Comparative genetics of a highly divergent DRB microsatellite in different macaque species
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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.
KeywordsMHC Nonhuman primates Evolution Microsatellites Macaques
The Mhc-DRB region in various primate species displays abundant levels of allelic variation (polymorphism) and diversity (gene copy number variation). However, the balance between these two phenomena can differ significantly, depending on the species studied (Brändle et al. 1992; Grahovac et al. 1992; Mayer et al. 1992; Schönbach et al. 1993; Gongora et al. 1997; Antunes et al. 1998; Doxiadis et al. 2000; de Groot et al. 2008). In humans, nine different genes have been characterized, designated HLA-DRB1–9 (Marsh et al. 2005), and equivalents have been detected in various nonhuman primate species (Kasahara et al. 1990; Klein et al. 1991; Brändle et al. 1992; Kenter et al. 1992; Mayer et al. 1992; Trtkova et al. 1993; Slierendregt et al. 1994; Bontrop et al. 1995; Leuchte et al. 2004; Doxiadis et al. 2006a). A region configuration is defined by the unique combination of distinct DRB genes present per haplotype. In humans, five major DRB region configurations are known (DR1, DR8, DR51, DR52, DR53), whereas in chimpanzees at least six and in rhesus macaques more than 30 different region configurations have been defined (Gyllensten et al. 1991; Mayer et al. 1992; Slierendregt et al. 1994; Gongora et al. 1997; Khazand et al. 1999; Doxiadis et al. 2000; Bontrop 2006; O’Connor et al. 2007; de Groot et al. 2008). In contrast, a New World primate species like the common marmoset (Callithrix jacchus) lacks region configuration polymorphism (Antunes et al. 1998; Doxiadis et al. 2006b).
Cynomolgus monkeys (Macaca fascicularis) are frequently used as animal models for immune-related diseases or in transplantation studies (Bosinger et al. 2004; Jonker et al. 2004; Langermans et al. 2005; Mueller et al. 2005; Wiseman et al. 2007; Wojcechowskyj et al. 2007). Resistance or susceptibility to certain immune-related diseases as for example HIV/SIV appears to be correlated to the geographic origin of rhesus as well as cynomolgus macaques (Wiseman et al. 2007; Goulder and Watkins 2008). Additionally, several studies have proven that cynomolgus macaques from different origins also vary concerning the genetic diversity of their mtDNA, Y chromosome, and autosomal markers with Mauritius animals being the most homogenous due to a founder effect (Smith et al. 2007; Tosi and Coke 2007; Blancher et al. 2008; Bonhomme et al. 2008). To get a first idea of the origin and the diversity of the cynomolgus monkeys selected for this study, mtDNA variation was analyzed as this has been proven to be a suitable method (Tosi et al. 2003; Smith and McDonough 2005; Kyes et al. 2006; Smith et al. 2007).
Although the Mafa-DR region is not as thoroughly analyzed as for the rhesus monkey (Macaca mulatta; Mamu-DR), recent studies have indicated that the levels of diversity at these regions are at least comparable (Blancher et al. 2006; Doxiadis et al. 2006a; O’Connor et al. 2007). Because of the complexity of the region, until now, laborious methods like cloning, followed by sequencing of the most polymorphic exon 2 or full-length DRB, are the only means available for accurate typing. It is clear that more straightforward typing protocols are needed that can easily be implemented by other laboratories.
A complex repeat, designated D6S2878 or DRB-STR, maps in close proximity to exon 2 and is present in the majority of all HLA- and Mamu-DRB genes (Andersson et al. 1987; Epplen et al. 1997; Bergstrom et al. 1999; Doxiadis et al. 2007). This entity is located at the beginning of intron 2 and has a compound character (Riess et al. 1990; Trtkova et al. 1995; Bergstrom et al. 1999; Kriener et al. 2000; Doxiadis et al. 2007). Genotyping of a large panel of cells, covering most of the known HLA- and Mamu-DRB specificities, resulted in the definition of unique DRB-STR patterns that appeared to be characteristic for a certain haplotype (Doxiadis et al. 2007). Therefore, this microsatellite may represent a promising marker for the DRB haplotyping of other macaque species. Consequently, a representative panel including several families was chosen to investigate whether it is possible to set up a high-resolution DRB haplotyping protocol for cynomolgus monkeys.
