Clinical and biochemical analysis of two families with type I and type II mannosidosis

We report on two unrelated patients with different presentations of mannosidosis. One patient was affected in early childhood with a severe phenotype characteristic of type I mannosidosis. The other was diagnosed with type II mannosidosis only after the onset of progressive neurologic deterioration in late adulthood. Both were detected by non-invasive urinary screening of oligosaccharides. Lymphoblasts transformed from both patients' blood cells had markedly reduced lysosomal α-mannosidase activity. Kinetic analyses showed that α-mannosidase from the type I patient had a 400-fold reduction in affinity while that from the type II patient was reduced 40-fold. Lymphoblasts from all 4 parents had reduced α-mannosidase activity, but there were overlapping activities among these type I and type II obligate heterozygotes. We conclude that screening urinary oligosaccharides will detect mannosidosis over a wide range of phenotypes, that lymphoblasts transformed from affected heterozygotes have decreased enzymatic activity, and that the severity of clinical expression is related to the degree of enzyme impairment. © 1995 Wiley-Liss, Inc.     

Investigation of the organelle pathology of skeletal muscle in chronic alcoholism.

The muscle abnormalities associated with chronic alcohol consumption were studied by applying histological and biochemical techniques to tissue obtained by percutaneous needle biopsy from the quadriceps muscles of 41 patients. Measurement of the fibre size showed atrophy of both type I (p less than 0.05) and type II (p less than 0.001) fibres. The degree of atrophy was more severe for type II fibres (33% reduction in median diameter) than type I (17%). Marker enzyme activities for the principal organelles were assayed. Compared with biopsy specimens from non-alcoholic controls, no differences were found in the activities of lysosomal, mitochondrial, peroxisomal, cytosolic, sarcolemmal, or sarcoplasmic reticulum enzymes, expressed per microgram DNA. A reduction in the protein to DNA ratio was evident in severely atrophic biopsies, and this was associated with a significant reduction of myofibrillary Ca2+-ATPase activity. These results suggest a selective loss of type II fibre myofibrillary protein and do not confirm earlier suggestions of specific mitochondrial damage.     

Inborn errors of lysosomal catabolism--principles of heterozygote detection.

Carriers of an inborn error of lysosomal catabolism can be recognized, as they have enzyme levels approximately half those of normal individuals. Of the various tissues readily available for assay, plasma and leukocytes and, in some situations, tears are preferred. Although mixed leukocytes have proved satisfactory in Tay-Sachs screening programs, purified preparations of granulocytes or lymphocytes will allow better discrimination in most situations. Enzymes are assayed relative to some other reference parameter which must be a constant or highly correlated with test enzyme activity. In the two mass screening programs in operation, beta-hexosaminidase A and alpha-mannosidase have both been assayed relative to total beta-hexosaminidase activity. Carrier detection is particularly important in X-linked diseases. The techniques used mostly involve hair roots or fibroblasts and depend on random inactivation of the X chromosome. In the mucolipidoses II and III, in which there are a number of deficient enzymes in cells, carriers may be identified on the basis of the ratio of beta-hexosaminidase I1 and I2 to total hexosaminidase.     

Mannosidosis in two brothers: prolonged survival in the severe phenotype

Two cases of mannosidosis are reported in brothers, one aged 41 years at death, the other aged 40 years and still alive. These patients are the oldest reported in the literature. Prolonged survival has previously been associated with the milder Type II phenotype. In addition to the characteristic clinical and radiological features of mannosidosis, both had severe joint destruction, which may be related to abnormal lysosomal enzymes in cartilage. The activity of acidic a-mannosidase was markedly reduced in plasma, leucocytes and fibroblasts, and the altered kinetic and physical properties are described.     

