Biochemical studies on the juvenile form of ceroid-lipofuscinosis.

Accumulation of oligosaccharyl diphosphodolichols (oligo-PP-Dol) in brains of patients with various forms of ceroid-lipofuscinoses (CL) is one of the most reproducible biochemical changes known so far. The objective of this study is to understand the biochemical basis of this observation. The biosynthesis of oligo-PP-Dol was studied by the incorporation of labelled glucose from UDP [14C]glucose into oligo-PP-Dol in cultured skin fibroblasts, and showed no changes in the level of synthesis. The level of labelled glucose incorporated into glycoproteins was also unchanged, suggesting that there is no decrease in the oligosaccharide transfer to proteins in this disorder. Since the biosynthesis and utilization of oligo-PP-dol are unaffected, a defect in the catabolism may be the only possibility for the storage of this compound in CL. Since terminal mannose residues are present in the accumulating oligo-PP-Dol, mannosidase activities at pH 4.4 and 6.0 were determined in cultured skin fibroblasts. Both mannosidase activities were unchanged in skin fibroblasts of juvenile CL. Endo-beta-N-acetylglucosaminidase-1 activities were determined in cultured skin fibroblasts using dansylated Man6GlcNAcGlcNAc-Asn as substrate. In three patients, a drastic reduction in the level of the pH 4.5 enzyme was shown, while the neutral pH enzyme activity was unaffected. A deficiency of the endo-beta-N-acetylglucosaminidase-1 will not only explain the accumulation of oligo-PP-Dol but also the known storage of high-mannose glycoproteins.     

Clinical and ultrastructural findings in three patients with geleophysic dysplasia

Geleophysic dysplasia, a rare disorder with autosomal-recessive inheritance, is characterized by short stature with a “happy-looking” facial appearance. Nonskeletal findings, particularly in an advanced stage, include hepatosplenomegaly and valvular cardiopathy. Based on the clinical picture and the detection of lysosome-like inclusions in hepatocytes, the underlying cause of the condition is considered to be a storage defect in the metabolism of glycoproteins. The clinical course, with progressive worsening of the condition favors this hypothesis. We report on 3 further cases, in which light and electron microscopic studies of iliac crest biopsies and cultured skin fibroblasts provided additional evidence that geleophysic dysplasia represents a lysosomal storage disease. The additional discovery of storage vacuoles in chondrocytes and skin fibroblasts strongly suggests that the condition is a generalized storage defect. To date, it has not yet been possible to identify the presumed biochemical defect in the metabolic pathways of glycoproteins. © 1996 Wiley-Liss, Inc.     

Inherited metabolic myopathy with storage of glycoproteins and glycosaminoglycans

Two related patients (mother and daughter, ages 28 and 5 years) showed mild to moderate weakness and atrophy of facial and shoulder muscles with congenital onset and minimal progression. Serum creatine kinase was elevated in the child. Muscle biopsy showed normal light-microscopic and histochemical findings, but scattered sarcoplasmic vacuoles with storage of granular material were evident on electron microscopy. Storage of granular material was also identified in fibroblasts which were weakly PAS-positive, stained metachromatically with toluidine blue and orthochromatically with alcian blue. Muscle glycogen values were lownormal. Repeated biochemical studies of cultured fibroblasts identified excessive storage of glycosaminoglycans and glycoproteins. The uptake of ³H-glucosamine in cultured fibroblasts was 1.7–3.4 times greater in the patients than in control individuals, while the rate of turnover of the radioisotope was normal. These findings suggest that the genetic defect in this inherited metabolic myopathy is related to excessive synthesis of glycosaminoglycans and glycoproteins.     

