B-cell-specific accumulation of inclusion bodies loaded with HLA class II molecules in patients with mucolipidosis II (I-cell disease).

BACKGROUND: I-cell disease is characterized by the presence of vacuole-like inclusions in lymphocytes. However, the nature and clinical significance of these inclusions have seldom been characterized. In this study, the authors tried to elucidate the distribution in different lymphocyte subpopulations, and the histological nature of the inclusions. METHODS: Blood samples from three unrelated patients were analyzed. Lymphocyte subpopulations were separated using monoclonal antibodies conjugated to immunomagnetic beads. Cytochemical studies were performed using FITC-conjugated lectins. The expressions of surface and cytoplasmic class II molecules were analyzed by flow cytometry. RESULTS: Virtually all B cells from the patients contained the inclusions. In contrast, CD4+ T cells, CD8+ T cells, natural killer cells, monocytes, or neutrophils did not contain the inclusions. Both fibroblasts and B cells from I-cell patients were stained intensely by multiple FITC-conjugated lectins with distinct binding profiles. The inclusions of B cells were stained intensely by fluorescence-conjugated antibodies against class II antigens. CONCLUSIONS: Inclusions in I-cell disease reflect the accumulation of HLA class II molecules within B cells. These results suggest a potential role for N-acetylglucosamine-1-phosphotransferase in immune functions. Furthermore, the fact that only B cells contain the inclusions provides a novel diagnostic aid for the diagnosis of I-cell disease.

Hereditary fructose intolerance mimicking a biochemical phenotype of mucolipidosis: A review of the literature of secondary causes of lysosomal enzyme activity elevation in serum.

We describe a patient with failure to thrive, hepatomegaly, liver dysfunction, and elevation of multiple plasma lysosomal enzyme activities mimicking mucolipidosis II or III, in whom a diagnosis of hereditary fructose intolerance (HFI) was ultimately obtained. She presented before introduction of solid foods, given her consumption of a fructose-containing infant formula. We present the most extensive panel of lysosomal enzyme activities reported to date in a patient with HFI, and propose that multiple enzyme elevations in plasma, especially when in conjunction with a normal plasma α-mannosidase activity, should elicit a differential diagnosis of HFI. We also performed a review of the literature on the different etiologies of elevated lysosomal enzyme activities in serum or plasma. © 2016 Wiley Periodicals, Inc.

Clinical and laboratory outcomes after umbilical cord blood transplantation in a patient with mucolipidosis II alpha/beta.

Mucolipidosis (ML) II alpha/beta is an autosomal recessive disease caused by reduced enzyme activity of N-acetylglucosamine-1-phosphotransferase. Clinical symptoms of ML II are severe psychomotor delay and dysostosis multiplex; death usually occurs by 5-8 years of age from cardiopulmonary complications. Allogeneic hematopoietic stem cell transplantation (HSCT) has been attempted for ML; however, few reports have documented the detailed outcomes of HSCT for ML. A 26-month-old girl received a human leukocyte antigen 3/6-allele-matched transplant from cord blood. The preparative regimen consisted of fludarabine, cyclophosphamide, 6-Gy total body irradiation, and rabbit antithymocyte globulin. Although comparing before and after cord blood transplantation results, we observed that lysosomal enzyme activities in the plasma decreased by approximately 20-40%. Low serum levels of immunoglobulin A, G2, and G4 were also observed before HSCT; however, these values normalized after transplantation. Despite undergoing HSCT, she was treated twice for bacterial pneumonia with acute respiratory distress syndrome at ages 37 and 38 months. Although HSCT effects on the clinical manifestations were limited, laboratory data including plasma lysosomal enzyme activities and serum levels of immunoglobulin showed improvement.

Mucolipidosis type III in an adolescent presenting with atypical facial features and skeletal deformities.

Mucolipidosis type III (MLIII) (MIM# 252600) is an uncommon autosomal recessive disorder that results from deficiency of the multimeric enzyme, UDP-N-acetylglucosamine-1-phosphotransferase. The enzymatic defect results in deficiencies of lysosomal degradative enzymes with concomitant intracellular accumulation of both partly degraded glycosaminoglycans and sphingolipids leading to clinical manifestations such as short stature, developmental delay and other structural abnormalities. The diagnosis is challenging since musculoskeletal presentation may mimic some of the rheumatic and metabolic disorders. We herein report on a 13-year-old adolescent who was admitted to our rheumatology clinic because of progressive joint stiffness and deformities of her hands. The clinical and radiological findings led us to the diagnosis of MLIII despite negative urinary aminoglycosyaminoglycans. Therefore we decided to check for the presence of elevated activities of alpha-mannosidase and beta-hexosaminidase A+B in the plasma which was actually the case and confirmed the clinical diagnosis ofMLIII.