Enzyme replacement therapy in alpha-mannosidosis guinea-pigs

alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of lysosomal alpha-mannosidase and is characterised by massive accumulation of mannose-containing oligosaccharides in affected individuals. Patients develop behaviour and learning difficulties, skeletal abnormalities, immune deficiency and hearing impairment. Disease in alpha-mannosidosis guinea-pigs resembles the clinical, histopathological, biochemical and molecular features of the human disease. We have used the guinea-pig model to investigate efficacy of enzyme replacement therapy as a treatment for alpha-mannosidosis. Intravenous recombinant human lysosomal alpha-mannosidase, administered at a dose of 1mg/kg, was cleared from circulation with a half-life of 53 h, with significant enzyme activity (1.4x normal levels) detected in circulation one week post-injection. alpha-Mannosidase administered to alpha-mannosidosis guinea-pigs at 1mg/kg (onset at birth or approximately 30 days) and 10mg/kg (at birth) was distributed widely amongst tissues, including to capillary depleted brain. By monitoring with tandem mass spectrometry, enzyme replacement therapy was found to be effective in reducing stored substrates in peripheral tissues at both dose rates, and in brain by up to 39% at the 10mg/kg dose, compared with untreated alpha-mannosidosis controls. Reductions of up to 60% of urinary mannose containing oligosaccharides were also observed. No histological improvements were seen in the brain at either dose, however marked decreases in lysosomal vacuolation in liver, kidney, spleen and endocrine pancreas, as well as a significant reduction in trigeminal ganglion neurons were observed. Multiple injections of 1mg/kg recombinant enzyme in alpha-mannosidosis guinea-pigs induced a very rapid humoral immune response precluding long-term intravenous treatment.     

A program of prevention of hereditary lysosomal diseases in the USSR]

The organization of genetic counselling for the families of patients with lysosomal storage diseases (LSD) was based on the interaction of the genetic counselling units of this country with a laboratory of inherited metabolic diseases of the National Research Center of Medical Genetics, USSR AMS. All the patients from 705 families at risk were examined using biochemical techniques and methods of somatic cell genetics. In total the loci differentiation was performed for 309 patients with mucopolysaccharidoses, glycoproteinoses, mucolipidoses, sphingolipidoses and other LSD. 53 families at risk (of 277) were prenatally diagnosed. 66 fetuses were diagnosed for mucopolysaccharidoses, type I, II, III, A and B, VI, Tay-Sachs disease, Sandhoff's disease, GM1-gangliosidosis, metachromatic leukodystrophy, mannosidosis, and multiple sulfatidosis. In total 18 affected fetuses were diagnosed and aborted. All the prenatal diagnoses were verified. The prevalence of mucopolysaccharidoses in two Central Asian republics was evaluated as 1:15,000. An Uneven ethnic distribution of different mucopolysaccharides in the USSR has also been shown.     

Novel mutations in lysosomal neuraminidase identify functional domains and determine clinical severity in sialidosis

Lysosomal neuraminidase is the key enzyme for the intralysosomal catabolism of sialylated glycoconjugates and is deficient in two neurodegenerative lysosomal disorders, sialidosis and galactosialidosis. Here we report the identification of eight novel mutations in the neuraminidase gene of 11 sialidosis patients with various degrees of disease penetrance. Comparison of the primary structure of human neuraminidase with the primary and tertiary structures of bacterial sialidases indicated that most of the single amino acid substitutions occurred in functional motifs or conserved residues. On the basis of the subcellular distribution and residual catalytic activity of the mutant neuraminidases we assigned the mutant proteins to three groups: (i) catalytically inactive and not lysosomal; (ii) catalytically inactive, but localized in lysosome; and (iii) catalytically active and lysosomal. In general, there was a close correlation between the residual activity of the mutant enzymes and the clinical severity of disease. Patients with the severe infantile type II disease had mutations from group I, whereas patients with a mild form of type I disease had at least one mutation from group III. Mutations from the second group were mainly found in juvenile type II patients with intermediate clinical severity. Overall, our findings explain the clinical heterogeneity observed in sialidosis and may help in the assignment of existing or new allelic combinations to specific phenotypes.     

