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.

Sphingomyelin lipidosis (Niemann-Pick disease) in a juvenile raccoon (Procyon lotor).

A wild caught juvenile male raccoon with neurological disease was humanely destroyed due to poor prognosis. Necropsy examination revealed hepatomegaly, splenomegaly and multicentric lymphadenomegaly with diffuse hepatic pallor and pulmonary consolidation with pinpoint pale subpleural foci. Microscopically, there was marked pale cytoplasmic swelling of the central and peripheral neurons as well as the glial cells in the brain, accompanied by multiorgan infiltration by abundant foamy macrophages. Ultrastructural investigation revealed accumulation of concentrically arranged lamellar material within lysosomes of the affected neurons, macrophages and endothelial cells. Biochemical enzymatic analysis detected sphingomyelinase deficiency and lysosomal storage disease consistent with sphingomyelin lipidosis (Niemann-Pick disease [NPD]) was diagnosed. This is the first report of NPD in a raccoon.

Sphingomyelinase deficiency (Niemann-Pick disease) in a Hereford calf.

A 5-month-old Hereford calf with neurologic disease was euthanatized, and a necropsy was done. No gross lesions were seen in the brain. Microscopically, neurons throughout the brain and spinal cord had distended, foamy vacuolated cytoplasm. Ultrastructure showed clear vacuoles filling the neuronal cytoplasm. A lysosomal storage disease was suspected. Sphingomyelinase deficiency was confirmed by biochemical analysis of liver and brain.

Abnormal neuronal metabolism and storage in mucopolysaccharidosis type VI (Maroteaux-Lamy) disease.

Mucopolysaccharidosis (MPS) type VI, also known as Maroteaux-Lamy disease, is an inherited disorder of glycosaminoglycan catabolism caused by deficient activity of the lysosomal hydrolase, N-acetylgalactosamine 4-sulphatase (4S). A variety of prominent visceral and skeletal defects are characteristic, but primary neurological involvement has generally been considered absent. We report here that the feline model of MPS VI exhibits abnormal lysosomal storage in occasional neurones and glia distributed throughout the cerebral cortex. Abnormal lysosomal inclusions were pleiomorphic with some resembling zebra bodies and dense core inclusions typical of other MPS diseases or the membranous storage bodies characteristic of the gangliosidoses. Pyramidal neurones were shown to contain abnormal amounts of GM2 and GM3 gangliosides by immunocytochemical staining and unesterified cholesterol by histochemical (filipin) staining. Further, Golgi staining of pyramidal neurones revealed that some possessed ectopic axon hillock neurites and meganeurites similar to those described in Tay-Sachs and other neuronal storage diseases with ganglioside storage. Some animals evaluated in this study also received allogeneic bone marrow transplants, but no significant differences in neuronal storage were noted between treated and untreated individuals. These studies demonstrate that deficiency of 4S activity can lead to metabolic abnormalities in the neurones of central nervous system in cats, and that these changes may not be readily amenable to correction by bone marrow transplantation. Given the close pathological and biochemical similarities between feline and human MPS VI, it is conceivable that children with this disease have similar neuronal involvement.

Peripheral neuropathy with hypomyelinating features in adult-onset Krabbe's disease.

We describe three brothers suffering from Krabbe's disease with onset in the fifth decade. The proband showed a complete deficiency of leukocyte enzyme galactocerebrosidase and was found to be heterozygous for two previously described mutations: G > A809 and 502T/del consisting of a 30 kb deletion. In all three brothers the neurological examination showed features of asymmetrical peripheral neuropathy associated with pyramidal signs and the electrophysiological examination showed a generalized slowing of nerve conduction velocities. Two patients died at 59 and 61 years of age due to respiratory failure. Both the proband and his brother underwent a sural nerve biopsy. In the former the most striking finding was the presence of uniformly thin myelin sheaths without evidence of demyelination; a complete absence of fibers was found in the latter. Our findings confirm that peripheral neuropathy may be the presenting feature of late-onset Krabbe's disease. Hypomyelination rather than demyelination may represent the distinguishing pathological finding of this condition.

Effects of dietary cholesterol restriction in a feline model of Niemann-Pick type C disease.

