Cutting Edge: Expansion of the KIR Locus by Unequal Crossing Over ¹ Free

Killer Ig-like receptor (KIR) ³ molecules regulate the activity of NK and some T cells through interaction with specific HLA class I molecules on target cells. Because HLA class I alleles are under continuous selection pressure from infectious disease morbidity and mortality, the KIR locus must also evolve to maintain and enhance beneficial interactions with HLA class I (1). A model asserting rapid evolution of the KIR locus is supported by the highly diverse nature of KIR haplotypes in terms of number and types of genes present on a given haplotype (2, 3). Segregation analysis within a limited number of families (4, 5, 6, 7) has indicated remarkable diversity in terms of the number and type of KIR genes present on independent KIR haplotypes (a compilation of distinct KIR haplotypes can be seen at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books (8)). In general, KIR haplotypes contain 7–12 genes plus two pseudogenes, although very short haplotypes that contain as few as three or four genes have been observed infrequently (Ref.3 and M. P. Martin, unpublished observations). The extent of KIR gene/haplotype diversity has only been appreciated over recent years, but already the influence of the presence/absence of specific KIR genes has been implicated in human disease (9, 10, 11).

The KIR genes map to 19q13.4 where they are arranged in a head to tail fashion spanning a region of roughly 150 Kb (12, 13). KIR genes are generally 80–90% identical, whereas allelic variants of a single KIR gene tend to differ by 2% or less (14, 15). Except for a unique 14-kb sequence in the center of the KIR gene cluster just upstream of the KIR2DL4 gene, intervening segments between adjacent KIR genes are consistently 2 kb in length and are highly conserved (16). Three prototypic KIR haplotypes have been sequenced in their entirety (5, 6), providing fundamental information regarding KIR gene order across the cluster. Additional information regarding gene order has been garnered from a sequence-specific priming (SSP) protocol in which forward and reverse PCR primers were designed from gene-specific segments near the 3′ end and 5′ end of each KIR gene, respectively (3), which we will refer to as “intergenic SSP-PCR.” By this approach, PCR products are produced only when the forward primer recognizing the 3′ end of one gene and the reverse primer recognizing the 5′ end of an immediately adjacent gene are used, thereby defining the pairwise order of KIR genes on that haplotype.

In this study, we describe an extended KIR haplotype in a family that contains two copies of both KIR2DL4 and KIR3DL1/S1, as well as a novel hybrid gene composed of half KIR2DL5A and half KIR3DP1. All individuals with the extended haplotype have three copies of both KIR2DL4 and KIR3DL1/S1, two of each on the extended haplotype and one of each on the homologous haplotype. We propose that the extended haplotype was generated by unequal crossing over, which represents a likely mechanism for the expansion and contraction of KIR haplotypes in general. Unequal crossing over may potentially maintain flux in the physical order of KIR genes within the set of KIR haplotypes present in a population.

Genomic DNA from a three-generation Center d’Etude du Polymorphisme Humaine family was genotyped for presence or absence of the following KIR genes: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. Genotyping was performed using PCR amplification with two pairs of locus-specific primers (PCR-SSP) as previously described (10). Internal control primers that amplify a 796-bp fragment of the third intron of DRB1 were also included in each PCR to validate proper amplifications. Additional primers that recognize 3DP1 and 2DS4, respectively, are as follows: 3DP1F, 5′-GCAGCACCATGTCGCTCATG-3′; 3DP1R, 5′-AACGGTGTTTCGGAATAC-3′; 3DP1Δexon 2F, 5′-CAGGGGGCCTGGCCACATGA-3′; 2DS4vF, 5′-GTTCAGGCAGGAGAGAAT-3′; 2DS4vR, 5′-GTTTGACCACTCGTAGGGAGC. Amplification was performed in a volume of 10 μl containing 200 μM dNTP, 500 nM primer, 1.5 mM MgCl₂, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 0.5U Platinum TaqDNA polymerase (Invitrogen, Carlsbad, CA), and 20 ng of DNA. Cycling was performed as follows: 2 min at 94°C; 5 cycles of 94°C for 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 for 30 s; 4 cycles of 94°C for 15 s, 55°C for 1 min, 72°C for 2 min, and a final extension step of 10 min at 72°C. PCR products were electrophoresed in 1.5% agarose gels containing ethidium bromide, and predicted size products were visualized under UV light.

