Diversity and locus specificity of chicken MHC B class I sequences

The major histocompatibility complex B (MHC B) region in a standard haplotype of Leghorn chickens contains two closely linked class I loci, B-FI and B-FIV. Few sequences of B-FI alleles are available, and therefore alleles of the two loci have not been compared with regard to sequence diversity or locus specificity. Here, we report eight new B-F alpha 1/alpha 2-coding sequences from broiler chicken MHC B haplotypes, and a unique recombinant between the two B-F loci. The new sequences were combined with existing B-F sequences from Leghorn and broiler haplotypes for analysis. On the basis of phylogenetic analysis and conserved sequence motifs, B-F sequences separated into two groups (Groups A and B), corresponding to B-FIV and B-FI locus, respectively. Every broiler haplotype had one B-F sequence in Group A and the second B-F sequence, if it existed, clustered in Group B. Group B (presumptive B-FI locus) sequences identified in broiler haplotypes resembled the human MHC class I HLA-C locus in their distinctive pattern of allelic polymorphism. Compared with B-FIV, B-FI alleles were less polymorphic and possessed a conserved locus-specific motif in the alpha1 helix, but nevertheless demonstrated evidence of diversifying selection. One B-FI alpha 1/alpha 2-coding nucleotide sequence was completely conserved in four different broiler haplotypes, but each allele differed in the exon encoding the alpha 3 domain.     

Antibody responses to sheep erythrocytes for White Leghorn chickens differing in haplotypes of the major histocompatibility complex (B)

Lines of White Leghorn chickens were developed by selection for high (HA) or low (LA) antibody response to sheep red blood cells (SRBC) and then backcrossed to provide individuals segregating for haplotypes B13 and B21 of the major histocompatibility complex (MHC) within each selected line. Although antibody response to SRBC was consistently higher in background genome HA than LA, there was a significant interaction between background genome and MHC haplotypes. The interaction resulted from higher antibody response in B13/B21 individuals of line HA and in B21/B21 individuals of line LA. Thus, response to SRBC was dependent on particular haplotype combinations present at the MHC as well as the background genome in which they were expressed.     

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility ( B ) complex. Nucleotide sequence data have been published for six class I ( B-F ) alleles and for class II ( B-Lβ ) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated B ^A4 and B ^A4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-Lβ fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-LβII family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-LβII family and B-F gene sequences from the B ^A4 haplotype were identical to the sequences obtained from the reference B ²¹ haplotype in Leghorns; however, in the B ^A4 haplotype the B-Lβ ²¹ and B-F ²¹ alleles were in linkage with B-G alleles that were not G ²¹. The nucleotide sequences from B ^A4variant were unique among the reported chicken B-LβII family and B-F alleles.     

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.     

Antibody responses to sheep erythrocytes for White Leghorn chickens differing in haplotypes of the major histocompatibility complex (B)

Summary. Lines of White Leghorn chickens were developed by selection for high (HA) or low (LA) antibody response to sheep red blood cells (SRBC) and then backcrossed to provide individuals segregating for haplotypes B ¹³ and B ²¹ of the major histocompatibility complex (MHC) within each selected line. Although antibody response to SRBC was consistently higher in background genome HA than LA, there was a significant interaction between background genome and MHC haplotypes. The interaction resulted from higher antibody response in B¹³/B²¹ individuals of line HA and in B²¹/ B ²¹ individuals of line LA. Thus, response to SRBC was dependent on particular haplotype combinations present at the MHC as well as the background genome in which they were expressed.     

Genetic diversity of the allodeterminant alr2 in Hydractinia symbiolongicarpus

Hydractinia symbiolongicarpus, a colonial cnidarian (class Hydrozoa) epibiont on hermit crab shells, is well established as a model for genetic studies of allorecognition. Recently, two linked loci, allorecognition (alr) 1 and alr2, were identified by positional cloning and shown to be major determinants of histocompatibility. Both genes encode putative transmembrane proteins with hypervariable extracellular domains similar to immunoglobulin (Ig)-like domains. We sought to characterize the naturally occurring variation at the alr2 locus and to understand the origins of this molecular diversity. We examined full-length cDNA coding sequences derived from a sample of 21 field-collected colonies, including 18 chosen haphazardly and two laboratory reference strains. Of the 35 alleles recovered from the 18 unbiased samples, 34 encoded unique gene products. We identified two distinct structural classes of alleles that varied over a large central region of the gene but both possessed highly polymorphic extracellular domains I, similar to an Ig-like V-set domain. The discovery of structurally chimeric alleles provided evidence that interallelic recombination may contribute to alr2 variation. Comparisons of the genomic region encompassing alr2 from two field-derived haplotypes and one laboratory reference sequence revealed a history of structural variation at the haplotype level as well. Maintenance of large numbers of equally rare alleles in a natural population is a hallmark of negative frequency-dependent selection and is expected to produce high levels of heterozygosity. The observed alr2 allelic diversity is comparable with that found in immune recognition molecules such as human leukocyte antigens, B cell Igs, or natural killer cell Ig-like receptors.     

