Equine neuroaxonal dystrophy/degenerative myeloencephalopathy in Gypsy Vanner horses.

BACKGROUND: Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a neurodegenerative disease that primarily affects young, genetically predisposed horses that are deficient in vitamin E. Equine NAD/EDM has not previously been documented in Gypsy Vanner horses (GVs). OBJECTIVES: To evaluate: (1) the clinical phenotype, blood vitamin E concentrations before and after supplementation and pedigree in a cohort of GV horses with a high prevalence of neurologic disease suspicious for eNAD/EDM and (2) to confirm eNAD/EDM in GVs through postmortem evaluation. ANIMALS: Twenty-six GVs from 1 farm in California and 2 cases from the Midwestern U.S. METHODS: Prospective observational study on Californian horses; all 26 GVs underwent neurologic examination. Pre-supplementation blood vitamin E concentration was assessed in 17- GVs. Twenty-three were supplemented orally with 10 IU/kg of liquid RRR-alpha-tocopherol once daily for 28 days. Vitamin E concentration was measured in 23 GVs after supplementation, of which 15 (65%) had pre-supplementation measurements. Two clinically affected GVs from California and the 2 Midwestern cases had necropsy confirmation of eNAD/EDM. RESULTS: Pre-supplementation blood vitamin E concentration was ≤2.0 µg/mL in 16/17 (94%) of GVs from California. Post-supplementation concentration varied, with a median of 3.39 µg/mL (range, 1.23-13.87 µg/mL), but only 12/23 (52%) were normal (≥3.0 µg/mL). Normalization of vitamin E was significantly associated with increasing age (P = .02). Euthanized horses (n = 4) had eNAD/EDM confirmed at necropsy. CONCLUSIONS AND CLINICAL IMPORTANCE: GVs could have a genetic predisposition to eNAD/EDM. Vitamin E supplementation should be considered and monitored in young GVs.

Measurement of 8-hydroxy-2'-deoxyguanosine in serum and cerebrospinal fluid of horses with neuroaxonal degeneration and other causes of proprioceptive ataxia.

BACKGROUND: Eight-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative damage evaluated in human neurodegenerative disease, has potential to correlate with postmortem diagnosis of neuroaxonal dystrophy/degenerative myeloencephalopathy (NAD/DM) in horses. HYPOTHESIS: We hypothesized that 8-OHdG will be higher in CSF and serum from NAD/DM horses compared with horses with other neurologic diseases (CVSM, EPM) and a control group of neurologically normal horses. We also hypothesized that 8-OHdG will be higher in CSF compared with serum from NAD/DM horses. ANIMALS: Fifty client-owned horses with postmortem diagnoses: 20 NAD/DM, 10 CVSM, 10 EPM, and 10 control horses. Serum and CSF samples were obtained between November 2010 and March 2022. METHODS: Case-control study using biobanked samples was performed and commercial competitive ELISA kit (Highly Sensitive 8-OHdG Check ELISA) utilized. Concentration of 8-OHdG was quantitated in both CSF and serum and compared between groups. RESULTS: No correlation was established between the measures of 8-OHdG in serum and CSF and group. CSF median [8-OHdG] for NAD/DM was 169.9 pg/mL (IQR25-75 : 67.18-210.6), CVSM 157.1 pg/mL (IQR25-75 : 132.1-229.1), EPM 131.4 pg/mL (IQR25-75 : 102.1-193.2), and control 149.8 pg/mL (IQR25-75 : 113.3-196.4). Serum median [8-OHdG] for NAD/DM was 130 pg/mL (IQR25-75 : 51.73-157.2), CVSM 125.8 pg/mL (IQR25-75 : 62.8-170.8), EPM 120.6 pg/mL (IQR25-75 : 87.23-229.7), and control 157.6 pg/mL (IQR25-75 : 97.15-245.6). Poisson regression analysis showed no difference established once confounding variables were considered. CONCLUSIONS: Eight-OHdG did not aid in antemortem diagnosis of NAD/DM in this cohort of horses. At the time of diagnosis horses with NAD/DM do not have ongoing oxidative stress.

Clinical and histopathological features in horses with neuroaxonal degeneration: 100 cases (2017-2021).