Diagnostic strategy for mucolipidosis II/III.

Overlapping clinical phenotypes are a diagnostic challenge to the clinician, especially in the cases of mucolipidosis (ML) and mucopolysaccharide disorders (MPS), due to overlapping phenotypes. Present study was carried out in 147 children suspected to have ML or MPS and 100 controls. They were screened for ML II/III by colorimetric method using substrate pNCS. Six children were found screen positive for ML II/III and further confirmatory study showed significantly raised activity in plasma confirming high specificity of the ML screening test. Forty-two (28.5%) children out of remaining 141 children that were screen negative, were found to have various MPS disorders, while rest 99 had normal enzyme activity in plasma and leucocytes. Present study demonstrates prompt and specific chemical method that can be used as a tool for estimating ML II/III, with high specificity.

Spectral optical coherence tomography in a patient with type I sialidosis.

BACKGROUND: The aim of our study was to analyze spectral optical coherence tomography (SD-OCT) findings in a patient with clinical signs of sialidosis. CASE REPORT: Fluorescein angiography and spectral optical coherence tomography was performed in a 37-year-old woman using a SD-OCT device with axial resolution of 6 µm. Enzyme assay followed. The patient was diagnosed with type I sialidosis by enzymatic assay. Besides a normal angiogram, a thickened nerve fiber layer was observed on spectral optical coherence tomography. CONCLUSIONS: The thickened nerve fiber layer was probably caused by accumulation of metabolic products such as sialylated oligosaccharides and glycopeptides, suggesting that SD- OCT, due to its enhanced resolution, can be a useful tool for diagnosis of rare neurological conditions.

Validation of disaccharide compositions derived from dermatan sulfate and heparan sulfate in mucopolysaccharidoses and mucolipidoses II and III by tandem mass spectrometry.

Glycosaminoglycans (GAGs) are accumulated in various organs in both mucopolysaccharidoses (MPS) and mucolipidoses II and III (ML II and III). MPS and ML II and III patients can not properly degrade dermatan sulfate (DS) and/or heparan sulfate (HS). HS storage occurs in the brain leading to neurological signs while DS storage involves mainly visceral and skeletal manifestations. Excessive DS and HS released into circulation and thus blood levels of both are elevated, therefore, DS and HS in blood could be critical biomarkers for MPS and ML. Such measurement can provide a potential early screening, assessment of the clinical course and efficacy of therapies. We here assay DS and HS levels in MPS and ML patients using liquid chromatography tandem mass spectrometry (LC/MS/MS). Plasma samples were digested by heparitinase and chondroitinase B to obtain disaccharides of DS and HS, followed by LC/MS/MS analysis. One hundred-twenty samples from patients and 112 control samples were analyzed. We found that all MPS I, II, III and VI patients had a significant elevation of all DS+HS compositions analyzed in plasma, compared with the controls (P<0.0001). Specificity and sensitivity was 100% if the cut off value is 800 ng/ml between control and these types of MPS group. All MPS I, II and III patients also had a significant elevation of plasma HS, compared with the controls (P<0.0001). All MPS VI patients had a significant elevation of plasma DS, compared with the controls (P<0.0001). These findings suggest measurement of DS and/or HS levels by LC/MS/MS is applicable to the screening for MPS I, II, III and VI patients.

Isolated ocular disease is associated with decreased mucolipin-1 channel conductance.

PURPOSE: To evaluate a 15-year-old boy with MLIV (mucolipidosis type IV) and clinical abnormalities restricted to the eye who also had achlorhydria with elevated blood gastrin levels. METHODS: In addition to a detailed neuro-ophthalmic and electrophysiological assessment, his mutant mucolipin-1 was experimentally expressed in liposomes and its channel properties studied in vitro. RESULTS: The patient was a compound heterzygote for c.920delT and c.1615delG. Detailed neuro-ophthalmic examination including electroretinography showed him to have a typical retinal dystrophy predominantly affecting rod and bipolar cell function. In vitro expression of MCOLN1 in liposomes showed that the c.1615delG mutated channel had significantly reduced conductance compared with wild-type mucolipin-1, whereas the inhibitory effect of low pH and amiloride remained intact. CONCLUSIONS: These findings suggest that reduced channel conductance is relatively well tolerated by the brain during development, whereas retinal cells and stomach parietal cells require normal protein function. MLIV should be considered in patients with retinal dystrophy of unknown cause and screened for using blood gastrin levels.