KIR haplotypes were determined by segregation analysis in the family (see Fig. 2). Because it was not always possible to define precisely the gene content of the haplotypes using segregation analysis, several assumptions were made in determining 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 present, 3) 3DS1 segregates as an allele of 3DL1, 4) 2DL2 and 2DL3 segregate as alleles of a single locus.

Order of the genes on the c haplotype (see Fig. 1 B) was determined by sequencing products derived from PCR in which forward primers recognized the 3′ end and reverse primers recognized the 5′ end of the various KIR genes (primers and annealing temperatures are provided in supplemental Tables I and II). In some 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 provided in http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = Books.

KIR genes that were suspected to be duplicated on haplotype c (see Fig. 2) were sequenced for allelic determination. The PCR product derived from exons 3–5 of 2DL4 was also cloned for sequencing because it was not possible to assign alleles after direct sequencing. The amplified product was cloned into the expression vector pcDNA2.1-TOPO (Invitrogen) and eight clones were sequenced. Primers used for sequencing of 2DL4 and 3DL1 are provided in supplemental Table II. Primers were designed to amplify all known alleles of the genes. The primers used for amplification and sequencing of the hybrid 2DL5A/3DP1 gene are provided in supplemental Tables I and II. The amplified products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA). Cycle sequencing was performed using the ABI BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA), followed by isopropanol precipitation. The samples were then run on an ABI 377 sequencer. Allele nomenclature is derived from Ref.17 .

Simultaneous detection of the target KIR2DL4 sequence and an internal single-copy gene control in the same sample material was achieved by dual-color detection using the Lightcycler (Roche Diagnostic Systems, Indianapolis, IN). The KIR2DL4 forward and reverse PCR primers, sited within exon 3, were 5′-TCAGGA CAAGCCCTTCTG-3′ and 5′-ACC CCATCT TTCTTG TACAGTG-3′, respectively. The penultimate nucleotide of the reverse primer (underlined) is a mismatch to all KIR gene sequences to prevent nonspecific priming. The KIR2DL4 hybridization probes were 5′-CTGTGGTGCCTCAAGGAGG-fluorescein-3′ and 5′-Red640-ACGTGACTCTTCGGTGTCAC-phosphate-3′. A proprietary internal control (β-globin gene; Roche Diagnostic Systems) was used. Final concentrations in 20-μl reaction volumes were 1× FastStart Reaction Mix (Roche Diagnostic Systems), 5 mM MgCl₂, 0.5 ng/μl DNA template, 500 nM of each primer, 0.1 μM of each fluorescein probe, and 0.2 μM of each Red fluorophore probe. Cycling was performed as follows: 10 min at 95°C followed by 45 cycles of 95°C for 3 s, 62°C for 5 s, and 72°C for 8 s. The results of duplicate experiments are expressed as the mean relative ratio of KIR2DL4 to the reference gene (Relative Quantification Software (Roche Diagnostic Systems) using a precreated coefficient file) with SDs (Table I). All samples were tested blindly.

While performing segregation analysis of KIR genes/haplotypes in a battery of Center d’Etude du Polymorphisme Humaine families, we identified a novel KIR gene sequence (termed KIR2DL5A/3DP1) that, in the 5′ region, is identical to the gene KIR2DL5A (accession no. AF217485), but is identical to another gene, the KIR3DP1 pseudogene (accession no. AL133414), from intron 2 to the end of the gene (Fig. 1,A; the novel sequence has been submitted to GenBank). We hypothesized that KIR2DL5A/3DP1 was derived by an unequal crossover between an ancestral KIR2DL5A gene (KIR2DL5A maps to the telomeric half of the KIR gene complex; Ref.3) and an ancestral KIR3DP1 gene (KIR3DP1 maps to the centromeric half of the complex; Ref.3). The ancestral KIR haplotypes in the model shown in Fig. 1 B have been observed at frequencies of 3.5 (red haplotype) and 14% (blue haplotype) in family studies (3, 4, 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.

