Analisis de curvas de fusion de alta resolucion para la tipificacion de la mutacion de la abiotrofia cerebelar equina / High resolution melting analysis for the equine cerebellar abiotrophy mutation typing

INTRODUCCIÓN: la abiotrofia cerebelar equina es una enfermedad neurodegenerativa de etiología genética autosómica y recesiva. Se presenta con más frecuencia en el Pura Raza Árabe. La enfermedad está causada por una mutación puntual que produce una degeneración progresiva de las células de Purkinje. OBJETIVOS: puesta a punto de una técnica sencilla, empleando el análisis de curvas de fusión de alta resolución (HRM), que permita identificar los individuos portadores de la mutación causante de la abiotrofia cerebelosa en los ancestros de un animal enfermo. MATERIAL Y MÉTODOS: extracción de ADN de 93 muestras de sangre provenientes de animales emparentados. Diseño y selección de una pareja de cebadores para la amplificación de una secuencia de 89 pares de bases que contiene la mutación. Amplificación de la secuencia y análisis de las curvas de fusión. RESULTADOS: el análisis genealógico confirmó el carácter autosómico recesivo de la enfermedad. La amplificación no generó fragmentos inespecíficos. La técnica de análisis HRM permitió diferenciar de forma inequívoca los genotipos homocigotos sano y enfermo, y también el genotipo heterocigoto de los portadores sanos. Los resultados coinciden con los obtenidos mediante la técnica de análisis de fragmentos publicada en 2011. CONCLUSIÓN: la técnica desarrollada permite detectar caballos portadores de la abiotrofia cerebelosa de forma sencilla

INTRODUCTION: the equine cerebellar abiotrophy is an autosomal recessive neurodegenerative disease. It has been more frequently described in the Arabian horse. The causative mutation produces a progressive degeneration of Purkinje cells. OBJECTIVES: developing a simple technique based on the high-resolution melt analysis (HRM) in order to identify the carriers of the mutation among a group of ancestors of an affected horse. MATERIAL AND METHODS: DNA extraction of 93 blood samples from a group of related animals. Primers designed to amplify an 89 base pair sequence that contains the mutation. Amplification of the sequence and melting curves analysis. RESULTS: the genealogical analysis confirms the autosomal recessive nature of disease. The amplification did not generate nonspecific fragments. The HRM analysis allowed the differentiation of healthy and affected homozygous genotypes, and also the differentiation of the carrier, heterozygous genotype. Our results concur with those obtained using the fragment analysis technique published in 2011. CONCLUSION: high resolution melting analysis is a simple technique that allows the detection of cerebellar abiotrophy carriers in horses

Variation in MUTYH expression in Arabian horses with Cerebellar Abiotrophy.

Cerebellar Abiotrophy (CA) is a neurodegenerative disease in Arabian horses affecting the cerebellum, more specifically the Purkinje neurons. Although CA occurs in several domestic species, CA in Arabian horses is unique in that a single nucleotide polymorphism (SNP) has been associated with the disease. Total RNA sequencing (RNA-seq) was performed on CA-affected horses to address the molecular mechanism underlying the disease. This research expands upon the RNA-seq work by measuring the impact of the CA-associated SNP on the candidate gene MutY homolog (MUTYH) and its regulation, isoform-specific expression and protein localization. We hypothesized that the CA-associated SNP compromises the promoter region of MUTYH, leading to differential expression of its isoforms. Our research demonstrates that the CA-associated SNP introduces a new binding site for a novel transcription factor (Myelin Transcription Factor-1 Like protein, MYT1L). In addition, CA-affected horses show differential expression of a specific isoform of MUTYH as well as different localization in the Purkinje and granular neurons of the cerebellum.

Defining Trends in Global Gene Expression in Arabian Horses with Cerebellar Abiotrophy.

