Mitral valve transcriptome analysis in thirty-four age-matched Cavalier King Charles Spaniels with or without congestive heart failure caused by myxomatous mitral valve disease.

We here report the results of a mitral valve transcriptome study designed to identify genes and molecular pathways involved in development of congestive heart failure (CHF) following myxomatous mitral valve disease (MMVD) in dogs. The study is focused on a cohort of elderly age-matched dogs (n = 34, age ~ 10 years) from a single breed-Cavalier King Charles Spaniels (CKCS)-with a high incidence of MMVD. The cohort comprises 19 dogs (10♀, 9♂) without MMVD-associated CHF, and 15 dogs (6♀, 9♂) with CHF caused by MMVD; i.e., we compare gene expression in breed and age-matched groups of dogs, which only differ with respect to CHF status. We identify 56 genes, which are differentially expressed between the two groups. In this list of genes, we confirm an enrichment of genes related to the TNFß-signaling pathway, extracellular matrix organization, vascular development, and endothelium damage, which also have been identified in previous studies. However, the genes with the greatest difference in expression between the two groups are CNTN3 and MYH1. Both genes encode proteins, which are predicted to have an effect on the contractile activity of myocardial cells, which in turn may have an effect on valvular performance and hemodynamics across the mitral valve. This may result in shear forces with impact on MMVD progression.

Identification of a novel QTL for lean meat percentage using imputed genotypes.

Lean meat percentage is a critical production trait in pig breeding systems with direct implications for the sustainability of the industry. In this study, we conducted a genome-wide association study for lean meat percentage using a cohort of 850 Duroc × (Landrace × Yorkshire) crossbred pigs and we identified QTL on SSC3 and SSC18. Based on the predicted effect of imputed variants and using the PigGTEx database of molecular QTL, we prioritized candidate genes and SNPs located within the QTL regions, which may be involved in the regulation of porcine leanness. Our results indicate that a nonsense mutation in ZC3HAV1L on SSC18 has a direct effect on lean meat percentage.

Towards identification of new genetic determinants for post-weaning diarrhea in piglets.

Post-weaning diarrhea in pigs is a considerable challenge in the pig farming industry due to its effect on animal welfare and production costs, as well as the large volume of antibiotics, which are used to treat diarrhea in pigs after weaning. Previous studies have revealed loci on SSC6 and SSC13 associated with susceptibility to specific diarrhea causing pathogens. This study aimed to identify new genetic loci for resistance to diarrhea based on phenotypic data. In depth clinical characterization of diarrhea was performed in 257 pigs belonging to two herds during the first 14 days post weaning. The daily diarrhea assessments were used for the classification of pigs into case and control groups. Pigs were assigned to case and control groups based only on the incidence of diarrhea in the second week of the study in order to differentiate between differences in etiology. Genome-wide association studies and metabolomics association analysis were performed in order to identify new biological determinants for diarrhea susceptibility. With the present work, we revealed a new locus for diarrhea resistance on SSC16. Furthermore, studies of metabolomics in the same pigs revealed one metabolite associated with diarrhea.

Evaluation of the value of genetic testing for cystinuria in the Danish population of English bulldogs.

Cystinuria is a genetic disease that can lead to cystine urolith formation. The English bulldog is the dog breed most frequently affected. In this breed, three missense mutations have been suggested to be associated with cystinuria: c.568A>G and c.2086A>G in SLC3A1 and c.649G>A in SLC7A9. In this study, the occurrence of these three mutations in the Danish population of English bulldogs was investigated. Seventy-one English bulldogs were genotyped using TaqMan assays. The dogs' owners were given questionnaires concerning the medical histories of their dogs. Allele frequencies of 0.40, 0.40, and 0.52 were found for the mutant alleles in the three loci: c.568A>G, c.2086A>G, and c.649G>A, respectively. For both mutations in SLC3A1, a statistically significant association was found between cystinuria and homozygosity for the G allele among male, English bulldogs. For the mutation in SLC7A9, there was no statistically significant association between homozygosity for the mutant allele and cystinuria. Due to high allele frequencies, limited genetic diversity, continued uncertainty about the genetic background of cystinuria, and more severe health problems in the breed, selection based on genetic testing for the mutations in SLC3A1 cannot be recommended in the Danish population of English bulldogs. However, results of the genetic test may be used as a guide to recommend prophylactic treatment.

