Temporospatial Development of Neuropathologic Findings in a Canine Model of Mucopolysaccharidosis IIIB.

Mucopolysaccharidosis (MPS) IIIB is a neuropathic lysosomal storage disease characterized by the deficient activity of a lysosomal enzyme obligate for the degradation of the glycosaminoglycan (GAG) heparan sulfate (HS). The pathogenesis of neurodegeneration in MPS IIIB is incompletely understood. Large animal models are attractive for pathogenesis and therapeutic studies due to their larger size, outbred genetics, longer lifespan, and naturally occurring MPS IIIB disease. However, the temporospatial development of neuropathologic changes has not been reported for canine MPS IIIB. Here we describe lesions in 8 brain regions, cervical spinal cord, and dorsal root ganglion (DRG) in a canine model of MPS IIIB that includes dogs aged from 2 to 26 months of age. Pathological changes in the brain included early microscopic vacuolation of glial cells initially observed at 2 months, and vacuolation of neurons initially observed at 10 months. Inclusions within affected cells variably stained positively with PAS and LFB stains. Quantitative immunohistochemistry demonstrated increased glial expression of GFAP and Iba1 in dogs with MPS IIIB compared to age-matched controls at all time points, suggesting neuroinflammation occurs early in disease. Loss of Purkinje cells was initially observed at 10 months and was pronounced in 18- and 26-month-old dogs with MPS IIIB. Our results support the dog as a replicative model of MPS IIIB neurologic lesions and detail the pathologic and neuroinflammatory changes in the spinal cord and DRG of MPS IIIB-affected dogs.

An exonic insertion in the NAGLU gene causing Mucopolysaccharidosis IIIB in Schipperke dogs.

Mucopolysaccharidosis (MPS) IIIB (Sanfilippo syndrome B; OMIM 252920), is a lysosomal storage disease with progressive neurological signs caused by deficient activity of alpha-N-acetylglucosaminidase (NAGLU, EC 3.2.1.50). Herein we report the causative variant in the NAGLU gene in Schipperke dogs and a genotyping survey in the breed. All six exons and adjacent regions of the NAGLU gene were sequenced from six healthy appearing and three affected Schipperkes. DNA fragment length and TaqMan assays were used to genotype privately owned Schipperkes. A single variant was found in exon 6 of MPS IIIB affected Schipperkes: an insertion consisting of a 40-70 bp poly-A and an 11 bp duplication of the exonic region preceding the poly-A (XM_548088.6:c.2110_2111ins[A(40_70);2100_2110]) is predicted to insert a stretch of 13 or more lysines followed by either an in-frame insertion of a repeat of the four amino acids preceding the lysines, or a frameshift. The clinically affected Schipperkes were homozygous for this insertion, and the sequenced healthy dogs were either heterozygous or homozygous for the wild-type allele. From 2003-2019, 3219 Schipperkes were genotyped. Of these, 1.5% were homozygous for this insertion and found to be clinically affected, and 23.6% were heterozygous for the insertion and were clinically healthy, the remaining 74.9% were homozygous for the wild-type and were also clinically healthy. The number of dogs homozygous and heterozygous for the insertion declined rapidly after the initial years of genotyping, documenting the benefit of a DNA screening program in a breed with a small gene pool. In conclusion, a causative NAGLU variant in Schipperke dogs with MPS IIIB was identified and was found at high frequency in the breed. Through genotyping and informed breeding practices, the prevalence of canine MPS IIIB has been drastically reduced in the Schipperke population worldwide.

In-vivo cortical thickness estimation from high-resolution T1w MRI scans in healthy and mucopolysaccharidosis affected dogs.

