Cutting Edge: Expansion of the KIR Locus by Maureen P. Martin, Arman Bashirova, James Traherne, John Trowsdale and Mary Carrington This information is current as of October 1, 2021. J Immunol 2003; 171:2192-2195; ; doi: 10.4049/jimmunol.171.5.2192 http://www.jimmunol.org/content/171/5/2192 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2003/08/15/171.5.2192.DC1 Material References This article cites 21 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/171/5/2192.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE

JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: Expansion of the KIR Locus by Unequal Crossing Over1 Maureen P. Martin,* Arman Bashirova,† James Traherne,‡ John Trowsdale,‡ and Mary Carrington2*

The killer Ig-like receptor (KIR) genes have high sequence The KIR genes map to 19q13.4 where they are arranged in a similarity and are organized in a head-to-tail fashion. head to tail fashion spanning a region of roughly 150 Kb (12, These properties may enhance misalignment of homolo- 13). KIR genes are generally 80–90% identical, whereas allelic gous during synapsis preceding meiotic re- variants of a single KIR gene tend to differ by 2% or less (14, combination, resulting in unequal crossing over. We have 15). Except for a unique 14-kb sequence in the center of the KIR gene cluster just upstream of the KIR2DL4 gene, interven- identified an extended KIR haplotype that contains a Downloaded from novel hybrid gene and two copies of each of two previously ing segments between adjacent KIR genes are consistently 2 kb in length and are highly conserved (16). Three prototypic KIR described KIR genes. A parsimonious mechanism for the haplotypes have been sequenced in their entirety (5, 6), provid- derivation of this haplotype invokes unequal crossing over ing fundamental information regarding KIR gene order across between two known ancestral KIR haplotypes. These data the cluster. Additional information regarding gene order has raise the possibility that unequal crossing over may be re- been garnered from a sequence-specific priming (SSP) protocol http://www.jimmunol.org/ sponsible in part for the expansion/contraction of KIR in which forward and reverse PCR primers were designed from haplotypes as well as other homologous gene families that gene-specific segments near the 3Ј end and 5Ј end of each KIR map in tandem. The Journal of Immunology, 2003, gene, respectively (3), which we will refer to as “intergenic SSP- 171: 2192–2195. PCR.” By this approach, PCR products are produced only Ј 3 when the forward primer recognizing the 3 end of one gene iller Ig-like receptor (KIR) molecules regulate the ac- and the reverse primer recognizing the 5Ј end of an immediately tivity of NK and some T cells through interaction adjacent gene are used, thereby defining the pairwise order of K with specific HLA class I molecules on target cells. Be- KIR genes on that haplotype. cause HLA class I alleles are under continuous selection pressure In this study, we describe an extended KIR haplotype in a by guest on October 1, 2021 from infectious disease morbidity and mortality, the KIR locus family that contains two copies of both KIR2DL4 and must also evolve to maintain and enhance beneficial interac- KIR3DL1/S1, as well as a novel hybrid gene composed of half tions with HLA class I (1). A model asserting rapid evolution of KIR2DL5A and half KIR3DP1. All individuals with the ex- the KIR locus is supported by the highly diverse nature of KIR tended haplotype have three copies of both KIR2DL4 and haplotypes in terms of number and types of genes present on a KIR3DL1/S1, two of each on the extended haplotype and one given haplotype (2, 3). Segregation analysis within a limited of each on the homologous haplotype. We propose that the ex- number of families (4–7) has indicated remarkable diversity in tended haplotype was generated by unequal crossing over, terms of the number and type of