Bruce A. Gordon

Deficiency of the Hexosaminidase A Activator Protein in a Case of GM2 Gangliosidosis; Variant AB

Summary: A patient is described whose clinical course and pathologic features, including massive brain storage of GM2 ganglioside in grey matter, are identical with those of classical Tay-Sachs disease despite normal levels of β-N-acetyl hexosaminidase and normal isozyme distribution. The kinetic properties and thermolability of the patients hexosaminidase are normal. Crude extracts of a postmortem sample of patients liver can catalyze the hydrolysis of 5.1 pmoles of labeled GM2 ganglioside/16 h/mg of protein (control liver = 69.9 pmoles/16 h/mg). Addition of partially purified human liver hexosaminidase A activator protein stimulated the hydrolysis of substrate by the patients liver extract by 27-fold, compared to 3-fold for control livers.Measurement of “activator” in enriched fractions of patients and control liver showed a reduced (25-30% of control) amount of stimulation of hexosaminidase A catalyzed hydrolysis of GM2 ganglioside as well as of Asialo-GM2 ganglioside. The addition of sphingomyelin to reaction mixtures, which is known to inhibit surfactant stimulation of hexosaminidase A, reduced activation of hexosaminidase A by patients liver preparation to undetectable levels.Polyacrylamide gel electrophoresis of enriched preparations of control and patients liver showed a rapidly migrating protein band in control liver corresponding to the activator protein and the absence of this protein band in the patients liver.Speculation: We believe that this patient is a genocopy of classical Tay-Sachs disease as a result of a mutation at a gene locus controlling the expression of the hexosaminidase A activator protein. The in vivo requirement of activator protein for hydrolysis of sphingolipid substrates by lysosomal hydrolases may explain the occurrence of other storage disease entities in which normal levels of enzyme are present in the homozygote when assayed using synthetic hydrophilic substrates.

Metabolic Changes in Alzheimer's Disease

Patients with Alzheimers disease (AD) and matched controls fasted for 24 hours, and serial glucose, pyruvate, lactate, β‐hydroxybutyrate, acetoacetate, insulin, and glucagon levels were measured. Patients with AD showed a glucose insulin correlation pattern over the 24 hours that differed from the control group. These differences may be secondary to weight loss or to other metabolic or nutritional factors affecting the AD patients.

Heteroallelic missense mutations of the galactosamine-6-sulfate sulfatase (GALNS) gene in a mild form of Morquio disease (MPS IVA).

Morquio disease (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Patients commonly present in early infancy with growth failure, spondyloepiphyseal dysplasia, corneal opacification, and keratan sulfaturia, but milder forms have been described. We report on a patient who grew normally until age 5 years. Her keratan sulfaturia was not detected until adolescence, and she now has changes restricted largely to the axial skeleton. She has experienced only mildly impaired vision. At age 22, thin-layer chromatography of purified glycosaminoglycans showed some keratan sulfaturia. GALNS activity in fibroblast homogenate supernatants was 20 +/- 5% of controls (as compared to 5 +/- 3% of controls in severe MPS IVA, P < .003). Kinetic analysis of residual fibroblast GALNS activity in patient and parents revealed decreased K(m) and increased Vmax in the mother and daughter, but not in the father, compatible with compound heterozygosity. GALNS exons were amplified from patient genomic DNA and screened by SSCP. Two missense mutations, a C to T transition at position 335 (predicting R94C) and a T to G transversion at position 344 (predicting F97V), were found on sequencing an abnormally migrating exon 3 amplicon. Digestion of the amplicon with FokI and AccI restriction enzymes (specific for the R94C and F97V mutations, respectively) confirmed heterozygosity. In fibroblast transfection experiments, heterozygous R94C and F97V mutants independently expressed as severe and mild GALNS deficiency, respectively. We interpret these findings to indicate that our patient bears heteroallelic GALNS missense mutations, leading to GALNS deficiency and mild MPS IVA. Our findings expand the clinical and biochemical phenotype of MPS IVA, but full delineation of the genotype-phenotype relationship requires further study of native and transfected mutant cell lines.

Acid hydrolases in the serum and liver in mucopolysaccharidoses types I and III

Summary 1. Results of the assay of several acid hydrolases in the serum and liver of control children and patients with types I and III mucopolysaccharidoses (Hurler and Sanfilippo syndromes) are described. 2. The activity of β-N-acetylglucosaminidase, β-glucuronidase, and aryl sulfatase A was elevated in the serum of patients with both types I and III disease. Serum acid phosphatase activity was depressed in type I patients. 3. Activities of α-mannosidase, α-fucosidase, β-N-acetylglucosaminidase, β-glucuronidase, hyaluronidase, and acid phosphatase were increased in the liver from patients with type I disease. In the liver from patients with type III disease activities of all of the above enzymes except acid phosphatase, and in addition aryl sulfatase A were elevated.

The mucopolysaccharidoses types I, II and III: Urinary findings in 23 cases*

Summary 1. Acid mucopolysaccharides (AMPS) were isolated from the urine of 23 patients with mucopolysaccharidoses, 8 with Type I disease (Hurler syndrome), 2 with Type II (Hunter syndrome) and 13 with Type III (Sanfilippo syndrome). 2. The AMPS-uronic acid excreted by all of the patients with mucopolysaccharidoses was significantly higher than that of normals and higher in Type I than in Type III patients. 3. The AMPS excreted were chromatographed on an anion exchange resin column. A distinctive pattern of AMPS distribution was seen with each Type of mucopolysaccharidoses permitting the differentiation of the various Types from each other as well as normals. 4. The fractions of AMPS isolated by anion exchange chromatography were characterized chemically and the size of AMPS was assessed by gel filtration chromatography. The dermatan sulfate and heparitin sulfate which appear in excess in the urine varied both in molecular size and degree of sulfation and they probably represent a series of degradation products of the native mucopolysaccharide polymers.

