Recombinant structures expand and contract inter and intragenic diversification at the KIR locus
© Pyo et al.; licensee BioMed Central Ltd. 2013
Received: 9 August 2012
Accepted: 26 January 2013
Published: 8 February 2013
The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total.
Towards that end, we developed a KIR haplotyping method that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available.
These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity of potential new KIR haplotypes derived from gene duplication, deletion, and hybridization.
KeywordsNatural killer cells Human KIR Recombinant structures
Natural killer (NK) cells are lymphocytes that act as central components of the innate immune response, providing immediate defense against infectious agents. As part of the mechanisms directing this function, these cells utilize a number of stimulatory and inhibitory receptors that react with major histocompatibility complex (MHC) class I antigens expressed by host cells. These NK receptors include variable killer cell immunoglobulin-like receptors (KIR) that in humans interact with the polymorphic HLA-A, B, and C ligands and the nearly invariant CD94/NKG2 family of receptors that interact with the conserved HLA-E ligand[3, 4]. For KIR, variation can be described at two levels, gene content and allelic variation. Early reports of gene content variation described two major haplotypes: group A, consisting of inhibitory KIR and one noninhibitory KIR; and group B, containing additional inhibitory and noninhibitory KIR not found in group A. The genomic structure of the KIR region has been extensively studied[6–8] and the complete genomic sequence of 24 KIR haplotypes has been delineated.
The genetic variation of KIR and the central role of NK cells in the immune response – including infectious disease and tumor immunity – have spurred investigation into the association of KIR genetics with a number of diseases and immunologic responses. Many of these studies have focused on the presence or absence of KIR genes and association with infectious disease. For example, the presence of KIR2DL3 and its HLA-C1 ligand directly influenced resolution of hepatitis C virus infection and herpes simplex type 1 infection was modified by the high-affinity receptor/ligand pair KIR2DL2/HLA-C1. Further, both genetic and functional data point to interactions between specific KIR genes and their HLA ligands in managing HIV infection. On the dysfunctional side of the immune response, several autoimmune diseases appear to be associated with the presence of a subset of activatory KIR genes, including KIR2DS2, which is associated with rhuematoid arthritis, and scleroderma. KIR was implicated in hematopoetic cell transplantation not long after the delineation of KIR genetic structures and more recently specific KIR haplotype substructures were associated with improved outcomes of unrelated transplants for acute myelogenous leukemia. Finally, KIR receptors and HLA ligands have been implicated in normal processes of pregnancy where allorecognition of paternal HLA-C by maternal KIR is postulated to be involved in essential trophoblast invasion and vascular remodeling and in turn potential derivative complications of pregnancy.
Our previous study of the KIR region yielded detailed information about KIR haplotype structures derived uniquely from complete phased genomic sequences, revealing substructures of known haplotypes and further defined linkage among the KIR genes. Taking into account the many reported correlations between KIR polymorphism and disease, there is some imperative to incorporate KIR haplotype structures, including potential intraregion epistasis, into the association studies so that causative variation at KIR can be identified. In addition, the repetitive gene content structure of the KIR region may contribute to rapid evolution through aberrant recombination mechanisms, possibly driven by the immune function of KIR and providing further impetus towards understanding overall genomic region variation. Towards these ends, we set out to establish methods for genotyping KIR that would yield the structures of phased haplotypes as defined by gene content. In the course of this development, we uncovered a number of new haplotype substructures and further defined genomic sequences that account for a large majority of those found in human populations. Stemming from the establishment of a comprehensive genotyping/haplotyping methodology, the results suggested that a number of different recombination events intertwined with strong linkage disequilibrium have been directly involved in the expansion and contraction of the KIR locus.
Cell lines/source DNAs
The DNAs used for library construction were extracted from a panel of cell lines chosen based on results of genotyping that indicated novel arrangements of KIR loci. DNA was prepared from B-LCLs using a Qiagen (Valencia, CA) genomic DNA extraction kit according to the manufacturers instructions. DNAs were obtained from the Research Cell Bank (RCB) at the Fred Hutchinson Cancer Research Center and are commercially available (http://www.ihwg.org). DNAs used in genotyping were derived from four sources, (1) a panel of 1,500 allogeneic hematopoietic cell transplantation (HCT) donors and recipients enrolled in a genome-wide association study and other reference DNAs from the IHWG.org totaling 3,229 samples, (2) a subset of the STEP HIV vaccine trials study samples described totaling 935, (3) DNAs from the UCLA DNA exchange for HLA typing, totaling 144 and (4) DNAs from the Carrington laboratory totaling 174. The ethnicity of these DNAs is approximately 85% Caucasian, 10% Asian, and 5% African and Hispanic. Data for finer breakdown of these ethnicities was not available, however a listing of haplotype frequencies for a panel of four ethnic groups used in this study is included in supplementary Additional file1: Table S3.
