Epidemiological and genetic study of exertional rhabdomyolysis in a Warmblood horse family in Switzerland

REASONS FOR PERFORMING STUDY: Exertional rhabdomyolysis (ER) and its familial basis in Warmblood horses is incompletely understood. OBJECTIVES: To describe the case details, clinical signs and management of ER-affected Warmblood horses from a family with a high prevalence of ER, to determine if histopathological signs of polysaccharide storage myopathy (PSSM) and the glycogen synthase (GYS1) mutation are associated with ER in this family, and to investigate potential risk factors for development of ER. METHODS: A family consisting of a sire with ER and 71 of his descendants was investigated. History of episodes of ER, husbandry, feeding and use was assessed by interviewing horse owners using a standardised questionnaire. All horses were genotyped for GYS1. In 10 ER-affected horses, muscle histopathology was evaluated. RESULTS: Signs of ER were reported in 39% of horses and 51% of the entire family possessed the GYS1 mutation. Horses possessing the GYS1 mutation had a 7.1-times increased risk for developing ER compared to those with the normal genotype (95% confidence interval [CI] 2.37-21.23, P = 0.0005). All muscle samples from horses in the family with ER showed polysaccharide accumulation typical for PSSM, amylase-resistant in 9/10 cases. There was evidence (odds ratio 5.6, CI 1.00-31.32, P = 0.05) that fat or oil feeding improved clinical signs of ER. No other effects of environmental factors associated with clinical signs of ER were identified. CONCLUSION AND POTENTIAL RELEVANCE: PSSM associated with the GYS1 mutation is one identifiable cause of ER in Warmblood horses. Signs of ER are not always manifest in GYS1 positive horses and there are also other causes for ER in Warmblood horses. Breeding animals with the GYS1 mutation results in a high prevalence of ER due to its dominant mode of inheritance.

IgE, IgGa, IgGb and IgG(T) serum antibody levels in offspring of two sires affected with equine recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is a chronic lower airway disease of the horse caused by hypersensitivity reactions to inhaled stable dust, including mould spores such as Aspergillus fumigatus. The goals of this study were to investigate whether total serum IgE levels and allergen-specific IgE and IgG subclasses are influenced by genetic factors and/or RAO and whether quantitative trait loci (QTL) could be identified for these parameters. The offspring of two RAO-affected sires (S1: n=56 and S2: n=65) were grouped by stallion and disease status, and total serum IgE levels and specific IgE, IgGa, IgGb and IgG(T) levels against recombinant Aspergillus fumigatus 7 (rAspf7) were measured by ELISA. A panel of 315 microsatellite markers covering the 31 equine autosomes were used to genotype the stallions and their offspring. A whole-genome scan using half-sib regression interval mapping was performed for each of the IgG and IgE subclasses. There was no significant effect of disease status or sire on total IgE levels, but there was a significant effect of gender and age. rAspf7-specific IgGa levels were significantly higher in RAO-affected than in healthy horses. The offspring of S1 had significantly higher rAspf7-specific IgGa and IgE levels than those of S2. Five QTLs were significant chromosome-wide (P<0.01). QTLs for rAspf7-specific IgGa and IgE were identified on ECA 1, for rAspf7-specific IgGa and IgGb on ECA 24 and for rAspf7 IgGa on ECA 26. These results provide evidence for effects of disease status and genetics on allergen-specific IgGa and IgE.

A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS: The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS: For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS: This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE: Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.

Mixed inheritance of equine recurrent airway obstruction

BACKGROUND: Mode of inheritance of equine recurrent airway obstruction (RAO) is unknown. HYPOTHESIS: Major genes are responsible for RAO. ANIMALS: Direct offspring of 2 RAO-affected Warmblood stallions (n = 197; n = 163) and a representative sample of Swiss Warmbloods (n = 401). METHODS: One environmental and 4 genetic models (general, mixed inheritance, major gene, and polygene) were tested for Horse Owner Assessed Respiratory Signs Index (1-4, unaffected to severely affected) by segregation analyses of the 2 half-sib sire families, both combined and separately, using prevalences estimated in a representative sample. RESULTS: In all data sets the mixed inheritance model was most likely to explain the pattern of inheritance. In all 3 datasets the mixed inheritance model did not differ significantly from the general model (P= .62, P= 1.00, and P= .27) but was always better than the major gene model (P < .01) and the polygene model (P < .01). The frequency of the deleterious allele differed considerably between the 2 sire families (P= .23 and P= .06). In both sire families the displacement was large (t= 17.52 and t= 12.24) and the heritability extremely large (h(2)= 1). CONCLUSIONS AND CLINICAL RELEVANCE: Segregation analyses clearly reveal the presence of a major gene playing a role in RAO. In 1 family, the mode of inheritance was autosomal dominant, whereas in the other family it was autosomal recessive. Although the expression of RAO is influenced by exposure to hay, these findings suggest a strong, complex genetic background for RAO.

Genetic diversity in the modern horse illustrated from genome-wide SNP data.

Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. F(ST) calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Whole genome scan identifies several chromosomal regions linked to equine sarcoids.

Despite the evidence for a genetic predisposition to develop equine sarcoids (ES), no whole genome scan for ES has been performed to date. The objective of this explorative study was to identify chromosome regions associated with ES. The studied population was comprised of two half-sibling sire families, involving a total of 222 horses. Twenty-six of these horses were affected with ES. All horses had been previously genotyped with 315 microsatellite markers. Quantitative trait locus (QTL) signals were suggested where the F statistic exceeded chromosome-wide significance at P < 0.05. The QTL analyses revealed significant signals reaching P < 0.05 on equine chromosome (ECA) 20, 23 and 25, suggesting a polygenic character for this trait. The candidate regions identified on ECA 20, 23 and 25 include genes regulating virus replication and host immune response. Further investigation of the chromosome regions associated with ES and of genes potentially responsible for the development of ES could form the basis for early identification of susceptible animals, breeding selection or the development of new therapeutic targets.

Genetic markers for stallion fertility - lessons from humans and mice

Our knowledge on the many aspects of mammalian reproduction in general and equine reproduction in particular has greatly increased during the last 15 years. Advances in the understanding of the physiology, cell biology, and biochemistry of reproduction have facilitated genetic analyses of fertility. Currently, there are more than 200 genes known that are involved in the production of fertile sperm cells. The completion of a number of mammalian genome projects will aid in the investigation of these genes in different species. Great progress has been made in the understanding of genetic aberrations that lead to male infertility. Additionally, the first genetic mechanisms are being discovered that contribute to the quantitative variation of fertility traits in fertile male animals. As artificial insemination (AI) represents a widespread technology in horse breeding, semen quality traits may eventually become an additional selection criterion for breeding stallions. Current research activities try to identify genetic markers that correlate to these semen quality traits. Here, we will review the current state of genetic research in male fertility and offer some perspectives for future research in horses.