title: Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.
abstract: BACKGROUND
  Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP.
  
  
  OBJECTIVE
  The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes.
  
  
  METHODS
  We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches.
  
  
  RESULTS
  The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes.
  
  
  CONCLUSIONS
  Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.
authors.affiliation: Department of Pediatrics, Division of Hematology and Oncology, MSC 10 5590, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA, KMatlawska-Wasowska@salud.unm.edu.
authors.forename: Ksenia
authors.initials: K
authors.lastname: Matlawska-Wasowska
authors.forename: James M
authors.initials: JM
authors.lastname: Gale
authors.forename: Christian K
authors.initials: CK
authors.lastname: Nickl
authors.forename: Parisa
authors.initials: P
authors.lastname: Khalili
authors.forename: Brian
authors.initials: B
authors.lastname: Shirley
authors.forename: Bridget S
authors.initials: BS
authors.lastname: Wilson
authors.forename: Mohammad A
authors.initials: MA
authors.lastname: Vasef
authors.forename: Stuart S
authors.initials: SS
authors.lastname: Winter
mesh_terms: D000483:Alleles; D003062:Codon / Q000235:genetics; D058851:GPI-Linked Proteins / Q000097:blood / Q000235:genetics; D005838:Genotype; D064368:Healthy Volunteers; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017452:Receptors, IgG / Q000097:blood / Q000235:genetics*
chemical_list: D003062:Codon; C496124:FCGR3A protein, human; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D017452:Receptors, IgG