Clinicopathological and pedigree investigation of a novel spinocerebellar neurological disease in juvenile Quarter Horses in North America.

BACKGROUND: In 2020, a novel neurologic disease was observed in juvenile Quarter Horses (QHs) in North America. It was unknown if this was an aberrant manifestation of another previously described neurological disorder in foals, such as equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM). HYPOTHESIS/OBJECTIVES: To describe the clinical findings, outcomes, and postmortem changes with Equine Juvenile Spinocerebellar Ataxia (EJSCA), differentiate the disease from other similar neurological disorders, and determine a mode of inheritance. ANIMALS: Twelve neurologically affected QH foals and the dams. METHODS: Genomic DNA was isolated and pedigrees were manually constructed. RESULTS: All foals (n = 12/12) had a history of acute onset of neurological deficits with no history of trauma. Neurological deficits were characterized by asymmetrical spinal ataxia, with pelvic limbs more severely affected than thoracic limbs. Clinicopathological abnormalities included high serum activity of gamma-glutamyl transferase and hyperglycemia. All foals became recumbent (median, 3 days: [0-18 days]), which necessitated humane euthanasia (n = 11/12, 92%; the remaining case was found dead). Histological evaluation at postmortem revealed dilated myelin sheaths and digestion chambers within the spinal cord, most prominently in the dorsal spinocerebellar tracts. Pedigree analysis revealed a likely autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL IMPORTANCE: EJSCA is a uniformly fatal, rapidly progressive, likely autosomal recessive neurological disease of QHs <1 month of age in North America that is etiologically distinct from other clinically similar neurological disorders. Once the causative variant for EJSCA is validated, carriers can be identified through genetic testing to inform breeding decisions.

Generalized myokymia, or neuromyotonia, or both in dogs with or without spinocerebellar ataxia.

BACKGROUND: KCNJ10 and CAPN1 variants cause "spinocerebellar" ataxia in dogs, but their association with generalized myokymia and neuromyotonia remains unclear. OBJECTIVE: To investigate the association between KCNJ10 and CAPN1 and myokymia or neuromyotonia, with or without concurrent spinocerebellar ataxia. ANIMALS: Thirty-three client-owned dogs with spinocerebellar ataxia, myokymia neuromytonia, or a combination of these signs. METHODS: Genetic analysis of a cohort of dogs clinically diagnosed with spinocerebellar ataxia, myokymia or neuromyotonia. KCNJ10 c.627C>G and CAPN1 c.344G>A variants and the coding sequence of KCNA1, KCNA2, KCNA6, KCNJ10 and HINT1 were sequenced using DNA extracted from blood samples. RESULTS: Twenty-four Jack Russell terriers, 1 Jack Russell terrier cross, 1 Dachshund and 1 mixed breed with spinocerebellar ataxia were biallelic (homozygous) for the KCNJ10 c.627C>G variant. Twenty-one of those dogs had myokymia, neuromyotonia, or both. One Parson Russell terrier with spinocerebellar ataxia alone was biallelic for the CAPN1 c.344G>A variant. Neither variant was found in 1 Jack Russell terrier with ataxia alone, nor in 3 Jack Russell terriers and 1 Yorkshire terrier with myokymia and neuromyotonia alone. No other causal variants were found in the coding sequence of the investigated candidate genes in these latter 5 dogs. CONCLUSION: The KCNJ10 c.627C>G variant, or rarely the CAPN1 c.344G>A variant, was confirmed to be the causal variant of spinocerebellar ataxia. We also report the presence of the KCNJ10 c.627C>G variant in the Dachshund breed. In dogs with myokymia and neuromyotonia alone the reported gene variants were not found. Other genetic or immune-mediated causes should be investigated to explain the clinical signs of these cases.

Spinocerebellar ataxia in the Bouvier des Ardennes breed is caused by a KCNJ10 missense variant.

