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Analysis of MHC class I genes across horse MHC haplotypes

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Abstract

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.

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Abbreviations

ARS:

Antigen recognition site

BAC:

Bacterial artificial chromosome

CTL:

Cytotoxic T lymphocyte

ELA:

Equine leukocyte antigen

HLA:

Human leukocyte antigen

MHC:

Major histocompatibility complex

NK:

Natural killer

UTR:

Untranslated region

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Acknowledgments

This research was funded by NIH grants HD-34086, HD-15799, and HD-049545, the Harry M. Zweig Memorial Fund for Equine Research, the Dorothy Russell Havemeyer Foundation, Inc., and the USDA Regional Grants Program. We thank Dr. Shirley Ellis, Dr. Ashley Moffett, Dr. John Trowsdale, and Dr. Stephan Beck for helpful discussions and Dr. Robert H. Mealey of Washington State University for generously providing samples from horses of known ELA serotypes.

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Authors and Affiliations

  1. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA

    Rebecca L. Tallmadge, Julie A. Campbell, Donald C. Miller & Douglas F. Antczak

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  1. Rebecca L. Tallmadge
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  2. Julie A. Campbell
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  3. Donald C. Miller
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  4. Douglas F. Antczak
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Corresponding author

Correspondence to Douglas F. Antczak.

Electronic supplementary material

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Supplementary Figure 1

Phylogenetic tree of horse nonclassical MHC class I cDNA sequences. The tree was constructed based on the coding sequences of putative nonclassical horse MHC class I genes (Tables 1; Supplementary Table 1) excluding residues of the antigen recognition site (ARS). Locus groupings are shown on the right. Sequence names exclude the prefix “Eqca”. Groups A, C, and E refer to phylogenetic analysis by Holmes and Ellis (1999). Sequences were analyzed using the neighbor-joining method (Saitou and Nei 1987) on the basis of Tamura–Nei distances (Tamura and Nei 1993) using the MEGA 3.1 program. Percent of supporting bootstrap replications (n = 1,000) are shown next to the branches. HLA-A*0101 sequence included as an outgroup to root the phylogenetic tree (PDF 12 kb)

Supplementary Figure 2

Phylogenetic tree of classical MHC class I sequences from MHC homozygous horses. Phylogenetic tree was constructed based on the A) coding sequence excluding residues of the antigen recognition site or B) 3′UTR sequence of classical horse MHC class I genes (Table 1). All of these sequences clustered with group B sequences defined by Holmes and Ellis (1999, data not shown). Allele names are followed by associated serological ELA haplotype and exclude the prefix “Eqca”. Putative ELA locus designations are shown on the right. Sequences were analyzed using the neighbor-joining method (Saitou and Nei 1987) on the basis of Tamura–Nei distances (Tamura and Nei 1993) using the MEGA 3.1 program. Percent of supporting bootstrap replications (n = 1,000) are shown next to the branches. HLA-A*0101 sequence included as an outgroup to root the phylogenetic tree. Provisional locus assignments are given in italics and followed by an asterisk (PDF 17.2 kb)

Supplementary Figure 3

Phylogenetic tree of horse MHC class I 3′UTR sequences from ten equine MHC haplotypes. The tree was constructed based on horse MHC class I 3′UTR sequences (Tables 1; Supplementary Table 1). All of these sequences clustered with group B sequences defined by Holmes and Ellis (1999). Allele names are followed by associated serological ELA haplotype and exclude the prefix “Eqca”. Putative locus assignments were shown on the right; provisional locus assignments are given in italics and followed by an asterisk. Sequences were analyzed using the neighbor-joining method (Saitou and Nei 1987) on the basis of Tamura–Nei distances (Tamura and Nei 1993) using the MEGA 3.1 program. Percent of supporting bootstrap replications (n = 1,000) are shown next to the branches. HLA-A*0101 sequence included as an outgroup to root the phylogenetic tree (PDF 15 kb)

Supplementary Figure 4

Nucleotide alignment of horse MHC class I 3′UTR sequences. Allele names are followed by associated serological ELA haplotype and exclude the prefix “Eqca”. The consensus sequence is shown at the top, dots represent identities, and dashes represent gaps inserted to optimize the alignment. Horizontal gray bars separate loci, listed on the right. Sequences are from Table 1; the reverse primer sequence is not included (PDF 18 kb)

Supplementary Figure 5

Phylogenetic tree of horse MHC class I intron 2 sequences from nonclassical loci 5, 6, and 7. The tree was constructed based on the intron 2 sequences from nonclassical loci 5, 6, and 7 (DQ145590-2, DQ083411-3), which correspond to groups A, C, and E, respectively, defined by Holmes and Ellis (1999). Allele names are followed by associated serological ELA haplotype and exclude the prefix “Eqca”. Locus assignments are shown on the right. Sequences were analyzed using the neighbor-joining method (Saitou and Nei 1987) on the basis of Tamura–Nei distances (Tamura and Nei 1993) using the MEGA 3.1 program. Percent of supporting bootstrap replications (n = 1,000) are shown next to the branches. HLA-A*0101 intron 2 sequence was included as an outgroup to root the phylogenetic tree (PDF 10 kb)

Supplementary Table 1

Previously published equine MHC class I sequences associated with serologically defined haplotypes included in this study (PDF 16 kb)

Supplementary Table 2

Oligonucleotides used for RT-PCR amplification of horse MHC class I genesa (PDF 13 kb)

Supplementary Table 3

Nomenclature for equine MHC class I alleles (PDF 32 kb)

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Tallmadge, R.L., Campbell, J.A., Miller, D.C. et al. Analysis of MHC class I genes across horse MHC haplotypes. Immunogenetics 62, 159–172 (2010). https://doi.org/10.1007/s00251-009-0420-9

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