Received: 22 July 2022 Accepted: 17 November 2022 DOI: 10.1111/jvim.16594 STANDARD ARTICLE Spinocerebellar ataxia in the Bouvier des Ardennes breed is caused by a KCNJ10 missense variant Kimberley Stee 1 | Mario Van Poucke 2 | Martí Pumarola 3 | Lise Geerinckx 4 | Iris Van Soens 5 | Sofie F. M. Bhatti 1 | Luc Peelman 2 | Ine Cornelis 1 1 Small Animals Department, Faculty of Veterinary Medicine, Ghent University, Abstract Merelbeke, Belgium Background: In Belgian Malinois, a KCNJ10 variant causes progressive 2 Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent spinocerebellar degeneration. University, Merelbeke, Belgium Hypothesis/Objectives: Describe the clinical, diagnostic, pathological and genetic 3 Mouse and Comparative Pathology Unit, features of spinocerebellar degeneration in the Bouvier des Ardennes breed. Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Universitat Animals: Five affected Bouvier des Ardennes puppies with spinocerebellar ataxia Autònoma de Barcelona, Bellaterra (SCA), 8 healthy related dogs, and 63 healthy unrelated Bouvier des Ardennes. (Barcelona), Spain 4 Methods: Sequential case study. Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Results: Clinical signs started at 6 weeks of age in 1 puppy with severe signs of cere- Medicine, University of Ghent, Ghent, Belgium 5 bellar disease, and at 7 to 10 weeks of age in the 4 remaining puppies with milder Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, signs of spinocerebellar disease. The first puppy displayed severe intention tremors University of Liège, Liège, Belgium and rapidly progressive generalized hypermetric ataxia, whereas the 4 others devel- Correspondence oped a milder progressive SCA. Euthanasia after progression to nonambulatory status Kimberley Stee, Small Animals Department, was performed by 8 weeks of age in the first puppy, and before 11 months of age in Faculty of Veterinary Medicine, Ghent University, 133 Salisburylaan, 9820 the 4 remaining puppies. Histopathology revealed cerebellar spongy degeneration Merelbeke, Belgium. and a focal symmetrical demyelinating myelopathy. All cases were homozygous for Email: kimberley.stee@ugent.be 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 cere- bellar ataxia (SDCA) 1 in Belgian Malinois dogs. All sampled parents were heterozy- gous 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 Abbreviations: ATP1B2, ATPase Na+/K+ transporting beta-2 polypeptide; BEAR, brainstem auditory evoked responses; CAPN1, calpain 1; DAB, diaminobenzidine; EDTA, ethylenediaminetetraacetic acid; GFAP, glial fibrillary acidic protein; GRM1, glutamate receptor metabotropic 1; HE, hematoxylin eosin; IHC, immunohistochemistry; ITPR1, inositol 1,4,5-triphosphate receptor, type 1; JRT, Jack Russell Terrier; KCNIP4, potassium channel-interacting protein 4; KCNJ10, potassium inwardly rectifying channel subfamily J member 10; LFB, luxol fast blue; MRI, magnetic resonance imaging; NF200, total neurofilaments; OMIA, Online Mendelian Inheritance in Animals; OMIM, Online Mendelian Inheritance in Man; PBS, phosphate- buffer saline; PRT, Parson Russell Terrier; SAMS, spinocerebellar ataxia with (or without) myokymia, seizures or both; SCA, spinocerebellar ataxia; SCN8A, sodium voltage gated channel, alpha subunit 8; SDCA, spongy degeneration and cerebellar ataxia; SLC12A6, solute carrier family 12 (potassium/chloride transporter), member 6. Kimberley Stee and Mario Van Poucke contributed equally to this manuscript. