Michelle L. Wagner

Evaluation of allele frequencies of inherited disease genes in subgroups of American Quarter Horses

OBJECTIVE To estimate allele frequencies of the hyperkalaemic periodic paralysis (HYPP), lethal white foal syndrome (LWFS), glycogen branching enzyme deficiency (GBED), hereditary equine regional dermal asthenia (HERDA), and type 1 polysaccharide storage myopathy (PSSM) genes in elite performance subgroups of American Quarter Horses (AQHs). DESIGN Prospective genetic survey. ANIMALS 651 elite performance AQHs, 200 control AQHs, and 180 control American Paint Horses (APHs). PROCEDURES Elite performance AQHs successful in 7 competitive disciplines (barrel racing, cutting, halter, racing, reining, western pleasure, and working cow horse) were geno- typed for 5 disease-causing alleles. Age-matched control AQHs and APHs were used to establish comparative whole-breed estimates of allele frequencies. RESULTS Highest allele frequencies among control AQHs were for type 1 PSSM (0.055) and GBED (0.054), whereas HERDA (0.021) and HYPP (0.008) were less prevalent. Control APHs uniquely harbored LWFS (0.107) and had high prevalence of HYPP (0.025), relative to AQHs. Halter horse subgroups had significantly greater allele frequencies for HYPP (0.299) and PSSM (0.155). Glycogen branching enzyme deficiency, HERDA, and PSSM were found broadly throughout subgroups; cutting subgroups were distinct for HERDA (0.142), and western pleasure subgroups were distinct for GBED (0.132). Racing and barrel racing subgroups had the lowest frequencies of the 5 disease genes. CONCLUSIONS AND CLINICAL RELEVANCE Accurate estimates of disease-causing alleles in AQHs and APHs may guide use of diagnostic genetic testing, aid management of genetic diseases, and help minimize production of affected foals.

A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse × hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse × hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19—a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution ofancestral HSA19 segments in eight mammalian orders involving about 50 species.

The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study

The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD–DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.

Genetic risk factors for insidious equine recurrent uveitis in Appaloosa horses

Appaloosa horses are predisposed to equine recurrent uveitis (ERU), an immune-mediated disease characterized by recurring inflammation of the uveal tract in the eye, which is the leading cause of blindness in horses. Nine genetic markers from the ECA1 region responsible for the spotted coat color of Appaloosa horses, and 13 microsatellites spanning the equine major histocompatibility complex (ELA) on ECA20, were evaluated for association with ERU in a group of 53 Appaloosa ERU cases and 43 healthy Appaloosa controls. Three markers were significantly associated (corrected P-value <0.05): a SNP within intron 11 of the TRPM1 gene on ECA1, an ELA class I microsatellite located near the boundary of the ELA class III and class II regions and an ELA class II microsatellite located in intron 1 of the DRA gene. Association between these three genetic markers and the ERU phenotype was confirmed in a second population of 24 insidious ERU Appaloosa cases and 16 Appaloosa controls. The relative odds of being an ERU case for each allele of these three markers were estimated by fitting a logistic mixed model with each of the associated markers independently and with all three markers simultaneously. The risk model using these markers classified ~80% of ERU cases and 75% of controls in the second population as moderate or high risk, and low risk respectively. Future studies to refine the associations at ECA1 and ELA loci and identify functional variants could uncover alleles conferring susceptibility to ERU in Appaloosa horses.

Estimated prevalence of the Type 1 Polysaccharide Storage Myopathy mutation in selected North American and European breeds.

The GYS1 gene mutation that is causative of Type 1 Polysaccharide Storage Myopathy (PSSM) has been identified in more than 20 breeds of horses. However, the GYS1 mutation frequency or Type 1 PSSM prevalence within any given breed is unknown. The purpose of this study was to determine the frequency of the GYS1 mutation and prevalence of genetic susceptibility to Type 1 PSSM in selected breeds from Europe and North America. The GYS1 mutation was detected in 11 breeds, including, in order of increasing allele frequency, Shires, Morgans, Appaloosas, Quarter Horses, Paints, Exmoor Ponies, Saxon-Thuringian Coldbloods, South German Coldbloods, Belgians, Rhenish German Coldbloods and Percherons. The prevalence of genetic susceptibility to Type 1 PSSM in these breeds varied from 0.5% to 62.4%. The GYS1 mutation was not found in the sampled Thoroughbreds, Akhal-Tekes, Connemaras, Clydesdales, Norwegian Fjords, Welsh Ponies, Icelandics, Schleswig Coldbloods or Hanoverians, but failure to detect the mutation does not guarantee its absence. This knowledge will help breed associations determine whether they should screen for the GYS1 mutation and will alert veterinarians to a possible differential diagnosis for muscle pain, rhabdomyolysis or gait abnormalities.

Allele Frequency and Likely Impact of the Glycogen Branching Enzyme Deficiency Gene in Quarter Horse and Paint Horse Populations

Glycogen Branching Enzyme Deficiency (GBED), a fatal condition recently identified in fetuses and neonatal foals of the Quarter Horse and Paint Horse lineages, is caused by a nonsense mutation in codon 34 of the GBE1 gene, which prevents the synthesis of a functional GBE protein and severely disrupts glycogen metabolism. The aims of this project were to determine the mutant GBE1 allele frequency in random samples from the major relevant horse breeds, as well as the frequency with which GBED is associated with abortion and early neonatal death using the tissue archives from veterinary diagnostic laboratories. The mutant GBE1 allele frequency in registered Quarter Horse, Paint Horse, and Thoroughbred populations was 0.041, 0.036, and 0.000, respectively. Approximately 2.5% of fetal and early neonatal deaths in Quarter Horse-related breeds submitted to 2 different US diagnostic laboratories were homozygous for the mutant GBE1 allele, with the majority of these being abortions. Retrospective histopathology of the homozygotes detected periodic acid Schiffs (PAS)-positive inclusions in the cardiac or skeletal muscle, which is characteristic of GBED, in 8 out of the 9 cases. Pedigree and genotype analyses supported the hypothesis that GBED is inherited as a simple recessive trait from a single founder. The frequency with which GBED is associated with abortion and neonatal mortality in Quarter Horse-related breeds makes the DNA-based test valuable in determining specific diagnoses and designing matings that avoid conception of a GBED foal.

A 1.3-Mb interval map of equine homologs of HSA2

A comparative approach that utilizes information from more densely mapped or sequenced genomes is a proven and efficient means to increase our knowledge of the structure of the horse genome. Human chromosome 2 (HSA2), the second largest human chromosome, comprising 243 Mb, and containing 1246 known genes, corresponds to all or parts of three equine chromosomes. This report describes the assignment of 140 new markers (78 genes and 62 microsatellites) to the equine radiation hybrid (RH) map, and the anchoring of 24 of these markers to horse chromosomes by FISH. The updated equine RH maps for ECA6p, ECA15, and ECA18 resulting from this work have one, two, and three RH linkage groups, respectively, per chromosome/chromosome-arm. These maps have a three-fold increase in the number of mapped markers compared to previous maps of these chromosomes, and an increase in the average marker density to one marker per 1.3 Mb. Comparative maps of ECA6p, ECA15, and ECA18 with human, chimpanzee, dog, mouse, rat, and chicken genomes reveal blocks of conserved synteny across mammals and vertebrates.

Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1

OBJECTIVE To develop a reliable method for converting cultured equine skin-derived fibroblasts into muscle cells. SAMPLE POPULATION Equine skin-derived fibroblasts. PROCEDURES The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin-derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid-Schiff staining. RESULTS eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen. CONCLUSIONS AND CLINICAL RELEVANCE Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.