Evolution of HLA-F and its orthologues in primate species: a complex tale of conservation, diversification and inactivation.

HLA-F represents one of the nonclassical MHC class I molecules in humans. Its main characteristics involve low levels of polymorphism in combination with a restricted tissue distribution. This signals that the gene product executes a specialised function, which, however, is still poorly understood. Relatively little is known about the evolutionary equivalents of this gene in nonhuman primates, especially with regard to population data. Here we report a comparative genetic analysis of the orthologous genes of HLA-F in various great ape, Old World monkey (OWM), and New World monkey (NWM) species. HLA-F-related transcripts were found in all subjects studied. Low levels of polymorphism were encountered, although the length of the predicted gene products may vary. In most species, one or two transcripts were discovered, indicating the presence of only one active F-like gene per chromosome. An exception was provided by a New World monkey species, namely, the common marmoset. In this species, the gene has been subject to duplication, giving rise to up to six F-like transcripts per animal. In humans, great apes, and OWM, and probably the majority of the NWM species, the evolutionary equivalents of the HLA-F gene experienced purifying selection. In the marmoset, however, the gene was initially duplicated, but the expansion was subjected afterwards to various mechanisms of genetic inactivation, as evidenced by the presence of pseudogenes and an array of genetic artefacts in a section of the transcripts.

MHC class I diversity of olive baboons (Papio anubis) unravelled by next-generation sequencing.

The olive baboon represents an important model system to study various aspects of human biology and health, including the origin and diversity of the major histocompatibility complex. After screening of a group of related animals for polymorphisms associated with a well-defined microsatellite marker, subsequent MHC class I typing of a selected population of 24 animals was performed on two distinct next-generation sequencing (NGS) platforms. A substantial number of 21 A and 80 B transcripts were discovered, about half of which had not been previously reported. Per animal, from one to four highly transcribed A alleles (majors) were observed, in addition to ones characterised by low transcripion levels (minors), such as members of the A*14 lineage. Furthermore, in one animal, up to 13 B alleles with differential transcription level profiles may be present. Based on segregation profiles, 16 Paan-AB haplotypes were defined. A haplotype encodes in general one or two major A and three to seven B transcripts, respectively. A further peculiarity is the presence of at least one copy of a B*02 lineage on nearly every haplotype, which indicates that B*02 represents a separate locus with probably a specialistic function. Haplotypes appear to be generated by recombination-like events, and the breakpoints map not only between the A and B regions but also within the B region itself. Therefore, the genetic makeup of the olive baboon MHC class I region appears to have been subject to a similar or even more complex expansion process than the one documented for macaque species.

A quick and robust MHC typing method for free-ranging and captive primate species.

Gene products of the major histocompatibility complex (MHC) of human and non-human primates play a crucial role in adaptive immunity, and most of the relevant genes not only show a high degree of variability (polymorphism) but also copy number variation (CNV) is observed. Due to this diversity, MHC proteins influence the capability of individuals to cope with various pathogens. MHC and/or MHC-linked gene products such as odorant receptor genes are thought to influence mate choice and reproductive success. Therefore, MHC typing of wild and captive primate populations is considered to be useful in conservation biology, which is, however, often hampered by the need of invasive and time-consuming methods. All intact Mhc-DRB genes in primates appear to possess a complex and highly divergent microsatellite, DRB-STR. A panel of 154 pedigreed olive baboons (Papio anubis) was examined for their DRB content by DRB-STR analysis of genomic DNA. Using the same methodology on DNA of feces samples, DRB variability of a silvery gibbon population (Hylobates moloch) (N = 24), an endangered species, could successfully be studied. In both species, length determination of the DRB-STR resulted in the definition of unique genotyping patterns that appeared to be specific for a certain chromosome. Moreover, the different STR lengths were shown to segregate with the allelic variation of the respective gene. The results obtained expand data gained previously on DRB-STR typing in macaques, great apes, and humans and strengthen the conclusion that this protocol is applicable in molecular ecology, conservation biology, and colony management, especially of endangered primate species.

No postcopulatory selection against MHC-homozygous offspring: Evidence from a pedigreed captive rhesus macaque colony.

