A mixed breed dog with neuronal ceroid lipofuscinosis is homozygous for a CLN5 nonsense mutation previously identified in Border Collies and Australian Cattle Dogs.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by progressive declines in neurological functions following normal development. The NCLs are distinguished from similar disorders by the accumulation of autofluorescent lysosomal storage bodies in neurons and many other cell types, and are classified as lysosomal storage diseases. At least 13 genes contain pathogenic sequence variants that underlie different forms of NCL. Naturally occurring canine NCLs can serve as models to develop better understanding of the disease pathologies and for preclinical evaluation of therapeutic interventions for these disorders. To date 14 sequence variants in 8 canine orthologs of human NCL genes have been found to cause progressive neurological disorders similar to human NCLs in 12 different dog breeds. A mixed breed dog with parents of uncertain breed background developed progressive neurological signs consistent with NCL starting at approximately 11 to 12 months of age, and when evaluated with magnetic resonance imaging at 21 months of age exhibited diffuse brain atrophy. Due to the severity of neurological decline the dog was euthanized at 23 months of age. Cerebellar and cerebral cortical neurons contained massive accumulations of autofluorescent storage bodies the contents of which had the appearance of tightly packed membranes. A whole genome sequence, generated with DNA from the affected dog contained a homozygous C-to-T transition at position 30,574,637 on chromosome 22 which is reflected in the mature CLN5 transcript (CLN5: c.619C > T) and converts a glutamine codon to a termination codon (p.Gln207Ter). The identical nonsense mutation has been previously associated with NCL in Border Collies, Australian Cattle Dogs, and a German Shepherd-Australian Cattle Dog mix. The current whole genome sequence and a previously generated whole genome sequence for an Australian Cattle Dog with NCL share a rare homozygous haplotype that extends for 87 kb surrounding 22: 30, 574, 637 and includes 21 polymorphic sites. When genotyped at 7 of these polymorphic sites, DNA samples from the German Shepherd-Australian Cattle Dog mix and from 5 Border Collies with NCL that were homozygous for the CLN5: c.619 T allele also shared this homozygous haplotype, suggesting that the NCL in all of these dogs stems from the same founding mutation event that may have predated the establishment of the modern dog breeds. If so, the CLN5 nonsence allele is probably segregating in other, as yet unidentified, breeds. Thus, dogs exhibiting similar NCL-like signs should be screened for this CLN5 nonsense allele regardless of breed.

Bottlenecks and selective sweeps during domestication have increased deleterious genetic variation in dogs.

Population bottlenecks, inbreeding, and artificial selection can all, in principle, influence levels of deleterious genetic variation. However, the relative importance of each of these effects on genome-wide patterns of deleterious variation remains controversial. Domestic and wild canids offer a powerful system to address the role of these factors in influencing deleterious variation because their history is dominated by known bottlenecks and intense artificial selection. Here, we assess genome-wide patterns of deleterious variation in 90 whole-genome sequences from breed dogs, village dogs, and gray wolves. We find that the ratio of amino acid changing heterozygosity to silent heterozygosity is higher in dogs than in wolves and, on average, dogs have 2-3% higher genetic load than gray wolves. Multiple lines of evidence indicate this pattern is driven by less efficient natural selection due to bottlenecks associated with domestication and breed formation, rather than recent inbreeding. Further, we find regions of the genome implicated in selective sweeps are enriched for amino acid changing variants and Mendelian disease genes. To our knowledge, these results provide the first quantitative estimates of the increased burden of deleterious variants directly associated with domestication and have important implications for selective breeding programs and the conservation of rare and endangered species. Specifically, they highlight the costs associated with selective breeding and question the practice favoring the breeding of individuals that best fit breed standards. Our results also suggest that maintaining a large population size, rather than just avoiding inbreeding, is a critical factor for preventing the accumulation of deleterious variants.

A homozygous PIGN missense mutation in Soft-Coated Wheaten Terriers with a canine paroxysmal dyskinesia.