A compendium of genetic regulatory effects across pig tissues.

The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.

DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

Analysis of merged transcriptomic and genomic datasets to identify genes and pathways underlying residual feed intake in growing pigs.

Improvement of feed efficiency (FE) in pigs is an important milestone in order to reduce the economic and environmental impact of pig production. The goal of finding biomarkers for FE has persisted for decades. However, due to the complexity of the FE trait, these goals have still not been met. Here, we search for quantitative trait loci (QTL), candidate genes, and biological pathways associated with FE using both genotype and RNA-seq data. We obtained genotype and colon epithelium RNA-seq data for 375 and 96 pigs, respectively. In total, a genome-wide association study (GWAS) and differential expression (DE) analysis led to detection of three QTL on SSC9 and 17 DE-genes associated with FE. Possible intersection points between genes located in QTL and DE-genes were found on levels of transcription factor-target interaction. Moreover, cis-eQTL analysis revealed associations between genotype and expression levels of three DE-genes and three genes located in the GWAS QTLs, which may establish the connection between genotype and phenotype through DE. Finally, single nucleotide polymorphism calling using RNA-seq data for genes located in GWAS QTLs revealed 53 polymorphisms of which eleven were missense variants.

Genome-wide association study in Dachshund: identification of a major locus affecting intervertebral disc calcification.

Intervertebral disc calcification and herniation commonly affects Dachshund where the predisposition is caused by an early onset degenerative process resulting in disc calcification. A continuous spectrum of disc degeneration is seen within and among dog breeds, suggesting a multifactorial etiology. The number of calcified discs at 2 years of age determined by a radiographic evaluation is a good indicator of the severity of disc degeneration and thus serves as a measure for the risk of developing intervertebral disc herniation. The aim of the study was to identify genetic variants associated with intervertebral disc calcification in Dachshund through a genome-wide association (GWA) study. Based on thorough radiographic examinations, 48 cases with ≥ 6 disc calcifications or surgically treated for disc herniation and 46 controls with 0-1 disc calcifications were identified. GWA using the Illumina CanineHD BeadChip identified a locus on chromosome 12 from 36.8 to 38.6 Mb with 36 markers reaching genome-wide significance (P(genome) = 0.00001-0.026). This study suggests that a major locus on chromosome 12 harbors genetic variations affecting the development of intervertebral disc calcification in Dachshund.

Identification of 2 loci associated with development of myxomatous mitral valve disease in Cavalier King Charles Spaniels.

Myxomatous mitral valve disease (MMVD) is the most common heart disease in dogs. It is characterized by chronic progressive degenerative lesions of the mitral valve. The valve leaflets become thickened and prolapse into the left atrium resulting in mitral regurgitation (MR). MMVD is most prevalent in small to medium sized dog breeds, Cavalier King Charles Spaniels (CKCS) in particular. The onset of MMVD is highly age dependent, and at the age of 10 years, nearly all CKCS are affected. The incidence of a similar disease in humans-mitral valve prolapse-is 1-5%. By defining CKCSs with an early onset of MMVD as cases and old dogs with no or mild signs of MMVD as controls, we conducted a genome-wide association study (GWAS) to identify loci associated with development of MMVD. We have identified a 1.58 Mb region on CFA13 (P(genome) = 4.0 × 10(-5)) and a 1.68 Mb region on CFA14 (P(genome) = 7.9 × 10(-4)) associated with development of MMVD. This confirms the power of using the dog as a model to uncover potential candidate regions involved in the molecular mechanisms behind complex traits.

Pig genome functional annotation enhances the biological interpretation of complex traits and human disease.

The functional annotation of livestock genomes is crucial for understanding the molecular mechanisms that underpin complex traits of economic importance, adaptive evolution and comparative genomics. Here, we provide the most comprehensive catalogue to date of regulatory elements in the pig (Sus scrofa) by integrating 223 epigenomic and transcriptomic data sets, representing 14 biologically important tissues. We systematically describe the dynamic epigenetic landscape across tissues by functionally annotating 15 different chromatin states and defining their tissue-specific regulatory activities. We demonstrate that genomic variants associated with complex traits and adaptive evolution in pig are significantly enriched in active promoters and enhancers. Furthermore, we reveal distinct tissue-specific regulatory selection between Asian and European pig domestication processes. Compared with human and mouse epigenomes, we show that porcine regulatory elements are more conserved in DNA sequence, under both rapid and slow evolution, than those under neutral evolution across pig, mouse, and human. Finally, we provide biological insights on tissue-specific regulatory conservation, and by integrating 47 human genome-wide association studies, we demonstrate that, depending on the traits, mouse or pig might be more appropriate biomedical models for different complex traits and diseases.

