Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.
We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.
Metes
D
D
Department of Surgery, Section of Cellular Transplantation, Thomas E. Starzl Transplantation Institute, E1551 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261, USA. metesdm@msx.upmc.edu
Gambotto
A A
AA
Nellis
J
J
Ruscin
A
A
Stewart-Akers
A M
AM
Morel
P A
PA
Rao
A S
AS
D000483:Alleles; D001483:Base Sequence; D015139:Blotting, Southern; D018388:Codon, Terminator; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes; D005091:Exons; D005434:Flow Cytometry; D005838:Genotype; D006801:Humans; D016130:Immunophenotyping; D007694:Killer Cells, Natural / Q000276:immunology; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D017452:Receptors, IgG / Q000235:genetics*
D018388:Codon, Terminator; D017931:DNA Primers; D017452:Receptors, IgG; D004262:DNA Restriction Enzymes