The heterozygosity status of polymorphic elements of the immune system, such as the major histocompatibility complex (MHC), is known to increase the potential to cope with a wider variety of pathogens. Pre- and postcopulatory processes may regulate MHC heterozygosity. In a population where mating occurs among individuals that share identical MHC haplotypes, postcopulatory selection may disfavour homozygous offspring or ones with two MHC haplotypes identical to its mother. We tested these ideas by determining the incidence of MHC-heterozygous and MHC-homozygous individuals in a pedigreed, partially consanguineous captive rhesus monkey colony. Bayesian statistics showed that when parents share MHC haplotypes, the distribution of MHC-heterozygous and MHC-homozygous individuals significantly fitted the expected Mendelian distribution, both for the complete MHC haplotypes, and for MHC class I or II genes separately. Altogether, we found in this captive colony no evidence for postcopulatory selection against MHC-homozygous individuals. However, the distribution of paternally and maternally inherited MHC haplotypes tended to differ significantly from expected. Individuals with two MHC haplotypes identical to their mother were underrepresented and offspring with MHC haplotypes identical to their father tended to be overrepresented. This suggests that postcopulatory processes affect MHC haplotype combination in offspring, but do not prevent low MHC heterozygosity.

Novel DRA alleles extracted from seven macaque cohorts.

In this document, we report the detection of 37 DRA alleles in macaque cohorts.

Mutational bias provides a model for the evolution of Huntington's disease and predicts a general increase in disease prevalence.

Huntington's disease (HD) correlates with abnormal expansion in a block of CAG repeats in the Huntington's disease gene. We have investigated HD evolution by typing CAG alleles in several human populations and in a variety of primates. We find that human alleles have expanded from a shorter ancestral state and exhibit unusual asymmetric length distributions. Computer simulations are used to show that the human state can be derived readily from a primate ancestor, without the need to invoke natural selection. The key element is a simple length-dependent mutational bias towards longer alleles. Our model can explain a number of empirical observations, and predicts an ever-increasing incidence of HD.

A high density of human communication-associated genes in chromosome 7q31-q36: differential expression in human and non-human primate cortices.

The human brain is distinguished by its remarkable size, high energy consumption, and cognitive abilities compared to all other mammals and non-human primates. However, little is known about what has accelerated brain evolution in the human lineage. One possible explanation is that the appearance of advanced communication skills and language has been a driving force of human brain development. The phenotypic adaptations in brain structure and function which occurred on the way to modern humans may be associated with specific molecular signatures in today's human genome and/or transcriptome. Genes that have been linked to language, reading, and/or autism spectrum disorders are prime candidates when searching for genes for human-specific communication abilities. The database and genome-wide expression analyses we present here revealed a clustering of such communication-associated genes (COAG) on human chromosomes X and 7, in particular chromosome 7q31-q36. Compared to the rest of the genome, we found a high number of COAG to be differentially expressed in the cortices of humans and non-human primates (chimpanzee, baboon, and/or marmoset). The role of X-linked genes for the development of human-specific cognitive abilities is well known. We now propose that chromosome 7q31-q36 also represents a hot spot for the evolution of human-specific communication abilities. Selective pressure on the T cell receptor beta locus on chromosome 7q34, which plays a pivotal role in the immune system, could have led to rapid dissemination of positive gene variants in hitchhiking COAG.

Methylation and expression analyses of the 7q autism susceptibility locus genes MEST , COPG2, and TSGA14 in human and anthropoid primate cortices.

The autism susceptibility locus on human chromosome 7q32 contains the maternally imprinted MEST and the non-imprinted COPG2 and TSGA14 genes. Autism is a disorder of the 'social brain' that has been proposed to be due to an overbalance of paternally expressed genes. To study regulation of the 7q32 locus during anthropoid primate evolution, we analyzed the methylation and expression patterns of MEST, COPG2, and TSGA14 in human, chimpanzee, Old World monkey (baboon and rhesus macaque), and New World monkey (marmoset) cortices. In all human and anthropoid primate cortices, the MEST promoter was hemimethylated, as expected for a differentially methylated imprinting control region, whereas the COPG2 and TSGA14 promoters were completely demethylated, typical for transcriptionally active non-imprinted genes. The MEST gene also showed comparable mRNA expression levels in all analyzed species. In contrast, COPG2 expression was downregulated in the human cortex compared to chimpanzee, Old and New World monkeys. TSGA14 either showed no differential regulation in the human brain compared to chimpanzee and marmoset or a slight upregulation compared to baboon. The human-specific downregulation supports a role for COPG2 in the development of a 'social brain'. Promoter methylation patterns appear to be more stable during evolution than gene expression patterns, suggesting that other mechanisms may be more important for inter-primate differences in gene expression.

Novel major histocompatibility complex class I alleles extracted from two rhesus macaque populations.

We report here the novel Mamu-A and -B alleles that were detected in two groups of rhesus monkeys.

TRIM5 allelic polymorphism in macaque species/populations of different geographic origins: its impact on SIV vaccine studies.

