Study of cynomolgus monkey (Macaca fascicularis) Mhc DRB gene polymorphism in four populations

The cynomolgus macaque (Macaca fascicularis) is currently used as an animal model in various fields of immunology especially in the development of innovative vaccines for the prevention and treatment of infectious diseases. The polymorphism of the major histocompatibility complex (MHC) influences the development of adaptive immune responses, and it is crucial to characterize the polymorphism of cynomolgus MHC genes. Among all macaque species, the cynomolgus macaque has the most diversified geographical area encompassing continental and insular populations. By the study of a large sample of animals from the Philippines (N = 359), we have characterized 20 DRB haplotypes. The DRB genotyping was performed by denaturing gradient gel electrophoresis (DGGE) sequencing of exon 2 and was confirmed by polymerase chain reaction-sequence-specific oligonucleotide. The DRB and DRA cDNA of 126 animals were characterized by cloning and sequencing. By means of DGGE sequencing, we characterized the polymorphism of genomic DRB exon 2 in three other cynomolgus macaque population samples (Java, Vietnam, and Mauritius), and we discuss about the origin of the founders of the Mauritian and the Filipino cynomolgus macaque populations.     

Identification of new Mamu-DRB alleles using DGGE and direct sequencing

 Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates. Received: 29 May 1996 / Revised: 8 August 1996     

Identification of New World monkey MHC-DRB alleles using PCR,DGGE and direct sequencing

Identification of New World monkey MHC-DRB alleles has previously relied upon labor-intensive cloning and sequencing techniques. Here we describe a rapid and unambiguous way to distinguish DRB alleles in New World monkeys using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of New World monkey DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. The validity of this typing procedure was confirmed by the identification of all DRB alleles previously characterized by cloning and sequencing techniques from an individual cotton-top tamarin. Importantly, our analysis revealed DRB alleles not previously identified in this reference animal. Following validation of our technique, the protocol was employed for the characterization of MHC-DRB alleles in four other species of New World monkey: the pygmy marmoset, white-faced saki monkey, long-haired spider monkey and owl monkey. Using this technique, we identified five alleles from the cotton-top tamarin, five alleles from the owl monkey, three alleles from the long-haired spider monkey, three alleles from the white-faced saki monkey and two alleles from the pygmy marmoset. On the basis of phylogenetic tree analyses, 13 new DRB alleles were assigned to eight different MHC-DRB lineages. Whereas traditional DRB typing via cloning and sequencing provides limited information, our new technique provides a simple and relatively rapid way of identifying New World monkey MHC-DRB alleles.Nucleotide sequence data reported are available in the GenBank/EMBL/DDBJ databases under the accession numbers AJ544165–AJ544177     

Convergent evolution of major histocompatibility complex molecules in humans and New World monkeys

  In both Old World and New World monkeys Mhc-DRB sequences have been found which resemble human DRB1*03 and DRB3 genes in their second exon. The resemblance is shared sequence motifs and clustering of the genes or the encoded proteins in phylogenetic trees. This similarity could be due to common ancestry, convergence at the molecular level, or chance. To test which of these three explanations applies, we sequenced segments of New World monkey and macaque genes which encompass the entire second exon and large parts of both flanking introns. The test strongly supports the monophyly of New World monkey DRB intron sequences. The phylogenies of introns 1 and 2 from DRB1*03-like and DRB3-like genes are congruent, but both are incongruent with the exon 2-based phylogeny. The matching of intron 1- and intron 2-based phylogenies with each other suggests that reciprocal recombination has not played a major role in exon 2 evolution. Statistical comparisons of exon 2 from different DRB1*03 and DRB3 lineages indicate that it was neither gene conversion (descent), nor chance, but molecular convergence that has shaped their characteristic motifs. The demonstration of convergence in anthropoid Mhc-DRB genes has implications for the classification, age, and mechanism of generation of DRB allelic lineages. Received: 30 August 1999 / Revised: 19 October 1999     

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.     

Evolution of MHC-<Emphasis Type="Italic">DRB</Emphasis> class II polymorphism in the genus <Emphasis Type="Italic">Apodemus</Emphasis> and a comparison of <Emphasis Type="Italic">DRB</Emphasis> sequences within the family Muridae (Mammalia: Rodentia)

Allelic diversity at major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection over long periods of time, even across multiple speciation events. Trans-species sharing of MHC alleles among genera has been supported by many studies on mammals and fish, but in rodents, the results are ambiguous. We investigated natural levels of MHC-DRB variability and evolutionary processes in the wood mouse (Apodemus sylvaticus) and the yellow-necked mouse (Apodemus flavicollis), which are common, sympatric murid rodents in European forests. Using single-strand conformation polymorphism analysis and DNA sequencing, 38 DRB exon 2 alleles were detected among 162 A. sylvaticus from nine different locations in Germany and Switzerland, and 15 DRB exon 2 alleles were detected among 60 A. flavicollis from three different locations in northern Germany. There was evidence for balancing selection in both species. Phylogenetic analysis, including additional murid taxa, showed that the DRB exon 2 sequences did not separate according to species, consistent with trans-species evolution of the MHC in these taxa.     

