The MHC haplotypes of the chicken

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as standard B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The standard haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.     

Ancestral and recombinant 16-locus HLA haplotypes in the Hutterites

 Prior studies in the Schmiedeleut Hutterites of South Dakota have demonstrated associations between human leukocyte antigen (HLA) haplotype matching and fetal loss (Ober et al. 1992) and mate preferences (Ober et al. 1997), as well as deficiencies of homozygotes for HLA haplotypes (Kostyu et al. 1993). These studies were based on the serologically-defined five-locus HLA-A, -C, -B, -DR, -DQ haplotype. To further elucidate the effects of specific major histocompatibility (MHC) loci or regions on fetal loss and mate choice, we genotyped a sample of Hutterites for 14 MHC loci by DNA or biochemical methods. Typing for additional loci in the HLA-A to HLA-DPB1 region increased the number of recognized Hutterite MHC haplotypes to 67, and further localized the site of cross-over in 9 of 15 recombinant haplotypes. Hutterite MHC haplotype sequences are similar to those observed in outbred Caucasians, suggesting that the influence of HLA haplotypes on fetal loss and mating structure may be general. Received: 1 May 1998 / Revised: 2 December 1998     

Ancestral organization of the MHC revealed in the amphibian Xenopus

With the advent of the Xenopus tropicalis genome project, we analyzed scaffolds containing MHC genes. On eight scaffolds encompassing 3.65 Mbp, 122 MHC genes were found of which 110 genes were annotated. Expressed sequence tag database screening showed that most of these genes are expressed. In the extended class II and class III regions the genomic organization, excluding several block inversions, is remarkably similar to that of the human MHC. Genes in the human extended class I region are also well conserved in Xenopus, excluding the class I genes themselves. As expected from previous work on the Xenopus MHC, the single classical class I gene is tightly linked to immunoproteasome and transporter genes, defining the true class I region, present in all nonmammalian jawed vertebrates studied to date. Surprisingly, the immunoproteasome gene PSMB10 is found in the class III region rather than in the class I region, likely reflecting the ancestral condition. Xenopus DMalpha, DMbeta, and C2 genes were identified, which are not present or not clearly identifiable in the genomes of any teleosts. Of great interest are novel V-type Ig superfamily (Igsf) genes in the class III region, some of which have inhibitory motifs (ITIM) in their cytoplasmic domains. Our analysis indicates that the vertebrate MHC experienced a vigorous rearrangement in the bony fish and bird lineages, and a translocation and expansion of the class I genes in the mammalian lineage. Thus, the amphibian MHC is the most evolutionary conserved MHC so far analyzed.     

Analysis of MHC class I genes across horse MHC haplotypes

The genomic sequences of 15 horse major histocompatibility complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and nonclassical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal and two to three nonclassical sequences. Phylogenetic analysis was applied to these sequences, and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The nonclassical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine major histocompatibility complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci.     

Three new MHC haplotypes in broiler breeder chickens

Six distinct serotypes of the chicken B blood group system (which encodes the major histocompatibility complex) were identified in a commercial broiler breeder line (Line C). The B serotypes were compared by B-G restriction fragment length polymorphism (RFLP) analysis, allele-specific PCR typing test for B-LBII family genes and nucleotide sequence analysis of expressed B-F and B-LBII family genes. The results indicated the existence of seven distinct B haplotypes. Nucleotide sequence analysis demonstrated that three of the Line C haplotypes encode new B-F and B-LB alleles.     

Y chromosome haplotypes reveal prehistorical migrations to the Himalayas

By using 19 Y chromosome biallelic markers and 3 Y chromosome microsatellite markers, we analyzed the genetic structure of 31 indigenous Sino-Tibetan speaking populations (607 individuals) currently residing in East, Southeast, and South Asia. Our results showed that a T to C mutation at locus M122 is highly prevalent in almost all of the Sino-Tibetan populations, implying a strong genetic affinity among populations in the same language family. Furthermore, the extremely high frequency of H8, a haplotype derived from M122C, in the Sino-Tibetan speaking populations in the Himalayas including Tibet and northeast India indicated a strong bottleneck effect that occurred during a westward and then southward migration of the founding population of Tibeto-Burmans. We, therefore, postulate that the ancient people, who lived in the upper-middle Yellow River basin about 10,000 years ago and developed one of the earliest Neolithic cultures in East Asia, were the ancestors of modern Sino-Tibetan populations.     