Mice lacking alpha/beta subunits of GlcNAc-1-phosphotransferase exhibit growth retardation, retinal degeneration, and secretory cell lesions.

PURPOSE: Mucolipidosis II and III (ML II; ML III) are lysosomal storage diseases characterized by a deficiency in GlcNAc-1-phosphotransferase. Patients with ML III have retinal disease, but in cases of the more clinically severe ML II, human ophthalmic studies are limited. In this study, retinal function and overall disease were assessed in mice lacking GNPTAB, the gene mutated in patients with ML II. METHODS: Mice deficient in GNPTAB were generated from Omnibank, a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones as part of a large-scale effort to knock out, phenotypically screen, and thereby validate pharmaceutically tractable genes for drug development. Routine diagnostics, expression analysis, histopathology, and ERG analyses were performed on mice lacking GNPTAB. In addition, measurements of serum lysosomal enzymes were performed. RESULTS: Severe retinal degeneration was observed in mice deficient in GNPTAB. Heterozygous mice were phenotypically normal and in situ hybridization showed expression across the neural retina. Compared to wild-type mice, the GNPTAB homozygous mice were smaller, had elevated levels of serum lysosomal enzymes, exhibited cartilage defects, and had cytoplasmic alterations in secretory cells of several exocrine glands. CONCLUSIONS: Mice deficient in GNPTAB exhibited severe retinal degeneration. Additional features observed in patients with ML II, a lysosomal storage disease, are also present in these mice. Understanding underlying mechanisms of this gene in the eye will increase its therapeutic potential for the treatment of retinal diseases.

Heparan sulfate levels in mucopolysaccharidoses and mucolipidoses.

Glycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.

Lysosomal inclusions in gastric parietal cells in mucolipidosis type IV: a novel cause of achlorhydria and hypergastrinemia.

Mucolipidosis type IV (ML-IV) is an autosomal recessive lysosomal storage disease that causes severe neurologic abnormalities. The brain disease is characterized by pigmented cytoplasmic granules in neurons and accumulation of lamellated membrane structures in lysosomes. The gastrointestinal disease in ML-IV was not previously recognized. Clinical examination of 20 patients with ML-IV (age range, 2-23 years) at the National Institutes of Health showed hypergastrinemia and constitutive achlorhydria. Endoscopic biopsy specimens from the gastric fundus, body, and antrum and from the duodenum of four such patients (ages 4, 6, 7, and 22 years) were evaluated histologically and by electron microscopy. Histologically, all gastric fundus and body biopsy specimens showed parietal cells in normal numbers. However, a striking cytoplasmic vacuolization of parietal cells was seen on hematoxylin and eosin stain. Electron microscopy showed the parietal cells to be markedly distended by large lysosomes containing lamellar, concentric, and cystic membranous inclusions. Additionally, chronic atrophic gastritis and enterochromaffin-like (ECL) cell hyperplasia were observed. Foveolar and chief cells in stomach and duodenum biopsy specimens were normal. We conclude that the cytoplasmic lysosomal inclusions in gastric parietal cells is a unique histologic feature of gastric biopsy in ML-IV.

Plasma methylumbelliferyl-tetra-N-acetyl-chitotetraoside hydrolase: further study of its characteristics as a chitinase and comparison with its activity on Remazol Brilliant Violet carboxymethyl chitin.

