CalifQrnia Medicine THE WESTERN JOUIINAL OF MEDICINE

Sphingolipidoses

FRANK M. YATSU, M.D., San Francisco

* Sphingolipidoses are an heterogeneous group of inherited disorders of lipid metabolism affecting primarily the . These disorders occur chiefly in the pediatric population, and the de- generative nature of the disease processes is generally characterized by diffuse and progressive involvement of neurones (gray matter) with psychomotor retardation and myoclQnus or of fiber tracts (white mat- ter) with weakness and . Biochemical research has identified the defects in the sphingolipi- doses to specific lysosomal enzymes. For example, Niemann-Pick dis- ease lacks sphingomyelinase; Krabbe's disease lacks galactocerebro- sidqse; Gaucher's disease lacks beta-D-glucosidase; metachromatic lacks sulfatase; Tqy-Sachs disease lacks hexosamini- dase A; and generalized lacks beta-galactosidase. Although there are no currently available modes of rendering cor- rective therapy in these disorders, a definitive diagnosis is possible both antepartum as well as postpartum. This information provides a sound and accurate basis for genetic counseling.

THIS REVIEW WILL FOCUS upon recent adva.nces mal brain function and structure. This review in neurochemistry of the sphingolipidoses. The will describe briefly the current neurochemical term sphingolipidoses refers to a group of in- information regarding the following sphingolip- herited disorders of lipid metabolism affecting idoses: Niemann-Pick disease, Krabbe's disease, chiefly the nervous system. The clinical prob- Gaucher's disease, metachromatic leukodystro- lems have stimulated a wealth of productive re- phy, Tay-Sachs disease, and generalized gangli- search in neurochemistry, particularly in the osidosis.14 (See Table 1.) areas of brain lipids and merpbranes. These in- vestigations have also provided a foundation for Clinical Aspects future research into the molecular basis of nor- The sphingolipidoses are a heterogeneous From the Department of Neurology, University of California, San group of diseases and the diagnosis on clinical Francisco, School of Medicine. The author is a John and Mary Markle Scholar in Academic Medi- grounds is oftentimes difficult. However, a clin- cine and Chief, Neurology Service and Neurochemistry Laboratory of the San Francisco General Hospital; and is supported by two grants ico-pathological approach is possible with these from the U.S. Public Health $ervice (NS 09128 and NS 07769). as well as other so-called diseases Presented in part at the Annual Meeting of the American Academy degenerative ,f Neurology, April 21, 1969, Washington, D.C. of the central nervous system. Submitted May 19, 1970. Reprint requests to: Neurology Service, Room 4521, San Francisco These diseases can be divided into those that General Hospital, 1001 Potrero Avenue, San Francisco, Ca. 94110 (Dr. F. M. Yatsu). have primarily gray matter symptoms and signs