Equine cerebellar abiotrophy (CA) is a hereditary neurodegenerative disease that affects the Purkinje neurons of the cerebellum and causes ataxia in Arabian foals. Signs of CA are typically first recognized either at birth to any time up to 6 months of age. CA is inherited as an autosomal recessive trait and is associated with a single nucleotide polymorphism (SNP) on equine chromosome 2 (13074277G>A), located in the fourth exon of TOE1 and in proximity to MUTYH on the antisense strand. We hypothesize that unraveling the functional consequences of the CA SNP using RNA-seq will elucidate the molecular pathways underlying the CA phenotype. RNA-seq (100 bp PE strand-specific) was performed in cerebellar tissue from four CA-affected and five age-matched unaffected horses. Three pipelines for differential gene expression (DE) analysis were used (Tophat2/Cuffdiff2, Kallisto/EdgeR, and Kallisto/Sleuth) with 151 significant DE genes identified by all three pipelines in CA-affected horses. TOE1 (Log2(foldchange) = 0.92, p = 0.66) and MUTYH (Log2(foldchange) = 1.13, p = 0.66) were not differentially expressed. Among the major pathways that were differentially expressed, genes associated with calcium homeostasis and specifically expressed in Purkinje neurons, CALB1 (Log2(foldchange) = -1.7, p < 0.01) and CA8 (Log2(foldchange) = -0.97, p < 0.01), were significantly down-regulated, confirming loss of Purkinje neurons. There was also a significant up-regulation of markers for microglial phagocytosis, TYROBP (Log2(foldchange) = 1.99, p < 0.01) and TREM2 (Log2(foldchange) = 2.02, p < 0.01). These findings reaffirm a loss of Purkinje neurons in CA-affected horses along with a potential secondary loss of granular neurons and activation of microglial cells.

Degenerative Encephalopathy in Nova Scotia Duck Tolling Retrievers Presenting with a Rapid Eye Movement Sleep Behavior Disorder.

BACKGROUND: Neurodegenerative diseases are a heterogeneous group of disorders characterized by loss of neurons and are commonly associated with a genetic mutation. HYPOTHESIS/OBJECTIVES: To characterize the clinical and histopathological features of a novel degenerative neurological disease affecting the brain of young adult Nova Scotia Duck Tolling Retrievers (NSDTRs). ANIMALS: Nine, young adult, related NSDTRs were evaluated for neurological dysfunction and rapid eye movement sleep behavior disorder. METHODS: Case series review. RESULTS: Clinical signs of neurological dysfunction began between 2 months and 5 years of age and were progressive in nature. They were characterized by episodes of marked movements during sleep, increased anxiety, noise phobia, and gait abnormalities. Magnetic resonance imaging documented symmetrical, progressively increasing, T2-weighted image intensity, predominantly within the caudate nuclei, consistent with necrosis secondary to gray matter degeneration. Abnormalities were not detected on clinicopathological analysis of blood and cerebrospinal fluid, infectious disease screening or urine metabolite screening in most cases. Postmortem examination of brain tissue identified symmetrical malacia of the caudate nuclei and axonal dystrophy within the brainstem and spinal cord. Genealogical analysis supports an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL IMPORTANCE: A degenerative encephalopathy was identified in young adult NSDTRs consistent with a hereditary disease. The prognosis is guarded due to the progressive nature of the disease, which is minimally responsive to empirical treatment.

Aspectos clínicos e laboratoriais em ovinos submetidos a dietas com níveis elevados de enxofre com objetivo de indução de polioencefalomalácia / Clinical and laboratory aspects of sheep supplemented with high levels of sulfur in diet to induce polioencephalomalacia

A polioencefalomalacia (PEM) é uma doença neurológica que acomete ruminantes e pode ser desencadeada por diversos fatores, dentre eles o consumo excessivo de enxofre. Este trabalho teve como objetivo verificar a relação entre dietas ricas em enxofre, altos níveis de gás sulfídrico ruminal e a ocorrência de polioencefalomalácia em ovinos. Foram utilizados 18 ovinos, divididos em três grupos (G1, G2 e G3) que receberam diferentes níveis de enxofre na dieta; 0,2%, 0,9% e 1,2%, respectivamente. Exames físicos (frequência cardíaca, frequência respiratória, temperatura retal e motricidade ruminal) e complementares (concentração de sulfeto de hidrogênio ruminal, hemogasometria venosa, pH do fluído ruminal, concentração de cobre sérico e hepático, tomografia computadorizada, necropsia e histopatologia) foram realizados. A temperatura retal, a hemogasometria venosa e o pH do fluido ruminal permaneceram dentro dos valores de referência para a espécie. A motricidade ruminal estava diminuída nos grupos G2 e G3 em comparação com o G1 (controle). Quanto maior a ingestão de enxofre, menores foram os níveis de cobre sérico e hepático. Valores elevados de sulfeto de hidrogênio ruminal foram detectados nos grupos G2 e G3. Nenhum animal apresentou sinais clínicos de PEM. Nos exames de tomografia computadorizada, necropsia e exame histopatológico do sistema nervoso central (SNC), não foram observadas alterações compatíveis com PEM. É provável que algum outro fator esteja associado ao excesso de enxofre na dieta para o desenvolvimento de PEM em ovinos.

