Refer to: Beutler E: Newer aspects of some interesting lipid storage diseases: Tay-Sachs and Gaucher's diseases-Seventh Annual Paul M. Aggeler Memorial Lecture-Medical Staff Conference, University of Califomia, San Francisco. West J Med 126:46-54, Jan 1977
Medical Staff Conference
Newer Aspects of Some Interesting Lipid Storage Diseases:
Tay-Sachs and Gaucher's Diseases Seventh Annual Paul M. Aggeler Memorial Lecture Delivered October 5, 1976, San Francisco General Hospital Medical Center
ERNEST BEUTLER, MD, Duarte, California
These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from transcriptions, they are prepared by Drs. David W. Martin, Jr., Associate Professor of Medicine, and H. David Watts, Assistant Professor of Medicine, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine. Requests for reprints should be sent to the Department of Medicine, University of California, San Francisco, CA 94143.
DR. WALLERSTEIN: * Dr. Beutler had his formal education at the University of Chicago where he earned his MD degree in 1950. He went through residency and fellowship there and stayed on the staff until 1959 when he assumed directorship of the Division of Medicine at the City of Hope. He is also Clinical Professor of Medicine at the University of Southern California. The most outstanding of his many prestigious lectureships is perhaps the Stratton Lecture at the American Society of Hematology in 1974. He is a member of the Central, the Western and the American Societies of Clinical Investigation, the Association of American Physicians, and the National Academy of Sciences. His bibliography comprises more than 300 publications, and he has written, edited or co*Ralph 0. Wallerstein, M, Chief of Clinical Hematology Division of the Medical Service, San Francisco General Hospital, Clinical Professor of Medicine and Clinical Pathology and Laboratory Medicine, University of California, San Francisco, and Chairman of the Paul M. Aggeler Memorial Committee. This work was supported in part by Grant AM 14755 from the National Institutes of Health, Bethesda, Maryland.
46
JANUARY 1977 * 126 * 1
edited six books. More important than the quantity of this output is the quality. He has been an important pioneer in the story of glucose-6-phosphate dehydrogenase deficiency and other red blood cell enzyme deficiencies causing hemolytic anemia. He has invented some useful fluorescent screening tests for these enzymopathies and has contributed considerable knowledge about iron metabolism. In 1957 he wrote an article on the clinical evaluation of iron stores that is still widely quoted. He has worked extensively on iron enzymes and was the first to show clinically that females are a mosaic of X chromosomes, some chromosomes being active and some not. His work has also included studies on galactosemia and preservation of red blood cells. He invented a new type of medical career in which he brings ideas and supervision to his superlative laboratory, supervises the work, puts it together on paper and presents it clearly at meetings. He has avoided being overburdened by clinical practice, formal teaching and administra-
TAY-SACHS AND GAUCHER'S DISEASES TAY-SACHS GANGLIOSIDE
~~~~~~A
t
GLUCOCERE BROSI DE GLUCOSE - GALACTOSE - N - ACETYLGALACTOSAMINE-GALACTOSE SPHINGOSINE- FATTY ACID
N- ACETYL NEURAMINIC ACID
CERAM IDE
Figure 1.-The structure of a ganglioside. Tay-Sachs ganglioside (GM2) is formed by removal of the terminal sugar. This ganglioside accumulates in patients with Tay-Sachs disease and Sandhoff's disease. If all but one of the sugars are removed, glucocerebroside, the glycolipid of Gaucher's disease, is formed. CERAMIDE TRIHEXOS IDE
GLUCOCERE BROSIDE GLUCOSE- GALACTOSE -GALACTOSE-N-ACETYLGALACTOSAMINE
SPHINGOSINE -FATTY ACID CERAM IDE
Figure 2.-The structure of a globoside. Removal of all but the first sugar produces glucocerebroside, the glycolipid stored in patients with Gaucher's disease. ABBREVIATIONS USED IN TEXT
Gm0 =Tay-Sachs ganglioside hex= hexosaminidase CRM = cross-reacting material
tion. Dr. Beutler has recently turned his attention to lipid storage diseases and will address us on recent developments in Tay-Sachs and Gaucher's diseases. DR. BEUTLER: * I feel very privileged to have been invited to present the Paul Aggeler Memorial Lecture. Paul Aggeler was one of our generation's great hematologists. He was one of those extraordinary persons, encountered only too rarely, whose ability to combine clinical observations and laboratory studies enabled him to advance significantly the frontiers of knowledge. I first met Paul Aggeler in the late 1950's, during my annual January pilgrimage to the clinical meetings in the charming setting provided by Carmel. Paul was always unassuming, friendly and soft spoken, yet his discovery of hemophilia B ranks as one of the milestones in the field of blood coagulation. Because of Paul's devotion to hematology, the privilege of presenting the annual Aggeler lecture has generally been accorded to a hematologist. It is, therefore, with considerable *Ernest Beutler,
MD, Chairman, Division of Medicine, and
Director, Department of Hematology, City of Hope Medical Center, Duarte, California.
hesitation that I selected the topic for my lecture today. The glycolipid storage diseases are not hematologic disorders in the usual sense of the word. However, because hematology touches so many fields, particularly genetics, the interests of hematologists generally are very broad. I reasoned that Paul would have approved. I first learned about storage diseases when I entered medical school 30 years ago. Each of these disorders was associated with storage of a chemical substance and with an eponym-the name of the person credited with describing the disease. Remembering which eponym belonged to which chemical was not always easy. Sometimes mnemonic devices were helpful. For example, I knew a young woman whose name rhymed with "Gaucher" and who seemed to have limited intelligence; this served as a reminder to me that Gaucher's disease was the disorder characterized by storage of cerebrosides. The situation is now very different; much has been added to our knowledge of the glycolipid storage diseases. In the past decade and a half there has been a virtual avalanche of progress in the field, and understanding of these disorders is now possible at a molecular level. All of the glycolipid storage disorders seem to have a similar pathogenesis. The complex glycolipids, gangliosides and globosides (Figures 1 and 2) are constantly turned over by normal cells. THE WESTERN JOURNAL OF MEDICINE
47
TAY-SACHS AND GAUCHER'S DISEASES
Their catabolism requires that the sugars that comprise their carbohydrate portion be hydrolyzed one at a time, starting at the distal end. Each sugar is removed by an enzyme that is specific both for the sugar and for the type of linkage (a or 8) in which it is attached. Deficiency of one of these hydrolytic enzymes results in accumulation of its substrate, the ganglioside or globoside degradation product it was intended to catabolize. I will now discuss the studies carried out in our laboratory concerning the biochemical genetics of two general types of glycolipid storage diseases, Tay-Sachs and Sandhoff's diseases in which Tay-Sachs ganglioside or ganglioside monosialate, 2 (GM2) accumulates, and Gaucher's disease in which glucocerebroside is stored.
