Unraveling molecular mechanisms involved in the development of leptin resistance using the pig as a model.

The increase in obesity worldwide underlines the need for research concerning its metabolic and genetic determinants. One of the most intriguing mechanisms regarding obesity involves leptin and its signaling cascade. Leptin is a key regulator contributing to the fine-tuned crosstalk between nutrient availability and appetite signaling in the central nervous system. Owing to ethical concerns, many human tissues are not readily available and pigs can serve as a good animal model owing to their comparable anatomy, metabolism and genetics. In the present study, we utilized the pig to investigate the possible impact of increased adiposity on the development of alterations within the leptin signaling pathway. Two divergent groups of pigs (High and Low) were defined based on a high and low amount of mesenteric fat. Cortex, cerebellum, hypothalamus, mesenteric, subcutaneous and retroperitoneal fat tissues were used to study changes in expression levels of 94 mRNA transcripts related to the leptin signaling pathway using the qPCR approach. No significant differences were found at the central nervous system, whereas the expression level of STAT1 was reduced in mesenteric fat and leptin (LEP) and interleukin 6 (IL6) were shown to be consistently increased in all analyzed fat compartments from pigs with a high amount of mesenteric fat. These results could imply the onset of leptin and pro-inflammatory cytokine overexpression at early stages of obesity in the analyzed pigs without affecting any key components in the central nervous system. Thus, these pigs showing a unique leptin deregulation in adipose tissues could be a useful translational resource for studies of obesity and leptin resistance phenotypes.

The first genome-wide association study concerning idiopathic epilepsy in Petit Basset Griffon Vendeen.

The dog breed Petit Basset Griffon Vendeen has a relatively high prevalence of idiopathic epilepsy compared to other dog breeds and previous studies have suggested a genetic cause of the disease in this breed. Based on these observations, a genome-wide association study was performed to identify possible epilepsy-causing loci. The study included 30 unaffected and 23 affected dogs, genotyping of 170K SNPs, and data analysis using plink and emmax. Suggestive associations at CFA13, CFA24 and CFA35 were identified with markers close to three strong candidate genes. However, subsequent sequencing of exons of the three genes did not reveal sequence variations, which could explain development of the disease. This is, to our knowledge, the first report on loci and genes with a possible connection to idiopathic epilepsy in Petit Basset Griffon Vendeen. However, further studies are needed to conclusively identify the genetic cause of idiopathic epilepsy in this dog breed.

Validation of DNA test for hip dysplasia failed in Danish Labrador Retrievers.

Canine hip dysplasia is characterized by poor hip joint conformation and laxity. The disease is a complex trait influenced by both genetics and environment. Diagnosis and quantification of hip dysplasia are performed by radiographic examination of the hip joint and the diagnosis is used for making breeding decisions in many breeds. A prognostic genetic test (the Dysgen test) based on seven associated SNPs has been developed in a study based on Spanish Labrador Retrievers. In our study this test has been evaluated in 39 Danish Labrador Retrievers with known radiographic hip score: 14 with hip dysplasia (grade D or E) and 25 without hip dysplasia (grade A or B). There was no significant correlation between the Dysgen test results and the radiographic hip status (P = 0.3203) in these dogs, indicating that Dysgen test results obtained for Danish Labrador Retrievers have no prognostic value.

Analysis of porcine body size variation using re-sequencing data of miniature and large pigs.

BACKGROUND: Domestication has led to substantial phenotypic and genetic variation in domestic animals. In pigs, the size of so called minipigs differs by one order of magnitude compared to breeds of large body size. We used biallelic SNPs identified from re-sequencing data to compare various publicly available wild and domestic populations against two minipig breeds to gain better understanding of the genetic background of the extensive body size variation. We combined two complementary measures, expected heterozygosity and the composite likelihood ratio test implemented in "SweepFinder", to identify signatures of selection in Minipigs. We intersected these sweep regions with a measure of differentiation, namely FST, to remove regions of low variation across pigs. An extraordinary large sweep between 52 and 61 Mb on chromosome X was separately analyzed based on SNP-array data of F2 individuals from a cross of Goettingen Minipigs and large pigs. RESULTS: Selective sweep analysis identified putative sweep regions for growth and subsequent gene annotation provided a comprehensive set of putative candidate genes. A long swept haplotype on chromosome X, descending from the Goettingen Minipig founders was associated with a reduction of adult body length by 3% in F2 cross-breds. CONCLUSION: The resulting set of genes in putative sweep regions implies that the genetic background of body size variation in pigs is polygenic rather than mono- or oligogenic. Identified genes suggest alterations in metabolic functions and a possible insulin resistance to contribute to miniaturization. A size QTL located within the sweep on chromosome X, with an estimated effect of 3% on body length, is comparable to the largest known in pigs or other species. The androgen receptor AR, previously known to influence pig performance and carcass traits, is the most obvious potential candidate gene within this region.