KIR genes present on indepen- which represents a likely mechanism for the expansion and con- dent KIR haplotypes (a compilation of distinct KIR haplotypes traction of KIR haplotypes in general. Unequal crossing over can be seen at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?dbϭ may potentially maintain flux in the physical order of KIR genes Books (8)). In general, KIR haplotypes contain 7–12 genes plus within the set of KIR haplotypes present in a population. two pseudogenes, although very short haplotypes that contain as few as three or four genes have been observed infrequently (Ref. 3 Materials and Methods and M. P. Martin, unpublished observations). The extent of KIR KIR genotyping gene/haplotype diversity has only been appreciated over recent Genomic DNA from a three-generation Center d’Etude du Polymorphisme years, but already the influence of the presence/absence of specific Humaine family was genotyped for presence or absence of the following KIR KIR genes has been implicated in human disease (9–11). genes: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5,

*Basic Research Program, Science Applications International Corporation-Frederick, and 1 This work was supported with federal funds from the National Cancer Institute, National †Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702; and Institutes of Health under contract no. NO1-CO-12400. The content of this publication ‡Immunology Division, Department of Pathology, University of Cambridge, Cambridge, does not necessarily reflect the views orpolicies of the Department of Health and Human United Kingdom Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Received for publication May 28, 2003. Accepted for publication July 11, 2003. 2 Address correspondence and reprint requests to Dr. Mary Carrington, National Cancer The costs of publication of this article were defrayed in part by the payment of page Institute, P.O. Box B, Frederick, MD 21702. E-mail address: [email protected] charges. This article must therefore be hereby marked advertisement in accordance 3 Abbreviations used in this paper: KIR, killer Ig-like receptor; SSP, sequence specific with 18 U.S.C. Section 1734 solely to indicate this fact. priming.

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 2193

Table I. Gene dosage determination of KIR2DL4 at the genomic level using haplotypes using segregation analysis, several assumptions were made in deter- quantitative real-time multiplex PCR mining the haplotypes based on published gene frequencies and patterns of linkage disequilibrium between pairs of KIR genes: 1) 3DL3, 3DP1, 2DL4, and 3DL2 are present on all haplotypes, 2) if 2DL1 is present, 2DP1 is always Genomic Copy Mean Relative Ratio Genotype Number (SD) present, 3) 3DS1 segregates as an allele of 3DL1,4)2DL2 and 2DL3 segregate as alleles of a single locus. bd 2 1.0 (0.01) bc 3 1.6 (0.1) Determination of KIR gene order; intergenic SSP-PCR ac 3 1.6 (0.04) Order of the genes on the c haplotype (see Fig. 1B) was determined by sequenc- ab 2 1.0 (0.04) ing products derived from PCR in which forward primers recognized the 3Ј end cd 3 1.6 (0.06) and reverse primers recognized the 5Ј end of the various KIR genes (primers and dg 2 1.1 (0.08) annealing temperatures are provided in supplemental Tables I and II). In some ch 3 1.6 (0.06) cases it was necessary to reamplify the initial PCR product because yield of the amplicon was inadequate for sequencing. In these cases, an internal primer was used in reamplification. KIR gene sequences are based on the alignment pro- vided in http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ϭ Books. 