Antenatal diagnosis of mucopolysaccharidosis type I (Hurler''s disease) is not possible by electron microscopy of uncultured amniotic fluid cells.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder characterised by the deficient activity of iduronidase and by the presence of MPS vacuoles in many tissues of affected patients. We studied whether these characteristics could be used for the antenatal diagnosis of the disease. We obtained amniotic fluid cells from two pregnancies at risk for MPS I, one pregnancy at risk for GSD II (another lysosomal disease), and eight normal control pregnancies. Measurements of iduronidase activity in cultured amniotic fluid cells indicated the presence of a MPS I fetus in one high risk pregnancy and an unaffected fetus in the other. This diagnosis was confirmed at delivery. On electron microscopy the uncultured amniotic fluid cells exhibited MPS-like vacuoles in the pregnancy with a GSD II fetus, in three of eight normal pregnancies, and in the pregnancy at risk for MPS I that had a normal fetus. No such vacuoles were seen in the pregnancy with the MPS I fetus. These false positive and false negative findings indicate that antenatal diagnosis of MPS I cannot be based on the electron microscopic presence or absence of MPS I vacuoles in uncultured amniotic fluid cells.     

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-l-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. © 1996 Wiley-Liss, Inc.     

Immunelectronmicroscopic characterization of T4 and T8 lymphocytes and natural killer cells in neuronal ceroid-lipofuscinosis

CD4+, CD8+, and CD56+ cells were isolated with the immunomagnetic separation technique from peripheral blood mononuclear cells (PBMC) of 3 patients with neuronal ceroid-lipofuscinosis: one patient each with infantile (INCL), late infantile (LINCL), and juvenile (JNCL) neuronal ceroid-lipofuscinoses, all studied by light (LM) and electron (EM) microscopy. To compare the pathology of these cells with affected cells in other types of lysosomal diseases, the separation was also performed with PBMC of 1 patient with mucolipidosis (ML) type II, 2 patients with mucopolysaccharidosis (MPS) type I, and 4 patients with MPS type III. Diseasespecific lysosomal inclusions were identified in CD4+, CD8+, and CD56+ cells. Subclass-specific storage products could not be found. T4 and T8 lymphocytes showed nearly the same ratio of affected to nonaffected cells. These results suggest that lysosomal storage can be found in most of the lymphocyte subclasses in these forms of neuronal ceroid-lipofuscinoses and other lysosomal diseases. © 1995 Wiley-Liss, Inc.     

Familial neuromuscular disease with "myotubes"

Van Wijngaarden et al. (1969) described a "myotubular myopathy" in a Dutch family, of which six male members were affected, indicating a recessive X-linked mode of transmission. They also thought that the morphology of a case reported by Engel et al. (1968) resembled that seen in their family.
The present case is a male sib of the patient reported by Engel and colleagues, who has similar clinical and morphologic features to his brother, and who died at 7 months of age. Muscle enzyme histochemistry of a quadriceps biopsy revealed that most type I fibers were small and had central pale areas or plump nuclei. A smaller percentage of type II fibers also showed the same features. Biopsies of muscle from the mother and father were histologically normal including fiber type composition. Karyotypic analyses, not reported in earlier cases, were performed on the family and showed no abnormalities in modal number or of individual chromosome structure. Pedigree analysis suggests an X-linked recessive inheritance pattern in this family.     

Correlation of lysosomal enzyme abnormalities in various forms of adult leukaemia

Lysosomal enzyme activities were studied in cells derived from the following types of leukaemia: chronic myeloid, acute myeloid, acute myelomonocytic, acute monocytic, non-T, non-B cell acute lymphoblastic, T-cell acute lymphoblastic, B-cell chronic lymphocytic and T-cell chronic lymphocytic. Activities of beta-hexosaminidase and alpha-mannosidase were significantly higher in cells from acute monocytic and acute myelomonocytic leukaemias, and somewhat higher in the other myeloid leukaemias, when compared with control granulocytes. Activities of beta-hexosaminidase, alpha-mannosidase, alpha-fucosidase, beta-glucuronidase and acid phosphatase were markedly lower in B cells of chronic lymphocytic leukaemia when compared with control or other leukaemic lymphoid cells. On isoelectric focusing abnormal patterns of beta-hexosaminidase, alpha-mannosidase and beta-glucuronidase activities were commonly found in myeloid and non-T, non-B cell leukaemias. All patients with acute myeloid leukaemia exhibited a relative decrease in the B form of beta-hexosaminidase activity. The results described show that studies on lysosomal enzymes may assist in the classification of different types of leukaemia.     