We have further studied some characteristics of human plasma specific chitinase by making use of the fluorescent substrate methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside (MU-TACT). The enzyme is also active towards the substrates MU-di-N-acetyl-beta-D-chitobioside (MU-DACB) and MU-N-acetyl-chitotrioside (MU-TRACT). MU-TACT hydrolase in plasma is very stable. It is inhibited by the substrate and the product of the reaction and by allosamidin and ethyleneglycolchitin. When the activity of plasma MU-TACT hydrolase was compared to Remazol Brilliant Violet carboxymethyl (RBV) chitin hydrolase (RBV chitinase), it appeared that another enzyme--lysozyme--is also active on RBV chitin and this enzyme represents about 50-60% of the total RBV chitinase activity. Highly increased activity of plasma MU-TACT hydrolase in plasma of Gaucher patients was reflected in a similar increase of RBV chitin hydrolase. In these patients, both MU-TACT hydrolase and RBV chitinase are totally inhibited by allosamidin indicating that specific chitinase is the increased enzyme. With the MU-TACT substrate, specific chitinase is measured and with RBV chitin as substrate the measured activity is a combination of specific chitinase (activity inhibited by allosamidin) as well as lysozyme (residual activity after allosamidin inhibition). For measurement of specific chitinase in human plasma and clinical applications, the di-, tri- or tetra-N-acetylglucosamine derivatives of MU are recommended. In order to avoid confusion, recommended names are either the total substrate followed by -ase, or chitinase.

Plasma hyaluronidase activity in mucolipidoses II and III: marked differences from other lysosomal enzymes.

A nearly pathognomonic finding of the lysosomal storage disorders mucolipidoses II and III is the marked increase of plasma lysosomal enzyme activities. The genetic lesion in ML II and III causes defective function of the enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. Defective function of this enzyme results in deficient phosphorylation of lysosomal enzyme asparagine-linked oligosaccharides and a consequent misrouting of many newly synthesized lysosomal enzymes. These enzymes are secreted from cells instead of being targeted to lysosomes, with resultant marked elevations of multiple lysosomal enzyme activities in plasma. We report here that plasma hyaluronidase activity, an endoglycosidase of presumably lysosomal origin, is not increased in the plasma from individuals with mucolipidoses II and III, unlike most lysosomal enzymes. Our data suggest the possibility that hyaluronidase is not targeted to lysosomes by a lysosomal enzyme phosphosmannosyl recognition mechanism. Alternatively, hyaluronidase activity may not be present in the cell type(s) responsible for the lysosomal enzyme hypersecretion in mucolipidoses II and III which, along with its deficiency in fibroblasts and leukocytes, would constitute an unusual tissue distribution of activity for a soluble lysosomal enzyme.

[Mucolipidosis II or "I-cell disease" in the newborn infant. 2 new cases]. / Mucolipidosis II o "I-cell disease" en el recién nacido. Dos nuevos casos.

Clinical and biochemical findings of two patients with neonatal mucolipidosis II are presented. Characteristic coarse facial features, shortness of stature, psychomotor retardation and vacuolated lymphocytes were observed in both cases. However, in one case symptoms presentation was earlier and course of the disease was markedly more severe, with death occurring at the age of 3 months. Biochemically similar increase of serum multiple lysosomal enzymes and decrease of fibroblast activities was demonstrated in both patients. Authors have found elevations of some acid hydrolases in sera from parents (obligate heterozygotes) of both patients and in individuals from two more families also obligated carries.

Carbohydrate composition of purified serum glycoproteins in mucolipidosis II and mucolipidosis III.

Mucolipidosis II (I-cell disease) and Mucolipidosis III (ML III) are inherited disorders in which the molecular defect may involve an abnormality in a common post-translational modification step (possibly glycosylation) shared by lysosomal hydrolases. We tested whether such an alteration might be a generalized defect in glycoprotein biosynthesis and, thus, be reflected in an abnormal carbohydrate composition of non-lysosomal glycoproteins. The apoprotein of low density lipoprotein (apo-LDL) and immunoglobulin G (IgG) were purified to apparent homogeneity. Gas liquid chromatographic (glc) analysis of the carbohydrate content of these glycoproteins from ML II, ML III and normal sera revealed no differences in the relative ratios and total amounts of mannose, galactose, N-acetylglucosamine and sialic acid. These results suggest that if the postulated post-translational defect in these disorders involves changes in carbohydrate composition, it is not a general defect in glycosylation and may be specific for lysosomal hydrolases.

Deficit in neuraminidase associated with mucolipidosis II (I-cell disease).

Using a tritiated sialyloligosaccharide as a substrate, the authors showed that mucolipidosis II is characterized by a lack of neuraminidase activity in leucocytes, while the other acidic hydrolases activities are normal. According to Ashwell, terminal galactose is the required signal for glycoproteins uptake by the cells. Thus, a neuraminidase deficit may explain the increase of sialylated hydrolases activities in the plasma and the non-recognition of these enzymes by cultured fibroblasts.