Reduced MIC Gene Repertoire Variation in West African Chimpanzees as Compared to Humans
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分子生物学进展 2005年第6期
* Departments of Comparative Genetics and Refinement, and Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands; and Anthony Nolan Research Institute and Royal Free and UCL School of Medicine, Royal Free Campus, London, United Kingdom
Correspondence: E-mail: groot@bprc.nl.
Abstract
The human major histocompatibility complex class I chain-related (MIC) genes are members of a multicopy family showing similarity to the classical HLA-A, HLA-B, and HLA-C genes. Only the MICA and MICB genes produce functional transcripts. In chimpanzees, however, only one MIC gene is expressed, showing an intermediate character, resulting from a deletion fusing the MICA and MICB gene segments together. The present population study illustrates that all chimpanzee haplotypes sampled possess the hybrid MICA/B gene. In contrast to the human situation this gene displays reduced allelic variation. The observed repertoire reduction of the chimpanzee MICA/B gene is in conformity with the severe repertoire condensation documented for Patr-B locus lineages, probably due to the close proximity of both genes.
Key Words: chimpanzee ? humans ? HIV ? MHC ? MIC
Introduction
The major histocompatibility complex class I chain-related gene (MIC), previously also called PERB11, was first described in 1994 (Bahram et al. 1994; Leelayuwat et al. 1994). The gene is located in the major histocompatibility complex (MHC) region. The MHC, playing an important role in the immune defense against pathogens, is characterized by its extensive degree of polymorphism at the population level. Also, in the relatively small sample size of chimpanzees that have been analyzed so far, a high degree of Patr class I diversity, with regard to allele numbers, has been detected (Adams et al. 2000; de Groot et al. 2000). At first sight, this agrees with the finding that chimpanzees display more mitochondrial DNA (mtDNA) variation than humans do (Gagneux et al. 1999; Ingman et al. 2000), and similar findings have been reported for particular nuclear genes (Kaessmann, Wiebe, and Paabo 1999; Zhao et al. 2000), although a recent publication shows that the variation in chimpanzees is not as high as a magnitude of four but more in the range of 1.5 compared to humans (Yu et al. 2003). A more detailed analysis of the MHC class I intron 2 sequences in chimpanzees showed that the MHC class I gene repertoire, with respect to the number of lineages, is severely reduced as compared to humans. This repertoire reduction is evident for Patr-A (McAdam et al. 1995) but is most prominent for the Patr-B locus lineages (de Groot et al. 2002).
Seven MIC genes are distinguished in the human genome. MICA and MICB produce functional transcripts, whereas MICC to MICG are pseudogenes (Bahram 2000). The MICA and MICB genes show a high degree of similarity to the classical MHC class I genes but are distinguished by their disparate organization of exons-introns. Moreover, they do not associate with ?2-microglobulin (?2m), and their expression is not induced by type I/II interferons. Furthermore, the MICA and MICB genes are predominantly expressed on fibroblast and epithelial cells (Bahram 2000). A part of the MICA and MICB promoter region shows similarity to heat shock protein gene promoters, and upregulation of the MICA and MICB molecules after heat shock has been reported (Groh et al. 1998). In conclusion, MICA and MICB seem to play a role in the detection of cell stress and appear to react preferentially with the ligands V1 TCR and NKG2D to induce an immune response (Groh et al. 1998; Bauer et al. 1999; Gleimer and Parham 2003). In chimpanzees only one functional MIC gene has been described (Steinle, Groh, and Spies 1998), and this gene appears to have an intermediate character as compared to human MICA and MICB genes (Cattley et al. 1999; Kulski et al. 2002). Studies on MIC gene polymorphism in chimpanzee populations are absent, and only one allele, Patr-MIC1, has been reported (Steinle, Groh, and Spies 1998). The human MICA and MICB genes show a considerable degree of polymorphism, and to date 54 MICA and 14 MICB alleles have been identified (Radosavljevic and Bahram 2003).
Compared to humans, chimpanzees have a severely reduced MHC class I gene repertoire caused by an ancient selective sweep (de Groot et al. 2002), and they appear to lack the equivalents of the HLA-DRB1*04 and HLA-DRB1*08 lineages (Bontrop et al. 1999). The present study was initiated to investigate whether the repertoire reduction, most prominent for the Patr-B locus lineages, is only restricted to the MHC class I and II genes or also extends to other genes located in the MHC region. For that reason we investigated the polymorphism at the chimpanzee MIC gene, which is located in the direct neighborhood of the Patr-B locus (Anzai et al. 2003). In humans, some of the MICA alleles and closely linked HLA-B alleles show a high degree of linkage disequilibrium (Bahram 2000). Here we report on MIC gene variation and linkage disequilibria in a West African chimpanzee population together with data on chimpanzees of other subspecies.
Materials and Methods
Animals
The chimpanzee (Pan troglodytes) colony (approximately 100 individuals) at the Biomedical Primate Research Centre (BPRC) started with 35 founder animals originating from Sierra Leone and belonging to the subspecies Pan troglodytes verus (West Africa). The animals are characterized at the molecular level for MHC class I and II gene polymorphisms (Bontrop et al. 1999; de Groot et al. 2000). Their offspring have been pedigreed based on segregation of Patr-A and Patr-B serotypes and molecularly defined Patr class I and II gene polymorphisms. Three animals of the Pan troglodytes troglodytes (Central Africa) and Pan troglodytes schweinfurthii (East Africa) subspecies have been included in this study. For the two bonobos (Pan paniscus) used in this study, the DNAs were analyzed with 12S primers to determine potential relationships (Kocher et al. 1989).
mtDNA Analysis
Genomic DNA (gDNA), obtained from Epstein-Barr virus-transformed B-cell lines, was used to amplify the mitochondrial D-loop (380 bp) sequences. To assign the subspecies, the nucleotide sequences were compared to published sequences (Morin et al. 1994; Gagneux et al. 1999). The polymerase chain reaction (PCR) mixture (50 μl) contained gDNA (50 ng), 1 μM of the primers (table 1), 1x PCR buffer + bovine serum albumin, 2 mM MgCl2, 0.2 mM of each deoxyribonucleoside triphosphate (dNTP), and 2.5 units (U) Taq polymerase Gold (Applied Biosystems, Foster City, Calif). A touchdown PCR consisting of the following cycles was run: 1 cycle of 15 min at 94°C, 2 cycles of 30 s at 94°C, 30 s at 63°C, 30 s at 72°C; after each two cycles the annealing temperature is decreased by 2°C until 55°C was reached, followed by 4 cycles of 30 s at 94°C, 30 s at 53°C, 30 s at 72°C, and 25 cycles of 30 s at 94°C, 30 s at 50°C, 30 s at 72°C, with a final extension of 5 min at 72°C. The PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and sequenced directly on an ABI 310 automatic sequencer using the ABI Prism dRhodamine Terminator Cycle sequencing Ready Reaction Kit (Applied Biosystems) to prepare the samples according to the manufacturer's instructions. The products were sequenced from the 5' and 3' ends. Sequences reported in this publication have been deposited in the EMBL database (accession numbers AJ863489–AJ863508).