Inclusion of endophenotypes in a standard GWAS facilitate a detailed mechanistic understanding of genetic elements that control blood lipid levels.

Dyslipidemia is the primary cause of cardiovascular disease, which is a serious human health problem in large parts of the world. Therefore, it is important to understand the genetic and molecular mechanisms that regulate blood levels of cholesterol and other lipids. Discovery of genetic elements in the regulatory machinery is often based on genome wide associations studies (GWAS) focused on end-point phenotypes such as total cholesterol level or a disease diagnosis. In the present study, we add endophenotypes, such as serum levels of intermediate metabolites in the cholesterol synthesis pathways, to a GWAS analysis and use the pig as an animal model. We do this to increase statistical power and to facilitate biological interpretation of results. Although the study population was limited to ~ 300 individuals, we identify two genome-wide significant associations and ten suggestive associations. Furthermore, we identify 28 tentative associations to loci previously associated with blood lipids or dyslipidemia associated diseases. The associations with endophenotypes may inspire future studies that can dissect the biological mechanisms underlying these previously identified associations and add a new level of understanding to previously identified associations.

Genomic diversity revealed by whole-genome sequencing in three Danish commercial pig breeds.

Whole-genome sequencing of 217 animals from three Danish commercial pig breeds (Duroc, Landrace [LL], and Yorkshire [YY]) was performed. Twenty-six million single-nucleotide polymorphisms (SNPs) and 8 million insertions or deletions (indels) were uncovered. Among the SNPs, 493,099 variants were located in coding sequences, and 29,430 were predicted to have a high functional impact such as gain or loss of stop codon. Using the whole-genome sequence dataset as the reference, the imputation accuracy for pigs genotyped with high-density SNP chips was examined. The overall average imputation accuracy for all biallelic variants (SNP and indel) was 0.69, while it was 0.83 for variants with minor allele frequency > 0.1. This study provides whole-genome reference data to impute SNP chip-genotyped animals for further studies to fine map quantitative trait loci as well as improving the prediction accuracy in genomic selection. Signatures of selection were identified both through analyses of fixation and differentiation to reveal selective sweeps that may have had prominent roles during breed development or subsequent divergent selection. However, the fixation indices did not indicate a strong divergence among these three breeds. In LL and YY, the integrated haplotype score identified genomic regions under recent selection. These regions contained genes for olfactory receptors and oxidoreductases. Olfactory receptor genes that might have played a major role in the domestication were previously reported to have been under selection in several species including cattle and swine.

Epigenetic and Transcriptomic Characterization of Pure Adipocyte Fractions From Obese Pigs Identifies Candidate Pathways Controlling Metabolism.

Reprogramming of adipocyte function in obesity is implicated in metabolic disorders like type 2 diabetes. Here, we used the pig, an animal model sharing many physiological and pathophysiological similarities with humans, to perform in-depth epigenomic and transcriptomic characterization of pure adipocyte fractions. Using a combined DNA methylation capture sequencing and Reduced Representation bisulfite sequencing (RRBS) strategy in 11 lean and 12 obese pigs, we identified in 3529 differentially methylated regions (DMRs) located at close proximity to-, or within genes in the adipocytes. By sequencing of the transcriptome from the same fraction of isolated adipocytes, we identified 276 differentially expressed transcripts with at least one or more DMR. These transcripts were over-represented in gene pathways related to MAPK, metabolic and insulin signaling. Using a candidate gene approach, we further characterized 13 genes potentially regulated by DNA methylation and identified putative transcription factor binding sites that could be affected by the differential methylation in obesity. Our data constitute a valuable resource for further investigations aiming to delineate the epigenetic etiology of metabolic disorders.

Genome-wide identification of quantitative trait loci in a cross between Hampshire and Landrace II: meat quality traits.

