Rapid single-step FCGR3A genotyping based on SYBR Green I fluorescence in real-time multiplex allele-specific PCR

Abstract

A polymorphism at position 559 in the cDNA of the FCGR3A gene encoding the FcγRIIIa generates two allotypes with either a valine (Val) or a phenylalanine (Phe) at amino acid position 158. This polymorphism is of major importance in immunopathology and in pharmacogenetics, especially monoclonal antibody treatments. In this study, we report a single-step and single-tube method for FCGR3A-158V/F genotyping by real-time multiplex allele-specific PCR and melting curve analysis in the presence of SYBR Green I fluorescent dye. Results obtained from 113 samples showed 100% accuracy compared to those obtained with a conventional PCR-based allele-specific restriction assay. Although this method requires expensive equipment, it is inexpensive in terms of consumables. It is also very rapid, reliable and suitable for large-scale analysis.

Introduction

Three classes of receptors for the Fc portion of IgG (FcγRI, FcγRII and FcγRIII) and their subclasses are encoded by eight genes in humans, all located on the long arm of chromosome 1. Some of these genes display an allelic polymorphism whose relevance to human disease has been widely studied (Rascu et al., 1997). The FCGR3A gene encodes FcγRIIIa which is expressed on NK cells and monocytes/macrophages and displays a dimorphism caused by a single nucleotide substitution (G→T) at nt 559 in the cDNA Koene et al., 1997, Wu et al., 1997. This polymorphism generates two FcγRIIIa allotypes with either a valine (Val) or a phenylalanine (Phe) at amino acid position 158 in the membrane-proximal Ig-like domain, a residue in close contact with IgG1 lower hinge region in IgG1-FcγRIIIa co-crystal structures Sondermann et al., 2000, Radaev et al., 2001. Interestingly, although the FCGR3B gene encoding FcγRIIIb expressed on neutrophils is highly homologous to FCGR3A, it displays a G at this position resulting in a Val in the FcγRIIIb sequence (Ravetch and Perussia, 1989). The FCGR3A-158 V/F dimorphism has been shown to be in linkage disequilibrium with the triallelic FCGR3A-48L/H/R polymorphism (Koene et al., 1997). Irrespective of the amino acid present at position 48, the FcγRIIIa-V/V genotype results in a more efficient binding of IgG1 or IgG3 to NK cells and in an increased NK cell activation in response to aggregated IgG as compared to the F/F genotype Koene et al., 1997, Wu et al., 1997.

The observed distribution in Caucasians and African Americans is 8–17% of homozygous V/V, 32–50% of homozygous F/F and 39–51% of heterozygous V/F individuals Koene et al., 1997, Wu et al., 1997, Lehrnbecher et al., 1999, Leppers-van de Straat et al., 2000. Over the last few years, several studies have reported an association between the FCGR3A-158V/F polymorphism and systemic lupus erythematosus Wu et al., 1997, Koene et al., 1998, Song et al., 1998, Salmon et al., 1999, Wegener's granulomatosis (Dijstelbloem et al., 1999), adult periodontitis (Sugita et al., 1999) and the development of Kaposi's sarcoma in HIV-infected men (Lehrnbecher et al., 2000). Very recently, we have shown an association between the FCGR3A genotype and the response to rituximab (Mabthera® and Rituxan®), a chimeric anti-CD20 IgG1 monoclonal antibody used in the treatment of non-Hodgkin lymphomas (Cartron et al., 2002). This finding would enable new therapeutic strategies in B lymphoproliferative disorders, based upon prior determination of the patient's FCGR3A genotype. Moreover, FCGR3A genotyping may also be of value in other therapeutic regimes based on intact IgG1 for the treatment of other malignancies, such as CAMPATH-1H®, Herceptin® and Erbitux®.

It follows from the above that the ability to carry out rapid and reliable FCGR3A genotyping could become an increasingly important task. Whatever the method employed, PCR reactions must take into account the difficulty of specifically amplifying FCGR3A without amplifying the highly homologous FCGR3B gene. Several methods have been previously described using PCR-based amplification of FCGR3A fragments followed by restriction fragment analysis Koene et al., 1997, Oh et al., 1999, allele-specific oligohybridization (Lehrnbecher et al., 1999), automatic sequencing (Sugita et al., 1999) or using allele-specific PCR Wu et al., 1997, Leppers-van de Straat et al., 2000. All those techniques require a laborious post-PCR processing step and, therefore, are not ideally suited to large-scale analysis. We describe here the development of a novel genotyping assay in which both alleles are co-amplified and discriminated in a single PCR reaction. Subsequent manipulation outside of the amplification plate is avoided by the use of SYBR Green I fluorescent dye which allows real-time detection of PCR products and analysis of their melting curves based on the length and nucleotide content. This single-step multiplex allele-specific real-time PCR assay is inexpensive in terms of consumables. It allows rapid and high-throughput FCGR3A genotyping.