Table 1 MIC-Specific PCR Primers and Internal Sequencing Primers
MIC Gene Characterization by Reference Strand Conformation Analysis
Reference strand conformation analysis (RSCA) was used to screen the chimpanzees for MIC gene polymorphisms. Locus-specific primers, amplifying human MICA, were used to independently amplify exon 2 and 3 in chimpanzees (Mendoza-Rincon et al. 1999). RSCA was performed at the Anthony Nolan Research Institute, London, as has been described in detail (Arguello et al. 1998).
MIC Gene Characterization by Direct Nucleotide Sequencing
gDNA was used for the PCR amplification. Two independent overlapping PCR fragments were generated, using MIC-specific primers (table 1), covering the MIC gene from exon 2 to exon 5. This resulted in a 1,046-bp PCR fragment (fragment A), covering exon 2 (255 bp), intron 2 (273 bp), and exon 3 (270 bp), and a part of introns 1 (101 bp) and 3 (100 bp). The second PCR product was 1,297-bp long (fragment B), containing complete intron 3 (596 bp), exon 4 (270 bp), intron 4 (102 bp), and exon 5 (141 bp), and a part of exon 3 (129 bp) and intron 5 (13 bp). The PCR for fragment A (25 μl) contained gDNA (100 ng), 0.8 μM of the primers (table 1), 2.5–3 mM MgCl2, 0.4 mM of each dNTP, and 5 U of Taq polymerase. A total of 33 cycles were run, each cycle consisting of 30 s at 95°C, 50 s at 61.3°C, and 30 s at 72°C with a final extension of 8 min at 72°C. PCR for fragment B (50 μl) contained gDNA (100 ng), 0.8 μM of the primers (table 1), 2.25–2.75 mM MgCl2, 0.4 mM of each dNTP, and 5 U of Taq polymerase. A total of 28 cycles were run, each cycle consisting of 30 s at 95°C, 30 s at 58.5°C, and 40 s at 72°C with a final extension of 7 min at 72°C. The PCR products were purified, and sequenced directly on an ABI 3100 genetic analyzer (Applied Biosystems) using the PCR primers and specific internal sequencing primers (table 1). Cycle sequencing reactions were carried out with ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using 0.2 μM primer, 1 μl BigDye terminator, and 5x sequencing dilution buffer (400 mM Tris-HCl, 10 mM MgCl2) in a 10 μl reaction. The sequences were analyzed using the Sequence Navigator program (Applied Biosystems). At least two independent PCR reactions were performed to confirm the allele.
MIC Allele Characterization by T/A-Cloning and Sequencing
The PCR for fragment B was performed as described above with the exception that in the PCR program the final extension step is changed into 30 min at 72°C. For MIC allele characterization the PCR products of fragment B were purified, ligated into the plasmid vector pTZ57R/T, and utilized in the transformation using the Ins T/A clone PCR product cloning Kit (Fermentas, St. Leon-Rot, Germany). Clones were isolated by the minipreparation protocol of Qiagen and sequenced as mentioned in the section above. Sequences reported in this publication have been deposited in the EMBL database (accession numbers AJ748822–AJ748831).
Phylogenetic Analysis
Neighbor-Joining (NJ) trees were constructed with the PAUP* program version 4.0b10 for Macintosh, using the method of Jukes-Cantor correction for multiple hits (Jukes 1969) for figure 1, and Kimura's two-parameter model (Swofford 1992) for figure 7. Bootstrap values were based on 1,000 resamplings. The PAUP* program was also used to construct phylogenetic trees using the maximum likelihood method (full heuristic search and quartet puzzling). These trees gave topologies similar to the NJ trees.
FIG. 1.— Phylogenetic tree of the different mtDNA D-loop sequences detected in the BPRC founder animals (indicated in bold), and in chimpanzees of the subspecies Pan troglodytes schweinfurthii (Joy, Pino, and Noah) and Pan troglodytes troglodytes (Pipi, Victoria, and Brigitte). For comparison some sequences from two other databases were added (Morin et al. 1994; Gagneux et al. 1999). From three founder animals (Marga, Tasja, and Tineke) no material was left, and in these cases the mtDNA D-loop was determined by analyzing the offspring. Brigitte is assigned to the P. t. troglodytes because specific nucleotides in the D-loop that are characteristic to the subspecies were found in the sequence of this animal. Relevant bootstrap values based on 1,000 resamplings are indicated.
FIG. 7.— Phylogenetic tree (exon 2) of the Patr-MICA/B alleles and human MICA and MICB alleles. Added are gorilla (AF045597 [GenBank] ), orangutan (AF045599 [GenBank] and AF045600 [GenBank] ), and rhesus macaque (AJ242438 [GenBank] , AJ242440 [GenBank] , AJ242441 [GenBank] , AC148666 [GenBank] , and AC148679 [GenBank] ) MICA-like and MICB-like sequences. The human MICE (AP000521) allele is taken as an out-group. The brackets indicate the division of the human MICA alleles into different lineages. The tree shows that the Mamu-MIC1*01 (Seo et al. 1999) and the Mamu-MICA(1) (Daza-Vamenta et al. 2004) alleles diversified. Relevant bootstrap values based on 1,000 resamplings are indicated.
Nomenclature
The Patr-MICA/B alleles received official designations. The first two digits identify the lineage, while the third and fourth digits reflect the order in which the alleles were found. The fifth digit reflects a mutation in the noncoding region. Sequences will be listed in the IMGT/MHC database (Robinson et al. 2003).
Results and Discussion
mtDNA and MIC Gene Analysis in Chimpanzees
From the 35 founder animals of the BPRC colony, 30 chimpanzees were selected for the present project. Phylogenetic analysis of the mtDNA D-loop sequences shows that most of the founder animals are not related (fig. 1). Although identical mtDNA D-loop sequences were detected in some chimpanzees, these animals appear to possess different MHC haplotypes (fig. 2).
FIG. 2.— List of the 30 founder animals of the BPRC chimpanzee colony, showing their mtDNA typing and MHC haplotypes. The capital letters indicate the different mtDNA sequences that are detected, whereas lowercase letters depict the different MHC haplotypes. The sharing of identical mtDNA typing or MHC haplotype between different animals is indicated by colors. "?" Indicates that this part of the haplotype is not known. "NT" means not typed.
Fifty-three chimpanzees (30 founder animals and 23 offspring) were screened by RSCA for MIC gene–associated polymorphisms. Three different fragments, covering exons 2 and 3 of the gene, were detected. The nucleotide sequence corresponding to the 2B/3B fragment is identical to the earlier published Patr-MIC1 allele (Steinle, Groh, and Spies 1998), which is renamed Patr-MICA/B*01011. The 2A/3A and 2C/3B fragments correspond with the Patr-MICA/B*0102 and *0103 alleles, respectively. Several additional chimpanzees were analyzed by direct sequencing, resulting in the detection of three other alleles, which are designated Patr-MICA/B*0104, *0105, and *0106 (figs. 3 and 4). The Patr-MICA/B*0104 and *0106 alleles are present in the subspecies P. t. troglodytes, whereas Patr-MICA/B*0105 is detected in P. t. schweinfurthii. All six alleles share an identical intron 2, reflecting their common ancestry.