Designation by restriction fragment length polymorphism of major histocompatibility complex class IV haplotypes in meat-type chickens

Major histocompatiblity complex (MHC) class IV haplotypes were identified in a population of meat-type chickens by restriction fragment length polymorphism (RFLP) analysis. Fourteen different haplotypes were designated on the basis of restriction patterns obtained from Southern blots of PvuII- or BglII-digested DNA, hybridized with the MHC class IV cDNA probe bg32.1. Digestion with each restriction enzyme yielded the same level of polymorphism among individuals. For each haplotype, 4–10 restriction fragments ranging from 0–8 to 8 kb were observed. Such a designation of meat-type chicken MHC class IV haplotypes enables a rapid recognition of previously defined haplotypes, is readily adjustable to additional, newly found restriction patterns and could prove useful in practical breeding programmes.     

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families

Summary We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.     

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.     

Gene organization of haplotypes expressing two different C4A allotypes

Summary The gene organization of C4 haplotypes expressing two different C4A allotypes with a C4B null allele (C4A3A2-BQ0 and C4A3A6BQO) was studied using Southern blot analysis with cDNA probes and restriction enzymes which give C4A and C4B locus-specific restriction fragments. These haplotypes were shown to have both a C4A and a C4B locus present, suggesting that the C4B locus expresses a C4A protein. The finding of a 21-OH A and a 21-OH B gene on the C4A3A6BQO haplotype further suggests that this haplotype has the common gene organization C4A, 21-OH A, C4B, 21-OH B. A model explaining C4 null alleles on haplotypes found to have two C4 loci is presented.     

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.     

Susceptibility locus for IgA deficiency and common variable immunodeficiency in the HLA-DR3, -B8, -A1 haplotypes.

BACKGROUND: A common genetic basis for IgA deficiency (IgAD) and common variable immunodeficiency (CVID) is suggested by their occurrence in members of the same family and the similarity of the underlying B cell differentiation defects. An association between IgAD/CVID and HLA alleles DR3, B8, and A1 has also been documented. In a search for the gene(s) in the major histocompatibility complex (MHC) that predispose to IgAD/CVID, we analyzed the extended MHC haplotypes present in a large family with 8 affected members. MATERIALS AND METHODS: We examined the CVID proband, 72 immediate relatives, and 21 spouses, and determined their serum immunoglobulin concentrations. The MHC haplotype analysis of individual family members employed 21 allelic DNA and protein markers, including seven newly available microsatellite markers. RESULTS: Forty-one (56%) of the 73 relatives by common descent were heterozygous and nine (12%) were homozygous for a fragment or the entire extended MHC haplotype designated haplotype 1 that included HLA- DR3, -C4A-0, -B8, and -A1. The remarkable prevalence of haplotype 1 was due in part to marital introduction into the family of 11 different copies of the haplotype, eight sharing 20 identical genotype markers between HLA-DR3 and HLA-B8, and three that contained fragments of haplotype 1. CONCLUSION: Crossover events within the MHC indicated a susceptibility locus for IgAD/CVID between the class III markers D821/D823 and HLA-B8, a region populated by 21 genes that include tumor necrosis factor alpha and lymphotoxins alpha and beta. Inheritance of at least this fragment of haplotype 1 appears to be necessary for the development of IgAD/CVID in this family.     

Polymorphic Alu insertions and their associations with MHC class I alleles and haplotypes in Han and Jinuo populations in Yunnan Province, southwest of China

The associations of polymorphic Alu insertions （POALINs） with major histocompatibility complex （MHC） class I genes enable us to better identify origins and evolution of MHC class I region haplotypes in different populations. For further studying origins and evolution of MHC class I region haplotypes in Han and Jinuo populations in Yunnan Province, we investigated frequencies of five POALINs, their associations with HLA-A and -B, the three-loci POALINs haplotype frequencies and HLA/POALIN four-loci haplotype frequencies within the alpha block of MHC class I region. We found that a strong positive association between AluHG and HLA-A＊02 is in Jinuo, but not in Yunnan Han. These results suggest that MHC class I region haplotypes of the two studied populations might derive from different progenitor haplotypes and MHC I-POALINs are informative genetic markers for investigating origins and evolution of MHC class I region haplotypes in different populations.     