A feline model of Niemann-Pick disease type C (NPC) was employed to evaluate the effect of dietary cholesterol restriction on progression of disease. Two NPC-affected treated cats were fed a cholesterol-restricted diet beginning at 8 weeks of age; the cats remained on the diet for 150 and 270 days respectively. The study goal was to lower the amount of low density lipoprotein (LDL) available to cells, hypothetically reducing subsequent lysosomal accumulation of unesterified cholesterol and other lipids. Neurological progression of disease was not altered and dietary cholesterol restriction did not significantly decrease storage in NPC-affected treated cats. One NPC-affected treated cat had decreased serum alkaline phosphatase activity (ALP) and decreased serum cholesterol concentration. Liver lipid concentrations of unesterified cholesterol, cholesterol ester and phospholipids in NPC-affected treated cats were similar to those seen in NPC-affected untreated cats. Ganglioside concentrations in the NPC-affected treated cats and NPC-affected untreated cats were similar. Histological findings in liver sections from NPC-affected treated cats showed a diffuse uniform microvacuolar pattern within hepatocytes and Kupffer cells, in contrast to a heterogeneous macro/microvacuolar pattern and prominent nodular fibrosis in NPC-affected untreated cats. Similar differences in vacuolar patterns were seen in splenic macrophages. Although some hepatic parameters were modified, dietary cholesterol restriction did not appear to alter disease progression in NPC-affected kittens.

Retrovirus-mediated gene transfer and galactocerebrosidase uptake into twitcher glial cells results in appropriate localization and phenotype correction.

Galactocerebrosidase (GALC) is deficient in all tissues from human patients and animal models with globoid cell leukodystrophy (GLD) or Krabbe disease. The deficiency results in decreased lysosomal catabolism of certain galactolipids including galactosylceramide and psychosine that are synthesized maximally during myelination. According to current theories, the accumulation of psychosine in humans and animals with GLD induces oligodendrocyte degeneration and myelination ceases. Transduction of oligodendrocytes from twitcher mice with a retroviral vector containing the GALC cDNA can correct the enzyme deficiency in these cells. Our data show that twitcher astrocytes and oligodendrocytes can internalize exogenous GALC, as well as donate the enzyme to the mutant glial cells. Antibodies against human GALC localized the GALC antigen in retrovirally transduced cells and cells receiving enzyme via cell to cell secretion and uptake to the lysosomal fraction. In fact immunocytochemical studies in transduced oligodendrocytes revealed that the GALC colocalizes in vesicles lysosomal-associated membrane protein-2 (LAMP2) (+). Moreover, labeling cells with anti-GALC and a marker for oligodendrocytes demonstrated that, upon differentiation, transduced, twitcher oligodendrocytes attained the normal branched process configuration, while untransduced cells show only abnormal morphology. Phenotype correction in mutant oligodendrocytes has also been observed after enzyme transfer. These studies indicate that GALC activity supplied to cultured oligodendrocytes from twitcher mice by different methods can correct the pathological phenotype of these cells.

Clinical course and biochemistry of sialuria.

Sialuria is a rare inborn error of metabolism in which excessive free sialic acid (N-acetylneuraminic acid, NeuAc) is synthesized. A defect in the feedback inhibition of UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase by the end-product of the sialic acid synthetic pathway, CMP-NeuAc, is the mechanism underlying this overproduction. Recent evidence suggests that sialuria is an autosomal dominant disorder. Only five patients have been documented to have such an enzymatic defect. We report a longitudinal study of one of the original sialuria patients, to age 11 years. Although he has coarse features and massive hepatomegaly, he has shown normal growth and relatively normal development. Pulmonary function testing showed minimal small airway obstruction. At 11 years, he developed intermittent abdominal pain and transient transaminase elevation above his baseline. Sialuria should be considered in the differential diagnosis of a patient with a phenotype suggestive of a mucopolysaccharidosis or oligosaccharidosis in the absence of developmental regression or prominent dysostosis multiplex. We recommend close monitoring of liver and pulmonary function in sialuria patients.

Generation of a mouse with low galactocerebrosidase activity by gene targeting: a new model of globoid cell leukodystrophy (Krabbe disease).