Hereditary paroxysmal dyskinesias (PxD) are a heterogeneous group of movement disorders classified by frequency, duration, and triggers of the episodes. A young-adult onset canine PxD has segregated as an autosomal recessive trait in Soft-Coated Wheaten Terriers. The medical records and videos of episodes from 25 affected dogs were reviewed. The episodes of hyperkinesia and dystonia lasted from several minutes to several hours and could occur as often as >10/day. They were not associated with strenuous exercise or fasting but were sometimes triggered by excitement. The canine PxD phenotype most closely resembled paroxysmal non-kinesigenic dyskinesia (PNKD) of humans. Whole genome sequences were generated with DNA from 2 affected dogs and analyzed in comparison to 100 control canid whole genome sequences. The two whole genome sequences from dogs with PxD had a rare homozygous PIGN:c.398C > T transition, which predicted the substitution of an isoleucine for a highly conserved threonine in the encoded enzyme. All 25 PxD-affected dogs were PIGN:c.398T allele homozygotes, whereas there were no c.398T homozygotes among 1185 genotyped dogs without known histories of PxD. PIGN encodes an enzyme involved in the biosynthesis of glycosylphosphatidylinositol (GPI), which anchors a variety of proteins including CD59 to the cell surface. Flow cytometry of PIGN-knockout HEK239 cells expressing recombinant human PIGN with the c.398T variant showed reduced CD59 expression. Mutations in human PIGN have been associated with multiple congenital anomalies-hypotonia-seizures syndrome-1 (MCAHS1). Movement disorders can be a part of MCAHS1, but this is the first PxD associated with altered GPI anchor function.

Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24.

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.

A mutation in the Warburg syndrome gene, RAB3GAP1, causes a similar syndrome with polyneuropathy and neuronal vacuolation in Black Russian Terrier dogs.

An autosomal recessive disease of Black Russian Terriers was previously described as a juvenile-onset, laryngeal paralysis and polyneuropathy similar to Charcot Marie Tooth disease in humans. We found that in addition to an axonal neuropathy, affected dogs exhibit microphthalmia, cataracts, and miotic pupils. On histopathology, affected dogs exhibit a spongiform encephalopathy characterized by accumulations of abnormal, membrane-bound vacuoles of various sizes in neuronal cell bodies, axons and adrenal cells. DNA from an individual dog with this polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV) was used to generate a whole genome sequence which contained a homozygous RAB3GAP1:c.743delC mutation that was absent from 73 control canine whole genome sequences. An additional 12 Black Russian Terriers with POANV were RAB3GAP1:c.743delC homozygotes. DNA samples from 249 Black Russian Terriers with no known signs of POANV were either heterozygotes or homozygous for the reference allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM), a severe developmental disorder characterized by abnormalities of the eye, genitals and nervous system including a predominantly axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, axonal transport, autophagy and synaptic transmission. The neuronal vacuolation and membranous inclusions and vacuoles in axons seen in this canine disorder likely reflect alterations of these processes. Thus, this canine disease could serve as a model for WARBM and provide insight into its pathogenesis and treatment.

A rare homozygous MFSD8 single-base-pair deletion and frameshift in the whole genome sequence of a Chinese Crested dog with neuronal ceroid lipofuscinosis.