Polioencephalomalacia (PEM) is a neuropathologic condition of ruminants that can be induced by a variety of factors including excessive sulfur intake. This study aimed to investigate the relationship between diets rich in sulfur, high levels of ruminal hydrogen sulfide and the occurrence of polioencephalomalacia in sheep. Eighteen sheep were divided into three groups (G1, G2, and G3) and supplemented with 0.2%, 0.9% and 1.2% sulfur in the diet respectively. Clinical evaluation (i.e. heart rate, respiratory rate, rectal temperature and rumen motility) and laboratory exams (i.e. ruminal hydrogen sulfide concentration, venous gas analysis, ruminal pH, serum and liver copper concentration, computed axial tomography, necropsy, and histopathological examination) were performed. Rectal temperature, venous gas and ruminal pH were within normal limits. Tachycardia and tachypnea were observed in sheep of the three groups. Rumen motility was decreased in animals of group G2 and G3 when compared with G1. The higher the sulfur intake, the lower was the serum and liver levels of copper. Increased ruminal hydrogen sulfide concentration was detected in G2 and G3 sheep. None of the animals had clinical signs of PEM. Computed axial tomography, macroscopic and histopathological examination of the central nervous system showed no evidence of PEM. It is suggested that other factors are associated with excessive sulfur consumption for a PEM outbreak to occur in sheep.

Genotyping assays for the canine degenerative myelopathy-associated c.118G>A (p.E40K) mutation of the SOD1 gene using conventional and real-time PCR methods: a high prevalence in the Pembroke Welsh Corgi breed in Japan.

Canine degenerative myelopathy is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds, particularly Pembroke Welsh Corgis. Recently, a degenerative myelopathy-associated mutation of the canine SOD1 gene was identified as c.118G>A (p.E40K). In the present study, genotyping assays using conventional and real-time PCR methods were developed, and a preliminary genotyping survey was performed on 122 randomly selected Pembroke Welsh Corgis without any degenerative myelopathy-related clinical signs to determine the current allele frequency in Japan. Both of the assays provided clear-cut genotyping. The survey demonstrated the frequencies of the G/G wild-type, G/A heterozygote and A/A homozygote to be 9.0, 42.6 and 48.4%, respectively, indicating that the prevalence of the mutant A allele (69.7%) in Pembroke Welsh Corgis is extremely high in Japan.

The frequency of the equine cerebellar abiotrophy mutation in non-Arabian horse breeds.

REASONS FOR PERFORMING STUDY: A putative mutation causative of cerebellar abiotrophy (CA), a genetic defect found almost exclusively in Arabian horses, was recently identified. OBJECTIVES AND HYPOTHESIS: To investigate the presence of the CA mutation in breeds other than Arabian and ascertain whether the mutation had been introduced into these breeds by Arabian ancestry. The CA mutation is present in breeds of horses with Arabian ancestry. METHODS: Allele-specific PCR was used to genotype 1845 non-Arabian horses for the CA mutation. For those breeds in which at least one carrier was identified, an additional 266 horses were genotyped to determine the frequency of the CA allele. Cerebellar abiotrophy carriers were further genotyped for a haplotype segregating with CA in Arabians. RESULTS: At least one CA carrier was identified in 3 breeds and the frequency of the CA allele calculated: Bashkir Curly Horses (2.8%), Trakehners (0.68%) and Welsh ponies (0.33%). Based on pedigree and haplotype analysis, CA was introduced into these breeds by Arabian ancestry. The Trakehner and Welsh pony carriers were at least half-Arabian, while the Bashkir Curly horses appeared to have had the CA allele introduced by a single Arabian stallion used for developing the breed in the 1960s. CONCLUSIONS: The CA mutation is present in breeds of horses that allow crossbreeding with Arabian horses and in breeds that have used Arabians as foundation stock during their development. POTENTIAL RELEVANCE: Breeds that allow registration of horses with Arabian ancestry should have any Arabian breeding stock tested for the mutation and breeds descended from Arabian ancestry should pursue genetic testing of breeding stock to prevent the occurrence of affected foals.