KIR gene content haplotyping
KIR haplotyping methodology – primary and secondary SNP positions
2DL2, 2DL3, 2DS2, 2DP1, (3DL2)
2DL5-58-G; -79-T; -103-C; -157-A
2DL1/2/3, 2DS2, 3DP1
2DS1, 2DS3/5, 3DP1, (2DL1)
2DS35-247-T; -251-G; -403-A; -404-T2DS3-446-G; -507-T2DS5-376-C
3DL1-157-C; -167-T3DS1-157-T; -167-G
2DL2/3, 2DS2, 2DP1
2DL2/3 2DP1 2DS2
2DS1, 2DL1, 2DS4 (2DS35)
2DS4-76-T; -86-T; -138-G; -187-A; -95-A; -179-A; -197-A; -225-T; -246-T; -345-G
2DS1, 2DL1, (2DS4)
3DL1, 3DS1, 3DL2
3DL1, 3DS1, 3DL2
KIR haplotyping methodology – SNP ratios for gene copy number
Gene copy determination using peak ratios
Genes amplified b
SNP ratios c
Observed peak ratios
2DL2, 2DL3, 2DS2, 2DP1 (3DL2)
C:T at 137
1:1, 2:1, 3:1, 3:2, or 4:1
A:C at 206
1:1 or 2:1
1:1 or 2:1
G:T at 136
1:1, 2:1, 1:2
G:C at 170
1 or multiple copies
No ratio data
2DL1/2/3, 2DS2, 3DP1
No ratio data
2DS1, 2DS3/5, 3DP1, (2DL1)
A:G at 446
1:1 or 1:2
1:1, 2:1, 1:2
T:G at 167
2DL2/3, 2DS2 2DP1
1:1 or 1:2
2DL2/3 2DP1 2DS2
No ratio data
2DS1, 2DL1, 2DS4 (2DS35)
A:C:T at 257
1:1:1, 1:2:1, 0:1:1, 0:2:1, or 0:1:2
2DS1, 2DL1, (2DS4)
No ratio data
3DL1, 3DS1, 3DL2
1:1, 0:2, 1:2 or 3:2
3DL1, 3DS1, 3DL2
1:1, 0:2, 1:2 or 3:2
Genomic DNA sequencing
Long-range PCR and fosmid clones were used to isolate phased genomic regions. Fosmid library construction and isolation was carried out as described in Raymond et al. with modifications. Fosmid libraries were produced from five cell lines and long-range PCR products from 3 additional DNAs as depicted in Additional file2: Figure S1. Briefly, fosmid library constructions used the Epicentre copy-control vector pCC1. Sheared, end-repaired inserts were size-selected to be 30–50 Kbp by pulsed-field-gel electrophoresis. Packaging was carried out with the Epicentre MaxPlax extracts and transfection was into Epicentre EPI300-T1RE. coli cells. Lysed aliquots of induced cultures were used directly as a source of PCR template during the STS-content mapping of 3,000-fosmid pools and at all subsequent stages of screening. For KIR screening, a panel of unlabeled primers designed locally and ordered from Sigma-Genosys were used to amplify each of the known KIR genes. Additional file1: Table S1 includes the sequences and gene specificity for the primer pairs used for all of the fosmid screening carried out and the copy number validation in some cases when the information of gene copy number was required. Samples were scored as positive or negative for a particular PCR assay based on SYBR green fluorescence (ABI). Data was collected on an ABI 7900 instrument operating in real-time (not end-point) mode.
All long-range PCRs were done using Platinum Taq High Fidelity (Invitrogen, Life Technologies Corporation). Reagents were added in the following order: 10 mM dNTPs, 10X High Fidelity PCR Buffer, 50 mM MgSO4, and 5 µM each of forward and reverse primers (Additional file1: Table S2) and mixed thoroughly before adding 2.5 U Platinum® Taq High Fidelity. PCR reactions used 250 ng genomic DNA in a total volume of 50 μl. An ABI 9600 thermal cycle was used to run the long-range PCR program: 94°C for 2 min; 15 cycles at 94°C for 15 sec, 65°C for 30 sec, 68°C for 20 min; 20 cycles at 94°C for 15 sec, 65°C for 30 sec, 68°C for 20 min 20 sec; and 68°C for 7 min.
Fosmids and long-range PCR products were sequenced using shotgun-sequencing protocols with dye-terminator sequencing as outlined previously[9, 25]. Sequences have been deposited in GenBank with the following accession numbers: JX008026 (HIP01829), JX008027 (HIP05435), JX008028 (HIP08327), JX008029 (HIP09410), JX008030 (HIP09453), JX008031 (HIP09498), JX008032 (2DS2 deletion), and JX008033 (3DL2-Fcar). To detect the 3DL1/L2 hybrid gene, we sequenced exons (exon3-5 and exon7-9) using gene specific sequencing primers (Additional file1: Table S1) on 3DL1/L2 hybrid long-range amplicons. For sequence analysis, we used full genomic sequence of the 3DL1/L2 hybrid from EU267269. Alleles and haplotypes have been named according to the guidelines established by the KIR Nomenclature Committee and deposited into IPD-KIR (http://www.ebi.ac.uk/ipd/kir/).
The finished genomic sequences were analyzed using cross-match of the Phred-Phrap-Consed package[27, 28], which was also used extensively for annotation. Annotation of the sequences used updated KIR cDNA and genomic sequences from the IPD-KIR database (http://www.ebi.ac.uk/ipd/kir/).