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine. 216 wileyonlinelibrary.com/journal/jvim J Vet Intern Med. 2023;37:216–222. STEE ET AL. 217 present with a spectrum of severity grades, ranging from severe cerebellar to milder spinocerebellar signs. KEYWORDS demyelinating myelopathy, hypermetric ataxia, intention tremors, SAMS, SDCA, spongy degeneration 1 | I N T RO DU CT I O N A 1,5 T magnetic resonance imaging (MRI) of the caudal cranial fossa, cervical and cranial thoracic spinal cord was performed in dog 3 Spinocerebellar ataxia (SCA) with myokymia, seizures or both (SAMS) (Siemens Magnetom, Siemens Healthcare). A video from 1 more is a form of hereditary ataxia reported in several breeds, including the related 5-month old Bouvier des Ardennes female puppy was Jack Russell Terrier (JRT),1 Parson Russell Terrier (PRT),2 Smooth assessed (dog 5). 3,4 5 Haired and Toy Fox Terriers, Patterdale Terrier and Belgian Malinois Shepherd (also termed “SeSAME/EAST homologue” in this breed).6 This syndrome is typically caused by variants in the KCNJ10 2.2 | Pathological examination gene, which encodes for a voltage-gated potassium channel that is highly expressed in the brain, spinal cord, inner ear and kidneys.7 Dogs A full necropsy was performed in dogs 1 and 2, immediately following affected with SAMS can present with SCA only, or alternatively have euthanasia. Samples from the brain, spinal cord, femoral nerve roots combinations of SCA, myokymia, neuromyotonia and epileptic sei- and nerves as well as skeletal muscle from the quadriceps were col- zures.2,4-6 Three different canine KCNJ10 variants have been lected, formalin-fixed paraffin-embedded and stained with 2,4 described so far, the c.627C>G variant, causal for SAMS in the JRT, hematoxylin-eosin (HE) as well as luxol fast blue (LFB) and Nissl stain. PRT, Smooth Haired and Toy Fox Terriers as well as the Patterdale Immunohistochemistry (IHC) was performed using neuronal (total Terrier5 breeds, the g.22141027insC variant,8 also causal for SAMS in neurofilaments, NF200) and glial (glial fibrillary acidic protein [GFAP] the JRT and PRT breeds, and the c.986T>C variant in the Belgian for astrocytes and Iba1 for microglia) markers. Technical details are 6 Malinois breed. In the Belgian Malinois, this genetic variant causes available in Video S1. Sections of 5 μm thicknesses were mounted on SAMS6 as well as spongy degeneration and cerebellar ataxia (SDCA) capillary glass slides, deparaffinized and rinsed with water. Primary 1,9,10 which likely represent different severity grades of the same dis- antibodies used are summarized in Table 1. Sections were pretreated ease. In this report, we describe the clinical, diagnostic, pathological in 10 mM citrate buffer pH 6.0 for 20 minutes at 96 C, cooled and genetic features of 5 related Bouvier des Ardennes puppies with 30 minutes at room temperature and rinsed in phosphate-buffer inherited SCA. saline (PBS). Previously, sections were treated 35 minutes with 3% peroxidase to block endogenous peroxydase activity. Nonspecific binding was blocked by normal goat or rabbit serum 30% diluted with 2 | MATERIAL AND METHODS PBS for 1 hour. Samples were incubated with primary antibodies 40 minutes at room temperature. Sections were therefore rinsed with 2.1 | Clinical examination PBS and incubated during 40 minutes with a labeled polymer accord- ing to the manufacturer's instructions (mouse kit K4007 or rabbit kit This case series was designed as a sequential hospital-based case K011, Dakocytomation). For Iba1, the incubation was performed using study with follow-up. Two 7-week old male intact Bouviers des a secondary goat anti-rabbit antibody and then with a standard ABC Ardennes siblings were presented at the Small Animal Department of Peroxidase staining kit (Thermo Scientific, kit number 32020) diluted the Faculty of Veterinary Medicine of Ghent University for an uncoor- with PBS for 1 hour at room temperature. Staining was completed by dinated gait since the age of 6 weeks (dog 1) and 7 weeks (dog 2). a 10-minute incubation with 3,30 -diaminobenzidine (DAB) and coun- Two related Bouviers des Ardennes