HLA class I variation in Iranian Lur and Kurd populations: high haplotype and allotype diversity with an abundance of KIR ligands.

HLA-A, -B and -C alleles of 285 individuals, representing three Iranian Lur populations and one Iranian Kurd population were sequenced completely, yielding human leukocyte antigen (HLA) class I genotypes at high resolution and filling four fields of the official HLA nomenclature. Each population has 87-99 alleles, evenly distributed between the three HLA class I genes, 145 alleles being identified in total. These alleles were already known, named and deposited in the HLA database. The alleles form 316 different HLA A-B-C haplotypes, with each population having between 80 and 112 haplotypes. The four Iranian populations form a related group that is distinguished from other populations, including other Iranians. All four KIR ligands - the A3/11, Bw4, C1 and C2 epitopes - are well represented, particularly Bw4, which is carried by three high-frequency allotypes: HLA-A*24:02, HLA-A*32:01 and HLA-B*51:01. In the Lur and Kurd populations, between 82% and 94% of individuals have the Bw4 epitope, the ligand for KIR3DL1. HLA-B*51:01 is likely of Neandertal origin and associated with Behcet's disease, also known as the Silk Road disease. The Lordegan Lur have the highest frequency of HLA-B*51:01 in the world. This allele is present on 46 Lur and Kurd haplotypes. Present at lower frequency is HLA-B*51:08, which is also associated with Behcet's disease. In the four Iranian populations, 31 haplotypes encode both Bw4(+) HLA-A and Bw4(+) HLA-B, a dual combination of Bw4 epitopes that is relatively rare in other populations, worldwide. This study both demonstrates and emphasizes the value of studying HLA class I polymorphism at highest resolution in anthropologically well-defined populations.

Description of the novel KIR2DL4*035 allele identified using high-throughput sequencing.

A newly identified allele of the KIR2DL4 natural killer cell receptor for human leukocyte antigen (HLA) class I.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets.

16(th) IHIW : review of HLA typing by NGS.

Human leucocyte antigen (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA-based genotyping methods, including Sanger sequence-based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high-resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next-generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to (i) template preparation including short- and long-range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele-calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Review: Immunogenetics of human placentation.

Natural killer (NK) cells are a population of lymphocytes that function in both immune defense and reproduction. Diversifying NK cell phenotype and function are interactions between NK cell receptors and major histocompatibility complex (MHC) class I ligands. As a consequence of strong and variable selection these ligand-receptor systems are polymorphic, rapidly evolving, and considerably species-specific. Counterparts to the human system of HLA class I ligands and killer cell immunoglobulin-like receptors (KIR) are present only in apes and Old World monkeys. HLA-C, the dominant ligand for human KIR and the only polymorphic HLA class I expressed by trophoblast, is further restricted to humans and great apes. Even then, the human system appears qualitatively different from that of chimpanzees, in that it has evolved a genetic balance between particular groups of receptors and ligands that favor reproductive success and other groups of receptors and ligands that have been correlated with disordered placentation. Human populations that have survived successive episodes of epidemic disease and population bottlenecks maintain a breadth of diversity for KIR and HLA class I, implying that loss of such diversity disfavors long-term survival of a human population.

An update to HLA nomenclature, 2010.

The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.

Analytic approximation of spatial epidemic models of foot and mouth disease.

The effect of spatial heterogeneity in epidemic models has improved with computational advances, yet far less progress has been made in developing analytical tools for understanding such systems. Here, we develop two classes of second-order moment closure methods for approximating the dynamics of a stochastic spatial model of the spread of foot and mouth disease. We consider the performance of such 'pseudo-spatial' models as a function of R(0), the locality in disease transmission, farm distribution and geographically-targeted control when an arbitrary number of spatial kernels are incorporated. One advantage of mapping complex spatial models onto simpler deterministic approximations lies in the ability to potentially obtain a better analytical understanding of disease dynamics and the effects of control. We exploit this tractability by deriving analytical results in the invasion stages of an FMD outbreak, highlighting key principles underlying epidemic spread on contact networks and the effect of spatial correlations.

Immunogenetics of killer-cell immunoglobulin-like receptors.

The immunogenetics of cell-surface antigens began with the study of red cells and then moved onto the white cells. HLA class I antigens were analyzed on leukocytes and HLA class II antigens on B cells. In the last decade the natural killer (NK) cell has become a target for immunogeneticists, in particular the family of genes encoding the killer-cell immunoglobulin-like receptors (KIRs).

