Associations between neurologic dysfunction and lesions in canine fucosidosis.

Canine fucosidosis in English Springer spaniels is the only animal model of the neurovisceral lysosomal storage disease fucosidosis available for preclinical therapeutic trials. For this reason, it is crucial to identify critical time points in disease progression, and if there are particular lesions associated with specific aspects of neurologic dysfunction. Historical records of 53 canine fucosidosis cases from 1979 to 2009 containing a neurologic dysfunction score assessing motor, behavioral and sensory dysfunction were interrogated by statistical analysis. Motor and behavioral dysfunction scores assessing gait deficits and apprehensive behavior first significantly increased at 12-17 months, and increased at each 6-month interval thereafter. Sensory dysfunction scores, assessing hearing loss, balance and vision deterioration, did not significantly increase until 18-23 months, and coincided with a rapid decline in neurologic function. Regression analysis incorporating published neuropathology data, measured by image analysis, identified neuroinflammation and apoptotic cell death as significant informative predictors of increasing neurologic dysfunction. These findings indicate that the level of neuropathology required to induce consistent and conspicuous clinical signs in canine fucosidosis is reached by approximately 12 months of age in the absence of other disease processes. Significant association between neuroinflammation and apoptotic cell death also suggests that specifically targeting these lesions combined with enzyme replacement in future studies may reduce disease burden in fucosidosis. Overall, examining this historical clinical data to identify associations between the extent of neuropathology and degree of clinical dysfunction provides a useful reference tool for monitoring disease and evaluating therapeutic trials conducted in canine fucosidosis.

Treatment of canine fucosidosis by intracisternal enzyme infusion.

The blood brain barrier is the major obstacle to treating lysosomal storage disorders of the central nervous system such as canine fucosidosis. This barrier was overcome by three, monthly injections of recombinant canine α-l-fucosidase enzyme were given intracisternally. In dogs treated from 8 weeks of age enzyme reached all areas of central nervous system as well as the cervical lymph node, bone marrow and liver. Brainstem and spinal cord samples from regions adjacent to the injection site had highest enzyme levels (39-73% of normal). Substantial enzyme activity (8.5-20% of normal controls) was found in the superficial brain compared to deeper regions (2.6-5.5% of normal). Treatment significantly reduced the fucosyl-linked oligosaccharide accumulation in most areas of CNS, liver and lymph node. In the surface and deep areas of lumbar spinal cord, oligosaccharide accumulation was corrected (79-80% reduction) to near normal levels (p<0.05). In the spinal meninges (thoracic and lumbar) enzyme activity (35-39% of normal control) and substrate reduction (58-63% affected vehicle treated samples) reached levels similar to those seen in phenotypically normal carriers (p<0.05).The procedure was safe and well-tolerated, treated (average 16%) dogs gained more weight (p<0.05) and there was no antibody formation or inflammatory reaction in plasma and CSF following treatments. The capacity of early ERT to modify progression of biochemical storage in fucosidosis is promising as this disease is currently only amenable to treatment by bone marrow transplantation which entails unacceptably high risks for many patients.

Canine fucosidosis: a neuroprogressive disorder.

The lysosomal storage disease, canine fucosidosis, is caused by the absence of the lysosomal enzyme canine α-L-fucosidase with storage of undegraded fucose-rich material in different organs. Canine fucosidosis is a severe, progressive, fatal neurological disease which results in death or euthanasia and is the only available animal model for this human disease. We analysed the progressive neuropathology from birth to severe clinical disease and related this to the clinical signs. At birth no vacuolation was observed in fucosidosis brain; however, a complex storage presence with vacuolation was well established by 4 months of age, before the clinical signs of motor dysfunction which occurred at 10-12 months of age. Purkinje cell loss, neuronal loss, gliosis, perivascular storage and demyelination accompanied disease progression. Increased vacuolation (15.3-fold increase compared to controls) coincided with advanced motor and mental deterioration in late-stage disease. Significant loss of myelin commenced early, with greatest impact in the cerebellum, and was severe in late disease (1.6- to 1.9-fold decrease) compared to controls (p < 0.05) contributing to clinical signs of motor and mental dysfunction. This detailed description and quantification of the CNS pathology in canine fucosidosis will inform monitoring of the onset, progression and response of this disease to therapy.

Fucosidosis in a domestic shorthair cat.

This paper documents the first reported case of fucosidosis in a cat. The cat presented with signs of forebrain and cerebellar dysfunction and a magnetic resonance imaging scan of the brain suggested a degenerative or metabolic disease process. A fine needle aspirate of grossly normal lymph nodes revealed vacuolated lymphocytes and a renal biopsy of an irregular shaped kidney identified vacuolated tubular epithelial cells. A white cell lysosomal enzyme screen revealed negligible α-fucosidase activity. Fucosidosis should be considered in the differential diagnosis of young cats with cerebellar dysfunction and must be added to the list of lysosomal storage diseases affecting the cat.

