Morphological and biochemical studies of human beta-mannosidosis: identification of a novel beta-mannosidase gene mutation.

BACKGROUND: There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. beta-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Since then, 13 cases of beta-mannosidase deficiency in ten families have been described. A human beta-mannosidase mutation has been reported only by Alkhayat et al. in 1998. OBJECTIVES: To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with beta-mannosidosis. METHODS: Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the beta-mannosidase gene. RESULTS: Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of beta-mannosidase activity. Sequencing the beta-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)-->G splice site mutation in intron 7. CONCLUSIONS: To our knowledge, this is the first case of beta-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of beta-mannose moieties in glycoproteins in basal keratinocytes is suggested.

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

Characterization of a novel alpha-mannosidosis-causing mutation and its use in leukocyte genotyping after bone marrow transplantation.

Alpha-Mannosidosis is a lysosomal storage disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). Major symptoms include mental retardation, skeletal changes and recurrent infections. Recently, a successful bone marrow transplantation (BMT) in an alpha-mannosidosis patient was reported. Here we show that this patient was homozygous for a novel mutation, a 1-bp insertion (1197-1198insA) in exon 9 of the LAMAN gene. By using this mutation as a marker, we demonstrate that 1 year post-BMT, the LAMAN genotype of the patient's leukocytes was identical to that of the donor. This method of genotyping blood cells is a fast and accurate way to monitor the colonization of donor bone marrow cells.

Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis.

Alpha-mannosidase-II (alphaM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of alphaM-II should abolish complex N-glycan production as reportedly does inhibition of alphaM-II by swainsonine. We find that mice lacking a functional alphaM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct alpha-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for alphaM-II in the formation of erythroid complex N-glycans. alphaM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

Purification of feline lysosomal alpha-mannosidase, determination of its cDNA sequence and identification of a mutation causing alpha-mannosidosis in Persian cats.

alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal alpha-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal alpha-mannosidases sequences respectively. A 4 bp deletion was identified in an alpha-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal alpha-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder alpha-mannosidosis phenotype than the Persian cat had a lysosomal alpha-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline alpha-mannosidosis.

Angiokeratoma corporis diffusum associated with beta-mannosidase deficiency.

BACKGROUND: Angiokeratoma corporis diffusum (ACD) was at one time thought to be synonymous with Anderson-Fabry disease. However, it is well known that widespread angiokeratomas may also be found in other lysosomal enzyme disorders, as well as in patients with normal enzyme activities. beta-Mannosidase deficiency was first described in humans in 1986; since then, only 11 cases of beta-mannosidase deficiency, which occurred in 8 families, have been reported. Although the clinical manifestations are varied, mental retardation and neurologic disorders are present in practically all patients. OBSERVATIONS: We describe a 22-year-old woman who, since the age of 12 years, presented with progressive ACD affecting the lower limbs and the buttocks. Enzymatic studies revealed beta-mannosidase deficiency in cultured fibroblasts and in samples of serum and leukocytes. The patients's parents in turn exhibited intermediate enzyme levels, thus confirming the recessive autosomal hereditary nature of the disease. With the exception of an introverted character, the patient demonstrated no other anomalies. CONCLUSIONS: This is the first case of beta-mannosidase deficiency diagnosed as a result of purely dermatologic findings, in the form of ACD. beta-Mannosidase deficiency should therefore be included when screening for enzyme abnormalities in patients with ACD, even in the absence of neurologic disorders or mental retardation.

Multiple suture synostosis, macrocephaly, and intracranial hypertension in a child with alpha-D-mannosidase deficiency. Case report.

The authors present an unusual case in which increased intracranial pressure developed because of multiple-suture craniosynostosis and megaloencephaly in a child with a previously undiagnosed lysosomal storage disease, alpha-D-mannosidase deficiency. This 3-year-old boy presented with a history of frequent naps, headaches, florid papilledema, enlarged head (> 95th percentile), elevated opening pressure by lumbar puncture, a "beaten copper" appearance on skull radiographs, and no hydrocephalus. Multiple synostectomies were performed. Postoperatively, the child's headaches and papilledema resolved and his level of physical activity increased dramatically. The authors discuss the paradoxical presentation of prematurely fused sutures and macrocrania in light of this lysosomal storage disease and its subsequent management.

Alpha-mannosidosis. Report of a case with morphologic, cytologic and immunohistochemical considerations.

We report the histopathologic features of the knee bone and synovium and the cytologic features of the synovial fluid from a patient with alpha-mannosidosis. The synovium showed marked papillary hyperplasia with infiltration of foamy histiocytes containing periodic acid-Schiff-positive, diastase-resistant material. Severe degenerative changes were seen in the knee bone. The synovial fluid showed increased numbers of macrophages containing periodic acid-Schiff-positive, diastase-resistant material. The differential diagnostic considerations in the synovial fluid are also discussed.

Congenital dyserythropoietic anaemia type II (HEMPAS) and its molecular basis.