Tripartite motif 5α (TRIM5α) is a potent antiretroviral immune factor present in the cytoplasm of cells of most tissue types. The rhesus macaque TRIM5 gene has been shown to display polymorphism, with different variants being divided into three groups (TRIM5(TFP), TRIM5(Q), and TRIM5(CypA)), which may have divergent retroviral effects on infection. Along with rhesus macaques, cynomolgus macaques are also used in simian immunodeficiency virus (SIV) infection studies. As a consequence, TRIM5 genotyping of these animals will contribute to interpreting the outcome of such studies. The present communication covers Burmese, Chinese, and a large cohort of Indian-origin rhesus macaques, and describes the first large cohort study on TRIM5 polymorphism in outbred cynomolgus macaques. We demonstrate the presence of the TRIM5(TFP) group in cynomolgus macaques. In addition, we have re-evaluated historical samples of rhesus macaques challenged with SIV(mac251), a virus that has been reported to be partially suppressed by particular rhesus macaque TRIM5 variants.

Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef.

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.

Major histocompatibility complex class II-restricted antigen presentation across a species barrier: conservation of restriction determinants in evolution.

The existence of at least three alleles of the HLA-DRB3 gene within the human population is evident. These alleles express DRw52 determinants and react with monoclonal antibody (mAb) 7.3.19.1. The polymorphic epitope recognized by 7.3.19.1 is not only present on human cells but is also expressed on chimpanzee (Pan troglodytes) class II-positive cells. The 7.3.19.1 determinant already existed before speciation of man and chimpanzee, and is at least 5,000,000 yr old. Two-dimensional gel electrophoresis demonstrated that the various HLA- and Patr-DRw52 molecules that are reactive with 7.3.19.1 exhibit isoelectric point differences due to primary amino acid heterogeneity, as was confirmed by sequencing data. Sequence comparison allowed us to map the binding site of mAb 7.3.19.1 to the alpha helix of the major histocompatibility complex (MHC) class II DRB1 domain surrounding the antigen-binding cleft. Despite MHC sequence variation, chimpanzee antigen-presenting cells can present antigen (purified protein derivative) to human T cell lines and vice versa. Only the HLA- and Patr-DRw52 molecules were shown to function as restriction elements for antigen presentation across this species barrier. It is concluded that these particular restriction determinants probably have been conserved in evolution. The HLA- and Patr-DRw52 molecules represent alleles displaying polymorphism that has been selected for in evolution. Such "biomutants" may thus be more useful to study the biological significance of MHC molecules than MHC variants that have been generated by in vitro mutagenesis experiments.

Evolutionary conservation of major histocompatibility complex-DR/peptide/T cell interactions in primates.

Many major histocompatibility complex (MHC) polymorphisms originate from ancient structures that predate speciation. As a consequence, members of the Mhc-DRB1*03 allelic lineage are not only present in humans but in chimpanzees and rhesus macaques as well. This emphasizes that Mhc-DRB1*03 members must have been present in a common ancestor of these primate species that lived about 30 million years ago. Due to the accumulation of genetic variation, however, alleles of the Mhc-DRB1*03 lineage exhibit species-unique sequences. To investigate the biological importance of such conservation and variation, we have studied both the binding and antigen presentation capacity of various trans-species Mhc-DRB1*03 lineage members. Here we show that p3-13 of the 65-kD heat-shock protein (hsp65) of Mycobacterium leprae and M. tuberculosis binds not only to HLA-DR17(3) but also to some chimpanzee and rhesus macaque class II-positive cells. Comparison of the corresponding human, chimpanzee, and rhesus macaque Mhc-DRB1*03 lineage members revealed the presence of uniquely shared amino acid residues, at positions 9-13 and 26-31, of the antigen-binding site that are critical for p3-13 binding. In addition it is shown that several nonhuman primate antigen-presenting cells that bind p3-13 can activate HLA-DR17-restricted T cells. Certain amino acid replacements, however, in Mhc-DRB1*03 lineage members did not influence peptide binding or T cell recognition. Therefore, these studies demonstrate that some polymorphic amino acid residues (motifs) within the antigen-binding site of MHC class II molecules that are crucial for peptide binding and recognition by the T cell receptor have been conserved for over 30 million years.

HLA-DO polymorphism associated with resistance to type I diabetes detected with monoclonal antibodies, isoelectric point differences, and restriction fragment length polymorphism.

