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Genetics and the Sphingolipidoses
Genetics and the Sphingolipidoses ROSCOE O. BRADY, M.D.*
The sphingolipidoses comprise a family of lipid storage diseases of importance to a wide spectrum of the medical profession. Knowledge of the manifestations, diagnosis, and pathological chemistry of hereditary sphingolipid storage diseases is relevant for physicians in general practice as well as those in a number of specialties. Patients with the infantile form of these diseases often show mental retardation and amaurosis early in life and are therefore encountered by pediatricians, ophthalmologists, and neurologists in their practice. If such patients survive infancy, they often appear in clinics with the signs and symptoms of splenomegaly, hepatomegaly, and an abnormal blood picture which may include thrombocytopenia, leukopenia, and mild anemia. Thus, surgeons, internists, and hematologists become involved in the care of these patients. In addition, these patients frequently have erosion of the cortices of long bones and the hip joint, which may be accompanied by pathological fractures and require the services of orthopedic surgeons. The skin of some of these patients becomes involved with pigmentations and maculopapular angiokeratomatous eruptions, and therefore the patients may be seen initially by dermatologists. Clinical chemists can now diagnose three of these lipid storage diseases using leukocytes obtained from small samples of venous blood. Procedures have just appeared on the horizon which hold great promise for the detection of these conditions in utero as well as for the determination of heterozygous carriers of these diseases. Exploitation of these exciting possibilities requires the participation of obstetricians and provides an increasingly important opportunity for genetic counseling.
GENERAL DESCRIPTION OF SPHINGOLIPIDS In this article we are concerned with a group of lipids which have certain structural features in common. They all contain the long chain amino alcohol sphingosine (Fig. 1 a) as a portion of their molecular structure. The biosynthesis of sphingosine occurs in many tissues of the "Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland Medical Clinics of North America- Vo!. 53, No. 4, July, 1969
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a. Sphingosine: CH,-(CH,),,-CH~CH-CH(OH)-CH(NH,)-CH,OH b. Ceramide: CH,,-(CH,),,-CH-=CH-CH-CH-C':'!1,OH
I
I
OHN-H
I I (CH,)""" I C~O
CH" ceramide-galactose c. Galactocerebroside: d. Glucocerebroside: ceramide-glucose e. Ceramide-Iactoside: ceramide-glucose-galactose Globoside: ceramide-glucose-galactose- galactose- N -acety Igalactosamine g. Ganglioside: ceramide-glucose-galactose- N -acetylgalactosamine-galactose
r
I
h.
i. j.
h.
N-acetylneuraminic acid Sphingomyelin: ceramide-phosphorylcholine Sulfatide: ceramide-galactose-3-sulfate Ceramide trihexoside: ceramide-glucose-galactose-galactose Tay-Sachs ganglioside: ceramide-glucose-galactose-N -acetylgalactosamine
I
N-acetylneuraminic acid ':'Point of attachment of various components. Figure l.
body. Carbon atoms 3 to 18 arise from palmitic acid, and carbon atoms 1 and 2 come from the amino acid serine. All of the materials to be discussed also have a long chain fatty acid linked through an amide bone to the nitrogen atom of sphingosine. This combination of fatty acid and sphingosine is called a ceramide (Fig. 1b). Various other molecules are linked to the primary alcoholic group on carbon 1 of the sphingosine portion of ceramide. For example, galactocerebroside, the largest single lipid component of the myelin sheath of nerves, contains the hexose galactose linked by a ,B-glycosidic bond to ceramide (Fig. le). The structure of various other sphingolipids under consideration in this article will be indicated where appropriate.
GAUCHER'S DISEASE SIGNS AND SYMPTOMS. Perhaps the most common lipid storage disease seen by clinicians is the inherited disorder first described by Phillipe Gaucher in 1882. There are two clinical forms of Gaucher's disease which have been designated as the infantile and adult varieties respectively. Both are characterized by the occurrence of splenomegaly, hepatomegaly, and erosion of the long bones. The central nervous system is affected in patients with the infantile form, and severe mental deterioration is usually present in these patients. In patients with the adult form of Gaucher's disease, there is generally very little or no central nervous system difficulty. Patients in the latter category often manifest a brownish pigmentation of the exposed areas of the skin. These patients frequently have a thrombocytopenia and mild hypochromic anemia which are generally improved after splenectomy. GENETICS. Gaucher's disease is the result of the inheritance of an autosomal recessive genetic defect, and hence both sexes may be af-
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fiicted. There has been one report of a family in which the passage of the Gaucher trait seemed to follow that of a dominant characteristic. H The rapidity of progression of the signs and symptoms of Gaucher's disease may vary over a wide range in patients with the adult form of Gaucher's disease. In some, the disease becomes manifest early in life and the enlargement of the spleen and bone involvement progress swiftly. In other patients, the hepatosplenomegaly is a late-appearing phenomenon, and the rate of hypertrophy of these organs is slow in comparison with the rate in other cases. In my opinion, these findings are clear manifestations of genetic heterogeneity and indicate that the genetically directed alteration definitely differs in such patients. This concept is strengthened by evidence obtained during recent biochemical investigations which have revealed the nature of the enzymatic defect in Gaucher's disease. PATHOLOGICAL CHEMISTRY. A characteristic histopathologic abnormality in patients with Gaucher's disease is the appearance of a large lipid-laden cell in the spleen, liver, and bone marrow. This cell stains for both fat and carbohydrate and is believed to be derived from cells of the reticuloendothelial system. The lipid which accumulates in these cells has been conclusively identified as glucocerebroside (Fig. Id). The biochemical defect in Gaucher's disease now is known to be a deficiency of an enzyme called glucocerebrosidase, which catalyzes the first step in the catabolism of glucocerebroside 2 • 5 (Reaction 1):
Table 1.
+ H"O
glucocerebrosidase
) glucose + ceramide This enzyme is normally present in most of the tissues of the body. The activity of this enzyme was found to be reduced to a mean level of about 15 per cent of the normal value in tissues of patients with the adult form of Gaucher's disease (Table 1). Patients whose tissues showed the least
1. Glucocerebroside
Enzymatic Lesions in the Sphingolipidoses MEAN ACTIVITY OF AFFECTED ENZYME (PER CENT OF NORMAL)
CONDITION
Gaucher's disease
ENZYME INVOLVED
Glucocerebrosidase
Adult form Infantile form
White blood cells 16%
1%
Sphingomyelinase
Metachromatic leukodystrophy
Arylsulfatase A (sulfatidase)
Fabry's disease
Ceramidetrihexosidase
"Not yet determined.
Spleen IS'/'.