KIR2DL5A/3DP1 was identified in a three generation Center d’Etude du Polymorphisme Humaine family. Extensive cloning, sequencing, and segregation analysis of KIR genes in the family indicated that two known alleles of both KIR2DL4 (X97229, AF034773) and KIR3DL1/S1 (AF262969, AF022044) segregated 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 a quantitative PCR technique to measure gene dosage, we confirmed that individuals with the c haplotype had three copies of KIR2DL4, and those without the c haplotype had two copies of this gene (Table I). The order of the genes on the c haplotype was then determined by sequencing products derived from PCR in which forward primers recognized the 3′ end and the reverse primers recognized the 5′ end of the various KIR genes. Every sequence obtained supported the order of genes shown on the extended haplotype in Fig. 1 B. Primer sequences used in this study and sequence of informative variant sites that allowed determination of gene order are provided in supplemental Tables I and II.

The gene duplication, gene order, and novel hybrid KIR2DL5A/3DP1 gene that characterize haplotype c strongly indicate that the mechanism by which this haplotype was derived involved unequal crossing over between two well-defined KIR haplotypes. We propose that this mechanism represents a common means by which expansion and contraction of KIR haplotypes occur, facilitating rapid evolution of the KIR gene complex. The truncated KIR haplotype that also would have been produced by the recombination event depicted in Fig. 1,B has not been observed in any family studies published to date (3, 4, 5, 6, 7). However, the sequence of the hybrid gene in this putative haplotype, KIR3DP1/2DL5A (Fig. 3), is virtually identical to the gene KIR2DL5B (7) (AF217486). It follows that the truncated haplotype (or one similar to it) containing a hybrid KIR3DP1/2DL5A (i.e., KIR2DL5B) gene has been generated previously and has circulated in the population. KIR2DL5A and KIR2DL5B are highly homologous but distinct genes that are sometimes located on the same haplotype (6, 7), indicating that an unequal recombination event occurred subsequent to that which gave rise to KIR2DL5B, placing KIR2DL5A and KIR2DL5B on a single haplotype. Interestingly, of the three defined KIR2DL5B alleles, two alleles, including the most common one, are not expressed due to a mutation in their promoter region (18), partially reverting this gene to the pseudogene status of its ancestor KIR3DP1 and hybrid counterpart KIR2DL5A/3DP1.

The KIR region does not fit comfortably with traditional genetic models (6, 15). Several distinct KIR genes, such as KIR2DL5A and KIR2DL5B, are highly related and because KIR haplotypes can have different numbers of loci, the distinction between genes and alleles is not always clear. KIR2DL4 and KIR3DL1/S1 are present on virtually all KIR haplotypes (3) and both genes have several alleles that are fairly evenly distributed (M. Carrington, unpublished observations). Although no functional significance has been assigned to the genetic variability at either of these loci, distinct beneficial phenotypes conferred by specific allotypes may exist, resulting in some level of balancing selection. KIR typing methods used currently distinguish only between presence and absence of each gene, and do not provide information regarding gene copy number. Thus, the frequency of individuals who have three (or more) copies of a single gene is not known and will require measurement of gene dosage, as described for KIR2DL4 in this report (Table I). If indeed the polymorphism at these loci is functionally significant, those individuals with three alleles are not encompassed by conventional genetic paradigms and “heterozygote advantage” is an inadequate description. Perhaps the term “polyzygote advantage” would more appropriately describe the polyallelic phenomenon observed within the KIR locus of some individuals. MHC haplotypes can contain one to three related copies of DRB sequences and provide another, albeit moderate, example of this phenomenon (19).

Evolution of tandem arrays of homologous genes might often occur through a mechanism involving unequal crossing over (20), which may underlie the “birth and death” of clustered genes (21). The mouse ly-49 region, the functional equivalent of KIR, behaves in a similar manner (22). Functional consequences of expanded (or truncated) KIR haplotypes in viral infections and cancer are quite plausible, and their characterization may illuminate our understanding of gene dosage effects in human disease.