Sequences of the individual genes were aligned and phylogenetic analysis was performed using the FSA multiple alignment server or MAFFT and manually corrected in BIOEDIT (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Alignments were viewed and analyzed on Geneious v5.5 (http://www.geneious.com). Recombination analysis was performed by using Recombination Detection Programs (RDP) version 3 with a sliding window of 20 bp, with Bonferroni correction and a threshold of P = 0.001. The neighbor joining (NJ) analysis was performed using MEGA version 5 (http://www.megasoftware.net/)) with 1000 replicates, pairwise deletion, midpoint rooting, and the Tamura-Nei method. The repeat sequence in the breakpoint was identified by using censor server.
Analysis of KIR haplotype structures
KIR haplotypes were determined based on the previously sequenced KIR haplotypes[6, 9, 34] and the copy number of each KIR gene in an individual. The linkage disequilibrium (LD) among the genes and the frequencies of KIR haplotypes were calculated by the maximum likelihood method using Arlequin v3.5. Primer sequences used in generating the haplotyping amplicons and are listed in Additional file1: Table S3.
KIR gene content haplotyping – methods development
Gene content haplotype mapping was carried out using PCR sequencing of amplicons, each designed to detect multiple KIR loci. These assays were initially based on the 27 published complete genomic haplotype sequences and used to test samples from a diversity panel consisting of 96 each of Asian, African, Caucasian, and Hispanic samples. Further development in methodology occurred over iterative applications. DNAs yielding results that did not correlate with any known haplotype structures were subjected to genomic sequence analysis to obtain detail on novel structures and assays were modified to incorporate new data obtained. Fourteen assays were eventually developed that met the following criteria: (i) each assay produced a PCR product regardless of the haplotype combination being queried, thus removing PCR failure as a source of false negative results; (ii) PCR sequencing of the collective products provided at least 2 independent confirmations of the presence of any particular locus; (iii) quantitative measurement of relative peak heights was available to predict copy number variation of related loci; and (iv) the sum of the results could be used – given a haplotype structure rule set – to predict unambiguously the diploid haplotypes within any individual examined.
Testing of the initial amplicon designs on a diversity panel of DNAs showed that in most cases results were consistent with expectations based on the known haplotype sequences. In assigning genes to specific haplotypes, the following assumptions were made, based in part on calculated LD values (relevant D’ and r2 are taken from Figure1): (i) The order and position of each KIR gene is invariant; (ii) The 2DL5-2DS35 gene block is located on either the centromere or telomere based on the presence and copy number of adjacent KIR genes (centromere-2DP1 and 2DL1; telomere-3DS1-2DS1); (iii) Some genes are completely segregated to discreet haplotypes (e.g. 2DL3 versus 2DS2-2DL2). If an individual has both 2DL3 (or 2DS1) and 2DS2-2DL2 (or 3DL1-2DS4) from the gene content measurement, each was assigned to a different haplotype; (iv) Blocks with strong LD (e.g. 2DS2-2DL2/2DP1-2DL1/3DP1-2DL4/3DL1-2DS4/3DS1-2DL5-2DS35-2DS1) were used as a secondary check of haplotype assignments; (v) The 2DP1-2DL1 block is connected with either 2DL3 or 2DL5-2DS35 (no case of a 2DS2-2DL2 connection was found); (vi) 3DP1-2DL4-3DLS1/3DP1-2DL4-3DS1-2DL5-2DS35 can be duplicated (−ins3, -ins4, -ins5) or deleted (−del5, -del6, -del7) in a haplotype.
DNAs that did not yield patterns consistent with these criteria were further analyzed using long-range PCR where the suspected regions could be amplified conveniently and fosmid cloning where the regions were larger or otherwise complex. Once new sequence data was acquired, the KIR genotyping amplicons were redesigned to incorporate the new genomic structures. This approach allowed our designs to converge on an optimal set of amplicon sequences and specific variant positions that could detect all KIR loci (Table 1). A somewhat more vexing issue arose when attempting to determine gene copy number due to limitations in our design and dye-terminator sequencing. This issue was resolved satisfactorily by comparing RT-PCR analysis of samples to unambiguously determined gene copy number and establish the identity of cell lines containing previously undetected duplications or deletions. After determining the genomic sequence of variant motifs, we were able to focus the design of amplicons using variant dye terminator peak heights at specific positions to determine gene copy numbers (Additional file2: Figure S6). These data provided additional confirmation of gene presence (Table 2).
KIR gene content haplotypes
Summary of KIR haplotype motif frequencies
hybd1–3DL1-3DL2 hybrid/2DS4 deletion
del7 or del8–2DP1(partial)-3DP1-2DL4-3DS1-2DL5-2DS35-2DS1(partial) deletion
ins5–3DP1-2DL4-3DS1-2DL5-2DS35-2DS1(partial) insertion and additional recombination with 2DP1
Deletion mutants in the cB01 motif
Recombination in the telomeric region generating hybrid genes
Using this LD relationship among the haplotypes, it was possible to predict the putative origins of the 3DL1/S1 hybrid genes described in this report and in previous studies. Sequence alignments pointed to a putative exchange segment in intron 5 flanked by repetitive elements in common with both recombinant structures. Pairwise identity plots more precisely pinpointed the recombination events that could have resulted in the 3DS1*013/L1 and 3DL1/L2 (3DL1*059) hybrid genes (Figure4B and Additional file2: Figure S3). These data may emphasize the importance of considering highly local linkage disequilibrium within KIR in genetic association studies because an association with a KIR locus or allele may indicate causation, linkage, or causation through epistatic interactions between linked loci.
