The pathology of feline GM2 gangliosidosis.

An 11-week-old and a 6-month-old kitten with feline GM2 gangliosidosis and deficiency in both A and B isoenzymes of beta-D-N-acetyl hexosaminidase were studied by light transmission (TEM), and scanning electron microscopy (SEM). Neurons throughout the nervous system contained cytoplasmic, membrane-bound inclusions which were PAS-positive at the fine structure level these inclusions were composed of membranous arrays in whorls, vesicles, or multilaminated stacks. Fusion of the bounding membranes of adjacent inclusions resulted in large inclusion-containing vacuoles. Hepatocytes and Kupffer cells contained inclusions slightly different from those in the central nervous system. SEM of cryofractured liver demonstrated their coalescence to form larger composite vacuoles. Vacuoles with inclusions were also seen in pancreatic acinar cells, endothelial cells, vascular smooth muscle, fibroblasts, myocardial cells, renal interstitial cells, corneal stromal cells, and R-E cells of bone marrow and spleen. The specific granules of eosinophils were swollen and took on bizarre forms. Pathologic manifestations of feline GM2 gangliosidosis differ from those seen in feline GM1 gangliosidosis but closely resemble those of Sandhoff disease in humans.     

Mosaic uniparental disomy results in GM1 gangliosidosis with normal enzyme assay

Inherited metabolic disorders are traditionally diagnosed using broad and expensive panels of screening tests, often including invasive skin and muscle biopsy. Proponents of next‐generation genetic sequencing have argued that replacing these screening panels with whole exome sequencing (WES) would save money. Here, we present a complex patient in whom WES allowed diagnosis of GM1 gangliosidosis, caused by homozygous GLB1 mutations, resulting in β‐galactosidase deficiency. A 10‐year‐old girl had progressive neurologic deterioration, macular cherry‐red spot, and cornea verticillata. She had marked clinical improvement with initiation of the ketogenic diet. Comparative genomic hybridization microarray showed mosaic chromosome 3 paternal uniparental disomy (UPD). GM1 gangliosidosis was suspected, however β‐galactosidase assay was normal. Trio WES identified a paternally‐inherited pathogenic splice‐site GLB1 mutation (c.75+2dupT). The girl had GM1 gangliosidosis; however, enzymatic testing in blood was normal, presumably compensated for by non‐UPD cells. Severe neurologic dysfunction occurred due to disruptive effects of UPD brain cells.     

GM2神经节苷脂沉积症发病的分子机理研究

目的：探讨GM2神经节苷脂沉积症发病的分子机理。方法：培养患者皮肤成纤维细胞，进行酶学、Western印迹杂交和免疫细胞化学分析。结果：4例GM2神经节苷脂沉积症患者成纤维细胞内β-氨基已糖苷酶（hexosaminidase,Hex）活性均显著降低，分别为正常人的12％、3％、15％和6％；两例Sandhoff病Hex成熟的α、β-亚基（αm、βm）明显减少，但婴儿型与成年型减少的程度不同，1例Tay-Sachs病只有αm明显减少，1例GM2激活蛋白缺陷症αm、βm与正常人无差异；4例患者成纤维细胞内均有不同程度的GM2神经节苷酯的堆积。结论：GM2神经节苷脂沉积症的发病与HEXA、HEXB、GM2A基因突变引起相应蛋白表达、成熟障碍有关。     

Krabbe''s globoid cell leucodystrophy. Studies on galactosylceramide beta-galactosidase and non-specific beta-galactosidase of leucocytes, cultured skin fibroblasts, and amniotic fluid cells.

