GM2 Gangliosidosis in Shiba Inu Dogs with an In‐Frame Deletion in HEXB

Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young‐adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL‐related variants were identified in a whole‐genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole‐genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3‐bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin‐layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3‐bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole‐genome sequencing can lead to the early identification of potentially disease‐causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Mucopolysaccharidosis type VI in a Miniature Poodle-type dog caused by a deletion in the arylsulphatase B gene

Abstract

AIM: To investigate and characterise an inborn error of metabolism in a dog with skeletal and ocular abnormalities.

METHODS: A 2.5-year-old small male Miniature Poodle-like dog was presented with gross joint laxity and bilateral corneal opacities. Clinical examination was augmented by routine haematology, serum chemistry, radiographs, pathology, enzymology and molecular genetic studies. Euthanasia was requested when the dog was 3 years of age because of progressively decreasing quality of life.

RESULTS: Radiology revealed generalised epiphyseal dysplasia, malformed vertebral bodies, luxation/subluxation of appendicular and lumbosacral joints with hypoplasia of the odontoid process and hyoid apparatus. These clinical and radiographic findings, together with a positive urinary Berry spot test for mucopolysaccharides, and metachromatic granules in leucocytes, were indicative of a mucopolysaccharidosis (MPS), a lysosomal storage disease. Histological lesions included vacuolation of stromal cells of the cornea, fibroblasts, chondrocytes, macrophages and renal cells. The brain was essentially normal except for moderate secondary Wallerian-type degeneration in motor and sensory tracts of the hind brain. Dermatan sulphate-uria was present and enzymology revealed negligible activity of N-acetylgalactosamine-4-sulphatase, also known as arylsulphatase B, in cultured fibroblasts and liver tissue. A novel homozygous 22 base pair (bp) deletion in exon 1 of this enzyme's gene was identified (c.103_124del), which caused aframe-shift and subsequent premature stop codon. The “Wisdom pure breed-mixed breed” test reported the dog as a cross between a Miniature and Toy Poodle.

CONCLUSIONS: The clinicopathological features are similar to those of MPS type VI as previously described in dogs, cats and other species, and this clinical diagnosis was confirmed by enzymology and molecular genetic studies. This is an autosomal recessively inherited lysosomal storage disease.

CLINICAL RELEVANCE: The prevalence of MPS VI in Miniature or Toy Poodles in New Zealand and elsewhere is currently unknown. Due to the congenital nature of the disorder, malformed pups may be subject to euthanasia without investigation and the potential genetic problem in the breed may not be fully recognised. The establishment of a molecular genetic test now permits screening for this mutation as a basis to an informed breeding policy.     

Dystrophin-deficient muscular dystrophy in a Toy Poodle with a single base pair insertion in exon 45 of the Duchenne muscular dystrophy gene

A 10-month-old, intact male Toy Poodle was referred for a postural abnormality. Blood biochemical tests revealed a marked increase in plasma creatine phosphokinase (CPK) concentration. The isoenzyme test showed that 99% of serum CPK consisted of CPK-MM. Histopathological evaluation of muscle biopsy samples confirmed scattered degeneration and necrosis of myofibers. Immunohistochemistry for dystrophin showed an absence of staining in muscle cells. Based on these findings, the dog was diagnosed with dystrophin-deficient muscular dystrophy. Whole genome sequencing using genomic DNA extracted from blood revealed a single base pair insertion in exon 45 of the Duchenne muscular dystrophy (DMD) gene. This is the first report on muscular dystrophy in Toy Poodles and identified a novel mutation in the DMD gene.     

Two‐Year Follow‐Up Magnetic Resonance Imaging and Spectroscopy Findings and Cerebrospinal Fluid Analysis of a Dog with Sandhoff's Disease

A 13‐month‐old female Toy Poodle was presented for progressive ataxia and intention tremors of head movement. The diagnosis of Sandhoff's disease (GM2 gangliosidosis) was confirmed by deficient β‐N‐acetylhexosaminidase A and B activity in circulating leukocytes and identification of the homozygous mutation (HEXB: c.283delG). White matter in the cerebrum and cerebellum was hyperintense on T2‐weighted and fluid‐attenuated inversion recovery magnetic resonance images. Over the next 2 years, the white matter lesions expanded, and bilateral lesions appeared in the cerebellum and thalamus, associated with clinical deterioration. Magnetic resonance spectroscopy showed progressive decrease in brain N‐acetylaspartate, and glycine‐myo‐inositol and lactate‐alanine were increased in the terminal clinical stage. The concentrations of myelin basic protein and neuron specific enolase in cerebrospinal fluid were persistently increased. Imaging and spectroscopic appearance correlated with histopathological findings of severe myelin loss in cerebral and cerebellar white matter and destruction of the majority of cerebral and cerebellar neurons.     