A KIR3DL2 deletion event
Unequal recombination and new telomeric motifs
A similar misaligned recombination event can be postulated to have given rise to a second expansion in the tA01 segment, duplicating the 3DP1-2DL4-3DS1-2DL5 gene block and portions of the flanking 2DL1 and 2DS1 genes to yield the ins5 mutant structure (Figure6B). This structure is found in combination with three different centromeric KIR segments and represents the second example of duplication of KIR3DLS1 with KIR3DL1 and KIR3DS1 separated in phase. The reciprocal recombinant from this postulated event would yield the cA01|tB01-del7 haplotype – missing the same gene block – previously described. A caveat for this postulated recombination event is the resultant 2DP1/L1 hybrid gene found in the ins5 structure, which is not reflected in the reciprocal del7 structure where the 2DL1/S1 hybrid is found. Sequence alignments and breakpoint analysis lend strong support to these hybrid structures (Additional file2: Figure S4). A complete sequence of the cA01|tA01-ins5 haplotype with overlapping fosmids covering the hybrid gene was obtained (Additional file2: Figure S1), most likely ruling out a sequence artifact. Therefore, in order to accommodate the recombination event modeled, a secondary gene conversion event between a 2DS1/L1 hybrid in the original tA01-ins5 chromosome and the 2DP1 exons 1–3 region is postulated.
Migration of the 2DL5-2DS35 gene block
One of the interesting substructures of the KIR locus includes the 2DL5-2DS35 gene block, found in both the cB01, cB03, and tB01 regions, with homology between the centromeric and telomeric versions at over 98.7% over the extended 26 kb region[50, 51]. Based on the genomic sequences from this and our previous study, we constructed a putative series of two successive unequal recombination events that could have moved the centromeric 2DL5-2DS35 gene block to the telomeric region (or vise versa). Phylogenetic analysis of the 2DL5 exons 1–2 genomic region showed clear differentiation of 2DL5A and 2DL5B alleles. Based on fully sequenced KIR haplotypes, the 2DL5A group is located on the telomere region and the 2DL5B group is located on the centromere region (Additional file2: Figure S5). Phylogenetic analysis of the intron3 to exon9 region showed that some 2DL5 alleles (2DL5A*00501 and 2DL5B*00201) associated with the 2DS3 gene while other alleles (2DL5A*001, B*00601, B*00801 and B*00401) associated with the 2DS5 gene. Thus, the exon1 to exon2 region might indicate the location of the 2DL5-2DS35 gene block (centromere or telomere) while the downstream region (intron3 to exon9) might be predictive of linkage association (2DS3 or 2DS5).
The human KIR locus, with its unique repetitive structure has facilitated numerous asymmetric recombinations that duplicated KIR genes, deleted KIR genes, and formed new hybrid KIR genes with novel ligand-binding and signaling functions. A unique sequence in the KIR locus is located in the 14 kb intergenic region that separates KIR3DP1 from KIR2DL4 and divides the locus into centromeric and telomeric segments of similar size[7, 9]. This unique sequence has been the site for events of reciprocal recombination to form new variant KIR haplotypes[6, 53]. Recombination at this site is evidently a major driver of KIR haplotype variation as it accounts for most of the major haplotypes, which involve combinations of the three common centromeric and two telomeric motifs. A minor percentage of recombinant haplotypes, including those newly reported in this study, may have arisen from other mechanisms of recombination including insertions and deletions of gene blocks that may have involved misalignment at repetitive sequences in common between different KIRs. The resources produced from this study include two new complete haplotype sequences and 6 partial sequences of duplicated or deleted regions representing submotifs found in one or more haplotypes.
The high mutability of the KIR region is likely driven by immunological processes, which contribute a central role in the human immune response for KIR receptors, including infection and reproduction. In line with this, KIR genetics have been widely studied in relation to complex disease. However, the genotyping methods used in most of those studies measured the presence or absence of specific KIR genes with reduced or absent ability to reference haplotype structures. Knowledge of the complete haplotype structure is likely important when attempting to identify causative KIR genetic factors associated with disease for a number of reasons, including LD and possible epistatic interactions among KIR. Given the complexity of KIR allelic polymorphism combined with gene content haplotype polymorphism, perhaps the optimal data set is the complete genomic sequences of each pair of haplotypes in an individual (i.e. all of the genetic variation present). However, until technology allows for such data to be derived economically, methods that determine haplotype structures, supplemented with allelic information, mark progress towards the identification of KIR genetic factors that are directly involved or causative of complex diseases.
The KIR haplotyping method described for this study is currently based on an ABI dye-terminator sequence-based approach that detects specific variant positions identifying KIR genes and ratios at SNP positions determining gene copy number. Our reliance on sequence-based methods stems from the desire to use uniform methodology in genotyping in our laboratory. However, this approach can be adapted to other sequencing methods that detect specific SNPs and allow for quantitative determinations at SNP positions. Regardless of the genotyping approach, robust information processing of the data is needed, especially when scaling studies into the thousands of samples. Again, although we developed in house software for this purpose, appropriate algorithms can be developed within a variety of different software frameworks. The overall cost of the assay is similar to existing KIR gene presence/absence methods while yielding potentially valuable more complete genetic information, including phase and copy number.