Sialidosis Type 1

Observations have been made on two brothers who had progressive ataxia, intention myoclonus and visual failure starting early in the third decade of life. Their parents were consanguineous. The brothers showed bilateral cherry red spots at the maculae and bilateral perinuclear cataracts; their intelligence was preserved. Urine was found to contain large amounts of sialylated oligosaccharides; cultured skin fibroblasts showed deficiency of the enzyme sialidase (neuraminidase). Studies on leucocytes and cultured skin fibroblasts showed aberrant electrophoretic mobilities of six enzymes all of which are known to be glycoproteins, and this has been attributed to excessive amounts of sialic acid on the enzyme molecules. The clinical features together with the biochemical findings indicate that these are further cases of the newly described condition Sialidosis Type 1 and it is suggested that the electrophoretic findings might be typical of the condition.     

Aspartylglucosaminuria: Deficiency of aspartylglucosaminidase in cultured fibroblasts of patients and their heterozygous parents

Aspartylglucosaminuria (AGU) is a genetic lysosomal storage disorder which probably affects the metabolism of glycoproteins. Earlier studies have shown a deficiency of a lysosomal hydrolase, N-aspartyl-β-glucosaminidase in the serum and seminal fluid, as well as in the brain and liver tissues of the patients. The present studies demonstrated a very low activity of N-aspartyl-β-glucosaminidase in cultured skin fibroblasts from AGU patients. The fibroblasts of the parents of the patients had a moderately low enzyme activity when conipared with control cultures. Thus, demonstration of the enzyme defect in fibroblasts offers possibilities both for detection of heterozygotes and for prenatal diagnosis of AGU.     

Combined sialidase (neuraminidase) and β-galactosidase deficiency. Clinical, morphological and enzymological observations in a patient

A patient with combined deficiency of sialidase and beta-galactosidase is described. This now 39-year-old man, who is of Japanese origin, showed gradually progressive clinical features from the age of six years. Many of these features are commonly found in sialidosis type 2 or in GM1-gangliosidosis. Both sialidase and beta-galactosidase activities were deficient in leucocytes and cultured fibroblasts. Leucocytes of his mother showed activities of both enzymes in the lower limit of the control range. Morphologically, the pattern of storage products in a skin biopsy resembled in many respects that seen in GM1-gangliosidosis. Moreover, storage products which could be typical of sialidosis were also observed. Since the patient showed angiokeratomata, the morphological findings were compared with those specific to Fabry's disease, but no similarities were found. An enzymological diagnosis of the disease is most reliable on cultured fibroblasts, discriminating it from sialidosis type 2 and GM1-gangliosidosis. In view of recent findings, leucocytes seem to be less suitable for the establishment of the diagnosis galactosialidosis.     

Diagnosis of lysosomal storage diseases. Pathomorphologic and biochemical possibilities

Optical light and electron microscopy were used in studies into two cases of infantile GM2-gangliosidosis. The results are reported in this paper. The correlation has been evident between histological and ultrastructural findings. Reliable delimitation between two different variants of infantile GM2-gangliosidosis was achieved through biochemical investigation of postmortally cultured skin fibroblasts. A classical form with isolated hexosaminidase-A defect (Tay-Sachs disease) was distinguished from a second variant with complete defect of both isoenzymes of hexosaminidase (Sandhoff's disease). Biochemical investigation of postmortally cultured fibroblasts today has become indispensable to enlargement of autopsy findings from other storage diseases, as well.     

Sterol concentrations in cultured Smith-Lemli-Opitz syndrome skin fibroblasts: Diagnosis of a biochemically atypical case of the syndrome