Homozygosity of single nucleotide polymorphisms in the 3′ region of the canine estrogen receptor 1 gene is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas

Differences in the distribution of single nucleotide polymorphisms (SNPs) and haplotypes in the estrogen receptor α gene (ESR1) were examined in Miniature Dachshunds (n = 48), Chihuahuas (n = 20) and Toy Poodles (n = 18). Five DNA fragments located in the 40‐kb region at the 3′ end of ESR1 were amplified by polymerase chain reaction and were directly sequenced. We compared allele, genotype and estimated haplotype frequencies at each SNP in the 3′ end of ESR1 for these three breeds of small dog. The frequency of the major allele and the genotype frequency of the major allele homozygotes, were significantly higher in Toy Poodles for five SNPs (SNP #5, #14–17) than in Miniature Dachshunds, and significantly higher in Toy Poodles than Chihuahuas for three SNPs (SNP #15–17). A common haplotype block was identified in an approximately 20‐kb region encompassing four SNPs (SNPs # 14–17). The frequencies of the most abundant estimated haplotype (GTTG) and GTTG homozygotes were significantly higher in Toy Poodles than in the other two breeds. These results imply that homozygosity for the allele, genotype and haplotype distribution within the block at the 3′ end of ESR1 is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.     

Intra‐thoracic gossypiboma (textiloma) in a 6‐year‐old cocker spaniel

This report documents the first case of gossypiboma (textiloma) identified within the thorax of a dog. CT findings, surgical removal and histopathology are described. Intra‐thoracic gossypiboma has not previously been reported in dogs and is rarely reported in the human medical literature, where it is most commonly associated with previous cardiac or pulmonary surgery. This dog had previously had a thoracotomy for attempted surgical correction of a persistent right aortic arch and left ligamentum arteriosum 6 years prior to presentation. A brief review of the previous literature and recommendations for prevention of this condition are provided.     

Mucopolysaccharidosis VII in a Cat Caused by 2 Adjacent Missense Mutations in the GUSB Gene

Background

Mucopolysaccharidoses (MPS) are common lysosomal storage disorders causing typically progressive skeletal and ocular abnormalities.

Objectives

To describe the clinic features, metabolic profile and a unique mutation in a domestic shorthair (DSH) kitten with MPS VII.

Animals

Affected kitten and 80 healthy cats.

Methods

Serum lysosomal enzyme activities and urinary glycosaminoglycan (GAG) accumulation were assessed. Exons of the β‐glucuronidase gene (GUSB) were sequenced from genomic DNA and genotyping was conducted.

Results

A 3‐month‐old DSH cat was presented for stunted growth, paresis, facial dysmorphia, multiple skeletal deformities, and corneal opacities. Evaluation of blood smears disclosed metachromatic granules in leukocytes and a urinary mucopolysaccharide spot test was positive. The proband had no GUSB activity but normal or increased activities for other lysosomal enzymes. Sequencing of the GUSB gene from the proband and comparison to the sequence of 2 healthy cats and the published feline genome sequence demonstrated 2 unique single base transitions (c.1421T>G and c.1424C>T) in exon 9, altering 2 adjacent codons (p.Ser475Ala and p.Arg476Trp). These amino acid changes are in a highly conserved domain of the GUSB protein and nontolerable to maintain function. Moreover, the p.Arg476Trp mutation previously has been identified in human patients. None of the other clinically healthy cats had these mutations.

Conclusions and Clinic Importance

The diagnostic approach to MPS disorders is delineated. This is only the second mutation known to cause MPS VII in cats. Similarly, 2 different mutations have been described in MPS VII dogs, thereby showing the molecular heterogeneity of MPS VII in companion animals.     

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.     

Nonsense mutation of feline beta-hexosaminidase beta-subunit (HEXB) gene causing Sandhoff disease in a family of Japanese domestic cats

G(M2) gangliosidoses are inherited metabolic disorders and are caused by severely reduced enzymatic activity of lysosomal beta-hexosaminidase. In the present study, the open reading frame (ORF) of the HEXB gene in a family of Japanese domestic cats with G(M2) gangliosidosis variant 0 (Sandhoff disease) was determined. Two types of abnormal cDNA clones were obtained from the liver of an affected cat tissue. One showed a single nucleotide substitution from C to T at nucleotide position 667 of the HEXB ORF. In the deduced amino acid sequence, the codon of arginine was altered to a stop codon. The genotyping, using PCR-primer introduced restriction analysis confirmed that Sandhoff disease in this family is associated with this nonsense mutation. Discovery of the nonsense mutation will permit the confirmation of the clinical diagnosis of Sandhoff disease in conjugation with the already established enzyme-based test.     