Niemann-Pick disease Type A Type C
Hemizygous males Heterozygous females
TISSUE
Liver 5% 17lj(
Brain
1-5%
Small intestine
28 c/r
8% 13C;:'r;,
lO(;{,
Skin fibroblasts 3'/'
<1 'Yc
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glucocerebrosidase activity had the most rapid progression of the disease, whereas those with attenuated but moderately higher residual glucocerebrosidase activity developed splenomegaly and hepatomegaly later in life. Tissues from patients with the infantile form of Gaucher's disease show virtually a complete absence of glucocerebrosidase activity. SOURCE OF ACCUMULATING GLUCOCEREBROSIDE. There appear to be several possible sources of the glucocerebroside which accumulates in the systemic organs and tissues of patients with this disease. Recent observations indicate that the preponderance of this material is produced during the catabolism of senescent leukocytes. 11 Ceramide lactoside (Fig. le) is a major lipid constituent of polymorphonuclear leukocytes. In normal individuals, nearly 400 mg. of glucocerebroside is derived from this source and must be disposed of each day. Another probable source of glucocerebroside in peripheral tissues appears to be the stroma of senescent erythrocytes. A glycolipid called globoside (Fig. If) is a major constituent of erythrocyte membranes, and glucocerebroside probably arises in the course of the sequential hydrolysis of the tetrahexoside portion of this material. Because of the deficiency of glucocerebrosidase in patients with Gaucher's disease, the degradation of glycolipids is impaired. Glucocerebroside is stored in various tissues, especially those involved in leuko- and erythrocytorrhexis. There are several reports which indicate that glucocerebroside accumulates to some extent in neuronal cells in patients with the infantile form of Gaucher's disease. It seems likely that the glucocerebroside in these cells is derived from materials called gangliosides (Fig. Ig). The turnover of gangliosides is rather active in the neonatal period; thereafter it occurs at a slower rate. Patients with the adult form of Gaucher's disease appear to have sufficient glucocerebrosidase activity in brain to catabolize the glucocerebroside which arises during tile critical period of rapid ganglioside metabolism. They thereby escape damage to the nervous system. DIAGNOSIS. The diagnosis of Gaucher's disease can usually be made clinically by the presence of splenomegaly, hepatomegaly, abnormalities of the long bones and pelvis, and an increase in tartrate-inhibitable serum acid phosphatase. The occurrence of the so-called "Gaucher cells" in marrow smears is pathognomonic of this condition. The diagnosis may be confirmed by direct assay of glucocerebrosidase activity in biopsy specimens of solid tissues or, more conveniently, by assaying the level of glucocerebrosidase activity in circulating leukocytes." The assay may be performed easily on leukocytes obtained from 5 ml. or less of venous blood. The estimation of enzymatic activity is conveniently determined with 14C-Iabeled glucocerebroside which is simply incubated with the washed white blood cells at pH 6.0. The radioactive glucose- 14 C which is liberated by the action of the enzyme is determined by liquid scintillation counting. The test is facile and reliable. Leukocytes obtained from patients with Gaucher's disease show a marked decrease in glucocerebrosidase activity compared with similar preparations from normal humans and patients with various other lipid storage diseases. Two additional diagnostic aids have recently been developed in our laboratory. Glucocerebrosidase activity is normally quite high in fibro-
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blasts derived from skin and grown in tissue culture. Preparations of this nature obtained from a patient with infantile Gaucher's disease showed an almost complete absence of glucocerebrosidase activity. A particularly interesting observation was the detection of intermediate levels of glucocerebrosidase activity in such tissue cultures obtained from heterozygous carriers of Gaucher's disease. If this finding can be confirmed by further experimentation, it will provide for the first time a procedure for detecting heterozygotes. The identification of these individuals is not possible through assays performed on solid tissue samples or leukocyte preparations. Such a determination should be of great usefulness for genetic counseling. From the points of view of the family physician and geneticist, another extremely important development appears imminent. Cells obtained by amniocentesis and subsequently grown in tissue culture also contain good glucocerebrosidase activity. Amniocentesis can be performed with safety about the fourth month of pregnancy, and sufficient cells can be grown for assay in about 5 weeks (see p. 773). Since the cells are of fetal origin, it seems very likely that glucocerebrosidase assays performed on these cells will enable the detection of this disease in utero. This information therefore should be available in sufficient time to permit a critical decision regarding the continuation of the pregnancy. I feel that many, if not all, of the heritable lipid storage diseases probably can be detected in a similar fashion once the nature of the enzymatic defect is known. This contribution alone emphasizes the importance of the painstaking and time-consuming research required for discovering the biochemical abnormalities in inherited metabolic diseases. TREATMENT. Another critical reason for the expenditure of so much effort in delineating the enzymatic defect in these diseases is that only when the explicit nature of the defect is known can rational therapeutic procedures be considered. As yet there is no specific therapy for any of the lipid storage diseases, although many potential remedial procedures are under active consideration. Included among these are trials of enzyme replacement therapy if sufficient pure and nonsensitizing preparations can be produced. Recent evidence suggests that exogenously administered enzymes can indeed pass through cell membranes, a condition which would appear to be required for the removal of intracellularly accumulated glucocerebroside. Perhaps this form of therapy can some day be supplemented or circumvented by transplanting a normal spleen to a patient with Gaucher's disease. Once the glucocerebrosidase has been obtained in a homogenous state, it should be possible to determine the amino acid sequence of the protein. Perhaps the enzyme can be synthesized by recently developed methods. Furthermore, information about the structure can in turn be extrapolated back to indicate the nucleotide sequence of the messenger RNA. It is conceivable that administration of messenger RNA may be efficacious and may cause production of active enzyme. Perhaps at some still further date it may be possible to modify the defective genetic apparatus of these patients by the administration of specific molecules or fragments of DNA (see p. 795). Recent experimental evidence indicates that DNA can be taken up and incorporated into nuclear material by
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intact cells. Still other therapeutic possibilities and modifications have been discussed in an earlier review. 3
NIEMANN-PICK DISEASE SIGNS AND SYMPTOMS. Patients with Niemann-Pick disease, another inherited lipid storage disease, also show systemic organomegaly. In this instance, hepatomegaly is especially predominant; some degree of splenomegaly is usually present as well. Bone involvement occurs, although to a lesser extent than in patients with Gaucher's disease. Niemann-Pick disease usually becomes manifest in patients early in infancy. These patients are quite severely retarded mentally. Bone marrow preparations from patients with Niemann-Pick disease contain large lipid-laden cells which have a waxy appearance and stain for both lipid and phosphorous. Some patients with Niemann-Pick disease have an olive-yellow color to their skin. About 30 per cent of the patients with this increasing number of cases of Niemann-Pick disease are being reported in which there is a less rapid progression of the disease process than in the classic infantile form. In contrast to the patients with the adult form of Gaucher's disease, some of the older patients with Niemann-Pick disease show evidence of slowly progressive brain damage. PATHOLOGICAL CHEMISTRY. The material which accumulates in tissues of patients with Niemann-Pick disease is a phospholipid called sphingomyelin (Fig. 1h). Studies with labeled sphingomyelin have demonstrated convincingly the nature of the metabolic defect in NiemannPick disease. 4 The lesion in these patients was shown to be a nearly complete absence in their tissues of an enzyme which catalyzes the first step in the catabolism of sphingomyelin (Reaction 2) (Table 1): 2. Sphingomyelin + H 2 0
sphingomyelinas~
phosphorylcholine +ceramide
Patients with the less rapidly progressing form of Niemann-Pick disease show higher residual sphingomyelinase activity in their tissues than those with the classic infantile form of the disease (type A). Once again it is necessary to consider potential sources of the sphingomyelin which accumulates in patients with this disease. Sphingomyelin is a quantitatively important component of the plasma membrane of cells and of the membranes of various subcellular organelles such as the endoplasmic reticulum and mitochondria. Sphingomyelin is also an important constituent of erythrocyte stroma. Thus it seems that sphingomyelin could arise in the course of the turnover of almost all cells and their intracellular components. Perhaps the continuous degradation and replacement of these latter structures in the nervous system accounts for the progressive involvement of the brain in the older patients with Niemann-Pick disease. GENETICS. Somewhat over 50 per cent of the patients with NiemannPick disease reported in the literature are of Ashkenazic Jewish ancestry; however, the disease has been reported in all races. This condition is due to the inheritance of a defective autosomal recessive trait. Most of the patients with Niemann-Pick disease die in early infancy, and one would
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expect that a deleterious mutation such as this would be self-extinguishing. Since it apparently is not, geneticists are forced to consider possible beneficial advantages conferred upon the heterozygous carriers of this trait. There may be a correlation between this heterozygous genetic state and survival or avoidance of malignant diseases. Significantly increased glucocerebrosidase and sphingomyelinase activity has been found in leukemic leukocytes. 1o Studies on the metabolism of membrane lipids in neoplastic diseases are just getting underway in several laboratories. My impression is that important discoveries will be made related to the metabolism of these materials in the transformation of cells with oncogenic viruses and ultimately in malignant diseases in humans. DIAGNOSIS. The diagnosis of Niemann-Pick disease can be made with reasonable certainty on the basis of clinical findings and the appearance of "foam" cells in the bone marrow. The diagnosis can be confirmed by direct analytical determination of the amount of sphingomyelin in tissue samples, e.g., from liver biopsies. A more facile method has recently become available for accurately diagnosing Niemann-Pick disease. It is based on the determination of the level of sphingomyelinase activity in sonicated leukocyte preparations. 9 The level of sphingomyelinase in white blood cell preparations obtained from patients with Niemann-Pick disease is markedly lower than normal. This test is conveniently performed with labeled sphingomyelin as substrate. Sphingomyelinase activity is very low in fibroblasts grown in tissue culture derived from biopsies of Niemann-Pick patients' skinY The enzyme is easily demonstrated in similar cultures from normal individuals and patients with other lipid storage diseases. Studies are underway with the hope of once again being able to detect heterozygous carriers of Niemann-Pick disease. Heterozygotes cannot be identified through the use of the leukocyte assay procedure. Fetal cells obtained by amniocentesis and grown in tissue culture also show very good sphingomyelinase activity. This finding suggests that it is quite likely that the diagnosis of Niemann-Pick disease can be made in utero.