siblings (a male and a female) terstained in hematoxylin for 3 seconds. The positive control used were presented at the Small Animal Clinic of the Faculty of Veterinary was normal canine brain tissue including gray matter and white mat- Medicine of the University of Liège (dogs 3 and 4) at 4 and 6 months ter. In all experiments, negative controls were done by omitting the of age respectively. A clinical and neurological examination was per- primary antibody. formed in all 4 dogs. A complete bloodwork including electrolytes, Toxoplasma gondii and Neospora caninum serologies was performed in dogs 1 and 4, and brainstem auditory evoked responses (BAER) were 2.3 | Genetic analysis recorded in dogs 1 and 2. The BAER test was performed under seda- tion with a commercially available electrophysiological unit (Natus An EDTA blood sample were obtained from the 5 affected puppies as Synergy UltraPro, Acertys Healthcare NV, Aartselaar, Belgium). well as 8 healthy closely related dogs (Figure 1), and from 63 healthy 218 STEE ET AL. TABLE 1 Overview of described canine variants potentially involved in the phenotype of the affected Bouviers des Ardennes Gene Gene OMIM OMIA ID12 ID symbol Variant Reference gene ID13 002110-9615 489479 ATP1B2a NC_006587.3(XM_546597.6):c.130_131insLT796559.1: Mauri et al, 201714 182331 g.50-276 001820-9615 483745 CAPN1a NC_006600.3(XM_540866.5):c.344G>A Forman et al, 201315 114 220 b 000078-9615 484024 GRM1 NC_006583.3(XM_005615490.3):c.2331_2332ins62bp Zeng et al, 201116 604473 b 17 002097-9615 476548 ITPR1 NC_006602.3:g.12880740CTT[7_651] Forman et al, 2015 147265 002240-9615 479129 KCNIP4b NC_006585.3(XM_005618660.3):c.436T>C Jenkins et al, 202018 608182 a 2 002089-9615 488635 KCNJ10 NC_006620.3(XM_545752.6):c.627C>G Gilliam et al, 2014 602208 002089-9615 488635 KCNJ10a NC_006620.3:g. 22141027_22141028insC Gast et al, 20168 602208 a 002089-9615 488635 KCNJ10 NC_006620.3(XM_545752.6):c.986T>C Van Poucke et al, 602208 20176 002194-9615 477604 SCN8Aa NC_006609.3(XM_022411522.1):c.4898G>T Letko et al, 201919 600702 002279-9615 478239 SLC12A6a NC_006612.3(XM_014109414.2): Van Poucke et al, 604878 c.178_181delinsCATCTCACTCAT 201920 a Known canine variants that were hypothesized to possibly account for the different phenotypes between the cases. b Additional known canine variants that could cause the earlier onset of abnormal gait observed in some of the cases. F I G U R E 1 Pedigree information of the affected Bouvier des Ardennes family, drawn with the kinship2 package in RStudio.11 Squares represent males, circles females, black icons sampled dogs, gray icons nonsampled dogs, shaded icons cases, nonshaded icons healthy dogs, strikethrough icons deceased dogs. The KCNJ10 XM_545752.6:c.986T>C genotype is added for all sampled dogs Belgian Bouviers des Ardennes with owner consent. Given the small pelvic limbs, severe intention tremors, bilateral absent patellar reflexes size of the Bouvier des Ardennes sample in Belgium, these 63 dogs and menace responses in dog 1, suggestive for cerebellar as well as are almost all related to some extent to the affected families. Geno- L4-L6 spinal cord segment or femoral nerve roots or nerves dysfunction. typing assays for variants known to be able to cause hereditary ataxia, Dog 2 showed a wide-based stance as well as bilateral absent patellar or affect the earlier onset and increased severity of the clinical signs reflexes and menace responses but only minimal ataxia (Video S1). as observed in some of the cases, were performed as described in Dog 1 was humanely euthanized a few days later because of the rapid their respective papers (Table 1). The complete KCNJ10 coding progression and severity of his clinical signs. By 4 months of age, the clin- sequence was also screened for new onset-effecting variants in the