Prevalence of AmpC‐ and Extended‐Spectrum β‐Lactamase‐Harbouring Enterobacteriaceae in Faecal Flora of a Healthy Domestic Canine Population

In order to estimate the prevalence of AmpC‐ and ESBL β‐lactamase‐producing Enterobacteriaceae in the faecal flora of a healthy domestic canine population, faecal samples were obtained from healthy dogs receiving routine parasitology screening at the Ohio State University Veterinary Medical Center, between January 2013 and April 2013. Samples were screened for the presence of AmpC and ESBL β‐lactamase phenotypes, and the clinically important genotypes, bla_CMY and bla_CTX‐M, were confirmed via conventional PCR. Minimum inhibitory concentrations were determined for isolates and plasmids were characterized. Two hundred and twelve canine faecal samples were screened, of which 30 harboured isolates carrying the AmpC bla_CMY, representing 14.2% of the population (95% CI: 9.4–18.9%). Nine samples harboured isolates that carried the ESBL bla_CTX‐M, representing 4.2% of the population (95% CI: 1.5–7.0%). Isolates containing bla_CMY harboured multiple plasmid replicon types, while isolates containing bla_CTX‐M harboured few plasmid replicon types. Our results suggest that domestic dogs may serve as a reservoir for extended‐spectrum cephalosporin resistance genes for other domestic animal populations as well as for their human companions. This represents a potential veterinary and public health risk that warrants further investigation and continued surveillance to ascertain the nature and extent of the risk. The high level of diversity of plasmid content among isolates harbouring bla_CMY suggests broader dissemination relative to bla_CTX‐M isolates.     

Immunization of Pups with Maternally Derived Antibodies to Canine Parvovirus (CPV) Using a Modified‐Live Variant(CPV‐2b)

The results of vaccination trials carried out on pups with maternally derived antibodies (MDA) to canine parvovirus (CPV), using a modified‐live CPV‐2b variant vaccine (29‐97/40 strain), are reported. The vaccine was able to overcome the obstacle of MDA, and to elicit protective immunity in 100% of the pups whose antibody titres were 1:10–1:40, 83% of the pups with titres of 1:80, 57% of the pups with titres of 1:160, and even in 60% of the pups with antibody titres of 1:320.     

Basset Hound thrombopathia in a 4‐month‐old female Ba‐Shar (Sharp Asset)

A 3‐month‐old female Basset Hound‐Shar Pei mix puppy (Ba‐Shar or Sharp Asset) presented with oral bleeding due to a cracked molar. On physical exam, an aural hematoma was also noted that the owner indicated was chronic. The puppy was hospitalized for over 24 h until the bleeding was brought under control. At 4 months of age, the puppy again presented with oral bleeding due to loss of deciduous teeth and was hospitalized until bleeding was controlled. Coagulation screening tests, platelet numbers, and von Willebrand Factor antigen levels were within reference limits. Based on the presence of platelet‐type bleeding in the face of normal screening test results, samples were submitted for DNA testing for Basset Hound thrombopathia. The puppy tested as affected for the calcium and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG‐GEFI) mutation causing this disorder. This is the first time thrombopathia has been diagnosed in a “designer” breed.     

The cardiopulmonary effects of a peripheral alpha‐2‐adrenoceptor antagonist,MK‐467, in dogs sedated with a combination of medetomidine and butorphanol

ObjectiveTo compare the cardiopulmonary effects of intravenous (IV) and intramuscular (IM) medetomidine and butorphanol with or without MK-467.Study designProspective, randomized experimental cross-over.AnimalsEight purpose–bred beagles (two females, six males), 3–4 years old and weighing 14.5 ±1.6 kg (mean ± SD).MethodsAll dogs received four different treatments as follows: medetomidine 20 μg kg^?1 and butorphanol tartrate 0.1 mg kg^?1 IV and IM (MB), and MB combined with MK-467,500 μg kg^?1 (MBMK) IV and IM. Heart rate (HR), arterial blood pressures (SAP, MAP, DAP), central venous pressure (CVP), cardiac output, respiratory rate (f_R), rectal temperature (RT) were measured and arterial blood samples were obtained for gas analysis at baseline and at 3, 10, 20, 30, 45 and 60 minutes after drug administration. The cardiac index (CI), systemic vascular resistance index (SVRI) and oxygen delivery index (DO₂I) were calculated. After the follow-up period atipamezole 50 μg kg^?1 IM was given to reverse sedation.ResultsHR, CI and DO₂I were significantly higher with MBMK after both IV and IM administration. Similarly, SAP, MAP, DAP, CVP, SVRI and RT were significantly lower after MBMK than with MB. There were no differences in f_R between treatments, but arterial partial pressure of oxygen decreased transiently after all treatments. Recoveries were uneventful following atipamezole administration after all treatments.Conclusions and clinical relevanceMK-467 attenuated the cardiovascular effects of a medetomidine-butorphanol combination after IV and IM administration.     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.