Michael Cantz

CORRECTIVE FACTORS FOR INBORN ERRORS OF MUCOPOLYSACCHARIDE METABOLISM

This paper will describe a “replacement therapy” for cultured fibroblasts which lack certain biologically active proteins because of genetic mutation. The defective fibroblasts in question are derived from patients with inborn errors of mucopolysaccharide metabolism: the Hurler, Hunter and Sanfilippo syndromes.‘ Genetically, the three disorders are distinct. Clinically, they are sufficiently similar so that pediatricians who are not specialists in this group of disorders may have trouble recognizing one from the other, particularly in the early stages; biochemically they belong to the same class, as all three are characterized by excessive quantities of mucopolysaccharide (dermatan sulfate and/or heparan monosulfate) excreted in the urine and deposited intracellularly in such tissues as liver and spleen. In 1965-1966, Danes and Beam’ and Matalon and Dorfman3 showed that fibroblasts cultured from the skin of such patients likewise accumulate mucopolysaccharide, subsequently identified as determatan ~ u l f a t e . ~ Whereas these rare disorders could previously be studied only in biopsy or autopsy material, it now became possible to perform biochemical studies in uitm on genetically marked cells, which could be grown in relatively large quantities. In 1967, this laboratory undertook a study of the kinetics of intracellular mucopolysaccharide accumulation, to determine if it occurred because of oversynthesis or reduced utilization. Using %04 as a marker for mucopolysaccharide, Fratantoni and associates’ noted that several processes went on simultaneously in all fibroblasts. Mucopolysaccharide is synthesized, and a portion (about 3/4) is promptly secreted into the medium; the remainder is diverted into a storage pool from which the only way out is by degradation. In addition, there is some reingestion and subsequent degradation of secreted mucopolysaccharide. The turnover of all the pools leading to synthesis and secretion (i.e., transport and activation of SO4, biosynthesis and sulfation of proteinpolysaccharide and its transport to the cell exterior) is very rapid, all these pools becoming saturated with %04 in less than an hour. On the other hand, the storage pool (lysosomal?) turns over on a time scale of days rather than minutes; it is much larger than the biosynthetic and secretory pools, and accounts for most of the intracellular mucopolysaccharide. By examining the rates of the various processes, it was concluded that the rates of synthesis, secretion and accumulation are not affected in the Hurler and Hunter syndromes, but that degradation is reduced. This does not mean that Hurler and Hunter fibroblasts do not degrade mucopolysaccharide; rather, they allow the storage pool to expand and turn it over more slowly with a half life of several days or even a week, compared to eight hours for normal fibroblasts. This is what one would expect if some enzyme in the degradative pathway had reduced activity or reduced affinity for its substrate. More recently, Wiesmann and coworkers have examined fibroblasts from patients with the Sanfilippo syndrome, and found that these also had an increased accumulation and lengthened turnover time of mucopolysaccharide, though not to the extent of Hurler and Hunter fibroblasts.6

A β-glucuronidase deficiency mucopolysaccharidosis: studies in cultured fibroblasts

Cultured skin fibroblasts derived from a patient with an atypical mucopolysaccharidosis are severely deficient in β-glucuronidase (β- d -glucuronide glucuronohydrolase). These fibroblasts display an, excessive accumulation and lengthened turnover time of sulfated mucopolysaccharide. The abnormal mucopolysaccharide metabolism can be corrected by addition of bovine β-glucuronidase to the culture medium. Identity of corrective factor and β-glucuronidase was demonstrated by coelectrophoresis of the two activities in polyacrylamide gel. Cells are able to take up β-glucuronidase in amounts far exceeding the requirement for full correction.

Biochemical heterogeneity of the sanfilippo syndrome: Preliminary characterization of two deficient factors

Abstract Fibroblasts cultured from the skin of Sanfilippo patients show excessive accumulation and prolonged turnover time of sulfated mucopolysaccharide. This abnormality can be corrected by a macromolecular factor contained in the secretions of fibroblasts of differing genotype, as well as in normal human urine. By cross-correction tests, the Sanfilippo fibroblasts can be subdivided into two groups, each deficient in a different factor. Analytical polyacrylamide gel electrophoresis shows the two factors, which are probably proteins, to have a similar molecular weight (ca. 200,000) but to differ in charge at pH 8.5.

The mucopolysaccharidoses: Biochemistry and clinical symptoms

SummaryThe mucopolysaccharidoses are a group of genetic diseases which are characterized by an excessive intralysosomal accumulation of partially degraded mucopolysaccharides. This storage is caused by the inactivity of one of eleven enzymes that are required for the degradation of the different types of mucopolysaccharides. There is a rough correlation between phenotype and chemical nature of the storage material. Similar clinical pictures, however, may be caused by an inactivity of different enzymes. Conversely, different clinical expressions of the defect of a single enzyme may be attributed to allelic mutations.The recent development of specific assay procedures for the respective enzymes allows 1. an early genotype-specific diagnosis of affected patients, 2. prenatal diagnosis of the metabolic defect in families at risk, 3. to prognosticate the course of the disease at least in some instances, and 4. genetic counselling for members of affected families. At present, there is no specific therapy. Attempts of enzyme replacement therapy are still at an experimental stage.ZusammenfassungDie Mucopolysaccharidosen stellen eine Gruppe genetischer Krankheiten dar, die durch eine intralysosomale Speicherung partiell abgebauter Mucopolysaccharide charakterisiert sind. Ursache der Speicherung ist die Inaktivität eines von elf verschiedenen Enzymen, die für den Abbau der verschiedenen Mucopolysaccharidtypen benötigt werden. Die Art des Speichermaterials korreliert annähernd mit dem klinischen Phänotyp. Ähnliche klinische Erscheinungsbilder können auf dem Defekt verschiedener Enzyme beruhen. Umgekehrt kann der Defekt eines einzelnen Enzyms beim Auftreten alleler Mutationen eine unterschiedliche klinische Expression zur Folge haben. Die Entwicklung spezifischer Methoden zur Aktivitätsbestimmung der betreffenden Enzyme ermöglicht 1. eine frühzeitige Genotyp-spezifische Diagnose erkrankter Patienten, 2. in Risikofamilien eine pränatale Erkennung des Stoffwechseldefektes, 3. in einzelnen Fällen eine Prognose über den Krankheitsverlauf und 4. eine genetische Beratung von Mitgliedern betroffener Familien. Eine kausale Therapie existiert zur Zeit nicht. Versuche zur Enzymsubstitution befinden sich noch im experimentellen Stadium.