Inotropic and lusitropic effects of levosimendan and milrinone assessed by strain echocardiography-A randomised trial.

BACKGROUND: We compared the direct inotropic and lusitropic effects of two inodilators, milrinone and levosimendan in patients after aortic valve replacement for aortic stenosis. METHODS: In this randomised, blinded study, 31 patients with normal LV function, were randomised to either levosimendan (0.1 and 0.2 µg/kg/min, n = 15) or milrinone (0.4 and 0.8 µg/kg/min, n = 16) after aortic valve replacement. The effects on LV performance, LV strain, systolic (SR-S) and early diastolic (SR-E) strain rate were assessed by a pulmonary artery catheter and transoesophageal two-dimensional speckle tracking echocardiography of the LV inferior wall. To circumvent the inodilator-induced hemodynamic changes on LV systolic and diastolic deformation, central venous pressure (CVP), systolic artery pressure (SAP), and heart rate were maintained constant by colloid infusion, phenylephrine-induced vasoconstriction and atrial pacing, respectively, during drug infusion. RESULTS: Both inotropic agents induced a dose-dependent increase in cardiac index and stroke volume index by approximately 20% at the highest infusion rates with no differences between groups (P = .139 and .249, respectively). CVP, pulmonary capillary wedge pressure, SAP and heart rate were maintained constant in both groups. LV strain and SR-S increased with both agents, dose-dependently, by 17%-18% and 25%-30%, respectively, at the highest infusion rates, with no difference between groups (P = .434 and .284, respectively). Both agents improved early LV relaxation with no differences between groups (P = .637). At the higher doses, both agents increased SR-E by 30%. CONCLUSIONS: At clinically relevant infusion rates and a certain increase in LV performance the direct inotropic and lusitropic of milrinone and levosimendan were comparable.

Identification of the mutation causing progressive retinal atrophy in Old Danish Pointing Dog.

Progressive retinal atrophy (PRA) is a common cause of blindness in many dog breeds. It is most often inherited as a simple Mendelian trait, but great genetic heterogeneity has been demonstrated both within and between breeds. In many breeds the genetic cause of the disease is not known, and until now, the Old Danish Pointing Dog (ODP) has been one of those breeds. ODP is one of the oldest dog breeds in Europe. Seventy years ago the breed almost vanished, but today a population still exists, primarily in Denmark but with some dogs in Germany and Sweden. PRA has been diagnosed in ODP since the late 1990s. It resembles late onset PRA in other dog breeds, and it is inherited as an autosomal recessive trait. In the present study, we performed whole-genome sequencing and identified a single base insertion (c.3149_3150insC) in exon 1 of C17H2orf71. This is the same mutation previously found to cause PRA in Gordon Setters and Irish Setters, and it was later found in Tibetan Terrier, Standard Poodle and the Polski Owczarek Nizinny. The presence of the mutation in such a diverse range of breeds indicates an origin preceding creation of modern dog breeds. Hence, we screened 262 dogs from 44 different breeds plus four crossbred dogs, and can subsequently add Miniature Poodle and another polish sheepdog, the Polski Owczarek Podhalanski, to the list of affected breeds.

A targeted genotyping approach enhances identification of variants in taste receptor and appetite/reward genes of potential functional importance for obesity-related porcine traits.