MET ACHROMATIC LEUKODYSTROPHY (MLD) SIGNS AND SYMPTOMS. The predominant manifestations of metachromatic leukodystrophy are severe impairment of the central nervous system and a decrease in nerve conduction velocity in peripheral nerves. These lesions cause motor incoordination and mental retardation. Two discrete clinical states seem to be seen in this disease. The first appears within the first 30 months of life and is characterized by the onset of weakness, ataxia, hypotonus, paralyses, and difficulties in speech and swallowing. The onset of the second form occurs later in life, and the initial symptoms are predominantly psychological difficulties which are followed by progressive dementia. PATHOLOGICAL CHEMISTRY. There is an accumulation of an acidic glycolipid called sulfatide (Fig. 1i) in nerve fibers, kidney, and bile ducts.
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These deposits cause some manifestations of renal and hepatic impairment. Some patients excrete increased quantities of material in the urinary sediment which stains metachromatically orange-brown when exposed to cresyl violet dye. The biochemical defect in MLD has been shown by Austin and coworkers I and Mehl and Jatzkewitzl~ to be a deficiency of a sulfuric acid esterase in these patients (Reaction 3):
3. Sulfatide
+
H20
sulfatase )
galactocerebroside + sulfuric acid
There are at least three sulfatases in mammalian tissues whose activity can be detected by measuring the hydrolysis of artificial substrates such as p-nitrocatechol sulfate. They have been designated as arylsulfatases A, B, and C, and they differ in their pH optima and subcellular distribution. Sulfatide is considered to be the natural substrate for arylsulfatase A, and the activity of only this sulfatase is diminished in patients with MLD. DIAGNOSIS. A convenient diagnostic procedure has been developed recently for the detection of patients with MLD. It is based on enzyme assays performed on circulating leukocytes. 15 Leukocytes contain both arylsulfatase A and B activity. Because of this, the level of arylsulfatase A activity in leukocytes is determined in the presence of sodium pyrophosphate to inhibit arylsulfatase B. Conversely, arylsulfatase B activity may be determined in the presence of arylsulfatase A by adding barium acetate to inhibit arylsulfatase A. Leukocytes obtained from patients with MLD showed a 90 per cent decrease in arylsulfatase A activity compared with normal individuals (Table 1). There was no change in arylsulfatase B activity in leukocytes obtained from patients with MLD. The test is reliable and facile and should lend itself readily to automated clinical laboratory procedures. GENETICS. MLD is also inherited as an autosomal recessive characteristic. It is hoped that enzyme assays on skin fibroblasts grown in tissue culture will provide a means for detecting the heterozygous carriers of this trait. Furthermore, it seems reasonable to expect that this condition will also lend itself to detection antenatally through enzyme assays performed on fetal cells grown in tissue culture. FABRY'S DISEASE SIGNS AND SYMPTOMS. Fabry's disease is characterized by the appearance of numerous purplish papules on the body, especially in the scrotal region and along the lateral iliac area. Male patients with Fabry's disease generally die of renal failure in the third or fourth decade of life. There is an accumulation of a glycolipid in the glomeruli of the kidney which causes a profound diminution of the function of this organ. Some patients show evidence of cardiac enlargement and electrocardiographic abnormalities. Involvement of the eyes is common and may occur in the form of corneal opacification or vascular dilatation. There may be pain in various joints in some patients. PATHOLOGICAL CHEMISTRY AND GENETICS. The lipid which accumulates in tissues of patients with Fabry's disease is a glycolipid called
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ceramidetrihexoside (Fig. ID. It was predicted that the metabolic defect in this disease would be found to be a deficiency of the enzyme which catalyzes the hydrolysis of the terminal molecule of galactose of ceramidetrihexoside (Reaction 4): 4. Ceramidetrihexoside + H 2 0
ceramide
trihexosidas~
ceramidelactoside
+ galactose
This hypothesis was shown to be correct through the use of the appropriately labeled compound.'; Male hemizygous patients with Fabry's disease have a total absence of this enzyme, ceramidetrihexosidase, in their tissues (Table 1). This disease has been shown to be due to the transmission of a sexlinked (X-chromosome) genetic defect. Since the affected males have only the defective X-chromosome in their cells, it is not surprising that the enzyme is entirely absent. Female carriers of this condition who have both a normal and a defective X-chromosome show intermediate levels of ceramidetrihexosidase activity in their tissues. This finding is exactly that which would be anticipated in a truly heterozygous state. DIAGNOSIS. The diagnosis can be made by determining the level of ceramidetrihexosidase activity in biopsy samples of small intestinal mucosa. Suitable specimens may be obtained by a Wangensteen tube with a small cutting device. In addition, biopsy samples of kidney tissues have been used successfully in our laboratory to establish the diagnosis of Fabry's disease. Once again the diagnostic enzymatic test is greatly facilitated through the use of the labeled compound. It may be possible to detect patients with Fabry's disease by quantitative analysis of ceramidetrihexoside in blood or urine. Such tests require the extraction and partial purification of ceramidetrihexoside from these sources and its subsequent chemical conversion to a derivative which is suitable for separation and identification by gas-liquid chromatography. The accuracy and simplicity of the radioassay procedure for ceramidetrihexosidase make this the diagnostic procedure of choice at the present time. TREATMENT. Specific enzyme replacement therapy has not yet been attempted in patients with Fabry's disease. Affected male patients benefit considerably from hemodialysis. It is conceivable that a renal transplant may be of some help for these patients. Although organ transplantation procedures are in the throes of harsh, and perhaps justified, self-criticism at this time, kidney transplantation under proper conditions has been successful in a number of pathologic conditions. It merits consideration for the treatment of patients with Fabry's disease.
GENERALIZED GANGLIOSIDOSIS Only a very few patients with generalized gangliosidosis have been detected and reported so far. These patients have some hepatomegaly, and there may be skeletal deformities. However, the hallmark is again a rapid, progressive degeneration of the central nervous system. Infants with this condition generally die before their second year of life. The
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metabolic abnormality in generalized gangliosidosis has recently been shown to be a total deficiency of l3-galactosidase activity in the tissues of these patients. 14, 16 This metabolic aberration causes a progressive accumulation of ganglioside (Fig. 19) in the brain and other tissues.
