A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism

Abstract

The FCGR3A gene encodes for the receptor important for antibody-dependent natural killer cell-mediated cytotoxicity. FCGR3A gene polymorphisms could affect the success of monoclonal antibody therapy. Although polymorphisms, such as the FcγRIIIA-V158F and -48L/R/H, have been studied extensively, an overview of other polymorphisms within this gene is lacking. To provide an overview of FCGR3A polymorphisms, we analysed the 1000 Genomes project database and found a total of 234 polymorphisms within the FCGR3A gene, of which 69%, 16%, and 15% occur in the intron, UTR, and exon regions respectively. Additionally, only 16% of all polymorphisms had a minor allele frequency (MAF) > 0.01. To facilitate (full-length) analysis of FCGR3A gene polymorphism, we developed a FCGR3A gene-specific amplification and sequencing protocol for Sanger sequencing and MinION (Nanopore Technologies). First, we used the Sanger sequencing protocol to study the presence of the V158F polymorphism in 76 individuals resulting in frequencies of 38% homozygous T/T, 7% homozygous G/G and 55% heterozygous. Next, we performed a pilot with both Sanger sequencing and MinION based sequencing of 14 DNA samples which showed a good concordance between Sanger- and MinION sequencing. Additionally, we detected 13 SNPs listed in the 1000 Genome Project, from which 11 had MAF > 0.01, and 10 SNPs were not listed in 1000 Genome Project. In summary, we demonstrated that FCGR3A gene is more polymorphic than previously described. As most novel polymorphisms are located in non-coding regions, their functional relevance needs to be studied in future functional studies.

Genetic Variation in Low-To-Medium-Affinity Fcγ Receptors: Functional Consequences, Disease Associations, and Opportunities for Personalized Medicine

“…Given the complexity of the locus and inaccuracies in the current databases holding reference sequences, research on the locus could benefit from a thorough genetic analysis that sequences through the entire region and can help to establish a correct and proper reference. Such an approach has recently been explored for FCGR3A using long-range sequencing with Nanopore MinION technology, and allowed a complete investigation of polymorphic sites within the gene (189). In any case, to use the full potential of genetic variation at the FCGR2/3 locus, a comprehensive analysis of all SNPs and CNVs together is warranted.…”

Is Polymorphism in the Apoptosis and Inflammatory Pathway Genes Associated With a Primary Response to Anti-TNF Therapy in Crohn’s Disease Patients?

“…Exploring their participation at the protein level requires functional testing. These variants could potentially affect RNA splicing by altering splice sites, branch points, or intronic enhancer/silencer motifs ( Mahaweni et al, 2018 ).…”

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity

“…Competition with plasma IgGs might be even more pronounced in disease conditions that are characterized by high levels of plasma IgGs, such as multiple myeloma. 43 In addition, CD16A polymorphisms in humans, 44 with the most prominent variants CD16A-158V and CD16A-158F differing in binding affinities to IgG and hence FcR functions like ADCC, result in different levels of efficacy of classical mAb therapy depending on the patient's individual CD16A genotype. 45,46 Leveraging our ROCK® platform, we created an anti-CD16A domain that recognizes a unique epitope on CD16A, 2 and that is virtually unaffected by plasma IgG competition and CD16A polymorphism.…”