Tay-Sachs and Sandhoff's Diseases Tay-Sachs disease and the clinically and pathologically almost identical Sandhoff's disease are progressive neuromuscular disorders that lead to blindness, severe mental retardation and death within the first few years of life. The glycolipid that is stored in this disorder is a ganglioside, GM2 (Figure 1). It is apparent that Gm2 has not just one terminal sugar but two, N-acetylneuraminic acid and N-acetylgalactosamine. Brady' and others attempted to resolve the question of which of the two enzymes involved was defective by synthesizing GM2 with labeled N-acetylgalactosamine and with labeled N-acetylneuraminic acid. Initially, it seemed likely that a deficiency of one of the neuraminidases was responsible for the disease, because studies with artificial substrates (simple analogs of the ganglioside such as p-nitrophenyl-N-acetylgalactosamine) had shown that normal N-acetylgalactosaminidase (hexosaminidase [hex]) activity was present in the tissues of children dying with Tay-Sachs disease. However, Robinson and Stirling2 showed that there were, in fact, two separable forms of hexosaminidase in human tissues, hex A and hex B. When Sandhoff3 and Okada and O'Brien4 examined hexosaminidase from children with Tay-Sachs disease for these two specific isoenzymes, striking results were obtained. Children with Tay-Sachs disease had increased activity of hex B, but hex A activity could not be detected. A few non-Jewish children with a particularly severe form of Tay-Sachs disease were found to lack both hex A and hex B; their disease was designated Sandhoff's disease. Recognition that Tay-Sachs disease was due to a deficiency of hex A was a finding of great poten48
JANUARY 1977 * 126 * 1
tial practical importance. Tay-Sachs disease is an autosomal recessive disorder, which has a high frequency among Jews of Eastern European ancestry. Measuring the hex A content of the serum of prospective parents could identify the heterozygous state and thereby make possible detection of couples whose fetuses were at risk of developing this dreadful disorder. Diagnosis by amniocentesis was possible, and selective abortion could be offered to the parents.5 Heterozygote detection, however, is not simple when it is necessary to depend solely on the level of activity of an enzyme. Others factors cause considerable variation, and overlap is encountered between the levels in putative normal and putative heterozygous persons. If the cutoff point for the differentiation of heterozygotes from normal persons is too high, many normal women might undergo amniocentesis unnecessarily. If it is too low, some parents who had been reassured by the results of prenatal testing would suffer the agony of having a child with Tay-Sachs disease. My own interest in the biochemical genetics of Tay-Sachs disease was stimulated by this vexing and potentially important problem. As a hematologist it occurred to me that a similar problem had been encountered in the diagnosis of hemophilia and that the solution might be applicable. Zimmerman, Ratnoff and Littell6 measured the ratio of Factor VIII procoagulant activity tp Factor VIII antigen in the plasma. Because patients with hemophilia usually produce immunologically active but functionally inactive Factor VIII, the plasma of carriers was found to have more immunologic than procoagulant activity. If patients with Tay-Sachs disease synthesized an immunologically detectable and catalytically inactive hex A, the ratio of hex A to hex A antigen would be a much more sensitive way to detect heterozygotes for this disorder. Another reason for undertaking studies of Tay-Sachs disease was a more academic one. How could it be that one autosomal recessive disorder produces a deficiency only of hex A, whereas in other families an autosomal recessive disease produces a deficiency of both hex A and B? There were several theoretic explanations, but which was the right one? Working with Dr. S. K. Srivastava in our laboratory we first purified hex A and hex B to homogeneity. Human placenta served as suitable starting material, and we were able to immunize rabbits against the homogeneous material. Im-
TAY-SACHS AND GAUCHER'S DISEASES
munoelectrophoresis showed us that little or no cross-reacting material (CRM) was present in the place of hex A in livers of children dying of TaySachs disease7 (Figure 3). This finding made it appear unlikely that we would be able to apply the strategy used in diagnosing hemophilia carriers to the problem of Tay-Sachs disease. However, our curiosity about the relationship between hex A and hex B had not yet been fulfilled. Study of the antibody against these two enzymes provided us with our first insight into their relationship. Not only were they related genetically, in that a single gene produced deficiency of both enzymes in Sandhoff's disease, but they were also closely related immunologically. The antiserum that we produced against hex A reacted with hex
Hex-B
B, and the antiserum that we produced against hex B reacted with hex A.8 We succeeded in absorbing anti-hex B activity from serum produced against hex A. The absorbed serum was still able to react against hex A (Figure 4). Thus, we concluded that hex A had antigenic determinants that were absent from hex B. On the other hand, we learned that anti-hex A activity could not be removed from antiserum raised against hex B without loss of its capacity to react with hex B as well. These studies led us to suggest that hex A had one subunit, which we designated "a," that was not present in hex B, and that hex A had another subunit, ",/," that was represented in hex B. Because the molecular weight of the two enzymes seemed to be identical,9 the simplest representa-
--
I
I
Hex-A -*
N
T,
N
T2
N
T3
N
S1
T,
S1
(
T, S2 Si
2
Figure 3.-Immunoelectrophoresis of normal liver extract (N), extract from livers of three unrelated patients with TaySachs disease (T,, T2, T3), and liver extracts from two unrelated patients with Sandhoff's disease (SI, S2). (Reproduced by permission from Srivastava and BeutlerT).
IL-
Figure 4.-Upper, Immunodiffusion of purified enzyme and liver extracts against antisera (central wells) against hex A, against hex B and against A treated with hex B (absorbed anti-A). Lower, same gel stained for hexosaminidase activity.
Center Well
Anti-A
Anti-B
absorbed Anti-A THE WESTERN JOURNAL OF MEDICINE
49
TAY-SACHS AND GAUCHER'S DISEASES
tion of their structure was that hex A was a/3 and that hex B was /,8. One previously published observation seemed to conflict with this interpretation, however. It had been reported that hex A could be converted to hex B by treatment with neuraminidase.10"11 Although we were unable to confirm this observation, we did note that as hex A solutions were incubated, traces of hex B appeared to form. The interpretation of these data was clarified when we saw a report that indicated that even heat-inactivated neuraminidase preparations had the capacity to convert hex A into hex B.'2 Apparently, something else in neuraminidase produced the conversion, and we quickly established that it was the Merthiolate' that had been added as a preservative.'3 p-Hydroxymercuribenzoate has long been used to dissociate hemoglobin chains, and it seemed reasonable that similar chain dissociation might be accomplished with other mercurial compounds such as Merthiolate. We proposed'4 the following schema to account for the effect of Merthiolate in producing hex B from hex A: a or alkylated a v- (a/3)n - (/3)n Inactive
hex A
hex B
If Merthiolate produces the conversion by dis-
sociating the a and /3 subunits of hex A, with subsequent reassociation of /3 subunits to form hex B, we should be able the find the a subunit. Indeed, we were able to separate an alkylated dimeric a subunit from pure hex A preparations that had been treated with Merthiolate or p-hydroxymercuribenzoic acid.'5 Recently, an anti-a chain serum has been produced by immunizing rabbits with such a-chains after their treatment with glutaraldehyde.'6 Definitive proof of the subunit structure of hex A and hex B was achieved through study of the tissues of a patient with Sandhoff's disease. As had previously been noted by others, a small amount of residual hexosaminidase activity could be identified in such persons and was designated hex S17,18 (Figure 5). According to our subunit model, patients with Sandhoff's disease are unable to form functionally adequate /l-chains, and thus the residual enzyme activity in Sandhoff's disease might be attributed to polymerized a-chains. The observation that hex S was made up of a-chains was verified immunologically; it reacted with antiA but not with anti-hex B serum.'8 Because hex S was a homopolymer of a-chains and hex B a homopolymer of /3-chains, it should be possible
+
-
Hex S
*---
Hex A
Origin -I *-IN%
Hex B
Cont. Pat. Cont. Pat. Cont. Pat. wbc wbc fib. fib. wbc wbc Figure 5.-Starch gel electrophoresis at pH 6.0 of fibroblasts (fib.) and leukocytes (wbc) from normal subjects (Cont.) and a patient (Pat.) with Sandhoff's disease.18 Extracts containing approximately equal amounts of hexosaminidase were placed in the slots, and the gels were stained for hexosaminidase activity. Hex A and hex B are missing from the cells obtained from a patient with Sandhoff's disease. Instead, a rapidly moving band, designated as hex S, was present. This band represents a-chain polymers.
50
JANUARY 1977 * 126 * 1
TAY-SACHS AND GAUCHER'S DISEASES
to produce hex B and hex S from hex A, and to MIXED CELLS
ILYMPHOCYTE- RICH
GRANULOCYTE-RICH
rAdE'SNRA NORMAL a"DG DISEASE
NORMAL NORMAL
NORMAL I%zS OSESE
0
120' 0
100-
I-
P.)
co
s0o .
