Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

Transcription and diversity of immunoglobulin lambda chain variable genes in the rat

The DRB region of the human major histocompatibility complex displays length polymorphism: Five major haplotypes differing in the number and type of genes they contain have been identified, each at appreciable frequency. In an attempt to determine whether this haplotype polymorphism, like the allelic polymorphism, predates the divergence of humansfrom great apes, we have worked out the organization of the DRB region of the chimpanzee Hugo using a combination of chromosome walking, pulsed-field gel electrophoresis, and sequencing. Hugo is a DRB homozygote whose single DRB haplotype is some 440 kilobases (kb) long and contains five genes. At least one and possibly two of these are pseudogenes, while three are presumably active genes. The genes are designated DRB ^* A0201, DRB2 ^* 0101, DRB3 ^* 0201, DRB6 ^* 0105, and DRB5 ^* 0301, and are arranged in this order on the chromosome. The DRB2 and DRB3 genes are separated by approximately 250 kb of sequence that does not seem to contain any additional DRB genes. The DRB ^* A0201 gene is related to the DRB1 gene of the human DR2 haplotype; the DRB2 ^* 0101 and DRB3 ^* 0201 genes are related to the DRB2 and DRB3 genes of the human DR3 haplotype, respectively; the DRB6 ^* 0105 and DRB5 ^* 0301 genes are related to the DRBVI and DRB5 genes of the human DR2 haplotype, respectively. Thus the Hugo haplotype appears to correspond to the entire human DR2 haplotype, into which a region representing a portion of the human DR3 haplotype has been inserted. Since other chimpanzees have their DRB regions organized in different ways, we conclude that, first, the chimpanzee DRB region, like the human DRB region, displays length polymorphism; second, some chimpanzee DRB haplotypes are longer than the longest known human DRB haplotypes; third, in some chimpanzee haplotypes at least, the DRB genes occur in combinations different from those of the human haplotypes; fourth, and most importantly, certain DRB gene combinations have been conserved in the evolution of chimpanzees and humans from their common ancestors. These data thus provide evidence that not only allelic but also haplotype polymorphism can be passed on from one species to another in a given evolutionary lineage.     

Mhc-DRB genes of platyrrhine primates

The two infraorders of anthropoid primates, Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys and the hominoids) are estimated to have diverged from a common ancestor 37 million years ago. The major histocompatibility complex class II DRB gene and haplotype polymorphism of the Catarrhini has been characterized in several recent studies. The present study was undertaken to obtain information on the DRB polymorphism of the Platyrrhini. Fifty-five complete exon 2 DRB sequences were obtained from six species of Platyrrhini representing both the Callitrichidae and the Cebidae families. Combined with the results of a parallel contig mapping study, our data indicate that at least three loci (DRB1*03, DRB3, and DRB5) are shared by the Catarrhini and the Platyrrhini. However, the three loci are occupied by functional genes in the former infraorder and mostly by pseudogenes in the latter. Instead of the pseudogenes, the Platyrrhini have evolved a new set of apparently functional genes — DRB11 and DRB*W12 through DRB*W19, which have thus far not been found in the Catarrhini. The DRB*W13, *W14, *W15, *W17, *W18, and *W19 genes seem to be restricted to the Cebidae family, whereas the DRB*W16 locus has so far been documented in the Callitrichidae family only. The DRB alleles of the cotton-top tamarin, and perhaps also those of the common marmoset (both members of the family Callitrichidae), are characterized by low nucleotide diversity, possibly indicating that they diverged from a common ancestral gene relatively recently. Correspondence to: J. Klein.     

Evolutionary relationship between human major histocompatibility complexHLA-DR haplotypes

 HLA-DR haplotypes of the human major histocompatibility complex are organized in five different groups. They can be identified based on the serological specificity expressed by the polymorphic DRB1 locus and by the presence of a characteristic set of DRB genes. The nucleotide sequences of introns 4 and 5 of the two DRB genes (DRB1 ^* 01 and DRB6 ^* 01) from a DR1 haplotype and the three DRB genes (DRB1 ^* 15, DRB6 ^* 15, and DRB5 ^* 15), from a DR51 haplotype were determined. This study identified endogenous retroviral long terminal repeat elements (ERV9 LTR) located at identical positions in intron 5 of the DRB1 genes in both the DR1 and DR51 haplotypes. Phylogenetic analyses revealed a close evolutionary relationship between these two haplotypes. The DRB5 gene, unique for the DR51 haplotype, may have been lost by a recent gene deletion event creating the DR1 haplotype. A model for the evolution of the human DR haplotypes involving separate duplication and contraction events is presented. Received: 10 October 1995 / Revised: 22 November 1995     