MHC Class II haplotypes of Colombian Amerindian tribes

We analyzed 1041 individuals belonging to 17 Amerindian tribes of Colombia, Chimila, Bari and Tunebo (Chibcha linguistic family), Embera, Waunana (Choco linguistic family), Puinave and Nukak (Maku-Puinave linguistic families), Cubeo, Guanano, Tucano, Desano and Piratapuyo (Tukano linguistic family), Guahibo and Guayabero (Guayabero Linguistic Family), Curripaco and Piapoco (Arawak linguistic family) and Yucpa (Karib linguistic family). for MHC class II haplotypes (HLA-DRB1, DQA1, DQB1). Approximately 90% of the MHC class II haplotypes found among these tribes are haplotypes frequently encountered in other Amerindian tribes. Nonetheless, striking differences were observed among Chibcha and non-Chibcha speaking tribes. The DRB1*04:04, DRB1*04:11, DRB1*09:01 carrying haplotypes were frequently found among non-Chibcha speaking tribes, while the DRB1*04:07 haplotype showed significant frequencies among Chibcha speaking tribes, and only marginal frequencies among non-Chibcha speaking tribes. Our results suggest that the differences in MHC class II haplotype frequency found among Chibcha and non-Chibcha speaking tribes could be due to genetic differentiation in Mesoamerica of the ancestral Amerindian population into Chibcha and non-Chibcha speaking populations before they entered into South America.     

Ancestral Asian source(s) of new world Y-chromosome founder haplotypes

Haplotypes constructed from Y-chromosome markers were used to trace the origins of Native Americans. Our sample consisted of 2,198 males from 60 global populations, including 19 Native American and 15 indigenous North Asian groups. A set of 12 biallelic polymorphisms gave rise to 14 unique Y-chromosome haplotypes that were unevenly distributed among the populations. Combining multiallelic variation at two Y-linked microsatellites (DYS19 and DXYS156Y) with the unique haplotypes results in a total of 95 combination haplotypes. Contra previous findings based on Y- chromosome data, our new results suggest the possibility of more than one Native American paternal founder haplotype. We postulate that, of the nine unique haplotypes found in Native Americans, haplotypes 1C and 1F are the best candidates for major New World founder haplotypes, whereas haplotypes 1B, 1I, and 1U may either be founder haplotypes and/or have arrived in the New World via recent admixture. Two of the other four haplotypes (YAP+ haplotypes 4 and 5) are probably present because of post-Columbian admixture, whereas haplotype 1G may have originated in the New World, and the Old World source of the final New World haplotype (1D) remains unresolved. The contrasting distribution patterns of the two major candidate founder haplotypes in Asia and the New World, as well as the results of a nested cladistic analysis, suggest the possibility of more than one paternal migration from the general region of Lake Baikal to the Americas.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

Structural relationships among the H-2 D-regions of murine MHC haplotypes

The number of genes encoding functional Ag-presenting molecules in the D region of the murine MHC differs among haplotypes. For example, the H-2b D region contains a single "D/L" gene, H-2Db, whereas the d-haplotype encodes two, H-2Dd and Ld. Using D/L specific oligonucleotide probes, we have found that, as with H-2d, the q- and v-haplotypes contain two D/L genes, whereas the other haplotype examined have one. Hybridization analysis using cloned probes that map between H-2Dd and Ld revealed similar structures in each of the three haplotypes (d, q, and v) which have "duplicated" D regions. Two approaches were used to examine allelic relationships among the D/L genes. First, the 5' region of the H-2Db gene was sequenced, and found to be more similar to H-2Ld than to H-2Dd. Second, oligonucleotide probes that distinguish H-2Ld from H-2Dd revealed H-2Ld-related genes in several haplotypes, including the duplicated haplotypes H-2q and H-2v. Analogous probes specific for H-2Dd, however, did not detect similar sequences in the other haplotypes. We interpret these results to mean that the three duplicated D regions arose from a common duplication event, and share the five gene structure of the D region cluster defined in H-2d. However, subsequent events have generated sequence divergence at the D-locus.     