Cortical thickness measurement estimated from high-resolution anatomical MRI scans may serve as a marker of cortical atrophy in clinical research applications. Most of the working algorithms and pipelines are optimized for human in-vivo data analyses that offer robust and reproducible measures. As animal-models are widely utilized in many preclinical phases of clinical trials the need for an optimized automated MRI data analysis to yield reliable data is warranted. We present a processing pipeline optimized for cortical thickness estimation of canine brains in native and template spaces. Preliminary results of 5 healthy and 5 mucopolysaccharidosis (MPS) dogs demonstrate single-canine mean/median cortical thickness in range of 2.69-3.58mm in native space and 3.26-4.15mm in template space. Our MRI generated values exceed previous histological measurements (observed mean about 2mm) in limited literature reports. Randomly selected manual measures corroborated the ranges defined by estimated cortical thickness probability density functions. Geometric transformations between native and template spaces change absolute mean/median cortical thickness values, but do not change the data nature and properties since the Pearson correlation coefficients between different space estimates were 0.84 for mean values and 0.89 for median values. No significant difference in total cortical thickness between MPS and age-and gender-matched dogs was observed.

Interference of an algal nutritional supplement with a urinary metabolic screening test for glycosaminoglycans in a dog suspected to have a storage disease.

The finding of excess urinary glycosaminoglycans (GAG) is the first step in the laboratory diagnosis of mucopolysaccharidosis (MPS). Urinary screening tests are based upon the binding of GAG to dimethylmethylene blue. Alternatively, paper spot tests using toluidine blue are used in human and veterinary laboratory medicine. Positive samples undergo GAG isolation for subsequent characterization. Here, we describe a 3-year-old English Cocker Spaniel with a positive urinary GAG test, but without other clinical signs of MPS. Urine samples were strongly positive with the dimethylmethylene blue test, and isolated GAG subjected to electrophoresis on cellulose acetate revealed a band co-migrating with dermatan sulfate. However, the isolated GAG were resistant to digestion with chondroitinase ABC, suggesting that the band did not represent dermatan sulfate. This was confirmed by mobility of the isolated GAG different from dermatan sulfate on agarose gel electrophoresis. MPS types VI and VII were excluded by enzyme assay. To test the hypothesis of a nutritional source, a healthy control dog was fed the same dog food as the index case. His urine showed a comparable abnormal GAG screening test and electrophoretic pattern. In addition, the analysis of an algal supplement present in the administered dog food showed a similar electrophoretic GAG pattern. The Cocker Spaniel was not available for further testing after withdrawal of the supplement. Algae contain highly sulfated fucans and galactans, and it appears that commercial dog food containing such algal, and possibly other, supplements can give rise to false-positive urinary MPS screening tests.

Diagnosis of caprine mucopolysaccharidosis type IIID by real-time polymerase chain reaction-based genotyping.

Mucopolysaccharidosis type IIID is caused by a deficiency of N-acetylglucosamine-6-sulfatase, which is one of the enzymes involved in the catabolism of heparin sulfate. Simple molecular marker assays underpin modern routine animal breeding and research activities worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important animal genetic disorders, the availability of routine assays for genotyping SNPs is of increased importance. In the current study, real-time polymerase chain reaction (PCR) is demonstrated to provide a valuable approach as a rapid and accurate alternative to a previously developed gel-based PCR as a straightforward and efficient assay for the diagnosis of caprine mucopolysaccharidosis IIID.

Radiographic evaluation of bones and joints in mucopolysaccharidosis I and VII dogs after neonatal gene therapy.