Galactosylceramide beta-galactosidase (cerebrosidase) and nonspecific beta-galactosidase activities were measured in both cultured skin fibroblasts and leucocytes from a family with Krabbe's globoid cell leucodystrophy (GLD). The activities of these enzymes were also determined in cultured skin fibroblasts of a patient with GM1 gangliosidosis and in cultured amniotic fluid cells. While cerebrosidase activity was deficient in GLD fibroblasts and leucocytes, its activity in GM1 gangliosidosis fibroblasts was increased. Two forms of each enzyme were found on isoelectric focusing, but in the GM1 gangliosidosis fibroblasts, cerebrosidase activity occurred as a single but intermediate peak. The use of cultured cells in assessing isoenzyme abnormalities associated with certain neurolipidoses is discussed.     

Ultrastructural findings of rectal and skin biopsies in adult GM1-gangliosidosis

We report the ultrastructural findings in rectal and skin biopsies in four cases with adult GM1-gangliosidosis. The rectal specimens taken under endoscopic control contained submucosal plexuses, which were easily identified on light-microscopic and electron-microscopic examination. In all cases the ganglion cells in submucosal plexus contained characteristic osmiophilic lamellar inclusions, some of which were typical membranous cytoplasmic bodies, were commonly observed in the ganglion cells, Schwann's cells, vascular endothelial cells, and perithelial cells in rectal biopsy, and membrane-bound clear vacuoles were occasionally seen in the cytoplasm of rectal and cutaneous fibroblasts. However, the axons of unmyelinated nerves in both biopsies showed a normal appearance, and there was no significant change in the eccrine glands in skin biopsy. Comparing rectal and skin biopsies, the former was found to be more valuable for morphological diagnosis of this disease, and our ultrastructural examination of rectal biopsy revealed that enteric nerve cells were involved even in adult GM1-gangliosidosis with a benign course and restricted cerebral lesions.     

Identification of a novel pseudodeficiency allele in the GLB1 gene in a carrier of GM1 gangliosidosis

The term 'pseudodeficiency' is used in lysosomal storage diseases to denote the situation in which individuals show greatly reduced enzyme activity but remain clinically healthy. Pseudodeficiencies have been reported for several lysosomal hydrolases. GM1 gangliosidosis is a rare autosomal recessive lysosomal storage disorder caused by beta-galactosidase hydrolase deficiency as a result of mutations in the GLB1 gene. Until now, two variants altering the beta-galactosidase activity have been described, p.Arg521Cys and p.Ser532Gly. Here we report the new variant p.Arg595Trp in the GLB1 gene, which markedly reduces beta-galactosidase activity when expressed in COS-1 cells. The variant was identified in the healthy father of a girl with GM1 gangliosidosis. He was a heterozygous compound with p.Arg595Trp in trans with one of the disease-causing mutations identified in his daughter; in leukocytes and plasma he showed lower beta-galactosidase activity than that observed in GM1 gangliosidosis carriers. As this family originated from the Basque Country in the north of Spain, we decided to analyse individuals of Basque and non-Basque origin, finding the p.Arg595Trp allele in 3.2% of Basque and in 0.8% of non-Basque alleles. The detection of the presence of alterations resulting in pseudodeficient activity in leukocytes and plasma is important for the correct diagnosis of GM1 gangliosidosis.     

Phenotype determining alleles in GM1 gangliosidosis patients bearing novel GLB1 mutations

