Copy number variation leads to considerable diversity for B but not A haplotypes of the human KIR genes encoding NK cell receptors

Abstract

The KIR complex appears to be evolving rapidly in humans, and more than 50 different haplotypes have been described, ranging from four to 14 KIR loci. Previously it has been suggested that most KIR haplotypes consist of framework genes, present in all individuals, which bracket a variable number of other genes. We used a new technique to type 793 families from the United Kingdom and United States for both the presence/absence of all individual KIR genes as well as copy number and found that KIR haplotypes are even more complex. It is striking that all KIR loci are subject to copy number variation (CNV), including the so-called framework genes, but CNV is much more frequent in KIR B haplotypes than KIR A haplotypes. These two basic KIR haplotype groups, A and B, appear to be following different evolutionary trajectories. Despite the great diversity, there are 11 common haplotypes, derived by reciprocal recombination near KIR2DL4, which collectively account for 94% of KIR haplotypes determined in Caucasian samples. These haplotypes could be derived from combinations of just three centromeic and two telomeric motifs, simplifying disease analysis for these haplotypes. The remaining 6% of haplotypes displayed novel examples of expansion and contraction of numbers of loci. Conventional KIR typing misses much of this additional complexity, with important implications for studying the genetics of disease association with KIR that can now be explored by CNV analysis.

Figure 1.

Frequency distributions of KIR haplotypes and motifs. Seventy-one unique haplotypes based on gene content were identified in 793 nuclear families. Eleven haplotypes with frequencies >1% (shaded) account for 94% of the total 2999 parental haplotypes. Haplotypes 01 and 02 (both Cen-A:Tel-A) are the two basic A haplotypes that differ by the type of 2DS4 present; 01 carries the 22-bp frameshift deletion variant and 02 carries the full-length form. Pie charts show the frequencies of unique KIR haplotypes, A (Cen-A:Tel-A) and B (Cen-A:Tel-B, Cen-B:Tel-A, Cen-B:Tel-B) haplotype groups, individual structural motifs, and motif combinations. KIR A haplotypes contain a Cen-A motif (defined by presence of 2DL3) and a Tel-A motif (2DS4 present). B haplotypes can be defined by the presence of a Cen-B (2DS2 present) and/or a Tel-B (2DS1 present); Cen-A motif 3DL3–2DL3–2DP1–2DL1-3DP1, Cen-B motif 3DL3–2DS2–2DL2–(2DL5C–2DS3S5C-2DP1–2DL1)–3DP1, Tel-A motif 2DL4–3DL1–2DS4 full-length or deletion variant–3DL2, Tel-B motif 2DL4–3DS1–2DL5T–2DS3S5T–2DS1–3DL2. Sixty haplotypes, together accounting for 6% of the 2999 resolvable haplotypes, have frequencies <1% and typically carry unconventional gene content. Haplotypes that did not strictly conform to standard A/B haplotypes or motifs because of altered structural composition are labeled “U.”

Figure 2.

Common KIR haplotypes. Depicted are the 11 common haplotypes that encompass 94% of Caucasian haplotypes studied. (Right) Haplotype frequencies, A/B group classification, and constituent motifs; for example, cA01 and tA01 denote centromeric-A haplotype 1 and telomeric-A haplotype 1, respectively. Gene and allele content were determined by segregation analysis. (Black spot) Site of the recombination hotspot between 3DP1 and 2DL4. The gene order is based on published sequences of KIR haplotypes that were sequenced in their entirety. Locations of 2DS3S5 may vary on unsequenced haplotypes. The boxed key identifies alleles, variants, and which genes are considered framework. 2DS4v represents the 22-bp frameshift deletion variant, and 2DS4f is the full-length form. 2DL2 and 2DL3 are allelic variants of 2DL2L3, 3DL1 and 3DS1 are variants of 3DL1S1, and 2DS3 and 2DS5 are variants of 2DS3S5. The motif annotation is at the resolution of gene content and does not distinguish allelic variation of individual genes. For example, the haplotype motif tA01 may have either form of 2DS4 (2DS4f or 2DS4v), and tB01 may have either variant of 2DS3S5 (2DS3 or 2DS5). T and C suffixes for 2DL5 (2DL5A or 2DL5B) and 2DS3S5 indicate the telomeric and centromeric versions of the genes, respectively (Gomez-Lozano et al. 2002; Ordonez et al. 2008; Pyo et al. 2010). For simplicity, 2DS1 and 2DS4 are shown at the same site because they showed a mutually exclusive relationship.