Congenital dyserythropoietic anaemia type II (CDA II) is a rare genetic anaemia in humans, inherited in an autosomally recessive mode. CDA II is also called HEMPAS as this disease is characterized by hereditary erythroblastic multinuclearity with positive acidified serum lysis test. Analyses of CDA II erythrocyte membranes showed that the band 3 glycoprotein is underglycosylated. An aberrant glycosylation pattern is seen in the polylactosamine carbohydrates which are normally attached to the band 3 and band 4.5 glycoproteins. The polylactosamines are, however, accumulated in the form of glycolipids. Therefore a genetic factor in CDA II appears to block the glycosylation of protein acceptors and shift these carbohydrates to the lipid acceptors. Structural analysis of CDA II band 3 carbohydrates identified truncated hybrid-type oligosaccharides and suggests that the Golgi glycosylation enzyme(s), alpha-mannosidase II or N-acetylglycosaminyltransferase II is defective in CDA II. By using a cDNA probe for alpha-mannosidase II, one CDA II case has been identified as being defective in the gene encoding alpha-mannosidase II. At present, it is not clear whether CDA II is a genetically heterogenous collection of glycosylation deficiencies, or genetically homogenous but apparently heterogenous in phenotype expression. Freeze-fracture electron microscopy and immunoelectron microscopy revealed that the band 3 glycoproteins are clustered in CDA II erythrocyte membranes. The abnormal distribution of band 3 might cause an unstable membrane organization. In CDA II erythroblasts, the membrane proteins might also be underglycosylated and abnormally distributed. When normal erythroblasts were cultured in vitro in the presence of swainsonine (alpha-mannosidase inhibitor) the erythroblasts became multinucleared. It is, therefore, quite possible that the enzymic defect of alpha-mannosidase II could cause various morphological anomalies including multinuclearity. Because the genes encoding glycosylation enzymes are housekeeping genes, the enzyme defect of CDA II is not restricted to erythroid cells and there is also an abnormal glycosylation of hepatocyte glycoproteins. On the other hand, there are many types of cells and tissues which appear not to be affected by the CDA II defect. A mechanism for the erythroid-specific manifestation of CDA II and its tissue specificity are also discussed.

Human chorionic beta-mannosidase: comparison with beta-mannosidase from human cultured fibroblasts.

The conditions for assay of beta-mannosidase activity in human chorionic villi (CV) were studied using the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannopyranoside. A comparison of the biochemical properties of the CV beta-mannosidase with those of the enzyme from human cultured fibroblasts showed their similarity. Like the enzyme from skin fibroblasts, the CV beta-mannosidase had rather high activity. Both enzymes had virtually the same pH optimum (4.2-4.7) and Km value. The data presented suggest that chorion biopsy specimens can be used for prenatal determination of beta-mannosidase activity at an early stage of development.

First-trimester diagnosis of an unusual case of alpha-mannosidosis.

Chorionic villi obtained in the first trimester from a pregnancy at risk for alpha-mannosidosis were shown to have reduced alpha-mannosidase (EC 3.2.1.24) activity. The pregnancy was terminated and subsequent enzyme studies of the fetal tissues were consistent with the diagnosis of alpha-mannosidosis. Like the enzyme in the child's fibroblast, alpha-mannosidase of the chorionic villi from a pregnancy at risk for alpha-mannosidosis was activated by high substrate concentration and by Zn2+, and displayed a Km value two-fold higher than normal. Our results confirm that chorionic villi can be used for early prenatal diagnosis of alpha-mannosidosis.

Otic pathology of caprine beta-mannosidosis.

Caprine beta-mannosidosis is an autosomal recessive defect of glycoprotein catabolism with a deficiency of tissue and plasma beta-mannosidase activity and tissue accumulation of oligosaccharides within lysosomes. This rapidly fatal genetic disorder of Nubian goats is expressed at birth by a variety of clinical signs including deafness. Affected goats had folded pinnas, and the tympanic cavity was decreased due to multiple, polypoid projections of bone covered by middle ear mucosa which obstructed the view of the cochlear promontory. Numerous cells of the cochlear duct including mesothelial and epithelial cells of Reissner's membrane, mesothelial cells lining the scala tympani, cells of the stria vascularis, numerous supportive cells of the organ of Corti, cochlear hair cells, endothelial cells, perithelial cells, fibroblasts, macrophages, and neurons of the spiral ganglion contained numerous nonstaining intracytoplasmic vacuoles which resulted in distention of affected cells and caused thickening of involved structures. Ultrastructurally, the vacuoles were membrane-bound and consistent with lysosomes. Vacuolated cells were desquamated into the scala vestibuli and scala tympani. This is one of few reports describing light and electron microscopic otic alterations of a storage disease. Goats with beta-mannosidosis appear to be good models of hearing loss in patients with storage disease.

Synthesis of lysosomal alpha-mannosidase in normal and mannosidosis fibroblasts.