CALIFORNIA MEDICINEE The Western Journal of Medicine TABLE I.-Sphingolipidoses Lipid Enzyme Deject Disease -P-choline =sphingo- - Niemann- (phosphorylcholine) myelin ase Pick** -galactose = galacto- galactocere- Krabbe's cerebro- brosidase side -glucose = glucocere- ,8-D-glucosidase Gaucher's broside + -gal-SO4 = sulfatase M.L.D. (sulfatide lipidosis) -Hexoses -trihexose = ceremide ceremide tri- Fabry's trihexoside hexosidase -hexoses + NANA* = : G Hexosamninidase A Tay-Sachs*** M2 G ganglioside ,B-galactosi- General- M. dase ized ganglio- sidcosis fucose fucosidase Fucosidosis CERAMIDE = palmitic acid + serine fatty acid *NANA=N-acetylneuramnsinic acid **There are 4 clinically separate forms of Niemann-Pick and only type A is referred to above. * * *There are now 5 gangliosidoses with varying ganglioside and lysosomal enzyme deficiencies. and those with primarily white matter symp- fantile form or Crocker's type A accounts for toms. With the former, the gray matter symp- approximately 85 percent of the cases of Nie- toms and signs are those of either "irritation," mann-Pick disease and definitive lipid abnormal- such as seizures, particularly myoclonic seizures, ities in brain are limited to this subgroup. The or "inhibition," such as apathy, lethargy and de- infantile form of Niemann-Pick disease is char- mentia. Further, if occipital neurons are in- acterized clinically by psychomotor retardation volved, cortical blindness will supervene. With with mental deterioration, seizures, spasticity, diseases affecting primarily the white matter, the and frequently the pres- symptoms and signs are those of long-tract in- ence of a "cherry red spot" in the fundi. (The volvement; for example, spastic weakness with "cherry red spot" is seen in several of the lipid involvement of the corticospinal tract, pseudo- storage diseases and is not diagnostic of any one bulbar palsy with damage to the corticobulbar entity.) tract, incoordination from destruction of cere- bellofugal fibers, and even cortical blindness, Pathological examination of enlarged visceral secondary to interruption of the optic radiations. organs, brain and bone marrow discloses the Tay-Sachs diseas.e would be a prototype of a dis- presence of "foam cells" laden with lipid mate- ease affecting primarily gray matter, while meta- rial. Analysis of the lipid reveals an abundance chromatic leukodystrophy would be more typical of sphingomyelin. An enzymatic mechanism that of disease predominantly of white matter. accotnts for the abnormal intracellular deposi- tion of sphingomyelin-that is, excessive synthe- Niemann-Pick Disease sis or inadequate breakdown-was established by Niemann-Pick disease5-7 is a genetic disorder the discovery that patients with this disease subdivided into four groups depending upon the lacked the hydrolytic enzyme sphingomyelinase.6 age of onset and clinical manifestations. The in- As with other similar disorders, it is anticipated

2 APRIL 1971 * 1 14 * 4 that future research will provide a rational Gaucher's Disease means by which to "induce" the deficient enzyme Gaucher's disease13-15 is an autosomal, recessive or prevent the abnormal accumulation of the genetic disorder that has its onset in infancy or lipid.4 adulthood and infrequently during adolescence. The infantile form characteristically affects the Krabbe's Disease central nervous system and is sometimes referred of disease. Krabbe's disease8-'2 is an autosomal, recessive, to as the "cerebral form" Gaucher's the Clinically this disease is similar to Niemann-Pick genetic disorder affecting primarily central disease in its presentation with psychomotor re- nervous system. Following normal early devel- to bulbar weakness, opment, the disease has its onset during the mid- tardation, poor feeding due those opisthotonus, spasticity, and striking hepatosple- dle of the first year of life. Symptoms are nomegaly. Death ensues in the first year of life of psychomotor retardation, tonic seizures, spas- state. ticity and blindness. Recently peripheral neu- in a decerebrate ropathy has been described. Histologically affected tissues contain large, multinucleated cells with a characteristic dull, Histological examination of brains disclose dif- waxy appearance. The lipid accumulation is due fuse denmelination of white matter with arcuate to cerebroside. Since the sugar mnoiety is glucose, fiber sparing. In addition, there is the character- it is designated a . istic accumulation of large epithelioid or "glo- Investigations for an enzyme defect have re- boid" cells in the white matter and hence its non- vealed an absence of lvsosomal beta-D-glucosi- eponymic title of "globoid cell leukodystrophy." dase, essential for the normal degradation of glu- Lipid analyses of brains affected with Krabbe's cocerebrosides. disease have given conflicting results, but sepa- ration and analysis of globoid cells indicate a Metachromatic Leukodystrophy higher relative concentration of cerebroside. This Metachromatic leukodystrophy16"^2 (MLD or sul- cerebroside's sugar moiety is galactose and hence fatide lipidosis) is an autosomal, recessive, ge- is termed galactocerebroside. The proposal that netic disorder known since 1910 that becomes a focal accumulation of galactocerebroside may manifest during childhood and occasionally in account for the disease is supported by experi- adulthood. The symptoms in childhood are those mental studies involving the intracerebral injec- of leukodystrophy, in that motor symptoms pre- tion of galactocerebroside. Dogs injected in this dominate. Muscular weakness and wasting, asso- fashion developed neurological deficits associated ciated with a stumbling gait or genu recurvatum, with typical epithelioid or globoid cells in white may be the initial symptom. matter. MLD is unique among the sphingolipidoses be- A postulated enzyme defect in this disorder is cause the peripheral nervous system is affected. unique, because it is in the synthetic as opposed Thus, evidence of a peripheral neuropathy with to the more common catabolic pathway. The de- sensory, motor, and reflex changes may precede ficient enzyme is the cytoplasmic cerebroside, or supersede the findings of a leukodystrophy. sulfotransferase. This enzyme transfers sulfate With progression of the disease, generalized cere- from the donor PAPS (phosphoadenosinephospho- bral dysfunction develops. Seizures occur in one- sulfate) to the 3-carbon of the galactose molecule half the patients, blindness in one-third, and of cerebroside. A genetic disorder in dogs mani- death follows a decerebrate or decorticate state. festing similar histological and biochemical alter- The adult form of MLD usually presents as an or- ations as globoid cell leukodystrophy should help ganic dementia or psychosis, frequently diag- to clarify the molecular pathogenesis of this dis- nosed as schizophrenia. These patients, however, ease and offer a laboratory model for possible eventually manifest diffuse central nervous system means of therapy. Recently, however, Suzuki'2 involvement with corticospinal, corticobulbar, has accumulated compelling evidence in human and cerebellar symptoms. brain tissue that the metabolic defect in Krabbe's Histochemical studies disclose the presence of disease is in fact a catabolic enzyme, namely, the intracellular lipids, not only in the central and lysosomal galactocerebroside-f - galactocerebro- peripheral nervous systems, but also within vis- sidase. ceral organs, such as kidney, liver, and gallblad-