The Smith-Lemli-Opitz syndrome is a common birth defect syndrome caused by a deficiency of 7-dehydrocholesterol Δ⁷-reductase, an essential enzyme in the biosynthesis of cholesterol. The syndrome can usually be diagnosed easily from the plasma markers of markedly elevated 7-dehydrocholesterol and reduced cholesterol concentrations. However, atypical cases with normal plasma levels of cholesterol with only moderately elevated 7-dehydrocholesterol have been reported. To establish a sensitive method for the biochemical diagnosis of the atypical cases of the syndrome, we measured sterol concentrations of cultured skin fibroblasts. 7-Dehydrocholesterol concentrations in patients' fibroblasts grown in the presence of 10% fetal bovine serum were significantly higher than those in controls and parents (P < 0.0005), but they were not elevated proportionately as much as in plasma. To re-produce the accumulation of 7-dehydrocholesterol, the cells were exposed to delipidated medium to induce sterol biosynthesis. After 4 weeks, 7-dehydrocholesterol concentrations in patients' fibroblasts increased from 2.8 ± 0.3% to 34 ± 3% of total sterols (cholesterol + 7-dehydrocholesterol + 8-dehydrocholesterol). The increase was also observed in fibroblasts from an atypical patient who has a normal plasma cholesterol level and a 7-dehydrocholesterol concentration of only 0.15 mg/dl. In contrast, cells from parents and controls accumulated very little 7-dehydrocholesterol (<1% of total sterols). These results demonstrate that cultured fibroblasts exhibit abnormally high accumulation of 7-dehydrocholesterol after cells are exposed to delipidated medium not only in typical patients, but also in an atypical case. The present method is a sensitive procedure for the biochemical diagnosis of this syndrome. Am. J. Med. Genet. 68:282–287, 1997. © 1997 Wiley-Liss, Inc.     

Infantile type of sialic acid storage disease with sialuria

We describe a male infant of Austrian ancestry, the main clinical features including attacks of dyspnea due to laryngomalacia, severe mental and motor retardation, pronounced splenohepatomegaly and vacuolisation of peripheral lymphocytes. The clinical condition deteriorated progressively and the child died at the age of 21 months. Phase and electron microscopy of cultured skin fibroblasts showed multiple vacuoles and inclusions suggestive of a lysosomal storage disorder. Increased excretion of free sialic acid was demonstrated by resorcinol staining after routine thin-layer screening for urinary oligosaccharides. Quantitative analyses of urine, leucocytes and cultured fibroblasts revealed 10 to 30-fold increased concentration of free sialic acid. In addition, 3-fold elevated amounts of sialyloligosaccharides were found in the urine. The activities of a variety of lysosomal enzymes, including sialidase, were normal. Our case is compared with infantile sialic acid storage disease recently observed by other authors. The close convergence of clinical, morphological and biochemical signs support the concept of a distinct lysosomal disease entity. The basic defect is so far unknown.     

Isolated acid neuraminidase deficiency: a distinct lysosomal storage disease

An 8-month-old female presented with coarse facies and hepatosplenomegaly at birth. Growth proceeded at an accelerated rate and mental development was normal. A pattern of dysostosis multiplex developed radiographically. Cytoplasmic inclusions consistent with lysosomal storage disease were demonstrated by electron microscopy in bone marrow, liver, and cartilage cells and in cultured skin fibroblasts. Assays of the fibroblasts revealed a specific deficiency of acid neuraminidase and 6-fold increase in intracellular bound sialic acid. An unidentified macromolecular compound rich in sialic acid was excreted in excessive amounts in the urine. The phenotype suggests defective degradation primarily of glycoproteins and possibly to a lesser extent of keratan sulfate and gangliosides.     

Phenylketonuria due to a deficiency of dihydropteridine reductase.

The onset of neurologic symptoms in a child who had markedly elevated blood phenylalanine levels during the first two weeks of life and who was promptly treated with a low phenylalanine diet, with excellent control of serum phenylalanine levels, suggested that this child had an unusual form of phenylketonuria. In assays of the components of the phenylalanine hydroxylating system (open liver biopsy at 14 months), the activity of phenylalanine hydroxylase was 20 per cent of the average normal adult value. By contrast, no dihydropteridine reductase activity was detected in the patient's liver, brain or cultured skin fibroblasts. Since dihydropteridine reductase is also essential for the biosynthesis of dopamine, norepinephrine, and serotonin, disturbed neurotransmitter function may be responsible for the patient's neurologic deterioration. On the basis of these results, assay of reductase in cultured skin fibroblasts may be advisable in the initial diagnosis of phenylketonuria.     

