Phenotypic and genetic aspects of hereditary ataxia in dogs

Abstract Hereditary ataxias are a large group of neurodegenerative diseases that have cerebellar or spinocerebellar dysfunction as core feature, occurring as an isolated sign or as part of a syndrome. Based on neuropathology, this group of diseases has so far been classified into cerebellar cortical degenerations, spinocerebellar degenerations, cerebellar ataxias without substantial neurodegeneration, canine multiple system degeneration, and episodic ataxia. Several new hereditary ataxia syndromes are described, but most of these diseases have similar clinical signs and unspecific diagnostic findings, wherefore achieving a definitive diagnosis in these dogs is challenging. Eighteen new genetic variants associated with these diseases have been discovered in the last decade, allowing clinicians to reach a definitive diagnosis for most of these conditions, and allowing breeding schemes to adapt to prevent breeding of affected puppies. This review summarizes the current knowledge about hereditary ataxias in dogs, and proposes to add a “multifocal degenerations with predominant (spino)cerebellar component” category regrouping canine multiple system degeneration, new hereditary ataxia syndromes that do not fit in 1 of the previous categories, as well as specific neuroaxonal dystrophies and lysosomal storage diseases that cause major (spino)cerebellar dysfunction.


| INTRODUCTION
Hereditary ataxias are defined as a large group of diseases that have inherited cerebellar or spinocerebellar ataxia (SCA) and dysfunction as a core feature. 1,2 It is thus a clinical definition for a group of inherited neurodegenerative diseases, where (spino)cerebellar dysfunction can occur as an isolated sign or be a key feature of a more complex syndrome.
In a previous review article about hereditary ataxias in dogs, 1 5 categories were defined based on neuropathology: cerebellar cortical degenerations (CCDs), spinocerebellar degenerations, cerebellar ataxias without substantial neurodegeneration, canine multiple system degeneration (CMSD), and episodic ataxia. These diseases are described in several dog breeds, each with different ages of onset and speed of progression, combinations of different clinical signs and lesion topography in the central nervous system (CNS). As opposed to former reviews, the most recent human hereditary ataxia reviews now include some storage diseases if the key feature of the clinical presentation of the disease is (spino)cerebellar ataxia. 2,3 Cerebellar ataxia most commonly causes symmetric hypermetria (a form of dysmetria) that is characterized by sudden bursts of motor activity with a marked overflexion of the limbs on protraction without loss of strength. 4 Spinocerebellar ataxia is described as more dancing or bouncing in quality. 5 One of the major challenges of the current review was thus to define the boundaries of this large and heterogeneous group of diseases. Moreover, some diseases in dogs present differently from their human counterpart, fitting the definition of canine but not human hereditary ataxias, and vice versa.
This review summarizes the current knowledge about hereditary ataxias in dogs. As in previous reviews of hereditary ataxia in dogs, 1,5 diseases will still be grouped according to their neuropathological features. A new category, multifocal degenerations with predominant (spino)cerebellar component, was added in order to adapt for new hereditary ataxia syndromes that do not fit the definition of the previously existing categories, encompassing some specific neuroaxonal dystrophies (NADs) and lysosomal storage diseases which cause major (spino)cerebellar dysfunction. Those for which an association to a genetic variant has been identified are discussed in this article and summarized in Table 1, specific information about the variants is provided in File S1. For each of these diseases, an overview of the clinical presentation, diagnostic, histopathological findings, and genetic variant is given. The terminology (associated or causal) used in this review conforms to the terminology used in the original articles describing the variants. A standardized terminology as per the American College of Medical Genetics and Genomics (ACMG) guidelines 37 is favored to describe variants when possible. Diseases and case reports without genetic information will not be discussed in this review but are listed in Table 2. 2 | CEREBELLAR CORTICAL DEGENERATIONS CCD, or cerebellar abiotrophies, are 1 of the most common neurodegenerative diseases in animals. Two forms of CCD are described: primary Purkinje cell degeneration (most common) and primary granule cell or "granuloprival" degeneration. Classic signs include hypermetric and dysmetric ataxia, wide-based stance, tendency to lean and fall towards both sides, difficulty negotiating stairs, loss of balance, intention tremors, and occasional nystagmus. Neurological examination can reveal postural reaction deficits, delayed hopping with an exaggerated response, bilateral absent menace responses, as well as positional spontaneous nystagmus and opsoclonus. [6][7][8]12,52,58,[67][68][69][70][71][72] In primary Purkinje cell degeneration, the functional disturbance caused by the underlying genetic variant results in spontaneous premature death of Purkinje neurons ( Figure 1). The clinical signs reflect the loss of the normal inhibitory function of the Purkinje neurons on cerebellar and brainstem nuclei. Signs can have a neonatal, juvenile, or sometimes adult onset, and can be progressive or static (after rapid cell destruction). On MRI, cerebellar atrophy is frequently seen in dogs with advanced clinical disease, being most obvious in the dorsal half of the cerebellum ( Figure 2). As there is no relevant difference in the percentage of the brain occupied by the cerebellum among breeds in normal 1 to 5 years old dogs, the ratio between the brainstem and cerebellum midsagittal cross-sectional area using a cut off value of 89% is a reliable tool to detect cerebellar atrophy. 73 On necropsy, a macroscopically decreased size of the cerebellum is also seen in chronic cases, with a cerebellum to brain ratio of 6% to 9.7% (normal: 10%-12%). Typical microscopic lesions for the primary Purkinje cell degeneration form of CCD include primary Purkinje cell loss, with secondary transneuronal retrograde degeneration of granule cells and shrinkage of the molecular layer. In the granuloprival degeneration form of CCD, the main lesions include a progressive loss of granule cells, thinning of the granule cell layer and gliosis, with relative sparing of Purkinje cells [6][7][8]12,52,58,[67][68][69][70][71][72] (Figure 3). Genetic variants have so far only been associated with Purkinje cell degenerations, and have been described in the Beagle, 6 Finnish Hound, 7 Gordon Setter, 8 Old English Sheepdog, 8 and Viszla. 9 Regions of interest have been identified in the Scottish Terrier 10 (primary Purkinje cell degeneration) and Australian Kelpie 11,12 (granuloprival degeneration).