ical signs of dog 2 had progressed to a clear hypermetric and dysmetric 6 affected dogs via Sanger sequencing as described previously. generalized ataxia worse on the hind limbs, truncal sway and mild inten- tion tremors (Video S2), which were similar to the clinical signs of dogs 3 (4 months of age) and 4 (6 months of age) at presentation, and on the 3 | RESULTS video of dog 5 (5 months of age). These clinical signs were suggestive of spinocerebellar as well as L4-L6 spinal cord segment or femoral nerve 3.1 | Clinical features roots or nerves dysfunction. The owners of dog 4 reported a 20-minute episode of rigidity and tremors in lateral recumbency with preserved Abnormalities were not detected on general clinical examination in all consciousness which caused the dog to develop a life threatening hyper- 4 examined puppies. Neurological examination at 7 weeks of age thermia of 42 C. Dogs 2 to 5 progressed to nonambulatory status revealed a severe generalized borderline ambulatory hypermetric ataxia because of their pronounced ataxia (Video S3) between 7 and 11 months with pronounced truncal sway, mildly decreased proprioception in both of age, and were therefore euthanized. STEE ET AL. 219 F I G U R E 2 BAER recordings from dog 1 showing disappearance of waves III/VI and V (A) (B) (C) (D) F I G U R E 3 Histopathology of dog 1 at 8 weeks of age: (A and B) HE stain of the cerebellar cortex, vacuoles are restricted mainly to granular layer or intraneuronal in Purkinje cells; (C) GFAP immunopositives reactive astrocytes in the cerebellar cortex; (D) LFB stain of the spinal cord shows pale areas in the lateral and ventral funiculi CBC and serum biochemistry including electrolytes (Na2+, K+, cerebellar nuclei. Purkinje cells were decreased in number and the  Cl , Ca2+) did not reveal any abnormalities, and N. caninum and remaining ones had a degenerating appearance. Associated gliosis was T. gondii serologies were negative in dogs 1 and 4. BAER recordings present and confirmed by IHC in all affected areas. A few differences revealed loss of waves III/VI and V in dogs 1 and 2 (Figure 2). The were noticed between these 2 dogs. In dog 1 (Figure 3), microgliosis MRI in dog 3 did not reveal any abnormalities at the level of the cere- was moderate and restricted to cerebellar spongiotic areas. Spinal spi- bellum nor the cervical spinal cord. nocerebellar tracts were also spongiotic, losing myelin and axonal components. The femoral ventral nerve roots showed a proliferative astrocytosis associated with spongiotic changes. 3.2 | Pathological features In dog 2 (Figure 4), Purkinje cells were diminished in number showing mild intracellular vacuoles. Degeneration of some neuronal Necropsy was performed on dog 1 and 2 at respectively 8 weeks and bodies in the cerebellar nuclei, as well as spheroids in the cerebellar 7 months of age. No macroscopic abnormalities were found. On histo- cortical white matter were evident. Cerebellar peduncles, pyramids pathology, cerebellar spongy degeneration as well as a bilateral sym- and pyramidal tracts, trapezoid body and associated tracts showed metrical demyelinating myelopathy targeting the dorsolateral marked spongiotic changes. All funiculi were affected showing a spinocerebellar tracts was found in both dogs. Middle to small, differ- marked spongiotic pattern mainly affecting the spinocerebellar, cune- ent sized vacuoles apparently empty or containing cellular debris, ate, and corticospinal tracts. In these areas, myelin vacuolation and were mainly found in the granular layer of the cerebellum, intracellu- loss and a markedly diminished number of axons were detected. A larly located in Purkinje cells and in the neuronal bodies of the strong and diffuse reactive microgliosis with microglial nodule 220 STEE ET AL. (A) (B) (C) (D) (E) (F) F