Diversity of the human LILRB3/A6 locus encoding a myeloid inhibitory and activating receptor pair

Abstract

Leukocyte immunoglobulin-like receptor (LILR)B3 and LILRA6 represent a pair of inhibitory/activating receptors with identical extracellular domains and unknown ligands. LILRB3 can mediate inhibitory signaling via immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail whereas LILRA6 can signal through association with an activating adaptor molecule, FcRγ, which bears a cytoplasmic tail with an immunoreceptor tyrosine-based activation motif. The receptors are encoded by two highly polymorphic neighboring genes within the leukocyte receptor complex on human chromosome 19. Here, we report that the two genes display similar levels of single nucleotide polymorphisms with the majority of polymorphic sites being identical. In addition, the LILRA6 gene exhibits copy number variation (CNV) whereas LILRB3 does not. A screen of healthy Caucasians indicated that 32 % of the subjects possessed more than two copies of LILRA6, whereas 4 % have only one copy of the gene per diploid genome. Analysis of mRNA expression in the major fractions of PBMCs showed that LILRA6 is primarily expressed in monocytes, similarly to LILRB3, and its expression level correlates with copy number of the gene. We suggest that the LILRA6 CNV may influence the level of the activating receptor on the cell surface, potentially affecting signaling upon LILRB3/A6 ligation.

Figure 1

Copy numbers of LILRA6 and LILRB3 determined by two independent assays. The Sybr Green and Taqman PCRs targeted distinct fragments in each gene and demonstrated identical results (see Table 1).

Figure 2

Electrophoregrams of a polymorphic LILRA6 genomic fragment sequenced in donors P1, 2, 3, 5, 7, 10 (a), P4 (b), P8 (c), P6 (d), and P9 (e). Nucleotide variants at positions 1322–1323 (exon 6, NM_024318) and corresponding alleles are shown under the electrophoregrams. One representative picture is presented in A. Trimorphism in P8 (c) as well as comparison of P6 (d) and P9 (e) support CNV.

Figure 3

LILRA6 CNV in 38 CEPH pedigrees. Cells with copy numbers >2 are grey and with one copy are balck. Copy numbers that did not pass high confidence criteria are underlined (8% of all samples). Haplotypes based on segregation analysis are shown in parentheses. Families 2 and 12 – French; 102 and 104 – Venezuelan; others – Utah. Three groups of families are related: 102/104, 1344/1375, 1328/13281/13291/13292/13293/13294 (www.ccr.coriell.org; Figure S3). Blank cells in parents denote absence of DNA samples.

Figure 4

Analysis of LILRA6 and LILRB3 transcription in PBMC by SYBR Green qPCR. Cell fractions were isolated using EasySep selection and included monocytes (CD14), B cells (CD19), T cells (CD4 and CD8) and NK cells (CD56). Expression was quantified using equimolar mixture of LILRB3/A6 PCR fragments as reference and normalized to the total RNA input (measured in the LabChip DX).

Figure 5

Relative level of LILRA6/B3 mRNA expression correlates with the CNV genotype. Total RNA was isolated from PBMC of healthy donors and the corresponding cDNA was used in SYBR Green qPCR. The PCR_mix is an equimolar mixture of LILRB3 and LILRA6 PCR fragments. Genomic DNA from an individual with two copies of LILRA6 was used as a reference control (gDNA, LILRA6/LILRB3=1). All reactions were performed in triplicate.

Figure 6

Schematic representation of NAHR between chromosomes possessing one copy of each gene. In the duplicated haplotype, LILRB3 stays intact and the newly formed hybrid gene contains the 3’ end of LILRA6 and is therefore defined as LILRA6 due to its “activating” 3’ end. The reciprocal haplotype retains only a hybrid gene that has the 3’ end of LILRB3, which defines the gene as inhibitory. If the recombinant chromosomes shown in the scheme were involved in further NAHR, the two daughter recombinant chromosomes will always have a single copy of LILRB3, and LILRA6 will either eliminated altogether or it will be present as one copy or multiple tandem copies.