BACKGROUND: Meat quality traits are important in pig breeding programs, but they are difficult to include in a traditional selection program. Marker assisted selection (MAS) of meat quality traits is therefore of interest in breeding programs and a Quantitative Trait Locus (QTL) analysis is the key to identifying markers that can be used in MAS. In this study, Landrace and Hampshire intercross and backcross families were used to investigate meat quality traits. Hampshire pigs are commonly used as the sire line in commercial pig breeding. This is the first time a pedigree including Hampshire pigs has been used for a QTL analysis of meat quality traits. RESULTS: In total, we analyzed 39 meat quality traits and identified eight genome-wide significant QTL peaks in four regions: one on chromosome 3, two on chromosome 6 and one on chromosome 16. At least two of the QTLs do not appear to have been detected in previous studies. On chromosome 6 we identified QTLs for water content in M. longissimus dorsi (LD), drip loss in LD and post mortem pH decline in LD. On chromosomes 3 and 16 we identified previously undetected QTLs for protein content in LD and for freezing and cooking loss respectively. CONCLUSION: We identified at least two new meat quality trait QTLs at the genome-wide significance level. We detected two QTLs on chromosome 6 that possibly coincide with QTLs detected in other studies. We were also able to exclude the C1843T mutation in the ryanodine receptor (RYR1) as a causative mutation for one of the chromosome 6 QTLs in this cross.

Haplotypes on pig chromosome 3 distinguish metabolically healthy from unhealthy obese individuals.

We have established a pig resource population specifically designed to elucidate the genetics involved in development of obesity and obesity related co-morbidities by crossing the obesity prone Göttingen Minipig breed with two lean production pig breeds. In this study we have performed genome wide association (GWA) to identify loci with effect on blood lipid levels. The most significantly associated single nucleotide polymorphisms (SNPs) were used for linkage disequilibrium (LD) and haplotype analyses. Three separate haploblocks which influence the ratio between high density lipoprotein cholesterol and total cholesterol (HDL-C/CT), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) levels respectively were identified on Sus Scrofa chromosome 3 (SSC3). Large additive genetic effects were found for the HDL-C/CT and LDL-C haplotypes. Haplotypes segregating from Göttingen Minipigs were shown to impose a positive effect on blood lipid levels. Thus, the genetic profile of the Göttingen Minipig breed seems to support a phenotype comparable to the metabolic healthy obese (MHO) phenotype in humans.

Gender and Obesity Specific MicroRNA Expression in Adipose Tissue from Lean and Obese Pigs.

Obesity is a complex condition that increases the risk of life threatening diseases such as cardiovascular disease and diabetes. Studying the gene regulation of obesity is important for understanding the molecular mechanisms behind the obesity derived diseases and may lead to better intervention and treatment plans. MicroRNAs (miRNAs) are short non-coding RNAs regulating target mRNA by binding to their 3'UTR. They are involved in numerous biological processes and diseases, including obesity. In this study we use a mixed breed pig model designed for obesity studies to investigate differentially expressed miRNAs in subcutaneous adipose tissue by RNA sequencing (RNAseq). Both male and female pigs are included to explore gender differences. The RNAseq study shows that the most highly expressed miRNAs are in accordance with comparable studies in pigs and humans. A total of six miRNAs are differentially expressed in subcutaneous adipose tissue between the lean and obese group of pigs, and in addition gender specific significant differential expression is observed for a number of miRNAs. The differentially expressed miRNAs have been verified using qPCR. The results of these studies in general confirm the trends found by RNAseq. Mir-9 and mir-124a are significantly differentially expressed with large fold changes in subcutaneous adipose tissue between lean and obese pigs. Mir-9 is more highly expressed in the obese pigs with a fold change of 10 and a p-value < 0.001. Mir-124a is more highly expressed in the obese pigs with a fold change of 114 and a p-value < 0.001. In addition, mir-124a is significantly higher expressed in abdominal adipose tissue in male pigs with a fold change of 119 and a p-value < 0.05. Both miRNAs are also significantly higher expressed in the liver of obese male pigs where mir-124a has a fold change of 12 and mir-9 has a fold change of 1.6, both with p-values < 0.05.

Comparative Analyses of QTLs Influencing Obesity and Metabolic Phenotypes in Pigs and Humans.