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. Genotyping was performed us- ing PCR amplification with two pairs of locus-specific primers (PCR-SSP) as Identification of KIR alleles previously described (10). Internal control primers that amplify a 796-bp frag- ment of the third intron of DRB1 were also included in each PCR to validate KIR genes that were suspected to be duplicated on haplotype c (see Fig. 2) were proper amplifications. Additional primers that recognize 3DP1 and 2DS4, re- sequenced for allelic determination. The PCR product derived from exons 3–5 spectively, are as follows: 3DP1F, 5Ј-GCAGCACCATGTCGCTCATG-3Ј; of 2DL4 was also cloned for sequencing because it was not possible to assign 3DP1R, 5Ј-AACGGTGTTTCGGAATAC-3Ј; 3DP1⌬exon 2F, 5Ј- alleles after direct sequencing. The amplified product was cloned into the ex- Downloaded from CAGGGGGCCTGGCCACATGA-3Ј; 2DS4vF, 5Ј-GTTCAGGCAG pression vector pcDNA2.1-TOPO (Invitrogen) and eight clones were se- GAGAGAAT-3Ј; 2DS4vR, 5Ј-GTTTGACCACTCGTAGGGAGC. Ampli- quenced. Primers used for sequencing of 2DL4 and 3DL1 are provided in sup- fication was performed in a volume of 10 ␮l containing 200 ␮M dNTP, 500 plemental Table II. Primers were designed to amplify all known alleles of the nM primer, 1.5 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and genes. The primers used for amplification and sequencing of the hybrid 0.5U Platinum TaqDNA polymerase (Invitrogen, Carlsbad, CA), and 20 ng of 2DL5A/3DP1 gene are provided in supplemental Tables I and II. The amplified DNA. Cycling was performed as follows: 2 min at 94°C; 5 cycles of 94°C for products were purified using the Qiaquick PCR purification kit (Qiagen, Va- 15 s, 65°C for 15 s, 72°C for 30 s; 21 cycles of 94°C for 15 s, 60°C for 15 s, 72°C lencia, CA). Cycle sequencing was performed using the ABI BigDye Termina- for 30 s; 4 cycles of 94°C for 15 s, 55°C for 1 min, 72°C for 2 min, and a final tor Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, http://www.jimmunol.org/ extension step of 10 min at 72°C. PCR products were electrophoresed in 1.5% CA), followed by isopropanol precipitation. The samples were then run on an agarose gels containing ethidium bromide, and predicted size products were ABI 377 sequencer. Allele nomenclature is derived from Ref. 17. visualized under UV light. Determination of 2DL4 copy number KIR haplotype determination Simultaneous detection of the target KIR2DL4 sequence and an internal single- KIR haplotypes were determined by segregation analysis in the family (see Fig. copy gene control in the same sample material was achieved by dual-color de- 2). Because it was not always possible to define precisely the gene content of the tection using the Lightcycler (Roche Diagnostic Systems, Indianapolis, IN). by guest on October 1, 2021

FIGURE 1. A recombinant gene product and its derivation. A, The KIR2DL5A/3DP1 gene is a product of recombination between KIR2DL5A (AF217485) and KIR3DP1 (AL133414). positions that differ between AF217485 and AL133414 are shown. B, The proposed ancestral haplotypes from which KIR2DL5A/3DP1 was derived have been observed previously in family studies and their frequencies are provided (4–7). The gene content and order on these ancestral haplotypes, joined as shown and forming the expanded haplotype, correspond precisely with those determined for haplotype c (see Fig. 2). Distinct alleles of the duplicated genes are indicated by superscript a and b. Both alleles of each duplicated gene have been observed previously. 2194 CUTTING EDGE: EXPANSION OF THE KIR LOCUS BY UNEQUAL CROSSING OVER

ancestral KIR3DP1 gene (KIR3DP1 maps to the centromeric half of the complex; Ref. 3). The ancestral KIR haplotypes in the model shown in Fig. 1B have been observed at frequencies of 3.5 (red haplotype) and 14% (blue haplotype) in family studies (3–5, 7). These haplotypes were chosen for the model because gene composition on the respective red centromeric and blue telomeric halves of the haplotypes corresponds precisely with those on the observed extended haplotype. We propose that during synapsis, misalignment of KIR genes on the two parental homologous chromosomes occurred, resulting in crossing over between the KIR2DL5A and KIR3DP1 