BACKGROUND: The neuronal ceroid lipofuscinoses are heritable lysosomal storage diseases characterized by progressive neurological impairment and the accumulation of autofluorescent storage granules in neurons and other cell types. Various forms of human neuronal ceroid lipofuscinosis have been attributed to mutations in at least 13 different genes. So far, mutations in the canine orthologs of 7 of these genes have been identified in DNA from dogs with neuronal ceroid lipofuscinosis. The identification of new causal mutations could lead to the establishment of canine models to investigate the pathogenesis of the corresponding human neuronal ceroid lipofuscinoses and to evaluate and optimize therapeutic interventions for these fatal human diseases. CASE PRESENTATION: We obtained blood and formalin-fixed paraffin-embedded brain sections from a rescue dog that was reported to be a young adult Chinese Crested. The dog was euthanized at approximately 19 months of age as a consequence of progressive neurological decline that included blindness, anxiety, and cognitive impairment. A diagnosis of neuronal ceroid lipofuscinosis was made based on neurological signs, magnetic resonance imaging of the brain, and fluorescence microscopic and electron microscopic examination of brain sections. We isolated DNA from the blood and used it to generate a whole genome sequence with 33-fold average coverage. Among the 7.2 million potential sequence variants revealed by aligning the sequence reads to the canine genome reference sequence was a homozygous single base pair deletion in the canine ortholog of one of 13 known human NCL genes: MFSD8:c.843delT. MFSD8:c.843delT is predicted to cause a frame shift and premature stop codon resulting in a truncated protein, MFSD8:p.F282Lfs13*, missing its 239 C-terminal amino acids. The MFSD8:c.843delT allele is absent from the whole genome sequences of 101 healthy canids or dogs with other diseases. The genotyping of archived DNA from 1478 Chinese Cresteds did not identify any additional MFSD8:c.843delT homozygotes and found only one heterozygote. CONCLUSION: We conclude that the neurodegenerative disease of the Chinese Crested rescue dog was neuronal ceroid lipofuscinosis and that homozygosity for the MFSD8:c.843delT sequence variant was very likely to be the molecular-genetic cause of the disease.

A CLN8 nonsense mutation in the whole genome sequence of a mixed breed dog with neuronal ceroid lipofuscinosis and Australian Shepherd ancestry.

The neuronal ceroid lipofuscinoses (NCLs) are hereditary neurodegenerative diseases characterized by seizures and progressive cognitive decline, motor impairment, and vision loss accompanied by accumulation of autofluorescent lysosomal storage bodies in the central nervous system and elsewhere in the body. Mutations in at least 14 genes underlie the various forms of NCL. One of these genes, CLN8, encodes an intrinsic membrane protein of unknown function that appears to be localized primarily to the endoplasmic reticulum. Most CLN8 mutations in people result in a form of NCL with a late infantile onset and relatively rapid progression. A mixed breed dog with Australian Shepherd and Blue Heeler ancestry developed neurological signs characteristic of NCL starting at about 8months of age. The signs became progressively worse and the dog was euthanized at 21months of age due to seizures of increasing frequency and severity. Postmortem examination of the brain and retinas identified massive accumulations of intracellular autofluorescent inclusions characteristic of the NCLs. Whole genome sequencing of DNA from this dog identified a CLN8:c.585G>A transition that predicts a CLN8:p.Trp195* nonsense mutation. This mutation appears to be rare in both ancestral breeds. All of our 133 archived DNA samples from Blue Heelers, and 1481 of our 1488 archived Australian Shepherd DNA samples tested homozygous for the reference CLN8:c.585G allele. Four of the Australian Shepherd samples tested heterozygous and 3 tested homozygous for the mutant CLN8:c.585A allele. All 3 dogs homozygous for the A allele exhibited clinical signs of NCL and in 2 of them NCL was confirmed by postmortem evaluation of brain tissue. The occurrence of confirmed NCL in 3 of 4 CLN8:c.585A homozygous dogs, plus the occurrence of clinical signs consistent with NCL in the fourth homozygote strongly suggests that this rare truncating mutation causes NCL. Identification of this NCL-causing mutation provides the opportunity for identifying dogs that can be used to establish a canine model for the CLN8 disease (also known as late infantile variant or late infantile CLN8 disease).

GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: a potential model for Tay Sachs disease.

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of ß-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a ß subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two ß subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.

A L2HGDH initiator methionine codon mutation in a Yorkshire terrier with L-2-hydroxyglutaric aciduria.

