Nanine de Groot

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.

Genetic Makeup of the DR Region in Rhesus Macaques: Gene Content, Transcripts, and Pseudogenes

In the human population, five major HLA-DRB haplotypes have been identified, whereas the situation in rhesus macaques (Macaca mulatta) is radically different. At least 30 Mamu-DRB region configurations, displaying polymorphism with regard to number and combination of DRB loci present per haplotype, have been characterized. Until now, Mamu-DRB region genes have been studied mainly by genomic sequencing of polymorphic exon 2 segments. However, relatively little is known about the expression status of these genes. To understand which exon 2 segments may represent functional genes, full-length cDNA analyses of -DRA and -DRB were initiated. In the course of the study, 11 cDRA alleles were identified, representing four distinct gene products. Amino acid replacements are confined to the leader peptide and cytoplasmatic tail, whereas residues of the α1 domain involved in peptide binding, are conserved between humans, chimpanzees, and rhesus macaques. Furthermore, from the 11 Mamu-DRB region configurations present in this panel, 28 cDRB alleles were isolated, constituting 12 distinct cDRA/cDRB configurations. Evidence is presented that a single configuration expresses maximally up to three -DRB genes. For some exon 2 DRB sequences, the corresponding transcripts could not be detected, rendering such alleles as probable pseudogenes. The full-length cDRA and cDRB sequences are necessary to construct Mhc class II tetramers, as well as transfectant cell lines. As the rhesus macaque is an important animal model in AIDS vaccine studies, the information provided in this communication is essential to define restriction elements and to monitor immune responses in SIV/simian human immunodeficiency virus-infected rhesus macaques.

A highly divergent microsatellite facilitating fast and accurate DRB haplotyping in humans and rhesus macaques

The DRB region of the MHC in primate species is known to display abundant region configuration polymorphism with regard to the number and content of genes present per haplotype. Furthermore, depending on the species studied, the different DRB genes themselves may display varying degrees of allelic polymorphism. Because of this combination of diversity (differential gene number) and polymorphism (allelic variation), molecular typing methods for the primate DRB region are cumbersome. All intact DRB genes present in humans and rhesus macaques appear to possess, however, a complex and highly divergent microsatellite. Microsatellite analysis of a sizeable panel of outbred rhesus macaques, covering most of the known Mamu-DRB haplotypes, resulted in the definition of unique genotyping patterns that appear to be specific for a given haplotype. Subsequent examination of a representative panel of human cells illustrated that this approach also facilitates high-resolution HLA-DRB typing in an easy, quick, and reproducible fashion. The genetic composition of this complex microsatellite is shown to be in concordance with the phylogenetic relationships of various HLA-DRB and Mamu-DRB exon 2 gene/lineage sequences. Moreover, its length variability segregates with allelic variation of the respective gene. This simple protocol may find application in a variety of research avenues such as transplantation biology, disease association studies, molecular ecology, paternity testing, and forensic medicine.

Pinpointing a selective sweep to the chimpanzee MHC class I region by comparative genomics

Chimpanzees experienced a reduction of the allelic repertoire at the major histocompatibility complex (MHC) class I A and B loci, which may have been caused by a retrovirus belonging to the simian immunodeficiency virus (SIV) family. Extended MHC haplotypes were defined in a pedigreed chimpanzee colony. Comparison of genetic variation at microsatellite markers mapping inside and outside the Mhc region was carried out in humans and chimpanzees to investigate the genomic extent of the repertoire reduction. Multilocus demographic analyses underscored that chimpanzees indeed experienced a selective sweep that mainly targeted the chromosomal segment carrying the Mhc class I region. Probably due to genetic linkage, the sweep also affected other polymorphic loci, mapping in the close vicinity of the Mhc class I region genes. Nevertheless, although the allelic repertoire at particular Mhc class I and II loci appears to be limited, naturally occurring recombination events allowed the establishment of haplotype diversity after the sweep. However, recombination did not have sufficient time to erase the signal of the selective sweep.

Genomic plasticity of the MHC class I A region in rhesus macaques: extensive haplotype diversity at the population level as revealed by microsatellites

The Mamu-A genes of the rhesus macaque show different degrees of polymorphism, transcription level variation, and differential haplotype distribution. Per haplotype, usually one “major” transcribed gene is present, A1 (A7), in various combinations with “minor” genes, A2 to A6. In silico analysis of the physical map of a heterozygous animal revealed the presence of similar Mamu-A regions consisting of four duplication units, but with dissimilar positions of the A1 genes on both haplotypes, and in combination with different minor genes. Two microsatellites, D6S2854 and D6S2859, have been selected as potential tools to characterize this complex region. Subsequent analysis of a large breeding colony resulted in the description of highly discriminative patterns, displaying copy number variation in concert with microsatellite repeat length differences. Sequencing and segregation analyses revealed that these patterns are unique for each Mamu-A haplotype. In animals of Indian, Burmese, and Chinese origin, 19, 15, or 9 haplotypes, respectively, could be defined, illustrating the occurrence of differential block duplications and subsequent rearrangements by recombination. The haplotypes can be assigned to 12 unique combinations of genes (region configurations). Although most configurations harbor two transcribed A genes, one or three genes per haplotype are also present. Additionally, haplotypes lacking an A1 gene or with an A1 duplication appear to exist. The presence of different transcribed A genes/alleles in monkeys from various origins may have an impact on differential disease susceptibilities. The high-throughput microsatellite technique will be a valuable tool in animal selection for diverse biomedical research projects.

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.

