Novel COL6A3 frameshift variant in American Staffordshire Terrier dogs with Ullrich-like congenital muscular dystrophy.

Two (male and female) 10-month-old American Staffordshire Terrier littermates presented for progressive weakness, joint contracture, and distal limb joint hyperlaxity beginning around 6 months of age. Neurological examination, serum creatine kinase activity, infectious disease titers, cerebrospinal fluid analysis, and electrodiagnostic testing were performed. Muscle biopsies were collected for histopathology and immunofluorescence staining for localization of dystrophy associated proteins. Whole-genome sequencing (WGS) was performed on 1 affected dog. Variants were compared to a database of 671 unaffected dogs of multiple breeds. Histopathology confirmed a dystrophic phenotype and immunofluorescence staining of muscle cryosections revealed an absence of staining for collagen-6. WGS identified a homozygous 1 bp deletion in the COL6A3 gene, unique to the first affected dog. Sanger sequencing confirmed the homozygous presence of the frameshift variant in both affected dogs. This report describes the clinical features and most likely genetic basis of an Ullrich-like recessively inherited form of congenital muscular dystrophy in American Staffordshire Terriers.

Congenital muscular dystrophy in a dog with a LAMA2 gene deletion.

A 2-year-old female spayed dog was presented with a chronic history of short-strided gait and inability to completely open the jaw. Clinical signs were present since the dog was adopted from a humane society at a few months of age. Serum creatine kinase activity was abnormally high. Neurological examination, electromyography, muscle biopsies with immunofluorescent staining, and whole genome sequencing (WGS) were performed. A dystrophic phenotype was identified histologically in muscle biopsies, deficiency of laminin α2 protein was confirmed by immunofluorescent staining, and a deletion in the LAMA2 gene was identified by analysis of the WGS data. Congenital muscular dystrophy associated with a disease variant in LAMA2 was identified.

Genome-Wide Analyses for Osteosarcoma in Leonberger Dogs Reveal the CDKN2A/B Gene Locus as a Major Risk Locus.

Dogs represent a unique spontaneous cancer model. Osteosarcoma (OSA) is the most common primary bone tumor in dogs (OMIA 001441-9615), and strongly resembles human forms of OSA. Several large- to giant-sized dog breeds, including the Leonberger, have a greatly increased risk of developing OSA. We performed genome-wide association analysis with high-density imputed SNP genotype data from 273 Leonberger cases with a median age of 8.1 [3.1-13.5] years and 365 controls older than eight years. This analysis revealed significant associations at the CDKN2A/B gene locus on canine chromosome 11, mirroring previous findings in other dog breeds, such as the greyhound, that also show an elevated risk for OSA. Heritability (h2SNP) was determined to be 20.6% (SE = 0.08; p-value = 5.7 × 10-4) based on a breed prevalence of 20%. The 2563 SNPs across the genome accounted for nearly all the h2SNP of OSA, with 2183 SNPs of small effect, 316 SNPs of moderate effect, and 64 SNPs of large effect. As with many other cancers it is likely that regulatory, non-coding variants underlie the increased risk for cancer development. Our findings confirm a complex genetic basis of OSA, moderate heritability, and the crucial role of the CDKN2A/B locus leading to strong cancer predisposition in dogs. It will ultimately be interesting to study and compare the known genetic loci associated with canine OSA in human OSA.

Sarcoglycan A mutation in miniature dachshund dogs causes limb-girdle muscular dystrophy 2D.

BACKGROUND: A cohort of related miniature dachshund dogs with exercise intolerance, stiff gait, dysphagia, myoglobinuria, and markedly elevated serum creatine kinase activities were identified. METHODS: Muscle biopsy histopathology, immunofluorescence microscopy, and western blotting were combined to identify the specific pathologic phenotype of the myopathy, and whole genome SNP array genotype data and whole genome sequencing were combined to determine its genetic basis. RESULTS: Muscle biopsies were dystrophic. Sarcoglycanopathy, a form of limb-girdle muscular dystrophy, was suspected based on immunostaining and western blotting, where α, ß, and γ-sarcoglycan were all absent or reduced. Genetic mapping and whole genome sequencing identified a premature stop codon mutation in the sarcoglycan A subunit gene (SGCA). Affected dachshunds were confirmed on several continents. CONCLUSIONS: This first SGCA mutation found in dogs adds to the literature of genetic bases of canine muscular dystrophies and their usefulness as comparative models of human disease.

A CNTNAP1 Missense Variant Is Associated with Canine Laryngeal Paralysis and Polyneuropathy.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

Genomic diversity and population structure of the Leonberger dog breed.