FIG. 3.— Deduced amino acid sequences of the Patr-MICA/B alleles. Human MICA*004 (X92841 [GenBank] ) is used as a reference, and human MICB (U65416 [GenBank] ) and Mamu-MICA(1) and B(2) (AC148666 [GenBank] and AC148679 [GenBank] ) are given for comparison. Identity to MIC*004 is indicated by dashes, whereas amino acid replacements are depicted by the conventional one-letter code. Dots indicate a deletion. The alleles Patr-MIC1 (AF055384 [GenBank] ) and Patr-MIC1*02 (AF322858 [GenBank] ) were renamed Patr-MICA/B*01011 and Patr-MICA/B*0102, respectively. The gray boxes indicate the part of the Patr-MICA/B gene that is more comparable to the human MICB gene, whereas exon 2 of the Patr-MICA/B gene is more related to human MICA. The open boxes in exon 5 indicate the alanine-repeat present in the human MICA and Mamu-MICA(1) gene. In humans this repeat can vary from 2 to 10 repeats per allele (Robinson et al. 2003).
FIG. 4.— Polymorphic nucleotide positions in different exons and introns of the Patr-MICA/B alleles. Intron 2 is identical for all the detected alleles. Identity to the consensus sequence (depicted at the top) is indicated by dashes. In the exons, a capital letter indicates a nonsynonymous substitution, whereas a lowercase letter indicates a synonymous substitution. The numbers above the consensus indicate the nucleotide substitution position. The introns are numbered individually, whereas the exons are numbered through.
In the classical MHC class I genes most polymorphism is confined to exons 2 and 3. In humans, however, MICA polymorphism is not exclusively restricted to these exons, but is also located in exon 4 (Fodil et al. 1996). Therefore the study was extended by sequencing intron 3 to exon 5, resulting in the definition of an additional allele, Patr-MICA/B*0107 (fig. 3), which is present in P. t. troglodytes. Furthermore, introns 3 and 4 show allelic variations that appear to be caused by point mutations (fig. 4). Patr-MICA/B*0101 is found in combination with four different intron 3 sequences, as reflected by the designations Patr-MICA/B*01011, *01012, *01013, and *01014, respectively (fig. 4). The Patr-MICA/B*01011 allele is observed in all three subspecies, Patr-MICA/B*01012 is present in the subspecies P. t. troglodytes and P. t. schweinfurthii, while Patr-MICA/B*01013 and *01014 are only detected in P. t. verus and P. t. troglodytes, respectively. Intron 3 of Patr-MICA/B*01011 is found in combination with other alleles, whereas Patr-MICA/B*0105 has its own unique intron 3 sequence (fig. 4). Strong conservative evolution must have operated on the Patr-MICA/B*0101 allele. This is supported by the fact that this allele is present in all subspecies, that it is the most frequently detected allele in the population (94% of the West African chimpanzees analyzed possess at least one copy of the allele), and that it exhibits more sequence variation in the introns than in the exons.
All presently known Patr-MICA/B alleles share the same characteristic hybrid sequence structure. The first part (exon 2 and intron 2) is comparable to the human MICA sequence, whereas the second part (exon 3 to 5) is roughly equivalent to the human MICB sequence (fig. 3). The recombinant character of the Patr-MICA/B sequences is further illustrated by the absence of the GCT repeat (alanine) in exon 5. The presence of this repeat is typical for the human MICA alleles but as such is absent in MICB (fig. 3).
The MIC Gene: A Comparison Between Humans, Chimpanzees, and Other Species
Figure 5A shows a schematic representation of the distribution of the MICA and MICB gene and their haplotypes in different species. For gorilla (Gogo), orangutan (Popy), and rhesus macaque (Mamu) a MICA-like gene has been documented (Pellet et al. 1999; Daza-Vamenta et al. 2004), additionally short tandem repeat analysis provided further evidence for the presence of human MICA gene orthologues in these species (data not shown). Recently, the genomic organization of the rhesus macaque MHC was published, showing the presence of a MIC1 and MIC2 gene that are situated on the chromosome on locations comparable to those of the human MICA and MICB gene (Daza-Vamenta et al. 2004). Furthermore, Mamu-MIC1 possesses the characteristic GCT-repeat coding for alanine residues (fig. 3), although the alanine residues are interrupted by one valine. Thus, MIC1 seems to be the orthologue of the human MICA gene. In Mamu-MIC2 the repeat is absent, and, additionally, intron 3 shows features that are characteristic for MICB (fig. 6, gray boxes). Despite evidence that an ancestor of humans, great apes, and Old World monkeys possessed a MICA-like and MICB-like gene tandem on a haplotype (fig. 5A), it is evident that these genes diversified considerably in humans versus rhesus macaques.
FIG. 5.— (A) Schematic representation of the relationship of the MICA and MICB gene in different species. The "?" indicates that it is not known if a MICB-like gene is present in this species. (B) A 95-kb genomic deletion in the ? block of the chimpanzee producing a Patr-MICA/B fusion product, the schematic representation is based on previously published work (Kulski et al. 2002), and compared to the human organization. MYA, million years ago.
FIG. 6.— Polymorphic nucleotide positions in intron 3. The Patr-MICA/B, human MICB, and Mamu-MICA(1) and B(2) are compared to the human MICA*004. Identity is indicated by dashes. Dots indicate deletions. For instance, "-/A" means that differences in the reported alleles are detected. The numbers above the human MICA*004 alignment indicate the nucleotide substitution positions. The open and gray boxes indicate the parts that show the differentiation between MICA and MICB, respectively.
It is not known whether a MICB-like gene is present in gorillas or orangutans. We therefore designed primers amplifying partial MICB intron 3 (nucleotide positions 235–513), including the characteristic insert (fig. 6). In orangutans we were able to amplify this sequence, indicating that at least a MICB-like gene segment is present in this species (fig. 5A). However, the primers did not produce a product with gorilla samples. This could indicate that a MICB-like gene is absent in gorillas, although the possibility that the primers are not working optimally cannot be excluded.
Exon 2 of the human MICA and MICB and the Patr-MICA/B nucleotide sequences were subjected to phylogenetic analysis together with the known gorilla, orangutan, and rhesus macaque MICA and MICB orthologues (fig. 7). The tree illustrates that this part of the Patr-MICA/B gene is distantly related to the human MICA gene but shows a reduced level of variation of one lineage versus four lineages, respectively. The second part of the Patr-MICA/B sequences, exons 3 to 5, showed a more intermediate character versus human MICA and MICB (data not shown). Comparison of the data shows that intron 3 of the Patr-MICA/B alleles possesses features more characteristic of the human MICB gene (fig. 6), and in addition exon 5 illustrates the more close genetic relation of the chimpanzee and human MICB sequences (fig. 3). The reported chimpanzee class I region is characterized by a 95-kb genomic deletion resulting in a Patr-MICA/B fusion product (fig. 5B) (Kulski et al. 2002; Anzai et al. 2003). The present communication shows that all chimpanzees analyzed so far possess haplotypes with the hybrid MICA/B gene. MICA polymorphism studies in humans resulted in the identification of 54 different alleles (Petersdorf et al. 1999; Visser et al. 1999; Yao et al. 1999; Bahram 2000; Robinson et al. 2001), whereas for MICB 14 different alleles are detected (Bahram and Spies 1996; Ando et al. 1997; Pellet et al. 1997; Visser et al. 1998). In the 30 unrelated West African chimpanzees studied, only four MICA/B alleles were identified (fig. 4), of which only three encode slightly distinct proteins (fig. 3). All variation can be explained by point mutations. Likewise, in the two other chimpanzee subspecies studied, only hybrid MICA/B sequences are detected. Taken together, 10 Patr-MICA/B alleles were identified, among which 7 encode for distinct proteins. Thus, it seems that West African chimpanzees have only one functional copy of a MIC gene, which possesses only one lineage and limited allelic variation, and the same trend seems to be observed in chimpanzees of other subspecies. Analysis of the Patr-MICA/B gene introns 3 and 4 revealed limited allelic variation, five and two different sequences, respectively. In the 595 nucleotide bases of intron 3, only one (Patr-MICA/B*01012 and Patr-MICA/B*01014) or three (Patr-MICA/B*0105) nucleotide substitutions are observed (fig. 4). The existence of these few nucleotide substitutions indicates that the alleles are probably of relatively recent origin.