600 .
40
.
20
* 0 00
O
.
I
I
I
I
*
0
pH 5.0 (CITRATE BUFFER)
Figure 6.-,8-Glucosidase activity of leukocytes from normal subjects and patients with Gaucher's disease. The assays were carried out at pH 5 with 4-methylumbelliferyl-,e-D-glucopyranoside as substrate. At this pH, poor discrimination between normal and Gaucher's disease subjects was achieved.
produce hex A from hex B plus hex S. This, too, was accomplished."' A striking similarity is apparent between the hexosaminidase system and the thalassemias. In both a-thalassemia and Tay-Sachs disease, a deficiency in a-subunits results in polymerization of B-subunits. In a-thalassemia, hemoglobin /8 chains form hemoglobin H; in Tay-Sachs disease, hexosaminidase ,B chains polymerize to form increased amounts of hex B. In both a-thalassemia and Sandhoff's disease, an excess of a-chains is found. The a-hemoglobin chains in /8-thalassemia apparently damage the cell membrane. The ahexosaminidase chains aggregate in Sandhoff's disease to form hex S. Gaucher's Disease Gaucher's disease is a particularly prevalent glycolipid storage disease, occurring at a frequency of 1:5,000 to 1:10,000 Jewish births. In the most common adult variety of Gaucher's disease, gradual progressive storage of glucocerebroside in liver, spleen and bone marrow results in pancytopenia, hepatic failure and pathologic fractures. The enzymatic defect in Gaucher's dis-
Figure7.-,B-GIucosldase
0
of leukocytes of normal subjects, patients with Gaucher's disease, and obligate heterozygotes. The assays were carried out at a pH of 4 with
4-methylumbelliferyl-,8-D(I)
:V
glucopyranoside as substrate. Excellent discrimination between the different genotypes was achieved at this pH. (Reproduced by permission from Beutler E and Kuhl W: J Lab Clin Med 76: 747-755, 1970.)
THE WESTERN JOURNAL OF MEDICINE
51
TAY-SACHS AND GAUCHER'S DISEASES
ease has been identified as a deficiency in glucocerebrosidase.20 Localization of the glycolipid in the reticuloendothelial system makes Gaucher's disease a particularly attractive target for replacement therapy. The first problem that we faced in 1969 as we began to study the biochemical genetics of Gaucher's disease was that the radioactively labeled glucocerebroside used to show enzyme deficiency existed in only one laboratory and was not available to us. Its synthesis was complex and it was by no means certain that it could be carried out successfully again. However, the use of an artificial analog of glucocerebroside, 4-methylumbelliferyl-/3-glucopyranoside for study of this disorder had recently been described,21 and this material was commercially available. Because it seemed possible that the artificial substrate measured enzymes other than glucocerebrosidase, W. Kuhl attempted to determine whether or not patients with Gaucher's disease lack this activity. Because leukocytes were the most readily available cells and because examination of these cells might also lead to detection of the heterozygous state, they were chosen for study. Our initial results were disappointing (Figure 6). Assaying ,8glucosidase at pH 5, the pH generally chosen for the assay of acid hydrolases, we found little differences in the p-glucosidase activity of normal leukocytes and those from Gaucher's patients. To determine whether or not a major qualitative difference existed between the ,B-glucosidase
Figure 8.-Partial splenectomy according to the procedure used by Dr. Robert Yonemoto, City of Hope Medical Center.
52
JANUARY 1977 * 126 * 1
of Gaucher's cells and normal cells, we investigated some of the properties of leukocyte /8-glucosidase using an approach similar to that employed in investigating a glucose-6-phosphate dehydrogenase mutant. The pH-activity curves of ,8-glucosidase from normal subjects showed a pH optimum of approximately 5, which was identical to that of patients with Gaucher's disease. We discovered, however, that a second and lower pH optimum was present in normal leukocytes and that the enzyme activity at this latter pH of 4.0 was missing from the leukocytes of patients with Gaucher's disease. If we assayed the enzyme at pH 4,22 clear distinction between the leukocytes from patients with Gaucher's disease and normal persons was possible (Figure 7). It was also possible to identify heterozygotes for Gaucher's disease with reasonable confidence by using a lymphocyte-rich preparation of leukocytes. Some overlap was present, due in large part to the higher enzyme activity in monocytes than in lymphocytes. The lymphocyte preparation also contained monocytes, and its activity was therefore influenced to a pronounced degree by the extent of contamination with these cells. Layering suspensions of the lymphocyte-rich fractions on plastic Petri dishes allowed us to separate lymphocytes from monocytes; the latter attach to the plastic surface. Now it was possible to better differentiate normal from heterozygous subjects.23 Similar results were obtained in a study of cultured fibroblasts. Here, too, use of the artificial substrate, 4-methylumbelliferyl-/3-glucopyranoside permitted differentiation of Gaucher's disease from normal and from heterozygous subjects. Amniotic cells also contain ,8-glucosidase activity, and the identification of fetuses with Gaucher's disease is now possible. The ultimate aim of our study, however, has been the development of improved methods for the treatment of patients with Gaucher's disease. No specific therapy is available and management of patients with this disorder is currently a frustrating experience. One of the most frequently encountered complications of Gaucher's disease is thrombocytopenia. Splenectomy invariably produces normalization of the platelet count but does not reduce the amount of globoside that the body must metabolize each year. Instead of being sequestered relatively harmlessly in a gradually enlarging spleen, the glycolipid now accumulates in liver and bone. Dr. Robert Yonemoto, of the Department of
TAY-SACHS AND GAUCHER'S DISEASES
Surgery at the City of Hope, has successfully carried out a partial splenectomy in a patient with Gaucher's disease. The portion of the upper pole of the spleen supplied by the short gastric vessels was preserved, and bleeding from the cut edges was controlled with fibrin foam and mattress sutures (Figure 8). Complete remission of the patient's thrombocytopenia was achieved. We hope that the small piece of spleen left behind will prosper and enlarge, continuing to accumulate glycolipid and thereby protecting the patient's liver and bones from damage. However, partial splenectomy may, at best, provide only a limited solution to some of the problems associated with Gaucher's disease. More promise lies in replacement of the missing enzyme. Unfortunately, glucocerebrosidase has proved to be extraordinarily difficult to purify, largely due to its firm attachment to the lysosomal membrane and our consequent inability to solubilize it. Pentchev and co-workers24 succeeded in carrying out partial purification of glucocerebrosidase in a detergent-soluble form. The enzyme was precipitated on serum albumin and administered to several patients.25 The yield was extremely low, limiting the amount that could be administered. Although transient decreases of glucocerebroside levels in red blood cells were observed, no clinical improvement was recorded. Dr. George Dale in our laboratory recently succeeded in developing a high-yield, relatively rapid method of purifying soluble 83-glucosidase from human placenta.26 Earlier attempts to solubilize the enzyme by preparing acetone powders from placenta had resulted in loss of all enzyme activity. Dr. Dale's discovery that ,-glucosidase has an absolute requirement for acidic phospholipid27 immediately explained our earlier failure. We found that treating placental extract with organic solvents such as acetone or ethanol does release the 8-glucosidase in soluble form, but that activity can only be measured in the presence of added phospholipid. With the solubilization of ,8-glucosidase, further purification by conventional and affinity techniques became possible.28 A delivery system is needed to bring the enzyme to the stored glycolipid. Several methods have been proposed, but one of the simplest appears to be enclosure of the enzyme in the patient's own red blood cells.29 When erythrocytes are lysed in hypotonic solution, resealing can be effected by incubation in isotonic saline. Such lysis and resealing is most easily carried out in dilute