BACKGROUND: Leonberger is a giant dog breed formed in the 1850s in Germany. Its post-World War II popularity has resulted in a current global population of ~ 30,000 dogs. The breed has predispositions to neurodegenerative disorders and cancer, which is likely due in large part to limited genetic diversity. However, to date there is no scientific literature on the overall demography and genomic architecture of this breed. RESULTS: We assessed extensive pedigree records, SNP array genotype data, and whole-genome sequences (WGS) on 142,072, 1203 and 39 Leonberger dogs, respectively. Pedigree analyses identified 22 founder animals and revealed an apparent popular sire effect. The average pedigree-based inbreeding coefficient of 0.29 and average kinship of 0.31 show a dramatic loss of genetic diversity. The observed average life span decreased over time from 9.4 years in 1989 to 7.7 years in 2004. A global health survey confirmed a high prevalence of cancer and neurological disorders. Analysis of SNP-based runs of homozygosity (ROH) identified 125,653 ROH with an average length of 5.88 Mb, and confirmed an average inbreeding coefficient of 0.28. Genome-wide filtering of the WGS data revealed 28 non-protein-changing variants that were present in all Leonberger individuals and a list of 22 potentially pathogenic variants for neurological disorders of which 50% occurred only in Leonbergers and 50% occurred rarely in other breeds. Furthermore, one of the two mtDNA haplogroups detected was present in one dog only. CONCLUSIONS: The increasing size of the Leonberger population has been accompanied by a considerable loss of genetic diversity after the bottleneck that occurred in the 1940s due to the intensive use of popular sires resulting in high levels of inbreeding. This might explain the high prevalence of certain disorders; however, genomic data provide no evidence for fixed coding variants that explain these predispositions. The list of candidate causative variants for polyneuropathy needs to be further evaluated. Preserving the current genetic diversity is possible by increasing the number of individuals for breeding while restricting the number of litters per sire/dam. In addition, outcrossing would help optimize long-term genetic diversity and contribute to the sustainability and health of the population.

NME5 frameshift variant in Alaskan Malamutes with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a hereditary defect of motile cilia in humans and several domestic animal species. Typical clinical findings are chronic recurrent infections of the respiratory tract and fertility problems. We analyzed an Alaskan Malamute family, in which two out of six puppies were affected by PCD. The parents were unaffected suggesting autosomal recessive inheritance. Linkage and homozygosity mapping defined critical intervals comprising ~118 Mb. Whole genome sequencing of one case and comparison to 601 control genomes identified a disease associated frameshift variant, c.43delA, in the NME5 gene encoding a sparsely characterized protein associated with ciliary function. Nme5-/- knockout mice exhibit doming of the skull, hydrocephalus and sperm flagellar defects. The genotypes at NME5:c.43delA showed the expected co-segregation with the phenotype in the Alaskan Malamute family. An additional unrelated Alaskan Malamute with PCD and hydrocephalus that became available later in the study was also homozygous mutant at the NME5:c.43delA variant. The mutant allele was not present in more than 1000 control dogs from different breeds. Immunohistochemistry demonstrated absence of the NME5 protein from nasal epithelia of an affected dog. We therefore propose NME5:c.43delA as the most likely candidate causative variant for PCD in Alaskan Malamutes. These findings enable genetic testing to avoid the unintentional breeding of affected dogs in the future. Furthermore, the results of this study identify NME5 as a novel candidate gene for unsolved human PCD and/or hydrocephalus cases.

The genetics of skeletal muscle disorders in horses.

Horses are remarkable athletes and a fascinating species in which to study the genetic bases of athletic performance, skeletal muscle biology, and neuromuscular disease. Genetic selection in horses has resulted in many breeds that possess anatomical, physiological, and metabolic variations linked to speed, power, and endurance that are beginning to be defined at the molecular level. Along with the concentration of positive traits, equine breeding programs have also inadvertently concentrated heritable muscle diseases for which mutations impacting electrical conduction, muscle contraction, and energy metabolism within and across breeds have been characterized. The study of heritable muscle diseases in horses has provided exciting insights into the normal structure and function of muscle and important diagnostic tools for veterinarians. Results empower breeders and breed associations to make difficult decisions about how to use this information to improve the overall health and well-being of horses.

A Gly98Val mutation in the N-Myc downstream regulated gene 1 (NDRG1) in Alaskan Malamutes with polyneuropathy.

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be "probably damaging" to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.

Evaluations of labrador retrievers with exercise-induced collapse, including response to a standardized strenuous exercise protocol.