BACKGROUND: Adult horses with proprioceptive ataxia and behavior changes that have histologic lesions consistent with neurodegenerative disease have been increasingly recognized. HYPOTHESIS/OBJECTIVES: Describe the history, clinical findings and histopathologic features of horses presented to a referral institution with neuroaxonal degeneration. ANIMALS: One hundred horses with a necropsy diagnosis of neuroaxonal degeneration compatible with neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM). METHODS: Retrospective study of horses presented to the University of Pennsylvania, New Bolton Center, between 2017 and 2021 with a necropsy diagnosis of eNAD/EDM. RESULTS: Affected horses had a median age of 8 years (range, 1-22), and the majority were Warmbloods (72). Sixty-eight horses had behavioral changes, and all 100 had proprioceptive ataxia (median grade, 2/5). Fifty-seven horses had abnormal findings on cervical vertebral radiographs, and 14 had myelographic findings consistent with compressive myelopathy. No antemortem diagnostic test results were consistently associated with necropsy diagnosis of neurodegenerative disease. All 100 horses had degenerative lesions characteristic of eNAD in the brainstem gray matter, and 24 had concurrent degenerative features of EDM in the spinal cord white matter. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical and histopathologic findings in this large group of horses with neurodegenerative disease were most consistent with eNAD/EDM, but with a different signalment and clinical presentation from earlier descriptions. The increasing occurrence of neurodegenerative disease in horses and the safety risk posed emphasize the importance of focused research in affected horses.

Genetic polymorphisms in vitamin E transport genes as determinants for risk of equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with vitamin E deficiency. In humans, polymorphisms in genes involved in vitamin E uptake and distribution determines individual vitamin E requirements. HYPOTHESIS/OBJECTIVES: Genetic polymorphisms in genes involved in vitamin E metabolism would be associated with an increased risk of eNAD/EDM in Quarter Horses (QHs). ANIMALS: Whole-genome sequencing: eNAD/EDM affected (n = 9, postmortem [PM]-confirmed) and control (n = 32) QHs. VALIDATION: eNAD/EDM affected (n = 39, 23-PM confirmed) and control (n = 68, 7-PM confirmed) QHs. Allele frequency (AF): Publicly available data from 504 horses across 47 breeds. METHODS: Retrospective, case control study. Whole-genome sequencing was performed and genetic variants identified within 28 vitamin E candidate genes. These variants were subsequently genotyped in the validation cohort. RESULTS: Thirty-nine confirmed variants in 15 vitamin E candidate genes were significantly associated with eNAD/EDM (P < .01). In the validation cohort, 2 intronic CD36 variants (chr4:726485 and chr4:731082) were significantly associated with eNAD/EDM in clinical (P = 2.78 × 10-4 and P = 4 × 10-4 , respectively) and PM-confirmed cases (P = 6.32 × 10-6 and 1.04 × 10-5 , respectively). Despite the significant association, variant AFs were low in the postmortem-confirmed eNAD/EDM cases (0.22-0.26). In publicly available equine genomes, AFs ranged from 0.06 to 0.1. CONCLUSIONS AND CLINICAL IMPORTANCE: Many PM-confirmed cases of eNAD/EDM were wild-type for the 2 intronic CD36 SNPs, suggesting either a false positive association or genetic heterogeneity of eNAD/EDM within the QH breed.

Cerebrospinal fluid and serum proteomic profiles accurately distinguish neuroaxonal dystrophy from cervical vertebral compressive myelopathy in horses.