BACKGROUND: In Belgian Malinois, a KCNJ10 variant causes progressive spinocerebellar degeneration. HYPOTHESIS/OBJECTIVES: Describe the clinical, diagnostic, pathological and genetic features of spinocerebellar degeneration in the Bouvier des Ardennes breed. ANIMALS: Five affected Bouvier des Ardennes puppies with spinocerebellar ataxia (SCA), 8 healthy related dogs, and 63 healthy unrelated Bouvier des Ardennes. METHODS: Sequential case study. RESULTS: Clinical signs started at 6 weeks of age in 1 puppy with severe signs of cerebellar disease, and at 7 to 10 weeks of age in the 4 remaining puppies with milder signs of spinocerebellar disease. The first puppy displayed severe intention tremors and rapidly progressive generalized hypermetric ataxia, whereas the 4 others developed a milder progressive SCA. Euthanasia after progression to nonambulatory status was performed by 8 weeks of age in the first puppy, and before 11 months of age in the 4 remaining puppies. Histopathology revealed cerebellar spongy degeneration and a focal symmetrical demyelinating myelopathy. All cases were homozygous for KCNJ10 XM_545752.6:c.986T>C(p.(Leu329Pro)), which is pathogenic for SCA with (or without) myokymia, seizures or both (SAMS) and spongy degeneration and cerebellar ataxia (SDCA) 1 in Belgian Malinois dogs. All sampled parents were heterozygous and none of the healthy dogs were homozygous for this recessive variant. This variant has an allele frequency of 15% in the 63 healthy dogs studied. CONCLUSIONS AND CLINICAL IMPORTANCE: Inherited spinocerebellar degeneration also affects the Bouvier des Ardennes breed and is caused by a KCNJ10 variant. It can present with a spectrum of severity grades, ranging from severe cerebellar to milder spinocerebellar signs.

Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Spinocerebellar ataxia in the Italian Spinone dog is associated with an intronic GAA repeat expansion in ITPR1.

Spinocerebellar ataxia in the Italian Spinone dog breed is characterised by a progressive gait abnormality that manifests from approximately 4 months of age. The disorder shows an autosomal recessive mode of inheritance, and affected individuals are usually euthanized by one year of age on welfare grounds due to an inability to ambulate. Using a homozygosity mapping technique with six cases and six controls, we mapped the disease locus to chromosome 20 of the canine genome. Linkage analysis across an extended pedigree confirmed the association, with microsatellite C20.374 achieving a maximal LOD score of 4.41. All five genes within the disease-associated interval were exon resequenced, although no exonic candidate mutations were identified. A targeted resequencing approach was therefore adopted to sequence the entire disease-associated interval. Analysis of the sequencing data revealed a GAA repeat expansion in intron 35 of ITPR1, which was homozygous in all cases and heterozygous in obligate carriers. Partial impairment of cerebellar ITPR1 expression in affected dogs was demonstrated by immunohistochemistry. Given the association of ITPR1 mutations with spinocerebellar ataxia (SCA) type 15 (also designated SCA16) in humans and that an intronic GAA repeat expansion has been shown to cause Friedreich ataxia, the repeat expansion is an excellent candidate for the cause of spinocerebellar ataxia in the Italian Spinone. This finding represents the first naturally occurring pathogenic intronic GAA repeat expansion in a non-human species and a novel mechanism for ITPR1 associated spinocerebellar ataxia.

A homozygous KCNJ10 mutation in Jack Russell Terriers and related breeds with spinocerebellar ataxia with myokymia, seizures, or both.