TAY -SACHS DISEASE GENETICS. Tay-Sachs disease is also inherited as an autosomal recessive metabolic defect. The frequency of the disease is about 1 in 6000 to 10,000 births of infants of Ashkenazic Jewish parentage. This figure means that 1 in 50 individuals in this group are carriers of the defect and that the actual gene frequency is 1 in 100. SIGNS AND SYMPTOMS. Infants with Tay-Sachs disease show severe progressive mental retardation and blindness. A cherry-red spot appears in the macula of most patients. There is relatively little organomegaly or skeletal involvement. Histological examination of the brain reveals the presence of cytoplasmic inclusions called membranous cytoplasmic bodies consisting of concentric layers of densely staining material in the ganglion cells. They are comprised of a mixture of gangliosides, cholesterol, and phospholipids. The involved ganglion cells are swollen and distorted. PATHOLOGICAL CHEMISTRY. The characteristic material which accumulates in brain tissue, and perhaps to a lesser extent in peripheral tissues as well in patients with Tay-Sachs disease, is an acidic glycolipid called Tay-Sachs ganglioside (Fig. 1k). This compound differs from the preponderant monosialoganglioside in brain (Fig. 19) in that the terminal molecule of galactose is missing. The absence of this terminal galactose has caused some speculation that the metabolic defect in Tay-Sachs disease is a deficiency of a galactose transferring enzyme required for the completion of the normal ganglioside structure. A defect of this nature is unlikely in Tay-Sachs disease because brain tissue from these individuals contains a normal complement of tetrahexoside gangliosides and only the quantity of trihexoside ganglioside is abnormal. This observation is not compatible with a defective step in ganglioside formation. I think that we are once again dealing with a missing catabolic enzyme. It has been very difficult to localize the specific metabolic defect in Tay-Sachs disease because of the extremely complicated chemistry involved in the synthesis of labeled Tay-Sachs ganglioside. There seem to be two alternative possibilities for a defect in Tay-Sachs disease. The first is a deficiency of an enzyme required for the hydrolysis of the terminal molecule of N-acetylgalactosamine of the Tay-Sachs ganglioside. The second is a deficiency of an enzyme which catalyzes the liberation of the N-acetylneuraminic acid residue. We have very recently prepared Tay-Sachs ganglioside labeled in either the N -acetylneuraminyl or N -acetylgalactosaminyl portion of the molecule. 12 It is hoped that biochemical investigations with these labeled substances will enable us to identify the metabolic lesion in Tay-Sachs disease. If the metabolic defect can be successfully identified in patients with Tay-Sachs disease, we will be in position to extend these studies in
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several important directions. We will attempt to devise simple and reliable diagnostic procedures to help in the detection of this condition. Other studies will be directed toward determining the heterozygous carriers of the trait. And finally, we hope to be able to devise a procedure for the antenatal diagnosis of Tay-Sachs disease.
SUMMARY The metabolic defects in Gaucher's disease, Niemann-Pick disease, metachromatic leukodystrophy, Fabry's disease, and generalized gangliosidosis have recently been identified. These critical developments have provided an opportunity to consider rational therapeutic procedures for the amelioration of these conditions. Consideration has been given to the potential sites of metabolic lesions in Tay-Sachs disease. Important contributions have been made for the detection and accurate diagnosis of the first four of these conditions. It is anticipated that tissue culture techniques coupled with the use of specifically labeled sphingolipid substrates will eventually provide the clinician with the means of detecting heterozygous carriers of the defective genes. We hope ultimately to be able to identify the occurrence of such diseases in utero. Remarkable opportunities for enlightened genetic counseling will become a reality when these developments occur.
REFERENCES l. Austin, J., Balasubramanian, A. S., Pattabiraman, T. N., Saraswathi, S., Basu, D. K. and Bachhawat, B. K.: A controlled study of enzymic activities on three human disorders of glycolipid metabolism. J. Neurochem., 10:805·816, 1963. 2. Brady, R. 0., Kanfer, J., and Shapiro, D.: Metabolism of glucocerebrosides. 11. Evidence for an enzymatic deficiency in Gaucher's disease. Biochem. Biophys. Res. Commun., 18:221-225,1965. 3. Brady, R. 0.: The sphingolipidoses. New Eng. J. Med., 275:312·318,1966. 4. Brady, R. 0., Kanfer, J. N., Mock, M. B., and Fredrickson, D. S.: The metabolism of sphin· gomyelin. 11. Evidence for an enzymatic deficiency in Niemann·Pick disease. Proc. Nat. Acad. Sci., 55:366·369,1966. 5. Brady, R. 0., Kanfer, J. N., Shapiro, D., and Bradley, R. M.: Demonstration of a deficiency of glucocerebroside-cleaving enzyme in Gaucher's disease. J. Clin. 1vest., 45: 1112·1115, 1966. 6. Brady, R. 0., Gal, A. E., Bradley, R. M., Martensson, E., Warshaw, A. L., and Laster, L.: Enzymatic defect in Fabry's disease: ceramidetrihexosidase deficiency. New Eng. J. Med., 276: 1163·1167, 1967. 7. Crocker, A. C.: The cerebral defect in Tay·Sachs disease and Niemann-Pick disease. J. Neurochem., 7:69·80, 19G1. 8. Hsia, D. Y·Y, Naylor, J., and Bigler, J. A., in Aronson, S. M., and Yolk, B. W., eds.: Cerebral Lipidoses. New York, Academic Press, 1962, pp. 327-342. 9. Kampine, J. P., Brady, R. 0., Kanfer, J. N., Feld, M., and Shapiro, D.: Diagnosis of Gaucher's disease and Niemann·Pick disease with small samples of venous blood. Science, 155 :86·88, 1967. 10. Kampine, J. P., Brady, R. 0., Yankee, R. A., Kanfer, J. N., Shapiro, D., and Gal, A. E.: Sphingolipid metabolism in leukemic leukocytes. Cancer Res., 27: 1312-1315, 1967. 1l. Kattlove, H. E., Williams, J. C, Gaynor, E., Spivack, M., Bradley, R. M., and Brady, R. 0.: Gaucher cells in chronic myelogenous leukemia: An acquired abnormality. Blood, 33:379-390, 1969. 12. Kolodny, E. H., Brady, R. 0., Quirk, J. M., and Kanfer, J. N.: Studies on the metabolism of Tay-Sachs ganglioside. Fed. Proc., 28:596, 1969. 13. Mehl, E., and Jatzkewitz, H.: Evidence for a genetic block in metachromatic leukodystrophy. Biochem. Biophys. Res. Commun., 19:407-411. 1965. 14. Okada, S., and O'Brien, J. S.: Generalized gangliosidosis: Beta galactosidase deficiencv. Science, 160: 1002·1 004, 1968. .
