Integrated DNA Technologies (IDTDNA, Coralville, Iowa). Specific method details can be found in Erbe AK et al, 2016 (33). Genotyping was conducted in a blinded manner, where those individuals that determined the genotype of the patients did not have access to the clinical outcome data.…”

Droplet-Digital PCR Provides a Rapid, Accurate and Cost-Effective Method for Identification of Biomarker FcγRIIIa-F158V Genotypes

“…Primers and probes utilized were generated by ABI/Thermo-Fisher Scientific for SNP analysis (rs396991) targeting FcγRIIIa nucleotide 559 [3,[21][22][23]. Final reaction mix consisted of: primers (900 nM each), probes (250 nM each) Bio-Rad ddPCR Supermix for Probes (no dUTP) (1X) and genomic DNA (10 to 50 ng) prior to loading into Bio-Rad QX200 droplet generator.…”