Mucopolysaccharidoses type I and IVA: clinical features and consanguinity in Tunisia

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by the deficiency of specific enzymes which leads to the lysosomal accumulation of glycosaminoglycanes. Mucopolysaccharidosis type I or Hurler disease is characterized by the deficiency of alpha-l-iduronidase enzyme. Mucopolysaccharidosis type IVA or Morquio A disease is due to the lack of N-acetylgalactosamine-6-sulfate-sulfatase. Theses deficiencies result in a progressive accumulation of the substrates: dermatan and heparan sulfates for Mucopolysaccharidosis type I and keratan sulfate for MPS type IVA. This process leads to progressive and chronic course for visceral attacks of the affected organs such as lungs and heart. In the Hurler disease, the nervous system is particularly affected while in Morquio a disease, a skeletal dysplasia and a normal intelligence are characteristic.     

Phenotypic variation in Meckel syndrome

Four sibs are described with Meckel syndrome, an autosomal recessive disorder with multiple abnormalities. Each sib manifested only two of the three cardinal signs of Meckel syndrome - encephalocoele and polycystic kidneys, lacking Polydactyly. The literature is examined to assess the phenotypic variation of the condition: 57 % of cases have all the three major abnormalities, 16% have the two found in this family, and the remainder exhibit other variations. In 9 of 17 families where more than one sib is affected, manifestation between sibs is the same, but in the only other two families with as many as four affected sibs, there is variation in expression between sibs.     

Deficient expression of the small proteoglycan decorin in a case of severe/lethal osteogenesis imperfecta

In osteogenesis imperfecta (OI) the effects of mutations in type I collagen genes generally reflect their nature and localization. Unrelated individuals sharing identical mutations present, in general, similar clinical phenotypes. However, in some such cases the clinical phenotype differs. This variable clinical expression could be the result of abnormalities in other connective tissue proteins. Since decorin is a component of connective tissue, binds to type I collagen fibrils and plays a role in matrix assembly, we studied decorin production in skin fibroblasts from OI patients. Cultured fibroblasts from one patient with extremely severe osteogenesis imperfecta (classified as type II/III) who has an α1(I)gly415ser mutation were found to secrete barely detectable amounts of decorin into culture medium. Western blotting using antibodies raised against decorin confirmed the reduction of the decorin core protein and Northern blot analysis showed decorin mRNA levels below the limit of detection. Cells from a patient, with a less severe phenotype, bearing a mutation in the same position of the triple helix (α1(I)gly415) expressed decorin normally. The different clinical phenotypes could be due to the differing genetic backgrounds of the patients so it is tempting to conclude that in our most severely affected patient the absence of decorin aggravates the clinical phenotype. © 1996 Wiley-Liss, Inc.     

Newborn screening for lysosomal storage disorders

Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected individuals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of individuals with an LSD and evaluated this assay in a retrospective study using blood-spots from; newborns subsequently diagnosed with an LSD (n=19, six different LSD), individuals sampled after diagnosis of an LSD (n=92, 11 different LSD), newborn controls (n=433), and adult controls (n=200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families.     

X-linked liver glycogenosis type II (XLG II) is caused by mutations in PHKA2, the gene encoding the liver alpha subunit of phosphorylase kinase

X-linked liver glycogenosis type II (XLG II) is a recently described X- linked liver glycogen storage disease, mainly characterized by enlarged liver and growth retardation. These clinical symptoms are very similar to those of XLG I. In contrast to XLG I patients, however, XLG II patients do not show an in vitro enzymatic deficiency of phosphorylase kinase (PHK). Recently, mutations were identified in the gene encoding the liver alpha subunit of PHK (PHKA2) in XLG I patients. We have now studied the PHKA2 gene of four unrelated XLG II patients and identified four different mutations in the open reading frame, including a deletion of three nucleotides, an insertion of six nucleotides and two missense mutations. These results indicate that XLG II is due to mutations in PHKA2. In contrast to XLG I, XLG II is caused by mutations that lead to minor structural abnormalities in the primary structure of the liver alpha subunit of PHK. These mutations are found in a conserved RXX(X)T motif, resembling known phosphorylation sites that might be involved in the regulation of PHK. These findings might explain why the in vitro PHK enzymatic activity is not deficient in XLG II, whereas it is in XLG I.      