genes. The progeny haplotype containing the observed novel hybrid gene, KIR2DL5A/3DP1, should theoretically contain two copies of both KIR2DL4 and KIR3DL1/S1. FIGURE 2. Segregation of the haplotype containing KIR2DL5A/3DP1 in a KIR2DL5A/3DP1 was identified in a three generation Center Center d’Etude du Polymorphisme Humaine family. KIR haplotypes were de- d’Etude du Polymorphisme Humaine family. Extensive cloning, termined by segregation analysis in all members of a three-generation family. sequencing, and segregation analysis of KIR genes in the family in- Allele designations correspond to HGNC nomenclature (http://www.gene. dicated that two known alleles of both KIR2DL4 (X97229, ucl.ac.uk/nomenclature/genefamily/kir.html). The position of the duplicated AF034773) and KIR3DL1/S1 (AF262969, AF022044) segregated Downloaded from block is shown by brackets and an arrow. on the c haplotype, whereas a single distinct allele of each of these loci segregated on each of the a, b, and d haplotypes (Fig. 2). Using The KIR2DL4 forward and reverse PCR primers, sited within exon 3, were a quantitative PCR technique to measure gene dosage, we con- 5Ј-TCAGGA CAAGCCCTTCTG-3Ј and 5Ј-ACC CCATCT TTCTTG TA firmed that individuals with the c haplotype had three copies of CAGTG-3Ј, respectively. The penultimate nucleotide of the reverse primer KIR2DL4, and those without the c haplotype had two copies of this (underlined) is a mismatch to all KIR gene sequences to prevent nonspecific http://www.jimmunol.org/ priming. The KIR2DL4 hybridization probes were 5Ј-CTGTGGTGCCT gene (Table I). The order of the genes on the c haplotype was then CAAGGAGG-fluorescein-3Ј and 5Ј-Red640-ACGTGACTCTTCGGTGT determined by sequencing products derived from PCR in which CAC-phosphate-3Ј. A proprietary internal control (␤-globin gene; Roche Di- forward primers recognized the 3Ј end and the reverse primers rec- agnostic Systems) was used. Final concentrations in 20-␮l reaction volumes Ј ϫ ognized the 5 end of the various KIR genes. Every sequence ob- were 1 FastStart Reaction Mix (Roche Diagnostic Systems), 5 mM MgCl2, 0.5 ng/␮l DNA template, 500 nM of each primer, 0.1 ␮M of each fluorescein tained supported the order of genes shown on the extended hap- probe, and 0.2 ␮M of each Red fluorophore probe. Cycling was performed as lotype in Fig. 1B. Primer sequences used in this study and sequence follows: 10 min at 95°C followed by 45 cycles of 95°Cfor3s,62°C for 5 s, and of informative variant sites that allowed determination of gene or- 72°C for 8 s. The results of duplicate experiments are expressed as the mean der are provided in supplemental Tables I and II. relative ratio of KIR2DL4 to the reference gene (Relative Quantification Soft- by guest on October 1, 2021 ware (Roche Diagnostic Systems) using a precreated coefficient file) with SDs The gene duplication, gene order, and novel hybrid (Table I). All samples were tested blindly. KIR2DL5A/3DP1 gene that characterize haplotype c strongly indicate that the mechanism by which this haplotype was de- Results and Discussion rived involved unequal crossing over between two well-defined While performing segregation analysis of KIR genes/haplotypes KIR haplotypes. We propose that this mechanism represents a in a battery of Center d’Etude du Polymorphisme Humaine common means by which expansion and contraction of KIR families, we identified a novel KIR gene sequence (termed haplotypes occur, facilitating rapid evolution of the KIR gene KIR2DL5A/3DP1) that, in the 5Ј region, is identical to the gene complex. The truncated KIR haplotype that also would have KIR2DL5A (accession no. AF217485), but is identical to an- been produced by the recombination event depicted in Fig. 1B other gene, the KIR3DP1 pseudogene (accession no. has not been observed in any family studies published to date AL133414), from intron 2 to the end of the gene (Fig. 1A; the (3–7). However, the sequence of the hybrid gene in this puta- novel sequence has been submitted to GenBank). We hypoth- tive haplotype, KIR3DP1/2DL5A (Fig. 3), is virtually identical esized that KIR2DL5A/3DP1 was derived by an unequal cross- to the gene KIR2DL5B (7) (AF217486). It follows that the over between an ancestral KIR2DL5A gene (KIR2DL5A maps truncated haplotype (or one similar to it) containing a hybrid to the telomeric half of the KIR gene complex; Ref. 3) and an KIR3DP1/2DL5A (i.e., KIR2DL5B) gene has been generated