BACKGROUND: Juvenile-onset spinocerebellar ataxia has been recognized in Jack Russell Terriers and related Russell group terriers (RGTs) for over 40 years. Ataxia occurs with varying combinations of myokymia, seizures, and other signs of neurologic disease. More than 1 form of the disease has been suspected. HYPOTHESIS/OBJECTIVES: The objective was to identify the mutation causing the spinocerebellar ataxia associated with myokymia, seizures, or both and distinguish the phenotype from other ataxias in the RGTs. ANIMALS: DNA samples from 16 RGTs with spinocerebellar ataxia beginning from 2 to 12 months of age, 640 control RGTs, and 383 dogs from 144 other breeds along with the medical records of affected dogs were studied. METHODS: This case-control study compared the frequencies of a KCNJ10 allele in RGTs with spinocerebellar ataxia versus control RGTs. This allele was identified in a whole-genome sequence of a single RGT with spinocerebellar ataxia and myokymia by comparison to whole-genome sequences from 81 other canids that were normal or had other diseases. RESULTS: A missense mutation in the gene coding for the inwardly rectifying potassium channel Kir4.1 (KCNJ10:c.627C>G) was significantly (P < .001) associated with the disease. Dogs homozygous for the mutant allele all had spinocerebellar ataxia with varying combinations of myokymia and seizures. CONCLUSIONS AND CLINICAL IMPORTANCE: Identification of the KCNJ10 mutation in dogs with spinocerebellar ataxia with myokymia, seizures, or both clarifies the multiple forms of ataxia seen in these breeds and provides a DNA test to identify carriers.

Missense mutation in CAPN1 is associated with spinocerebellar ataxia in the Parson Russell Terrier dog breed.

Spinocerebellar ataxia (SCA) in the Parson Russell Terrier (PRT) dog breed is a disease of progressive incoordination of gait and loss of balance. Clinical signs usually become notable between 6 and 12 months of age with affected dogs presenting with symmetric spinocerebellar ataxia particularly evident in the pelvic limbs. The degree of truncal ataxia, pelvic limb hypermetria and impaired balance is progressive, particularly during the initial months of disease. A certain degree of stabilisation as well as intermittent worsening may occur. At the later stages of the disease ambulation often becomes difficult, with owners often electing to euthanise affected dogs on welfare grounds. Using a GWAS approach and target-enriched massively-parallel sequencing, a strongly associated non-synonymous SNP in the CAPN1 gene, encoding the calcium dependent cysteine protease calpain1 (mu-calpain), was identified. The SNP is a missense mutation causing a cysteine to tyrosine substitution at residue 115 of the CAPN1 protein. Cysteine 115 is a highly conserved residue and forms a key part of a catalytic triad of amino acids that are crucial to the enzymatic activity of cysteine proteases. The CAPN1 gene shows high levels of expression in the brain and nervous system and roles for the protein in both neuronal necrosis and maintenance have been suggested. Given the functional implications and high level of conservation observed across species, the CAPN1 variant represents a provocative candidate for the cause of SCA in the PRT and a novel potential cause of ataxia in humans.

Genome-wide mRNA sequencing of a single canine cerebellar cortical degeneration case leads to the identification of a disease associated SPTBN2 mutation.

BACKGROUND: Neonatal cerebellar cortical degeneration is a neurodegenerative disease described in several canine breeds including the Beagle. Affected Beagles are unable to ambulate normally from the onset of walking and the main pathological findings include Purkinje cell loss with swollen dendritic processes. Previous reports suggest an autosomal recessive mode of inheritance. The development of massively parallel sequencing techniques has presented the opportunity to investigate individual clinical cases using genome-wide sequencing approaches. We used genome-wide mRNA sequencing (mRNA-seq) of cerebellum tissue from a single Beagle with neonatal cerebellar cortical degeneration as a method of candidate gene sequencing, with the aim of identifying the causal mutation. RESULTS: A four-week old Beagle dog presented with progressive signs of cerebellar ataxia and the owner elected euthanasia. Histopathology revealed findings consistent with cerebellar cortical degeneration. Genome-wide mRNA sequencing (mRNA-seq) of RNA from cerebellum tissue was used as a method of candidate gene sequencing. After analysis of the canine orthologues of human spinocerebellar ataxia associated genes, we identified a homozygous 8 bp deletion in the ß-III spectrin gene, SPTBN2, associated with spinocerebellar type 5 in humans. Genotype analysis of the sire, dam, ten clinically unaffected siblings, and an affected sibling from a previous litter, showed the mutation to fully segregate with the disorder. Previous studies have shown that ß-III spectrin is critical for Purkinje cell development, and the absence of this protein can lead to cell damage through excitotoxicity, consistent with the observed Purkinje cell loss, degeneration of dendritic processes and associated neurological dysfunction in this Beagle. CONCLUSIONS: An 8 bp deletion in the SPTBN2 gene encoding ß-III spectrin is associated with neonatal cerebellar cortical degeneration in Beagle dogs. This study shows that mRNA-seq is a feasible method of screening candidate genes for mutations associated with rare diseases when a suitable tissue resource is available.