Globoid cell leukodystrophy (Krabbe disease) is a severe leukodystrophy caused by mutations in the galactocerebrosidase (GALC) gene leading to extremely low (less than 5% of normal activity) GALC activity. Human patients include primarily severely affected infants as well as patients with a later onset of symptoms. The infants usually die before 2 years of age, but it is difficult to predict the clinical course in older patients. In addition to these patients, additional individuals identified in this laboratory have 10--20% of normal GALC activity measured in accessible tissues. These individuals have a wide range of clinical presentations involving neurological degeneration. On molecular analysis of the GALC gene they all have three or more mutations considered to be normal polymorphisms resulting in amino acid changes in the two copies of the GALC gene. In order to investigate the role these amino acid changes may play on clinical, biochemical, and pathological findings, a new transgenic mouse was generated by homologous recombination. After preliminary studies determined what effect each amino acid change had on mouse GALC activity in transient transfection experiments, mice containing a cysteine residue at codon 168 instead of histidine (H168C) were produced. These mice developed symptoms, but they were delayed by 10--15 days from the well-characterized twitcher (twi) mouse. They accumulated psychosine slightly slower than twi mice, showed pathological changes less severe than twi mice in the central and peripheral nervous systems, and live about 15 days longer than twi mice. They have large litters and will play a role in therapy trials using new procedures currently under development.

Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop.

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.

Addressing multiple problems in the family practice office visit.

OBJECTIVES: The purpose of the study was to describe the number of problems addressed during family practice outpatient visits, the nature of additional problems raised, how they affect the duration of the visit, and how well they are reflected in the billing record. STUDY DESIGN: Cross-sectional. POPULATION: We studied a total 266 randomly selected adult patient encounters representing 37 physicians. OUTCOMES MEASURED: A problem was defined as an issue requiring physician action in the form of a decision, diagnosis, treatment, or monitoring. Visit duration and the number of billing diagnoses were also assessed. RESULTS: On average, 2.7 problems and 8 physician actions were observed during an encounter. More than one problem was addressed during 73% of the encounters; 36% of these additional problems were raised by the physician and 58% by the patient. On average, each additional problem increased the length of the visit by 2.5 minutes (P<.001). The concordance between the number of problems observed and the number of problems on the billing sheet indicated a trend toward underbilling the number of problems addressed. CONCLUSIONS: Multiple problems are commonly addressed during family practice outpatient visits and are raised by both the physicians and the patients. Our findings suggest that current views of physician productivity and the billing record are poor indicators of the reality of providing primary care.

Niemann-Pick disease type C: two cases and an update.

We describe two patients with juvenile-onset Niemann-Pick disease type C (NPC) to illustrate the variable neurologic features of this condition. One presented with hypersplenism at age 10 and was misdiagnosed with Gaucher disease. He developed complex partial seizures in his teens but remained otherwise neurologically asymptomatic until his mid 30s. At age 45, he had mild dementia and dysarthria, vertical supranuclear ophthalmoplegia, axonal sensorimotor polyneuropathy, and cerebellar ataxia. The second patient presented with rapidly progressive dystonia at age 8, and mild hepatosplenomegaly, vertical supranuclear ophthalmoplegia, severe behavioral disorder, and dementia by age 14. The diagnosis of NPC was based on deficient cholesterol esterification and excessive lysosomal filipin staining in cultured skin fibroblasts. Current notions about diagnosis and pathogenesis of NPC are reviewed.

Murine, canine and non-human primate models of Krabbe disease.