Mhc-DRB Genes and the Origin of New World Monkeys

The major histocompatibility complex (Mhc) is a family of loci characterized by its relatively rapid evolutionary turnover, large genetic distances between genes, and long persistence of allelic lineages effected by balancing selection. These features render the Mhc highly suitable for answering questions concerning speciation and adaptive radiation. The aim of the present study was to use Mhc-DRB genes to make inferences about the founding population of the Platyrrhini. Three segments, each approximately 300 base pairs in length, of the platyrrhine DRB genes were amplified by the polymerase chain reaction and sequenced. The segments were derived from intron 2, exon 3, and exon 6 of DRB genes from different species of New World monkeys. The results of the study have revealed that on a phylogenetic tree, all of the tested platyrrhine genes appear to form a single cluster, while all catarrhine DRB genes form a distinct cluster, although the bootstrap values fail to provide statistically significant support for the separation of these two clades. This observation suggests that the multiple platyrrhine genes originated from a single ancestral gene after the divergence of the Platyrrhini and Catarrhini and thus contradicts the results of an earlier study in which some exon 2 DRB sequences appeared to predate the split of the two primate groups. The inconsistency in the DRB gene phylogeny can be explained by postulating convergent evolution for the peptide-binding region of the DRB exon 2 sequences. The phylogeny of the platyrrhine DRB genes (except for exon 2) is relatively "shallow"; the distances between genes are relatively short (in comparison to the catarrhine DRB genes), and there is a tendency for sequences of individual species to cluster together. The phylogeny of the platyrrhine DRB genes is consistent with the postulate that a small population founded the group and that there is an ongoing adaptive radiation from small, relatively isolated founding populations.     

Characterization of Mhc-DRB allelic diversity in white-tailed deer (Odocoileus virginianus) provides insight into Mhc-DRB allelic evolution within Cervidae

 Although white-tailed deer (Odocoileus virginianus) are one of North America's best studied mammals, no information is available concerning allelic diversity at any locus of the major histocompatibility complex in this taxon. Using the polymerase chain reaction, single-stranded conformation polymorphism analysis, and DNA sequencing techniques, 15 DRB exon 2 alleles were identified among 150 white-tailed deer from a single population in southeastern Oklahoma. These alleles represent a single locus and exhibit a high degree of nucleotide and amino acid polymorphism, with most amino acid variation occurring at positions forming the peptide binding sites. Furthermore, twenty-seven amino acid residues unique to white-tailed deer DRB alleles were detected, with 19 of these occurring at residues forming contact points of the peptide binding region. Significantly higher rates of nonsynonymous than synonymous substitutions were detected among these DRB alleles. In contrast to other studies of Artiodactyla DRB sequences, interallelic recombination does not appear to be playing a significant role in the generation of allelic diversity at this locus in white-tailed deer. To examine evolution of white-tailed deer (Odvi-DRB) alleles within Cervidae, we performed a phylogenetic analysis of all published red deer (Ceel-DRB), roe deer (Caca-DRB), and moose (Alal-DRB) DRB alleles. The phylogenetic tree clearly shows a trans-species persistence of DRB lineages among these taxa. Moreover, this phylogenetic tree provides insight into evolution of DRB allelic lineages within Cervidae and may aid in assignment of red deer DRB alleles to specific loci. Received: 25 June 1998 / Revised: 2 September 1998     

Demonstration of threeDRB locl in a domestic horse family

  Single-strand conformational polymorphism (SSCP) gel electrophoresis and DNA sequencing were used to characterize the second exon of the horse DRB homologue as well as to identify eight new DRB alleles. The SSCP gels presented a complex pattern, with phenotypes exhibiting between 4 and 13 bands. The DRB SSCP patterns were studied for two families (6 to 13 bands per pattern). For both families, the patterns showed simple Mendelian inheritance. The polymerase chain reaction products from two individuals possessing homozygous major histocompatibility complex (MHC) alleles by descent were cloned and retested on SSCP gels. All bands derived from the genomic DNA amplification could be accounted for with bands derived from the cloned DNA amplification products. The results were consistent with three DRB loci, though this number may be variable within the domestic horse population. Gene sequences were variable among the different products, and we were unable to assign locus designations for particular sequences. Amplification of cDNA library material derived from one of the individuals who is MHC homozygous by descent showed an SSCP profile suggesting that all three DRB loci are transcribed into mRNA. Received: 10 April 1996 / Revised: 2 July 1996     