Deficiency of galactosylhydroxylysyl glucosyltransferase, an enzyme of collagen synthesis, in a family with dominant epidermolysis bullosa simplex

Members of a family with dominant epidermolysis bullosa simplex were found to have a deficiency of galactosylhydroxylysyl glucosyltransferase (GGT), an enzyme catalyzing the glucosylation of galactosylhydroxylysyl residues in the biosynthesis of collagen. The enzyme's activity was low in serum, skin tissue, and cultured skin fibroblasts, although no abnormality was found in three other intracellular enzymes of collagen biosynthesis. Mixtures of serum samples from patients and healthy controls gave the expected GGT activity, indicating that the low values were not due to inhibitors. GGT deficiency was accompanied by decreased product formation in vivo, as shown by a markedly decreased urinary excretion of glucosylgalactosylhydroxylysine. Six of 12 affected members had definite GGT deficiency, and five had some evidence suggestive of this abnormality; 13 of 15 unaffected members had no such manifestations. No similar GGT deficiency was found in three other families with the same disease. We conclude that GGT deficiency may be etiologically related to this disease in some families, but that different defects must be the cause in other cases.     

Hepatic storage of bis(monoacylglycerol) phosphate without concomitant storage of sphingomyelin in a 72-year-old patient with a partial deficiency of sphingomyelinase

A 72-year-old patient with marked splenomegaly and low sphingomyelinase (6% of lowest control value) in peripheral blood leukocytes is described. Much higher but variable residual sphingomyelinase activity was observed in cultured skin fibroblasts (40-67% of lowest control value); reduced activity was also found in a liver biopsy sample. Excess storage of sphingomyelin was not observed in a liver biopsy; instead, a lipid tentatively identified as bis(monoacylglycerol) phosphate was present in amounts at least 20 times greater than in age-matched control livers. The biochemical relationship of this patient to patients with sphingomyelin storage disease (Niemann-Pick disease) and phospholipidosis Type II is discussed.     

Properties of sulfatases in cultured skin fibroblasts of multiple sulfatase deficient patients

Various sulfatase activities were assayed in cultured skin fibroblasts from patients with multiple sulfatase deficiency (MSD). MSD cell lines displayed deficiencies of arylsulfatase A and iduronate sulfatase, but activities of arylsulfatase B, N-acetylgalactosamine 6-sulfate sulfatase and N-acetylglucosamine 6-sulfate sulfatase were within normal ranges, but not consistently. Arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD cell lines had similar Km, pH optima, inhibitory or activator sensitivity to that of normal skin fibroblasts. Arylsulfatase B in MSD cell lines also had properties similar to that of normal skin fibroblasts, except an abnormal heat stability. From our results, we conclude that properties of arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD fibroblasts were intact. On the other hand, arylsulfatase B in MSD might be a functionally abnormal enzyme.     

Acid carboxypeptidase deficiency in galactosialidosis

Carboxypeptidase activity with an optimal pH at 5.7 was found to be deficient in cultured lymphoblastoid cells and skin fibroblasts from 16 galactosialidosis patients of Japanese origin. The amounts of residual enzyme activities did not correlate with clinical phenotypes (early infantile and juvenile/adult). Four parents of the patients from different families showed enzyme activities at an intermediate level between the patients and normal controls. It was concluded that this enzyme deficiency is closely connected to the genetic defect of "protective protein." Further characterization with various protease inhibitors indicated that the enzyme deficient in galactosialidosis cells is a serine carboxypeptidase with histidine and cysteine residues at or near the active site.     

Loss of Co2+ sensitivity of monkey brain cytosolic neutral alpha-D-mannosidase by cyclic AMP dependent phosphorylation.

alpha-D-Mannosidase that exhibits a pH optimum close to neutrality (neutral mannosidase) purified from monkey brain cytosol is known to be stimulated by Co2+ and this stimulation is suggested to be mediated through a Co2+ activated aminopeptidase that is inseparable from the neutral mannosidase and that cleaves amino acids from the neutral mannosidase. In the present studies, the phosphorylation on serine residues of the neutral mannosidase by cyclic AMP dependent protein kinase is demonstrated. After phosphorylation the mannosidase activity remained unchanged, but it was not stimulated by Co2+. The aminopeptidase activity, although it retained its response to stimulation by Co2+, showed a drastic reduction in its activity after phosphorylation. It is suggested that the loss of Co2+ sensitivity of the neutral mannosidase after phosphorylation is mediated through the aminopeptidase.     