BACKGROUND: L-2-hydroxyglutaric aciduria is a metabolic repair deficiency characterized by elevated levels of L-2-hydroxyglutaric acid in urine, blood and cerebrospinal fluid. Neurological signs associated with the disease in humans and dogs include seizures, ataxia and dementia. CASE PRESENTATION: Here we describe an 8 month old Yorkshire terrier that presented with episodes of hyperactivity and aggressive behavior. Between episodes, the dog's behavior and neurologic examinations were normal. A T2 weighted MRI of the brain showed diffuse grey matter hyperintensity and a urine metabolite screen showed elevated 2-hydroxyglutaric acid. We sequenced all 10 exons and intron-exon borders of L2HGDH from the affected dog and identified a homozygous A to G transition in the initiator methionine codon. The first inframe methionine is at p.M183 which is past the mitochondrial targeting domain of the protein. Initiation of translation at p.M183 would encode an N-terminal truncated protein unlikely to be functional. CONCLUSIONS: We have identified a mutation in the initiation codon of L2HGDH that is likely to result in a non-functional gene. The Yorkshire terrier could serve as an animal model to understand the pathogenesis of L-2-hydroxyglutaric aciduria and to evaluate potential therapies.

Genome-wide association analysis reveals a SOD1 mutation in canine degenerative myelopathy that resembles amyotrophic lateral sclerosis.

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.

Novel Homozygous ADAMTS2 Variants and Associated Disease Phenotypes in Dogs with Dermatosparactic Ehlers-Danlos Syndrome.

Tissue fragility, skin hyperextensibility and joint hypermobility are defining characteristics of Ehlers-Danlos syndrome (EDS). Human EDS is subclassified into fourteen types including dermatosparactic EDS, characterized by extreme skin fragility and caused by biallelic ADAMTS2 mutations. We report two novel, ADAMTS2 variants in DNA from EDS-affected dogs. Separate whole-genome sequences from a Pit Bull Terrier and an Alapaha Blue Blood Bulldog each contained a rare, homozygous variant (11:2280117delC, CanFam3.1), predicted to produce a frameshift in the transcript from the first coding ADAMTS2 exon (c.10delC) and a severely truncated protein product, p.(Pro4ArgfsTer175). The clinical features of these dogs and 4 others with the same homozygous deletion included multifocal wounds, atrophic scars, joint hypermobility, narrowed palpebral fissures, skin hyperextensibility, and joint-associated swellings. Due to severe skin fragility, the owners of all 6 dogs elected euthanasia before the dogs reached 13 weeks of age. Cross sections of collagen fibrils in post-mortem dermal tissues from 2 of these dogs showed hieroglyphic-like figures similar to those from cases of severe dermatosparaxis in other species. The whole-genome sequence from an adult Catahoula Leopard Dog contained a homozygous ADAMTS2 missense mutation, [11:2491238G>A; p.(Arg966His)]. This dog exhibited multifocal wounds, atrophic scars, and joint hypermobility, but has survived for at least 9 years. This report expands the spectrum of clinical features of the canine dermatosparactic subtype of EDS and illustrates the potential utility of subclassifying canine EDS by the identity of gene harboring the causal variant.

A truncating mutation in ATP13A2 is responsible for adult-onset neuronal ceroid lipofuscinosis in Tibetan terriers.

A recessive, adult-onset neuronal ceroid-lipofuscinosis (NCL) occurs in Tibetan terriers. A genome-wide association study restricted this NCL locus to a 1.3Mb region of canine chromosome 2 which contains canine ATP13A2. NCL-affected dogs were homozygous for a single-base deletion in ATP13A2, predicted to produce a frameshift and premature termination codon. Homozygous truncating mutations in human ATP13A2 have been shown by others to cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disease. These findings suggest that KRS is also an NCL, although analysis of KRS brain tissue will be needed to confirm this prediction. Generalized brain atrophy, behavioral changes, and cognitive decline occur in both people and dogs with ATP13A2 mutations; however, other clinical features differ between the species. For example, Tibetan terriers with NCL develop cerebellar ataxia not reported in KRS patients and KRS patients exhibit parkinsonism and pyramidal dysfunction not observed in affected Tibetan terriers. To see if ATP13A2 mutations could be responsible for some cases of human adult-onset NCL (Kufs disease), we resequenced ATP13A2 from 28 Kufs disease patients. None of these patients had ATP13A2 sequence variants likely to be causal for their disease, suggesting that mutations in this gene are not common causes of Kufs disease.