Diversity and duplication of DQB and DRB-like genes of the MHC in baleen whales (suborder: Mysticeti)

The molecular diversity and phylogenetic relationships of two class II genes of the baleen whale major histocompatibility complex were investigated and compared to toothed whales and out-groups. Amplification of the DQB exon 2 provided sequences showing high within-species and between-species nucleotide diversity and uninterrupted reading frames consistent with functional class II loci found in related mammals (e.g., ruminants). Cloning of amplified products indicated gene duplication in the humpback whale and triplication in the southern right whale, with average nucleotide diversity of 5.9 and 6.3%, respectively, for alleles of each species. Significantly higher nonsynonymous divergence at sites coding for peptide binding (32% for humpback and 40% for southern right) suggested that these loci were subject to positive (overdominant) selection. A population survey of humpback whales detected 23 alleles, differing by up to 21% of their inferred amino acid sequences. Amplification of the DRB exon 2 resulted in two groups of sequences. One was most similar to the DRB3 of the cow and present in all whales screened to date, including toothed whales. The second was most similar to the DRB2 of the cow and was found only in the bowhead and right whales. Both loci showed low diversity among species and apparent loss of function or altered function including interruption of reading frames. Finally, comparison of inferred protein sequence of the DRB3-like locus suggested convergence with the DQB, perhaps resulting from intergenic conversion or recombination.Electronic Supplementary Material Supplementary material is available for this article at     

Introgression from Domestic Goat Generated Variation at the Major Histocompatibility Complex of Alpine Ibex

The major histocompatibility complex (MHC) is a crucial component of the vertebrate immune system and shows extremely high levels of genetic polymorphism. The extraordinary genetic variation is thought to be ancient polymorphisms maintained by balancing selection. However, introgression from related species was recently proposed as an additional mechanism. Here we provide evidence for introgression at the MHC in Alpine ibex (Capra ibex ibex). At a usually very polymorphic MHC exon involved in pathogen recognition (DRB exon 2), Alpine ibex carried only two alleles. We found that one of these DRB alleles is identical to a DRB allele of domestic goats (Capra aegagrus hircus). We sequenced 2489 bp of the coding and non-coding regions of the DRB gene and found that Alpine ibex homozygous for the goat-type DRB exon 2 allele showed nearly identical sequences (99.8%) to a breed of domestic goats. Using Sanger and RAD sequencing, microsatellite and SNP chip data, we show that the chromosomal region containing the goat-type DRB allele has a signature of recent introgression in Alpine ibex. A region of approximately 750 kb including the DRB locus showed high rates of heterozygosity in individuals carrying one copy of the goat-type DRB allele. These individuals shared SNP alleles both with domestic goats and other Alpine ibex. In a survey of four Alpine ibex populations, we found that the region surrounding the DRB allele shows strong linkage disequilibria, strong sequence clustering and low diversity among haplotypes carrying the goat-type allele. Introgression at the MHC is likely adaptive and introgression critically increased MHC DRB diversity in the genetically impoverished Alpine ibex. Our finding contradicts the long-standing view that genetic variability at the MHC is solely a consequence of ancient trans-species polymorphism. Introgression is likely an underappreciated source of genetic diversity at the MHC and other loci under balancing selection.     

Exonic polymorphism vs intronic simple repeat hypervariability in MHC-DRB genes

Gene products encoded by the major histocompatibility complex often exhibit a high degree of polymorphism. In humans the HLA-DR polymorphism is due to more than 50 alleles with varying exon 2 sequences. Each group of DRB alleles contains a certain form of the basic simple repeat motif (gt)_n(ga)_m in intron 2. Identical alleles can be differentiated on the basis of the hypervariable repeat. In this study focused on cattle (Bos taurus) we identified different Bota-DRB alleles in a limited survey by amplification via polymerase chain reaction and sequencing. In addition DRB exon 2 sequences were also obtained from eight additional hoofed animal species (seven horned artiodactyls and one pig) revealing artiodactyl-specific polymorphic and nonpolymorphic substitutions. In the genus Bos the intronic simple repeat variability was compared with exonic DRB polymorphism. As in humans all Bota-DRB exons were always associated with specifically organized basic simple repeat structures. Yet the extent of simple repeat variability was lower in cattle compared to humans. Selective breeding in the process of domestication might be responsible for the diminished intronic hypervariability. Nevertheless, the hypermutable simple repeat sequences have been preserved in the same position and with the same principal structure for at least 70 × 10⁶ years of evolution. Unexpectedly, the rate of intronic simple repeat and exonic changes appear quite similar.     

