Distinct frequency patterns of LILRB3 and LILRA6 allelic variants in Europeans

Abstract

The leukocyte immunoglobulin-like receptor (LILR)B3 and LILRA6 genes encode homologous myeloid inhibitory and activating orphan receptors, respectively. Both genes exhibit a strikingly high level of polymorphism at the amino acid level and LILRA6 (but not LILRB3) displays copy number variation (CNV). Although multiple alleles have been reported for both genes, limited data is available on frequencies of these alleles among humans. We have sequenced LILRB3/A6 exons encoding signal peptides and ectodomains in 91 healthy blood donors of European descent who carry one or two copies of LILRA6 per diploid genome. Analysis of haplotypes among individuals with two LILRA6 copies, representing the majority in this cohort (N = 86), shows that common LILRB3 and LILRA6 alleles encode some distinct amino acid sequences in homologous regions of the receptors, which could potentially impact their respective functions differentially. Comparison of sequences in individuals with one vs. two copies of LILRA6 supports non-allelic homologous recombination between LILRB3 and LILRA6 as a mechanism for generating LILRA6 CNV and LILRB3 diversity. These data characterize LILRB3/LILRA6 genetic variation in more detail than previously described and underscore the need to determine their ligands.

Keywords: Copy number variation; LILR; Non-allelic homologous recombination; Polymorphism.

Fig. 1

Polymorphism in LILRB3 and LILRA6 observed in individuals with two copies of LILRA6. (A) The heatmap shows nonsynonymous SNPs in exons encoding the signal peptide (SP) and ectodomain, including Ig-like domains D1-D4. Only SNPs with frequencies > 5% in at least one of the genes are included. The nucleotide variants and corresponding amino acids (in parentheses) are shown on the left. Protein domains are depicted on the right. Frequencies provided represent the second variant (e.g., the frequency of A is shown for G10A). Nucleotide positions are relative to the ATG start codon and amino acid positions correspond to the mature protein. Amino acid positions that have variants with > 5% frequency in both genes are highlighted in blue and those that have variants with > 5% only in one gene are shown in red. (B) Ribbon diagrams of signal peptides and ectodomains in LILRB3 and LILRA6 depicting polymorphic sites highlighted in blue and red as defined in (A). The 3D structures correspond to 421 amino acid–long fragments of the AlphaFold models O75022 for human LILRB3 (https://alphafold.ebi.ac.uk/entry/O75022). Rendering was performed with a custom script in PyMOL 2.4 software package by Schrodinger (https://pymol.org/2/)

Fig. 2

LILRB3 and LILRA6 allotypes in individuals with two copies of LILRA6. Phase of the nonsynonymous amino acid variants was estimated using Haploview (first 436 amino acids). Only haplotypes with frequencies > 3% are included. Dashes indicate identical amino acids to the reference sequence LILRB3_1 encoded by transcript ENST00000445347. Domains corresponding to amino acid positions are shown on top. Matching variants from previous work are shown on the right: ref 1 – Bashirova et al. (2014), ref 2 – Lopez-Alvarez et al. (2014), ref 3 – Hirayasu et al. (2021)

Fig. 3

Phylogenetic relationship between LILRB3 and LILRA6 allotypes estimated in individuals with two copies of LILRA6. A neighbor-joining tree was constructed based on alignment of homologous fragments of the proteins (first 436 amino acids). Poisson-corrected evolutionary distances between sequences were computed from amino acid identities. The tree is rooted at its midpoint and the scale indicates the proportion of amino acid divergence between sequences

Fig. 4

LILRB3 amino acid sequence and predicted allotypes in individuals with one copy of LILRA6. (A) LILRB3 amino acid sequences encoded by genotypes of five individuals (HD87–HD91) are shown in bold. Gray highlights indicate positions that are fixed or rarely polymorphic in LILRB3 among 86 individuals who have two LILRA6 copies (i.e., one per chromosome). Predicted allotypes (allotype 1p and allotype 2p) encoded by the two LILRB3 alleles in each individual were determined by comparing the amino acid variants present in each of the five donors with common allotypes shown in Fig. 2. This comparison suggests the presence of a recombinant LILRB3 gene product (allotype 2p) containing LILRA6_1-like sequences in HD87, HD88, and HD90 and LILRA6_3-like sequences in HD89 and HD91 in the respective signal peptide/ectodomains. (B) Schematic representation of the predicted LILRB3/LILRA6 genotype in donor HD91. Genes are shown as boxes. Labels above the boxes depict the corresponding protein variants. The top chromosome has a “normal” configuration of the locus containing both LILRB3 and LILRA6 that has not undergone NAHR. The homologous chromosome on the bottom is theoretically derived from an ancestral NAHR, as it is missing the LILRA6 gene and carries a recombinant LILRB3 gene encoding a molecule identical to LILRA6_3 in its N-terminal fragment (the SP and most of the ectodomain) and LILRB3_5 in its C-terminal fragment (remainder of the ectodomain and the transmembrane/cytoplasmic domains), the latter of which defines this receptor as inhibitory LILRB3