The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice) do not completely recapitulate key pathophysiological features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564) designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35) from birth to slaughter (242 ± 48 days), including body composition determined at about two months of age (63 ± 10 days) via dual-energy x-ray absorptiometry (DXA) scanning. All pigs were genotyped using Illumina Porcine 60k SNP Beadchip and a combined linkage disequilibrium-linkage analysis was used to identify genome-wide significant associations for collected phenotypes. We identified 229 QTLs which associated with adiposity- and metabolic phenotypes at genome-wide significant levels. Subsequently comparative analyses were performed to identify the extent of overlap between previously identified QTLs in both humans and pigs. The combined analysis of a large number of obesity phenotypes has provided insight in the genetic architecture of the molecular mechanisms underlying these traits indicating that QTLs underlying similar phenotypes are clustered in the genome. Our analyses have further confirmed that genetic heterogeneity is an inherent characteristic of obesity traits most likely caused by segregation or fixation of different variants of the individual components belonging to cellular pathways in different populations. Several important genes previously associated to obesity in human studies, along with novel genes were identified. Altogether, this study provides novel insight that may further the current understanding of the molecular mechanisms underlying human obesity.

A hereditary disposition for bovine peripheral nerve sheath tumors in Danish Holstein cattle.

BACKGROUND: Peripheral nerve sheath tumors (PNSTs) are frequently found in Danish cattle at slaughter. Bovine PNSTs share several gross and histopathological characteristics with the PNSTs in humans with heritable neurofibromatosis syndromes. The aim of the present study was to investigate a possible hereditary disposition to PNSTs in dairy cattle by statistical analysis performed on data from 567 cattle with PNSTs. Furthermore, a preliminary genome-wide association study (GWAS) was performed on DNA isolated from 28 affected and 28 non-affected Holstein cows to identify loci in the bovine genome involved in the development of PNSTs. RESULTS: PNSTs were significantly more common in the Danish Holstein breed than in other breeds with 0.49% of Danish Holsteins slaughtered during an eight-year-period having PNSTs. PNSTs also occurred significantly more frequently in the offspring of some specific Holstein sires. Examination of three generation pedigrees showed that these sires were genetically related through a widely used US Holstein sire. The PNSTs included in GWAS were histologically classified as neurofibroma-schwannoma (43%), schwannoma (36%) and neurofibroma (21%) and derived from Holstein cows with multiple PNSTs. A single SNP on chromosome 27 reached genome-wide significance. CONCLUSIONS: Gross and histological characteristics of bovine PNSTs are comparable to PNSTs in humans (schwannomatosis). Danish Holsteins are genetically disposed to develop PNSTs but the examined materials are insufficient to allow determination of the mode of inheritance.

An f2 pig resource population as a model for genetic studies of obesity and obesity-related diseases in humans: design and genetic parameters.

Obesity is a rising worldwide public health problem. Difficulties to precisely measure various obesity traits and the genetic heterogeneity in human have been major impediments to completely disentangle genetic factors causing obesity. The pig is a relevant model for studying human obesity and obesity-related (OOR) traits. Using founder breeds divergent with respect to obesity traits we have created an F2 pig resource population (454 pigs), which has been intensively phenotyped for 36 OOR traits. The main rationale for our study is to characterize the genetic architecture of OOR traits in the F2 pig design, by estimating heritabilities, genetic, and phenotypic correlations using mixed- and multi-trait BLUP animal models. Our analyses revealed high coefficients of variation (15-42%) and moderate to high heritabilities (0.22-0.81) in fatness traits, showing large phenotypic and genetic variation in the F2 population, respectively. This fulfills the purpose of creating a resource population divergent for OOR traits. Strong genetic correlations were found between weight and lean mass at dual-energy x-ray absorptiometry scanning (0.56-0.97). Weight and conformation also showed strong genetic correlations with slaughter traits (e.g., r g between abdominal circumference and leaf fat at slaughtering: 0.66). Genetic correlations between fat-related traits and the glucose level vary between 0.35 and 0.74 and show a strong correlation between adipose tissue and impaired glucose metabolism. Our power calculations showed a minimum of 80% power for QTL detection for all phenotypes. We revealed genetic correlations at population level, for the first time, for several difficult to measure and novel OOR traits and diseases. The results underpin the potential of the established F2 pig resource population for further genomic, systems genetics, and functional investigations to unravel the genetic background of OOR traits.