BACKGROUND: Cervical vertebral compressive myelopathy (CVCM) and equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) are leading causes of spinal ataxia in horses. The conditions can be difficult to differentiate, and there is currently no diagnostic modality that offers a definitive antemortem diagnosis. OBJECTIVE: Evaluate novel proteomic techniques and machine learning algorithms to predict biomarkers that can aid in the antemortem diagnosis of noninfectious spinal ataxia in horses. ANIMALS: Banked serum and cerebrospinal fluid (CSF) samples from necropsy-confirmed adult eNAD/EDM (n = 47) and CVCM (n = 25) horses and neurologically normal adult horses (n = 45). METHODS: . A subset of serum and CSF samples from eNAD/EDM (n = 5) and normal (n = 5) horses was used to evaluate the proximity extension assay (PEA). All samples were assayed by PEA for 368 neurologically relevant proteins. Data were analyzed using machine learning algorithms to define potential diagnostic biomarkers. RESULTS: Of the 368 proteins, 84 were detected in CSF and 146 in serum. Eighteen of 84 proteins in CSF and 30/146 in serum were differentially abundant among the 3 groups, after correction for multiple testing. Modeling indicated that a 2-protein test using CSF had the highest accuracy for discriminating among all 3 groups. Cerebrospinal fluid R-spondin 1 (RSPO1) and neurofilament-light (NEFL), in parallel, predicted normal horses with an accuracy of 87.18%, CVCM with 84.62%, and eNAD/EDM with 73.5%. MAIN LIMITATIONS: Cross-species platform. Uneven sample size. CONCLUSIONS AND CLINICAL IMPORTANCE: Proximity extension assay technology allows for rapid screening of equine biologic matrices for potential protein biomarkers. Machine learning analysis allows for unbiased selection of highly accurate biomarkers from high-dimensional data.

Vitamin E depletion is associated with subclinical axonal degeneration in juvenile horses.

BACKGROUND: Phosphorylated neurofilament heavy, a marker of neuroaxonal damage, is increased in horses with equine neuroaxonal dystrophy. However, the temporal dynamics of this biomarker during the post-natal risk period are not understood. OBJECTIVE: To measure serum and cerebrospinal fluid phosphorylated neurofilament heavy concentrations in juvenile foals across the post-natal window of susceptibility for equine neuroaxonal dystrophy. STUDY DESIGN: Case-control in vivo experimental study. METHODS: Concentrations of phosphorylated neurofilament heavy were measured using frozen serum and cerebrospinal fluid collected from 13 foals raised in a vitamin E deficient environment from 1 to 6 months of age. Four of these foals were produced by equine neuroaxonal dystrophy-affected dams, developed clinical signs consistent with equine neuroaxonal dystrophy and had a diagnosis confirmed by histopathology. The remaining nine foals, produced by healthy mares, were vitamin E depleted and remained clinically healthy. An additional cohort of foals, produced by healthy mares, were supplemented with vitamin E (α-tocopherol; α-TOH) from birth and sampled similarly. RESULTS: Serum α-TOH concentrations were significantly higher in vitamin E supplemented healthy foals. Serum phosphorylated neurofilament heavy concentrations did not differ significantly between groups at any time point. Cerebrospinal fluid phosphorylated neurofilament heavy concentrations increased with age in healthy vitamin E depleted foals (p < 0.001); an effect that was not observed in healthy vitamin E supplemented foals. MAIN LIMITATIONS: A genetically susceptible cohort supplemented with vitamin E was not available for comparison. CONCLUSION: We demonstrate that vitamin E depletion may elevate cerebrospinal fluid phosphorylated neurofilament heavy in otherwise healthy juvenile foals by 6 months of age. We highlight an important cofactor to consider when interpreting cerebrospinal fluid phosphorylated neurofilament heavy concentrations in juvenile horses.

Equine Neuroaxonal Dystrophy and Degenerative Myeloencephalopathy.

Neuroaxonal degenerative disease in the horse is termed equine neuroaxonal dystrophy (eNAD), when pathologic lesions are localized to the brainstem and equine degenerative myeloencephalopathy (EDM) and degenerative changes extend throughout the spinal cord. Both pathologic conditions result in identical clinical disease, most commonly characterized by the insidious onset of ataxia during early development. However, later onset of clinical signs and additional clinical features, such as behavior changes, is also observed. A definitive diagnosis of eNAD/EDM requires histologic evaluation of the caudal medulla and cervicothoracic spinal cord. Strong evidence has suggested that eNAD/EDM is an inherited disorder and there seems to be a role for vitamin E acting as an environmental modifier to determine the overall severity of the phenotype of horses affected with eNAD/EDM.

Serum and cerebrospinal fluid phosphorylated neurofilament heavy protein concentrations in equine neurodegenerative diseases.