The biosynthesis and secretion of lysosomal alpha-mannosidase was studied in metabolically labelled fibroblasts from controls and two patients with mannosidosis. Normal fibroblasts secrete alpha-mannosidase as a 110kDa polypeptide. Intracellularly alpha-mannosidase is represented by several polypeptides with apparent Mrs ranging from 40 to 67kDa. In two mannosidosis cell lines none of intra- and extracellular polypeptides of alpha-mannosidase were detectable. The mannosidosis fibroblasts secreted acid alpha-mannosidase activity at one third of the normal rate. In contrast to normal cells the secretion was not enhanced by NH4C1 and the secreted activity was not immunoprecipitable, indicating that the acid alpha-mannosidase activity secreted by mannosidosis fibroblasts is not related to the lysosomal alpha-mannosidase.

Pancytopenia in mannosidosis.

A case of autoimmune pancytopenia is described in a patient with mannosidosis who developed anti-platelet and anti-neutrophil antibodies and a low haptoglobin level. Bone marrow biopsy and 111InCl3 bone marrow scanning demonstrated hypoplasia of the marrow with a paucity of "storage cells." Pathogenic mechanisms and implications for other inborn errors of metabolism are discussed.

Genetic heterogeneity of I-cell disease is demonstrated by complementation of lysosomal enzyme processing mutants.

I-cell disease (mucolipidosis II) is a fatal childhood disorder affecting the expression of multiple lysosomal acid hydrolases. The disorder is characterized by clinical and biochemical heterogeneity which may reflect different mutants with a similar phenotype. Genetic complementation studies demonstrating genetic heterogeneity within this disorder are described utilizing cultured fibroblasts from 11 different patients. Fibroblasts from I-cell disease (ICD) and from five different lysosomal storage diseases with single structural gene enzyme deficiencies were fused in different combinations, and fractions enriched for multinucleated heterokaryons were isolated and tested for acid hydrolase activity and electrophoretic mobility. In fusions of ICD fibroblasts and various single lysosomal enzyme-deficient fibroblasts, the activity of the deficient enzyme and of the other ICD hydrolases were restored, demonstrating that ICD is not a lysosomal enzyme structural gene defect and that the ICD defect, and not just the single enzyme deficiency, is corrected. In fusions involving only I-cell fibroblasts, at least two complementation groups were identified by the recovery of activities of all lysosomal enzymes tested in heterokaryons. These results demonstrate the existence of genetic heterogeneity within the disorder and suggest that different mutations can result in the I-cell clinical and biochemical phenotype. The data support an altered post-translational processing of lysosomal enzymes as the cause of ICD and suggest that at least two genes participate in this pathway.

Electronmicroscopy of conjunctival biopsy in mannosidosis.

Electronmicroscopic examination was performed on conjunctival biopsies from two adolescent siblings with mannosidosis. Fibroblasts and endothelial cells contained membrane-bound vacuoles and vesicles that contained homogeneous osmiophilic globules. These vesicles seem to be pathognomonic of mannosidosis. Plasma cells also contained membrane-bound vacuoles, suggesting inhibition of the immunoglobulin production.

Unusual clinical and ultrastructural features in a boy with biochemically typical mannosidosis.

A 4 1/2-year-old boy with a history of recurring respiratory tract infections and seizures, and evidence of severe retardation of psychomotor development and growth, lacked the coarse facial features, skeletal changes and other clinical stigmata generally associated with mannosidosis, but the total alpha-mannosidase activity in his leukocytes, cultured fibroblasts and liver were no more than 10% of the control mean. Studies of the residual alpha-mannosidase enzyme suggest a specific deficiency of the thermostable isoenzyme with an acidic pH optimum. The alpha-mannosidase in the fibroblasts of our and another (control) patient with mannosidosis had a reduced affinity for the substrate 4-methylumbelliferyl-alpha-D-mannoside. Light microscopy of the liver biopsy showed an increase in connective tissue often distorting the hepatic architecture; numerous tiny vacuoles, small dense and lipid bodies in most hepatocytes, and similar but more extensive changes in sinusoidal cells; and sinusoidal pools of hepatocytic debris. Electron microscopy of hepatocytes revealed vacuoles similar but not identical to those described in reported mannosidosis patients, and in addition several forms of secondary lysosomes; prominent peroxisomes (microbodies); increased numbers of profiles of smooth endoplasmic reticulum; dilated rough endoplasmic reticulum containing traces of fine granulo-fibrillar material; increased numbers of rosettes of alpha particles of glycogen and reduced numbers of mitochondria with alterations in their distribution, size and configuration. It is believed that the usual clinical and hepatic ultrastructural features in our patient reflect another variant of mannosidosis.

Urinary oligosaccharide excretion in disorders of glycolipid, glycoprotein and glycogen metabolism. A review of screening for differential diagnosis.

In recent years great interest has centered around metabolic disorders in which excessive oligosacchariduria is a prominent feature. This review describes the methods of both structural and diagnostic investigations of oligosaccharides in a number of these diseases. Special emphasis has been laid upon simple screening methods which would avail themselves to the clinical chemistry laboratory