Identification and characterization of 20 novel pathogenic variants in 60 unrelated Indian patients with mucopolysaccharidoses type I and type II

Mucopolysaccharidoses (MPS), a subgroup of lysosomal storage disorders, are caused due to deficiency of specific lysosomal enzyme involved in catabolism of glycosaminoglycans. To date more than 200 pathogenic variants in the alpha‐l ‐iduronidase (IDUA) for MPS I and ～500 pathogenic variants in the iduronate‐2‐sulphatase (IDS) for MPS II have been reported worldwide. The mutation spectrum of MPS type I and MPS type II disorders in Indian population is not characterized yet. In this study, we carried out clinical, biochemical, molecular and in silico analyses to establish the mutation spectrum of MPS I and MPS II in the Indian population. We conducted molecular analysis for 60 MPS‐affected patients [MPS I (n = 30) (Hurler syndrome = 17, Hurler–Scheie syndrome = 13), and MPS II (n = 30) (severe = 18, attenuated = 12)] and identified a total of 44 [MPS I (n = 22) and MPS II (n = 22)] different pathogenic variants comprising missense, nonsense, frameshift, gross deletions and splice site variants. A total of 20 [MPS I (n = 14), and MPS II (n = 6)] novel pathogenic sequence variants were identified in our patient cohort. We found that 32% of pathogenic variants detected in IDUA were recurrent and 25% in MPS II. This is the first study revealing the mutation spectrum of MPS I and MPS II patients in the Indian population.     

An in vitro model for abnormal skeletal development in the lysosomal storage diseases

Lysosomal storage diseases such as G_M1-gangliosidosis are associated with skeletal abnormalities. Radiological and histological studies, both in human and corresponding animal models, indicate retarded bone formation. Since cartilage maturation leads to bone formation, we developed an in vitro system to study and compare the biological features of cartilage from dogs affected with G_M1-gangliosidosis with age-matched controls. Costochondral chondrocytes were grown in monolayer and in agarose culture. Both affected and control cells dedifferentiated in monolayer; however, in agarose culture they re-expressed the chondrocytic phenotype. Cells from affected dogs were enlarged and contained numerous large vaculoes when compared with control cells. This morphology was similar to that seen in vivo. In addition, the affected cells appeared to have a reduction in mitosis and alcian blue staining proteoglycans. Cultures from affected animals contained fewer cells positive for alkaline phosphatase activity. Both affected and control cells expressed collagen types I and II and were positive for the lectin Ricinus communis agglutinin-I. However, the staining of the control culture for type II collagen was more prominent than in the affected cells. These findings suggest that culture of chondrocytes in agarose may be a useful method for studying the biology of cartilage which leads to skeletal abnormalities in lysosomal storage diseases.     

Deficient Kupffer cell phagocytosis and lysosomal enzymes in the endotoxin-low-responsive C3H/HeJ mouse

Various substances, including lysosomal enzymes, are produced by Kupffer cells and other macrophages; their release has been implicated in the toxic response to endotoxins. C3H/HeJ mice exhibit little or no response to doses of endotoxin that are lethal in syngeneic C3HeB/FeJ mice. To explore the nature of this deficient response, the Kupffer cells of these mice were studied using in vivo microscopic as well as histochemical and electron microscopical methods. In vivo, the rate of phagocytosis of single 0.8 micron latex particles was measured in individual Kupffer cells as was the number of phagocytic cells per microscopic field. Frozen sections of livers were stained for a variety of lysosomal enzymes and liver specimens also were processed for electron microscopy. In comparison to the endotoxin-sensitive C3HeB/FeJ mice, the livers of the C3H/HeJ mice contained 60% fewer Kupffer cells that phagocytosed latex. However, the rate of phagocytosis by these cells was not statistically different and ranged from 19-26 sec. The volume density of acid-phosphatase-positive Kupffer cells was 40% less in the C3H/HeJ mice. Similar differences were observed with other lysosomal enzymes including cathepsins B and H and dipeptidyl peptidases I and II. However, light and electron microscopy revealed a relatively normal number of Kupffer cells in livers stained for peroxidase, a nonlysosomal enzyme. The results suggest that the insensitivity of C3H/HeJ mice to endotoxin may be related in part to a lysosomal enzyme deficiency and a paucity of phagocytic Kupffer cells in these animals.