Haplotype diversity generated by ancient recombination-like events in the MHC of Indian rhesus macaques

The Mamu-A, Mamu-B, and Mamu-DRB genes of the rhesus macaque show several levels of complexity such as allelic heterogeneity (polymorphism), copy number variation, differential segregation of genes/alleles present on a haplotype (diversity) and transcription level differences. A combination of techniques was implemented to screen a large panel of pedigreed Indian rhesus macaques (1,384 individuals representing the offspring of 137 founding animals) for haplotype diversity in an efficient and inexpensive manner. This approach allowed the definition of 140 haplotypes that display a relatively low degree of region variation as reflected by the presence of only 17 A, 18 B and 22 DRB types, respectively, exhibiting a global linkage disequilibrium comparable to that in humans. This finding contrasts with the situation observed in rhesus macaques from other geographic origins and in cynomolgus monkeys from Indonesia. In these latter populations, nearly every haplotype appears to be characterised by a unique A, B and DRB region. In the Indian population, however, a reshuffling of existing segments generated “new” haplotypes. Since the recombination frequency within the core MHC of the Indian rhesus macaques is relatively low, the various haplotypes were most probably produced by recombination events that accumulated over a long evolutionary time span. This idea is in accord with the notion that Indian rhesus macaques experienced a severe reduction in population during the Pleistocene due to a bottleneck caused by geographic changes. Thus, recombination-like processes appear to be a way to expand a diminished genetic repertoire in an isolated and relatively small founder population.

Impact of Endogenous Intronic Retroviruses on Major Histocompatibility Complex Class II Diversity and Stability

ABSTRACT The major histocompatibility complex (MHC) represents a multigene family that is known to display allelic and gene copy number variations. Primate species such as humans, chimpanzees (Pan troglodytes), and rhesus macaques (Macaca mulatta) show DRB region configuration polymorphism at the population level, meaning that the number and content of DRB loci may vary per haplotype. Introns of primate DRB alleles differ significantly in length due to insertions of transposable elements as long endogenous retrovirus (ERV) and human ERV (HERV) sequences in the DRB2, DRB6, and DRB7 pseudogenes. Although the integration of intronic HERVs resulted sooner or later in the inactivation of the targeted genes, the fixation of these endogenous retroviral segments over long time spans seems to have provided evolutionary advantage. Intronic HERVs may have integrated in a sense or an antisense manner. On the one hand, antisense-oriented retroelements such as HERV-K14I, observed in intron 2 of the DRB7 genes in humans and chimpanzees, seem to promote stability, as configurations/alleles containing these hits have experienced strong conservative selection during primate evolution. On the other hand, the HERVK3I present in intron 1 of all DRB2 and/or DRB6 alleles tested so far integrated in a sense orientation. The data suggest that multigenic regions in particular may benefit from sense introgressions by HERVs, as these elements seem to promote and maintain the generation of diversity, whereas these types of integrations may be lethal in monogenic systems, since they are known to influence transcript regulation negatively.

The chimpanzee Mhc-DRB region revisited: gene content, polymorphism, pseudogenes, and transcripts.

In humans, great apes, and different monkey species, the major histocompatibility complex (MHC) class II DRB region is known to display considerable copy number variation. The microsatellite D6S2878 has been shown to be a valuable marker for haplotyping the DR region in humans and macaque species. The present report illustrates that chimpanzee haplotypes also can be discriminated with this marker. The analyses resulted in the description of nine different region configurations, of which seven are present within the West African chimpanzee population studied. The region configurations vary in gene content from two up to five DRB genes. Subsequent cDNA sequencing increased the number of known full-length Patr-DRB sequences from 3 to 32, and shows that one to three Patr-DRB genes per haplotype apparently produce functional transcripts. This is more or less comparable to humans and rhesus macaques. Moreover, microsatellite analysis in concert with full-length DRB gene sequencing showed that the Patr-DRB*W9 and -DRB3*01/02 lineages most likely arose from a common ancestral lineage: hence, the Patr-DRB*W9 lineage was renamed to Patr-DRB3*07. Overall, the data demonstrate that the D6S2878 microsatellite marker allows fast and accurate haplotyping of the Patr-DRB region. In addition, the limited amount of allelic variation observed at the various Patr-DRB genes is in agreement with the fact that chimpanzees experienced a selective sweep that may have been caused by an ancient retroviral infection.

Extensive DRB region diversity in cynomolgus macaques: recombination as a driving force.

The DR region of primate species is generally complex and displays diversity concerning the number and combination of distinct types of DRB genes present per region configuration. A highly variable short tandem repeat (STR) present in intron 2 of nearly all primate DRB genes can be utilized as a quick and accurate high through-put typing procedure. This approach resulted previously in the description of unique and haplotype-specific DRB-STR length patterns in humans, chimpanzees, and rhesus macaques. For the present study, a cohort of 230 cynomolgus monkeys, including self-sustaining breeding groups, has been examined. MtDNA analysis showed that most animals originated from the Indonesian islands, but some are derived from the mainland, south and north of the Isthmus of Kra. Haplotyping and subsequent sequencing resulted in the detection of 118 alleles, including 28 unreported ones. A total of 49 Mafa-DRB region configurations were detected, of which 28 have not yet been described. Humans and chimpanzees possess a low number of different DRB region configurations in concert with a high degree of allelic variation. In contrast, however, allelic heterogeneity within a given Mafa-DRB configuration is even less frequently observed than in rhesus macaques. Several of these region configurations appear to have been generated by recombination-like events, most probably propagated by a retroviral element mapping within DRB6 pseudogenes, which are present on the majority of haplotypes. This undocumented high level of DRB region configuration-associated diversity most likely represents a species-specific strategy to cope with various pathogens.