Detection of the FCGR3a polymorphism using a real-time polymerase chain reaction assay

The fragment crystallizable (Fc) region of the immunoglobulin G, low affinity III A receptor (FCGR3a, also known as CD16) belongs to the Fc gamma receptor family (FCGR), which plays an important role in immunoinflammatory processes. It is a low affinity, transmembrane receptor that is mainly expressed in monocytes, natural killer cells, and macrophages. It has been implicated in various inflammatory conditions, and recently a polymorphism (rs396991) in this gene has been shown to influence response to rituximab (anti-CD20) therapy in various disorders. We evaluated two molecular methods to genotype this polymorphism. Archived, formalin-fixed, paraffin-embedded samples from 26 biopsies of diffuse large B-cell lymphoma were retrieved and DNA was extracted. The samples were tested for the FCGR3a polymorphism using real-time polymerase chain reaction (PCR) followed by melt curve analysis or by a standard TaqMan allelic discrimination assay using the ABI 7500 FAST real-time PCR instrument. With the TaqMan allelic discrimination assay, we found that 16 cases were the wild type genotype, homozygous phenylalanine (F/F), for the FCGR3a receptor, whereas two cases had the homozygous valine (V/V) polymorphism and eight cases were heterozygous with a V/F genotype. Results with the real-time PCR followed by melt curve analysis were similar for 25 cases; however, four samples did not have sufficient DNA for the melt curve analysis method, and the result from one sample was discordant. The new TaqMan assay offers several advantages over previously published assays, such as faster turnaround time and ease of interpretation. These performance characteristics make it highly suitable for use in a clinical laboratory.