Leading the way: finding genes for neurologic disease in dogs using genome-wide mRNA sequencing.

Because of dogs' unique population structure, human-like disease biology, and advantageous genomic features, the canine system has risen dramatically in popularity as a tool for discovering disease alleles that have been difficult to find by studying human families or populations. To date, disease studies in dogs have primarily employed either linkage analysis, leveraging the typically large family size, or genome-wide association, which requires only modest-sized case and control groups in dogs. Both have been successful but, like most techniques, each requires a specific combination of time and money, and there are inherent problems associated with each. Here we review the first report of mRNA-Seq in the dog, a study that provides insights into the potential value of applying high-throughput sequencing to the study of genetic diseases in dogs. Forman and colleagues apply high-throughput sequencing to a single case of canine neonatal cerebellar cortical degeneration. This implementation of whole genome mRNA sequencing, the first reported in dog, is additionally unusual due to the analysis: the data was used not to examine transcript levels or annotate genes, but as a form of target capture that revealed the sequence of transcripts of genes associated with ataxia in humans. This approach entails risks. It would fail if, for example, the relevant transcripts were not sufficiently expressed for genotyping or were not associated with ataxia in humans. But here it pays off handsomely, identifying a single frameshift mutation that segregates with the disease. This work sets the stage for similar studies that take advantage of recent advances in genomics while exploiting the historical background of dog breeds to identify disease-causing mutations.

Neuromyotonia in a dachshund with clinical and electrophysiological signs of spinocerebellar ataxia.

A 10-month-old dachshund was presented with a recent history of episodic muscle rippling and generalised stiffness. An uncoordinated gait was present since eight weeks of age. On presentation the dog showed cerebellar-like ataxia and poor menace responses. Myokymic contractions were visible in the appendicular and truncal muscles and neuromyotonic discharges were detected by electromyography. Central components of the brain auditory evoked potentials were absent and the onset latencies of the tibial sensory-evoked potentials recorded at the lumbar intervertebral level were delayed. Response to slow-release phenytoin was temporary. The clinical picture together with the electrophysiological findings in this dachshund are identical to the findings in Jack Russell terriers with hereditary ataxia and neuromyotonia. This is the first description of neuromyotonia associated with clinical and electrophysiological signs of spinocerebellar ataxia in a breed other than the Jack Russell terrier. This case also strengthens the theory that spinocerebellar ataxia and neuromyotonia are related. An ion channel dysfunction is presumed to link both disorders.

Episodic cerebellar dysfunction in a bichon frise: a canine case of episodic ataxia?

A four-year-old neutered male bichon frise was presented for the evaluation of chronic, progressive yet episodic neurological dysfunction that was predominantly cerebellar in nature. Diagnostic testing including haematology, serum chemistry, magnetic resonance imaging, cerebrospinal fluid analysis and urine organic acid screening was normal. Trial therapies with phenobarbital, prednisone and acetazolamide were unsuccessful. Treatment with 4-aminopyridine led to complete resolution of the signs.