Clinical and metabolic variables were evaluated in 14 Labrador retrievers with exercise-induced collapse (EIC) before, during, and following completion of a standardized strenuous exercise protocol. Findings were compared with previously reported variables from 14 normal Labrador retrievers that participated in the same protocol. Ten of 14 dogs with EIC developed an abnormal gait during evaluation, and these dogs were significantly more tachycardic and had a more severe respiratory alkalosis after exercise compared to the normal dogs. Muscle biopsy characteristics and sequential lactate and pyruvate concentrations were normal. Genetic testing and linkage analysis excluded malignant hyperthermia as the cause of EIC. Common causes of exercise intolerance were eliminated, but the cause of collapse in EIC was not determined.

Microsatellite instability in canine mammary gland tumors.

BACKGROUND: Genomic instability is a hallmark of cancer and may be required for the accumulation of cancer-causing mutations within cells. One form of genomic instability occurs in tandem nucleotide repeats and is known as microsatellite instability (MSI). HYPOTHESIS: We hypothesized that MSI can be observed in canine mammary gland tumors (MGT) and represents a potential carcinogenic mechanism in dogs. ANIMALS: Thirty-five dogs with MGTs and 9 dogs with other tumors were recruited from the University of Minnesota Veterinary Medical Center and referring veterinary clinics. METHODS: A panel of 21 canine microsatellite (MS) markers was amplified by polymerase chain reaction (PCR) from deoxyribonucleic acid obtained from blood and from fresh or formalin-fixed, paraffin-embedded tumor tissues. PCR products were evaluated by using capillary electrophoresis, and the chromatograms were analyzed by using genotyping software. MS genotypes obtained from fresh and formalin-fixed tumor tissues were compared, as were MS genotypes from normal tissue and tumor tissue. RESULTS: Genotypes obtained from formalin-fixed and fresh tissues were identical for all MS in 9 tumors evaluated, suggesting excellent concordance between the 2 sample types. For the 35 canine mammary tumors evaluated, 13 (37%) had stable genotypes; 22 (63%) exhibited aberrations in 1 or 2 MS; and 4 tumors (11%) demonstrated high-level instability, with aberrations in 29 to 61% of MS. CONCLUSIONS AND CLINICAL IMPORTANCE: Although some low-level MSI often is observed, high-level MSI is an infrequent finding in canine mammary tumors. Further evaluations are required to better characterize this phenomenon and to determine its relevance to canine carcinogenesis.

Biochemical and genetic evaluation of the role of AMP-activated protein kinase in polysaccharide storage myopathy in Quarter Horses.

OBJECTIVE: To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses. ANIMALS: 30 PSSM-affected and 30 unaffected (control) Quarter Horses. PROCEDURES: By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK gamma subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA. RESULTS: No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK gamma subunit genes were also not associated with PSSM. CONCLUSIONS AND CLINICAL RELEVANCE: These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.

Exclusion of linkage of the RYR1, CACNA1S, and ATP2A1 genes to recurrent exertional rhabdomyolysis in Thoroughbreds.

OBJECTIVE: To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation. ANIMALS: 34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed. PROCEDURES: Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified. Genotypes were obtained for all horses in the 4 families affected by RER, and data were used to test for linkage of these 3 loci to the RER phenotype. RESULTS: Analysis of the RYR1, CACNA1S, and ATP2A1 microsatellites excluded a link between those markers and the RER trait. CONCLUSIONS AND CLINICAL RELEVANCE: It is likely that the heritable alterations in muscle contractility that are characteristic of RER are caused by a gene that is not yet known to cause related muscle disease in other species.

Inheritance of recurrent exertional rhabdomyolysis in thoroughbreds.

OBJECTIVE: To develop a diagnostic test for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds that relied on in vitro contracture of muscle biopsy specimens and determine whether the inheritance pattern of RER diagnosed on the basis of this contracture test was consistent with an autosomal dominant trait. DESIGN: Clinical trial. ANIMALS: 8 adult horses with RER and 16 control adult horses for development of the contracture test; 23 foals for inheritance of RER. PROCEDURE: External intercostal muscle biopsy specimens from the 24 adult horses were tested for contracture in response to halothane and caffeine, and criteria for a positive test result were determined. These criteria were then applied to results for the 23 foals to determine whether they had RER. Simple segregation analysis was performed to determine whether results were consistent with a dominant pattern of inheritance. RESULTS: Results of the contracture test were positive for 5 of the 12 colts and 4 of the 11 fillies. Results of segregation analysis were consistent with an auto-. somal dominant pattern of inheritance. Two sires with RER produced colts with RER, supporting the hypothesis that RER had an autosomal, rather than an X-linked, inheritance pattern. In addition, in 1 instance, an unaffected colt was produced by 2 affected parents, which was not consistent with a recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: Although the expression of the RER trait is influenced by sex, temperament, and diet, among other factors, results from the in vitro muscle contracture test and this breeding trial suggest that RER in Thoroughbreds can be modeled as a genetic trait with an autosomal dominant pattern of inheritance.