One question to be answered is: when did the 95-kb deletion resulting in a Patr-MICA/B fusion product take place? Most likely this deletion occurred after the human-chimpanzee split (fig. 5A). The presence of an equivalent in bonobos indicates that the deletion happened before the speciation of chimpanzee and bonobo approximately 2 MYA. Thus, the deletion took place in the interval of 2 to 5–6 MYA (fig. 5A). The alternative explanation, that the human genome experienced a 95-kb insertion generating the MICA and MICB genes, is highly unlikely. Such an assumption is also in disagreement with the fact that the existence of the MICA gene predates the speciation of humans, chimpanzees, gorillas, orangutans (Pellet et al. 1999), and rhesus macaques (fig. 5A) (Daza-Vamenta et al. 2004). This would imply that all ancestral chimpanzee haplotypes with the original Patr-MICA and Patr-MICB genes have been subject to a strong negative selection because these genes are apparently absent in the contemporary population. Earlier we reported that both the MHC class I Patr-A and Patr-B loci have suffered a severe repertoire reduction with regard to lineages, as compared to the human population. The intron 2 analysis suggested that the selective sweep in the MHC region occurred approximately 2 to 3 MYA (de Groot et al. 2002). Due to the close vicinity of the Patr-B and Patr-MICA/B loci it is possible that the hybrid Patr-MICA/B gene was selected based on a piggyback effect.
In humans, four candidate genes are found within the 95-kb section that is deleted: namely two uncharacterized transcripts, 3.8-1.1 and P5-1 (class I–like transcript), and two pseudogenes, HCGIX-1 and HLA-X (Kulski et al. 2002; Anzai et al. 2003). Based on the knowledge that humans and chimpanzees shared a common ancestor, it is highly probable that equivalents of these genes were also present in chimpanzees before the deletion. However, because the function of the 3.8-1.1 and P5-1 transcripts is unknown, we cannot exclude the possibility that the deletion of these genes may have given a selective advantage to chimpanzee haplotypes with the hybrid Patr-MICA/B gene.
Linkage Between the MHC-B Locus and the Human MICA/Patr–MICA/B Locus
In humans, particular MICA alleles are strongly associated to particular HLA-B alleles. From an evolutionary point of view one could expect that particular Patr-MICA/B alleles also show an association with certain Patr-B alleles. Genomic mapping studies demonstrated that the Patr-MICA/B gene and the human MICA gene are located 45.2 and 46.4 kb centromeric from the MHC-B locus, respectively. However, in the well-characterized BPRC chimpanzee population, specific association between particular Patr-MICA/B alleles and particular Patr-B alleles is not observed. The most frequently detected MIC allele, Patr-MICA/B*01011, is present in all subspecies. Furthermore, all the Patr-MICA/B alleles that are detected seem to belong to one lineage. Again, it is possible that the Patr-B lineages/alleles that survived the ancient selective sweep were all linked to the frequently detected Patr-MICA/B*01011 allele and that the contemporary limited allelic variation seen in the Patr-MICA/B locus was generated after the selective sweep. Support for this observation is found in the Patr-MICA/B intron 3 sequences (fig. 4), where only a few nucleotide substitutions distinguish the different alleles. This most probably reflects their more recent origin.
Could Patr-MICA/B Contribute to AIDS Resistance in Chimpanzees?
Chimpanzees can be naturally infected with Simian immunodeficiency virus from chimpanzees (SIVcpz) but are also susceptible to infection with Human immunodeficiency virus type 1 (HIV-1). Although natural SIVcpz infections in West African chimpanzees have never been observed, the animals are susceptible to HIV-1 infection in captivity (Rutjens et al. 2003). In contrast to humans, chimpanzees normally do not develop symptoms of acquired immunodeficiency syndrome (AIDS). We have put forward the hypothesis that in the past chimpanzees may have been decimated by an AIDS-like pandemic caused by an HIV-1/SIVcpz-like retrovirus and that the contemporary population represents the offspring of the survivors (de Groot et al. 2002). In this respect, the contemporary chimpanzee population may have been enriched for resistance genes, and a candidate group that was identified is the group of the MHC molecules, which play an important role in controlling infections.
Chimpanzees are often described as herbivores, but they also hunt and consume other nonhuman primate species (Stanford et al. 1994). As most of these Old World monkeys have natural SIV infections, chimpanzees may have become infected through predating on infected monkeys. Probably chimpanzees have been infected multiple times and by various SIV-like strains (Courgnaud et al. 2002; Bailes et al. 2003). To date little is known about the transmission of SIVcpz between different chimpanzees. The initial infections with HIV-1/SIVcpz-like viruses in humans probably happened through blood contact during the hunting of infected chimpanzees. The routes of HIV-1 infection in humans are now known to be via intimate sexual contact, contaminated blood or blood products, or by transmission from mother to child (Levy, Scott, and Mackewicz 2003). Nonetheless, the intestine may be the major site for HIV replication and depletion of CD4+ T cells (Veazey and Lackner 2004). In this perspective, it is possible that the hybrid Patr-MICA/B gene is highly efficient in detecting stress mediated by infection, and as such may induce efficient natural killer (NK) cell–mediated killing.
The human MICA and probably also the closely related MICB molecules are ligands for the NKG2D receptor, which is expressed on NK cells, T cells, and CD8+ ? T cells. After cellular stress, triggered for instance by a viral infection or malignant transformation, MIC is upregulated and may provoke an immune response (Bauer et al. 1999; Gleimer and Parham 2003). Recent work, for instance, showed that the activating KIR gene KIR3DS1 is associated with a delay in progression to AIDS in HIV-1–infected individuals (Martin et al. 2002). The chimpanzee MICA/B molecule is able to recognize human V1 T cells specific for MICA and MICB, suggesting a conserved recognition site (Steinle, Groh, and Spies 1998). Indeed, also the NKG2D receptor shows a high degree of similarity, approximately 98.9%, between humans and chimpanzees (Shum et al. 2002). Based upon these observations and the knowledge that humans and chimpanzees are closely related, a similar kind of immune response as described in humans is plausible.
The possible role of MIC-elicited anti-HIV-1/SIVcpz NK effector responses remains to be proved, and subsequent studies will be required to elucidate the functional significance of the Patr-MICA/B gene in virus infections.