MHC heterozygosity and survival in red junglefowl

Genes of the major histocompatibility complex (MHC) form a vital part of the vertebrate immune system and play a major role in pathogen resistance. The extremely high levels of polymorphism observed at the MHC are hypothesised to be driven by pathogen‐mediated selection. Although the exact nature of selection remains unclear, three main hypotheses have been put forward; heterozygote advantage, negative frequency‐dependence and fluctuating selection. Here, we report the effects of MHC genotype on survival in a cohort of semi‐natural red junglefowl (Gallus gallus) that suffered severe mortality as a result of an outbreak of the disease coccidiosis. The cohort was followed from hatching until 250 days of age, approximately the age of sexual maturity in this species, during which time over 80% of the birds died. We show that on average birds with MHC heterozygote genotypes survived infection longer than homozygotes and that this effect was independent of genome‐wide heterozygosity, estimated across microsatellite loci. This MHC effect appeared to be caused by a single susceptible haplotype (CD_c) the effect of which was masked in all heterozygote genotypes by other dominant haplotypes. The CD_c homozygous genotype had lower survival than all other genotypes, but CD_c heterozygous genotypes had survival probabilities equal to the most resistant homozygote genotype. Importantly, no heterozygotes conferred greater resistance than the most resistant homozygote genotype, indicating that the observed survival advantage of MHC heterozygotes was the product of dominant, rather than overdominant processes. This pattern and effect of MHC diversity in our population could reflect the processes ongoing in similarly small, fragmented natural populations.     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

Mitochondrial DNA and Y‐chromosome diversity in East Adriatic sheep

Variation in mitochondrial DNA (mtDNA) and Y‐chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon ( Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt‐3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt‐3 respectively. In contrast, Y‐chromosome analyses revealed extraordinary paternal allelic richness: HY‐6, 89.3%; HY‐8, 5.0%; HY‐18, 3.1%; HY‐7, 1.3%; and HY‐5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY‐18 (A‐oY1/135‐SRYM18), identified here for the first time, provides a link between the haplotype HY‐12 (A‐oY1/139‐SRYM18) found in a few rams in Turkey and haplotype HY‐9 (A‐oY1/131‐SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt‐55), and all were paternally monomorphic for haplotype HY‐6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y‐chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to ‘upgrade’ local populations using rams with different paternal origins.     

The major histocompatability complex (MHC) contains conserved polymorphic genomic sequences that are shuffled by recombination to form ethnic-specific haplotypes

The major histocompatibility complex (MHC) consists of polymorphic frozen blocks (PFBs) that are linked to form megabase haplotypes. These blocks consist of polymorphic sequences and define regions where recombination appears to be inhibited. We have been able to show, using a highly polymorphic sequence centromeric of HLA-B (within the beta block), that PFBs are conserved and contain specific insertions/deletions and substitutions that are the same for individuals with the same MHC haplotype but that differ between at least most different haplotypes. A sequence comparison between ethnic-specific haplotypes shows that these sequences have remained stable and predate the formation of these haplotypes. To determine whether the same conserved block has been involved in the generation of multiple haplotypes, we compared the block typing profiles of different ethnic specific haplotypes. Block typing profiles have previously been shown to be identical in individuals with the same MHC haplotype but, generally, to differ between different haplotypes. It was found that some PFBs are common to more than one haplotype, implying a common ancestry. Subsequently, haplotypes have been generated by the shuffling and exchange of these PFBs. The regions between these PFBs appear to permit the recombination sites and therefore could be expected to exhibit either low polymorphism or a localized ``hotspot.' Received: 20 January 1997 / Accepted: 11 March 1997     

Molecular analysis of the HLA class II genes in two DRw6-related haplotypes, DRw13 DQw1 and DRw14 DQw3

We have compared the sequence polymorphism of HLA class II genes of two distinct DRw6 haplotypes. cDNA libraries were constructed from two lymphoblastoid cell lines: CB6B (10w9060) which types as DRw13 DQw1, and AMALA (10w9064) which types as DRw14 DQw3. Multiple sequence differences were found at the DR beta I, DQ alpha, and DQ beta loci when these two haplotypes were compared. The DR beta I allele found in the DRw14 DQw3 haplotype appears to have diverged primarily as a result of a gene conversion event with a DR1 allele acting as donor. In contrast, the DRw13 DQw1 haplotype appears to have arisen by means of a recombination event between the DR and DQ subregions. Thus, multiple genetic mechanisms, including point mutation, gene conversion, and recombination, have generated diversity among DRw6 haplotypes.     

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