Taste receptors (TASRs) and appetite and reward (AR) mechanisms influence eating behaviour, which in turn affects food intake and risk of obesity. In a previous study, we used next generation sequencing to identify potentially functional mutations in TASR and AR genes and found indications for genetic associations between identified variants and growth and fat deposition in a subgroup of animals (n = 38) from the UNIK resource pig population. This population was created for studying obesity and obesity-related diseases. In the present study we validated results from our previous study by investigating genetic associations between 24 selected single nucleotide variants in TASR and AR gene variants and 35 phenotypes describing obesity and metabolism in the entire UNIK population (n = 564). Fifteen variants showed significant association with specific obesity-related phenotypes after Bonferroni correction. Six of the 15 genes, namely SIM1, FOS, TAS2R4, TAS2R9, MCHR2 and LEPR, showed good correlation between known biological function and associated phenotype. We verified a genetic association between potentially functional variants in TASR/AR genes and growth/obesity and conclude that the combination of identification of potentially functional variants by next generation sequencing followed by targeted genotyping and association studies is a powerful and cost-effective approach for increasing the power of genetic association studies.

Load-dependence of myocardial deformation variables - a clinical strain-echocardiographic study.

BACKGROUND: The effects of left ventricular (LV) loading on myocardial deformation variables are not well-studied in the clinical setting. In the present study, we evaluated the effects of isolated changes in preload, afterload and heart rate on LV longitudinal strain, systolic (SR-S) and early diastolic strain rate (SR-E) in post-cardiac surgery patients. METHODS: Twenty-one patients were studied early after cardiac surgery. Longitudinal myocardial strain and SR were analysed off-line using 2-D speckle echocardiography. The experimental protocol consisted of three consecutive interventions: (1) preload was increased by passive leg elevation, (2) afterload was increased by an infusion of phenylephrine to increase arterial blood pressure by 10-15% and (3) heart rate was increased 10% and 20% by atrial pacing. During both the preload and afterload challenges heart rate was kept constant by atrial pacing. Central venous pressure was kept constant during pacing by infusion of hetastarch/albumin. RESULTS: The increase in preload increased LV strain, SR-S and SR-E by 20%, 11% and 17%, respectively. The phenylephrine-induced increase in afterload, did not affect LV strain, SR-S or SR-E. LV strain was not affected while SR-S and SR-E increased by pacing-induced heart rate increase. CONCLUSION: After cardiac surgery, systolic and early diastolic strain rate are dependent on both preload and heart rate, while neither of these variables was afterload-dependent. LV strain was preload-dependent but not affected by atrial pacing. When evaluating the direct effects of various pharmacological or other interventions on myocardial contractility and relaxation, preload and heart rate must be controlled.

Discrepancy in compliance between the clinical and genetic diagnosis of choroidal hypoplasia in Danish Rough Collies and Shetland Sheepdogs.

Collie eye anomaly (CEA) is a congenital, inherited ocular disorder which is widespread in herding breeds. Clinically, the two major lesions associated with CEA are choroidal hypoplasia (CH) and coloboma, and both lesions are diagnosed based on ophthalmological examination. A 7.8-kb intronic deletion in the gene encoding non-homologous end-joining factor 1 (NHEJ1) has been reported to be the causative mutation underlying CH when present in the homozygous state. In this study, we have investigated the compliance between the clinical and genetic diagnosis of CH in the Danish Rough Collie and Shetland Sheepdog populations. Our results show that the deletion in NHEJ1 is not predictive for CH in the Danish Rough Collie population, whereas the clinical and genetic diagnosis is in accordance with each other in the Shetland Sheepdog population. Based on these results, it can be concluded that the intronic deletion in NHEJ1 is not the causative mutation but, rather, a marker linked to the locus underlying the trait in some populations but linked to both the wild-type and CH-causing locus in most dogs in the Danish Rough Collie population.

Identifying QTL and genetic correlations between fur quality traits in mink (Neovison vison).