Figure 3.

The 11 common haplotypes are derived from recombination of a small number of haplotypes. The figure shows the putative mechanism for formation of common recombinant haplotypes from parental haplotypes by reciprocal recombination. All 11 haplotypes can be accounted for by recombination at the recombination hotspot near 2DL4. The mechanism effectively swaps the centromeric and telomeric motifs (identification in key above) between haplotypes by reciprocal recombination. Genes are not duplicated or deleted by this process, but haplotype motifs are interchanged. Some recombinations generate equivalent recombinant haplotypes to the others, in terms of gene content and not considering alleles. (Right) Constituent haplotype motifs based on gene content; (f) frequency. The numbers in the parentheses refer to haplotypes defined in Figure 1. Where there are two haplotypes in parentheses, e.g., (01/02), then these haplotypes only differ by 2DS4 type (2DS4f or 2DS4v; see below). (Gray) Cen-B and Tel-B motif genes; (white) Cen-A and Tel-A motif genes. cA01 and tA01 denote Cen-A haplotype motif 1 and Tel-A haplotype motif 1, respectively. tA01 may have either form of 2DS4 (2DS4f or 2DS4v). tB01 may have either variant of 2DS3S5 (2DS3 or 2DS5). The arrows point both up and down because the precursor and recombinant haplotypes are not known and the process could occur in either direction, exchanging parental and progeny haplotypes.

Figure 4.

Unconventional KIR haplotypes and their frequencies. Fifteen altered A haplotypes and 56 B haplotypes all with frequencies <1%. Duplicated genes are not depicted in order, but for simplicity, the paralog has been arbitrarily placed adjacent to the ancestral gene. In some cases, the order of all of the genes has not been determined. Genes suffixed with “v” have parts altered or deleted including fused genes resulting from NAHR or a novel allele. 2DS4v represents the 22-bp frameshift deletion variant, and 2DS4f is the full-length form. 3DL1v on haplotypes 63 and 48 tested negative for exon 4 and on haplotypes 59, 61, 71, and 43 negative for exon 9. 3DL2v on haplotypes 64 and 26 were negative for exon 4 and on haplotype 32 negative for exon 9. Nineteen haplotypes, marked with a black dot, were missing a copy or part of a single locus but otherwise could be the same as standard haplotypes (common arrangements of genes). Apart from haplotypes 15 and 21, which carried a hybrid gene (2DS2/2DS3; 2DS2*005), these marked haplotypes may not reflect true copy number variation because in these cases, we could not rule out the possibility that the gene was present but was erroneously scored as missing due to sequence variation that did not complement our generic primers (see Methods; Supplemental Material). To help resolve novel KIR haplotypes from unphased genotypes of unrelated individuals, we developed a tool for imputing haplotype pairs given observed copy number for each KIR loci. This tool is provided at http://www.bioinformatics.cimr.cam.ac.uk/haplotypes/.

Figure 5.

Gene duplication:absence ratios in the 71 unique haplotypes. CNV is spatially biased within the KIR locus, as illustrated by this plot showing the proportion of the 71 haplotypes showing duplication or absence for each respective gene.

Figure 6.

The fusion gene 2DL5/3DP1 is carried on four different KIR haplotypes. The precise chromosome breakage in 2DL5/3DP1 (3DP1*004) is shared by these four haplotypes, suggesting a common ancestry rather than independent formation of the fusion gene by NAHR between different haplotypes. Instead, the haplotype structures are consistent with recombination occurring with different haplotypes, i.e., motif interchange, at the recombination hotspots sited between 3DP1 and 2DL4, after the fusion gene was created. Constituent haplotype motifs based on gene content are boxed. (f) Frequency. The numbers in the parentheses refer to the haplotypes defined in Figure 1.