Diversity associated with the second expressed H L A - D R B locus in the human population

Although diversity within the HLA-DRB region is predominantly focused in the DRB1 gene, the second expressed DRB loci, DRB3, DRB4, and DRB5, also exhibit variation. Within DRB1 ^* 15 or DRB1 ^* 16 haplotypes, four new variants were identified: 1) two new DRB5 alleles, DRB5 ^* 0104 and DRB5 ^* 0204, 2) a haplotype carrying a DRB1 ^* 15 or ^* 16 allele without the usual accompanying DRB5 allele, and 3) a haplotype carrying a DRB5^* 0101 allele without a DRB1 ^* 15 or ^* 16 allele. The evolutionary origins of these haplotypes were postulated based on their associations with the DRB6 pseudogene. Within HLA haplotypes which carry DRB3, a new DRB3 ^* 0205 allele and one unusual DRB3 association were identified. Finally, two new null DRB4 alleles are described: DRB4 ^* 0201N, which exhibits a deletion in the second exon, and a second allele, DRB4 ^* null, which lacks the second exon completely. Gene conversion-like events and variation in the number of functional genes through reciprocal recombination and inactivation contribute to the diversity observed in the second expressed HLA-DRB loci. Received: 2 November 1996 / Revised: 23 December 1996     

Trans-species origin of Mhc-DRB polymorphism in the chimpanzee

Trans-specific evolution of allelic polymorphism at the major histocompatibility complex loci has been demonstrated in a number of species. Estimating the substitution rates and the age of trans-specifically evolving alleles requires detailed information about the alleles in related species. We provide such information for the chimpanzee DRB genes. DNA fragments encompassing exon 2 were amplified in vitro from genomic DNA of ten chimpanzees. The nucleotide sequences were determined and their relationship to the human DRB alleles was evaluated. The alleles were classified according to their positioni in dendrograms and the presence of lineage-specific motifs. Twenty alleles were found at the expressed loci Patr-DRB1,-DRB3, -DRB4, -DRB5, and at the pseudogenes Patr-DRB6, -DRB7; of these, 13 are new alleles. Two other chimpanzee sequences were classified as members of a new lineage tentatively designated DRBX. Chimpanzee counterparts of HLA-DRB1 ^* 01 and ^* 04 were not detected. The number of alleles found at individual loci indicates asymmetrical distribution of polymorphism between humans and chimpanzees. Estimations of intra-lineage divergence times suggest that the lineages are more than 30 million year old. Predictions of major chimpanzee DRB haplotypes are made.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M94937-M94954.     

The cotton-top tamarin revisited: Mhc class I polymorphism of wild tamarins,and polymorphism and allelic diversity of the class II DQA1, DQB1, and DRB loci

Cotton-top tamarins (Saguinus oedipus) in captivity are unusual in that they exhibit low levels of polymorphism and allelic diversity at the major histocompatibility complex (Mhc) class I loci. Since the polymorphism has previously only been examined in captive tamarins, we analyzed the Mhc class I alleles of a population of wild tamarins. These wild tamarins, like their captive counterparts, exhibited limited class I polymorphism. We also assessed the levels of polymorphism and allelic diversity at the Mhc class II DQA1, DQB1, DQB2, and the DRB loci in captive populations of cotton-top tamarins. In contrast to the extensive polymorphism in Old World monkeys, only two alleles were detected at each of DQA1 and DQB1. Also, the DQB2 locus was monomorphic and conserved between New and Old World monkeys. Sequences derived from four putative DRB loci were obtained, and extensive polymorphism was found at all four loci. Phylogenetic analysis did not indicate that any of the tamarin DRB loci, with the possible exception of Saoe-DRB3, were orthologous to the human DRB loci. At three of the DRB loci (Saoe-DRB11, Saoe-DRB ^* W12, Saoe-DRB ^* W22), the number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites, indicative of positive selection. We found no support for a restriction on the polymorphism at the cotton-top tamarin class II loci. However, the allelic diversity at some of the Saoe-DRB loci is more limited than for the HLA-DRB1, consistent with a restriction imposed by the bone marrow-chimerical lifestyle.     

