Additional file 4: of eQTL discovery and their association with severe equine asthma in European Warmblood horses

Association of ATF7IP2, and GLIPR1L2 gene expression to RAO disease status. Html output of an R markdown document. The file contains one multiple logistic regression for each gene ATF7IP2, and GLIPR1L2. These models quantify the association between gene expression in ATF7IP2, or GLIPR1L2 and disease status. Four significant surrogate variables were calculated for the HDE treatment by SVA and therefore none were included in the model. An R markdown document that generated this html file is available on GitHub: https://github.com/VCMason . (HTML 866 kb

Additional file 2: of eQTL discovery and their association with severe equine asthma in European Warmblood horses

Linear mixed models jointly modeling MCK and HDE. Linear mixed models with random intercepts for each individual model the association between the top fifteen RAO associated SNPs that were also eSNPs in either MCK or HDE (chr13.32843309, chr13.32844446, chr13.33460982, chr13.33502488, chr28.3692072, chr21.52625145) and the gene expression of the genes they regulated (DEXI, NSUN2, ATF7IP2, GLIPR1L2) with reduced maximum likelihood (REML) and maximum likelihood (ML). An R markdown document that generated this html file is available on GitHub: https://github.com/VCMason . (HTML 5545 kb

Additional file 3: of eQTL discovery and their association with severe equine asthma in European Warmblood horses

Association of DEXI and NSUN2 gene expression to RAO disease status. Html output of an R markdown document. The file contains two multiple logistic regressions and one simple logistic regression showing the association between DEXI gene expression and disease status. Multiple logistic regression with known confounders as independent variables, and the simple logistic regression only has the independent variable of interest (DEXI or NSUN2 gene expression) as the single covariate. An R markdown document that generated this html file is available on GitHub: https://github.com/VCMason . (HTML 844 kb

DataSheet_1_Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.pdf

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.</p

Table_2_Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.xlsx

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.</p

Mapping of congenital hepatic fibrosis to chromosome 20 with 5 cases and 12 controls.

<p>(<b>A</b>) Manhattan plot of the genome-wide allelic association study (GWAS). (<b>B</b>) Detailed view of the associated region. A single SNP at position 49,643,575 exceeded the genome-wide significance threshold. (<b>C</b>) Homozygosity mapping. The blue bars represent homozygous regions with shared alleles in the 5 affected foals. The common identical by descent segment among the 5 affected animals delineates a critical interval ranging from Chr20∶49,164,218–50,115,936.</p

Genotype distribution of 2 strongly associated <i>PKHD1</i> variants.

a<p>This horse was slaughtered before one year of age and the phenotype must be considered as unknown.</p>b<p>This table summarizes a subset of all horses that were analyzed. In this table we list only those horses for which the genotypes at both variants were available.</p><p>Genotype distribution of 2 strongly associated <i>PKHD1</i> variants.</p

Congenital liver fibrosis in Franches-Montagnes horses.

<p>(A) Affected, 2 month old foal. The foal was small for its age and displayed a potbelly. (B) Enlarged and grey colored liver of the affected foal with macroscopically visible cysts. (C) Histological image of liver tissue from a foal with congenital hepatic fibrosis. Note the marked porto-portal bridging fibrosis and the abundant dilated bile ductules surrounded by some inflammatory cells within the fibrotic tissue (H&E, 400x). (D) Histological image of liver tissue from a non-affected foal. Note the small bile ducts in the portal areas and the absence of fibrosis (H&E 400x).</p

Allele-specific quantification of <i>PKHD1</i> transcripts.

<p>We sequenced a PCR product derived from genomic DNA and an RT-PCR product derived from liver RNA of a heterozygous stallion, which had already sired an affected foal and was therefore considered an obligate carrier of the disease causing variant. The electropherograms show comparable ratios of both alleles on genomic DNA and cDNA. This indicates that both alleles are transcribed at similar levels and that there is no nonsense-mediated decay of the transcript derived from the disease-associated A-allele.</p

Pedigree of five Franches-Montagnes horses with congenital hepatic fibrosis.

<p>Males are represented by squares, females by circles. Affected animals are shown with black filled symbols, the year of birth is shown for all animals, and FM laboratory numbers are given for animals from which DNA was available. The causative genetic defect was probably mainly spread into the population by the stallion Elu born in 1964. All affected foals are inbred to this stallion and have him both as paternal and maternal ancestor.</p