suspensions of erythrocytes, but this would be much too wasteful of the precious enzyme purified from many kilograms of placenta. We therefore added the purified enzyme to packed erythrocytes in a cell dialysis bag, dialyzing first against a 5 mM potassium phosphate buffer, pH 7.4. The dialysis bath was then replaced with a phosphate buffer containing 0.9 percent sodium chloride, and the mixture was warmed to 25°C. After two hours, the resealed erythrocytes were washed. Usually 15 to 30 percent of the enzyme was entrapped within erythrocytes. We have undertaken experimental enzyme replacement therapy in one patient with far-advanced Gaucher's disease. The first course of treatment, three injections of resealed cells containing a total of 560 mUnits of enzyme, was carried out in April 1976. In July 3,700 mUnits of enzyme were directly administered intravenously as two injections. Finally, in September another 2,200 mUnits of enzyme were administered, again in erythrocytes-but those which were coated with anti-Rh antibody to facilitate their clearance from the circulation. In the complex setting of far-advanced Gaucher's disease, the results of therapy are not easy to evaluate. Intercurrent infections and other complications tend to obfuscate the results. However, it was clear that enzyme therapy was well tolerated; no untoward reactions were experienced by the patient during any of the courses of treatment. At the onset of therapy, the patient's liver was the same size as had been recorded eight years previously. During the six months of enzyme therapy, some decrease in the size of the liver appeared to occur; the hepatic margin, identified by palpation, regressed 3 to 6 cm. Visualization of the liver with sulfur-technetium colloid in 1968 showed there to be major areas in which the radionuclide was not taken up. These areas had enlarged somewhat before enzyme replacement therapy was started. At the end of six months of treatment, hepatic uptake of colloid had greatly improved, so that the liver scan had a much more normal homogeneous appearance. Each course of enzyme therapy was associated with a rapid increase in the platelet count which lasted for several weeks. There seemed to be some increase in the overall sense of well-being. Further observation of this patient and other patients whom we plan to treat will be required to delineate the possible role of enzyme replacement in the management of patients with Gaucher's disease. THE WESTERN JOURNAL OF MEDICINE
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TAY-SACHS AND GAUCHER'S DISEASES
The treatment of glycolipid storage diseases by the administration of exogenous enzyme represents the type of interaction between basic science and clinical medicine that was one of the outstanding features of Paul Aggeler's professional career. It is for this reason that I considered these studies to be an appropriate theme for this year's Paul Aggeler Memorial Lecture. REFERENCES 1. Brady RO: Tay-Sachs disease. N Engl J Med 281:1243-1244, Nov 27, 1969 2. Robinson D, Stirling JL: N-acetyl-f1-glucosaminidase in human spleen. Biochem J 107:321-327, 1968 3. Sandhoff K: Variation of 83-N-acetylhexosaminidase-pattern in Tay-Sachs disease. FEBS Lett 4:351-354, 1969 4. Okada S, O'Brien JS: Tay-Sachs disease: Generalized absence of a j8-D-N-acetylhexosaminidase component. Science 165: 698-700, 1969 5. O'Brien JS, Okada S, Fillerup DL, et al: Tay-Sachs disease: Prenatal diagnosis. Science 172:61-64, 1971 6. Zimmerman TS, Ratnoff OL, Littell AS: Detection of carriers of classic hemophilia using an immunologic assay for antihemophilic factor (Factor VIII). J Clin Invest 50:255-258, 1971 7. Srivastava SK, Beutler E: Studies on human j8-D-N-acetylhexosaminidases-III. Biochemical genetics of Tay-Sachs and Sandhoff's diseases. J Biol Chem 249:2054-2057, 1974 8. Srivastava SK, Beutler E: Antibody against purified human hexosaminidase B cross-reacting with human hexosaminidase A. Biochem Biophys Res Commun 47:753-759, 1972 9. Srivastava SK, Yoshida A, Awasthi YC, et al: Studies on human 8-D-N-acetylhexosaminidases-II. Kinetic and structural properties. J Biol Chem 249:2049-2053, 1974 10. Goldstone A, Konecny P, Koenig H: Lysosomal hydrolases: Conversion of acidic to basic forms by neuraminidase. FEBS Lett 13:68-72, 1971 11. Murphy JV, Craig L: Effect of human cerebral neuraminidase on hexosaminidase A: Clin Chim Acta 51:67-73, 1974 12. Carmody PJ, Rattazzi MC: Is neuraminidase responsible for the in vitro conversion of human hexosaminidase A to hexosaminidase B? Am J Hum Genet 25:19A, 1973 13. Beutler E, Villacorte D, Srivastava SK: Non-enzymatic conversion of hexosaminidase-A to hexosaminidase-B by merthiolate. IRCS:2:1090, 1974
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JANUARY 1977 * 126 * 1
14. Beutler E, Villacorte D, Kuhl W, et al: Nonenzymatic conversion of human hexosaminidase A. J Lab Clin Med 86:195-203, 1975 15. Beutler E, Yoshida A, Kuhl W, et al: The subunits of human hexosaminidase A. Biochem J 159:541-543, 1976 16. Srivastava SK, Wiktorowicz JE, Awasthi YC: Interrelationship of hexosaminidase A and B-Confirmation of the common and the unique subunit theory. Proc Natl Acad Sci USA 73:28332837, 1976 17. Ikonne JU, Rattazzi MC, Desnick RJ: Characterization of hex S, the major residual /1-hexosaminidase activity in type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease). Am J Hum Genet 27:639-650, 1975 18. Beutler E, Kuhl W, Comings D: Hexosaminidase isozyme in type 0 Gii2 gangliosidosis (Sandhoff-Jatzkewitz disease). Am J Hum Genet 27:628-638, 1975 19. Beutler E, Kuhl W: Subunit structure of human hexosaminidase verified: Interconvertibility of hexosaminidase isozymes. Nature 258: 262-264, 1975 20. Brady RO, Kanfer J, Shapiro D: The metabolism of gltcocerebrosides-I. Purification and properties of a glucocerebrosidecleaving enzyme from spleen tissue. J Biol Chem 240:39-43, 1965 21. Ockerman PA, Kohlin P: Tissue acid hydrolase activities in Gaucher's disease. Scand J Clin Lab Invest 22:62-64, 1968 22. Beutler E, Kuhl W: Detection of the defect of Gaucher's disease and its carrier state in peripheral-blood leukocytes. Lancet 1:612-613, 1970 23. Beutler E, Kuhl W, Matsumoto F, et al: Acid hydrolases in leukocytes and platelets of normal subjects and in patients with Gaucher's and Fabry's disease. J Exp Med 143:975-980, 1976 24. Pentchev PG, Brady RO, Hibbert SR, et al: Isolation and characterization of glucocerebrosidase from human placental tissue. J Biol Chem 248:5256-5261, 1973 25. Pentchev PG, Brady RO, Gal AE, et al: Replacement therapy for inherited enzyme deficiency-Sustained clearance of accumulated glucocerebroside in Gaucher's disease following infusion of purified glucocerebrosidase. J Molecular Med 1: 73-78, 1975 26. Dale GL, Beutler E: Enzyme replacement therapy in Gaucher's disease-I. A rapid, high yield method for purification of glucocerebrosidase. Proc Natl Acad Sci USA (in press), 1976 27. Dale GL, Villacorte D, Beutler E: Solubilization of glucocerebrosidase from human placenta and demonstration of a phospholipid requirement for its catalytic activity. Biochem Biophys Res Commun 71:1048-1053, 1976 28. Dale GL, Beutler E: Enzyme replacement in Gaucher's disease: A rapid high-yield method for purification of glucocerebrosidase. Proc Natl Acad Sci USA 73: Dec 1976 29. Ihler GM, Glew RH, Schnure FW: Enzyme loading of erythrocytes. Proc Natl Acad Sci USA 70:2663-2666, 1973
Medical Staff Conference
Newer Aspects of Some Interesting Lipid Storage Diseases:
Tay-Sachs and Gaucher's Diseases Seventh Annual Paul M. Aggeler Memorial Lecture Delivered October 5, 1976, San Francisco General Hospital Medical Center
ERNEST BEUTLER, MD, Duarte, California
These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from transcriptions, they are prepared by Drs. David W. Martin, Jr., Associate Professor of Medicine, and H. David Watts, Assistant Professor of Medicine, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine. Requests for reprints should be sent to the Department of Medicine, University of California, San Francisco, CA 94143.
