Mamu-B genes and their allelic repertoires in different populations of Chinese-origin rhesus macaques

Since rhesus monkeys of Chinese origin have gained greater utilization in recent years, it is urgent to investigate the major histocompatibility complex (MHC) immunogenetics of Chinese rhesus macaques. In this study, we identified 81 Mamu-B sequences using complementary DNA cloning and sequencing on a cohort of 58 rhesus monkeys derived from three local populations of China. Twenty of these Mamu-B alleles are novel and four of them represent new lineages. Although more alleles are shared among different populations than Mamu-A locus, the Mamu-B allelic repertoires found in these three populations of Chinese macaques are largely independent, which underscores the MHC polymorphism among different populations of Chinese rhesus macaques. Our results are an important addition to the limited MHC immunogenetic information available for rhesus macaques of Chinese origin.     

Diversity of MHC class I genes in Burmese-origin rhesus macaques

Rhesus macaques (Macaca mulatta) are widely used in developing a strategy for vaccination against human immunodeficiency virus by using simian immunodeficiency virus infection as a model system. Because the genome diversity of major histocompatibility complex (MHC) is well known to control the immune responsiveness to foreign antigens, MHC loci in Indian- and Chinese-origin macaques used in the experiments have been characterized, and it was revealed that the diversity of MHC in macaques was larger than the human MHC. To further characterize the diversity of Mamu-A and Mamu-B loci, we investigated a total of 73 different sequences of Mamu-A, 83 sequences of Mamu-B, and 15 sequences of Mamu-I cDNAs isolated from Burmese-origin macaques. It was found that there were one to five expressing genes in each locus. Among the Mamu-A, Mamu-B, and Mamu-I sequences, 44 (60.2%), 45 (54.2%), and 8 (53.3%), respectively, were novel, and most of the other known alleles were identical to those reported from Chinese- or Indian-origin macaques, demonstrating a genetic mixture between the geographically distinct populations of present day China and India. In addition, it was found that a Mamu haplotype contained at least two highly transcribed Mamu-A genes, because multiple Mamu-A1 cDNAs were obtained from one haplotype. These findings further revealed the diversity and complexity of MHC locus in the rhesus macaques.     

Unique peptide-binding motif for Mamu-B*037:01: an MHC class I allele common to Indian and Chinese rhesus macaques

Indian and Chinese rhesus macaques are often used in biomedical research. Genetic analyses of the major histocompatibility class I region have revealed that these macaques display a substantial level of polymorphism at Mamu-A and Mamu-B loci, which have been subject to duplication. Only a few Mamu class I allotypes are characterised for their peptide-binding motifs, although more information of this nature would contribute to a better interpretation of T cell-mediated immune responses. Here, we present the results of the characterisation of the functional properties of Mamu-B*037:01, an allotype commonly encountered in rhesus macaques of Indian and Chinese origin. Mamu-B*037:01 is seen to have a strong preference for acidic amino acids at the third residue, and for arginine, lysine, and tyrosine at the carboxyl terminus. This peptide-binding motif is not described in the human population.     

Comparative genetics of a highly divergent DRB microsatellite in different macaque species

The DRB region of the major histocompatibility complex (MHC) of cynomolgus and rhesus macaques is highly plastic, and extensive copy number variation together with allelic polymorphism makes it a challenging enterprise to design a typing protocol. All intact DRB genes in cynomolgus monkeys (Mafa) appear to possess a compound microsatellite, DRB-STR, in intron 2, which displays extensive length polymorphism. Therefore, this STR was studied in a large panel of animals, comprising pedigreed families as well. Sequencing analysis resulted in the detection of 60 Mafa-DRB exon 2 sequences that were unambiguously linked to the corresponding microsatellite. Its length is often allele specific and follows Mendelian segregation. In cynomolgus and rhesus macaques, the nucleotide composition of the DRB-STR is in concordance with the phylogeny of exon 2 sequences. As in humans and rhesus monkeys, this protocol detects specific combinations of different DRB-STR lengths that are unique for each haplotype. In the present panel, 22 Mafa-DRB region configurations could be defined, which exceeds the number detected in a comparable cohort of Indian rhesus macaques. The results suggest that, in cynomolgus monkeys, even more frequently than in rhesus macaques, new haplotypes are generated by recombination-like events. Although both macaque species are known to share several identical DRB exon 2 sequences, the lengths of the corresponding microsatellites often differ. Thus, this method allows not only fast and accurate DRB haplotyping but may also permit discrimination between highly related macaque species.     

Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

Background

Cynomolgus macaques (Macaca fascicularis) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.

Results

We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.

Conclusion

BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.     

Genetic diversity and population structure in wild Sichuan rhesus macaques

Because wild rhesus macaque (Macaca mulatta) populations have suffered major declines, there is a growing need to characterize their genetic and population structure in order to protect the genetic integrity of this species. In this study, we genotyped a sample comprising 120 wild rhesus macaques from six sites in Sichuan Province for 30 nuclear microsatellite (STR) loci using an ABI 3130xl genetic analyzer. Bayesian analyses and PCA clearly differentiated monkeys from Heishui from those at other sites. The samples from all six sites exhibited high gene diversity suggesting that the Sichuan wild rhesus macaque populations are not threatened by a lack of genetic diversity. Deviation from Hardy–Weinberg equilibrium was more frequent in the Danba and Heishui populations. This may be due to the more fragmented habitat and less disturbance by humans in this area that foster greater subpopulation structuring than occurs in eastern China. We suggest that this population subdivision is the result of both long-term geographic barriers and human activity.     

Comprehensive identification of MHC class II alleles in a cohort of Chinese rhesus macaques

Rhesus macaque is a very important animal model for various human diseases, especially for AIDS and vaccine research. The susceptibility and/or resistance to some of these diseases are related to the major histocompatibility complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, Mamu-DPB1, Mamu-DQB1, and Mamu-DRB alleles were investigated in 30 Chinese rhesus macaques through gene cloning and sequencing. A total of 66 alleles were identified in this study, including 14 Mamu-DPB1, 20 Mamu-DQB1, and 30 Mamu-DRB alleles as well as 2 high-frequency Mamu-DPB1 alleles. Interestingly, one of the high-frequency Mamu-DPB1 alleles had been undocumented in earlier studies. Eleven of the other alleles, including four Mamu-DPB1, three Mamu-DQB1, and four Mamu-DRB alleles were also novel. Importantly, like MHC-DRB, more than two Mamu-DPB1 sequences per animal were detected in 13 monkeys, which suggested that they might represent gene duplication. Our data also indicated quite a few differences in the distribution of MHC class II alleles between the Chinese rhesus macaques and the previously reported Indian rhesus macaques. To our knowledge, our results revealed comprehensively the combination of MHC II alleles. This information will not only promote the understanding of Chinese rhesus macaque MHC polymorphism but will also facilitate the use of Chinese rhesus macaques in studies of human disease.     

Microsatellite typing of the rhesus macaque MHC region

To improve the results gained by serotyping rhesus macaque major histocompatibility complex (MHC) antigens, molecular typing techniques have been established for class I and II genes. Like the rhesus macaque Mamu-DRB loci, the Mamu-A and -B are not only polymorphic but also polygenic. As a consequence, sequence-based typing of these genes is time-consuming. Therefore, eight MHC-linked microsatellites, or short tandem repeats (STRs), were evaluated for their use in haplotype characterization. Polymorphism analyses in rhesus macaques of Indian and Chinese origin showed high STR allelic diversity in both populations but different patterns of allele frequency distribution between the groups. Pedigree data for class I and II loci and the eight STRs allowed us to determine extended MHC haplotypes in rhesus macaque breeding groups. STR sequencing and comparisons with the complete rhesus macaque MHC genomic map allowed the exact positioning of the markers. Strong linkage disequilibria were observed between Mamu-DR and -DQ loci and adjacent STRs. Microsatellite typing provides an efficient, robust, and quick method of genotyping and deriving MHC haplotypes for rhesus macaques regardless of their geographical origin. The incorporation of MHC-linked STRs into routine genetic tests will contribute to efforts to improve the genetic characterization of the rhesus macaque for biomedical research and can provide comparative information about the evolution of the MHC region.     