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15. Percy, A. K., and Brady, R. 0.: Metachromatic leukodystrophy: Diagnosis with samples of venous blood. Science, 161 :594, 1968. 16. Sacrez, R., Juif, J. G., Gigonnet,.I. M., and Gruner, J. E.: La maladie de Landing ou idiotie amaurotique infantile precoce avec gangliosidose generalisee de type GM. Pediatrie, 22:143-162,1967. 17. Sloan, H. R., Uhlendorf, B. W., Kanfer, J. N., Brady, R. 0., and Fredrickson, D. S.: Deficiency of sphingomyelin-cleaving enzyme activity in tissue cultures derived from patients with Niemann-Pick disease. Biochem. Biophys. Res. Commun., 34:582-588,1969. National Institute of Neurological Diseases and Stroke National Institutes of Health Bethesda, Maryland 20014
The sphingolipidoses comprise a family of lipid storage diseases of importance to a wide spectrum of the medical profession. Knowledge of the manifestations, diagnosis, and pathological chemistry of hereditary sphingolipid storage diseases is relevant for physicians in general practice as well as those in a number of specialties. Patients with the infantile form of these diseases often show mental retardation and amaurosis early in life and are therefore encountered by pediatricians, ophthalmologists, and neurologists in their practice. If such patients survive infancy, they often appear in clinics with the signs and symptoms of splenomegaly, hepatomegaly, and an abnormal blood picture which may include thrombocytopenia, leukopenia, and mild anemia. Thus, surgeons, internists, and hematologists become involved in the care of these patients. In addition, these patients frequently have erosion of the cortices of long bones and the hip joint, which may be accompanied by pathological fractures and require the services of orthopedic surgeons. The skin of some of these patients becomes involved with pigmentations and maculopapular angiokeratomatous eruptions, and therefore the patients may be seen initially by dermatologists. Clinical chemists can now diagnose three of these lipid storage diseases using leukocytes obtained from small samples of venous blood. Procedures have just appeared on the horizon which hold great promise for the detection of these conditions in utero as well as for the determination of heterozygous carriers of these diseases. Exploitation of these exciting possibilities requires the participation of obstetricians and provides an increasingly important opportunity for genetic counseling.
GENERAL DESCRIPTION OF SPHINGOLIPIDS In this article we are concerned with a group of lipids which have certain structural features in common. They all contain the long chain amino alcohol sphingosine (Fig. 1 a) as a portion of their molecular structure. The biosynthesis of sphingosine occurs in many tissues of the "Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland Medical Clinics of North America- Vo!. 53, No. 4, July, 1969
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a. Sphingosine: CH,-(CH,),,-CH~CH-CH(OH)-CH(NH,)-CH,OH b. Ceramide: CH,,-(CH,),,-CH-=CH-CH-CH-C':'!1,OH
I
I
OHN-H
I I (CH,)""" I C~O
CH" ceramide-galactose c. Galactocerebroside: d. Glucocerebroside: ceramide-glucose e. Ceramide-Iactoside: ceramide-glucose-galactose Globoside: ceramide-glucose-galactose- galactose- N -acety Igalactosamine g. Ganglioside: ceramide-glucose-galactose- N -acetylgalactosamine-galactose
r
I
h.
i. j.
h.
N-acetylneuraminic acid Sphingomyelin: ceramide-phosphorylcholine Sulfatide: ceramide-galactose-3-sulfate Ceramide trihexoside: ceramide-glucose-galactose-galactose Tay-Sachs ganglioside: ceramide-glucose-galactose-N -acetylgalactosamine
I
N-acetylneuraminic acid ':'Point of attachment of various components. Figure l.
body. Carbon atoms 3 to 18 arise from palmitic acid, and carbon atoms 1 and 2 come from the amino acid serine. All of the materials to be discussed also have a long chain fatty acid linked through an amide bone to the nitrogen atom of sphingosine. This combination of fatty acid and sphingosine is called a ceramide (Fig. 1b). Various other molecules are linked to the primary alcoholic group on carbon 1 of the sphingosine portion of ceramide. For example, galactocerebroside, the largest single lipid component of the myelin sheath of nerves, contains the hexose galactose linked by a ,B-glycosidic bond to ceramide (Fig. le). The structure of various other sphingolipids under consideration in this article will be indicated where appropriate.
GAUCHER'S DISEASE SIGNS AND SYMPTOMS. Perhaps the most common lipid storage disease seen by clinicians is the inherited disorder first described by Phillipe Gaucher in 1882. There are two clinical forms of Gaucher's disease which have been designated as the infantile and adult varieties respectively. Both are characterized by the occurrence of splenomegaly, hepatomegaly, and erosion of the long bones. The central nervous system is affected in patients with the infantile form, and severe mental deterioration is usually present in these patients. In patients with the adult form of Gaucher's disease, there is generally very little or no central nervous system difficulty. Patients in the latter category often manifest a brownish pigmentation of the exposed areas of the skin. These patients frequently have a thrombocytopenia and mild hypochromic anemia which are generally improved after splenectomy. GENETICS. Gaucher's disease is the result of the inheritance of an autosomal recessive genetic defect, and hence both sexes may be af-
829
GENETICS AND THE SPHINGOLlPIDOSES
fiicted. There has been one report of a family in which the passage of the Gaucher trait seemed to follow that of a dominant characteristic. H The rapidity of progression of the signs and symptoms of Gaucher's disease may vary over a wide range in patients with the adult form of Gaucher's disease. In some, the disease becomes manifest early in life and the enlargement of the spleen and bone involvement progress swiftly. In other patients, the hepatosplenomegaly is a late-appearing phenomenon, and the rate of hypertrophy of these organs is slow in comparison with the rate in other cases. In my opinion, these findings are clear manifestations of genetic heterogeneity and indicate that the genetically directed alteration definitely differs in such patients. This concept is strengthened by evidence obtained during recent biochemical investigations which have revealed the nature of the enzymatic defect in Gaucher's disease. PATHOLOGICAL CHEMISTRY. A characteristic histopathologic abnormality in patients with Gaucher's disease is the appearance of a large lipid-laden cell in the spleen, liver, and bone marrow. This cell stains for both fat and carbohydrate and is believed to be derived from cells of the reticuloendothelial system. The lipid which accumulates in these cells has been conclusively identified as glucocerebroside (Fig. Id). The biochemical defect in Gaucher's disease now is known to be a deficiency of an enzyme called glucocerebrosidase, which catalyzes the first step in the catabolism of glucocerebroside 2 • 5 (Reaction 1):
Table 1.
+ H"O
glucocerebrosidase
) glucose + ceramide This enzyme is normally present in most of the tissues of the body. The activity of this enzyme was found to be reduced to a mean level of about 15 per cent of the normal value in tissues of patients with the adult form of Gaucher's disease (Table 1). Patients whose tissues showed the least
1. Glucocerebroside
Enzymatic Lesions in the Sphingolipidoses MEAN ACTIVITY OF AFFECTED ENZYME (PER CENT OF NORMAL)
CONDITION
Gaucher's disease
ENZYME INVOLVED
Glucocerebrosidase
Adult form Infantile form
White blood cells 16%
1%
Sphingomyelinase
Metachromatic leukodystrophy
Arylsulfatase A (sulfatidase)
Fabry's disease
Ceramidetrihexosidase
"Not yet determined.
Spleen IS'/'.