Polioencefalomalacia experimental em bovinos induzida por toxicose por enxofre / Experimental polioencephalomalacia in cattle induced by sulfur toxicosis

O presente trabalho teve como objetivos avaliar os sinais clínicos, as concentrações do sulfeto de hidrogênio ruminal e as alterações anatomopatológicas associadas à intoxicação experimental por enxofre em bovinos. Foram utilizados dez bezerros mestiços leiteiros, sendo que quatro bovinos ingeriram ração sem sulfato de sódio (G1) e seis consumiram ração com sulfato de sódio (G2). Exames clínicos (temperatura retal, frequência cardíaca e respiratória e motricidade ruminal) e laboratoriais (hemograma, fibrinogênio, proteína plasmática, pH do fluido ruminal, concentração do sulfeto de hidrogênio ruminal, líquido cerebrospinal e histopatológico) foram realizados. A temperatura retal, frequência cardíaca, hemograma, fibrinogênio, proteína plasmática, pH do fluido ruminal e os valores do líquido cerebrospinal estavam dentro dos valores de referência para a espécie. Taquipnéia, hipomotricidade ruminal e elevados valores de sulfeto de hidrogênio ruminal foram observados nos bezerros do grupo G2. Um bezerro do grupo G2 apresentou sinais neurológicos e lesões histopatológicas de PEM. Dois animais de cada grupo foram eutanasiados. Lesões microscópicas foram observadas nos bezerros do G2. Histologicamente as alterações observadas foram necrose neuronal cortical e lesões hemorrágicas nos núcleos basais, tálamo, mesencéfalo, ponte e bulbo. O protocolo experimental constituído por uma dieta rica em carboidrato de alta fermentação, baixa quantidade de fibra efetiva e altos níveis de enxofre (0,52%) ocasionou alterações clinicas e histológicas e elevadas concentrações de sulfeto de hidrogênio ruminal compatíveis com quadro de intoxicação por enxofre.

The aims of this study were to evaluate the clinical signs, the ruminal hydrogen sulfide concentration and the histological lesions induced by sulfur toxicosis in cattle. Ten crossbred calves were fed an experimental diet, four without sodium sulfate (G1) and six with (G2). The calves were submitted to clinical (rectal temperature, cardiac and respiratory rate and ruminal motricity) and laboratorial (hemogram, fibrinogen, total plasma protein, ruminal fluid pH, ruminal hydrogen sulfide concentration, cerebrospinal fluid and histopathological) evaluations. Rectal temperature, cardiac rate, hemogram, fibrinogen, total plasma protein, ruminal fluid pH and cerebrospinal fluid values were within normal reference ranges in animals from both groups. Ruminal hypomotricity and increased respiratory rate and ruminal hydrogen sulfide concentration occurred in G2 animals. One out of six calves in G2 developed neurological signs and lesions of PEM. Two calves of each Group were euthanized. Microscopic lesions of PEM were observed in G2 animals. Histologically there were cortical neuronal necrosis and hemorrhagic lesions in basal nuclei, thalamus, midbrain, pons and medulla oblongata. The experimental model consisting of a diet with high carbohydrate and low in long fiber content with high sulfur concentrations (0.52%) resulted in clinical and histological abnormalities and high ruminal hydrogen sulfide concentration consistent with sulpur toxicosis in cattle.

Degenerative progressive hereditary mieloencefalopathy (Weaver Syndrome) in Gir calves / Mieloencefalopatia degenerativa progressiva hereditária (Síndrome da Oscilação) em bezerros Gir

Degenerative progressive hereditary mieloencefalopathy is a disease described in young Brown Swiss cattle. In these animals the disease occurs in calves between six months and two years old showing mainly neurological signs. Two Zebu Gir calves were examined at the Veterinary Hospital of Uberaba, both with balance deficits. The neurological examination has shown that they were mentally alert, with appropriate conscience, head tremor, normal sensitive reflexes, normal sensitivity for superficial and deep pain, motor deficit of the pelvic limbs with severe ataxia. The calves were sacrificed. Based on the clinical case and necroscopic and histopathological findings, it could be suggested that the neuropathy, which affected both animals described above, is very similar to the degenerative progressive hereditary mieloencefalopathy observed in Brown Swiss calves.