Comprehensive characterization of MHC class II haplotypes in Mauritian cynomolgus macaques

There are currently no nonhuman primate models with fully defined major histocompatibility complex (MHC) class II genetics. We recently showed that six common MHC haplotypes account for essentially all MHC diversity in cynomolgus macaques (Macaca fascicularis) from the island of Mauritius. In this study, we employ complementary DNA cloning and sequencing to comprehensively characterize full length MHC class II alleles expressed at the Mafa-DPA, -DPB, -DQA, -DQB, -DRA, and -DRB loci on the six common haplotypes. We describe 34 full-length MHC class II alleles, 12 of which are completely novel. Polymorphism was evident at all six loci including DPA, a locus thought to be monomorphic in rhesus macaques. Similar to other Old World monkeys, Mauritian cynomolgus macaques (MCM) share MHC class II allelic lineages with humans at the DQ and DR loci, but not at the DP loci. Additionally, we identified extensive sharing of MHC class II alleles between MCM and other nonhuman primates. The characterization of these full-length-expressed MHC class II alleles will enable researchers to generate MHC class II transferent cell lines, tetramers, and other molecular reagents that can be used to explore CD4+ T lymphocyte responses in MCM.     

Large-scale comparative mapping of the MHC class I region of predominant haplotypes in Japanese

 In order to further our understanding of major histocompatibility complex (MHC) class I gene organization, we began a comparative analysis of the large scale organization of the class I region in diverse haplotypes. For these studies, the MHC in healthy Japanese donors who have the predominant MHC haplotypes and/or HLA-A or -B alleles was examined by pulsed field gel electrophoresis and Southern analysis using probes spanning the class I region. Hybridization with probes from the HLA-A to HLA-G region revealed that individuals expressing HLA-A30, -A31, or -A33 have an approximately 70 kilobase (kb) insertion near the HLA-A gene as compared with haplotypes containing the HLA-A11 or -A26 allele. Conversely, HLA-A24-containing haplotypes appear to have an approximately 50 kb deletion from the same region. Further, it appears that chromosomes carrying closely related alleles are similar to each other in this region, consistent with their presumed evolutionary relationship. While little is known about the gene content between the HLA-A and HLA-G region, it will be interesting to examine the prospect that functional genes do in fact reside within the inserted or deleted portions, thereby raising the possibility that distinct functional differences are conferred by different haplotypes. Overall, the results reported here should contribute to furthering our understanding of the association between diseases and HLA as well as provide new insights into the evolution of the MHC. Received: 11 December 1996     

Variation in the number of expressed MHC genes in different cattle class I haplotypes

 Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes. Received: 15 February 1999 / Revised: 23 June 1999     

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex.

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 25 degrees C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCl to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system.     

Characterization of the class III region in different MHC haplotypes by pulsed-field gel electrophoresis

The class III region of the human major histocompatibility complex (MHC) in sevenHLA haplotypes has been analyzed using pulsed-field gel electrophoresis (PFGE), restriction enzymes that cut genomic DNA infrequently, and Southern blotting. In particular, extensive mapping with the enzymeBss HII, which generates fragments in the size range 8–280 kilobases (kb), has revealed that in the haplotypes studied the DNA content of the class III region does not appear to vary other than as previously observed at theC4 andCYP21 loci.     

Mitochondrial haplotypes reveal olive fly (Bactrocera oleae) population substructure in the Mediterranean

The olive fly (Bactrocera oleae) is the most important olive tree (Olea europaea) pest. In the Mediterranean basin, where 98?% of its main hosts are concentrated, it causes major agricultural losses, due to its negative effect on production and quality of both olive and olive oil. Previous phylogeographic analyses have established that Mediterranean olive fly populations are distinct from other Old World populations, but did not agree on the specific population substructure within this region. In order to achieve a higher resolution of the diversity of olive fly populations, particularly in Central and Western Mediterranean (home to 70?% of the world production), we comparatively analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the species' worldwide range. Strong evidence of population substructure was found in the Central and Western Mediterranean area, with two clearly separate phylogenetic branches. Together with previously published data, our results strongly support the existence of at least three distinct Mediterranean populations of the olive fly, raise the possibility of additional regional substructure and suggest specific avenues for future research. This knowledge can be instrumental in the development of better management and control strategies for a major pest of Mediterranean agriculture.     

Multilocus definition of MHC haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

Cynomolgus macaques (Macaca fascicularis) are used widely in biomedical research, and the genetics of their MHC (Mhc-Mafa) has become the focus of considerable attention in recent years. The cohort of Indonesian pedigreed macaques that we present here was typed for Mafa-A, -B, and -DR, by sequencing, as described in earlier studies. Additionally, the DRB region of these animals was characterised by microsatellite analyses. In this study, full-length sequencing of Mafa-DPA/B and -DQA/B in these animals was performed. A total of 75 different alleles were observed; 22 of which have not previously been reported, plus 18 extended exon 2 alleles that were already known. Furthermore, two microsatellites, D6S2854 and D6S2859, were used to characterise the complex Mafa-A region. Sequencing and segregation analyses revealed that the length patterns of these microsatellites are unique for each Mafa-A haplotype. In this work, we present a pedigreed colony of approximately 120 cynomolgus macaques; all of which are typed for the most significant polymorphic MHC class I and class II markers. Offspring of these pedigreed animals are easily characterised for their MHC by microsatellite analyses on the Mafa-A and -DRB regions, which makes the cumbersome sequencing analyses redundant.     

