A novel method for KIR-ligand typing by pyrosequencing to predict NK cell alloreactivity.
Studies have shown that KIR-ligand mismatching to predict NK cell alloreactivity may result in less relapse and better survival in patients with AML. KIR-ligands are distinguished by single nucleotide polymorphisms (SNPs) from HLA-B and HLA-C sequences. We hypothesized that pyrosequencing to determine KIR-ligand status by direct sequencing of the ligand epitope can be done as an alternative to high-resolution HLA-typing. Pyrosequencing is rapid and would be particularly useful in analysis of retrospective cohorts where high-resolution HLA-typing is unavailable or too expensive. To validate this assay, RNA and DNA from 70 clinical samples were tested for KIR-ligand by pyrosequencing. Primer binding to invariant regions without known SNPs was critical for KIR-ligand assignment by pyrosequencing to be in full concordance with high-resolution HLA-typing. Pyrosequencing is sensitive, specific, high-throughput, inexpensive, and can rapidly screen KIR-ligand status to evaluate potential alloreactive NK cell or transplant donors.
Yun
Gong
G
Division of Adult Hematology, Oncology, and Transplantation, University of Minnesota Cancer Center, MMC 806, Harvard Street at East River Road, Minneapolis, MN 55455, USA, and Anthony Nolan Research Institute, Royal Free Hospital, London, UK.
Tolar
Jakub
J
Yerich
Anton K
AK
Marsh
Steven G E
SG
Robinson
James
J
Noreen
Harriet
H
Blazar
Bruce R
BR
Miller
Jeffrey S
JS
D000483:Alleles; D001483:Base Sequence; D030561:Databases, Nucleic Acid; D015235:HLA-B Antigens / Q000235:genetics* / Q000276:immunology; D015236:HLA-C Antigens / Q000235:genetics* / Q000276:immunology; D006650:Histocompatibility Testing; D006801:Humans; D020407:Internet; D007519:Isoantigens / Q000235:genetics / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D008024:Ligands; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011971:Receptors, Immunologic / Q000276:immunology*; D054340:Receptors, KIR; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D014181:Transplantation Immunology / Q000235:genetics / Q000276:immunology; D014584:User-Computer Interface
D015235:HLA-B Antigens; D015236:HLA-C Antigens; D007519:Isoantigens; D008024:Ligands; D011971:Receptors, Immunologic; D054340:Receptors, KIR