| Beagle
Signs in Beagles are noticed at the onset of walking (3 weeks of age).
Progression of signs is minimal, but the puppies are severely affected: falling frequently while trying to walk and having difficulties eating, frequently missing the food bowl when lowering their head to prehend the food. 69,70 An autosomal recessive 8-bp deletion in SPTBN2 (XM_038424853.1:c.5855_5862del (p.[Ile1952Argfs*28])) is associated with this disease, which compromises the functional expression of the spectrin beta, non-erythrocytic 2 protein (SPTBN2), a cytoskeletal protein that is highly expressed in Purkinje neurons. 6

| Finnish Hound
The onset of signs in Finnish Hounds is described around 9 weeks

| Gordon Setter and Old English Sheepdog
Clinical signs in Gordon Setters become apparent between 6 and 10 months of age, whereas a later onset of signs, between 6 and 40 months of age occurs in Old English Sheepdogs. The disease is slowly progressive (over several years) in both breeds, with milder severity in the Old English Sheepdog. 67,68,71 In Gordon Setters, an increased extensor muscle tone is also reported with disease progression. 67

| Scottish Terrier
Affected Scottish Terriers typically start showing signs between 2 months and 6 years of age (median: 7 months), with 76% of the dogs having an onset of signs before 1 year of age. The progression of the disease is very slow (over several years) in most dogs (74%) and signs can sometimes even stabilize after 1 year of age. The severity of the disease is variable in this breed, but very few affected dogs are euthanized because of their CCD. 74,75 On histopathology polyglucosan body accumulation is identified in the molecular layer of the cerebellum. 76 The disease trait maps to a 4-Mb region on chromosome X (CFAX) in this breed, but an association to a variant has not yet been identified. 10

| Australian Working Kelpie
Onset of clinical signs in Australian Working Kelpies usually occurs between 5 and 12 weeks of age. Clinical severity is quite variable in this breed, ranging from mild nonprogressive ataxia and intention tremors to severe ataxia and seizures. 11,77,78 Histology in this breed reveals granuloprival degeneration, with secondary loss of Purkinje cells in severely affected individuals. The disease trait maps to a 3-Mb region on chromosome 3 (CFA3), but an association to a variant could not be identified. 11 More recently, another study found 2 more loci associated with CCD in this breed on CFA9 and CFA20, respectively.
Interestingly, dogs homozygous for the risk haplotype on CFA20 show clinical signs before 10 weeks of age, whereas dogs homozygous for the risk haplotype on CFA9 have later onset ataxia. 12 3 | SPINOCEREBELLAR DEGENERATIONS

| Late onset ataxia in the Jack and Parson Russell Terriers
This disease is most prominent in the Parson Russell Terrier breed but occurs in a few Jack Russell Terriers. Signs of dysfunction have an onset between 6 and 12 months of age, hence "late onset." Clinically,  reported to still be alive by 4 years of age. 16,86,90 The size and weight of the dog probably has an influence on its ability to cope with the severe ataxia, hence its ability to walk and the owner's decision to consider euthanasia.
Myokymia is seen in up to 70% to 75% of affected Jack and Par-  (Figure 4). Epileptic seizures occur in 12.5% to 50% of dogs with SAMS. 14,18,83,84 Brainstem auditory evoked recordings of these dogs show loss of waves III, IV, and V, as well as in most cases mildly increased latencies that is mainly expressed in the brain, spinal cord, inner ear, and kidneys. Several breed-specific variants have been described in dogs.
Phenotypic heterogeneity is well documented with KCNJ10 variants in dogs. 15,18,19,21,84  suggesting that at least 1 additional variant is segregating in the Jack and Parson Russell Terrier breeds. An additional KCNJ10 variant in the 3 0 -UTR region (a C-insertion with a predicted regulatory effect on the expression) was described in some of these unexplained cases, 90 but this variant was also homozygously present in a clinically healthy control dog of more than 4 years old.