Of the 9,024 chromosomes examined, 10 samples showed unique genotyping patterns, each different from one another and from the 37 haplotypes described. It was not possible to assign a unique structure to these haplotypes using the existing set of insertion, deletion, and hybridization submotifs. In addition, we did not pursue proving these structures through sequencing due to their single occurrence. There are likely other rare recombinant types not yet described with frequencies in the very rare range, perhaps similar to the frequency of new HLA alleles still being discovered. In addition, our survey populations did not include substantial numbers of African, Asian or Hispanic individuals (in the hundreds each), leaving much of the frequencies to be determined for those populations and with numerous new haplotypes likely to be found in the more diverse African populations. Excluding these 10 very rare haploytpes, the sequence-validated rare haplotypes – those with submotif patterns (Table 3) – collectively account for almost 7% of the total we examined here. These numbers may be significant in studies when the copy number or tandem arrangement of genes is important. In addition, when combined with allelic variants that significantly alter expression of KIR genes the exceptional cases can collectively amount to significant percentages. This may prove to be an important perspective for KIR and complex disease, especially in light of studies where rare variants collectively may be causative of complex diseases.
Among the rare mutations that may deserve unique attention are those that delete the framework genes KIR3DL3, KIR2DL4, and KIR3DL2, which may perform essential functions given their near invariant presence in all KIR haplotypes compared with more variable presence of other KIR within a haplotype. In this study, while no cases of a KIR3DL3 deletion were observed, the KIR2DL4 deletion found in the cB02|tB01-del6 haplotype accounted for about 2.3% of the haplotypes surveyed. The KIR2DL4 protein has been implicated as the receptor for the HLA-G ligand and may function in the pregnant environment although it may not play an essential role there as maternal homozygous deletion variants can apparently achieve a successful pregnancy. The KIR3DL2 protein has been shown to interact with specific HLA-A ligands and may act as an inhibitory receptor[60, 61]. As a framework KIR locus, KIR3DL2 shares with KIR2DL4 a rare variant type. The cA01|cB01-del7 haplotype is the only haplotype found where both genes are deleted. Functional studies performed on lymphocytes derived from individuals with this haplotype in a homozygous or hemizygous state could be revealing.
No doubt additional functional data about individual KIR receptors and their ligands are needed before conclusive causative genetic associations of KIR can be made with complex diseases. However, as new functional understandings are revealed, and as KIR population genetics is more comprehensively defined and genotyping becomes more extensive, refined, and economical, we can fully expect to achieve this long sought goal.
During the course of submitting this paper, a parallel study on KIR haplotypes was published where 72 haplotype structures were reported. Three differences between our reporting of haplotypes and that study account for the numerical difference. First, in our descriptions we did not consider haplotypes that carried the 2DS3 or 2DS5 genes or the 2DS4L and 2DS4S groups as distinguishing and instead considered the 2DS3/5 genes and the 2DS4L/S allele groups as sets of alleles. Also, among the 72 reported, 31 were single occurrence, none validated by sequencing, and none of which intersected with haplotypes from our samples. Although we detected 10 haplotypes for which we could estimate structures in addition to the 37 we reported in Figure2, each had only a single occurrence in our population set, and we chose not to fully characterize them through sequence analysis. Our standard for reporting new haplotypes was determination by our genotyping assay and sequencing of genomic DNA (fosmids or LR-PCR) when new structures were uncovered.
Knowledge of the complete haplotype structure of KIR is critical for association studies between KIR genetic variation and complex diseases. We developed a method to detect phased KIR gene-content haplotypes based on PCR sequencing of amplicons. Using this method, we defined a total of 37 KIR haplotypes from genotyping 9,024 chromosomes. An additional 10 haplotypes were detected in single copy but were not confirmed by sequencing. The 37 KIR haplotypes were sorted into 10 types of structural alterations, including gene deletions, insertions, and hybridizations, which together suggest a number of recombination events that might have occurred during KIR evolution. This haplotyping method is an important step towards the identification of KIR genetic factors that are associated with complex diseases.
Hemapoietic Cell Transplantation
Killer cell Immunoglobulin-like Receptor
Major Histocompatability Complex
Natural killer cell.
This work was supported by the National Institutes of Health [RR018669 to D.E.G.]. The expert contributions of Dr. Shu Shen in the preparation of the manuscript are gratefully acknowledged. We thank the organizers of the UCLA International Cell Exchange for the KIR reference panel (http://www.hla.ucla.edu/cellDna.htm).