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessively inherited neurological disease caused by mutations in the gene coding for the lysosomal enzyme galacto-cerebrosidase (GALC). GALC is responsible for the degradation of specific galactolipids, including several that are important in the production of compact, stable myelin. A failure to adequately degrade galactosylceramide and psychosine (galactosylsphingosine) results in the characteristic pathological findings observed in tissue from humans and animals affected with GLD. These galactosphingolipids are normally synthesized during active myelination, and psychosine accumulates in individuals with very low GALC activity. Psychosine is highly toxic to the myelin-forming oligodendrocytes, causing their death and the paucity of myelin found on autopsy. While most human patients present with symptoms before six months of age and die before 18 months of age, older children and adults can also be diagnosed with GLD[1,2]. The cloning of both the human GALC cDNA and the GALC gene opened the way for the identification of mutations causing GLD in humans and animals and the development of novel strategies to treat this severe and fatal disease[3]. The pheno-typic differences between human patients result from the wide range of mutations identified, as well as additional unknown factors. Treatment of late-onset patients and pre-symptomatic individuals (identified either because prenatal testing was not requested or a fetus predicted to be affected was not aborted) by hemato-poietic stem cell transplantation (HSCT) resulted in a less severe phenotype than was predicted and, in some cases, a significant delay in the onset of symptoms[4]. Attempts to treat this disorder by in utero HSCT have not been successful[5].GLD in dogs

Mucopolysaccharidosis VII in a cat.

Mucopolysaccharidosis VII was diagnosed in a domestic shorthair cat from California. The cat was small and had multiple abnormalities, including a small body disproportionate to the size of the skull, angular deformities of the ribs, abnormally short forelimbs, luxating patellas, generalized epiphyseal dysplasia involving the vertebrae and long bones, cuboidal vertebrae, pectus excavatum, subluxation of both hips, osteosclerosis of the tentorium cerebelli and left petrous temporal bone, tracheal hypoplasia, and corneal clouding. Beta-glucuronidase activity was markedly decreased in peripheral blood leukocytes. The cat died at 21 months of age, and a complete necropsy was performed. Tissues were examined by light and transmission electron microscopy. Large clear, round vacuoles representing distended lysosomes were present in many epithelial and connective tissue cells, including fibrocytes, chondrocytes, smooth muscle cells, hepatocytes, astrocytes, and macrophages.

MR imaging and proton MR spectroscopy in adult Krabbe disease.

We present the MR imaging findings in four patients (two pairs of siblings from two unrelated families) with adult Krabbe disease. In the first family, clinical presentation mimicked familial spastic paraplegia. Their MR images showed selective, increased signal intensity on T2-weighted sequences along the corticospinal tracts, most prominently in the proband and barely detectable in her brother. Proton MR spectroscopy showed increased choline and myo-inositol in the affected white matter. In the second family, the clinical presentation differed in that the signs of pyramidal tract involvement were asymmetrical, with concomitant asymmetry on MR images in one. In adults, Krabbe disease may present on MR imaging with selective pyramidal fiber involvement.

Krabbe disease: genetic aspects and progress toward therapy.

Krabbe disease or globoid cell leukodystrophy is a disorder involving the white matter of the peripheral and central nervous systems. Mutations in the gene for the lysosomal enzyme galactocerebrosidase (GALC) result in low enzymatic activity and decreased ability to degrade galactolipids found almost exclusively in myelin. The pathological changes observed, including the presence of globoid cells and decreased myelin, appear to result from the toxic nature of psychosine and accumulation of galactosylceramide that cannot be degraded due to the GALC deficiency. Over 60 mutations have been identified in this gene. The great majority are disease-causing; however, a few are considered polymorphisms. While most patients present with symptoms within the first 6 months of life, others present later in life including adulthood. Even patients with the same genotype can have very different clinical presentations and course. The reason for this is not known. Treatment at this time is limited to hematopoietic stem cell transplantation that appears to slow the progression of the disease and improve the magnetic resonance images. Studies using stem cells and viral vectors to transduce transplantable cells are under way in model systems. In culture, oligodendrocytes from the twitcher mouse model can assume a normal appearance after differentiation if GALC activity is provided via viral transduction or uptake from donor cells. Therefore continued myelination and/or remyelination in patients will require supplying GALC activity by transplanted cells or viral vectors to still functional endogenous oligodendrocytes or transplantation of normal oligodendrocytes or stem cells that can differentiate into oligodendrocytes. Using the animal models these options can be explored.

Investigating demyelination in the brain in a canine model of globoid cell leukodystrophy (Krabbe disease) using magnetization transfer contrast: preliminary results.