Mapping of QTL affecting fur quality traits (guard hair length, guard hair thickness, density of wool, surface of the fur and quality) and skin length was performed in a three-generation mink population (F2 design). In the parental generation, Nordic Brown mink were crossed reciprocally with American Black short nap mink. In all, 1082 mink encompassing three generations were used for the analyses. The mink were genotyped for 104 microsatellites covering all 14 autosomes. The QTL analyses were performed by least-square regression implemented in gridqtl software. Genetic and phenotypic correlations and heritabilities were estimated using the average information-restricted maximum-likelihood method. Evidence was found for QTL affecting fur quality traits on nine autosomes. QTL were detected for guard hair thickness on chromosomes 1, 2, 3, 6 and 13; for guard hair length on chromosomes 2, 3 and 6; for wool density on chromosomes 6 and 13; for surface on chromosomes 7, 12 and 13; for quality on chromosomes 6, 7, 11 and 13; and for skin length on chromosomes 7 and 9. Proximity of locations of QTL for guard hair length, guard hair thickness and for wool density and quality suggests that some of the traits are in part under the influence of the same genes. Traits under the influence of QTL at close or identical positions also were traits that were strongly genotypically correlated. Based on the results of correlation analyses, the most important single traits influencing the quality were found to be density of wool, guard hair thickness and appearance of the surface.

Expression studies of six human obesity-related genes in seven tissues from divergent pig breeds.

Obesity has reached epidemic proportions globally and has become the cause of several major health risks worldwide. Presently, more than 100 loci have been related to obesity and metabolic traits in humans by genome-wide association studies. The complex genetic architecture behind obesity has triggered a need for the development of better animal models than rodents. The pig has emerged as a very promising biomedical model to study human obesity traits. In this study, we have characterized the expression patterns of six obesity-related genes, leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor (MC4R), fat mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin (ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production pigs and Göttingen minipigs) that deviate phenotypically and genetically from each other with respect to obesity traits. We observe significant differential expression for LEP, LEPR and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP expression is only detected in the fat minipigs. FTO shows significant differential expression in all tissues analyzed, and NEGR1 shows significant differential expression in muscle, pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be detected only in hypothalamus. In general, the expression profiles of the investigated genes are in accordance with those observed in human studies. Our study shows that both the differences between the investigated breeds and the phenotypic state with respect to obesity/leanness play a large role for differential expression of the obesity-related genes.

Potential role of the porcine superoxide dismutase 1 (SOD1) gene in pig reproduction.

In a recent study we confirmed that QTL regions on pig chromosomes 11, 13, and 15 are associated with reproduction traits in the pig. Within these regions the genetic variation was largest on chromosome 13. The QTL region on this chromosome was therefore studied further to identify genes known to contribute to litter size. The superoxide dismutase (SOD1) gene localized at around 200 Mb in the pig (Sscrofa10) was the most obvious candidate gene. In the present study, we have cloned and sequenced the porcine SOD1 gene. The SOD1 amino acid sequence is highly conserved between human, mouse, rat, and pig. Expression studies by quantitative PCR showed differential levels of the SOD1 transcript in all tissues investigated. Sequence comparison between sows with high and low estimated breeding value (EBV) for litter size, revealed a total of eight single nucleotide polymorphisms (SNPs) in the noncoding sequence and no SNPs in the coding region. One of the intronic SNPs was genotyped in 248 sows with high and low EBV for litter size. Allele frequency differed significantly between the two group of sows indicating that polymorphism in the chromosome 13 locus has an impact on litter size. The sows homozygous for the A/A genotype conceive three piglets more compared to the A/T genotype, making this SNP a possible marker for litter size. However, this genotype was negatively correlated with other important traits under selection in the Danish pig production.

A molecular study of congenital erythropoietic porphyria in cattle.

Previous studies have shown that congenital erythropoietic porphyria (CEP) in cattle is caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS) encoded by the UROS gene. In this study, we have established the pedigree of an extended Holstein family in which the disease is segregating in a manner consistent with autosomal recessive inheritance. Biochemical analyses demonstrated accumulation of uroporphyrin, thus confirming that it is indeed insufficient activity of UROS which is the cause of the disease. We have therefore sequenced all nine exons of UROS in affected and non-affected individuals without detecting any potential causative mutations. However, a single nucleotide polymorphism (SNP) located within the spliceosome attachment region in intron 8 of UROS is shown to segregate with the disease allele. Our study supports the hypothesis that CEP in cattle is caused by a mutation affecting UROS; however, additional functional studies are needed to identify the causative mutation.