FIGURE 3. The origin of KIR2DL5B. The KIR2DL5B gene (AF217486) appears to be the reciprocal of the novel hybrid gene KIR2DL5A/3DP1, derived from an unequal crossover event between KIR3DP1 (AL133414) and KIR2DL5A (AF217485). Nucleotide positions that differ among the genes are shown. The Journal of Immunology 2195 previously and has circulated in the population. KIR2DL5A References and KIR2DL5B are highly homologous but distinct genes that 1. Khakoo, S. I., R. Rajalingam, B. P. Shum, K. Weidenbach, L. Flodin, D. G. Muir, F. Canavez, S. L. Cooper, N. M. Valiante, L. L. Lanier, and P. Parham. 2000. Rapid are sometimes located on the same haplotype (6, 7), indicating evolution of NK cell receptor systems demonstrated by comparison of chimpanzees that an unequal recombination event occurred subsequent to and humans. 12:687. 2. Uhrberg, M., N. M. Valiante, B. P. Shum, H. G. Shilling, K. Lienert-Weidenbach, that which gave rise to KIR2DL5B, placing KIR2DL5A and B. Corliss, D. Tyan, L. L. Lanier, and P. Parham. 1997. Human diversity in killer cell KIR2DL5B on a single haplotype. Interestingly, of the three de- inhibitory receptor genes. Immunity 7:753. 3. Hsu, K. C., S. Chida, B. Dupont, and D. E. Geraghty. 2002. The killer cell immu- fined KIR2DL5B alleles, two alleles, including the most com- noglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and allelic mon one, are not expressed due to a in their promoter polymorphism. Immunol. Rev. 190:40. region (18), partially reverting this gene to the pseudogene sta- 4. Shilling, H. G., L. A. Guethlein, N. W. Cheng, C. M. Gardiner, R. Rodriguez, D. Tyan, and P. Parham. 2002. Allelic polymorphism synergizes with variable gene tus of its ancestor KIR3DP1 and hybrid counterpart content to individualize human KIR genotype. J. Immunol. 168:2307. KIR2DL5A/3DP1. 5. Uhrberg, M., P. Parham, and P. Wernet. 2002. Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain The KIR region does not fit comfortably with traditional ge- between seven and eleven KIR genes. Immunogenetics 54:221. netic models (6, 15). Several distinct KIR genes, such as 6. Hsu, K. C., X. R. Liu, A. Selvakumar, E. Mickelson, R. J. O’Reilly, and B. Dupont. 2002. Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic KIR2DL5A and KIR2DL5B, are highly related and because KIR diversity with a minimum of six basic framework haplotypes, each with multiple sub- haplotypes can have different numbers of loci, the distinction sets. J. Immunol. 169:5118. 7. Gomez-Lozano, N., C. M. Gardiner, P. Parham, and C. Vilches. 2002. Some human between genes and alleles is not always clear. KIR2DL4 and KIR haplotypes contain two KIR2DL5 genes: KIR2DL5A and KIR2DL5B. Immu- KIR3DL1/S1 are present on virtually all KIR haplotypes (3) and nogenetics 54:314. 8. Carrington, M., and P. J. Norman. 2003. The KIR Gene Cluster, Vol. 2003. National both genes have several alleles that are fairly evenly distributed Library of Medicine (U.S.), National Center for Biotechnology Information, Be- Downloaded from (M. Carrington, unpublished observations). Although no func- thesda, MD. 9. Yen, J. H., B. E. Moore, T. Nakajima, D. Scholl, D. J. Schaid, C. M. Weyand, and tional significance has been assigned to the genetic variability at J. J. Goronzy. 2001. Major histocompatibility complex class I-recognizing receptors either of these loci, distinct beneficial phenotypes conferred by are disease risk genes in . J. Exp. Med. 193:1159. 