- Lanier LL: NK cell recognition. Annu Rev Immunol. 2005, 23: 225-274. 10.1146/annurev.immunol.23.021704.115526.View ArticlePubMed
- Parham P: MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005, 5 (3): 201-214. 10.1038/nri1570.View ArticlePubMed
- Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE: HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci U S A. 1998, 95 (9): 5199-5204. 10.1073/pnas.95.9.5199.PubMed CentralView ArticlePubMed
- Gunturi A, Berg RE, Forman J: The role of CD94/NKG2 in innate and adaptive immunity. Immunol Res. 2004, 30 (1): 29-34. 10.1385/IR:30:1:029.View ArticlePubMed
- Valiante NM, Uhrberg M, Shilling HG, Lienert-Weidenbach K, Arnett KL, D'Andrea A, Phillips JH, Lanier LL, Parham P: Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity. 1997, 7 (6): 739-751. 10.1016/S1074-7613(00)80393-3.View ArticlePubMed
- Hsu KC, Chida S, Geraghty DE, Dupont B: The killer cell immunoglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and allelic polymorphism. Immunol Rev. 2002, 190: 40-52. 10.1034/j.1600-065X.2002.19004.x.View ArticlePubMed
- Wilson MJ, Torkar M, Haude A, Milne S, Jones T, Sheer D, Beck S, Trowsdale J: Plasticity in the organization and sequences of human KIR/ILT gene families. Proc Natl Acad Sci U S A. 2000, 97 (9): 4778-4783. 10.1073/pnas.080588597.PubMed CentralView ArticlePubMed
- Martin AM, Freitas EM, Witt CS, Christiansen FT: The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster. Immunogenetics. 2000, 51 (4–5): 268-280.View ArticlePubMed
- Pyo CW, Guethlein LA, Vu Q, Wang R, Abi-Rached L, Norman PJ, Marsh SG, Miller JS, Parham P, Geraghty DE: Different patterns of evolution in the centromeric and telomeric regions of group A and B haplotypes of the human killer cell Ig-like receptor locus. PLoS One. 2010, 5 (12): e15115-10.1371/journal.pone.0015115.PubMed CentralView ArticlePubMed
- Kulkarni S, Martin MP, Carrington M: The Yin and Yang of HLA and KIR in human disease. Semin Immunol. 2008, 20 (6): 343-352. 10.1016/j.smim.2008.06.003.PubMed CentralView ArticlePubMed
- Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, Cheng J, Goedert JJ, Vlahov D, Hilgartner M: HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science. 2004, 305 (5685): 872-874. 10.1126/science.1097670.View ArticlePubMed
- Moraru M, Cisneros E, Gomez-Lozano N, de Pablo R, Portero F, Canizares M, Vaquero M, Roustan G, Millan I, Lopez-Botet M: Host genetic factors in susceptibility to herpes simplex type 1 virus infection: contribution of polymorphic genes at the interface of innate and adaptive immunity. J Immunol. 2012, 188 (9): 4412-4420. 10.4049/jimmunol.1103434.View ArticlePubMed
- Bashirova AA, Thomas R, Carrington M: HLA/KIR restraint of HIV: surviving the fittest. Annu Rev Immunol. 2011, 29: 295-317. 10.1146/annurev-immunol-031210-101332.PubMed CentralView ArticlePubMed
- Majorczyk E, Pawlik A, Luszczek W, Nowak I, Wisniewski A, Jasek M, Kusnierczyk P: Associations of killer cell immunoglobulin-like receptor genes with complications of rheumatoid arthritis. Genes Immun. 2007, 8 (8): 678-683. 10.1038/sj.gene.6364433.View ArticlePubMed
- Momot T, Koch S, Hunzelmann N, Krieg T, Ulbricht K, Schmidt RE, Witte T: Association of killer cell immunoglobulin-like receptors with scleroderma. Arthritis Rheum. 2004, 50 (5): 1561-1565. 10.1002/art.20216.View ArticlePubMed
- Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, Posati S, Rogaia D, Frassoni F, Aversa F: Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002, 295 (5562): 2097-2100. 10.1126/science.1068440.View ArticlePubMed
- Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Le CT, Marsh SG, Geraghty D, Spellman S, Haagenson MD: Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood. 2010, 116 (14): 2411-2419. 10.1182/blood-2010-05-283051.PubMed CentralView ArticlePubMed
- Chazara O, Xiong S, Moffett A: Maternal KIR and fetal HLA-C: a fine balance. J Leukoc Biol. 2011, 90 (4): 703-716. 10.1189/jlb.0511227.View ArticlePubMed
- Chien JW, Zhang XC, Fan W, Wang H, Zhao LP, Martin PJ, Storer BE, Boeckh M: Warren EH. 2012, Hansen JA: Evaluation of published single nucleotide polymorphisms associated with acute graft versus host disease. Blood
- Fellay J, Frahm N, Shianna KV, Cirulli ET, Casimiro DR, Robertson MN, Haynes BF, Geraghty DE, McElrath MJ, Goldstein DB: Host genetic determinants of T cell responses to the MRKAd5 HIV-1 gag/pol/nef vaccine in the step trial. J Infect Dis. 2011, 203 (6): 773-779. 10.1093/infdis/jiq125.PubMed CentralView ArticlePubMed