Structure and Phylogenetic Analysis of an Endogenous Retrovirus Inserted into the Human Growth Factor Gene Pleiotrophin

A human endogenous retrovirus-like element (HERV), flanked by long terminal repeats of 502 and 495 nucleotides is inserted into the human pleiotrophin (PTN) gene upstream of the open reading frame. Based on its Glu-tRNA primer binding site specificity and the location within the PTN gene, we named this element HERV-E.PTN. HERV-E.PTN appears to be a recombined viral element based on its high homology (70 to 86%) in distinct areas to members of two distantly related HERV type C families, HERV-E and retrovirus-like element I (RTVL-I). Furthermore, its pseudogene region is organized from 5′ to 3′ into gag-, pol-, env-, pol-, env-similar sequences. Interestingly, full-length and partial HERV-E.PTN-homologous sequences were found in the human X chromosome, the human hereditary haemochromatosis region, and the BRCA1 pseudogene. Finally, Southern analyses indicate that the HERV-E.PTN element is present in the PTN gene of humans, chimpanzees, and gorillas but not of rhesus monkeys, suggesting that genomic insertion occurred after the separation of monkeys and apes about 25 million years ago.     

Identification of MHC class I sequences in Chinese-origin rhesus macaques

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population.     

High levels of diversity characterize mandrill (Mandrillus sphinx) Mhc-DRB sequences

The major histocompatibility complex (MHC) is highly polymorphic in most primate species studied thus far. The rhesus macaque (Macaca mulatta) has been studied extensively and the Mhc-DRB region demonstrates variability similar to humans. The extent of MHC diversity is relatively unknown for other Old World monkeys (OWM), especially among genera other than Macaca. A molecular survey of the Mhc-DRB region in mandrills (Mandrillus sphinx) revealed extensive variability, suggesting that other OWMs may also possess high levels of Mhc-DRB polymorphism. In the present study, 33 Mhc-DRB loci were identified from only 13 animals. Eleven were wild-born and presumed to be unrelated and two were captive-born twins. Two to seven different sequences were identified for each individual, suggesting that some mandrills may have as many as four Mhc-DRB loci on a single haplotype. From these sequences, representatives of at least six Mhc-DRB loci or lineages were identified. As observed in other primates, some new lineages may have arisen through the process of gene conversion. These findings indicate that mandrills have Mhc-DRB diversity not unlike rhesus macaques and humans.     

Blood groups of bonnet macaques (Macaca radiata), with a brief introduction to seroprimatology

The human-type A-B-O blood groups of 52 bonnet macaques (Macaca radiata) were determined. Application of method of population genetics indicated the gene frequences to be O = 0.173, A = 0.480 and B = 0.347. Cross testing of sera and red cells of the bonnet macaques revealed two blood-type-specific isoagglutinins, one of them strong enough for use as a blood typing reagent. No blood group polymorphism was revealed by testing bonnet macaque red cells with isoantisera produced in rhesus monkeys (M. mulatta) and in crab-eating macaques (M. fascicularis). The rhesus and crab-eating macaque isoantisera reacted either with all or with none of the bonnet macaque red cells tested.     

Structure of tumor necrosis factor-alpha haploblocks in European populations

DNA variants in the tumor necrosis factor-α (TNF) and linked lymphotoxin-α genes, and specific alleles of the highly polymorphic human leukocyte antigen B (HLA-B) gene have been implicated in a plethora of immune and infectious diseases. However, the tight linkage disequilibrium characterizing the central region of the human major histocompatibility complex (MHC) containing these gene loci has made difficult the unequivocal interpretation of genetic association data. To alleviate these difficulties and facilitate the design of more focused follow-up studies, we investigated the structure and distribution of HLA-B-specific MHC haplotypes reconstructed in a European population from unphased genotypes at a set of 25 single nucleotide polymorphism sites spanning a 66-kilobase long region across TNF. Consistent with the published data, we found limited genetic diversity across the so-called TNF block, with the emergence of seven common MHC haplotypes, termed TNF block super-haplotypes. We also found that the ancestral haplotype 8.1 shares a TNF block haplotype with HLA-B*4402. HLA-B*5701, a known protective allele in HIV-1 pathogenesis, occurred in a unique TNF block haplotype.     