Materials and methods
Samples and genomic DNA isolation
All of the 71 cynomolgus macaques but two unrelated males (Cyn81 and Cyn83) used for studying Mafa-DRB belong to an outbred colony that is housed at the University of Utrecht, The Netherlands, and are members of four different pedigreed families (Juanita, Rastafa, Sayonara, and Alfa). For mtDNA analysis, representatives of these four families have been analyzed, and members of other cynomolgus families (Epha, Harpo, Bilboa, Cleo, Hoeba, and Cornea) and some unrelated monkeys (Cyn80, Vip, Clint, K66, and K95) have been included. Genomic DNA was extracted from EDTA blood samples or from immortalized B-cell lines using a standard salting out procedure.
DNA was obtained as described above, or was extracted from feces in 96% ethanol using the QIAamp DNA stool mini kit (Qiagen, GmbH, Germany) according to the manufacturer’s recommendations. In particular, the 3′ part of the 12S rRNA gene is helpful in distinguishing species and origin-related variability (van der Kuyl et al. 1995, 2000; Doxiadis et al. 2003; Tosi et al. 2003). Amplification of part of the mitochondrial 12S rRNA gene, purification, and sequencing was performed essentially according to published methods (Kocher et al. 1989; Doxiadis et al. 2003). The data were analyzed using the Sequence Navigator program (Applied Biosystems). Unreported sequences resulting from at least two independent PCR reactions have been deposited in the EMBL/GeneBank database (accession numbers FM179743–FM179751).
Phylogenetic analysis of mtDNA
Multiple sequence alignments of the 12S rRNA sequences were obtained of newly generated mtDNA sequences (accession numbers see above) together with published mtDNA sequences of animals of documented origin (Doxiadis et al. 2003; Tosi et al. 2003) using MacVector v9.5.2 (MacVector, Inc., Cambridge, UK). Mr Bayes v3.1.2 was used for Bayesian interference of the phylogenetic relationships of the published (Tosi et al. 2003; Doxiadis et al. 2006a) and newly detected mtDNA sequences (373 bp). Four Monte Carlo Markov chains (MCMCs) were run simultaneously for 1 × 106 generations, with posterior sampling of the trees after every 100 generations and a burn-in after 1,250 generations (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003).
Amplification of the relevant DNA segment in cynomolgus macaques was performed as described for rhesus macaques using the same primer sets (Doxiadis et al. 2007). Briefly, the relevant DNA segment in macaques was amplified with a forward primer located at the end of exon 2 (5′ Mamu-DRB-STR—TTC ACA GTG CAG CGG CGA GGT) and two labeled reverse primers in intron 2 (3′ Mamu-DRB-STR_VIC—ACA CCT GTG CCC TCA GAA CT and 3′ Mamu-DRB-STR_FAM_1007—ACA TCT GTG TCC TCA GAC CT). The labeled primers were synthesized by Applied Biosystems (Foster City, USA) and the unlabelled primers by Invitrogen (Paisley, Scotland). The PCR reaction was performed in a 25-μl reaction volume containing 1 U of Taq polymerase (Invitrogen) with 0.6 μM of the unlabeled forward primer, 0.4 μM of the VIC labeled reversed primer, 0.2 μM of the FAM-labeled reversed primer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 1× PCR buffer II (Invitrogen), and 100 ng DNA. The cycling parameters were a 5-min 94°C initial denaturation step, followed by five cycles of 1 min at 94°C, 45 s at 58°C, and 45 s at 72°C. Then the program was followed by 25 cycles of 45 s at 94°C, 30 s at 58°C, and 45 s at 72°C. A final extension step was performed at 72°C for 30 min. The amplified DNA was prepared for genotyping according to the manufacturer’s guidelines and analyzed on an ABI 3130 genetic analyzer (Applied Biosystems). STR analysis was performed with the Genemapper program (Applied Biosystems) and all samples were analyzed at least twice.