Acknowledgements
The authors wish to thank D. Devine and H. van Westbroek for expert assistance, J. Heeney for discussion, and S. Langehuizen for technical assistance. This study was financed in part by the European Union Project, IMGT-QLG2-2000-01287.
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Correspondence: E-mail: groot@bprc.nl.
Abstract
The human major histocompatibility complex class I chain-related (MIC) genes are members of a multicopy family showing similarity to the classical HLA-A, HLA-B, and HLA-C genes. Only the MICA and MICB genes produce functional transcripts. In chimpanzees, however, only one MIC gene is expressed, showing an intermediate character, resulting from a deletion fusing the MICA and MICB gene segments together. The present population study illustrates that all chimpanzee haplotypes sampled possess the hybrid MICA/B gene. In contrast to the human situation this gene displays reduced allelic variation. The observed repertoire reduction of the chimpanzee MICA/B gene is in conformity with the severe repertoire condensation documented for Patr-B locus lineages, probably due to the close proximity of both genes.
Key Words: chimpanzee ? humans ? HIV ? MHC ? MIC
Introduction
The major histocompatibility complex class I chain-related gene (MIC), previously also called PERB11, was first described in 1994 (Bahram et al. 1994; Leelayuwat et al. 1994). The gene is located in the major histocompatibility complex (MHC) region. The MHC, playing an important role in the immune defense against pathogens, is characterized by its extensive degree of polymorphism at the population level. Also, in the relatively small sample size of chimpanzees that have been analyzed so far, a high degree of Patr class I diversity, with regard to allele numbers, has been detected (Adams et al. 2000; de Groot et al. 2000). At first sight, this agrees with the finding that chimpanzees display more mitochondrial DNA (mtDNA) variation than humans do (Gagneux et al. 1999; Ingman et al. 2000), and similar findings have been reported for particular nuclear genes (Kaessmann, Wiebe, and Paabo 1999; Zhao et al. 2000), although a recent publication shows that the variation in chimpanzees is not as high as a magnitude of four but more in the range of 1.5 compared to humans (Yu et al. 2003). A more detailed analysis of the MHC class I intron 2 sequences in chimpanzees showed that the MHC class I gene repertoire, with respect to the number of lineages, is severely reduced as compared to humans. This repertoire reduction is evident for Patr-A (McAdam et al. 1995) but is most prominent for the Patr-B locus lineages (de Groot et al. 2002).
Seven MIC genes are distinguished in the human genome. MICA and MICB produce functional transcripts, whereas MICC to MICG are pseudogenes (Bahram 2000). The MICA and MICB genes show a high degree of similarity to the classical MHC class I genes but are distinguished by their disparate organization of exons-introns. Moreover, they do not associate with ?2-microglobulin (?2m), and their expression is not induced by type I/II interferons. Furthermore, the MICA and MICB genes are predominantly expressed on fibroblast and epithelial cells (Bahram 2000). A part of the MICA and MICB promoter region shows similarity to heat shock protein gene promoters, and upregulation of the MICA and MICB molecules after heat shock has been reported (Groh et al. 1998). In conclusion, MICA and MICB seem to play a role in the detection of cell stress and appear to react preferentially with the ligands V1 TCR and NKG2D to induce an immune response (Groh et al. 1998; Bauer et al. 1999; Gleimer and Parham 2003). In chimpanzees only one functional MIC gene has been described (Steinle, Groh, and Spies 1998), and this gene appears to have an intermediate character as compared to human MICA and MICB genes (Cattley et al. 1999; Kulski et al. 2002). Studies on MIC gene polymorphism in chimpanzee populations are absent, and only one allele, Patr-MIC1, has been reported (Steinle, Groh, and Spies 1998). The human MICA and MICB genes show a considerable degree of polymorphism, and to date 54 MICA and 14 MICB alleles have been identified (Radosavljevic and Bahram 2003).
Compared to humans, chimpanzees have a severely reduced MHC class I gene repertoire caused by an ancient selective sweep (de Groot et al. 2002), and they appear to lack the equivalents of the HLA-DRB1*04 and HLA-DRB1*08 lineages (Bontrop et al. 1999). The present study was initiated to investigate whether the repertoire reduction, most prominent for the Patr-B locus lineages, is only restricted to the MHC class I and II genes or also extends to other genes located in the MHC region. For that reason we investigated the polymorphism at the chimpanzee MIC gene, which is located in the direct neighborhood of the Patr-B locus (Anzai et al. 2003). In humans, some of the MICA alleles and closely linked HLA-B alleles show a high degree of linkage disequilibrium (Bahram 2000). Here we report on MIC gene variation and linkage disequilibria in a West African chimpanzee population together with data on chimpanzees of other subspecies.
Materials and Methods
Animals
The chimpanzee (Pan troglodytes) colony (approximately 100 individuals) at the Biomedical Primate Research Centre (BPRC) started with 35 founder animals originating from Sierra Leone and belonging to the subspecies Pan troglodytes verus (West Africa). The animals are characterized at the molecular level for MHC class I and II gene polymorphisms (Bontrop et al. 1999; de Groot et al. 2000). Their offspring have been pedigreed based on segregation of Patr-A and Patr-B serotypes and molecularly defined Patr class I and II gene polymorphisms. Three animals of the Pan troglodytes troglodytes (Central Africa) and Pan troglodytes schweinfurthii (East Africa) subspecies have been included in this study. For the two bonobos (Pan paniscus) used in this study, the DNAs were analyzed with 12S primers to determine potential relationships (Kocher et al. 1989).
mtDNA Analysis
Genomic DNA (gDNA), obtained from Epstein-Barr virus-transformed B-cell lines, was used to amplify the mitochondrial D-loop (380 bp) sequences. To assign the subspecies, the nucleotide sequences were compared to published sequences (Morin et al. 1994; Gagneux et al. 1999). The polymerase chain reaction (PCR) mixture (50 μl) contained gDNA (50 ng), 1 μM of the primers (table 1), 1x PCR buffer + bovine serum albumin, 2 mM MgCl2, 0.2 mM of each deoxyribonucleoside triphosphate (dNTP), and 2.5 units (U) Taq polymerase Gold (Applied Biosystems, Foster City, Calif). A touchdown PCR consisting of the following cycles was run: 1 cycle of 15 min at 94°C, 2 cycles of 30 s at 94°C, 30 s at 63°C, 30 s at 72°C; after each two cycles the annealing temperature is decreased by 2°C until 55°C was reached, followed by 4 cycles of 30 s at 94°C, 30 s at 53°C, 30 s at 72°C, and 25 cycles of 30 s at 94°C, 30 s at 50°C, 30 s at 72°C, with a final extension of 5 min at 72°C. The PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and sequenced directly on an ABI 310 automatic sequencer using the ABI Prism dRhodamine Terminator Cycle sequencing Ready Reaction Kit (Applied Biosystems) to prepare the samples according to the manufacturer's instructions. The products were sequenced from the 5' and 3' ends. Sequences reported in this publication have been deposited in the EMBL database (accession numbers AJ863489–AJ863508).
Table 1 MIC-Specific PCR Primers and Internal Sequencing Primers
MIC Gene Characterization by Reference Strand Conformation Analysis
Reference strand conformation analysis (RSCA) was used to screen the chimpanzees for MIC gene polymorphisms. Locus-specific primers, amplifying human MICA, were used to independently amplify exon 2 and 3 in chimpanzees (Mendoza-Rincon et al. 1999). RSCA was performed at the Anthony Nolan Research Institute, London, as has been described in detail (Arguello et al. 1998).