Identification of MhcMafa-DRB Alleles in a Cohort of Cynomolgus Macaques of Vietnamese Origin

Cynomolgus macaques have been used widely to build a research model of infectious and chronic diseases, as well as in transplantation studies, where disease susceptibility and/or resistance are associated with the major histocompatibility complex (MHC). To better elucidate polymorphisms and genetic differences in the Mafa‐DRB locus, and facilitate the experimental use of cynomolgus macaques, we used pool screening combined with cloning and direct sequencing of polymerase chain reaction products to characterize MhcMafa‐DRB gene alleles in 153 Vietnamese cynomolgus macaques. We identified 30 Mafa‐DRB alleles belonging to 17 allelic lineages, including four novel sequences that had not been documented in earlier reports. The highest frequency allele was Mafa‐DRB*W27:04, which was present in 7 of 35 (20%) monkeys. The next most frequent alleles were Mafa‐DRB*3:07 and Mafa‐DRB*W7:01, which were detected in 5 of 35 (14.3%) and 4 of 35 (11.4%) of the monkeys, respectively. The high‐frequency alleles in this Vietnamese population may be high priority targets for additional characterization of immune functions. Only the DRB1*03 and DRB1*10 lineages were also present in humans, whereas the remaining alleles were monkey‐specific lineages. We found 25 variable sites by aligning the deduced amino acid sequences of 29 identified alleles. Evolutionary and population analyses based on these sequences showed that human, rhesus, and cynomolgus macaques share several Mhc‐DRB lineages and the shared polymorphisms in the DRB region may be attributable to the existence of interbreeding between rhesus and cynomolgus macaques. This information will promote the understanding of MHC diversity and polymorphism in cynomolgus macaques and increase the value of this species as a model for biomedical research. Am. J. Primatol. 74:958‐966, 2012. © 2012 Wiley Periodicals, Inc.     

MHC polymorphism in Caribbean African green monkeys

African green monkeys (AGM) are among the most widely used nonhuman primate models used in various fields of medical research. One species of AGM that originated from West Africa, Chlorocebus sabaeus, was introduced three centuries ago in the Caribbean islands. We present here a systematic study of the major histocompatibility complex (MHC) polymorphism of Caribbean AGM which is currently frequently used as an animal model. We studied 54 animals originated from Barbados (N?=?25) or Saint Kitts (N?=?29). The MHC polymorphism was characterized by means of 17 MHC microsatellites spread across MHC and DRB genotyping by DGGE sequencing. We defined nine frequent MHC haplotypes of which two were found in the two insular populations suggesting either past exchanges between the two populations or a common origin of the founders of the two populations. By the analysis of a previously described EST library, we characterized 38 MHC cDNA sequences (17 class I and 21 class II). In conclusion, we characterized for the first time the MHC polymorphism of Barbados and Saint Kitts AGM. We found a restricted polymorphism due to a founding effect, which is responsible for a strong bottleneck. The poorness of MHC polymorphism observed in the Caribbean AGM populations is similar to that observed in the Mauritian cynomolgus macaque population.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Sequence diversity of MHC class-II <Emphasis Type="BoldItalic">DRB</Emphasis> gene in gazelles (<Emphasis Type="BoldItalic">Gazella subgutturosa</Emphasis>) raised in Sanliurfa of Turkey

In this study, we aimed to assess the sequence diversity of major histocompatibility complex (MHC) class-II DRB gene at exon 2 in gazelles raised in Sanliurfa Province of Turkey. Twenty DNA samples isolated from gazelles (Gazella subgutturosa) were used for sequencing exon 2 of MHC class-II DRB gene. Target region was amplified by polymerase chain reaction (PCR) and their products were directly sequenced. Nine of these 20 samples yielded unambiguously readable sequences. Three of the nine samples were homozygotes and each showed different sequences. A 262-bp sequence obtained from the three homozygote samples were submitted to GenBank (accession numbers: KC309405, KC309406 and KC309407). Using an allele specific PCR, we detected 10 additional haplotypes. Among 13 haplotypes, 45 nucleotide positions were polymorphic and most of the polymorphic nucleotide positions localized at peptide-binding region (PBR). Rates of nonsynonymous substitutions were significantly higher than synonymous substitutions at PBR. Phylogenetic analysis of the haplotypes showed that 10 haplotypes of the gazelles were clustered together while three were clustered with ovine and bovine haplotypes. The results indicated that at least 13 haplotypes at exon 2 of MHC class-II DRB gene were showing high degree of nucleotide and amino acid diversity, and certain haplotypes of G. subgutturosa were more similar to haplotypes from sheep or cattle than to each other. Rates of synonymous and nonsynonymous substitutions suggested that positive selection was a driving force for diversity at this locus in G. subgutturosa.