10. Martin, M. P., G. Nelson, J. H. Lee, F. Pellett, X. Gao, J. Wade, M. J. Wilson, specific allotypes may exist, resulting in some level of balancing J. Trowsdale, D. Gladman, and M. Carrington. 2002. Cutting edge: susceptibility to selection. KIR typing methods used currently distinguish only psoriatic arthritis: influence of activating killer Ig-like receptor genes in the absence of specific HLA-C alleles. J. Immunol. 169:2818. http://www.jimmunol.org/ between presence and absence of each gene, and do not provide 11. Martin, M. P., X. Gao, J. H. Lee, G. W. Nelson, R. Detels, J. J. Goedert, information regarding gene copy number. Thus, the frequency S. Buchbinder, K. Hoots, D. Vlahov, J. Trowsdale, et al. 2002. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat. Genet. 31:429. of individuals who have three (or more) copies of a single gene 12. Borges, L., M. L. Hsu, N. Fanger, M. Kubin, and D. Cosman. 1997. A family of is not known and will require measurement of gene dosage, as human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I described for KIR2DL4 in this report (Table I). If indeed the molecules. J. Immunol. 159:5192. 13. Wagtmann, N., S. Rojo, E. Eichler, H. Mohrenweiser, and E. O. Long. 1997. A new polymorphism at these loci is functionally significant, those in- human gene complex encoding the killer cell inhibitory receptors and related mono- dividuals with three alleles are not encompassed by conven- cyte/macrophage receptors. Curr. Biol. 7:615. 14. Valiante, N. M., M. Uhrberg, H. G. Shilling, K. Lienert-Weidenbach, K. L. Arnett, A. tional genetic paradigms and “heterozygote advantage” is an in- D’Andrea, J. H. Phillips, L. L. Lanier, and P. Parham. 1997. Functionally and struc- adequate description. Perhaps the term “polyzygote advantage” turally distinct NK cell receptor repertoires in the peripheral blood of two human by guest on October 1, 2021 donors. Immunity 7:739. would more appropriately describe the polyallelic phenomenon 15. Selvakumar, A., U. Steffens, and B. Dupont. 1997. Polymorphism and domain vari- observed within the KIR locus of some individuals. MHC hap- ability of human killer cell inhibitory receptors. Immunol. Rev. 155:183. 16. Wilson, M. J., M. Torkar, A. Haude, S. Milne, T. Jones, D. Sheer, S. Beck, and lotypes can contain one to three related copies of DRB se- J. Trowsdale. 2000. Plasticity in the organization and sequences of human KIR/ILT quences and provide another, albeit moderate, example of this gene families. Proc. Natl. Acad. Sci. USA 97:4778. 17. Marsh, S. G. E., P. Parham, B. Dupont, D. E. Geraghty, J. Trowsdale, D. Middleton, phenomenon (19). C. Vilches, M. Carrington, C. Witt, L. A. Guethlein, et al. 2003. Killer immunoglob- Evolution of tandem arrays of homologous genes might often ulin-like receptor (KIR) nomenclature report, 2002. Tissue 62:79. 18. Vilches, C., C. M. Gardiner, and P. Parham. 2000. Gene structure and promoter occur through a mechanism involving unequal crossing over variation of expressed and nonexpressed variants of the KIR2DL5 gene. J. Immunol. (20), which may underlie the “birth and death” of clustered 165:6416. 19. Trowsdale, J. 2001. Genetic and functional relationships between MHC and NK re- genes (21). The mouse ly-49 region, the functional equivalent ceptor genes. Immunity 15:363. of KIR, behaves in a similar manner (22). Functional conse- 20. Jeffreys, A. J., V. Wilson, and S. L. Thein. 1985. Individual-specific “fingerprints” of human DNA. Nature 316:76. quences of expanded (or truncated) KIR haplotypes in viral in- 21. Nei, M., X. Gu, and T. Sitnikova. 1997. Evolution by the birth-and-death process in fections and cancer are quite plausible, and their characteriza- multigene families of the vertebrate . Proc. Natl. Acad. Sci. USA 94:7799. tion may illuminate our understanding of gene dosage effects in 22. Yokoyama, W. M. 1998. Natural killer cell receptors. Curr. Opin. Immunol. human disease. 10:298.