Mucopolysaccharidosis I (MPS I) and MPS VII are due to deficient activity of the glycosaminoglycan-degrading lysosomal enzymes alpha-L-iduronidase and beta-glucuronidase, respectively, and result in abnormal bones and joints. Here, the severity of skeletal disease in MPS I and MPS VII dogs and the effects of neonatal gene therapy were evaluated. For untreated MPS VII dogs, the lengths of the second cervical vertebrae (C2) and the femur were only 56% and 84% of normal, respectively, and bone dysplasia and articular erosions, and joint subluxation were severe. Previously, we reported that neonatal intravenous injection of a retroviral vector (RV) with the appropriate gene resulted in expression in liver and blood cells, and high serum enzyme activity. In this study, we demonstrate that C2 and femurs of RV-treated MPS VII dogs were longer at 82% and 101% of normal, respectively, and there were partial improvements of qualitative abnormalities. For untreated MPS I dogs, the lengths of C2 and femurs (91% and 96% of normal, respectively) were not significantly different from normal dogs. Qualitative changes in MPS I bones and joints were generally modest and were partially improved with RV treatment, although cervical spine disease was severe and was difficult to correct with gene therapy in both models. The greater severity of skeletal disease in MPS VII than in MPS I dogs may reflect accumulation of chondroitin sulfate in cartilage in MPS VII, or could relate to the specific mutations. Neonatal RV-mediated gene therapy ameliorates, but does not prevent, skeletal disease in MPS I and MPS VII dogs.

Radiographic features of feline joint diseases.

Feline joint disorders are often overlooked, possibly because cats are extremely agile and appear to cope with pathologic changes within their joints better than their canine counterparts. There is a growing awareness that osteoarthritis occurs more frequently than previously anticipated in cats, and recently we have seen the emergence of hip dysplasia as an entity of concern in some purebred cats. There are also several poorly understood conditions that affect the joints of cats, such as synovial osteochondromatosis, that invite further study. In recent years we have seen an expansion in the knowledge of immune-mediated and infectious arthropathies and their inter-relationship with infective agents. This article describes the radiographic changes seen in many of the currently recognized joint disorders of domestic cats.

Hepatic storage of glycosaminoglycans in feline and canine models of mucopolysaccharidoses I, VI, and VII.

Livers from normal cats and dogs, cats with mucopolysaccharidoses (MPS) I and VI, and dogs with MPS VII were analyzed biochemically and morphometrically to determine the lysosomal storage of glycosaminoglycans (GAG) in these animal models of human genetic disease. Analyses were performed on liver samples from seven normal cats ranging in age from 13 weeks to 15 months; six MPS I-affected cats ranging in age from 10 weeks to 26 months; four MPS VI-affected cats ranging in age from 9 months to 32 months; four normal dogs ranging in age from 1 month to 47 months; and three MPS VII-affected dogs, 5 days, 11 days, and 14 months of age. All of the animals were from the breeding colony at the University of Pennsylvania School of Veterinary Medicine and were maintained in accordance with national standards for the care and use of laboratory animals. Each GAG subclass was quantitated, and total GAG concentration was determined. Liver from cats with MPS I had the highest total GAG concentration (5.7 times that of the control), followed by liver from dogs with MPS VII (1.8 times) and cats with MPS VI (1.5 times). These data were very closely correlated (R2 = 0.982) with the results of the morphometric analyses of hepatocyte and Kupffer cell vacuolation associated with lysosomal storage and support the validity of both methods. This is particularly important for the quantification of total and individual GAG concentrations in tissue preparations. The values obtained should prove useful in future assessments of therapeutic regimes, such as enzyme replacement, bone marrow transplantation, and gene therapy, for these genetic diseases.

Morphology of leukocytes from cats affected with alpha-mannosidosis and mucopolysaccharidosis VI (MPS VI).

The morphology and ultrastructure of circulating white blood cells from six Persian and from five Russian Blue/Siamese cats deficient in lysosomal activity of alpha-mannosidase and arylsulfatase B, respectively, were studied and compared to cells from corresponding normal and carrier cats. In cats with mannosidosis, light microscopic examination revealed vacuoles in lymphocytes and monocytes, whereas electron microscopic studies demonstrated additional vacuoles in neutrophils, eosinophils, and basophils. In cats with mucopolysaccharidosis VI (MPS VI), vacuoles containing metachromatic granules were observed in lymphocytes, neutrophils, eosinophils, and monocytes. Ultrastructural studies of these cells identified the accumulation of fibrillar material, which often was associated with lamellated membrane structures.