A new HLA-DQ-related genetic system with two alleles, 2B3 and TA10, defined serologically by MAbs and alloantisera, showed an almost perfect correlation with charge differences on DQ beta molecules, as well as with two polymorphic DNA fragments hybridizing with a DQ beta probe and various restriction enzymes on a panel of 14 DR4+ homozygous typing cells. It was therefore concluded that the serologically defined alleles 2B3 and TA10 are coded by the DQ beta gene and situated on the HLA-DQ beta chain. This 2B3/TA10 polymorphism is independent of HLA-D and segregates with HLA in families. The TA10 allele appears to be a new marker for resistance to type I diabetes, which is independent from the known resistance marker DR2, whereas no association was observed between this DQ beta polymorphism and rheumatoid arthritis.

Resistance to collagen-induced arthritis in a nonhuman primate species maps to the major histocompatibility complex class I region.

Type II collagen-induced arthritis (CIA) is an experimentally inducible autoimmune disorder that is, just like several forms of human arthritis, influenced by a genetic background. Immunization of young rhesus monkeys (Macaca mulatta) with type II collagen (CII) induced CIA in about 70% of the animals. One major histocompatibility complex (MHC) class I allele was present only in young animals resistant to CIA and absent in arthritic animals. This strong association suggests that the MHC class I allele itself, or a closely linked gene, determines resistance to CIA. The mechanism controlling the resistance to CIA becomes less efficient in aged animals since older rhesus monkeys, which were positive for the resistance marker, developed a mild form of arthritis. At the cellular level it is demonstrated that resistance to CIA is reflected by a low responsiveness of T cells to CII. This association between a specified MHC class I allele and resistance to an autoimmune disease points at the importance of the MHC class I region in the regulation of the immune response to an autoantigen.

Major histocompatibility complex class I-associated vaccine protection from simian immunodeficiency virus-infected peripheral blood cells.

To evaluate the effectiveness of vaccine protection from infected cells from another individual of the same species, vaccinated rhesus macaques (Macaca mulatta) were challenged with peripheral blood mononuclear cells from another animal diagnosed with acquired immune deficiency syndrome (AIDS). Half of the simian immunodeficiency virus (SIV)-vaccinated animals challenged were protected, whereas unprotected vaccinates progressed as rapidly to AIDS. Protection was unrelated to either total antibody titers to human cells, used in the production of the vaccine, to HLA antibodies or to virus neutralizing activity. However, analysis of the serotype of each animal revealed that all animals protected against cell-associated virus challenge were those which were SIV vaccinated and which shared a particular major histocompatibility complex (MHC) class I allele (Mamu-A26) with the donor of the infected cells. Cytotoxic T lymphocytes (CTL) specific for SIV envelope protein were detected in three of four protected animals vs. one of four unprotected animals, suggesting a possible role of MHC class I-restricted CTL in protection from infected blood cells. These findings have possible implications for the design of vaccines for intracellular pathogens such as human immunodeficiency virus (HIV).

Patterns of diversity in HIV-related loci among subspecies of chimpanzee: concordance at CCR5 and differences at CXCR4 and CX3CR1.

Human immunodeficiency virus type 1 (HIV-1) arose in humans via zoonotic transmissions of simian immunodeficiency viruses (SIV(cpz)) from common chimpanzees, Pan troglodytes. Despite the close relatedness of the two viruses and their hosts, we do not yet understand what causes SIV(cpz) to be nonpathogenic in chimpanzees, and HIV/AIDS to be one of the most devastating infectious diseases to have emerged in humans. There have been a number of genes identified in humans that confer disease resistance/susceptibility toward HIV-1, but little is known about the evolution and diversity of most of these chemokine receptor genes in chimpanzees. Here we show that genetic variation in chimpanzees differs across the various loci related to HIV-1, and that the pattern of variation differs among the chimpanzee subspecies. For all three subspecies, low diversity at CCR5 is confined to a small area of chromosome 3, suggesting that a selective sweep at this locus may have predated subspeciation. In contrast, diversity and neutrality tests suggest differing evolutionary forces among subspecies at CXCR4 and CX(3)CR1, with directional selection (in Pan troglodytes vellerosus) and demographic expansion (Pan troglodytes troglodytes) offering the most likely scenarios. These are some of the first data demonstrating differentiation in functional loci among chimpanzee subspecies.

High resolution definition of HLA-DRB haplotypes by a simplified microsatellite typing technique.

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2-positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing; thus, it is suitable for a variety of researchers in diverse research areas.

Fifty-one full-length major histocompatibility complex class II alleles in the olive baboon (Papio anubis).

Here we report 51 novel major histocompatibility complex (MHC) class II alleles in a group of related olive baboons.