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Evaluation of a DLA-79 allele associated with multiple immune-mediated diseases in dogs

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Abstract

Immune-mediated diseases are common and life-threatening disorders in dogs. Many canine immune-mediated diseases have strong breed predispositions and are believed to be inherited. However, the genetic mutations that cause these diseases are mostly unknown. As many immune-mediated diseases in humans share polymorphisms among a common set of genes, we conducted a candidate gene study of 15 of these genes across four immune-mediated diseases (immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis (IMPA), and atopic dermatitis) in 195 affected and 206 unaffected dogs to assess whether causative or predictive polymorphisms might exist in similar genes in dogs. We demonstrate a strong association (Fisher’s exact p = 0.0004 for allelic association, p = 0.0035 for genotypic association) between two polymorphic positions (10 bp apart) in exon 2 of one allele in DLA-79, DLA-79*001:02, and multiple immune-mediated diseases. The frequency of this allele was significantly higher in dogs with immune-mediated disease than in control dogs (0.21 vs. 0.12) and ranged from 0.28 in dogs with IMPA to 0.15 in dogs with atopic dermatitis. This allele has two non-synonymous substitutions (compared with the reference allele, DLA-79*001:01), resulting in F33L and N37D amino acid changes. These mutations occur in the peptide-binding pocket of the protein, and based upon our computational modeling studies, are likely to affect critical interactions with the peptide N-terminus. Further studies are warranted to confirm these findings more broadly and to determine the specific mechanism by which the identified variants alter canine immune system function.

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Notes

  1. http://agenabio.com/sites/default/files/iPLEX%20Application%20Note.pdf

  2. The PyMOL Molecular Graphics System, version 1.7.4 Schrödinger, LLC.

  3. Note that the numbering scheme of the cDNA starts at the initiation codon in the canine sequence; however, the numbering scheme of the protein is adapted to align with human MHC class I molecules such that the first amino acid in exon 2 is counted as number 1 (Graumann et al. 1998).

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Authors

Corresponding author

Correspondence to Steven G. Friedenberg.

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Funding

This study was funded in part by a National Institutes of Health T32 training award provided to Dr. Friedenberg (5T32OD011130-07). Some DNA samples and associated phenotypic data were provided by the Cornell Veterinary Biobank, a resource built with the support of NIH grant R24-GM082910 and the Cornell University College of Veterinary Medicine.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Informed consent

Informed consent was obtained from the owners of all animal participants included in the study.

Electronic supplementary material

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Online Resource 1

Detailed breakdown of dogs, breeds, genotyping cohorts, and diseases used as cases and controls for evaluation of the association of DLA-79*001:02 with various autoimmune diseases. Only dogs in the top table were included in the final analysis; dogs in the bottom table were included in the original targeted resequencing or validation cohort 1 but were eliminated from the final analysis in order to balance cases and controls within breeds. Genotyping cohorts labels are as follows: TR, dogs genotyped with targeted resequencing; 1, dogs genotyped by custom Sequenom array; 2, dogs genotyped by Sanger sequencing or endpoint genotyping. Abbreviations: AD atopic dermatitis, IMHA immune-mediated hemolytic anemia, ITP immune-mediated thrombocytopenia, IMPA immune-mediated polyarthritis, TR targeted resequencing. (PDF 21 kb)

Online Resource 2

Predicted DLA-79 binding peptides with a binding affinity less than 200 nM as determined by NetMHCpan 2.8. Peptides were derived from protein sequences of antigens implicated in the pathogenesis of canine IMHA and ITP. Abbreviations: SLC4A1 Solute Carrier 4, Member 1, ITGA2B Integrin α-Chain 2B. Note that SLC4A1 is also known as Erythrocyte Membrane Protein Band 3 (EMPB3). (PDF 11 kb)

Online Resource 3

Detailed breakdown of the effect of variants identified for each gene from the targeted resequencing experiment of 16 dogs with IMHA. Numbers in the table represent the number of variants identified. (PDF 16 kb)

Online Resource 4

Ramachandran plots for the DLA-79 wild type (a), mutant (b), and 1Q94 template (c). In the wild-type model, 92.3 % of residues are in the favored portion of the Ramachandran plot and 1.5 % are outliers. Two outliers (254, 255) are near the C-terminus, and the third (228) is outside of the peptide binding region. In the mutant model, 92.0 % of residues are within the favored region and 2.0 % are outliers. Three of the outliers are also outliers in the wild-type model and a fourth outlier (189) is also outside the peptide binding domain. Ramachandran plot of the 1Q94 template model is shown for comparison. None of the Ramachandran outliers occur in the peptide binding domain (1–181). All Ramachandran plots were calculated in MolProbity, as implemented in Phenix. (GIF 59 kb)

High-resolution image (TIF 1082 kb)

Online Resource 5

DLA-79 has a conserved MHC class I aromatic motif. The protein model developed using the DLA-79 reference allele (dark gray) was superimposed on the template structure PDB 1Q94 (gray). The four MHC class I conserved tyrosine residues (7, 59, 159, 171) are shown as gray sticks. The four aromatic substitutions in DLA-79 are colored magenta. The additional amino acid in DLA-79 (S82) is also colored magenta. DLA-79F33 is colored dark blue, and conserved residues surrounding the F33L substitution site are colored cyan (W51, W167). Residue numbering in the figure is according to the reference structure with the exception of S82. (GIF 331 kb)

High-resolution image (TIF 381 kb)

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Friedenberg, S.G., Buhrman, G., Chdid, L. et al. Evaluation of a DLA-79 allele associated with multiple immune-mediated diseases in dogs. Immunogenetics 68, 205–217 (2016). https://doi.org/10.1007/s00251-015-0894-6

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  • DOI: https://doi.org/10.1007/s00251-015-0894-6

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