DR. WALLERSTEIN: * Dr. Beutler had his formal education at the University of Chicago where he earned his MD degree in 1950. He went through residency and fellowship there and stayed on the staff until 1959 when he assumed directorship of the Division of Medicine at the City of Hope. He is also Clinical Professor of Medicine at the University of Southern California. The most outstanding of his many prestigious lectureships is perhaps the Stratton Lecture at the American Society of Hematology in 1974. He is a member of the Central, the Western and the American Societies of Clinical Investigation, the Association of American Physicians, and the National Academy of Sciences. His bibliography comprises more than 300 publications, and he has written, edited or co*Ralph 0. Wallerstein, M, Chief of Clinical Hematology Division of the Medical Service, San Francisco General Hospital, Clinical Professor of Medicine and Clinical Pathology and Laboratory Medicine, University of California, San Francisco, and Chairman of the Paul M. Aggeler Memorial Committee. This work was supported in part by Grant AM 14755 from the National Institutes of Health, Bethesda, Maryland.
46
JANUARY 1977 * 126 * 1
edited six books. More important than the quantity of this output is the quality. He has been an important pioneer in the story of glucose-6-phosphate dehydrogenase deficiency and other red blood cell enzyme deficiencies causing hemolytic anemia. He has invented some useful fluorescent screening tests for these enzymopathies and has contributed considerable knowledge about iron metabolism. In 1957 he wrote an article on the clinical evaluation of iron stores that is still widely quoted. He has worked extensively on iron enzymes and was the first to show clinically that females are a mosaic of X chromosomes, some chromosomes being active and some not. His work has also included studies on galactosemia and preservation of red blood cells. He invented a new type of medical career in which he brings ideas and supervision to his superlative laboratory, supervises the work, puts it together on paper and presents it clearly at meetings. He has avoided being overburdened by clinical practice, formal teaching and administra-
TAY-SACHS AND GAUCHER'S DISEASES TAY-SACHS GANGLIOSIDE
~~~~~~A
t
GLUCOCERE BROSI DE GLUCOSE - GALACTOSE - N - ACETYLGALACTOSAMINE-GALACTOSE SPHINGOSINE- FATTY ACID
N- ACETYL NEURAMINIC ACID
CERAM IDE
Figure 1.-The structure of a ganglioside. Tay-Sachs ganglioside (GM2) is formed by removal of the terminal sugar. This ganglioside accumulates in patients with Tay-Sachs disease and Sandhoff's disease. If all but one of the sugars are removed, glucocerebroside, the glycolipid of Gaucher's disease, is formed. CERAMIDE TRIHEXOS IDE
GLUCOCERE BROSIDE GLUCOSE- GALACTOSE -GALACTOSE-N-ACETYLGALACTOSAMINE
SPHINGOSINE -FATTY ACID CERAM IDE
Figure 2.-The structure of a globoside. Removal of all but the first sugar produces glucocerebroside, the glycolipid stored in patients with Gaucher's disease. ABBREVIATIONS USED IN TEXT
Gm0 =Tay-Sachs ganglioside hex= hexosaminidase CRM = cross-reacting material
tion. Dr. Beutler has recently turned his attention to lipid storage diseases and will address us on recent developments in Tay-Sachs and Gaucher's diseases. DR. BEUTLER: * I feel very privileged to have been invited to present the Paul Aggeler Memorial Lecture. Paul Aggeler was one of our generation's great hematologists. He was one of those extraordinary persons, encountered only too rarely, whose ability to combine clinical observations and laboratory studies enabled him to advance significantly the frontiers of knowledge. I first met Paul Aggeler in the late 1950's, during my annual January pilgrimage to the clinical meetings in the charming setting provided by Carmel. Paul was always unassuming, friendly and soft spoken, yet his discovery of hemophilia B ranks as one of the milestones in the field of blood coagulation. Because of Paul's devotion to hematology, the privilege of presenting the annual Aggeler lecture has generally been accorded to a hematologist. It is, therefore, with considerable *Ernest Beutler,
MD, Chairman, Division of Medicine, and
Director, Department of Hematology, City of Hope Medical Center, Duarte, California.
hesitation that I selected the topic for my lecture today. The glycolipid storage diseases are not hematologic disorders in the usual sense of the word. However, because hematology touches so many fields, particularly genetics, the interests of hematologists generally are very broad. I reasoned that Paul would have approved. I first learned about storage diseases when I entered medical school 30 years ago. Each of these disorders was associated with storage of a chemical substance and with an eponym-the name of the person credited with describing the disease. Remembering which eponym belonged to which chemical was not always easy. Sometimes mnemonic devices were helpful. For example, I knew a young woman whose name rhymed with "Gaucher" and who seemed to have limited intelligence; this served as a reminder to me that Gaucher's disease was the disorder characterized by storage of cerebrosides. The situation is now very different; much has been added to our knowledge of the glycolipid storage diseases. In the past decade and a half there has been a virtual avalanche of progress in the field, and understanding of these disorders is now possible at a molecular level. All of the glycolipid storage disorders seem to have a similar pathogenesis. The complex glycolipids, gangliosides and globosides (Figures 1 and 2) are constantly turned over by normal cells. THE WESTERN JOURNAL OF MEDICINE
47
TAY-SACHS AND GAUCHER'S DISEASES
Their catabolism requires that the sugars that comprise their carbohydrate portion be hydrolyzed one at a time, starting at the distal end. Each sugar is removed by an enzyme that is specific both for the sugar and for the type of linkage (a or 8) in which it is attached. Deficiency of one of these hydrolytic enzymes results in accumulation of its substrate, the ganglioside or globoside degradation product it was intended to catabolize. I will now discuss the studies carried out in our laboratory concerning the biochemical genetics of two general types of glycolipid storage diseases, Tay-Sachs and Sandhoff's diseases in which Tay-Sachs ganglioside or ganglioside monosialate, 2 (GM2) accumulates, and Gaucher's disease in which glucocerebroside is stored.