A missense mutation in canine CLN6 in an Australian shepherd with neuronal ceroid lipofuscinosis.

The childhood neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases that are progressive and ultimately fatal. An Australian Shepherd that exhibited a progressive neurological disorder with signs similar to human NCL was evaluated. The cerebral cortex, cerebellum, and retina were found to contain massive accumulations of autofluorescent inclusions characteristic of the NCLs. Nucleotide sequence analysis of DNA from the affected dog identified a T to C variant (c.829T>C) in exon 7 of CLN6. Mutations in the human ortholog underlie a late-infantile form of NCL in humans. The T-to-C transition results in a tryptophan to arginine amino acid change in the predicted protein sequence. Tryptophans occur at homologous positions in the CLN6 proteins from all 13 other vertebrates evaluated. The c.829T>C transition is a strong candidate for the causative mutation in this NCL-affected dog. Dogs with this mutation could serve as a model for the analogous human disorder.

International veterinary canine dyskinesia task force ECVN consensus statement: Terminology and classification.

Movement disorders are a heterogeneous group of clinical syndromes in humans and animals characterized by involuntary movements without changes in consciousness. Canine movement disorders broadly include tremors, peripheral nerve hyperexcitability disorders, paroxysmal dyskinesia, and dystonia. Of these, canine paroxysmal dyskinesias remain one of the more difficult to identify and characterize in dogs. Canine paroxysmal dyskinesias include an array of movement disorders in which there is a recurrent episode of abnormal, involuntary, movement. In this consensus statement, we recommend standard terminology for describing the various movement disorders with an emphasis on paroxysmal dyskinesia, as well as a preliminary classification and clinical approach to reporting cases. In the clinical approach to movement disorders, we recommend categorizing movements into hyperkinetic vs hypokinetic, paroxysmal vs persistent, exercise-induced vs not related to exercise, using a detailed description of movements using the recommended terminology presented here, differentiating movement disorders vs other differential diagnoses, and then finally, determining whether the paroxysmal dyskinesia is due to either inherited or acquired etiologies. This consensus statement represents a starting point for consistent reporting of clinical descriptions and terminology associated with canine movement disorders, with additional focus on paroxysmal dyskinesia. With consistent reporting and identification of additional genetic mutations responsible for these disorders, our understanding of the phenotype, genotype, and pathophysiology will continue to develop and inform further modification of these recommendations.

A mutation in canine PPT1 causes early onset neuronal ceroid lipofuscinosis in a Dachshund.

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases characterized by progressive neurodegeneration and accumulation of autofluorescent storage granules. A 9-month-old Miniature Dachshund presented with NCL-like signs that included disorientation, ataxia, weakness, visual impairment, and behavioral changes. Neurons throughout the CNS contained autofluorescent lysosomal inclusions with granular osmiophilic deposit (GROD) ultrastructure characteristic of classical infantile NCL (INCL). Human INCL is an autosomal recessive disorder that results from mutations in PPT1, a gene that encodes the enzyme palmitoyl protein thioesterase 1 (PPT1; EC 3.1.22). Resequencing of PPT1 from the affected dog revealed that the dog was homozygous for a single nucleotide insertion in exon 8 (PPT1 c.736_737insC), upstream from the His289 active site. Brain tissue from this dog lacked PPT1 activity. The sire and dam of the propositus were heterozygous for the c.736_737insC mutation; whereas, 127 unrelated Dachshunds were homozygous for the wild-type allele. This is the first reported instance of canine NCL caused by a mutation in PPT1.