An initial genetic linkage map of the rhesus macaque (Macaca mulatta) genome using human microsatellite loci

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate species in biomedical research. To create new opportunities for genetic and genomic studies using rhesus monkeys, we constructed a genetic linkage map of the rhesus genome. This map consists of 241 microsatellite loci, all previously mapped in the human genome. These polymorphisms were genotyped in five pedigrees of rhesus monkeys totaling 865 animals. The resulting linkage map covers 2048 cM including all 20 rhesus autosomes, with average spacing between markers of 9.3 cM. Average heterozygosity among those markers is 0.73. This linkage map provides new comparative information concerning locus order and interlocus distances in humans and rhesus monkeys. The map will facilitate whole-genome linkage screens to locate quantitative trait loci (QTLs) that influence individual variation in phenotypic traits related to basic primate anatomy, physiology, and behavior, as well as QTLs relevant to risk factors for human disease.     

Serotonin Transporter Genotype Modulates Social Reward and Punishment in Rhesus Macaques

Background

Serotonin signaling influences social behavior in both human and nonhuman primates. In humans, variation upstream of the promoter region of the serotonin transporter gene (5-HTTLPR) has recently been shown to influence both behavioral measures of social anxiety and amygdala response to social threats. Here we show that length polymorphisms in 5-HTTLPR predict social reward and punishment in rhesus macaques, a species in which 5-HTTLPR variation is analogous to that of humans.

Methodology/Principal Findings

In contrast to monkeys with two copies of the long allele (L/L), monkeys with one copy of the short allele of this gene (S/L) spent less time gazing at face than non-face images, less time looking in the eye region of faces, and had larger pupil diameters when gazing at photos of a high versus low status male macaques. Moreover, in a novel primed gambling task, presentation of photos of high status male macaques promoted risk-aversion in S/L monkeys but promoted risk-seeking in L/L monkeys. Finally, as measured by a “pay-per-view” task, S/L monkeys required juice payment to view photos of high status males, whereas L/L monkeys sacrificed fluid to see the same photos.

Conclusions/Significance

These data indicate that genetic variation in serotonin function contributes to social reward and punishment in rhesus macaques, and thus shapes social behavior in humans and rhesus macaques alike.     

Designing new microsatellite markers for linkage and population genetic analyses in rhesus macaques and other nonhuman primates

Identification of polymorphic microsatellite loci in nonhuman primates is useful for various biomedical and evolutionary studies of these species. Prior methods for identifying microsatellites in nonhuman primates are inefficient. We describe a new strategy for marker development that uses the available whole genome sequence for rhesus macaques. Fifty-four novel rhesus-derived microsatellites were genotyped in large pedigrees of rhesus monkeys. Linkage analysis was used to place 51 of these loci into the existing rhesus linkage map. In addition, we find that microsatellites identified this way are polymorphic in other Old World monkeys such as baboons. This approach to marker development is more efficient than previous methods and produces polymorphisms with known locations in the rhesus genome assembly. Finally, we propose a nomenclature system that can be used for rhesus-derived microsatellites genotyped in any species or for novel loci derived from the genome sequence of any nonhuman primate.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

Evolutionary patterns of killer cell Ig-like receptor genes in Old World monkeys

Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of Natural Killer cells. KIR genes are encoded in the Leucocyte Receptor Complex and are characterized by their high haplotypic diversity and polymorphism. The KIR system has been studied in only three species of Old World monkeys, the rhesus macaque, the cynomolgus macaque, and the sabaeus monkey, displaying a complexity rivaling that of hominids (human and apes). Here we analyzed bacterial artificial chromosome draft sequences spanning the KIR haplotype of three other Old World monkeys, the vervet monkey (Chlorocebus aethiops), the olive baboon (Papio anubis) and the colobus monkey (Colobus guereza). A total of 25 KIR gene models were identified in these species, predicted to encode receptors with 1, 2, and 3 extracellular Ig domains, all of them with long cytoplasmic domains having two putative ITIMs, although three had a positively charged residue in the transmembrane domain. Sequence and phylogenetic analyses showed that most Old World monkeys shared five classes of KIR loci: i) KIR2DL5/3DL20 in the most centromeric region, followed by ii) the single Ig domain-encoding locus KIR1D, iii) the pseudogene KIR2DP, iv) the conserved KIR2DL4, and v) the highly diversified KIR3DL/H loci in the telomeric half of the cluster. An exception to this pattern was the KIR haplotype of the colobus monkey that lacked the KIR1D, KIR2DP, and KIR2DL4 loci of the central region of the cluster. Thus, Old World monkeys display a broad spectrum of KIR haplotype variation that has been generated upon an ancestral haplotype architecture by gene duplication, gene deletion, and non-homologous recombination.