Niemann-Pick disease Type A Type C
Hemizygous males Heterozygous females
TISSUE
Liver 5% 17lj(
Brain
1-5%
Small intestine
28 c/r
8% 13C;:'r;,
lO(;{,
Skin fibroblasts 3'/'
<1 'Yc
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glucocerebrosidase activity had the most rapid progression of the disease, whereas those with attenuated but moderately higher residual glucocerebrosidase activity developed splenomegaly and hepatomegaly later in life. Tissues from patients with the infantile form of Gaucher's disease show virtually a complete absence of glucocerebrosidase activity. SOURCE OF ACCUMULATING GLUCOCEREBROSIDE. There appear to be several possible sources of the glucocerebroside which accumulates in the systemic organs and tissues of patients with this disease. Recent observations indicate that the preponderance of this material is produced during the catabolism of senescent leukocytes. 11 Ceramide lactoside (Fig. le) is a major lipid constituent of polymorphonuclear leukocytes. In normal individuals, nearly 400 mg. of glucocerebroside is derived from this source and must be disposed of each day. Another probable source of glucocerebroside in peripheral tissues appears to be the stroma of senescent erythrocytes. A glycolipid called globoside (Fig. If) is a major constituent of erythrocyte membranes, and glucocerebroside probably arises in the course of the sequential hydrolysis of the tetrahexoside portion of this material. Because of the deficiency of glucocerebrosidase in patients with Gaucher's disease, the degradation of glycolipids is impaired. Glucocerebroside is stored in various tissues, especially those involved in leuko- and erythrocytorrhexis. There are several reports which indicate that glucocerebroside accumulates to some extent in neuronal cells in patients with the infantile form of Gaucher's disease. It seems likely that the glucocerebroside in these cells is derived from materials called gangliosides (Fig. Ig). The turnover of gangliosides is rather active in the neonatal period; thereafter it occurs at a slower rate. Patients with the adult form of Gaucher's disease appear to have sufficient glucocerebrosidase activity in brain to catabolize the glucocerebroside which arises during tile critical period of rapid ganglioside metabolism. They thereby escape damage to the nervous system. DIAGNOSIS. The diagnosis of Gaucher's disease can usually be made clinically by the presence of splenomegaly, hepatomegaly, abnormalities of the long bones and pelvis, and an increase in tartrate-inhibitable serum acid phosphatase. The occurrence of the so-called "Gaucher cells" in marrow smears is pathognomonic of this condition. The diagnosis may be confirmed by direct assay of glucocerebrosidase activity in biopsy specimens of solid tissues or, more conveniently, by assaying the level of glucocerebrosidase activity in circulating leukocytes." The assay may be performed easily on leukocytes obtained from 5 ml. or less of venous blood. The estimation of enzymatic activity is conveniently determined with 14C-Iabeled glucocerebroside which is simply incubated with the washed white blood cells at pH 6.0. The radioactive glucose- 14 C which is liberated by the action of the enzyme is determined by liquid scintillation counting. The test is facile and reliable. Leukocytes obtained from patients with Gaucher's disease show a marked decrease in glucocerebrosidase activity compared with similar preparations from normal humans and patients with various other lipid storage diseases. Two additional diagnostic aids have recently been developed in our laboratory. Glucocerebrosidase activity is normally quite high in fibro-
GENETICS AND THE SPHINGOLIPIDOSES
831
blasts derived from skin and grown in tissue culture. Preparations of this nature obtained from a patient with infantile Gaucher's disease showed an almost complete absence of glucocerebrosidase activity. A particularly interesting observation was the detection of intermediate levels of glucocerebrosidase activity in such tissue cultures obtained from heterozygous carriers of Gaucher's disease. If this finding can be confirmed by further experimentation, it will provide for the first time a procedure for detecting heterozygotes. The identification of these individuals is not possible through assays performed on solid tissue samples or leukocyte preparations. Such a determination should be of great usefulness for genetic counseling. From the points of view of the family physician and geneticist, another extremely important development appears imminent. Cells obtained by amniocentesis and subsequently grown in tissue culture also contain good glucocerebrosidase activity. Amniocentesis can be performed with safety about the fourth month of pregnancy, and sufficient cells can be grown for assay in about 5 weeks (see p. 773). Since the cells are of fetal origin, it seems very likely that glucocerebrosidase assays performed on these cells will enable the detection of this disease in utero. This information therefore should be available in sufficient time to permit a critical decision regarding the continuation of the pregnancy. I feel that many, if not all, of the heritable lipid storage diseases probably can be detected in a similar fashion once the nature of the enzymatic defect is known. This contribution alone emphasizes the importance of the painstaking and time-consuming research required for discovering the biochemical abnormalities in inherited metabolic diseases. TREATMENT. Another critical reason for the expenditure of so much effort in delineating the enzymatic defect in these diseases is that only when the explicit nature of the defect is known can rational therapeutic procedures be considered. As yet there is no specific therapy for any of the lipid storage diseases, although many potential remedial procedures are under active consideration. Included among these are trials of enzyme replacement therapy if sufficient pure and nonsensitizing preparations can be produced. Recent evidence suggests that exogenously administered enzymes can indeed pass through cell membranes, a condition which would appear to be required for the removal of intracellularly accumulated glucocerebroside. Perhaps this form of therapy can some day be supplemented or circumvented by transplanting a normal spleen to a patient with Gaucher's disease. Once the glucocerebrosidase has been obtained in a homogenous state, it should be possible to determine the amino acid sequence of the protein. Perhaps the enzyme can be synthesized by recently developed methods. Furthermore, information about the structure can in turn be extrapolated back to indicate the nucleotide sequence of the messenger RNA. It is conceivable that administration of messenger RNA may be efficacious and may cause production of active enzyme. Perhaps at some still further date it may be possible to modify the defective genetic apparatus of these patients by the administration of specific molecules or fragments of DNA (see p. 795). Recent experimental evidence indicates that DNA can be taken up and incorporated into nuclear material by
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intact cells. Still other therapeutic possibilities and modifications have been discussed in an earlier review. 3
NIEMANN-PICK DISEASE SIGNS AND SYMPTOMS. Patients with Niemann-Pick disease, another inherited lipid storage disease, also show systemic organomegaly. In this instance, hepatomegaly is especially predominant; some degree of splenomegaly is usually present as well. Bone involvement occurs, although to a lesser extent than in patients with Gaucher's disease. Niemann-Pick disease usually becomes manifest in patients early in infancy. These patients are quite severely retarded mentally. Bone marrow preparations from patients with Niemann-Pick disease contain large lipid-laden cells which have a waxy appearance and stain for both lipid and phosphorous. Some patients with Niemann-Pick disease have an olive-yellow color to their skin. About 30 per cent of the patients with this increasing number of cases of Niemann-Pick disease are being reported in which there is a less rapid progression of the disease process than in the classic infantile form. In contrast to the patients with the adult form of Gaucher's disease, some of the older patients with Niemann-Pick disease show evidence of slowly progressive brain damage. PATHOLOGICAL CHEMISTRY. The material which accumulates in tissues of patients with Niemann-Pick disease is a phospholipid called sphingomyelin (Fig. 1h). Studies with labeled sphingomyelin have demonstrated convincingly the nature of the metabolic defect in NiemannPick disease. 4 The lesion in these patients was shown to be a nearly complete absence in their tissues of an enzyme which catalyzes the first step in the catabolism of sphingomyelin (Reaction 2) (Table 1): 2. Sphingomyelin + H 2 0
sphingomyelinas~
phosphorylcholine +ceramide
Patients with the less rapidly progressing form of Niemann-Pick disease show higher residual sphingomyelinase activity in their tissues than those with the classic infantile form of the disease (type A). Once again it is necessary to consider potential sources of the sphingomyelin which accumulates in patients with this disease. Sphingomyelin is a quantitatively important component of the plasma membrane of cells and of the membranes of various subcellular organelles such as the endoplasmic reticulum and mitochondria. Sphingomyelin is also an important constituent of erythrocyte stroma. Thus it seems that sphingomyelin could arise in the course of the turnover of almost all cells and their intracellular components. Perhaps the continuous degradation and replacement of these latter structures in the nervous system accounts for the progressive involvement of the brain in the older patients with Niemann-Pick disease. GENETICS. Somewhat over 50 per cent of the patients with NiemannPick disease reported in the literature are of Ashkenazic Jewish ancestry; however, the disease has been reported in all races. This condition is due to the inheritance of a defective autosomal recessive trait. Most of the patients with Niemann-Pick disease die in early infancy, and one would
GENETICS AND THE SPHINGOLIPIDOSES
833
expect that a deleterious mutation such as this would be self-extinguishing. Since it apparently is not, geneticists are forced to consider possible beneficial advantages conferred upon the heterozygous carriers of this trait. There may be a correlation between this heterozygous genetic state and survival or avoidance of malignant diseases. Significantly increased glucocerebrosidase and sphingomyelinase activity has been found in leukemic leukocytes. 1o Studies on the metabolism of membrane lipids in neoplastic diseases are just getting underway in several laboratories. My impression is that important discoveries will be made related to the metabolism of these materials in the transformation of cells with oncogenic viruses and ultimately in malignant diseases in humans. DIAGNOSIS. The diagnosis of Niemann-Pick disease can be made with reasonable certainty on the basis of clinical findings and the appearance of "foam" cells in the bone marrow. The diagnosis can be confirmed by direct analytical determination of the amount of sphingomyelin in tissue samples, e.g., from liver biopsies. A more facile method has recently become available for accurately diagnosing Niemann-Pick disease. It is based on the determination of the level of sphingomyelinase activity in sonicated leukocyte preparations. 9 The level of sphingomyelinase in white blood cell preparations obtained from patients with Niemann-Pick disease is markedly lower than normal. This test is conveniently performed with labeled sphingomyelin as substrate. Sphingomyelinase activity is very low in fibroblasts grown in tissue culture derived from biopsies of Niemann-Pick patients' skinY The enzyme is easily demonstrated in similar cultures from normal individuals and patients with other lipid storage diseases. Studies are underway with the hope of once again being able to detect heterozygous carriers of Niemann-Pick disease. Heterozygotes cannot be identified through the use of the leukocyte assay procedure. Fetal cells obtained by amniocentesis and grown in tissue culture also show very good sphingomyelinase activity. This finding suggests that it is quite likely that the diagnosis of Niemann-Pick disease can be made in utero.
