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. 2014 Mar 5;6(3):20.
doi: 10.1186/gm537. eCollection 2014.

Killer-cell Immunoglobulin-like Receptor gene linkage and copy number variation analysis by droplet digital PCR

Chrissy H Roberts  1 , Wei Jiang  2 , Jyothi Jayaraman  2 , John Trowsdale  2 , Martin J Holland  1 , James A Traherne  2
Affiliations

Killer-cell Immunoglobulin-like Receptor gene linkage and copy number variation analysis by droplet digital PCR

Chrissy H Roberts et al. Genome Med. .

Abstract

The Killer-cell Immunoglobulin-like Receptor (KIR) gene complex has considerable biomedical importance. Patterns of polymorphism in the KIR region include variability in the gene content of haplotypes and diverse structural arrangements. Droplet digital PCR (ddPCR) was used to identify different haplotype motifs and to enumerate KIR copy number variants (CNVs). ddPCR detected a variety of KIR haplotype configurations in DNA from well-characterized cell lines. Mendelian segregation of ddPCR-estimated KIR2DL5 CNVs was observed in Gambian families and CNV typing of other KIRs was shown to be accurate when compared to an established quantitative PCR method.

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Figures

Figure 1
Figure 1
Organization of the KIR locus on the human chromosome region 19q13.4. KIR haplotypes are composed of different centromeric (cA01, cB01 and cB02) and telomeric motifs (tA01, tB01) [10]. Each motif has a different content and arrangement of genes. Framework genes, which are found at the ends and near the middle of the locus on nearly all haplotypes [11] are shaded grey. The KIR2DL5 gene can be located in either the centromeric or telomeric motif, adjacent to KIR2DL2 and KIR3DS1, respectively, or on both motifs of a single haplotype. Different centromeric motifs can be paired with different telomeric motifs through the central reciprocal recombination hotspot between KIR3DP1 and KIR2DL4, as indicated by different dashed lines. KIR haplotypes can be classified into two categories [12]: group A haplotypes (shaded grey), composed of cA01 and tA01 motifs only, have fixed gene-content with one activating gene (KIR2DS4); group B haplotypes (unshaded), comprising at least one motif of type cB01, cB02 or tB01, have variable gene content between framework genes and more than one activating KIR locus.
Figure 2
Figure 2
Experimentally determined signatures of linkage between (A) KIR2DL2 and KIR2DL5 and (B) KIR3DS1 and KIR2DL5 for the 10 IHW cell lines. Evidence of linkage (black bars) in DNA sample preparations (fragment size >23 kb), expressed as the percentage of KIR2DL2 or KIR3DS1 genes that were linked to a KIR2DL5 gene. Linkage was abrogated by EcoRI restriction endonuclease digestion (gray bars).
Figure 3
Figure 3
KIR CNV estimates in (A) UCLA International KIR exchange samples and (B) Gambian family samples. Colour-coded points indicate cluster membership (0 copies (n = 3), 1 copy (n = 10), 2 copies (n = 12), 3 copies (n = 2)).
Figure 4
Figure 4
Segregation of KIR2DL5 in five Gambian families. Patterns of segregation are consistent with Mendelian inheritance and with segregation of the linked KIR2DS3 and KIR2DS5 genes. In most world populations, KIR2DL5 is in perfect linkage disequilibrium with KIR2DS3 and KIR2DS5. In Gambia, these genes are in almost perfect linkage disequilibrium (Roberts C.h., Molina, S., Makalo, P., Joof, H., Harding-Esch, E.M., Burr, S.E., Mabey, D.W., Bailey, R.L., Burton, M.J. and Holland, M.J., in press). The absence of both KIR2DS3 and KIR2DS5 implies that KIR2DL5 will be absent. The presence of both genes indicates that at least two KIR2DL5 copies are present. The co-segregation of KIR2DL5 with the KIR2DS3 and KIR2DS5 genes is expected and was observed in these pedigrees. To protect the anonymity of participant families, all F1 subjects have been changed to ‘male’.
Figure 5
Figure 5
The predicted gene carriage and resolved arrangements for WJR076 haplotypes. Showing the constituent haplotype motifs: cB01:tA01 within the extended KIR B haplotype and cA01:tA01 forming a standard KIR A haplotype. The extended haplotype has the 2DL5/3DP1 hybrid gene (KIR3DP1*004) and duplication of the KIR2DL4 and KIR3DL1/S1 loci. Linkage analysis supported close relationship of KIR2DL2 ~ KIR2DL5 (indicated by a solid black line) and long-distance linkage between KIR2DL5 and KIR3DS1 (dashed line) on the extended haplotype.
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