Hofer D, Paul K, Fantur K, Beck M, Roubergue A, Vellodi A, Poorthuis BJ, Michelakakis H, Plecko B, Paschke E. Phenotype determining alleles in GM1 gangliosidosis patients bearing novel GLB1 mutations. GM1 gangliosidosis manifests with progressive psychomotor deterioration and dysostosis of infantile, juvenile, or adult onset, caused by alterations in the structural gene coding for lysosomal acid ß‐galactosidase (GLB1). In addition, allelic variants of this gene can result in Morquio B disease (MBD), a phenotype with dysostosis multiplex and entire lack of neurologic involvement. More than 100 sequence alterations in the GLB1 gene have been identified so far, but only few could be proven to be predictive for one of the GM1 gangliosidosis subtypes or MBD. We performed genotype analyses in 16 GM1 gangliosidosis patients of all phenotypes and detected 28 different genetic lesions. Among these, p.I55FfsX16, p.W65X, p.F107L, p.H112P, p.C127Y, p.W161X, p.I181K, p.C230R, p.W273X, p.R299VfsX5, p.A301V, p.F357L, p.K359KfsX23, p.L389P, p.D448V, p.D448GfsX8, and the intronic mutation IVS6‐8A>G have not been published so far. Due to their occurrence in homozygous patients, four mutations could be correlated to a distinct GM1 gangliosidosis phenotype. Furthermore, the missense mutations from heteroallelic patients and three artificial nonsense mutations were characterized by overexpression in COS‐1 cells, and the subcellular localization of the mutant proteins in fibroblasts was assessed. The phenotype specificity of 10 alleles can be proposed on the basis of our results and previous data.     

Late onset GM2 gangliosidosis: an α-locus genetic compound with near normal hexosaminidase activity

A non-Jewish child with late onset GM2 gangliosidosis is described. Tissues from the patient had near normal hexosaminidase A (hex A) activity using 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (MU-glcNAc) as substrate, and deficient activity when assayed with the 6-sulphate derivative of MU-glcNAc (MU-glcNAcS) or GM2 in the presence of activator. We present evidence that this patient is a genetic compound for different alpha-subunit mutations. The father's tissues have hex A activity in the heterozygote range when assayed with MU-glcNAcS, but normal activity using MU-glcNAc; the mother's tissues have activities toward both substrates in the heterozygote range. These results emphasize the pitfalls of using only MU-glcNAc for the diagnosis of unusual variants of GM2 gangliosidosis.     

Intramuscular distribution of nerves supplying the soleus muscle in the chimpanzee

The human soleus muscle is considered a specialized muscle in terms of its origin, insertion and muscle fibre architecture (especially with regard to the existence of the bipenniform part). Its peculiarities have been understood as results of erect posture and bipedal walking (Frey, 1913). Sekiya (1991) pointed out that another feature of the human soleus muscle the nerve supply, i.e. the muscle received two kinds of nerves, the anterior branch (R. anterior) and the posterior branch (R. posterior); the former supplied the bipenniform part at the anterior surface of the muscle and communicated with the R. posterior within the muscle. In nonhuman primates, the soleus muscle has no bipenniform part and the nerve, identical with the R. anterior to the human soleus muscle, is unknown. The purpose of the present study is to clarify the pattern of the nerve supply to the soleus muscle in the chimpanzee, with special reference to the intramuscular distribution of the nerves and to discuss the origin of the R. anterior to the human soleus muscle from a comparative anatomical point of view. Six soleus muscles from three chimpanzees (Pan troglodytes) were examined under a stereomicroscope to clarify the intramuscular distribution of nerves supplying these muscles. The nerves supplying the soleus muscle were classified into three types according to the sites of their entry into the muscle. The first group nerve was the thickest of all nerves innervating the muscle, entered the muscle at the posterior surface of the proximal third and was considered as homologous with the R. posterior in the human.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of 14 novel GLB1 mutations, including five deletions, in 19 patients with GM1 gangliosidosis from South America

GM1 gangliosidosis is a lysosomal storage disorder caused by the absence or reduction of lysosomal beta-galactosidase activity because of mutations in the GLB1 gene. Three major clinical forms have been established: type I (infantile), type II (late infantile/juvenile) and type III (adult). A mutational analysis was performed in 19 patients with GM1 gangliosidosis from South America, mainly from Argentina. Two of them were of Gypsy origin. Main clinical findings of the patients are presented. All 38 mutant alleles were identified: of the 22 different mutations found, 14 mutations are described here for the first time. Among the novel mutations, five deletions were found. Four of them are relatively small (c.435_440delTCT, c.845_846delC, c.1131_1145del15 and c.1706_1707delC), while the other one is a deletion of 1529 nucleotides that includes exon 5 and is caused by an unequal crossover between intronic Alu sequences. All the described patients with GM1 gangliosidosis were affected by the infantile form, except for four unrelated patients classified as type II, III, and II/III (two cases). The two type II/III patients bore the previously described p.R201H mutation, while the adult patient bore the new p.L155R. The juvenile patient bore two novel mutations: p.S434L and p.G554E. The two Gypsy patients are homozygous for the p.R59H mutation as are all Gypsy patients previously genotyped.     

GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: A potential model for Tay Sachs disease

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of β-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a β subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two β subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G_M2 gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.     

Ultrastructural classification of paracrystalline inclusions in untreated and estrogen treated human Leydig cells

Ultrastructural examination of untreated and estrogen-treated human testes revealed for the first time five types of paracrystalline inclusions in the cytoplasm of Leydig cells. The distinctions between each type of inclusion were based on longitudinal and cross-sectional ultrastructure. The inclusions were not found in Leydig cells containing Reinke crystals, nor were different types of inclusions found in the cytoplasm of the same cell. One form of inclusion or Reinke crystals were occasionally found in the nucleus. Some Leydig cells with intranuclear inclusions contained the same or a different type of inclusion in the cytoplasm. The present study revealed five types of paracrystalline inclusion in contrast to the one or two types described in previous reports. A possible explanation for this difference is that earlier investigators based their observations on small pieces of tissue obtained from biopsy, while the present data were obtained by studying many regions of each testis. All five types of paracrystalline inclusions were found in relatively normal as well as azoospermic (estrogen-treated) testes.     

The natural history of Type 1 infantile GM1 gangliosidosis: A literature-based meta-analysis

IntroductionType 1 GM1 gangliosidosis is an ultra-rare, rapidly fatal lysosomal storage disorder, with life expectancy of <3 years of age. To date, only one prospective natural history study of limited size has been reported. Thus, there is a need for additional research to provide a better understanding of the progression of this disease. We have leveraged the past two decades of medical literature to conduct the first comprehensive retrospective study characterizing the natural history of Type 1 GM1 gangliosidosis.ObjectivesThe objectives of this study were to establish a large sample of patients from the literature in order to identify: 1) clinically distinguishing factors between Type 1 and Type 2 GM1 gangliosidosis, 2) age at first symptom onset, first hospital admission, diagnosis, and death, 3) time to onset of common clinical findings, and 4) timing of developmental milestone loss.MethodsPubMed was searched with the keyword “GM1 Gangliosidosis” and for articles from the year 2000 onwards. A preliminary review of these results was conducted to establish subtype classification criteria for inclusion of only Type 1 patients, resulting in 44 articles being selected to generate the literature dataset of 154 Type 1 GM1 gangliosidosis patients. Key clinical events of these patient cases were recorded from the articles.ResultsComprehensive subtyping criteria for Type 1 GM1 gangliosidosis were created, and clinical events, including onset, diagnosis, death, and symptomology, were mapped over time. In this dataset, average age of diagnosis was 8.7 months, and average age of death was 18.9 months.DiscussionThis analysis demonstrates the predictable clinical course of this disease, as almost all patients experienced significant multi-organ system dysfunction and neurodevelopmental regression, particularly in the 6- to 18-month age range. Patients were diagnosed at a late age relative to disease progression, indicating the need for improved public awareness and screening.ConclusionThis study highlights the significant burden of illness in this disease and provides critical natural history data to drive earlier diagnosis, inform clinical trial design, and facilitate family counseling.     