CALIFORNIA MEDICINE 3 The Western Journal of Medicine der. Impairment of these organs does not become findings would support in vitro biochemical data clinically apparent. Characteristic staining is met- of defective catabolism of sulfatide. achromasia-that is, an alternate color to the dye Attempts to restrict sulfur intake and thereby used, such as toluicline blue staining red or cresyl the available precursor pool for sulfatide synthe- violet staining brown. In addition to the deposi- sis or the infusion of the missing enzyme sulfatase tion of sulfatide within white matter cells, there have been unsuccessful in altering progression of is symmetrical demyelination of white matter the disease in clinical trials. Knowledge that vita- with U-fiber or arcuate fiber sparing. Intraneu- min A is a required cofactor in the synthesis of ronal collection of metachromasia is not charac- PAPS (phosphoadenosinephosphosulfate) has teristic except for few Betz cells and for certain raised the possibility of inducing vitamin A de- subcortical neurones, notably the cerebellar den- ficiency in MLD to decrease the available activated tate nucleus. sulfate for sulfatide synthesis. Identification of sulfatide as the lipid responsible Clinical application of this proposal would ap- for metachromasia in MLD was first demonstrated pear to have impracticalities, but information to in 1958 by two independent workers, Austin in date is insufficient. the United States and Jatzkewitz in Germany. Lipid analyses in MU)D reveal a ten-fold increase Tay-Sachs Disease in brain sulfatide concentration with an inversion Tay-Sachs disease22-28 or infantile amaurotic of the sulfatide: cerebroside ratio to 4:1. Nor- familial idiocy (AI) is a genetic disease occur- mally the ratio is approximately 0.25 to 1. Analy- ring primarily in Jewish infants. The disorder sis of the lipid composition of myelin in MLD dem- commences during the middle of the first year of onstrates a preponderance of sulfatide; this has life after an apparently normal early develop- led to the speculation that demyelination in this ment. Death ensues after a period of two to four disease may be due to formation of an unstable years. The brunt of the disease is upon the cen- membrane. O'Brien reasoned that the preponder- tral nervous system, particularly the grey matter. ance of sulfatide in the myelin membrane could As the name implies, amaurosis develops, and lead to a less cohesive membrane because of ab- the so-called "cherry-red spot" of the macular re- normalities in the surface charge of the mem- gion is to be seen on funduscopic examination brane, due to an excess of electronegatively and idiocy is concomitant. In addition, grey charged sulfate groups. MLD appears to be one matter involvement gives rise to a cortical ir- of the first examples of a disease resulting from a ritative phenomenon, manifested by myoclonic molecular defect in membrane structure.18 seizures. Pathological examination reveals "bal- Although the clinical diagnosis of MLD iS sug- looned" neurones filled with lipid staining gested by the presence of leukodystrophic symp- material. Electron microscopic studies of the in- toms and signs combined with a peripheral neu- volved neurones disclose the cytoplasm to be filled ropathy, the definitive diagnosis is established by with membranous, lamellated bodies termed one of several techniques. The older technique "membranous cytoplasmic bodies" or MCBs. Lipid of demonstrating metachromatic granules in the analysis of these bodies, obtained by differential urine with toluidine blue can give false-positive centrifugation, reveals an increase of monosialo- results, since other substances besides ganglioside. This ganglioside which lacks the ter- (mucopolysaccharides, for example), are meta- minal galactose is normally found in very small chromatic. A sensitive and accurate test devised quantities and is referred to as the "Tay-Sachs by Austin is detection of arylsulfatase in urine. ganglioside" or GM, ganglioside (Svennerholm's Affected persons have no enzyme activity. Alter- nomenclature). The enzymatic defect in Tay- natively, biopsy of a nerve, such as sural nerve, Sachs disease is now known to be an absence of with demonstration of metachromasia would es- a lysosomal beta-D-N-acetylhexosaminidase.8 An tablish the diagnosis of MLD. Biochemical studies as yet unexplained and unconfirmed finding in on tissues from MLD have identified an absence of Tay-Sachs disease is decreased activity of serum sulfatase, the lysosomal enzyme which hydrolyses fructose-l-phosphate-aldolase. 3 Recently, O'Brien the sulfate group from sulfatide. Clinical investi- and Okada have taken amniotic fluid of suspected gations of sulfated compound reveal a slow turn- carrier mothers and applied tissue culture tech- over in MLD compared to normal subjects. These niques to grow fibroblasts upon which enzyme