Atypical expression of ß-galactosidase deficiency in a child with Hurler-like features but without neurological abnormalities

A 28-month-old child was found to have several clinical features of lysosomal storage diseases, including: coarse facies, hepatosplenomegaly, lumbar kyphosis due to hypoplastic beaked L1 and L2 vertebral bodies, vacuolated lymphocytes in blood smears and rare foamy hystiocytes in bone marrow. However, no signs of neurological or ocular abnormalities were detected. A beta-galactosidase deficiency was demonstrated in leukocytes and cultured skin fibroblasts, with a residual activity toward 4-methylumbelliferyl-beta-galactopyranoside ranging between 5 and 15% of the normal mean. Normal activities were found for several other lysosomal acid hydrolases. beta-Galactosidase activities in leukocytes and cultured skin fibroblasts from both parents were within the normal ranges. The patient seems to represent an atypical expression of acid beta-galactosidase deficiency, since his clinical picture does not exaclty correspond to that of either the two classical types of GM1-gangliosidosis or other atypical patients reported in the literature havining beta-galactosidase deficiency.     

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.     

Prenatal diagnosis for severe methylenetetrahydrofolate reductase deficiency by linkage analysis and enzymatic assay

Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is characterized by varying degrees of developmental delay, motor and gait abnormalities, seizures, and thrombosis. Biochemical abnormalities include homocystinuria and hyperhomocysteinemia. Clinical severity correlates with MTHFR activity in cultured fibroblasts; activity can also be assayed in cultured amniocytes and chorionic villus cells (CVC). Forty-four private mutations have been identified, limiting the use of direct mutation detection for prenatal diagnosis. However, intragenic polymorphisms have been identified, making prenatal diagnosis by linkage analysis a possible option, even without knowledge of deleterious mutations. Prenatal diagnosis for severe MTHFR deficiency has been available by biochemical methodologies, but molecular genetic approaches have not yet been reported. We performed prenatal diagnosis for severe MTHFR deficiency in 11 at-risk pregnancies in seven families. A combined approach of linkage analysis and enzymatic assays was used in six pregnancies; linkage analysis alone was performed in one pregnancy. Linkage analysis for the 677C > T or 1298A > C polymorphisms predicted that all seven fetuses were unaffected. For six of these seven fetuses, enzymatic activities were also measured and demonstrated concordant results. Of the 10 pregnancies in which enzymatic assays were performed, activities in cultured amniocytes predicted six unaffected fetuses (1.4-7.1 nmol CHO/mg prot/h (U)) and one affected fetus (0.24 U [control 3.1-9.6 U]). Three pregnancies assessed via CVCs demonstrated two unaffected fetuses (3.6 and 7.7 U) and 1 affected fetus (0 U [control 4.5-7.8 U]). These values were compared to those of the probands (range = 0.02-0.7 U (control 2.4-11.7 U)) in cultured fibroblasts. Our findings suggest that linkage analysis for severe MTHFR deficiency can be a practical approach for prenatal diagnosis.     

溶酶体α-甘露糖苷贮积症基因治疗的初步研究

目的探讨溶酶体α－甘露糖苷贮积症基因治疗的可行性。方法以逆转录病毒为载体,将编码人溶酶体α－甘露糖苷酶ｃＤＮＡ导入病猫皮肤成纤维细胞。结果人源ｃＤＮＡ在病猫细胞内表达高活性α－甘露糖苷酶,重组酶的ｐＨ活性范围与正常酶相同,定位于细胞的溶酶体,分泌到细胞外后能通过甘露糖－６－磷酸受体介导的胞饮作用被其他细胞吸收。在体内,由重组病毒感染细胞组成的类器官表达有活性酶可达６周。结论此表达系统是研究溶酶体α－甘露糖苷贮积症基因治疗的有用模型。