Tay-Sachs and Sandhoff's Diseases Tay-Sachs disease and the clinically and pathologically almost identical Sandhoff's disease are progressive neuromuscular disorders that lead to blindness, severe mental retardation and death within the first few years of life. The glycolipid that is stored in this disorder is a ganglioside, GM2 (Figure 1). It is apparent that Gm2 has not just one terminal sugar but two, N-acetylneuraminic acid and N-acetylgalactosamine. Brady' and others attempted to resolve the question of which of the two enzymes involved was defective by synthesizing GM2 with labeled N-acetylgalactosamine and with labeled N-acetylneuraminic acid. Initially, it seemed likely that a deficiency of one of the neuraminidases was responsible for the disease, because studies with artificial substrates (simple analogs of the ganglioside such as p-nitrophenyl-N-acetylgalactosamine) had shown that normal N-acetylgalactosaminidase (hexosaminidase [hex]) activity was present in the tissues of children dying with Tay-Sachs disease. However, Robinson and Stirling2 showed that there were, in fact, two separable forms of hexosaminidase in human tissues, hex A and hex B. When Sandhoff3 and Okada and O'Brien4 examined hexosaminidase from children with Tay-Sachs disease for these two specific isoenzymes, striking results were obtained. Children with Tay-Sachs disease had increased activity of hex B, but hex A activity could not be detected. A few non-Jewish children with a particularly severe form of Tay-Sachs disease were found to lack both hex A and hex B; their disease was designated Sandhoff's disease. Recognition that Tay-Sachs disease was due to a deficiency of hex A was a finding of great poten48
JANUARY 1977 * 126 * 1
tial practical importance. Tay-Sachs disease is an autosomal recessive disorder, which has a high frequency among Jews of Eastern European ancestry. Measuring the hex A content of the serum of prospective parents could identify the heterozygous state and thereby make possible detection of couples whose fetuses were at risk of developing this dreadful disorder. Diagnosis by amniocentesis was possible, and selective abortion could be offered to the parents.5 Heterozygote detection, however, is not simple when it is necessary to depend solely on the level of activity of an enzyme. Others factors cause considerable variation, and overlap is encountered between the levels in putative normal and putative heterozygous persons. If the cutoff point for the differentiation of heterozygotes from normal persons is too high, many normal women might undergo amniocentesis unnecessarily. If it is too low, some parents who had been reassured by the results of prenatal testing would suffer the agony of having a child with Tay-Sachs disease. My own interest in the biochemical genetics of Tay-Sachs disease was stimulated by this vexing and potentially important problem. As a hematologist it occurred to me that a similar problem had been encountered in the diagnosis of hemophilia and that the solution might be applicable. Zimmerman, Ratnoff and Littell6 measured the ratio of Factor VIII procoagulant activity tp Factor VIII antigen in the plasma. Because patients with hemophilia usually produce immunologically active but functionally inactive Factor VIII, the plasma of carriers was found to have more immunologic than procoagulant activity. If patients with Tay-Sachs disease synthesized an immunologically detectable and catalytically inactive hex A, the ratio of hex A to hex A antigen would be a much more sensitive way to detect heterozygotes for this disorder. Another reason for undertaking studies of Tay-Sachs disease was a more academic one. How could it be that one autosomal recessive disorder produces a deficiency only of hex A, whereas in other families an autosomal recessive disease produces a deficiency of both hex A and B? There were several theoretic explanations, but which was the right one? Working with Dr. S. K. Srivastava in our laboratory we first purified hex A and hex B to homogeneity. Human placenta served as suitable starting material, and we were able to immunize rabbits against the homogeneous material. Im-
TAY-SACHS AND GAUCHER'S DISEASES
munoelectrophoresis showed us that little or no cross-reacting material (CRM) was present in the place of hex A in livers of children dying of TaySachs disease7 (Figure 3). This finding made it appear unlikely that we would be able to apply the strategy used in diagnosing hemophilia carriers to the problem of Tay-Sachs disease. However, our curiosity about the relationship between hex A and hex B had not yet been fulfilled. Study of the antibody against these two enzymes provided us with our first insight into their relationship. Not only were they related genetically, in that a single gene produced deficiency of both enzymes in Sandhoff's disease, but they were also closely related immunologically. The antiserum that we produced against hex A reacted with hex
Hex-B
B, and the antiserum that we produced against hex B reacted with hex A.8 We succeeded in absorbing anti-hex B activity from serum produced against hex A. The absorbed serum was still able to react against hex A (Figure 4). Thus, we concluded that hex A had antigenic determinants that were absent from hex B. On the other hand, we learned that anti-hex A activity could not be removed from antiserum raised against hex B without loss of its capacity to react with hex B as well. These studies led us to suggest that hex A had one subunit, which we designated "a," that was not present in hex B, and that hex A had another subunit, ",/," that was represented in hex B. Because the molecular weight of the two enzymes seemed to be identical,9 the simplest representa-
--
I
I
Hex-A -*
N
T,
N
T2
N
T3
N
S1
T,
S1
(
T, S2 Si
2
Figure 3.-Immunoelectrophoresis of normal liver extract (N), extract from livers of three unrelated patients with TaySachs disease (T,, T2, T3), and liver extracts from two unrelated patients with Sandhoff's disease (SI, S2). (Reproduced by permission from Srivastava and BeutlerT).
IL-
Figure 4.-Upper, Immunodiffusion of purified enzyme and liver extracts against antisera (central wells) against hex A, against hex B and against A treated with hex B (absorbed anti-A). Lower, same gel stained for hexosaminidase activity.
Center Well
Anti-A
Anti-B
absorbed Anti-A THE WESTERN JOURNAL OF MEDICINE
49
TAY-SACHS AND GAUCHER'S DISEASES
tion of their structure was that hex A was a/3 and that hex B was /,8. One previously published observation seemed to conflict with this interpretation, however. It had been reported that hex A could be converted to hex B by treatment with neuraminidase.10"11 Although we were unable to confirm this observation, we did note that as hex A solutions were incubated, traces of hex B appeared to form. The interpretation of these data was clarified when we saw a report that indicated that even heat-inactivated neuraminidase preparations had the capacity to convert hex A into hex B.'2 Apparently, something else in neuraminidase produced the conversion, and we quickly established that it was the Merthiolate' that had been added as a preservative.'3 p-Hydroxymercuribenzoate has long been used to dissociate hemoglobin chains, and it seemed reasonable that similar chain dissociation might be accomplished with other mercurial compounds such as Merthiolate. We proposed'4 the following schema to account for the effect of Merthiolate in producing hex B from hex A: a or alkylated a v- (a/3)n - (/3)n Inactive
hex A
hex B
If Merthiolate produces the conversion by dis-
sociating the a and /3 subunits of hex A, with subsequent reassociation of /3 subunits to form hex B, we should be able the find the a subunit. Indeed, we were able to separate an alkylated dimeric a subunit from pure hex A preparations that had been treated with Merthiolate or p-hydroxymercuribenzoic acid.'5 Recently, an anti-a chain serum has been produced by immunizing rabbits with such a-chains after their treatment with glutaraldehyde.'6 Definitive proof of the subunit structure of hex A and hex B was achieved through study of the tissues of a patient with Sandhoff's disease. As had previously been noted by others, a small amount of residual hexosaminidase activity could be identified in such persons and was designated hex S17,18 (Figure 5). According to our subunit model, patients with Sandhoff's disease are unable to form functionally adequate /l-chains, and thus the residual enzyme activity in Sandhoff's disease might be attributed to polymerized a-chains. The observation that hex S was made up of a-chains was verified immunologically; it reacted with antiA but not with anti-hex B serum.'8 Because hex S was a homopolymer of a-chains and hex B a homopolymer of /3-chains, it should be possible
+
-
Hex S
*---
Hex A
Origin -I *-IN%
Hex B
Cont. Pat. Cont. Pat. Cont. Pat. wbc wbc fib. fib. wbc wbc Figure 5.-Starch gel electrophoresis at pH 6.0 of fibroblasts (fib.) and leukocytes (wbc) from normal subjects (Cont.) and a patient (Pat.) with Sandhoff's disease.18 Extracts containing approximately equal amounts of hexosaminidase were placed in the slots, and the gels were stained for hexosaminidase activity. Hex A and hex B are missing from the cells obtained from a patient with Sandhoff's disease. Instead, a rapidly moving band, designated as hex S, was present. This band represents a-chain polymers.
50
JANUARY 1977 * 126 * 1
TAY-SACHS AND GAUCHER'S DISEASES
to produce hex B and hex S from hex A, and to MIXED CELLS
ILYMPHOCYTE- RICH
GRANULOCYTE-RICH
rAdE'SNRA NORMAL a"DG DISEASE
NORMAL NORMAL
NORMAL I%zS OSESE
0
120' 0
100-
I-
P.)
co
s0o .