- Geraghty DE, Daza R, Williams LM, Vu Q, Ishitani A: Genetics of the immune response: identifying immune variation within the MHC and throughout the genome. Immunol Rev. 2002, 190: 69-85. 10.1034/j.1600-065X.2002.19006.x.View ArticlePubMed
- Pyo CW, Williams LM, Moore Y, Hyodo H, Li SS, Zhao LP, Sageshima N, Ishitani A, Geraghty DE: HLA-E, HLA-F, and HLA-G polymorphism: genomic sequence defines haplotype structure and variation spanning the nonclassical class I genes. Immunogenetics. 2006, 58 (4): 241-251. 10.1007/s00251-005-0076-z.View ArticlePubMed
- Smith WP, Vu Q, Li SS, Hansen JA, Zhao LP, Geraghty DE: Toward understanding MHC disease associations: partial resequencing of 46 distinct HLA haplotypes. Genomics. 2006, 87 (5): 561-571. 10.1016/j.ygeno.2005.11.020.View ArticlePubMed
- Raymond CK, Subramanian S, Paddock M, Qiu R, Deodato C, Palmieri A, Chang J, Radke T, Haugen E, Kas A: Targeted, haplotype-resolved resequencing of long segments of the human genome. Genomics. 2005, 86 (6): 759-766. 10.1016/j.ygeno.2005.08.013.View ArticlePubMed
- Daza-Vamenta R, Glusman G, Rowen L, Guthrie B, Geraghty DE: Genetic divergence of the rhesus macaque major histocompatibility complex. Genome Res. 2004, 14 (8): 1501-1515. 10.1101/gr.2134504.PubMed CentralView ArticlePubMed
- Marsh SG, Parham P, Dupont B, Geraghty DE, Trowsdale J, Middleton D, Vilches C, Carrington M, Witt C, Guethlein LA: Killer-cell immunoglobulin-like receptor (KIR) nomenclature report, 2002. Immunogenetics. 2003, 55 (4): 220-226. 10.1007/s00251-003-0571-z.View ArticlePubMed
- Gordon D, Abajian C, Green P: Consed: a graphical tool for sequence finishing. Genome Res. 1998, 8 (3): 195-202.View ArticlePubMed
- Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998, 8 (3): 175-185.View ArticlePubMed
- Bradley RK, Roberts A, Smoot M, Juvekar S, Do J, Dewey C, Holmes I, Pachter L: Fast statistical alignment. PLoS Comput Biol. 2009, 5 (5): e1000392-10.1371/journal.pcbi.1000392.PubMed CentralView ArticlePubMed
- Katoh K, Misawa K, Kuma K, Miyata T: MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002, 30 (14): 3059-3066. 10.1093/nar/gkf436.PubMed CentralView ArticlePubMed
- Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P: RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics. 2010, 26 (19): 2462-2463. 10.1093/bioinformatics/btq467.PubMed CentralView ArticlePubMed
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28 (10): 2731-2739. 10.1093/molbev/msr121.PubMed CentralView ArticlePubMed
- Kohany O, Gentles AJ, Hankus L, Jurka J: Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor. BMC Bioinformatics. 2006, 7: 474-10.1186/1471-2105-7-474.PubMed CentralView ArticlePubMed
- Traherne JA, Martin M, Ward R, Ohashi M, Pellett F, Gladman D, Middleton D, Carrington M, Trowsdale J: Mechanisms of copy number variation and hybrid gene formation in the KIR immune gene complex. Hum Mol Genet. 2010, 19 (5): 737-751. 10.1093/hmg/ddp538.PubMed CentralView ArticlePubMed
- Excoffier L, Lischer HE: Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour. 2010, 10 (3): 564-567. 10.1111/j.1755-0998.2010.02847.x.View ArticlePubMed
- Robinson J, Mistry K, McWilliam H, Lopez R, Parham P, Marsh SG: The IMGT/HLA database. Nucleic Acids Res. 2011, 39: 1171-1176. 10.1093/nar/gkq998.View Article
- Smit AF: Interspersed repeats and other mementos of transposable elements in mammalian genomes. Curr Opin Genet Dev. 1999, 9 (6): 657-663. 10.1016/S0959-437X(99)00031-3.View ArticlePubMed
- Rajalingam R, Gardiner CM, Canavez F, Vilches C, Parham P: Identification of seventeen novel KIR variants: fourteen of them from two non-Caucasian donors. Tissue Antigens. 2001, 57 (1): 22-31. 10.1034/j.1399-0039.2001.057001022.x.View ArticlePubMed
- Ordonez D, Gomez-Lozano N, Rosales L, Vilches C: Molecular characterisation of KIR2DS2*005, a fusion gene associated with a shortened KIR haplotype. Genes Immun. 2011, 12 (7): 544-551. 10.1038/gene.2011.35.View ArticlePubMed
- Guethlein LA, Older Aguilar AM, Abi-Rached L, Parham P: Evolution of killer cell Ig-like receptor (KIR) genes: definition of an orangutan KIR haplotype reveals expansion of lineage III KIR associated with the emergence of MHC-C. J Immunol. 2007, 179 (1): 491-504.View ArticlePubMed
- Maxwell LD, Wallace A, Middleton D, Curran MD: A common KIR2DS4 deletion variant in the human that predicts a soluble KIR molecule analogous to the KIR1D molecule observed in the rhesus monkey. Tissue Antigens. 2002, 60 (3): 254-258. 10.1034/j.1399-0039.2002.600307.x.View ArticlePubMed
- Hou L, Chen M, Ng J, Hurley CK: Conserved KIR allele-level haplotypes are altered by microvariation in individuals with European ancestry. Genes Immun. 2012, 13 (1): 47-58. 10.1038/gene.2011.52.PubMed CentralView ArticlePubMed