Human KIR sequences 2003.

We have compiled the nucleotide sequences and their amino acid translations from a total of 89 Killer Immunoglobulin-like Receptor (KIR) alleles, derived from 17 different KIR genes. The alignments use the KIR3DL2*001 allele as a reference sequence. Each of the KIR sequences included in these alignments has been checked and where discrepancies have arisen between reported sequences, the original authors have been contacted where possible, and necessary amendments to published sequences have been incorporated into this alignment. Future sequencing may identify errors in this list and we would welcome any evidence that helps to maintain the accuracy of this compilation.

Cloning and sequencing full-length HLA-B and -C genes.

Currently most available HLA-A, -B and -C DNA sequences cover exons 2 and 3 with a limited number extending to include other exons and introns. We have developed a method for the accurate determination of full-length genomic DNA sequences for HLA-A, -B and -C alleles. The method involves cloning of PCR amplified full-length HLA genes to separate alleles at heterozygous loci. The approach avoids any ambiguities from sequencing heterozygous PCR products directly and also avoids ambiguities from sequencing overlapping PCR products to achieve full-length sequence. To date we have sequenced full-length genomic sequences from representatives of all the major HLA-B and -C allele groups.

Exon 5 encoding the transmembrane region of HLA-A contains a transitional region for the induction of nonsense-mediated mRNA decay.

HLA class I alleles containing premature termination codons (PTCs) are increasingly being found. To understand their effects on MHC class I expression, HLA-A*2402 mutants containing PTCs were transfected into class I-deficient cells, and expression of HLA-A mRNA and protein was determined. In exons 2, 3, and 4, and in the 5' part of exon 5, PTCs reduced mRNA levels by up to 90%, whereas in the 3' part of exon 5 and in exons 6 and 7 they had little effect. Transition in the extent of nonsense-mediated mRNA decay occurred within a 48-nt segment of exon 5, placed 58 nt upstream from the exon 5/exon 6 junction. This transition did not conform to the positional rule obeyed by other genes, which predicted it to be approximately 50-55 nt upstream of the exon 7/exon 8 junction and thus placing it in exon 6. Mutants containing extra gene segments showed the difference is caused by the small size of exons 5 and 6, which renders them invisible to the surveillance machinery. For the protein, a transition from secretion to membrane association occurs within a 26-nt segment of exon 5, 17 nt upstream of the exon 5/exon 6 junction. Premature termination in exon 5 can produce secreted and membrane-associated HLA-A variants expressed at high levels.

Comparison of chimpanzee and human leukocyte Ig-like receptor genes reveals framework and rapidly evolving genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.

A novel, nonclassical MHC class I molecule specific to the common chimpanzee.

All expressed human MHC class I genes (HLA-A, -B, -C, -E, -F, and -G) have functional orthologues in the MHC of the common chimpanzee (Pan troglodytes). In contrast, a nonclassical MHC class I gene discovered in the chimpanzee is not present in humans or the other African ape species. In exons and more so in introns, this Patr-AL gene is similar to the expressed A locus in the orangutan, Popy-A, suggesting they are orthologous. Patr-AL/Popy-A last shared a common ancestor with the classical MHC-A locus >20 million years ago. Population analysis revealed little Patr-AL polymorphism: just three allotypes differing only at residues 52 and 91. Patr-AL is expressed in PBMC and B cell lines, but at low level compared with classical MHC class I. The Patr-AL polypeptide is unusually basic, but its glycosylation, association with beta(2)-microglobulin, and antigenicity at the cell surface are like other MHC class I. No Patr-AL-mediated inhibition of polyclonal chimpanzee NK cells was detected. The Patr-AL gene is present in 50% of chimpanzee MHC haplotypes, correlating with presence of a 9.8-kb band in Southern blots. The flanking regions of Patr-AL contain repetitive/retroviral elements not flanking other class I genes. In sequenced HLA class I haplotypes, a similar element is present in the A*2901 haplotype but not the A*0201 or A*0301 haplotypes. This element, 6 kb downstream of A*2901, appears to be the relic of a human gene related to Patr-AL. Patr-AL has characteristics of a class I molecule of innate immunity with potential to provide common chimpanzees with responses unavailable to humans.