PCR, cloning, and sequencing of Mafa-DRB
Sixty different Mafa-DRB alleles were sequenced from exon 2 to intron 2, including the microsatellite. Therefore, we used the same primers and PCR reaction as described for rhesus macaques (Doxiadis et al. 2007). The 18 unreported Mafa-DRB sequences have been deposited in the EMBL/GeneBank database (accession numbers AM910925–AM910929, AM911049, AM911051, AM911053–AM911060, AM911062, AM911063, and AM911065) and are also available via the IPD/MHC database (www.ebi.ac.uk/ipd/mhc/nhp; European Bioinformatics Institute, Cambridge, UK). Alleles have been named according to a standardized nomenclature protocol (Klein et al. 1990; Robinson et al. 2003; Ellis et al. 2006).
Phylogenetic analysis of DRB exon 2 sequences
Multiple sequence alignments of exon 2 of Mafa-DRB sequences generated in this study together with published Mafa- and Mamu-DRB sequences from the NCBI database were created using MacVectorTM version 8.1.1 (Oxford Molecular Group), followed by a phylogenetic analysis performed using PAUP version 4.0b.10 (Swofford 2002). Pairwise distances were calculated using the Kimura two-parameter model for creating a phylogram. Confidence estimates of grouping were calculated according to the bootstrap method generated from 1,000 replicates.
Results and discussion
Origin of cynomolgus macaques, defined by mtDNA analysis
Mafa-DRB haplotype definition by DRB-STR genotyping
Based on the unique combinations of certain DRB-STR/exon 2 alleles and subsequent segregation analyses, 22 different Mafa-DRB haplotypes characterized by the presence of two to four distinct DRB genes were defined (Fig. 2). As in humans and rhesus macaques, the combination of different DRB-STR markers appears to be unique for a given haplotype. DRB haplotypes of all but two monkeys could be determined. The two exceptions represent animals that are unrelated to the rest of the panel. Thus, this technique can be used to study other Old World monkeys that have complex DRB regions such as mandrills (Abbott et al. 2006) and baboons (Huchard et al. 2006); this approach is especially powerful if family samples are available.
Mamu- versus Mafa-DRB: allelic variation, diversity, and region configurations/haplotypes
The present study shows that numbers of DRB alleles, lineages, and region configurations observed in the cynomolgus macaques studied are comparable to the rhesus monkey system that was described earlier (Doxiadis et al. 2007) (Fig. 2). One particular Mafa-DRB region configuration has also been observed in Chinese rhesus macaques, indicating that this configuration may predate speciation, and thus represent an evolutionarily old entity (Fig. 2, haplotype 3). However, introgression of rhesus macaques into cynomolgus macaques near the isthmus of Kra has been described, and therefore the possibility of a mixture of both species cannot be excluded (Tosi et al. 2002). Both panels comprised animals of pedigreed macaque families. However, in the former study, twice the number of rhesus macaques was analyzed. Furthermore, the Mamu-DRB region was mainly studied in monkeys of Indian origin, which, however, made up about 60% of the region configurations determined (Doxiadis et al. 2007). Based on these comparisons, it can be concluded that cynomolgus macaques possess more allelic variation as well as a higher level of DRB region configuration polymorphism in comparison to Indian rhesus macaques. These results are in agreement with mtDNA analyses, which suggest that Indian rhesus macaques were reproductively isolated from Chinese monkeys during much of the Pleistocene and may have experienced a severe genetic bottleneck (Smith and McDonough 2005; Smith et al. 2007). However, Indian and Chinese rhesus macaques show higher levels of gene copy number variation, as two to six DRB loci are present per haplotype (Doxiadis et al. 2007). In contrast, in cynomolgus macaques, most of the region configurations are composed of three DRB loci, and more than four loci per haplotype have not been observed (Fig. 2). The composition of the DRB region configurations of the two macaque species also shows marked differences. Some DRB region configurations of the rhesus monkey display limited levels of allelic polymorphism, whereas no allelic variation can be observed for the Mafa-DRB region configurations (Khazand et al. 1999; Doxiadis et al. 2000; Doxiadis et al. 2007). It is noteworthy that the allelic polymorphism of rhesus macaques has only been observed between either monkeys of different origin or between animals of Indian origin. Since the Indian rhesus macaques seem to have run through a bottleneck, it may be speculated that these monkeys built up their genetic repertoire afterwards by the creation of allelic variation instead of haplotype diversity (Hernandez et al. 2007; Smith et al. 2007).
