ARTICLE
Defining KIR and HLA Class I Genotypes
at Highest Resolution via High-Throughput Sequencing
Paul J. Norman,
1
,
*
Jill A. Hollenbach,
2
Neda Nemat-Gorgani,
1
Wesley M. Marin,
2
Steven J. Norberg,
3
Elham Ashouri,
1
Jyothi Jayaraman,
4
Emily E. Wroblewski,
1
John Trowsdale,
4
Raja Rajalingam,
5
Jorge R. Oksenberg,
2
Jacques Chiaroni,
6
Lisbeth A. Guethlein,
1
James A. Traherne,
4
Mostafa Ronaghi,
3
and Peter Parham
1
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 contain-
ing 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 trans-
plantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, se-
quences, 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.
Introduction
The human leukocyte antigen (HLA) complex of chromo-
some 6 is the most polymorphic region of the human
genome.
1
This variation is driven by pressure to resist
diverse pathogens but also underlies susceptibility to auto-
immunity and other inflammatory diseases of major
importance to human health.
2
HLA class I molecules are
expressed on the surface of most tissue cells, where they
interact with receptors on the surface of lymphocytes,
effector cells of the immune system.
3
Natural killer (NK)
cells are innate and adaptive lymphocytes that destroy in-
fected or tumor cells with aberrant expression of HLA class
I; they also regulate trophoblast invasion during early preg-
nancy.
4
NK cell activity is genetically modulated through
differential expression of polymorphic killer cell immuno-
globulin-like receptors (KIRs) that recognize HLA class I
molecules.
5
Only recently has the KIR genomic region
been characterized to high resolution.
6
Consequently, re-
examination of diseases with long-established associations
with specific HLA polymorphisms is revealing a strong and
collective influence from KIR polymorphism.
7–10
The KIR locus in chromosomal region 19q13.4 is charac-
terized by unusually high diversity in the numbers of both
genes and their alleles.
11
The region varies in size from 100
to 350 kb as a result of structurally diverse haplotypes with
duplicated segments, large deletions, and gene fusions.
12,13
As a consequence of this plasticity, the 13 distinct KIR genes
(KIR2DL1 [MIM: 604936], KIR2DL2 [MIM: 604937],
KIR2DL4 [MIM: 604945], KIR3DL1 [MIM: 604946],
KIR3DL2
[MIM: 604947], KIR2DS1 [MIM: 604952],
KIR2DS2 [MIM: 604953], KIR2DS3 [MIM: 604954], KIR2DS4
[MIM: 604955], KIR2DS5 [MIM: 604956], KIR2DL5 [MIM:
605305], KIR3DL3 [MIM: 610095], and KIR3DP1 [MIM:
610604]) are combined in numerous ways. Haplotypes
have between 4 and 20 KIR genes, and the most common
KIR region haplotype has seven genes.
14
To varying degrees,
each KIR gene is polymorphic, and more than 600 KIR
alleles are currently defined.
15
KIR and HLA class I (HLA-A
[MIM: 142800], HLA-B [MIM: 142830], and HLA-C [MIM:
142840]) polymorphism are actively co-evolving,
16
sug-
gesting that many more KIR alleles and haplotypes await
discovery. During the last three decades, over 10,000 HLA
class I alleles have been characterized in specialized
clinical HLA laboratories,
15
and similar intensive study
will be needed for characterizing KIR diversity.
The function of an HLA class I molecule is to bind a pep-
tide, usually a nonamer, inside a cell and take it to the cell
surface, where the complex of peptide and HLA class I is
engaged by KIRs and other lymphocyte receptors.
17,18
On
healthy cells, the peptides bound by HLA class I molecules
derive from normal human proteins and do not stimulate
1
Departments of Structural Biology and Microbiology & Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA;
2
Department of
Neurology, School of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA;
3
Illumina Inc., 5200 Illumina Way, San Diego, CA
92122, USA;
4
Division of Immunology, Department of Pathology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2
1QP, UK;
5
Immunogenetics and Transplantation Laboratory, University of California, San Francisco, San Francisco, CA 94143, USA;
6
UMR 7268 ADE´S, Aix-
Marseille Universite´, l’Etablissement Franc¸ais du Sang, Centre National de la Recherche Scientifique, 13344 Marseille, France
*Correspondence:
paul.norman@stanford.edu
http://dx.doi.org/10.1016/j.ajhg.2016.06.023
.
The American Journal of Human Genetics 99, 375–391, August 4, 2016
375
Ó 2016 American Society of Human Genetics.