MIC Gene Characterization by Direct Nucleotide Sequencing
gDNA was used for the PCR amplification. Two independent overlapping PCR fragments were generated, using MIC-specific primers (table 1), covering the MIC gene from exon 2 to exon 5. This resulted in a 1,046-bp PCR fragment (fragment A), covering exon 2 (255 bp), intron 2 (273 bp), and exon 3 (270 bp), and a part of introns 1 (101 bp) and 3 (100 bp). The second PCR product was 1,297-bp long (fragment B), containing complete intron 3 (596 bp), exon 4 (270 bp), intron 4 (102 bp), and exon 5 (141 bp), and a part of exon 3 (129 bp) and intron 5 (13 bp). The PCR for fragment A (25 μl) contained gDNA (100 ng), 0.8 μM of the primers (table 1), 2.5–3 mM MgCl2, 0.4 mM of each dNTP, and 5 U of Taq polymerase. A total of 33 cycles were run, each cycle consisting of 30 s at 95°C, 50 s at 61.3°C, and 30 s at 72°C with a final extension of 8 min at 72°C. PCR for fragment B (50 μl) contained gDNA (100 ng), 0.8 μM of the primers (table 1), 2.25–2.75 mM MgCl2, 0.4 mM of each dNTP, and 5 U of Taq polymerase. A total of 28 cycles were run, each cycle consisting of 30 s at 95°C, 30 s at 58.5°C, and 40 s at 72°C with a final extension of 7 min at 72°C. The PCR products were purified, and sequenced directly on an ABI 3100 genetic analyzer (Applied Biosystems) using the PCR primers and specific internal sequencing primers (table 1). Cycle sequencing reactions were carried out with ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using 0.2 μM primer, 1 μl BigDye terminator, and 5x sequencing dilution buffer (400 mM Tris-HCl, 10 mM MgCl2) in a 10 μl reaction. The sequences were analyzed using the Sequence Navigator program (Applied Biosystems). At least two independent PCR reactions were performed to confirm the allele.
MIC Allele Characterization by T/A-Cloning and Sequencing
The PCR for fragment B was performed as described above with the exception that in the PCR program the final extension step is changed into 30 min at 72°C. For MIC allele characterization the PCR products of fragment B were purified, ligated into the plasmid vector pTZ57R/T, and utilized in the transformation using the Ins T/A clone PCR product cloning Kit (Fermentas, St. Leon-Rot, Germany). Clones were isolated by the minipreparation protocol of Qiagen and sequenced as mentioned in the section above. Sequences reported in this publication have been deposited in the EMBL database (accession numbers AJ748822–AJ748831).
Phylogenetic Analysis
Neighbor-Joining (NJ) trees were constructed with the PAUP* program version 4.0b10 for Macintosh, using the method of Jukes-Cantor correction for multiple hits (Jukes 1969) for figure 1, and Kimura's two-parameter model (Swofford 1992) for figure 7. Bootstrap values were based on 1,000 resamplings. The PAUP* program was also used to construct phylogenetic trees using the maximum likelihood method (full heuristic search and quartet puzzling). These trees gave topologies similar to the NJ trees.
FIG. 1.— Phylogenetic tree of the different mtDNA D-loop sequences detected in the BPRC founder animals (indicated in bold), and in chimpanzees of the subspecies Pan troglodytes schweinfurthii (Joy, Pino, and Noah) and Pan troglodytes troglodytes (Pipi, Victoria, and Brigitte). For comparison some sequences from two other databases were added (Morin et al. 1994; Gagneux et al. 1999). From three founder animals (Marga, Tasja, and Tineke) no material was left, and in these cases the mtDNA D-loop was determined by analyzing the offspring. Brigitte is assigned to the P. t. troglodytes because specific nucleotides in the D-loop that are characteristic to the subspecies were found in the sequence of this animal. Relevant bootstrap values based on 1,000 resamplings are indicated.
FIG. 7.— Phylogenetic tree (exon 2) of the Patr-MICA/B alleles and human MICA and MICB alleles. Added are gorilla (AF045597 [GenBank] ), orangutan (AF045599 [GenBank] and AF045600 [GenBank] ), and rhesus macaque (AJ242438 [GenBank] , AJ242440 [GenBank] , AJ242441 [GenBank] , AC148666 [GenBank] , and AC148679 [GenBank] ) MICA-like and MICB-like sequences. The human MICE (AP000521) allele is taken as an out-group. The brackets indicate the division of the human MICA alleles into different lineages. The tree shows that the Mamu-MIC1*01 (Seo et al. 1999) and the Mamu-MICA(1) (Daza-Vamenta et al. 2004) alleles diversified. Relevant bootstrap values based on 1,000 resamplings are indicated.
Nomenclature
The Patr-MICA/B alleles received official designations. The first two digits identify the lineage, while the third and fourth digits reflect the order in which the alleles were found. The fifth digit reflects a mutation in the noncoding region. Sequences will be listed in the IMGT/MHC database (Robinson et al. 2003).
Results and Discussion
mtDNA and MIC Gene Analysis in Chimpanzees
From the 35 founder animals of the BPRC colony, 30 chimpanzees were selected for the present project. Phylogenetic analysis of the mtDNA D-loop sequences shows that most of the founder animals are not related (fig. 1). Although identical mtDNA D-loop sequences were detected in some chimpanzees, these animals appear to possess different MHC haplotypes (fig. 2).
FIG. 2.— List of the 30 founder animals of the BPRC chimpanzee colony, showing their mtDNA typing and MHC haplotypes. The capital letters indicate the different mtDNA sequences that are detected, whereas lowercase letters depict the different MHC haplotypes. The sharing of identical mtDNA typing or MHC haplotype between different animals is indicated by colors. "?" Indicates that this part of the haplotype is not known. "NT" means not typed.
Fifty-three chimpanzees (30 founder animals and 23 offspring) were screened by RSCA for MIC gene–associated polymorphisms. Three different fragments, covering exons 2 and 3 of the gene, were detected. The nucleotide sequence corresponding to the 2B/3B fragment is identical to the earlier published Patr-MIC1 allele (Steinle, Groh, and Spies 1998), which is renamed Patr-MICA/B*01011. The 2A/3A and 2C/3B fragments correspond with the Patr-MICA/B*0102 and *0103 alleles, respectively. Several additional chimpanzees were analyzed by direct sequencing, resulting in the detection of three other alleles, which are designated Patr-MICA/B*0104, *0105, and *0106 (figs. 3 and 4). The Patr-MICA/B*0104 and *0106 alleles are present in the subspecies P. t. troglodytes, whereas Patr-MICA/B*0105 is detected in P. t. schweinfurthii. All six alleles share an identical intron 2, reflecting their common ancestry.