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

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

Primate DRB genes from the DR3 and DR8 haplotypes contain ERV9 LTR elements at identical positions

The HLA-DRB genes of the human major histocompatibility complex constitute a multigene family with a varying number of DRB genes in different haplotypes. To gain further knowledge concerning the evolutionary relationship, the complete nucleotide sequence was determined for a region spanning introns 4 and 5 of the three DRB genes (DRB1*0301, DRB2 and DRB3*0101) from a DR52 haplotype and the single DRB gene (DRB1*08021) in the DR8 haplotype. These analyses identified an endogenous retroviral long terminal repeat element (ERV9 LTR3), inserted at identical positions in intron 5 of the functional DRB genes in these two haplotypes. Comparison of the nucleotide sequence from introns 4 and 5 including the ERV9 LTR elements revealed a strong similarity between the three expressed DRB genes. The DRB3*0101 and DRB1*08021 genes were most similar in this comparison. These findings provide further evidence for a separate duplication in a primordial DR52 haplotype followed by a gene contraction event in the DR8 haplotype. A homologous element was found in a chimpanzee DRB gene from a DR52 haplotype. This represents the first characterized ERV9 LTR element in a nonhuman species. The corresponding introns of the DRB genes in the DR4 haplotype contain no ERV9 LTRs. In contrast, these genes have insertions of distinct Alu repeats, implying distinct evolutionary histories of DR52 and DR53 haplotypes, respectively. Phylogenetic analyses of DRB introns from DR52, DR53, and DR8 haplotypes showed a close relationship between the DRB2 and DRB4 genes. Thus, the ancestral DR haplotype that evolved to generate the DR52 and DR53 haplotypes most likely shared a primordial common DRB gene.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X82660–X82663     

Multiplication of Mhc-DRB5 loci in the orangutan: implications for the evolution of DRB haplotypes

The chain-encoding (B) class II genes of the primate major histocompatibility complex belong to several families. The DRB family of class II genes is distinguished by the occurrence of haplotype polymorphism—the existence of multiple chromosomal forms differing in length, gene number, and gene combinations, each form occurring at an appreciable frequency in the population. Some of the haplotypes, or fragments thereof, are shared by humans, chimpanzees, and gorillas. In an effort to follow the DRB haplotype polymorphism further back in time, we constructed DRB contig maps of the two chromosomes present in the orangutan cell line CP81. Two types of genes were found in the two haplotypes, Popy-DRB5 and Popy-DRB1 ^*03, the former occurring in two copies and one gene fragment in each haplotype, so that the CP81 cell line contains four complete DRB5 genes and two DRB5 fragments altogether. Since the four genes are more closely related to one another than they are to other DRB5 genes, they must have arisen from a single ancestral copy by multiple duplications. At the same time, however, the two CP81 haplotypes differ considerably in their restriction enzyme sites and in the presence of Alu elements at different positions, indicating that they have been separated for a length of time that exceeds the lifespan of a primate species. Moreover, a segment of about 100 kilobase pairs is shared between the orangutan CP81-1 and the human HLA-DR2 haplotype. These findings indicate that part of the haplotype polymorphism may have persisted for more than 13 million years, which is the estimated time of human-orangutan divergence.     

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

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

HLA class II diversity in Australian Aborigines: unusual HLA-DRB1 alleles

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M77670 (DRB1 ^* AB3), M77671 (DRB1 ^* AB4), MM77672 (DRB1 ^* AB2), MM77673 (DRB1*A01), M81670 (DRB1*0410), and M81700 (DRB1 ^* 0411).     

A phylogenetic analysis of cattle DRB3 alleles with a deletion of codon 65

 One of the most common cattle major histocompatibility complex DRB3 alleles, ^* 0201, includes a deletion of codon 65 encoding one residue in the α-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, ^* 3301 and ^* 4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11–17 nucleotide substitutions causing 8–11 amino acid substitutions) we found clear indications of a common ancestry. The α-helical region was very similar or identical among the alleles whereas the β-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles. Received: 20 March 1997 / Revised: 17 June 1997     