PARTIAL DEFICIENCY OF GLYCOPROTEIN NEURAMINIDASE IN SOME PATIENTS WITH MORQUIO DISEASE TYPE A

SUMMARY: A deficiency of glycoprotein neuraminidase (sialidase, acylneuraminyl hydrolase, EC 3.2.1.18) activity was found in fibroblasts from a patient with the clinical symptoms of Morquio disease type A (mucopolysaccharidosis IV A ). Residual neuraminidase activity was about 5 % of the mean normal activity. N-Acetylgalactosamine-6-sulfate (GalNAc-6-S) sulfatase activity was reduced to less than 1 % of normal with a pH-optimum of 3.0 as expected for the severe form of Morquio disease. In peripheral leucocytes of the patient, however, neuraminidase activity but not GalNAc-6-S sulfatase activity was in the normal range. Mixing experiments excluded the presence of excessive amounts of inhibitors of neuraminidase activity.In the urine of the patient, the content of bound sialic acid was with 0.357 μmol/mg creatinine increased, but amounted for only about 10 % of that of a sialidosis patient.Somatic cell hybridization of the patients fibroblasts with those from a Morquio A patient did not result in complementation of GalNAc-6-S sulfatase activity. Fusion with sialidosis fibroblasts, on the other hand, yielded a significant increase in neuraminidase activity.Reinvestigation of 10 different Morquio A cell lines revealed two of them to have neuraminidase activities below the normal range, whereas the activity of 6 cell lines was in the low normal range.

72: The diagnosis of mucolipidosis II in a pregnancy at risk by studies on amniotic materials

Mucolipidosis II (ML II, I-cell disease) is a fatal inborn error of metabolism with altered levels of certain lysosomal hydrolases. In a pregnancy at risk, in which ML II was suspected, we were able to monitor the activity of several lysosomal hydrolases in cultured amniotic fluid cells and in amniotic fluid. In amniotic fluid an increase in hydrolase activity was observed, whereas cultured amniotic cells showed a decrease, together with an accumulation of 35S-labeled glycosaminoglycans (35S GAGs). The pregnancy was therefore interrupted in the 22nd gestational week. The diagnosis was clearly confirmed by analysis of material from the aborted fetus. In fetal serum the activities of several lysosomal hydrolases were elevated approximately 10 to 70-fold, whereas the enzyme levels in fetal fibroblasts were considerably reduced. There was also an excessive accumulation of 35S GAGs in fetal fibroblasts. Our results, and those of others (Aula et al, J.Pediat. 87, 1975; Huijing et al, Clin.Chim.Acta 44, 1973;Matsuda et al, Humangenetik 30 1975), indicate that increased levels of these enzymes in amniotic fluid may suffice as a prenatal diagnosis of ML II. However, due to considerable variations between individual samples, the diagnosis should also be confirmed by analyses of these enzymes and by 35S incorporation studies in cultured amniotic cells.

Alpha-L-iduronidase deficiency: a spectrum of clinical disorders

Hurler and Scheie diseases (mucopolysaccharidosis I-H and I-S) are both manifestations of the defective action of alpha-L-iduronidase. Fourteen and nine year old sisters are presented showing excessive 35S-mucopolysaccharide storage and deficient activity of alpha-L-iduronidase in their cultured fibroblasts. Their pattern of clinical and radiographic findings differed from that of both Hurler and Scheie diseases. The intelligence was only moderately impaired. Microscopic and ultrastructural studies demonstrated excessive mucopolysaccharide storage in leucocytes, bone marrow cells and liver cells. Increased amounts of mucopolysaccharides with characteristics of dermatan sulfate and chondroitin sulfate were found in the urine. The condition may be caused by a state of double heterozygosity for the Hurler and Scheie gene (so-called Hurler-Scheie compound).

Beta-D-glucuronidase deficiency mucopolysaccharidosis

A two year old girl presented with mild facial dysmorphism reminiscent of Hurler disease, moderate mental retardation, and hepatosplenomegaly. Hercorneae were clear. Radiologically, there was moderate dysostosis multiplex. Peripheral granulocytes contained abundant, coarse Alder-Reilly granulations. There was an increased urinary excretion of acid mucopolysaccharides. Determination of lysosomal glycosidases showed a markedly decreased activity of β-D-glucuronidase in cultured fibroblasts, leudocytes, and serum. In the parents, in one grandmother and in one sister, β-D-glucuronidase activity in serum and leucocytes was decreased to between 30 and 50%. The disorder has been classified as mucopolysaccharidosis,type VII. Our results indicate that a reliable diagnosis of the homozygous and heterozygous deficient state can be made by determining β-D-glucuronidase in serum.

Deficiency of Specific Proteins in the Inborn Errors of Mucopolysaccharide Metabolism

This paper will summarize recent studies on the nature and function of those proteins which are deficient in the mucopolysaccharidoses.