Tay-Sachs disease: B1 variant

This first child of non-Jewish parents had nystagmus at 4 months of age, bilateral cherry-red macular spots at 7 months of age, and hyperacusis at 8 months of age; the patient has deteriorated progressively following a clinical course typical of Tay-Sachs disease B variant. Total beta-N-acetylhexosaminidase assayed with 4-methylumbelliferyl-beta-glucosamine (4 MU GlcNAc) as substrate was within the normal range in plasma and cultured dermal fibroblasts and 2/3 the normal mean in leukocytes. The hexosaminidase A activity, assayed with the same substrate in plasma and cultured fibroblasts, approximated Tay-Sachs disease heterozygote levels; however, the activity of hexosaminidase A assayed with 4 MU Glc NAc-6-sulfate in the plasma, leukocytes, and cultured fibroblasts was less than 8, 2, and 1%, respectively of the control mean. This female infant with the B1 variant of Tay-Sachs disease demonstrated an earlier onset and more rapidly progressive course than was observed in 4 of the 5 previously reported patients with this Tay-Sachs disease variant.

Heparitin Sulfate Mucopolysaccharidosis |[lpar]|Sanfilippo Disease|[rpar]|: A Case Study with Ultrastructural, Biochemical, and Radiological Findings

Extract: A case study of a 14-year-old female child who developed normally for 18 months but subsequently deteriorated rapidly in mental status and social behavior is reported. Histochemical studies of tissues removed at biopsy from liver, skin, and bone marrow showed changes similar to those found in the Hurler syndrome. Ultrastructural changes of the liver were also similar to those found in the above syndrome with the exception of the presence of mitochondrial crystalloids not observed to date in the Hurler disease. Heparitin sulfate constituted the bulk of the acid mucopolysaccharides in the urine with only traces of chondroitin sulfate B present. In view of these observations and the lack of clinical and radiological findings generally associated with the Hunter and Hurler syndromes the diagnosis of Sanfilippo disease was made.Speculation: On the basis of the finding that the pattern of acid mucopolysaccharide (AMPS) excreted in urine in our patient differed from that of 12 other children with Sanfilippo disease [12], it is speculated that two subtypes of this disease exist characterized by different patterns of excretion of the AMPS in urine. Whether this difference will prove to be extended to other manifestations is not known at present, but it is conceivable that the difference in radiological findings (in our case minimal and nonspecific, in other cases resembling those of the Hurler and Hunter syndromes), degree of mental retardation, or morphological features, may parallel these biochemical differences. It would be important, therefore, to study other children with Sanfilippo disease in detail, to determine whether other variables are consistent with varying patterns of excretion of AMPS in urine.

Hepatic acid mucopolysaccharides in the mucopolysaccharidoses type I, II and III*

Summary 1. Acid mucopolysaccharides (AMPS) were isolated from hepatic tissue from 3 patients with Hurler syndrome (type I mucopolysaccharidoses), 1 with the Hunter syndrome (type II) and 2 with the Sanfilippo syndrome (type III). 2. The AMPS which constituted 4 to 6% of the dry liver weight were almost exclusively heparan sulfate in the type III patients and a mixture of heparan and dermatan sulfate in the type I and II patients with dermatan sulfate predominant in 1 of the type I patients but heparan sulfate dominant in each of the others. 3. The AMPS were divided into a series of fractions by anion exchange chromatography: the distribution was dependent on the type of AMPS, i.e. heparan or dermatan sulfate and the extent of their sulfation. 4. The fractions of either AMPS on gel filtration chromatography tended to be polydisperse with a mean size generally considerably smaller than that of either heparan sulfate from human placental tissue or dermatan sulfate from human umbilical cords. Dermatan sulfate eluted from the type I or II livers from the anion exchange resin with 2.0 M saline contained components whose molecular weight approximated that of the native material.

UNUSUAL CLINICAL AND ULTRASTRUCTURAL FEATURES IN A BOY WITH BIOCHEMICALLY TYPICAL MANNOSIDOSIS

Abstract. Gordon, B. A., Carson, R. and Haust, M. Daria (Departments of Biochemistry, Paediatrics and Pathology, the Childrens Psychiatric Research Institute, and the University of Western Ontario, London, Ontario, Canada). Unusual clinical and ultrastructural features in a boy with biochemically typical mannosidosis. Acta Paediatr Scand, 69: 787, 1980.—A 4½‐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 α‐mannosidase activity in his leukocytes, cultured fibroblasts and liver were no more than 10% of the control mean. Studies of the residual α‐mannosidase enzyme suggest a specific deficiency of the thermostable isoenzyme with an acidic pH optimum. The α‐mannosidase in the fibroblasts of our and another (control) patient with mannosidosis had a reduced affinity for the substrate 4‐methylumbelliferyl‐α‐D‐mannoside. Light micros‐copy 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 α 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.

Ultrastructure and Peroxidase of Leucocytes in Five Patients with Juvenile form of Ceroid Lipofuscinoses

Peripheral leucocytes obtained from five patients with clinical histories and funduscopic findings typical of the juvenile form of the so-called neuronal ceroid lipofuscinosis (NCLF) (synonym: Spielmeyer-Vogt disease) were assayed for peroxidase activity and examined by electron microscopy. The peroxidase levels were considerably lower in three but normal in two patients. Ultrastructurally, the lymphocytes of all five patients showed the presence of tubulo-membranous cytosomes many displaying the fingerprint images at present regarded as being typical for the NCLF. The possible implications of the discrepancy between the morphological observations and the enzymatic findings are discussed.