600 .
40
.
20
* 0 00
O
.
I
I
I
I
*
0
pH 5.0 (CITRATE BUFFER)
Figure 6.-,8-Glucosidase activity of leukocytes from normal subjects and patients with Gaucher's disease. The assays were carried out at pH 5 with 4-methylumbelliferyl-,e-D-glucopyranoside as substrate. At this pH, poor discrimination between normal and Gaucher's disease subjects was achieved.
produce hex A from hex B plus hex S. This, too, was accomplished."' A striking similarity is apparent between the hexosaminidase system and the thalassemias. In both a-thalassemia and Tay-Sachs disease, a deficiency in a-subunits results in polymerization of B-subunits. In a-thalassemia, hemoglobin /8 chains form hemoglobin H; in Tay-Sachs disease, hexosaminidase ,B chains polymerize to form increased amounts of hex B. In both a-thalassemia and Sandhoff's disease, an excess of a-chains is found. The a-hemoglobin chains in /8-thalassemia apparently damage the cell membrane. The ahexosaminidase chains aggregate in Sandhoff's disease to form hex S. Gaucher's Disease Gaucher's disease is a particularly prevalent glycolipid storage disease, occurring at a frequency of 1:5,000 to 1:10,000 Jewish births. In the most common adult variety of Gaucher's disease, gradual progressive storage of glucocerebroside in liver, spleen and bone marrow results in pancytopenia, hepatic failure and pathologic fractures. The enzymatic defect in Gaucher's dis-
Figure7.-,B-GIucosldase
0
of leukocytes of normal subjects, patients with Gaucher's disease, and obligate heterozygotes. The assays were carried out at a pH of 4 with
4-methylumbelliferyl-,8-D(I)
:V
glucopyranoside as substrate. Excellent discrimination between the different genotypes was achieved at this pH. (Reproduced by permission from Beutler E and Kuhl W: J Lab Clin Med 76: 747-755, 1970.)
THE WESTERN JOURNAL OF MEDICINE
51
TAY-SACHS AND GAUCHER'S DISEASES
ease has been identified as a deficiency in glucocerebrosidase.20 Localization of the glycolipid in the reticuloendothelial system makes Gaucher's disease a particularly attractive target for replacement therapy. The first problem that we faced in 1969 as we began to study the biochemical genetics of Gaucher's disease was that the radioactively labeled glucocerebroside used to show enzyme deficiency existed in only one laboratory and was not available to us. Its synthesis was complex and it was by no means certain that it could be carried out successfully again. However, the use of an artificial analog of glucocerebroside, 4-methylumbelliferyl-/3-glucopyranoside for study of this disorder had recently been described,21 and this material was commercially available. Because it seemed possible that the artificial substrate measured enzymes other than glucocerebrosidase, W. Kuhl attempted to determine whether or not patients with Gaucher's disease lack this activity. Because leukocytes were the most readily available cells and because examination of these cells might also lead to detection of the heterozygous state, they were chosen for study. Our initial results were disappointing (Figure 6). Assaying ,8glucosidase at pH 5, the pH generally chosen for the assay of acid hydrolases, we found little differences in the p-glucosidase activity of normal leukocytes and those from Gaucher's patients. To determine whether or not a major qualitative difference existed between the ,B-glucosidase
Figure 8.-Partial splenectomy according to the procedure used by Dr. Robert Yonemoto, City of Hope Medical Center.
52
JANUARY 1977 * 126 * 1
of Gaucher's cells and normal cells, we investigated some of the properties of leukocyte /8-glucosidase using an approach similar to that employed in investigating a glucose-6-phosphate dehydrogenase mutant. The pH-activity curves of ,8-glucosidase from normal subjects showed a pH optimum of approximately 5, which was identical to that of patients with Gaucher's disease. We discovered, however, that a second and lower pH optimum was present in normal leukocytes and that the enzyme activity at this latter pH of 4.0 was missing from the leukocytes of patients with Gaucher's disease. If we assayed the enzyme at pH 4,22 clear distinction between the leukocytes from patients with Gaucher's disease and normal persons was possible (Figure 7). It was also possible to identify heterozygotes for Gaucher's disease with reasonable confidence by using a lymphocyte-rich preparation of leukocytes. Some overlap was present, due in large part to the higher enzyme activity in monocytes than in lymphocytes. The lymphocyte preparation also contained monocytes, and its activity was therefore influenced to a pronounced degree by the extent of contamination with these cells. Layering suspensions of the lymphocyte-rich fractions on plastic Petri dishes allowed us to separate lymphocytes from monocytes; the latter attach to the plastic surface. Now it was possible to better differentiate normal from heterozygous subjects.23 Similar results were obtained in a study of cultured fibroblasts. Here, too, use of the artificial substrate, 4-methylumbelliferyl-/3-glucopyranoside permitted differentiation of Gaucher's disease from normal and from heterozygous subjects. Amniotic cells also contain ,8-glucosidase activity, and the identification of fetuses with Gaucher's disease is now possible. The ultimate aim of our study, however, has been the development of improved methods for the treatment of patients with Gaucher's disease. No specific therapy is available and management of patients with this disorder is currently a frustrating experience. One of the most frequently encountered complications of Gaucher's disease is thrombocytopenia. Splenectomy invariably produces normalization of the platelet count but does not reduce the amount of globoside that the body must metabolize each year. Instead of being sequestered relatively harmlessly in a gradually enlarging spleen, the glycolipid now accumulates in liver and bone. Dr. Robert Yonemoto, of the Department of
TAY-SACHS AND GAUCHER'S DISEASES
Surgery at the City of Hope, has successfully carried out a partial splenectomy in a patient with Gaucher's disease. The portion of the upper pole of the spleen supplied by the short gastric vessels was preserved, and bleeding from the cut edges was controlled with fibrin foam and mattress sutures (Figure 8). Complete remission of the patient's thrombocytopenia was achieved. We hope that the small piece of spleen left behind will prosper and enlarge, continuing to accumulate glycolipid and thereby protecting the patient's liver and bones from damage. However, partial splenectomy may, at best, provide only a limited solution to some of the problems associated with Gaucher's disease. More promise lies in replacement of the missing enzyme. Unfortunately, glucocerebrosidase has proved to be extraordinarily difficult to purify, largely due to its firm attachment to the lysosomal membrane and our consequent inability to solubilize it. Pentchev and co-workers24 succeeded in carrying out partial purification of glucocerebrosidase in a detergent-soluble form. The enzyme was precipitated on serum albumin and administered to several patients.25 The yield was extremely low, limiting the amount that could be administered. Although transient decreases of glucocerebroside levels in red blood cells were observed, no clinical improvement was recorded. Dr. George Dale in our laboratory recently succeeded in developing a high-yield, relatively rapid method of purifying soluble 83-glucosidase from human placenta.26 Earlier attempts to solubilize the enzyme by preparing acetone powders from placenta had resulted in loss of all enzyme activity. Dr. Dale's discovery that ,-glucosidase has an absolute requirement for acidic phospholipid27 immediately explained our earlier failure. We found that treating placental extract with organic solvents such as acetone or ethanol does release the 8-glucosidase in soluble form, but that activity can only be measured in the presence of added phospholipid. With the solubilization of ,8-glucosidase, further purification by conventional and affinity techniques became possible.28 A delivery system is needed to bring the enzyme to the stored glycolipid. Several methods have been proposed, but one of the simplest appears to be enclosure of the enzyme in the patient's own red blood cells.29 When erythrocytes are lysed in hypotonic solution, resealing can be effected by incubation in isotonic saline. Such lysis and resealing is most easily carried out in dilute