Complotypes in individuals of African origin: frequencies and possible extended MHC haplotypes

We analyzed the frequency distribution of 106 complotypes [four allele sets of the major histocompatibility complex (MHC) genes for the complement proteins factor B, C2, C4A, and C4B] from 32 Black families residing in Boston and Washington, DC. Twenty-five different complotypes were identified, among which there were four complotypes that had not been previously observed in our large database of complotypes compiled from family studies of Boston Caucasians and that are, presumably, unique to individuals of African origin. These four African-derived complotypes areFC(1,90)0, FC63, S1C2,17, andSC(3,2,90)0. The frequencies of two of these four unique Black complotypes,FC(1,90)0 andFC63, were increased significantly when compared to Caucasians (p_corr <0.00042, p_corr=0.00294, respectively). The complotypeFC(1,90)0 was in positive linkage disequilibrium withHLA-DR3 haplotypes containing theB locus antigens Bw42, Bw52, Bw53, and Bw58, whileFC63 was associated withHLA-Bw70,-DR5. These findings demonstrate the extensive polymorphism of complotypes in Blacks, and also suggest that it may be possible to define unique extended haplotypes of African origin.     

High-resolution Y chromosome haplotypes of Israeli and Palestinian Arabs reveal geographic substructure and substantial overlap with haplotypes of Jews

High-resolution Y chromosome haplotype analysis was performed in 143 paternally unrelated Israeli and Palestinian Moslem Arabs (I&P Arabs) by screening for 11 binary polymorphisms and six microsatellite loci. Two frequent haplotypes were found among the 83 detected: the modal haplotype of the I&P Arabs (approximately 14%) was spread throughout the region, while its one-step microsatellite neighbor, the modal haplotype of the Galilee sample (approximately 8%), was mainly restricted to the north. Geographic substructuring within the Arabs was observed in the highlands of Samaria and Judea. Y chromosome variation in the I&P Arabs was compared to that of Ashkenazi and Sephardic Jews, and to that of North Welsh individuals. At the haplogroup level, defined by the binary polymorphisms only, the Y chromosome distribution in Arabs and Jews was similar but not identical. At the haplotype level, determined by both binary and microsatellite markers, a more detailed pattern was observed. Single-step microsatellite networks of Arab and Jewish haplotypes revealed a common pool for a large portion of Y chromosomes, suggesting a relatively recent common ancestry. The two modal haplotypes in the I&P Arabs were closely related to the most frequent haplotype of Jews (the Cohen modal haplotype). However, the I&P Arab clade that includes the two Arab modal haplotypes (and makes up 32% of Arab chromosomes) is found at only very low frequency among Jews, reflecting divergence and/or admixture from other populations.     

Quantitative variation of C4 variant proteins associated with many MHC haplotypes

C4 protein variants were analyzed in 64 individuals, of which 51 were either homozygous or heterozygous for an extended major histocompatibility complex (MHC) haplotype (a fixed combination of MHC alleles). The relative amount of each C4 variant was measured by densitometric scanning of stained immunofixed electrophoretic patterns of neuraminidase- and carboxypeptidase-treated samples. The relative concentrations of C4 variants on any haplotype were stable and inherited in families. In five of the eight extended haplotypes investigated, the amount of one of the C4 variants relative to others in the same pattern was increased:[HLA-B8, SC01, DR3] and[HLA-B7, SC31, DR2] produced an approximately doubled amount of C4B1;[HLA-B18, S042, DR2] an increased amount of C4B2; and[HLA-B44, SC30, DR4] a double amount of C4A3. The extended haplotype[HLA-Bw57, SC61, DR7] gave rise to two to three times as much C4B1 as C4A6. In the extended haplotypes[HLA-B44, FC31, DR7] and[HLA-Bw62, SC33, DR4], the results did not clearly indicate differences in expression of the C4 isotypes. DNA analysis possibly supported an actual gene duplication only for the haplotype[HLA-B7, SC31, DR2]. The results suggest that, in addition to variation in the number of structural genes, other MHC-linked mechanisms may be involved in the regulation of the relative amounts of C4A or C4B protein specified by any haplotype.