Exonization of a LINE1 fragment implicated in X-linked hypohidrotic ectodermal dysplasia in cattle.

A case of X-linked hypohidrotic ectodermal dysplasia (XHED) was identified in a family of Danish Red Holstein cattle. The ectodysplasin-signalling protein (EDA) is known to be central in the normal development of ectodermal structures, and mutations in the ectodysplasin A (EDA) gene have been reported to cause XHED. In this study, we analysed different EDA transcript variants in affected and unaffected cattle and identified a new transcript variant including a LINE1-derived pseudoexon between EDA exons 1 and 2. The 161-bp-long pseudoexon introduces a shift in reading frame and a premature stop codon early in EDA exon 2 and is probably the cause of XHED in this Danish Red Holstein family.

Diet-Dependent Changes of the DNA Methylome Using a Göttingen Minipig Model for Obesity.

Objective: Environmental factors can influence obesity by epigenetic mechanisms. The aim of this study was to investigate obesity-related epigenetic changes and the potential for reversal of these changes in the liver of Göttingen minipigs subjected to diet interventions. Methods: High-throughput liquid hybridization capture-based bisulfite sequencing (LHC-BS) was used to quantify the methylation status of gene promotor regions in liver tissue in three groups of male castrated Göttingen minipigs: a standard chow group (SD, N = 7); a group fed high fat/fructose/cholesterol diet (FFC, N = 10) and a group fed high fat/fructose/cholesterol diet during 7 months and reversed to standard diet for 6 months (FFC/SD, N = 12). Expression profiling by qPCR of selected metabolically relevant genes was performed in liver tissue from all pigs. Results: The pigs in the FFC diet group became morbidly obese. The FFC/SD diet did not result in a complete reversal of the body weight to the same weight as in the SD group, but it resulted in reversal of all lipid related metabolic parameters. Here we identified widespread differences in the patterning of cytosine methylation of promoters between the different feeding groups. By combining detection of differentially methylated genes with a rank-based hypergeometric overlap algorithm, we identified 160 genes showing differential methylation in corresponding promoter regions in the FFC diet group when comparing with both the SD and FFC/SD groups. As expected, this differential methylation under FFC diet intervention induced de-regulation of several metabolically-related genes involved in lipid/cholesterol metabolism, inflammatory response and fibrosis generation. Moreover, five genes, of which one is a fibrosis-related gene (MMP9), were still perturbed after diet reversion. Conclusion: Our findings highlight the potential of exploring diet-epigenome interactions for treatment of obesity.

Refined candidate region specified by haplotype sharing for Escherichia coli F4ab/F4ac susceptibility alleles in pigs.

Infection of the small intestine by enterotoxigenic Escherichia coli F4ab/ac is a major welfare problem and financial burden for the pig industry. Natural resistance to this infection is inherited as a Mendelian recessive trait, and a polymorphism in the MUC4 gene segregating for susceptibility/resistance is presently used in a selection programme by the Danish pig breeding industry. To elucidate the genetic background involved in E. coli F4ab/ac susceptibility in pigs, a detailed haplotype map of the porcine candidate region was established. This region covers approximately 3.7 Mb. The material used for the study is a three generation family, where the founders are two Wild boars and eight Large White sows. All pigs have been phenotyped for susceptibility to F4ab/ac using an adhesion assay. Their haplotypes are known from segregation analysis using flanking markers. By a targeted approach, the candidate region was subjected to screening for polymorphisms, mainly focusing on intronic sequences. A total of 18 genes were partially sequenced, and polymorphisms were identified in GP5, CENTB2, APOD, PCYT1A, OSTalpha, ZDHHC19, TFRC, ACK1, MUC4, MUC20, KIAA0226, LRCH3 and MUC13. Overall, 227 polymorphisms were discovered in the founder generation. The analysis revealed a large haplotype block, spanning at least 1.5 Mb around MUC4, to be associated with F4ab/ac susceptibility.

Genetic mapping of quantitative trait loci for growth and fatness in pigs.