BACKGROUND: Currently, there is little information regarding the concentrations of phosphorylated neurofilament heavy protein (pNfH) in the serum and cerebrospinal fluid (CSF) of horses with neurodegenerative diseases. Specifically, pNfH concentrations have not yet been evaluated in horses with equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM). OBJECTIVES: To determine pNfH concentrations using a commercial enzyme-linked immunosorbent assay (ELISA) in serum and CSF from control horses and horses with eNAD/EDM, cervical vertebral compressive myelopathy (CVCM) and Shivers. STUDY DESIGN: Case-control study using biobanked samples from diseased horses and prospective or biobanked samples from control horses. METHODS: The pNfH ELISA was performed on samples from horses diagnosed with eNAD/EDM (n = 64), CVCM (n = 26) and Shivers (n = 9) and 51 neurologically normal control horses. RESULTS: Median and 95% confidence interval (CI) serum pNfH concentrations in control, CVCM, and eNAD/EDM horses were 0.08 ng/mL (0.07-0.15), 0.07 ng/mL (0.07-0.15) and 0.07 ng/mL (0.07-1.13), respectively. Serum pNfH concentrations were below the limit of detection (<0.07 ng/mL) for all Shivers horses. CSF pNfH concentrations in control, CVCM-, eNAD/EDM- and Shivers-affected horses were 1.26 ng/mL (1.06-1.5), 3.07 ng/mL (1.15-29.9), 1.78 ng/mL (1.5-2.28) and 1.39 ng/mL (0.74-3.89), respectively. CSF pNfH concentrations were significantly higher in CVCM (P = .001) and eNAD/EDM (P  = .01) affected horses compared to control horses. Serum pNfH concentrations >1 ng/mL were significantly associated with eNAD/EDM (P = .01) with only 12% sensitivity but 99% specificity. CSF pNfH concentrations >3 ng/mL were significantly associated with CVCM (P = .0002), with 50% sensitivity and 86% specificity. MAIN LIMITATIONS: A limited number of control horses tested were <1 year of age. CONCLUSIONS: Serum pNfH concentrations are specifically increased (>1 ng/mL) in some horses with eNAD/EDM. Increased CSF pNfH concentrations (>3 ng/mL) can be observed with eNAD/EDM or CVCM.

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. HYPOTHESIS/OBJECTIVES: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses. ANIMALS: Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses. METHODS: The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog. RESULTS: Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

Postmortem diagnoses of spinal ataxia in 316 horses in California.

OBJECTIVE: To determine period prevalences of postmortem diagnoses for spinal cord or vertebral column lesions as underlying causes of ataxia (spinal ataxia) in horses. ANIMALS: 2,861 client-owned horses (316 with ataxia [ataxic group] and 2,545 without ataxia [control group]). PROCEDURES: The medical records database of the University of California-Davis Veterinary Medical Teaching Hospital was searched to identify horses necropsied between January 1, 2005, and December 31, 2017. Results were compared between the ataxic and control groups and between various groups of horses in the ataxic group. Period prevalences were determined for the most common causes of ataxia. RESULTS: 2,861 horses underwent full necropsy, and the period prevalences for the most common definitive diagnoses for ataxia were 2.7% (77/2,861) for cervical vertebral compressive myelopathy (CVCM), 1.3% (38/2,861) for equine neuroaxonal dystrophy or equine degenerative myeloencephalopathy (eNAD-EDM), and 0.9% (25/2,861) for trauma; the period prevalence of ataxia of unknown origin was 2.0% (56/2,861). Horses in the ataxic group (vs the control group) were more likely to have been warmblood horses (OR, 2.70) and less likely to have been Arabian horses (OR, 0.53). In the ataxic group, horses < 5 (vs ≥ 5) years of age had greater odds of CVCM (OR, 2.82) or eNAD-EDM (OR, 6.17) versus trauma or ataxia of unknown origin. Horses in the ataxic group with CVCM were more likely Thoroughbreds (OR, 2.54), whereas horses with eNAD-EDM were more likely American Quarter Horses (OR, 2.95) and less likely Thoroughbreds (OR, 0.11). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that breed distributions differed for horses with CVCM versus eNAD-EDM; therefore, breed should be considered in the clinical evaluation of spinal ataxia in horses.

Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry.

Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated following acidic hydrolysis and solid phase extraction to quantitate vitamin E and its metabolites in equine urine. A blank control horse urine matrix was used and spiked with different concentrations of analytes to form a standard curve using either alpha-tocopherol-d6 or chlorpropamide as the internal standard. Inter-day and intra-day statistics were performed to evaluate the method for accuracy (90% to 116%) and precision (0.75% to 14%). Matrix effects, percent recovery, and stability were also assessed. The method successfully analyzed alpha-carboxyethyl hydroxychroman (α-CEHC), alpha-carboxymethylbutyl hydroxychromans (α-CMBHC), gamma-carboxyethyl hydroxychroman γ-CEHC, and α-TP concentrations in urine to determine a baseline levels of analytes in healthy horses, and can be used to determine concentrations of vitamin E metabolites in equine urine allowing for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.

Compound heterozygous PLA2G6 loss-of-function variants in Swaledale sheep with neuroaxonal dystrophy.

Sporadic occurrences of neurodegenerative disorders including neuroaxonal dystrophy (NAD) have been previously reported in sheep. However, so far no causative genetic variant has been found for ovine NAD. The aim of this study was to characterize the phenotype and the genetic aetiology of an early-onset neurodegenerative disorder observed in several lambs of purebred Swaledale sheep, a native English breed. Affected lambs showed progressive ataxia and stiff gait and subsequent histopathological analysis revealed the widespread presence of axonal spheroid indicating neuronal degeneration. Thus, the observed clinical phenotype could be explained by a novel form of NAD. After SNP genotyping and subsequent linkage mapping within a paternal half-sib pedigree with a total of five NAD-affected lambs, we identified two loss-of-function variants by whole-genome sequencing in the ovine PLA2G6 gene situated in a NAD-linked genome region on chromosome 3. All cases were carriers of a compound heterozygous splice site variant in intron 2 and a nonsense variant in exon 8. Herein we present evidence for the occurrence of a familial novel form of recessively inherited NAD in sheep due to allelic heterogeneity at PLA2G6. This study reports two pathogenic variants in PLA2G6 causing a novel form of NAD in Swaledale sheep which enables selection against this fatal disorder.

Genome-Wide Association Study and Subsequent Exclusion of ATCAY as a Candidate Gene Involved in Equine Neuroaxonal Dystrophy Using Two Animal Models.

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder of unknown etiology. Clinical signs of neurological deficits develop within the first year of life in vitamin E (vitE) deficient horses. A genome-wide association study (GWAS) was carried out using 670,000 SNP markers in 27 case and 42 control Quarter Horses. Two markers, encompassing a 2.5 Mb region on ECA7, were associated with the phenotype (p = 2.05 × 10-7 and 4.72 × 10-6). Within this region, caytaxin (ATCAY) was identified as a candidate gene due to its known role in Cayman Ataxia and ataxic/dystonic phenotypes in mouse models. Whole-genome sequence data in four eNAD/EDM and five unaffected horses identified 199 associated variants within the ECA7 region. MassARRAY® genotyping was performed on these variants within the GWAS population. The three variants within ATCAY were not concordant with the disease phenotype. No difference in expression or alternative splicing was identified using qRT-PCR in brainstem across the ATCAY transcript. Atcayji-hes mice were then used to conduct functional analysis in a second animal model. Histologic lesions were not identified in the central nervous system of Atcayji-hes mice. Additionally, supplementation of homozygous Atcayji-hes mice with 600 IU/day of dl-α-tocopheryl acetate (vitE) during gestation, lactation, and adulthood did not improve the phenotype. ATCAY has therefore been excluded as a candidate gene for eNAD/EDM.

Spontaneous Incidental Brain Lesions in C57BL/6J Mice.

Genetically engineered mouse lines on a C57BL/6J background are widely employed as preclinical models to study neurodegenerative human disorders and brain tumors. However, because of the lack of comprehensive data on the spontaneous background neuropathology of the C57BL/6J strain, discriminating between naturally occurring changes and lesions caused by experimental mutations can be challenging. In this context, this study aims at defining the spectrum and frequency of spontaneous brain changes in a large cohort of C57BL/6J mice and their association with specific biological variables, including age and sex. Brains from 203 experimentally naive and clinically unremarkable C57BL/6J mice were collected and analyzed by means of histopathology and immunohistochemistry. Mice ranged in age from 3 to 110 weeks with 89 females, 111 males, and 3 unknowns. Sixteen different spontaneous lesion categories were described in this cohort. Age-related neurodegenerative and/or neuroinflammatory findings represented the most common pathologic changes and included (1) Hirano-like inclusions in the thalamic neurons, (2) neuroaxonal dystrophy in the medulla oblongata, (3) periodic acid-Schiff-positive granular deposits in the neuropil of the hippocampus, and (4) progressive neuroinflammation characterized by microgliosis and astrogliosis. Neoplastic conditions, developmental abnormalities, and circulatory disorders were rarely observed incidental findings. In conclusion, this study describes spontaneous age-related brain lesions of the C57BL/6J mouse and provides a reference for evaluating and interpreting the neuropathological phenotype in genetically engineered mouse models developed and maintained on this congenic background.