suspensions of erythrocytes, but this would be much too wasteful of the precious enzyme purified from many kilograms of placenta. We therefore added the purified enzyme to packed erythrocytes in a cell dialysis bag, dialyzing first against a 5 mM potassium phosphate buffer, pH 7.4. The dialysis bath was then replaced with a phosphate buffer containing 0.9 percent sodium chloride, and the mixture was warmed to 25°C. After two hours, the resealed erythrocytes were washed. Usually 15 to 30 percent of the enzyme was entrapped within erythrocytes. We have undertaken experimental enzyme replacement therapy in one patient with far-advanced Gaucher's disease. The first course of treatment, three injections of resealed cells containing a total of 560 mUnits of enzyme, was carried out in April 1976. In July 3,700 mUnits of enzyme were directly administered intravenously as two injections. Finally, in September another 2,200 mUnits of enzyme were administered, again in erythrocytes-but those which were coated with anti-Rh antibody to facilitate their clearance from the circulation. In the complex setting of far-advanced Gaucher's disease, the results of therapy are not easy to evaluate. Intercurrent infections and other complications tend to obfuscate the results. However, it was clear that enzyme therapy was well tolerated; no untoward reactions were experienced by the patient during any of the courses of treatment. At the onset of therapy, the patient's liver was the same size as had been recorded eight years previously. During the six months of enzyme therapy, some decrease in the size of the liver appeared to occur; the hepatic margin, identified by palpation, regressed 3 to 6 cm. Visualization of the liver with sulfur-technetium colloid in 1968 showed there to be major areas in which the radionuclide was not taken up. These areas had enlarged somewhat before enzyme replacement therapy was started. At the end of six months of treatment, hepatic uptake of colloid had greatly improved, so that the liver scan had a much more normal homogeneous appearance. Each course of enzyme therapy was associated with a rapid increase in the platelet count which lasted for several weeks. There seemed to be some increase in the overall sense of well-being. Further observation of this patient and other patients whom we plan to treat will be required to delineate the possible role of enzyme replacement in the management of patients with Gaucher's disease. THE WESTERN JOURNAL OF MEDICINE
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TAY-SACHS AND GAUCHER'S DISEASES
The treatment of glycolipid storage diseases by the administration of exogenous enzyme represents the type of interaction between basic science and clinical medicine that was one of the outstanding features of Paul Aggeler's professional career. It is for this reason that I considered these studies to be an appropriate theme for this year's Paul Aggeler Memorial Lecture. REFERENCES 1. Brady RO: Tay-Sachs disease. N Engl J Med 281:1243-1244, Nov 27, 1969 2. Robinson D, Stirling JL: N-acetyl-f1-glucosaminidase in human spleen. Biochem J 107:321-327, 1968 3. Sandhoff K: Variation of 83-N-acetylhexosaminidase-pattern in Tay-Sachs disease. FEBS Lett 4:351-354, 1969 4. Okada S, O'Brien JS: Tay-Sachs disease: Generalized absence of a j8-D-N-acetylhexosaminidase component. Science 165: 698-700, 1969 5. O'Brien JS, Okada S, Fillerup DL, et al: Tay-Sachs disease: Prenatal diagnosis. Science 172:61-64, 1971 6. Zimmerman TS, Ratnoff OL, Littell AS: Detection of carriers of classic hemophilia using an immunologic assay for antihemophilic factor (Factor VIII). J Clin Invest 50:255-258, 1971 7. Srivastava SK, Beutler E: Studies on human j8-D-N-acetylhexosaminidases-III. Biochemical genetics of Tay-Sachs and Sandhoff's diseases. J Biol Chem 249:2054-2057, 1974 8. Srivastava SK, Beutler E: Antibody against purified human hexosaminidase B cross-reacting with human hexosaminidase A. Biochem Biophys Res Commun 47:753-759, 1972 9. Srivastava SK, Yoshida A, Awasthi YC, et al: Studies on human 8-D-N-acetylhexosaminidases-II. Kinetic and structural properties. J Biol Chem 249:2049-2053, 1974 10. Goldstone A, Konecny P, Koenig H: Lysosomal hydrolases: Conversion of acidic to basic forms by neuraminidase. FEBS Lett 13:68-72, 1971 11. Murphy JV, Craig L: Effect of human cerebral neuraminidase on hexosaminidase A: Clin Chim Acta 51:67-73, 1974 12. Carmody PJ, Rattazzi MC: Is neuraminidase responsible for the in vitro conversion of human hexosaminidase A to hexosaminidase B? Am J Hum Genet 25:19A, 1973 13. Beutler E, Villacorte D, Srivastava SK: Non-enzymatic conversion of hexosaminidase-A to hexosaminidase-B by merthiolate. IRCS:2:1090, 1974
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JANUARY 1977 * 126 * 1
14. Beutler E, Villacorte D, Kuhl W, et al: Nonenzymatic conversion of human hexosaminidase A. J Lab Clin Med 86:195-203, 1975 15. Beutler E, Yoshida A, Kuhl W, et al: The subunits of human hexosaminidase A. Biochem J 159:541-543, 1976 16. Srivastava SK, Wiktorowicz JE, Awasthi YC: Interrelationship of hexosaminidase A and B-Confirmation of the common and the unique subunit theory. Proc Natl Acad Sci USA 73:28332837, 1976 17. Ikonne JU, Rattazzi MC, Desnick RJ: Characterization of hex S, the major residual /1-hexosaminidase activity in type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease). Am J Hum Genet 27:639-650, 1975 18. Beutler E, Kuhl W, Comings D: Hexosaminidase isozyme in type 0 Gii2 gangliosidosis (Sandhoff-Jatzkewitz disease). Am J Hum Genet 27:628-638, 1975 19. Beutler E, Kuhl W: Subunit structure of human hexosaminidase verified: Interconvertibility of hexosaminidase isozymes. Nature 258: 262-264, 1975 20. Brady RO, Kanfer J, Shapiro D: The metabolism of gltcocerebrosides-I. Purification and properties of a glucocerebrosidecleaving enzyme from spleen tissue. J Biol Chem 240:39-43, 1965 21. Ockerman PA, Kohlin P: Tissue acid hydrolase activities in Gaucher's disease. Scand J Clin Lab Invest 22:62-64, 1968 22. Beutler E, Kuhl W: Detection of the defect of Gaucher's disease and its carrier state in peripheral-blood leukocytes. Lancet 1:612-613, 1970 23. Beutler E, Kuhl W, Matsumoto F, et al: Acid hydrolases in leukocytes and platelets of normal subjects and in patients with Gaucher's and Fabry's disease. J Exp Med 143:975-980, 1976 24. Pentchev PG, Brady RO, Hibbert SR, et al: Isolation and characterization of glucocerebrosidase from human placental tissue. J Biol Chem 248:5256-5261, 1973 25. Pentchev PG, Brady RO, Gal AE, et al: Replacement therapy for inherited enzyme deficiency-Sustained clearance of accumulated glucocerebroside in Gaucher's disease following infusion of purified glucocerebrosidase. J Molecular Med 1: 73-78, 1975 26. Dale GL, Beutler E: Enzyme replacement therapy in Gaucher's disease-I. A rapid, high yield method for purification of glucocerebrosidase. Proc Natl Acad Sci USA (in press), 1976 27. Dale GL, Villacorte D, Beutler E: Solubilization of glucocerebrosidase from human placenta and demonstration of a phospholipid requirement for its catalytic activity. Biochem Biophys Res Commun 71:1048-1053, 1976 28. Dale GL, Beutler E: Enzyme replacement in Gaucher's disease: A rapid high-yield method for purification of glucocerebrosidase. Proc Natl Acad Sci USA 73: Dec 1976 29. Ihler GM, Glew RH, Schnure FW: Enzyme loading of erythrocytes. Proc Natl Acad Sci USA 70:2663-2666, 1973