MET ACHROMATIC LEUKODYSTROPHY (MLD) SIGNS AND SYMPTOMS. The predominant manifestations of metachromatic leukodystrophy are severe impairment of the central nervous system and a decrease in nerve conduction velocity in peripheral nerves. These lesions cause motor incoordination and mental retardation. Two discrete clinical states seem to be seen in this disease. The first appears within the first 30 months of life and is characterized by the onset of weakness, ataxia, hypotonus, paralyses, and difficulties in speech and swallowing. The onset of the second form occurs later in life, and the initial symptoms are predominantly psychological difficulties which are followed by progressive dementia. PATHOLOGICAL CHEMISTRY. There is an accumulation of an acidic glycolipid called sulfatide (Fig. 1i) in nerve fibers, kidney, and bile ducts.
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These deposits cause some manifestations of renal and hepatic impairment. Some patients excrete increased quantities of material in the urinary sediment which stains metachromatically orange-brown when exposed to cresyl violet dye. The biochemical defect in MLD has been shown by Austin and coworkers I and Mehl and Jatzkewitzl~ to be a deficiency of a sulfuric acid esterase in these patients (Reaction 3):
3. Sulfatide
+
H20
sulfatase )
galactocerebroside + sulfuric acid
There are at least three sulfatases in mammalian tissues whose activity can be detected by measuring the hydrolysis of artificial substrates such as p-nitrocatechol sulfate. They have been designated as arylsulfatases A, B, and C, and they differ in their pH optima and subcellular distribution. Sulfatide is considered to be the natural substrate for arylsulfatase A, and the activity of only this sulfatase is diminished in patients with MLD. DIAGNOSIS. A convenient diagnostic procedure has been developed recently for the detection of patients with MLD. It is based on enzyme assays performed on circulating leukocytes. 15 Leukocytes contain both arylsulfatase A and B activity. Because of this, the level of arylsulfatase A activity in leukocytes is determined in the presence of sodium pyrophosphate to inhibit arylsulfatase B. Conversely, arylsulfatase B activity may be determined in the presence of arylsulfatase A by adding barium acetate to inhibit arylsulfatase A. Leukocytes obtained from patients with MLD showed a 90 per cent decrease in arylsulfatase A activity compared with normal individuals (Table 1). There was no change in arylsulfatase B activity in leukocytes obtained from patients with MLD. The test is reliable and facile and should lend itself readily to automated clinical laboratory procedures. GENETICS. MLD is also inherited as an autosomal recessive characteristic. It is hoped that enzyme assays on skin fibroblasts grown in tissue culture will provide a means for detecting the heterozygous carriers of this trait. Furthermore, it seems reasonable to expect that this condition will also lend itself to detection antenatally through enzyme assays performed on fetal cells grown in tissue culture. FABRY'S DISEASE SIGNS AND SYMPTOMS. Fabry's disease is characterized by the appearance of numerous purplish papules on the body, especially in the scrotal region and along the lateral iliac area. Male patients with Fabry's disease generally die of renal failure in the third or fourth decade of life. There is an accumulation of a glycolipid in the glomeruli of the kidney which causes a profound diminution of the function of this organ. Some patients show evidence of cardiac enlargement and electrocardiographic abnormalities. Involvement of the eyes is common and may occur in the form of corneal opacification or vascular dilatation. There may be pain in various joints in some patients. PATHOLOGICAL CHEMISTRY AND GENETICS. The lipid which accumulates in tissues of patients with Fabry's disease is a glycolipid called
835
GENETICS AND THE SPHINGOLIPIDOSES
ceramidetrihexoside (Fig. ID. It was predicted that the metabolic defect in this disease would be found to be a deficiency of the enzyme which catalyzes the hydrolysis of the terminal molecule of galactose of ceramidetrihexoside (Reaction 4): 4. Ceramidetrihexoside + H 2 0
ceramide
trihexosidas~
ceramidelactoside
+ galactose
This hypothesis was shown to be correct through the use of the appropriately labeled compound.'; Male hemizygous patients with Fabry's disease have a total absence of this enzyme, ceramidetrihexosidase, in their tissues (Table 1). This disease has been shown to be due to the transmission of a sexlinked (X-chromosome) genetic defect. Since the affected males have only the defective X-chromosome in their cells, it is not surprising that the enzyme is entirely absent. Female carriers of this condition who have both a normal and a defective X-chromosome show intermediate levels of ceramidetrihexosidase activity in their tissues. This finding is exactly that which would be anticipated in a truly heterozygous state. DIAGNOSIS. The diagnosis can be made by determining the level of ceramidetrihexosidase activity in biopsy samples of small intestinal mucosa. Suitable specimens may be obtained by a Wangensteen tube with a small cutting device. In addition, biopsy samples of kidney tissues have been used successfully in our laboratory to establish the diagnosis of Fabry's disease. Once again the diagnostic enzymatic test is greatly facilitated through the use of the labeled compound. It may be possible to detect patients with Fabry's disease by quantitative analysis of ceramidetrihexoside in blood or urine. Such tests require the extraction and partial purification of ceramidetrihexoside from these sources and its subsequent chemical conversion to a derivative which is suitable for separation and identification by gas-liquid chromatography. The accuracy and simplicity of the radioassay procedure for ceramidetrihexosidase make this the diagnostic procedure of choice at the present time. TREATMENT. Specific enzyme replacement therapy has not yet been attempted in patients with Fabry's disease. Affected male patients benefit considerably from hemodialysis. It is conceivable that a renal transplant may be of some help for these patients. Although organ transplantation procedures are in the throes of harsh, and perhaps justified, self-criticism at this time, kidney transplantation under proper conditions has been successful in a number of pathologic conditions. It merits consideration for the treatment of patients with Fabry's disease.
GENERALIZED GANGLIOSIDOSIS Only a very few patients with generalized gangliosidosis have been detected and reported so far. These patients have some hepatomegaly, and there may be skeletal deformities. However, the hallmark is again a rapid, progressive degeneration of the central nervous system. Infants with this condition generally die before their second year of life. The
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metabolic abnormality in generalized gangliosidosis has recently been shown to be a total deficiency of l3-galactosidase activity in the tissues of these patients. 14, 16 This metabolic aberration causes a progressive accumulation of ganglioside (Fig. 19) in the brain and other tissues.