The European wild boar was crossed with the domesticated Large White pig to genetically dissect phenotypic differences between these populations for growth and fat deposition. The most important effects were clustered on chromosome 4, with a single region accounting for a large part of the breed difference in growth rate, fatness, and length of the small intestine. The study is an advance in genome analyses and documents the usefulness of crosses between divergent outbred populations for the detection and characterization of quantitative trait loci. The genetic mapping of a major locus for fat deposition in the pig could have implications for understanding human obesity.

High heritability for Ascaris and Trichuris infection levels in pigs.

Aggregated distributions of macroparasites within their host populations are characteristic of most natural and experimental infections. We designed this study to measure the amount of variation that is attributable to host genetic factors in a pig-helminth system. In total, 195 piglets were produced after artificial insemination of 19 sows (Danish Landrace-Yorkshire crossbreds) with semen selected from 13 individual Duroc boars (1 or 2 sows per boar; mean litter size: 10.3; 5-14 piglets per litter). Starting at 10 weeks of age, piglets were repeatedly infected with the gastrointestinal helminths Trichuris suis and Ascaris suum by administering eggs in the feed for 14 weeks until necropsy. Faecal egg counts (FECs) were estimated regularly and A. suum worm burden was obtained at necropsy. Heritability calculations for log (FEC+1) at weeks 7-10 post-infection (p.i.) showed that 0.32-0.73 of the phenotypic variation for T. suis could be attributed to genetic factors. For A. suum, heritabilities of 0.29-0.31 were estimated for log (FEC+1) at weeks 7-14 p.i., whereas the heritability of log worm counts was 0.45. Strong positive genetic correlations (0.75-0.89) between T. suis and A. suum FECs suggest that resistance to both infections involves regulation by overlapping genes. Our data demonstrate that there is a strong genetic component in resistance to A. suum and T. suis infections in pigs. Identification of responsible genes would enhance our understanding of the host immune response to these common nematodes and for the closely related species (T. trichiura and A. lumbricoides) in man infecting more than a billion people.

Population dynamics of Trichuris suis in trickle-infected pigs.

The population dynamics of Trichuris suis in pigs was studied during long-term experimental infections. Twenty-three 10-week-old pigs were inoculated with 5 T. suis eggs/kg/day. Seven, 8, and 8 pigs were necropsied at weeks 4, 8, and 14 post-start of infection (p.i.), respectively. The median numbers of worms in the colon were 538 (min-max: 277-618), 332 (14-1140) and 0 (0-4) at 4, 8, and 14 weeks p.i. respectively, suggesting an increased aggregation of the worms with time and acquisition of nearly sterile immunity. The serum levels of T. suis specific antibodies (IgG1, IgG2 and IgA) peaked at week 8 p.i. By week 14 p.i. the IgG2 and IgA antibody levels remained significantly elevated above the level of week 0. The population dynamics of T. suis trickle infections in pigs is discussed with focus on interpretation of diagnostic and epidemiological data of pigs, the use of pigs as a model for human Trichuris trichiura infections and the novel approach of using T. suis eggs in the treatment of patients with inflammatory bowel disease.

The dynamics of genetically marked Ascaris suum infections in pigs.

SUMMARY: The genotypes of both host and parasite may influence the outcome of parasitic infections, but few attempts have been made to quantify the effect of parasite genotype on macroparasite infections of socio-economic importance. We examined variation in particular traits during the infection in pigs with the parasitic nematode Ascaris suum. We infected 26 pigs with mixtures of equal proportions of embryonated eggs from 4 single female worms each with a unique mtDNA haplotype--the eggs from each female worm were a mixture of siblings and half-siblings. Pigs were necropsied on days 14, 17 and 28 following inoculation, which corresponded to time-points before, during and after the main immune responses against the nematode. A total of approximately 11,000 worms were recovered at necropsy. The location in the small intestine was recorded for all worms and the length and mtDNA haplotype were determined for about 4200 individual worms. There were significant differences in the distribution and abundance of the 4 individual haplotypes among individual pigs demonstrating strong interactions between parasite and host. We found significant differences in the abundance and position in the small intestine as well as the size of worms among haplotypes. We conclude that both parasite and host effects as well as the interplay between them play important roles in determining the characteristics and outcome of infection.