Neuronal Ceroid Lipofuscinosis in a Domestic Cat Associated with a DNA Sequence Variant That Creates a Premature Stop Codon in CLN6.

A neutered male domestic medium-haired cat presented at a veterinary neurology clinic at 20 months of age due to progressive neurological signs that included visual impairment, focal myoclonus, and frequent severe generalized seizures that were refractory to treatment with phenobarbital. Magnetic resonance imaging revealed diffuse global brain atrophy. Due to the severity and frequency of its seizures, the cat was euthanized at 22 months of age. Microscopic examination of the cerebellum, cerebral cortex and brainstem revealed pronounced intracellular accumulations of autofluorescent storage material and inflammation in all 3 brain regions. Ultrastructural examination of the storage material indicated that it consisted almost completely of tightly-packed membrane-like material. The clinical signs and neuropathology strongly suggested that the cat suffered from a form of neuronal ceroid lipofuscinosis (NCL). Whole exome sequence analysis was performed on genomic DNA from the affected cat. Comparison of the sequence data to whole exome sequence data from 39 unaffected cats and whole genome sequence data from an additional 195 unaffected cats revealed a homozygous variant in CLN6 that was unique to the affected cat. This variant was predicted to cause a stop gain in the transcript due to a guanine to adenine transition (ENSFCAT00000025909:c.668G > A; XM_003987007.5:c.668G > A) and was the sole loss of function variant detected. CLN6 variants in other species, including humans, dogs, and sheep, are associated with the CLN6 form of NCL. Based on the affected cat's clinical signs, neuropathology and molecular genetic analysis, we conclude that the cat's disorder resulted from the loss of function of CLN6. This study is only the second to identify the molecular genetic basis of a feline NCL. Other cats exhibiting similar signs can now be screened for the CLN6 variant. This could lead to establishment of a feline model of CLN6 disease that could be used in therapeutic intervention studies.

A neonatal encephalopathy with seizures in standard poodle dogs with a missense mutation in the canine ortholog of ATF2.

Neonatal encephalopathy with seizures (NEWS) is a previously undescribed autosomal recessive disease of standard poodle puppies. Affected puppies are small and weak at birth. Many die in their first week of life. Those surviving past 1 week develop ataxia, a whole-body tremor, and, by 4 to 6 weeks of age, severe generalized clonic-tonic seizures. None have survived to 7 weeks of age. Cerebella from affected puppies were reduced in size and often contained dysplastic foci consisting of clusters of intermixed granule and Purkinje neurons. We used deoxyribonucleic acid samples from related standard poodles to map the NEWS locus to a 2.87-Mb segment of CFA36, which contains the canine ortholog of ATF2. This gene encodes activating transcription factor 2 (ATF-2), which participates in the cellular responses to a wide variety of stimuli. We amplified and sequenced all coding regions of canine ATF2 from a NEWS-affected puppy and identified a T > G transversion that predicts a methionine-to-arginine missense mutation at amino acid position 51. Methionine-51 lies within a hydrophobic docking site for mitogen-activated protein kinases that activate ATF-2 so the arginine substitution is likely to interfere with ATF-2 activation. All 20 NEWS-affected puppies in the standard poodle family were homozygous for the mutant G allele. The 58 clinically normal family members were either G/T heterozygotes or homozygous for the ancestral T allele. There are no previous reports of spontaneous ATF2 mutations in people or animals; however, atf2-knockout mice have cerebellar lesions that are similar to those in puppies with NEWS.

Retinal pathology in a canine model of late infantile neuronal ceroid lipofuscinosis.