Several Mafa-DRB haplotypes share alleles/loci that are identical for their exon 2 sequences, whereas the other DRB genes on the chromosome are different (Fig. 2, bordered). An example is provided by haplotypes 9, 10, and 11, which all share the Mafa-DRB1*0401 allele together with a DRB5*03 lineage member. Furthermore, haplotypes 9 and 11 encode a DRB4*01 allele, whereas haplotype 10 possesses a DRB*W3 allele instead. Haplotype 9, however, has an additional DRB6 locus that is absent on haplotype 11. This sharing of haplotype segments suggests that, in cynomolgus monkeys, even more frequently than in rhesus macaques, new region configurations are generated by recombination-like events.
Evolutionary history of the DRB-STR in macaques
Comparison of DRB sequences obtained from different species indicates that the ancestral structure of the DRB-STR most likely must have been a (GT)x(GA)y dinucleotide repeat (Riess et al. 1990; Bergstrom et al. 1999; Kriener et al. 2000; Doxiadis et al. 2007). Nearly all HLA- and Mamu-DRB gene-associated microsatellites thus far encountered are constructed of four sections, namely (GT)x(GA)z-mix(GA)y(GC)n, exhibiting differential evolutionary stabilities (Doxiadis et al. 2007). The 5′(GT) repeat represents habitually the longest and uninterrupted part, and evolves most rapidly. The second (GA)z part is often shorter and interrupted by other dinucleotides; its composition correlates well with different DRB loci/lineages. In the case of HLA-DRB1, the length of the third (GA)y segment often correlates with lineages as well, whereas in rhesus macaques and for other HLA-DRB loci, such a correlation is less prominent. The 3′ end of the repeat is formed by a short (GC)n part.
Identical Mamu and Mafa-DRB alleles and their STR compositions
Mafa class II alleles detected and their identity to Mamu orthologue
The DRB region of humans as well as macaque species has been subjected to several rounds of duplication and contraction processes caused by a complex series of recombination-like events. Different mechanisms have been proposed for the instability of the macaque DRB region. For instance, sense- or antisense integration of intronic endogenous retroviruses may influence the stability or instability of primate DRB genes/region configurations (Doxiadis et al. 2008a). Here, a recombination event is reported that maps within a compound microsatellite. It is even possible that the microsatellite itself influences the stability of the DR region by promoting recombination-like events. At this stage, it is difficult to determine whether highly related genes located on different region configurations represent separate loci or whether these sequences have an allelic affiliation. Bearing in mind the results described above, this microsatellite will be helpful in sorting out paralogous and orthologous relationships.
The authors wish to thank Donna Devine for editing the manuscript and Henk van Westbroek for preparing the figures. This work was supported in part by NIH/NIAID contract numbers HHSN266200400088C/NOI-AI-0088 and 5R24RR016038-05 (CFA: 03.9389).
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