A mieloencefalopatia degenerativa progressiva hereditária é uma doença descrita em bovinos Pardo Suíço. Nesses animais, a doença ocorre em bezerros de seis meses a dois anos de idade, e os principais sinais clínicos são nervosos. Dois bezerros Gir foram atendidos no Hospital Veterinário de Uberaba, ambos com dificuldades de equilíbrio motor. O exame clínico revelou estado mental alerta, consciência apropriada, tremores de cabeça, reflexos sensitivos normais, sensibilidade à dor profunda e superficial normal, déficit motor dos membros posteriores com intensa incoordenação. Os bezerros foram sacrificados. Baseado no quadro clínico e nos achados necroscópicos e histopatológicos, pode-se sugerir que a neuropatia que acometeu os dois animais descritos em muito se assemelha à mieloencefalopatia degenerativa progressiva hereditária, observada em bovinos da raça Pardo Suíço.

Genetic mapping of canine multiple system degeneration and ectodermal dysplasia loci.

We characterized a movement disorder of Chinese Crested dogs clinically and pathologically indistinguishable from canine multiple system degeneration (CMSD) previously recognized in Kerry Blue Terriers. This fatal disease segregated as an autosomal recessive in a 51-dog pedigree of both breeds and their crosses. The occurrence of affected dogs among first-generation crosses demonstrated that the mutations causing multiple system degeneration in these breeds are allelic. The CMSD locus maps to CFA1 (LOD > 18) and haplotype analysis narrowed the CFA1 target region to a 15-Mb segment that contains orthologs of genes on HSA6, including PARK2, the gene for the ubiquitin ligase parkin. Mutations in human PARK2 cause the most common form of familial Parkinson's disease, autosomal recessive juvenile parkinsonism, which has clinical and pathological similarities to canine multiple system degeneration. A second phenotype, canine ectodermal dysplasia (CED), segregated in the pedigree as an autosomal dominant with homozygous lethality. Dogs with ectodermal dysplasia have a sparse hair coat and abnormal dentition that is characteristic of the "hairless" variety of Chinese Cresteds. CED mapped to a region of CFA17 (LOD > 14) containing orthologs from HSA2. EDAR, the gene for the ectodysplasin A1 receptor, occurs on HSA2 but was excluded as the cause of canine ectodermal dysplasia.

Inner ear histopathology in "nervous Pointer dogs" with severe hearing loss.

Ten puppy dogs (82, 131 or 148 days-old) from a Pointer cross-colony, exhibiting a juvenile severe hearing loss transmitted as an autosomal recessive trait, were used for histopathological characterization of the inner ear lesion. Immunostaining with calbindin, Na,K-ATPase, cytokeratins, S100, S100A1 and S100A6 antisera were helpful in identifying the different cell types in the degenerated cochleae. Lesions, restricted to the Corti's organ and spiral ganglion, were bilateral but sometimes slightly asymmetrical. Mild to severe lesions of the Corti's organ were unevenly distributed among the different parts of the middle and basal cochlear turns while the apical turn remained unaffected at 148 days. In 82 day-old puppies (n = 2), severe lesions of the Corti's organ, meaning that it was replaced by a layer of unidentifiable cells, involved the lower middle and upper basal turns junction area, extending in the upper basal turn. Mild lesions of the Corti's organ, with both hair and supporting cells abnormalities, involved the lower middle turn and extended from the rest of upper basal turn into the lower basal turn. The outer hair cells (ohc) were more affected than the inner hair cell (ihc). The lesions extended towards the basal end of the cochlea in the 131 (n = 5) and 148 (n = 3) day-old puppies. Additionally, the number of spiral ganglion neurons was reduced in the 131 and 148 day-old puppies; it is earlier than observed in most other canine hereditary deafness. These lesions were interpreted as a degeneration of the neuroepithelial type. This possible animal model might provide information about progressive juvenile hereditary deafness and neuronal retrograde degeneration investigations in human.

Degenerative neurological and neuromuscular disease in young rottweilers.

A number of idiopathic degenerative diseases affecting the central nervous system, peripheral nerves and muscles of immature and young adult rottweilers are reported. Tetraparesis or ataxia causing abnormalities in gait and posture are clinical findings common to these conditions. The current knowledge about these syndromes is presented in this review, with an emphasis on the clinical characteristics. Knowledge of these syndromes and a methodical approach to neurological diagnosis can help the veterinarian to identify the underlying disease and establish a prognosis when presented with a tetraparetic or ataxic young rottweiler.