Fatal infantile neuromuscular presentation of glycogen storage disease type IV.

Glycogen storage disease type IV or Andersen disease is an autosomal recessive disorder due to deficiency of glycogen branching enzyme. Typically, glycogen storage disease type IV presents with rapidly progressive liver cirrhosis and death in childhood. Variants include a cardiopathic form of childhood, a relatively benign myopathic form of young adults, and a late-onset neurodegenerative disorder (adult polyglucosan body disease). A severe neuromuscular variant resembling Werdnig-Hoffmann disease has also been described in two patients. The objective was to describe two additional infants with the neuromuscular variant and novel mutations in the GBE1 gene. Branching enzyme assay, Western blot, RT-PCR and sequencing were performed in muscle biopsies from both patients. The cDNA of patient 1 was subcloned and sequenced to define the mutations. Muscle biopsies showed accumulation of periodic acid Schiff-positive, diastase-resistant storage material in both patients and increased lysosomal enzyme activity in patient 1. Branching enzyme activity in muscle was negligible in both patients, and Western blot showed decreased branching enzyme protein. Patient 1 had two single base pair deletions, one in exon 10 (1238delT) and the other in exon 12 (1467delC), and each parent was heterozygous for one of the deletions. Patient 2 had a large homozygous deletion that spanned 627 bp and included exons 8-12. Patient 1, who died at 41 days, had neurophysiological and neuropathological features of Spinal Muscular Atrophy. Patient 2, who died at 5(1/2) weeks, had a predominantly myopathic process. The infantile neuromuscular form of glycogen storage disease type IV is considered extremely rare, but our encountering two patients in close succession suggests that the disease may be underdiagnosed.

Exceptional conservation of horse-human gene order on X chromosome revealed by high-resolution radiation hybrid mapping.

Development of a dense map of the horse genome is key to efforts aimed at identifying genes controlling health, reproduction, and performance. We herein report a high-resolution gene map of the horse (Equus caballus) X chromosome (ECAX) generated by developing and typing 116 gene-specific and 12 short tandem repeat markers on the 5,000-rad horse x hamster whole-genome radiation hybrid panel and mapping 29 gene loci by fluorescence in situ hybridization. The human X chromosome sequence was used as a template to select genes at 1-Mb intervals to develop equine orthologs. Coupled with our previous data, the new map comprises a total of 175 markers (139 genes and 36 short tandem repeats, of which 53 are fluorescence in situ hybridization mapped) distributed on average at approximately 880-kb intervals along the chromosome. This is the densest and most uniformly distributed chromosomal map presently available in any mammalian species other than humans and rodents. Comparison of the horse and human X chromosome maps shows remarkable conservation of gene order along the entire span of the chromosomes, including the location of the centromere. An overview of the status of the horse map in relation to mouse, livestock, and companion animal species is also provided. The map will be instrumental for analysis of X linked health and fertility traits in horses by facilitating identification of targeted chromosomal regions for isolation of polymorphic markers, building bacterial artificial chromosome contigs, or sequencing.

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain.

Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.

Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis.

OBJECTIVE: To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER). ANIMALS: 6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses. PROCEDURES: Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in myoplasmic calcium. In addition, intact intercostal muscle fiber bundles were prepared from both types of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined. RESULTS: Myotubes of RER-affected and control horses had identical resting myoplasmic calcium concentrations. Myotubes from RER-affected horses had significantly higher myoplasmic calcium concentrations than myotubes from control horses following the addition of > or = 2mM caffeine; however, there was no difference in their response to 4-CMC (> or = 1 mM). Caffeine contracture thresholds for RER and control intact muscle cell bundles (2 vs 10mM, respectively) were significantly different, but 4-CMC contracture thresholds of muscle bundles from RER-affected and control horses (500 microM) did not differ. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in caffeine sensitivity of muscle cells derived from a family of related RER-affected horses was detected in vitro by use of cell culture with calcium imaging and by use of fiber bundle contractility techniques. An alteration in muscle cell calcium regulation is a primary factor in the cause of this heritable myopathy.