PURPOSE: This study was designed to examine the use of quantitative magnetization transfer imaging (MTI) in naturally occurring globoid cell leukodystrophy (GLD) in the Cairn terrier. METHOD: A model of GLD was established via a breeding colony, and a total of seven animals were studied with MTI, including two dogs with GLD, one of which underwent whole-body irradiation (725 cGy) and bone marrow transplantation from a genotypically normal littermate. The remaining dogs served as untreated, irradiated, and unirradiated controls. RESULTS: Region-of-interest (ROI) analysis of the MTI showed a decrease in MT ratio (MTR) in the internal capsule of the untreated/affected dog compared with age-matched controls but revealed similar results in the two other study animals. On MT contour plotting, inside-to-out gradients of MTR mimicked the demyelination pathology of the disease in the untreated/affected dog. CONCLUSION: MT contour plotting demonstrated patterns of MT abnormality in the untreated/affected dog that were consistent with histopathology, establishing a clear relationship between pathology-proven demyelination and MTR as well as a striking contrast to the patterns of radiation damage.

Niemann-Pick C1 disease: the I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype.

Niemann-Pick type C (NPC) disease is an autosomal recessive lipid-storage disorder usually characterized by hepatosplenomegaly and severe progressive neurological dysfunction, resulting from mutations affecting either the NPC1 gene (in 95% of the patients) or the yet-to-be-identified NPC2 gene. Our initial study of 25 patients with NPC1 identified a T3182-->C transition that leads to an I1061T substitution in three patients. The mutation, located in exon 21, affects a putative transmembrane domain of the protein. PCR-based tests with genomic DNA were used to survey 115 unrelated patients from around the world with all known clinical and biochemical phenotypes of the disease. The I1061T allele constituted 33 (14.3%) of the 230 disease-causing alleles and was never found in controls (>200 alleles). The mutation was particularly frequent in patients with NPC from Western Europe, especially France (11/62 alleles) and the United Kingdom (9/32 alleles), and in Hispanic patients whose roots were in the Upper Rio Grande valley of the United States. The I1061T mutation originated in Europe and the high frequency in northern Rio Grande Hispanics results from a founder effect. All seven unrelated patients who were homozygous for the mutation and their seven affected siblings had a juvenile-onset neurological disease and severe alterations of intracellular LDL-cholesterol processing. The mutation was not found (0/40 alleles) in patients with the severe infantile neurological form of the disease. Testing for this mutation therefore has important implications for genetic counseling of families affected by NPC.

Protracted course of Krabbe disease in an adult patient bearing a novel mutation.

BACKGROUND: Krabbe disease, or globoid cell leukodystrophy, is an autosomal recessive disorder caused by the deficiency of galactocerebrosidase (GALC) activity. Although most cases are diagnosed in infancy and show a fatal outcome in childhood, adult patients have been identified, showing progressive spastic hemiparesis to tetraparesis, followed by optic atrophy, dementia, and neuropathy. The disease can be diagnosed by detecting the deficiency of GALC activity (less than 5% of normal) in any available tissue sample. The cloning of the human GALC gene allowed the molecular characterization of newly diagnosed patients. More than 75 disease-causing mutations and polymorphisms in this gene have been identified. OBJECTIVE: To describe a 28-year-old woman with Krabbe disease, correlating clinical and biochemical abnormalities to a novel mutation on the GALC gene. METHODS: Clinical investigation was enriched by neurophysiological and neuroimaging data. The activity of GALC was assayed in white blood cells using radiolabeled natural substrate. Genomic DNA was isolated from peripheral blood, and the GALC gene was sequenced. The mutated gene was expressed and GALC activity was measured in transfected COS-1 cells. RESULTS: The patient had progressive and bilateral amaurosis starting at 8 years of age. Although she was experiencing weakness in all her extremities, her intellect remained intact. She was found to be homozygous for a previously unreported missense mutation (T1886G), which leads to low, but not totally deficient, GALC activity. CONCLUSIONS: Expression of this mutation in COS-1 cells using the pcDNA3 expression vector (Invitrogen, Carlsbad, Calif) resulted in low, although not null, GALC activity, which can explain the protracted clinical course in this patient. Patients carrying the mutation described herein might be potential candidates for therapeutic trials, such as bone marrow transplantation or gene therapy.