TAY -SACHS DISEASE GENETICS. Tay-Sachs disease is also inherited as an autosomal recessive metabolic defect. The frequency of the disease is about 1 in 6000 to 10,000 births of infants of Ashkenazic Jewish parentage. This figure means that 1 in 50 individuals in this group are carriers of the defect and that the actual gene frequency is 1 in 100. SIGNS AND SYMPTOMS. Infants with Tay-Sachs disease show severe progressive mental retardation and blindness. A cherry-red spot appears in the macula of most patients. There is relatively little organomegaly or skeletal involvement. Histological examination of the brain reveals the presence of cytoplasmic inclusions called membranous cytoplasmic bodies consisting of concentric layers of densely staining material in the ganglion cells. They are comprised of a mixture of gangliosides, cholesterol, and phospholipids. The involved ganglion cells are swollen and distorted. PATHOLOGICAL CHEMISTRY. The characteristic material which accumulates in brain tissue, and perhaps to a lesser extent in peripheral tissues as well in patients with Tay-Sachs disease, is an acidic glycolipid called Tay-Sachs ganglioside (Fig. 1k). This compound differs from the preponderant monosialoganglioside in brain (Fig. 19) in that the terminal molecule of galactose is missing. The absence of this terminal galactose has caused some speculation that the metabolic defect in Tay-Sachs disease is a deficiency of a galactose transferring enzyme required for the completion of the normal ganglioside structure. A defect of this nature is unlikely in Tay-Sachs disease because brain tissue from these individuals contains a normal complement of tetrahexoside gangliosides and only the quantity of trihexoside ganglioside is abnormal. This observation is not compatible with a defective step in ganglioside formation. I think that we are once again dealing with a missing catabolic enzyme. It has been very difficult to localize the specific metabolic defect in Tay-Sachs disease because of the extremely complicated chemistry involved in the synthesis of labeled Tay-Sachs ganglioside. There seem to be two alternative possibilities for a defect in Tay-Sachs disease. The first is a deficiency of an enzyme required for the hydrolysis of the terminal molecule of N-acetylgalactosamine of the Tay-Sachs ganglioside. The second is a deficiency of an enzyme which catalyzes the liberation of the N-acetylneuraminic acid residue. We have very recently prepared Tay-Sachs ganglioside labeled in either the N -acetylneuraminyl or N -acetylgalactosaminyl portion of the molecule. 12 It is hoped that biochemical investigations with these labeled substances will enable us to identify the metabolic lesion in Tay-Sachs disease. If the metabolic defect can be successfully identified in patients with Tay-Sachs disease, we will be in position to extend these studies in
GENETICS AND THE SPHINGOLIPIDOSES
837
several important directions. We will attempt to devise simple and reliable diagnostic procedures to help in the detection of this condition. Other studies will be directed toward determining the heterozygous carriers of the trait. And finally, we hope to be able to devise a procedure for the antenatal diagnosis of Tay-Sachs disease.
SUMMARY The metabolic defects in Gaucher's disease, Niemann-Pick disease, metachromatic leukodystrophy, Fabry's disease, and generalized gangliosidosis have recently been identified. These critical developments have provided an opportunity to consider rational therapeutic procedures for the amelioration of these conditions. Consideration has been given to the potential sites of metabolic lesions in Tay-Sachs disease. Important contributions have been made for the detection and accurate diagnosis of the first four of these conditions. It is anticipated that tissue culture techniques coupled with the use of specifically labeled sphingolipid substrates will eventually provide the clinician with the means of detecting heterozygous carriers of the defective genes. We hope ultimately to be able to identify the occurrence of such diseases in utero. Remarkable opportunities for enlightened genetic counseling will become a reality when these developments occur.
REFERENCES l. Austin, J., Balasubramanian, A. S., Pattabiraman, T. N., Saraswathi, S., Basu, D. K. and Bachhawat, B. K.: A controlled study of enzymic activities on three human disorders of glycolipid metabolism. J. Neurochem., 10:805·816, 1963. 2. Brady, R. 0., Kanfer, J., and Shapiro, D.: Metabolism of glucocerebrosides. 11. Evidence for an enzymatic deficiency in Gaucher's disease. Biochem. Biophys. Res. Commun., 18:221-225,1965. 3. Brady, R. 0.: The sphingolipidoses. New Eng. J. Med., 275:312·318,1966. 4. Brady, R. 0., Kanfer, J. N., Mock, M. B., and Fredrickson, D. S.: The metabolism of sphin· gomyelin. 11. Evidence for an enzymatic deficiency in Niemann·Pick disease. Proc. Nat. Acad. Sci., 55:366·369,1966. 5. Brady, R. 0., Kanfer, J. N., Shapiro, D., and Bradley, R. M.: Demonstration of a deficiency of glucocerebroside-cleaving enzyme in Gaucher's disease. J. Clin. 1vest., 45: 1112·1115, 1966. 6. Brady, R. 0., Gal, A. E., Bradley, R. M., Martensson, E., Warshaw, A. L., and Laster, L.: Enzymatic defect in Fabry's disease: ceramidetrihexosidase deficiency. New Eng. J. Med., 276: 1163·1167, 1967. 7. Crocker, A. C.: The cerebral defect in Tay·Sachs disease and Niemann-Pick disease. J. Neurochem., 7:69·80, 19G1. 8. Hsia, D. Y·Y, Naylor, J., and Bigler, J. A., in Aronson, S. M., and Yolk, B. W., eds.: Cerebral Lipidoses. New York, Academic Press, 1962, pp. 327-342. 9. Kampine, J. P., Brady, R. 0., Kanfer, J. N., Feld, M., and Shapiro, D.: Diagnosis of Gaucher's disease and Niemann·Pick disease with small samples of venous blood. Science, 155 :86·88, 1967. 10. Kampine, J. P., Brady, R. 0., Yankee, R. A., Kanfer, J. N., Shapiro, D., and Gal, A. E.: Sphingolipid metabolism in leukemic leukocytes. Cancer Res., 27: 1312-1315, 1967. 1l. Kattlove, H. E., Williams, J. C, Gaynor, E., Spivack, M., Bradley, R. M., and Brady, R. 0.: Gaucher cells in chronic myelogenous leukemia: An acquired abnormality. Blood, 33:379-390, 1969. 12. Kolodny, E. H., Brady, R. 0., Quirk, J. M., and Kanfer, J. N.: Studies on the metabolism of Tay-Sachs ganglioside. Fed. Proc., 28:596, 1969. 13. Mehl, E., and Jatzkewitz, H.: Evidence for a genetic block in metachromatic leukodystrophy. Biochem. Biophys. Res. Commun., 19:407-411. 1965. 14. Okada, S., and O'Brien, J. S.: Generalized gangliosidosis: Beta galactosidase deficiencv. Science, 160: 1002·1 004, 1968. .
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15. Percy, A. K., and Brady, R. 0.: Metachromatic leukodystrophy: Diagnosis with samples of venous blood. Science, 161 :594, 1968. 16. Sacrez, R., Juif, J. G., Gigonnet,.I. M., and Gruner, J. E.: La maladie de Landing ou idiotie amaurotique infantile precoce avec gangliosidose generalisee de type GM. Pediatrie, 22:143-162,1967. 17. Sloan, H. R., Uhlendorf, B. W., Kanfer, J. N., Brady, R. 0., and Fredrickson, D. S.: Deficiency of sphingomyelin-cleaving enzyme activity in tissue cultures derived from patients with Niemann-Pick disease. Biochem. Biophys. Res. Commun., 34:582-588,1969. National Institute of Neurological Diseases and Stroke National Institutes of Health Bethesda, Maryland 20014