FIG. 3.— Deduced amino acid sequences of the Patr-MICA/B alleles. Human MICA*004 (X92841 [GenBank] ) is used as a reference, and human MICB (U65416 [GenBank] ) and Mamu-MICA(1) and B(2) (AC148666 [GenBank] and AC148679 [GenBank] ) are given for comparison. Identity to MIC*004 is indicated by dashes, whereas amino acid replacements are depicted by the conventional one-letter code. Dots indicate a deletion. The alleles Patr-MIC1 (AF055384 [GenBank] ) and Patr-MIC1*02 (AF322858 [GenBank] ) were renamed Patr-MICA/B*01011 and Patr-MICA/B*0102, respectively. The gray boxes indicate the part of the Patr-MICA/B gene that is more comparable to the human MICB gene, whereas exon 2 of the Patr-MICA/B gene is more related to human MICA. The open boxes in exon 5 indicate the alanine-repeat present in the human MICA and Mamu-MICA(1) gene. In humans this repeat can vary from 2 to 10 repeats per allele (Robinson et al. 2003).
FIG. 4.— Polymorphic nucleotide positions in different exons and introns of the Patr-MICA/B alleles. Intron 2 is identical for all the detected alleles. Identity to the consensus sequence (depicted at the top) is indicated by dashes. In the exons, a capital letter indicates a nonsynonymous substitution, whereas a lowercase letter indicates a synonymous substitution. The numbers above the consensus indicate the nucleotide substitution position. The introns are numbered individually, whereas the exons are numbered through.
In the classical MHC class I genes most polymorphism is confined to exons 2 and 3. In humans, however, MICA polymorphism is not exclusively restricted to these exons, but is also located in exon 4 (Fodil et al. 1996). Therefore the study was extended by sequencing intron 3 to exon 5, resulting in the definition of an additional allele, Patr-MICA/B*0107 (fig. 3), which is present in P. t. troglodytes. Furthermore, introns 3 and 4 show allelic variations that appear to be caused by point mutations (fig. 4). Patr-MICA/B*0101 is found in combination with four different intron 3 sequences, as reflected by the designations Patr-MICA/B*01011, *01012, *01013, and *01014, respectively (fig. 4). The Patr-MICA/B*01011 allele is observed in all three subspecies, Patr-MICA/B*01012 is present in the subspecies P. t. troglodytes and P. t. schweinfurthii, while Patr-MICA/B*01013 and *01014 are only detected in P. t. verus and P. t. troglodytes, respectively. Intron 3 of Patr-MICA/B*01011 is found in combination with other alleles, whereas Patr-MICA/B*0105 has its own unique intron 3 sequence (fig. 4). Strong conservative evolution must have operated on the Patr-MICA/B*0101 allele. This is supported by the fact that this allele is present in all subspecies, that it is the most frequently detected allele in the population (94% of the West African chimpanzees analyzed possess at least one copy of the allele), and that it exhibits more sequence variation in the introns than in the exons.
All presently known Patr-MICA/B alleles share the same characteristic hybrid sequence structure. The first part (exon 2 and intron 2) is comparable to the human MICA sequence, whereas the second part (exon 3 to 5) is roughly equivalent to the human MICB sequence (fig. 3). The recombinant character of the Patr-MICA/B sequences is further illustrated by the absence of the GCT repeat (alanine) in exon 5. The presence of this repeat is typical for the human MICA alleles but as such is absent in MICB (fig. 3).
The MIC Gene: A Comparison Between Humans, Chimpanzees, and Other Species
Figure 5A shows a schematic representation of the distribution of the MICA and MICB gene and their haplotypes in different species. For gorilla (Gogo), orangutan (Popy), and rhesus macaque (Mamu) a MICA-like gene has been documented (Pellet et al. 1999; Daza-Vamenta et al. 2004), additionally short tandem repeat analysis provided further evidence for the presence of human MICA gene orthologues in these species (data not shown). Recently, the genomic organization of the rhesus macaque MHC was published, showing the presence of a MIC1 and MIC2 gene that are situated on the chromosome on locations comparable to those of the human MICA and MICB gene (Daza-Vamenta et al. 2004). Furthermore, Mamu-MIC1 possesses the characteristic GCT-repeat coding for alanine residues (fig. 3), although the alanine residues are interrupted by one valine. Thus, MIC1 seems to be the orthologue of the human MICA gene. In Mamu-MIC2 the repeat is absent, and, additionally, intron 3 shows features that are characteristic for MICB (fig. 6, gray boxes). Despite evidence that an ancestor of humans, great apes, and Old World monkeys possessed a MICA-like and MICB-like gene tandem on a haplotype (fig. 5A), it is evident that these genes diversified considerably in humans versus rhesus macaques.
FIG. 5.— (A) Schematic representation of the relationship of the MICA and MICB gene in different species. The "?" indicates that it is not known if a MICB-like gene is present in this species. (B) A 95-kb genomic deletion in the ? block of the chimpanzee producing a Patr-MICA/B fusion product, the schematic representation is based on previously published work (Kulski et al. 2002), and compared to the human organization. MYA, million years ago.
FIG. 6.— Polymorphic nucleotide positions in intron 3. The Patr-MICA/B, human MICB, and Mamu-MICA(1) and B(2) are compared to the human MICA*004. Identity is indicated by dashes. Dots indicate deletions. For instance, "-/A" means that differences in the reported alleles are detected. The numbers above the human MICA*004 alignment indicate the nucleotide substitution positions. The open and gray boxes indicate the parts that show the differentiation between MICA and MICB, respectively.
It is not known whether a MICB-like gene is present in gorillas or orangutans. We therefore designed primers amplifying partial MICB intron 3 (nucleotide positions 235–513), including the characteristic insert (fig. 6). In orangutans we were able to amplify this sequence, indicating that at least a MICB-like gene segment is present in this species (fig. 5A). However, the primers did not produce a product with gorilla samples. This could indicate that a MICB-like gene is absent in gorillas, although the possibility that the primers are not working optimally cannot be excluded.
Exon 2 of the human MICA and MICB and the Patr-MICA/B nucleotide sequences were subjected to phylogenetic analysis together with the known gorilla, orangutan, and rhesus macaque MICA and MICB orthologues (fig. 7). The tree illustrates that this part of the Patr-MICA/B gene is distantly related to the human MICA gene but shows a reduced level of variation of one lineage versus four lineages, respectively. The second part of the Patr-MICA/B sequences, exons 3 to 5, showed a more intermediate character versus human MICA and MICB (data not shown). Comparison of the data shows that intron 3 of the Patr-MICA/B alleles possesses features more characteristic of the human MICB gene (fig. 6), and in addition exon 5 illustrates the more close genetic relation of the chimpanzee and human MICB sequences (fig. 3). The reported chimpanzee class I region is characterized by a 95-kb genomic deletion resulting in a Patr-MICA/B fusion product (fig. 5B) (Kulski et al. 2002; Anzai et al. 2003). The present communication shows that all chimpanzees analyzed so far possess haplotypes with the hybrid MICA/B gene. MICA polymorphism studies in humans resulted in the identification of 54 different alleles (Petersdorf et al. 1999; Visser et al. 1999; Yao et al. 1999; Bahram 2000; Robinson et al. 2001), whereas for MICB 14 different alleles are detected (Bahram and Spies 1996; Ando et al. 1997; Pellet et al. 1997; Visser et al. 1998). In the 30 unrelated West African chimpanzees studied, only four MICA/B alleles were identified (fig. 4), of which only three encode slightly distinct proteins (fig. 3). All variation can be explained by point mutations. Likewise, in the two other chimpanzee subspecies studied, only hybrid MICA/B sequences are detected. Taken together, 10 Patr-MICA/B alleles were identified, among which 7 encode for distinct proteins. Thus, it seems that West African chimpanzees have only one functional copy of a MIC gene, which possesses only one lineage and limited allelic variation, and the same trend seems to be observed in chimpanzees of other subspecies. Analysis of the Patr-MICA/B gene introns 3 and 4 revealed limited allelic variation, five and two different sequences, respectively. In the 595 nucleotide bases of intron 3, only one (Patr-MICA/B*01012 and Patr-MICA/B*01014) or three (Patr-MICA/B*0105) nucleotide substitutions are observed (fig. 4). The existence of these few nucleotide substitutions indicates that the alleles are probably of relatively recent origin.