Allelic diversity at the primate major histocompatibility complex DRB6 locus

The HLA-DRB6 gene (also called DRB/V1) has been found only in about 26% of human HLA haplotypes, i.e.; DR1, DRw10, and DR2-bearing ones (Corell et al. 1991). In contrast, exon-2 DRB6 sequences have been obtained from all tested primates: nine chimpanzees (Pan troglodytes), three gorillas (Gorilla gorilla) and three orangutans (Pongo pygmaeus); other apes which had already been sequenced (one gorilla and one chimpanzee) also had the DRB6 gene. Thus, all apes tested from three different species, some of them evolutionary separated by at least 14–16 million years, bear the DRB6 gene. In addition, more than one gene copy per haplotype has been found in one chimpanzee; this, together with the apparent loss of this gene in some of the human DR haplotypes, may indicate that the DR genome has undergone evolutionary changes more recently and more actively than class I or III genes. In addition, ten different and presumably allelic DRB6 exon-2 sequences have been obtained, and some of them coming from different species are more similar to each other than the one from the same species; this finding goes in favor of the trans-species theory of major histocompatibility complex polymorphism generation. Also, data are presented supporting that DRB6 may be one of the eldest genes of the DRB family, thus one of the first to diverge from the ancestral DRB gene.The contribution to this paper by A. Corell and P. Morales is equal, and the order of the authorship is arbitrary.     

The evolutionary origin of the HLA-DR3 haplotype

The human HLA-DR3 haplotype consists of two functional genes (DRB1*03 and DRB3*01) and one pseudogene (DRB2), arranged in the order DRB1... DRB2... DRB3 on the chromosome. To shed light on the origin of the haplotype, we sequenced 1480 nucleotides of the HLA-DRB2 gene and aong stretches of two other genes, Gogo-DRB2 from a gorilla, Sylvia and Patr-DRB2 from a chimpanzee, Hugo. All three sequences (HLA-DRB2, Gogo-DRB2, Patr-DRB2) are pseudogenes. The HLA-DRB2 and Gogo-DRB2 pseudogenes lack exon 2 and contain a twenty-nucleotide deletion in exon 3, which destroys the correct translational reading frame and obliterates the highly conserved cysteine residue at position 173. The Patr-DRB2 pseudogene lacks exons 1 and 2; it does not contain the twenty-nucleotide deletion, but does contain a characteristic duplication of that part of exon 6 which codes for the last four amino acid residues of the cytoplasmic region. When the nucleotide sequences of these three genes are compared to those of all other known DRB genes, the HLA-DRB2 is seen as most closely related to Gogo-DRB2, indicating orthologous relationship between the two sequences. The Patr-DRB2 gene is more distantly related to these two DRB2 genes and whether it is orthologous to them is uncertain. The three genes are in turn most closely related to HLA-DRBVI (the pseudogene of the DR2 haplotype) and Patr-DRB6 (another pseudogene of the Hugo haplotype), followed by HLA-DRB4 (the functional but nonpolymorphic gene of the DR4 haplotype). These relationships suggest that these six genes evolved from a common ancestor which existed before the separation of the human, gorilla, and chimpanzee lineages. The DRB2 and DRB6 have apparently been pseudogenes for at least six million years (myr). In the human and the gorilla haplotype, the DRB2 pseudogene is flanked on each side by what appear to be related genes. Apparently, the DR3 haplotype has existed in its present form for more than six myr.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M86691–94.     

Primate DRB6 pseudogenes: clue to the evolutionary origin of the HLA-DR2 haplotype

The HLA-DR2 haplotype contains three \-chain encoding DRB genes and one -chain encoding DRA gene. Of the three DRB genes, two are presumably functional (HLA-DRB1 and HLA-DRB5), whereas the third (HLA-DRBV1) is a pseudogene. A pseudogene closely related to HLA-DRBVI is present in the chimpanzee (Patr-DRB6) and in the gorilla (Gogo-DRB6). We sequenced the HLA-DRBVI and Patr-DRB6 pseudogenes (all exons and most of the introns), and compared the sequence to that of the Gogo-DRB6 gene (of which only the exon sequence is available). All three pseudogenes seem to lack exon 1 and contain other deletions responsible for shifts in the translational reading frame. At least the HLA-DRBVI pseudogene, however, seems to be transcribed nevertheless. The chimpanzee pseudogene contains two inserts in intron 2, one of which is an Alu repeat belonging to the Sb subfamily, while the other remains unidentified. These inserts are lacking in the human gene. A comparison with sequences published by other investigators revealed the presence of the HLA-DRBVI pseudogene also in the DRI and DRw10 haplotypes. Measurements of genetic distances indicate DRB6 to be closely related to the DRB2 pseudogene and to the HLA-DRB4 functional gene. In humans, gorillas, and chimpanzees, the DRB6 pseudogene is associated with the same functional gene (DRB5) indicating that this linkage disequilibrium is at least six million years old and that DR2 is one of the oldest DR haplotypes in higher primates.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M77284-M77295. Address correspondence and offprint requests to: J. Klein.