Characterization of the defective beta-glucuronidase activity in canine mucopolysaccharidosis type VII.

Canine mucopolysaccharidosis type VII results from deficient activity of lysosomal beta-glucuronidase. Residual enzymatic activity (0.2-1.7% of normal) was detected in tissue homogenates from affected dogs. In contrast, serum and urine from affected animals had up to 15% residual activity. To further characterize the nature of the defective enzyme, hepatic beta-glucuronidase was partially purified from normal and MPS VII dogs for determination of their physical and kinetic properties. About 65% of the total beta-glucuronidase in normal canine liver required detergent for solubilization (i.e., membrane-associated), whereas only 22% of the residual activity in canine MPS VII liver was membrane-associated. Compared to the normal hepatic enzyme, the Km towards 4-methylumbelliferyl-beta-glucuronide was markedly increased in MPS VII dogs (i.e., 0.48 versus greater than 2.5 mmol/l). In contrast, the thermo-, cryo-, and pH stability properties, as well as the pH optimum (approximately 4.6), were essentially unaffected. In addition, the canine MPS VII hepatic residual activity was unresponsive to sulfhydryl reducing reagents and divalent cations, despite the fact that incubation of normal canine beta-glucuronidase with dithiothreitol and magnesium and/or calcium enhanced the activity more than 15-fold.

Long-term neurological effects of bone marrow transplantation in a canine lysosomal storage disease.

A naturally occurring disease in Plott hound dogs, caused by deficiency of the lysosomal enzyme alpha-L-iduronidase, was used to study the feasibility of bone marrow transplantation therapy in a neurodegenerative storage disease. Three long-term survivors of transplantation with littermate marrow at 5 months of age (before clinical signs) had CNS enzyme activity, glycosaminoglycan storage, and light microscopic and ultrastructural changes evaluated 594, 628, and 740 days after treatment. Iduronidase activity in small amounts (1-3% of donor values) was detectable in brain tissue. Cerebrospinal fluid had higher iduronidase activity after transplantation (7-15% of donor values). Enzyme activity within the CNS resulted in significant reductions in stored glycosaminoglycans and resolution, to a large extent, of light microscopic and ultrastructural lesions observed in affected, untreated littermate control dogs.

Disease expression in cultured pigment epithelium. Feline mucopolysaccharidosis VI.

Primary cultures of retinal pigment epithelial (RPE) cells from cats with mucopolysaccharidosis VI (MPS VI) have been initiated from mixed populations of cells (ie, derived from the entire eyecup and represented by both pigmented and nonpigmented RPE cells). The cells were enzymatically dissociated from the eyecup and seeded at 6 X 10(4) cells/cm2. Cells from normal and affected cats formed confluent monolayers of polygonal cells between 5-10 days in culture and maintained most of their in vivo morphologic characteristics. The only abnormality observed in the MPS VI-affected cultures was the accumulation of vacuolated intracytoplasmic inclusions; when numerous, these vacuoles caused cellular hypertrophy. Hypertrophy was present only in cells devoid of pigment. Pigmented cells adjacent to or near the hypertrophied cells exhibited little or no accumulation of vacuoles. The inclusions were indistinguishable from those observed in vivo in terms of size, distribution, and appearance. The MPS VI-affected RPE exhibited deficient arylsulfatase B (ASB) activity (RPE-ASB activity: normal = 506 nmol/hr/mg protein; affected = 22 nmol/hr/mg protein), whereas the activities of two other lysosomal enzymes, arylsulfatase A and alpha-L-iduronidase, were normal. A method was developed to initiate primary cultures of RPE cells from defined regions of normal and MPS VI-affected eyes. Studies indicated that cultures initiated from superior-equatorial regions (RPE nonpigmented) contained more vacuolated cytoplasmic inclusion than those initiated from inferior-equatorial regions (RPE pigmented). These findings indicated that the spatial distribution characteristic of the disease in vivo was maintained in culture and that disease expression was inversely correlated with pigmentation.