Previously Identified Genetic Variants in ADGRL3 Are not Associated with Risk for Equine Degenerative Myeloencephalopathy across Breeds.

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is a neurologic disease that has been reported in young horses from a wide range of breeds. The disease is inherited and associated with vitamin E deficiency during the first two years of life, resulting in bilateral symmetric ataxia. A missense mutation (chr3:71,917,591 C > T) within adhesion G protein-coupled receptor L3 (ADGRL3) was recently associated with risk for EDM in the Caspian breed. In order to confirm these findings, genotyping of this missense mutation, along with the three other associated single nucleotide polymorphisms (SNPs) in the genomic region, was carried out on 31 postmortem-confirmed eNAD/EDM cases and 43 clinically phenotyped controls from various breeds. No significant association was found between eNAD/EDM confirmed cases and genotype at any of the four identified SNPs (P > 0.05), including the nonsynonymous variant (EquCab2.0 chr3:71,917,591; allelic P = 0.85). These findings suggest that the four SNPs, including the missense variant in the ADGRL3 region, are not associated with risk for eNAD/EDM across multiple breeds of horses.

Neuroaxonal Dystrophy in a mule: first case reported / Distrofia neuroaxonal em muar: primeiro relato de caso

A 12-month-old mule (sterile hybrid equine species) presented unspecific neurological changes (symmetric ataxia, dysmetria, conscious proprioceptive deficit and weakness). Due to poor prognosis and to the fact that a sibling from the previous generation exhibited similar clinical signs that were not definitively diagnosed, the animal was euthanized. Diagnosis of neuroaxonal dystrophy was confirmed by anatomohistopathological analysis. This is the first clinical case of neuronal dystrophy in a mule reported in the world. The clinical and histopathological characteristics of this disease were very similar to those reported for several equine breeds. Therefore, the disease should also be considered in the diagnosis of neurological conditions in mules and donkeys.(AU)

Relata-se o caso de uma mula de 12 meses que apresentou alterações neurológicas inespecíficas (ataxia simétrica, dismetria, déficit proprioceptivo consciente e fraqueza). Devido ao mau prognóstico e ao fato de um irmão da geração anterior apresentar sinais clínicos similares sem diagnóstico conclusivo, o animal foi eutanasiado. O diagnóstico de distrofia neuroaxonal foi confirmado por análise anátomo-histopatológica. Esse é o primeiro caso clínico de distrofia neuroaxonal em muar relatado no mundo. As características clínicas e histopatológicas dessa doença foram muito semelhantes às relatadas em várias raças de equinos. Portanto, a doença também deve ser considerada no diagnóstico de condições neurológicas em muares e asininos.(AU)