Genomic screening for fucosidosis in English Springer Spaniels.

OBJECTIVE: To develop a robust molecular genetic test for alpha-L-fucosidosis in English Springer Spaniels and to screen dogs from the United Kingdom and United States for the mutant allele. ANIMALS: 35 English-bred English Springer Spaniels, 60 American-bred English Springer Spaniels, and 1 affected dog and its parents from a family of English Springer Spaniels in Colorado. PROCEDURE: Polymerase chain reaction analysis was used to amplify the mutated region in the gene encoding alpha-L-fucosidase. High guanine-cytosine (GC) content of the region required use of an amplification buffer with high pH. Mutant and normal alleles were separated by polyacrylamide gel electrophoresis. Molecular genetic test results were compared with enzyme data. RESULTS: A 262-bp PCR product was amplified from normal dogs and compared with a 248-bp product from affected dogs. Carriers had 1 copy of each allele, distinguishable by the 14-bp size difference. Two carriers among the English-bred dogs were identified by use of enzyme and genomic DNA analyses. The molecular defect in dogs from Colorado was proven to be the same as that in British and Australian dogs. None of the other 60 American-bred dogs carried the mutant allele. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR method that can be used to identify dogs affected with or carriers of the autosomal recessive disease fucosidosis was established. Amplification was achieved within a GC-rich region, using a method that may be useful in overcoming amplification problems in GC-rich areas within other genes. Using this test, fucosidosis can be controlled and ultimately eradicated from the English Springer Spaniel population.

Deficiency of fucosidase results in acrosomal dysgenesis and impaired sperm maturation.

Although a variety of glycosyltransferases and glycosidases have been implicated in spermatogenesis and posttesticular sperm maturation, the biological role of these enzymes in these processes is largely unknown. We describe reproductive sequelae in a cohort of male dogs suffering from fucosidosis, a heritable lysosomal storage disorder caused by a severe deficiency of alpha-L-fucosidase. There was a reduction in the total number of sperm in the ejaculate. Only 3-5% of sperm were motile. None of the sperm were found to be morphologically normal. The predominant morphological defects observed were malformed acrosomes (56%) and retained proximal cytoplasmic droplets (92%), indicating that spermiogenesis and sperm maturation were impaired. The cytoplasm of all cellular components of the testis and excurrent ducts were vacuolated. The vacuolation resulted from enlargement of lysosomes caused by accumulation of compounds that are otherwise cleaved/degraded when lysosomal hydrolases are present normally. It is possible that impairment in spermatogenesis, particularly morphogenesis of the acrosome, is due to physical damage caused by anomalous enlargement of lysosomes. Although an unambiguous causal relationship could not be established, it is evident from the available information that the derangement in events associated with epididymal sperm maturation, namely acquisition of motility and shedding of the cytoplasmic droplet, is likely due to lack of fucosidase leading to impaired sperm membrane modification. This heritable condition in dogs may serve as a spontaneously occurring knock-out model for further elucidating the role of alpha-L-fucosidase in spermatogenesis and sperm maturation.

Fucosidosis in a family of American-bred English Springer Spaniels.

Fucosidosis was detected in a family of English Springer Spaniels in the United States. To our knowledge, these are the first reported cases of this disease in American-bred dogs. Affected and carrier status of dogs were determined by measuring the activity of the enzyme alpha-L-fucosidase in plasma and in leukocytes. Fucosidosis results in neurologic signs, particularly changes in behavior, in adolescent and adult dogs. Late onset of signs may result in misdiagnosis as a primary behavior problem or acquired neurologic disease. Fucosidosis is inherited in an autosomal recessive manner, and carrier dogs are clinically normal. Thus, the abnormal gene can become widespread in a population before homozygous-affected dogs are produced.

The molecular defect underlying canine fucosidosis.

Fucosidosis is a lysosomal storage disease which affects humans and English springer spaniel dogs. The disease is recessively inherited in both species and results from a deficiency of the enzyme alpha-L-fucosidase. We have recently cloned and sequenced the canine fucosidase gene (EMBL sequence admission number X92448 (cDNA) and X92671-X92678 (individual exonic data)). The gene spans 12 kb and consists of eight exons. SSCP based mutation analysis of affected animals was carried out on the coding region of this gene both with exonic primers, and intronic primer pairs for each exon. A 14 base pair deletion of the cDNA was identified at the 3' end of exon 1 in fucosidosis affected animals. Surprisingly, PCR based genomic cloning of DNA from these animals showed an identical deletion in this DNA, ending at the start of intron 1. This change causes a frameshift and, in consequence, 25 novel codons are transcribed in exon 2 before the first of two adjacent premature stop codons is encountered.

Isolation of the canine alpha-L-fucosidase cDNA and definition of the fucosidosis mutation in English Springer Spaniels.