| Belgian Malinois Shepherd and Bouvier des Ardennes
In both breeds, spinocerebellar degeneration can present with a spectrum of phenotypic severity, ranging from the classical SAMS phenotype 18,21 to a severe cerebellar phenotype reported as SDCA1. 19,21,89,92 In both phenotypes, the wide-based stance and gen-

| SCA8 in the Alpine Dachsbracke
This disease has an onset of signs when puppies start to move,

| Canine multiple system degeneration in the Kerry Blue Terrier and Chinese Crested dog
This disease is also called striatonigral and cerebello-olivary degeneration because of the extrapyramidal nuclei (olivary nuclei, substantia nigra, putamen, and caudate nuclei) degeneration next to the CCD.
This disease has an onset of signs between 9 weeks and 6 months of age. Dogs initially present a mild intention tremor and stiffness in thoracic limb gait, which progresses within 3 to 4 months to severe hypermetric ataxia, spasticity, truncal sway, wide-based stance, delayed postural reactions, and decreased menace response. Signs progress to akinesia, inability to stand and euthanasia by 1 to 2 years of age. 4,95-98 Cerebellar atrophy and T2W hyperintensity at the level of the caudate nuclei, putamen, and substantia nigra are visible on MRI in dogs affected for several weeks. 4,98 Macroscopic changes in advanced cases include a decreased cerebellar size (6%-9% of total brain weight) and necrosis of the caudate nuclei, putamen, and substantia nigra. Microscopically, an ischemic degeneration of Purkinje cells is seen first, followed by Purkinje cell and secondary granule cell loss. With chronicity, degeneration occurs in the

| SDCA2 in the Belgian Malinois Shepherd
Onset This gene encodes the ATPase Na + /K + transporting subunit beta 2 protein (ATP1B2), an Na + /K + ATPase from which the main role of which is to restore extracellular K + homeostasis after neuronal depolarization and is predominantly expressed in the cerebellum. 27

| CNS atrophy with cerebellar ataxia in the Belgian Malinois Shepherd
Clinical signs have an onset around 2 weeks of age and are rapidly progressive but of variable severity. They include intention tremors, truncal wobbling, increased muscle tone with short episodes of spastic fits, reduced swallow reflex, difficulties walking as well as a lower body weight gain compared with healthy littermates. Euthanasia is performed in most dogs around 4 weeks of age because of severe ataxia, even though 1 dog was able to reach 10 years of age. Total selenium concentration in the blood of affected puppies is 30% lower than in healthy lit- [His391Phefs*4])) frameshift variant was found to be associated with this disease. 29 The patatin-like phospholipase domain containing 8 protein (PNPLA8) contributes to mitochondrial function through its role in mitochondrial energy production. 99 The allele frequency in Australian Shepherds was found to be 4.7% in the studied sample from the population in France. 29

| Hereditary ataxia in Black Norwegian Elkhound
This disease has an onset of signs around 3 to 4 weeks of age and is  of signs (range: 6 months to 6.5 years). MRI demonstrates diffuse cerebellar atrophy ( Figure 9) and cerebrospinal fluid can show a mild increase in protein. 105 Brainstem auditory evoked recordings can show decreased amplitudes of waves I and II and increased inter-wave latency for waves III to V. 106 On histopathology, obvious cerebellar atrophy is present macroscopically (cerebellum to brain ratio: 5%-7% [normal 10%-12%]). 108   On histopathology, a pale granular cytoplasm (GM1-ganglioside) is present in neurons throughout the cerebral cortex, cerebellum, basal nuclei, brainstem, and spinal cord. 113 Numerous cytoplasmic vacuoles can also be found in selected cells of various organs as the eyes, lymph nodes, thymus, lungs, liver, pancreas, spleen, kidneys, adrenal glands, and ovaries. 114 (Table 1), which explains why they have such extensive consequences on the CNS of these dogs. This review aims to provide an overview of currently described hereditary ataxia syndromes. As new diseases and genetic variants are constantly being recognized and discovered, this classification proposal will likely need to evolve and be updated in time, as our understanding of diseases continuously progresses.

ACKNOWLEDGMENT
No funding was received for this study.