title: Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing.
abstract: The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrum of diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes.
authors.affiliation: Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address: paul.norman@stanford.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Norman
authors.affiliation: Department of Neurology, School of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jill A
authors.initials: JA
authors.lastname: Hollenbach
authors.affiliation: Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
authors.forename: Neda
authors.initials: N
authors.lastname: Nemat-Gorgani
authors.affiliation: Department of Neurology, School of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Wesley M
authors.initials: WM
authors.lastname: Marin
authors.affiliation: Illumina Inc., 5200 Illumina Way, San Diego, CA 92122, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Norberg
authors.affiliation: Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
authors.forename: Elham
authors.initials: E
authors.lastname: Ashouri
authors.affiliation: Division of Immunology, Department of Pathology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 1QP, UK.
authors.forename: Jyothi
authors.initials: J
authors.lastname: Jayaraman
authors.affiliation: Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Wroblewski
authors.affiliation: Division of Immunology, Department of Pathology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 1QP, UK.
authors.forename: John
authors.initials: J
authors.lastname: Trowsdale
authors.affiliation: Immunogenetics and Transplantation Laboratory, University of California, San Francisco, San Francisco, CA 94143, USA.
authors.forename: Raja
authors.initials: R
authors.lastname: Rajalingam
authors.affiliation: Department of Neurology, School of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jorge R
authors.initials: JR
authors.lastname: Oksenberg
authors.affiliation: UMR 7268 ADÉS, Aix-Marseille Université, l'Etablissement Français du Sang, Centre National de la Recherche Scientifique, 13344 Marseille, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Chiaroni
authors.affiliation: Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
authors.forename: Lisbeth A
authors.initials: LA
authors.lastname: Guethlein
authors.affiliation: Division of Immunology, Department of Pathology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 1QP, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Traherne
authors.affiliation: Illumina Inc., 5200 Illumina Way, San Diego, CA 92122, USA.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ronaghi
authors.affiliation: Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Parham
mesh_terms: D000483:Alleles; D018628:Gene Dosage; D005805:Genes, MHC Class I / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics; D005838:Genotype; D015234:HLA-A Antigens / Q000235:genetics; D015235:HLA-B Antigens / Q000235:genetics; D015236:HLA-C Antigens / Q000235:genetics; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D011110:Polymorphism, Genetic; D054340:Receptors, KIR / Q000235:genetics*
chemical_list: D015234:HLA-A Antigens; D015235:HLA-B Antigens; D015236:HLA-C Antigens; D054340:Receptors, KIR
keywords: HLA class I; KIR; gene content diversity; highly polymorphic; immunity