Fucosidosis is a lysosomal storage disorder caused by deficiency of alpha-L-fucosidase. A biochemically and clinically well characterized canine model of fucosidosis exists in a colony of English Springer Spaniels. To facilitate its use as a model for gene therapy and enzyme replacement therapy in lysosomal storage disorders displaying neurological symptoms, isolation of the canine alpha-L-fucosidase cDNA was undertaken. Both the nucleotide sequence and the predicted amino acid sequence of canine fucosidase show high levels of identity with the human and rat sequences. Fucosidosis dogs were found to have a greatly reduced level of alpha-L-fucosidase mRNA when compared with normal dogs by Northern blot analysis. Direct PCR sequencing of products generated from cDNA demonstrated a 14-bp deletion in mRNA from affected dogs. This deletion creates a frameshift mutation and introduces a premature translation termination codon at amino acid position 152 and was shown to correspond to a deletion of the last 14 base pairs of exon 1 of the canine alpha-L-fucosidase gene. Rapid PCR-based screening for the mutation has now been performed on genomic DNA from dogs within the colony, enabling detection of both carriers and homozygotes.

Inherited lysosomal storage disease in an English springer spaniel.

Alpha-L-fucosidosis was diagnosed in a 17-month-old English Springer Spaniel with a history of slow development and progressive visual impairment. Lymphocytes and mononuclear cells with vacuolated cytoplasm were seen in a blood smear and in CSF, respectively. A severe deficiency of alpha-L-fucosidase activities in plasma and leukocytes was determined. Histologic examination revealed vacuolation of neurons, macrophages, and epithelial cells in most organ tissues. Canine fucosidosis is a progressive and fatal lysosomal storage disease in English Springer Spaniels. Affected dogs develop a neurologic disorder characterized by progressive motor and mental deterioration. Visual impairment is an unusual primary sign in a dog with alpha-L-fucosidosis.

Reproductive abnormalities in canine fucosidosis.

Male English springer spaniel dogs affected with fucosidosis, a lysosomal storage disorder, were found to be infertile while females with the disease reproduced successfully. Ejaculates of semen collected from affected dogs had reduced total sperm output and morphologically abnormal spermatozoa. A high proportion of ejaculated spermatozoa had midpiece droplets, bent tails and poor motility. Severely vacuolated epididymal epithelial cells were observed by light microscopy. Electron microscopic examination revealed membrane-bound vacuoles of variable size containing scanty amounts of granular to fibrillar material in epididymal epithelial cells, smooth muscle, myoid cells and Sertoli cells. Male infertility is believed to result from lysosomal storage of fucosyl-linked substrates in cells of the reproductive system. The extensive lesions in the epididymis may have interfered with maturation and transport of spermatozoa. Also, deficiency of alpha-L-fucosidase activity could have impaired the shedding of cytoplasmic droplets from spermatozoa and altered the surface glycoprotein composition of the sperm during epididymal transit.

Fucosidosis in an English springer spaniel presenting as a malabsorption syndrome.

A case of alpha-fucosidosis in a 2-year-old male English Springer Spaniel presented as a malabsorption syndrome without any clinical neurological abnormalities. The dog had a history of chronic weight loss, diarrhoea, mild anaemia, hypoproteinemia and reduced jejunal absorption of D-xylose. A diagnosis of fucosidosis with intestinal malabsorption was based on these findings, markedly reduced plasma fucosidase levels and the diffuse infiltration of the lamina propria and submucosa of the stomach, small intestine, Peyer's patches and mesenteric lymph nodes by macrophages with finely vacuolated cytoplasm. Cytoplasmic vacuolation was also a feature of cells of the pancreas, thryroid, parathyroid and adenohypophysis and the epithelia lining respiratory airways and the urogenital tract. Neurons of the autonomic plexuses of the gastrointestinal tract and the urinary bladder as well as those of the brain, spinal cord, spinal ganglia and retina were also vacuolated. The profound decrease in sigma-fucosidase activity in the brain, liver and kidney was accompanied by a marked increase in 6 other lysosomal enzymes, especially beta-n-acetyl glucosaminidase.

Human and canine fucosidosis: a comparative lectin histochemistry study.

Selected formalin-fixed, paraffin-embedded tissues of human and canine fucosidosis were stained with nine different lectins. Neurons, splenic sinusoidal cells, hepatic Kupffer cells, tissue macrophages, and capillary endothelium from human patients with fucosidosis stained intensely with Ulex europaeus agglutinin-I (UEA-I), but the same cells were unstained in tissues from canine fucosidosis. Since UEA-I specifically binds to terminal fucose residues, and fucose-rich undegraded metabolites are stored in affected cells of both human and canine fucosidosis, the variable lectin staining pattern demonstrates an unexpected species-specific histochemical variability. This finding highlights the fact that although both species have decreased fucosidase activity, the precursor substrates, undegraded stored metabolites, and particular cells affected by this enzyme deficiency are different.