Lipid peroxidation biomarkers for evaluating oxidative stress in equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is a neurodegenerative disorder affecting genetically predisposed foals maintained on an α-tocopherol (α-TOH) deficient diet. Currently no antemortem diagnostic test for eNAD/EDM is available. HYPOTHESIS: Because α-TOH deficiency is associated with increased lipid peroxidation, it was hypothesized that F2 -isoprostanes (F2 IsoP), F4 -neuroprostanes (F4 NP) and oxysterols derived from free radical oxidation would be increased in the cerebrospinal fluid (CSF) and neural tissue of eNAD/EDM affected horses and could serve as potential biomarkers for disease. ANIMALS: Isoprostane Study A: 14 Quarter horse foals (10 healthy foals and 4 eNAD/EDM affected foals) at 1 and 6 months of age. Isoprostane Study B: 17 eNAD/EDM affected and 10 unaffected horses ≥ 1-4 years of age. Oxysterol study: eNAD/EDM affected (n = 14, serum; n = 11, CSF; n = 10, spinal cord [SC]) and unaffected horses 1-4 years of age (n = 12, serum; n = 10, CSF; n = 7, SC). PROCEDURES: Cerebrospinal fluid [F2 IsoP] and [F4 NP] were assessed using gas chromatography-negative ion chemical ionization mass spectrometry. Serum, CSF, and cervical SC [oxysterols] were quantified using high performance liquid chromatography mass spectrometry. Results were compared with respective α-TOH concentrations. RESULTS: Spinal cord [7-ketocholesterol], [7-hydroxycholesterol], and [7-keto-27-hydrocholesterol] were higher in eNAD/EDM horses whereas [24-ketocholesterol] was lower. No significant difference was found in CSF [F2 IsoP] and [F4 NP], serum [oxysterols] and CSF [oxysterols] between eNAD/EDM affected and unaffected horses. No correlation was found between [F2 IsoP], [F4 NP], or [oxysterols] and respective [α-TOH]. CONCLUSIONS AND CLINICAL IMPORTANCE: In the SC, targeted markers of cholesterol oxidation were significantly increased in horses with eNAD/EDM.

A Missense Mutation in the Vacuolar Protein Sorting 11 (VPS11) Gene Is Associated with Neuroaxonal Dystrophy in Rottweiler Dogs.

Canine neuroaxonal dystrophy (NAD) is a recessive, degenerative neurological disease of young adult Rottweiler dogs (Canis lupus familiaris) characterized pathologically by axonal spheroids primarily targeting sensory axon terminals. A genome-wide association study of seven Rottweilers affected with NAD and 42 controls revealed a significantly associated region on canine chromosome 5 (CFA 5). Homozygosity within the associated region narrowed the critical interval to a 4.46 Mb haplotype (CFA5:11.28 Mb - 15.75 Mb; CanFam3.1) that associated with the phenotype. Whole-genome sequencing of two histopathologically confirmed canine NAD cases and 98 dogs unaffected with NAD revealed a homozygous missense mutation within the Vacuolar Protein Sorting 11 (VPS11) gene (g.14777774T > C; p.H835R) that was associated with the phenotype. These findings present the opportunity for an antemortem test for confirming NAD in Rottweilers where the allele frequency was estimated at 2.3%. VPS11 mutations have been associated with a degenerative leukoencephalopathy in humans, and VSP11 should additionally be included as a candidate gene for unexplained cases of human NAD.

Ultrastructural features of canine neuroaxonal dystrophy in a Papillon dog.

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by severe axonal swelling (spheroids) throughout the nervous system. In dogs, NAD has been reported in several breeds and a missense mutation in PLA2G6 gene has recently been identified in the Papillon dog NAD. Here we performed ultrastructural analysis to clarify the detailed ultrastructural features of the Papillon dog NAD. Dystrophic axons consisted of accumulation of filamentous materials, tubulovesicular structures, and swollen edematous mitochondria with degenerated inner membranes were often observed in the central nervous system. At axonal terminals, degeneration of presynaptic membrane was also detected. As reported in Pla2g6 knockout mice, mitochondrial and presynaptic degeneration may be related with the pathogenesis of NAD in Papillon dogs.

Primary, congenital neuroaxonal dystrophy with peripheral nerve demyelination in Merino-Border Leicester × Polled Dorset lambs.

CASE REPORT: Clinicopathological features of neuroaxonal dystrophy (NAD) in newborn, Merino-Border Leicester × Polled Dorset lambs are described. The affected lambs were unable to walk at birth and microscopic examination of brainstem and spinal cord sections revealed bilaterally symmetrical accumulations of axonal swellings (spheroids), the histological hallmark of primary NAD. The neurological deficit was also exacerbated by myelin loss and secondary axonal degeneration, particularly in the spinal cord and sciatic nerves, but also, to a more limited extent, in brainstem and spinal nerves. CONCLUSIONS: Although lambs previously diagnosed with NAD have ranged in age from 2 days to 7 months, this is believed to be the first report of congenital NAD in this species. Moreover, the present cases are the only ones in which peripheral nerve demyelination has been found.