PURPOSE: Late infantile neuronal ceroid lipofuscinosis (NCL) is an inherited disorder characterized by progressive vision loss. The disease results from mutations in the TPP1 (CLN2) gene. Studies were undertaken to characterize the effects of a TPP1 frameshift mutation on the retina in Dachshunds. METHODS: A litter of four puppies consisting of one homozygous affected dog, two heterozygotes, and one homozygous normal dog were monitored for neurologic and retinal changes through 10 months of age. The affected and homozygous normal dogs, as well as one of the heterozygotes, were then euthanatized, and the retinas were examined morphologically. RESULTS: The affected dog exhibited normal visual behavior and retinal function at 3 months of age, but vision was clearly impaired by 7 months, with markedly reduced ERG b-wave amplitudes. Beyond 7 months of age, the affected dog was functionally blind, and pupillary light reflexes and ERG response amplitudes continued to decline through 10 months of age. Both rod and cone system functions were severely impaired. The retina exhibited accumulation of autofluorescent storage bodies with distinctive curvilinear contents. Substantial cell loss occurred in the inner nuclear layer, with a smaller reduction in photoreceptor cell density. CONCLUSIONS: The canine TPP1 mutation results in progressive vision loss and retinal degeneration similar to that which occurs in human late infantile NCL. With the canine model, the natural history of disease progression in the retina provides a better understanding of the pathologic course of the disease and provides objective markers that can be used to assess the efficacy of therapeutic interventions.

Feline dysautonomia in the Midwestern United States: a retrospective study of nine cases.

Dysautonomia of domestic animals is pathologically characterized by chromatolytic degeneration of the neurons in the autonomic nervous ganglia that results in clinical signs related to dysfunction or failure of the sympathetic and parasympathetic nervous systems. The exact cause is unknown. It has a poor prognosis among all species reported and no definitive treatment is available currently. To date, most reported feline cases have occurred in the United Kingdom and Scandinavia. The cases reported here highlight the clinical signs, physical examination findings, and results of autonomic nervous system function testing in nine cats with dysautonomia in the US. Feline dysautonomia is uncommon in the US, but may have a regional prevalence, as is seen in dogs with most cases reported in Missouri and Kansas.

Clinical characterization of a familial degenerative myelopathy in Pembroke Welsh Corgi dogs.

BACKGROUND: Adult dogs with degenerative myelopathy (DM) have progressive ataxia and paresis of the pelvic limbs, leading to paraplegia and euthanasia. Although most commonly reported in German Shepherd dogs, high disease prevalence exists in other breeds. OBJECTIVE: Our aim was the clinical and histopathologic characterization of familial degenerative myelopathy (FDM) in Pembroke Welsh Corgi (PWC) dogs. ANIMALS: Twenty-one PWCs were prospectively studied from initial diagnosis until euthanasia. METHODS: Neurologic examination, blood tests, cerebrospinal fluid (CSF) analysis, electrodiagnostic testing, and spinal imaging were performed. Concentrations of 8-iso-prostaglandin F2alpha (8-isoprostane) were measured in CSF. Routine histochemistry was used for neuropathology. Deoxyribonucleic acid and pedigrees were collected from 110 dogs. RESULTS: Median duration of clinical signs before euthanasia was 19 months. Median age at euthanasia was 13 years. All dogs were nonambulatory paraparetic or paraplegic, and 15 dogs had thoracic limb weakness at euthanasia. Electrodiagnostic testing and spinal imaging were consistent with noncompressive myelopathy. No significant difference was detected in 8-isoprostane concentrations between normal and FDM-affected dogs. Axonal and myelin degeneration of the spinal cord was most severe in the dorsal portion of the lateral funiculus. Pedigree analysis suggested a familial disease. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical progression of FDM in PWC dogs was similar to that observed in other breeds but characterized by a longer duration. Spinal cord pathology predominates as noninflammatory axonal degeneration. Oxidative stress injury associated with 8-isoprostane production is not involved in the pathogenesis of FDM-affected PWC dogs. A familial disease is suspected.