- Norman PJ, Abi-Rached L, Gendzekhadze K, Korbel D, Gleimer M, Rowley D, Bruno D, Carrington CV, Chandanayingyong D, Chang YH: Unusual selection on the KIR3DL1/S1 natural killer cell receptor in Africans. Nat Genet. 2007, 39 (9): 1092-1099. 10.1038/ng2111.View ArticlePubMed
- Shilling HG, Guethlein LA, Cheng NW, Gardiner CM, Rodriguez R, Tyan D, Parham P: Allelic polymorphism synergizes with variable gene content to individualize human KIR genotype. J Immunol. 2002, 168 (5): 2307-2315.View ArticlePubMed
- Jiang W, Johnson C, Jayaraman J, Simecek N, Noble J, Moffatt MF, Cookson WO, Trowsdale J, Traherne JA: Copy number variation leads to considerable diversity for B but not A haplotypes of the human KIR genes encoding NK cell receptors. Genome Res. 2012, 22 (10): 1845-1854. 10.1101/gr.137976.112.PubMed CentralView ArticlePubMed
- Martin MP, Bashirova A, Traherne J, Trowsdale J, Carrington M: Cutting edge: expansion of the KIR locus by unequal crossing over. J Immunol. 2003, 171 (5): 2192-2195.View ArticlePubMed
- Gomez-Lozano N, Estefania E, Williams F, Halfpenny I, Middleton D, Solis R, Vilches C: The silent KIR3DP1 gene (CD158c) is transcribed and might encode a secreted receptor in a minority of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes are duplicated. Eur J Immunol. 2005, 35 (1): 16-24. 10.1002/eji.200425493.View ArticlePubMed
- Norman PJ, Carrington CV, Byng M, Maxwell LD, Curran MD, Stephens HA, Chandanayingyong D, Verity DH, Hameed K, Ramdath DD: Natural killer cell immunoglobulin-like receptor (KIR) locus profiles in African and South Asian populations. Genes Immun. 2002, 3 (2): 86-95. 10.1038/sj.gene.6363836.View ArticlePubMed
- Gomez-Lozano N, de Pablo R, Puente S, Vilches C: Recognition of HLA-G by the NK cell receptor KIR2DL4 is not essential for human reproduction. Eur J Immunol. 2003, 33 (3): 639-644. 10.1002/eji.200323741.View ArticlePubMed
- Gomez-Lozano N, Gardiner CM, Parham P, Vilches C: Some human KIR haplotypes contain two KIR2DL5 genes: KIR2DL5A and KIR2DL5B. Immunogenetics. 2002, 54 (5): 314-319. 10.1007/s00251-002-0476-2.View ArticlePubMed
- Ordonez D, Meenagh A, Gomez-Lozano N, Castano J, Middleton D, Vilches C: Duplication, mutation and recombination of the human orphan gene KIR2DS3 contribute to the diversity of KIR haplotypes. Genes Immun. 2008, 9 (5): 431-437. 10.1038/gene.2008.34.View ArticlePubMed
- Abi-Rached L, Parham P: Natural selection drives recurrent formation of activating killer cell immunoglobulin-like receptor and Ly49 from inhibitory homologues. J Exp Med. 2005, 201 (8): 1319-1332. 10.1084/jem.20042558.PubMed CentralView ArticlePubMed
- Yawata M, Yawata N, Abi-Rached L, Parham P: Variation within the human killer cell immunoglobulin-like receptor (KIR) gene family. Crit Rev Immunol. 2002, 22 (5–6): 463-482.PubMed
- Trowsdale J, Moffett A: NK receptor interactions with MHC class I molecules in pregnancy. Semin Immunol. 2008, 20 (6): 317-320. 10.1016/j.smim.2008.06.002.View ArticlePubMed
- Pelak K, Need AC, Fellay J, Shianna KV, Feng S, Urban TJ, Ge D, De Luca A, Martinez-Picado J, Wolinsky SM: Copy number variation of KIR genes influences HIV-1 control. PLoS Biol. 2011, 9 (11): e1001208-10.1371/journal.pbio.1001208.PubMed CentralView ArticlePubMed
- Norman PJ, Abi-Rached L, Gendzekhadze K, Hammond JA, Moesta AK, Sharma D, Graef T, McQueen KL, Guethlein LA, Carrington CV: Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes. Genome Res. 2009, 19 (5): 757-769. 10.1101/gr.085738.108.PubMed CentralView ArticlePubMed
- McClellan J, King MC: Genetic heterogeneity in human disease. Cell. 2010, 141 (2): 210-217. 10.1016/j.cell.2010.03.032.View ArticlePubMed
- Rajagopalan S, Bryceson YT, Kuppusamy SP, Geraghty DE, van der Meer A, Joosten I, Long EO: Activation of NK cells by an endocytosed receptor for soluble HLA-G. PLoS Biol. 2006, 4 (1): e9-10.1371/journal.pbio.0040009.PubMed CentralView ArticlePubMed
- Nowak I, Majorczyk E, Ploski R, Senitzer D, Sun JY, Kusnierczyk P: Lack of KIR2DL4 gene in a fertile Caucasian woman. Tissue Antigens. 2011, 78 (2): 115-119. 10.1111/j.1399-0039.2011.01711.x.View ArticlePubMed
- Dohring C, Colonna M: Human natural killer cell inhibitory receptors bind to HLA class I molecules. Eur J Immunol. 1996, 26 (2): 365-369. 10.1002/eji.1830260215.View ArticlePubMed
- Pende D, Biassoni R, Cantoni C, Verdiani S, Falco M, di Donato C, Accame L, Bottino C, Moretta A, Moretta L: The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. J Exp Med. 1996, 184 (2): 505-518. 10.1084/jem.184.2.505.View ArticlePubMed
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