Correction of feline arylsulphatase B deficiency (mucopolysaccharidosis VI) by bone marrow transplantation.

Feline and human mucopolysaccharidosis VI (MPS VI or Maroteaux-Lamy syndrome) are inherited autosomal recessive deficiencies of lysosomal enzyme arylsulphatase B. Affected cats and children exhibit lesions caused by incompetent degradation, retinal atrophy and excessive urinary excretion of dermatan facial dysmorphia, corneal stromal opacities, leukocyte granulation, retinal atrophy and excessive urinary excretion of dermatan sulphate--and usually die before adulthood. Most attempts to treat humans affected with MPS VI or other mucopolysaccharidoses have been ineffective or logistically prohibitive, but allogeneic bone marrow transplantation (BMT) offers promise for cure of certain inborn errors of metabolism. Engraftment of normal donor marrow may endow the enzyme-deficient recipient with a continuous source of enzyme-competent blood cells and tissue macrophages to facilitate degradation of stored substrate and to prevent genesis of further malformations. To test this hypothesis, we performed allogeneic BMT in a 2-year-old male Siamese cat with advanced MPS VI. Here we describe BMT-induced correction of this hereditary enzyme deficiency.

Feline mucopolysaccharidosis VI: General ocular and pigment epithelial pathology.

Feline mucopolysaccharidosis VI (MPS VI) is a recessively inherited deficiency of arylsulfatase B (ASB). In the eye, the disease is expressed by the intracytoplasmic accumulation of vacuolated inclusions. These are present in connective tissue cells in the cornea, conjunctiva, sclera, choroid, and the stroma of the iris and ciliary body. In the iris and ciliary body epithelia, only the nonpigmented cells of the latter show presence of the disease. In the retinal pigment epithelium (RPE), a spatial and temporal distribution of the disease has been noted. In general, the nonpigmented RPE in the posterior pole is affected to a greater extent earlier in the disease; the peripheral pigmented RPE remains normal. Although hypertrophy of nonpigmented RPE cells causes disarray of the photoreceptor outer segments, their internal disc organization is not disrupted. Normal outer segment renewal rates and the presence of RPE phagosomes suggest that the diseased RPE is still able to function normally.

Spinal cord compression and hindlimb paresis in cats with mucopolysaccharidosis VI.

Six cats with mucopolysaccharidosis VI had hindlimb paresis and other clinical signs associated with compression of the thoracolumbar spinal cord. In 5 cats, the neurologic abnormality progressed over 2 to 4 weeks to loss of thoracolumbar spinal cord function. In 1 cat, the hindlimb paresis remained stable for 18 months. In the cats with progressive worsening of hindlimb function, the abnormality was caused by compression of the spinal cord from proliferation of bony tissue in the thoracolumbar region. In all affected cats, the compression occurred from T12 to L2. In 1 cat, an attempt to relieve the clinical signs by surgery was unsuccessful.

Feline mucopolysaccharidosis VI: purification and characterization of the resident arylsulfatase B activity.

Hepatic arylsulfatase B (ASB) from normal and mucopolysaccharidosis VI (MPS VI) cats was purified over 2,800- and 1,800-fold, respectively, and their physical and kinetic properties were characterized. In contrast to the normal feline enzyme, the partially purified MPS VI residual activity had a 100-fold greater Km value and was markedly less stable to thermal, cryo-, and pH-inactivation. In addition, the MPS VI enzyme had a more negative charge as determined by its migration on polyacrylamide gel electrophoresis and its elution profile on cation exchange chromatography. Finally, the MPS VI activity had approximately half the apparent molecular weight of the normal feline enzyme, which was a homodimer, suggesting that the genetic mutation in feline MPS VI altered the subunit association as well as the kinetic and stability properties of the mutant protein.