Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection - PubMed (original) (raw)

Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection

Yun Young Go et al. J Virol. 2011 Dec.

Abstract

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3+ T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3+ T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.

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Figures

Fig. 1.

Fig. 1.

Schematic representation of the study design. A total of 310 horses of different breeds, American Saddlebred (ASB; n = 60), Standardbred (STB; n = 60), Thoroughbred (TB; n = 137), and Quarter Horse (QH n = 53), were used in the study. Horses were phenotyped first and grouped into the susceptible or resistant phenotype group based on their CD3+ T cell infectivity to EAV. To identify the region associated with EAV-susceptible/resistant phenotype, gDNA from 37 TB horses was isolated and analyzed using Illumina Equine SNP50. Results from the initial study were confirmed by genotyping 267 additional horses, including TB horses tested with Illumina Equine SNP50, with MassARRAY system technologies.

Fig. 2.

Fig. 2.

Effect of breeds in prevalence of T cell susceptible/resistant phenotypes. (A) Representative dot plots from flow cytometry analysis for each breed are shown. The mean percentage of CD3+ T cells with intracellular EAV NSP1 antigen is indicated in the right upper quadrant for the susceptible phenotype. (B) The percentages of susceptible (black) and resistant (white) phenotypes for each breed are indicated in a bar graph. Seroprevalences of each breed (red) are indicated below the bar, representing phenotypic prevalence where available. TB, Thoroughbred (n = 137); STB, Standardbred (n = 60); ASB, American Saddlebred (n = 60); QH, Quarter Horse (n = 53).

Fig. 3.

Fig. 3.

Manhattan plot showing the distribution of probability values (−log10 transformed) for the 42,506 SNPs investigated using the 37 Thoroughbred horses (16 were susceptible and 21 were resistant). Genomic positions are indicated by chromosomes with different colors.

Fig. 4.

Fig. 4.

Linkage disequilibrium (LD) plots for all breeds, showing the region used for defining the EAV susceptibility haplotype (ECA11 positions 49572804 to 49643932). The haplotype block is highlighted with a black line.

Fig. 5.

Fig. 5.

Frequency of the GGGGAGGT haplotype found for selected SNPs between ECA11 positions 49572804 to 49643932 among horses susceptible and resistant for the EAV in vitro infection phenotype. The blue area in the pie chart represents the proportion of the GGGGAGGT haplotype, and the red area represents the allelic haplotypes. The frequency represented by each section of the pie chart is shown on the pie chart in white. Data are represented for all horses together (All) and for the individual breeds (Thoroughbred, American Saddlebred, Quarter Horse, and Standardbred). The statistical significance for the frequency differences of the GGGGAGGT haplotype between susceptible and resistant horses is shown to the left of each set of pie charts (P“GGGGAGGT”).

Fig. 6.

Fig. 6.

(A) Three top networks retrieved using unsupervised IPA were merged into a single interaction network. Red nodes represent molecules encoded by genes found in the region of ECA11; white nodes are genes identified by IPA with direct or indirect interactions with genes of ECA11. The blue nodes represent molecules/complexes identified by IPA involved in canonical pathways. Classification of candidate molecules based on their molecular functions (B) and biological processes (C).

Fig. 7.

Fig. 7.

Subcellular location of candidate genes and EAV life cycle. The function and subcellular location of candidate molecules were manually curated based on PANTHER, IPA, and published literature search. Subsequently, molecules were mapped along with stages of the EAV life cycle at the position most likely to interact with the virus life cycle. Green circles, molecules encoded by genes located in the haplotype block; red circles, putative molecules encoded by genes found within 500 kb upstream and downstream of the haplotype block; blue circles, molecules in the vicinity interacting with genes located in the region retrieved in the network analysis; black circles, viral proteins.

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