I G U R E 4 Histopathology of dog 2 at 7 months of age: (A and B) HE stain shows spongiosis in the cortical white matter, and infiltrating the cortical cerebellar granular layer. (C) IHC for Iba1 shows reactive microglial cells associated to spongiotic cerebellar area; (D) LFB stain of the spinal cord shows marked pallor of lateral and ventral funiculi. (E and F) IHC of the dorsal spinocerebellar tract of the cervical spinal cord. Absence or decreased immune positivity for NF200 (E) evidence loose of axonal component, some spheroids are also present. Marked immune positivity for Iba1 (F) in that area corresponding to reactive microgliosis is also evident T A B L E 2 Overview of the KCNJ10 c.986T>C genotype T/T T/C C/C Total C-allele frequency XM_545752.6:c.986T>C genotypes in Cases 0 0 5 5 100% the 5 affected dogs, 8 healthy family Healthy (family members) 4 4 0 8 25% members and 63 other healthy Belgian Healthy (other) 44 19 0 63 15% Bouviers des Ardennes, as well as the calculated C-allele frequencies formation was also associated with all spinal spongiotic areas. The 4 | DI SCU SSION femoral ventral nerve roots showed moderate to mild astrocytosis associated to spongiotic areas. Two clinical presentations were seen in our 5 affected dogs. Dog 1 had very severe early-onset and fast progressing cerebellar signs, which much resembles SDCA1 in Belgian Malinois dogs. SDCA1 3.3 | Genetics is characterized by severe generalized cerebellar (hypermetric) ataxia, stumbling and falling as well as severe intention tremors, with an All 5 affected dogs were homozygous for the KCNJ10 onset at 4 to 8 weeks of age and euthanasia of all puppies by 13 to XM_545752.6:c.986T>C missense variant, previously reported to 17 weeks of age. Also the histopathological findings of dog 1 are simi- be pathogenic for SAMS and SDCA1 in the Belgian Malinois lar to the ones described in SDCA1, where the main lesions are local- dog breed.6,10 All sampled parents were heterozygous and none of ized in the cerebellum and include vacuolization of the cerebellar the healthy dogs were homozygous for this recessive variant nuclei, Purkinje cells, granular cell layer and white matter, whereas (Figure 1 and Table 2). This variant has an estimated allele fre- much less severe or no lesions are found in the spinal cord.9,10 quency of 15% in the Belgian Bouviers des Ardennes study sample Dogs 2 to 5 presented with much milder signs at onset, and (Table 2). showed a much slower progression, more resembling SAMS (also No additional variant was found in the KCNJ10 coding sequence termed “SeSAME/EAST homologue” in this breed) which was also of any of the 5 affected cases. No other tested variant known to be described in the Belgian Malinois breed. This disease is characterized able to cause the observed spinocerebellar degeneration, or affect the by generalized (hypermetric) SCA with an onset at 6 to 8 weeks of earlier onset and increased severity of the clinical signs as observed in age, a consistent feature in these patients is the absence of patellar some of the cases (Table 1), was present in any of the 5 cases. reflexes. In some cases, the SCA is associated with a variable STEE ET AL. 221 combination of myokymia, neuromyotonia and epileptic seizures. No DATA AVAILABILITY STAT EMEN T myokymia nor seizures were seen in our Bouvier des Ardennes Some or all data generated or analyzed during this study are patients, even though the description of the episode of rigidity and included in this published article or in the data repositories listed in hyperthermia with preserved consciousness in dog 4 could have been References. compatible with an episode of neuromyotonia. In the Belgian Malinois dog, progression of the ataxia results in nonambulatory status and OF F-LABEL ANTIMI CROBIAL DECLARATION euthanasia before 