One question to be answered is: when did the 95-kb deletion resulting in a Patr-MICA/B fusion product take place? Most likely this deletion occurred after the human-chimpanzee split (fig. 5A). The presence of an equivalent in bonobos indicates that the deletion happened before the speciation of chimpanzee and bonobo approximately 2 MYA. Thus, the deletion took place in the interval of 2 to 5–6 MYA (fig. 5A). The alternative explanation, that the human genome experienced a 95-kb insertion generating the MICA and MICB genes, is highly unlikely. Such an assumption is also in disagreement with the fact that the existence of the MICA gene predates the speciation of humans, chimpanzees, gorillas, orangutans (Pellet et al. 1999), and rhesus macaques (fig. 5A) (Daza-Vamenta et al. 2004). This would imply that all ancestral chimpanzee haplotypes with the original Patr-MICA and Patr-MICB genes have been subject to a strong negative selection because these genes are apparently absent in the contemporary population. Earlier we reported that both the MHC class I Patr-A and Patr-B loci have suffered a severe repertoire reduction with regard to lineages, as compared to the human population. The intron 2 analysis suggested that the selective sweep in the MHC region occurred approximately 2 to 3 MYA (de Groot et al. 2002). Due to the close vicinity of the Patr-B and Patr-MICA/B loci it is possible that the hybrid Patr-MICA/B gene was selected based on a piggyback effect.
In humans, four candidate genes are found within the 95-kb section that is deleted: namely two uncharacterized transcripts, 3.8-1.1 and P5-1 (class I–like transcript), and two pseudogenes, HCGIX-1 and HLA-X (Kulski et al. 2002; Anzai et al. 2003). Based on the knowledge that humans and chimpanzees shared a common ancestor, it is highly probable that equivalents of these genes were also present in chimpanzees before the deletion. However, because the function of the 3.8-1.1 and P5-1 transcripts is unknown, we cannot exclude the possibility that the deletion of these genes may have given a selective advantage to chimpanzee haplotypes with the hybrid Patr-MICA/B gene.
Linkage Between the MHC-B Locus and the Human MICA/Patr–MICA/B Locus
In humans, particular MICA alleles are strongly associated to particular HLA-B alleles. From an evolutionary point of view one could expect that particular Patr-MICA/B alleles also show an association with certain Patr-B alleles. Genomic mapping studies demonstrated that the Patr-MICA/B gene and the human MICA gene are located 45.2 and 46.4 kb centromeric from the MHC-B locus, respectively. However, in the well-characterized BPRC chimpanzee population, specific association between particular Patr-MICA/B alleles and particular Patr-B alleles is not observed. The most frequently detected MIC allele, Patr-MICA/B*01011, is present in all subspecies. Furthermore, all the Patr-MICA/B alleles that are detected seem to belong to one lineage. Again, it is possible that the Patr-B lineages/alleles that survived the ancient selective sweep were all linked to the frequently detected Patr-MICA/B*01011 allele and that the contemporary limited allelic variation seen in the Patr-MICA/B locus was generated after the selective sweep. Support for this observation is found in the Patr-MICA/B intron 3 sequences (fig. 4), where only a few nucleotide substitutions distinguish the different alleles. This most probably reflects their more recent origin.
Could Patr-MICA/B Contribute to AIDS Resistance in Chimpanzees?
Chimpanzees can be naturally infected with Simian immunodeficiency virus from chimpanzees (SIVcpz) but are also susceptible to infection with Human immunodeficiency virus type 1 (HIV-1). Although natural SIVcpz infections in West African chimpanzees have never been observed, the animals are susceptible to HIV-1 infection in captivity (Rutjens et al. 2003). In contrast to humans, chimpanzees normally do not develop symptoms of acquired immunodeficiency syndrome (AIDS). We have put forward the hypothesis that in the past chimpanzees may have been decimated by an AIDS-like pandemic caused by an HIV-1/SIVcpz-like retrovirus and that the contemporary population represents the offspring of the survivors (de Groot et al. 2002). In this respect, the contemporary chimpanzee population may have been enriched for resistance genes, and a candidate group that was identified is the group of the MHC molecules, which play an important role in controlling infections.
Chimpanzees are often described as herbivores, but they also hunt and consume other nonhuman primate species (Stanford et al. 1994). As most of these Old World monkeys have natural SIV infections, chimpanzees may have become infected through predating on infected monkeys. Probably chimpanzees have been infected multiple times and by various SIV-like strains (Courgnaud et al. 2002; Bailes et al. 2003). To date little is known about the transmission of SIVcpz between different chimpanzees. The initial infections with HIV-1/SIVcpz-like viruses in humans probably happened through blood contact during the hunting of infected chimpanzees. The routes of HIV-1 infection in humans are now known to be via intimate sexual contact, contaminated blood or blood products, or by transmission from mother to child (Levy, Scott, and Mackewicz 2003). Nonetheless, the intestine may be the major site for HIV replication and depletion of CD4+ T cells (Veazey and Lackner 2004). In this perspective, it is possible that the hybrid Patr-MICA/B gene is highly efficient in detecting stress mediated by infection, and as such may induce efficient natural killer (NK) cell–mediated killing.
The human MICA and probably also the closely related MICB molecules are ligands for the NKG2D receptor, which is expressed on NK cells, T cells, and CD8+ ? T cells. After cellular stress, triggered for instance by a viral infection or malignant transformation, MIC is upregulated and may provoke an immune response (Bauer et al. 1999; Gleimer and Parham 2003). Recent work, for instance, showed that the activating KIR gene KIR3DS1 is associated with a delay in progression to AIDS in HIV-1–infected individuals (Martin et al. 2002). The chimpanzee MICA/B molecule is able to recognize human V1 T cells specific for MICA and MICB, suggesting a conserved recognition site (Steinle, Groh, and Spies 1998). Indeed, also the NKG2D receptor shows a high degree of similarity, approximately 98.9%, between humans and chimpanzees (Shum et al. 2002). Based upon these observations and the knowledge that humans and chimpanzees are closely related, a similar kind of immune response as described in humans is plausible.
The possible role of MIC-elicited anti-HIV-1/SIVcpz NK effector responses remains to be proved, and subsequent studies will be required to elucidate the functional significance of the Patr-MICA/B gene in virus infections.
Acknowledgements
The authors wish to thank D. Devine and H. van Westbroek for expert assistance, J. Heeney for discussion, and S. Langehuizen for technical assistance. This study was financed in part by the European Union Project, IMGT-QLG2-2000-01287.
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