Lacunar dilatations of intrafusal and extrafusal terminal cisternae, annulate lamellae, confronting cisternae and tubulofilamentous inclusions within the spectrum of muscle and nerve fiber changes in myotonic dystrophy

In 3 out of 5 muscle spindles available in skeletal muscle biopsy specimens from 30 patients with myotonic dystrophy (MD) unusually large lacunar dilatations of terminal cisternae were observed that had thus far only been reported in extrafusal muscle fibers. Cytoplasmic annulate lamellae, confronting cisternae and regularly proliferated terminal cisternae, as well as intranuclear tubulovesicular inclusions were found in extrafusal muscle fibers that in combination with concentric membranous bodies seen in perineurial cells and Schwann cells generally emphasize an involvement of the endoplasmic reticulum in the pathogenesis of MD. In addition, a nuclear inclusion body was observed composed of tubulofilamentous structures with close similarity to those thought to be rather specific for inclusion body myositis. Vesicles filled with amorphous material originating from outer spindle capsule cells were suggested to indicate matrical lipidic debris leading to "ghost bodies" and calcifying globules. Light microscopical evaluation of 8 sural nerve specimens revealed a neuropathy in only 2 patients that was predominantly axonal in type and of slight to moderate severity with a secondary demyelinating component in 1 patient. These findings add to the large spectrum of muscle and nerve fiber changes in MD underlining the phenotypic multiplicity of a well defined genetic defect.     

人股二头肌肌内神经分布和神经入肌点定位

目的查清人股二头肌长头和短头的肌内神经分布和神经入肌点定位,为其肌移植提供形态学依据。方法(1)观察20具尸体股二头肌长头和短头的神经分支数量,拟将坐骨结节与股骨外上髁连线分4等份,观测神经入肌点水平。(2)用3具童尸股二头肌做Sihler's肌神经染色,观察肌内神经分布。结果(1)长头神经来自坐骨神经胫侧,神经肌支一支型占22.5%,两支型72.5%,三支型5.0%,入肌点位于1/4区占22.1%,2/4区57.1%,3/4区20.8%。短头神经来自坐骨神经腓侧,一支型占95.0%,两支型5.0%,入肌点在肌的近部浅面。(2)长、短头肌内神经分支各形成一条神经支配带,横过各肌束中段。结论股二头肌长头和短头有单独神经支配。长头神经支配多见于两支型,神经入肌点多见于2/4区。     

Bone marrow transplantation in canine GM1 gangliosidosis

Allogeneic bone marrow transplantation was carried out in an 81-day-old Portuguese water dog with GM1 gangliosidosis using a DLA identical sibling as donor. Engraftment was complete and beta-galactosidase activity in leukocytes of the transplanted dog were similar to those in the donor. Over the next 2.5 months neurological deterioration in the transplanted dog was similar to that in untreated dogs with GM1 gangliosidosis. Cerebral ganglioside GM1 concentrations were not diminished by bone marrow transplantation and cerebral beta-galactosidase activity was negligible. We conclude that allogeneic bone marrow transplantation early in life is ineffective in canine GM1 gangliosidosis.     

Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm

Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.     

Adult Gmi gangliosidosis: Clinical and biochemical studies on two patients and comparison to other patients called variant or adult Gm1 gangliosidosis

Two young adult siblings were diagnosed as having a deficiency of acid beta-galactosidase activity in leukocytes and fibroblasts. The parents had enzyme levels approximately half of the normal level, consistent with this being the primary enzymatic lesion. Sialidose activities measured with natural and synthetic substrates in the patient's skin fibroblast cultures were normal. Hybridization of one of these patient's cells with cells from a patient with GM1 gangliosidosis, Type 1 did not show complementation of beta-galactosidase activity. However, when the cells from the patient were hybridized with cells from a patient with combined sialidase and beta-galactosidase deficiency, complementation was observed. These two siblings have ataxia, mild intellectual deterioration, slurred speech, mild vertebral changes and little, if any, visceromegaly. They do not have myoclonus, seizures or cherry-red spots, which are found in most patients with combined sialidase and beta-galactosidase deficiency. These patients are discussed with regard to other patients in the literature called variant or adult GM1 gangliosidosis.     

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

Deficiencies of lysosomal β-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant β-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal β-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.