6 months of age in most cases, which is a bit earlier Authors declare no off-label use of antimicrobials. than in our patients (7-11 months of age). The predominant histopath- ological lesion found in dogs with SAMS is a bilateral myelopathy with INSTITU TIONAL ANIMAL C AR E AND USE COMMITTEE a predominant axonopathy and myelin vacuolization, most severe in (IACUC) OR OTHER APPROVAL DECLARATION the corticospinal tracts, whereas similar but less severe lesions are Approval was granted by the local ethical (Faculty of Veterinary Medi- found in the cerebellar white matter,6 which resembles the histopath- cine, Ghent University, Belgium) and deontological (Federal Public ological findings of dog 2. Service Health, Food Chain Safety and Environment, Brussels, SDCA1 and SAMS are believed to be part of a continuous spec- Belgium) committees (EC2017/86) for the use of all left-over samples trum of severity grade of one same disease, as demonstrated in this study. genetically by the fact that they are caused by the same KCNJ10 c.986T>C (p.(Leu329Pro)) variant in the Belgian Malinois breed. HUMAN E THICS APPROVAL DECLARATION The KCNJ10 c.986T>C variant was considered a strong genetic Authors declare human ethics approval was not needed for this study. candidate after recognition of these different severity phenotypes in our Bouvier des Ardennes patients. Phenotypic variability has OR CID been reported in dogs and humans with KCNJ10 vari- Kimberley Stee https://orcid.org/0000-0002-5208-2605 ants.1,6,9,10,21-23 In humans, the presence of residual channel func- Mario Van Poucke https://orcid.org/0000-0002-4316-8984 tion or compensatory mechanisms in certain organs have been shown to contribute to this phenotypic heterogeneity within RE FE RE NCE S KCNJ10 variants.24 It was therefore hypothesized that this differ- 1. Bhatti SF, Vanhaesebrouck AE, Van Soens I, et al. Myokymia and ent phenotypic severity could be influenced by a combination of neuromyotonia in 37 Jack Russell terriers. Vet J. 2011;189: the KCNJ10 variant with another variant. Testing of several other 284-288. variants known to cause hereditary ataxia and resequencing the 2. Gilliam D, O'Brien DP, Coates JR, et al. A homozygous KCNJ10 muta- tion in Jack Russell terrier and related breeds with spinocerebellar complete KCNJ10 coding sequence was therefore conducted in our ataxia with myokymia, seizures or both. J Vet Intern Med. 2014;28: 5 affected dogs, but no additional variant was found. 871-877. The Bouvier des Ardennes breed was well established in Belgium 3. Rohdin C, Lüdtke L, Wohlstein P, et al. New aspects of hereditary in the 19th century. The breed almost became extinct at the begin- ataxia in smooth-haired fox terriers. Vet Rec. 2010;166:557-560. ning of the 20th century wherefore the current, limited Bouvier des 4. Rohdin C, Gilliam D, O'Leary CA, et al. A KCNJ10 mutation previously identified in the Russell group of terriers also occurs in smooth-haired Ardennes population (estimated to be only few hundreds individuals fox terriers with hereditary ataxia an in related breeds. Acta Vet in the world [unpublished data, Club Belge du Bouvier des Flandres et Scand. 2015;57:26. des Ardennes]) originates from just a few individuals that were discov- 5. Fisher C, Liebel F. Neuromyotonia, myokymia and spinocerebellar ered in Belgian farms in 1985.25,26 Investigating the breeding schemes ataxia in two Patterdale terrier littermates. Vet Rec. 2022; 10(4):e439. 6. Van Poucke M, Stee K, Bhatti SFM, et al. The novel homozygous revealed that some Bouvier des Flandres and Belgian Malinois dogs KCNJ10 c.986T>C (p.(Leu329Pro)) variant is pathogenic for the were used to strengthen the Bouvier des Ardennes blood lines in the SeSAME/EAST homologue in Malinois dogs. 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