4 APRIL 1971 * 114 * 4 TABLE 2.-Diagnostic Tests for Sphingolipidosis Disease Blood Tissues Other Laboratory Diagnosis Niemann-Pick vacuolated lymphocytes foam cells in marrow, involved viscera Krabbe's brain* and peripheral elevated CSF nerve biopsy protein Gaucher's deficient Beta- Gaucher's cells in glucosidase in WBC marrow and involved or skin viscera WILD ( sulfatide decreased WBC sulfatase peripheral nerve biopsy urinary sulfatide lipidosis) showing metachromasia increase or sulfa- tase decrease Tay-Sachs absent rectal and/or brain* elevated CSF protein A in serum & WBC biopsy; fibroblast from skin; prenatally by amniotic fluid cell assay Generalized ganglio- deficient brain* and/or visceral deficient beta- sidosis beta-galactosidase biopsy; foam cells galactosidase in in WBC in marrow urine *Brain Biopsy. Establishment of a definitive diagnosis with the use of brain biopsy is justified for genetic counseling provided the patient demonstrates irreversible dementia and that adequate neurosurgical, histological and biochemical facilities are available. analyses are made. The diagnosis of Tay-Sacbs three to ten years. Although beta-galactosidase disease may be possible before birth by finding is also absent in this disease, it is phenotypically a deficiency of hexosaminidase.25 This technique distinct from generalized gangliosidosis because of amniocentesis holds great promise as a power- of the absence of visual disturbances, cherry red ful diagnostic tool and opens doors for its utiliza- spot of the macula, hepatosplenomegaly, and sig- tion for antepartum diagnosis in other genetic nificant bony deformities. (Late infantile amau- disorders.26'28 rotic idiocy or Jansky-Bielschowsky disease is phenotypically distinct from generalized ganglio- Generalized Gangliosidosis sidosis and does not represent a sphingolipidosis.) Generalized gangliosidosis (or GM1 gangliosi- Table 2 shows diagnostic tests for the'identifi- dosis, type l)2931 is an acute infantile disease char- cation of various kinds of sphingolipidoses. acterized by psychomotor retardation, hepato- splenomegaly, and coarse features similar to those Neurochemistry of Brain Lipids of Hurler's disease. Generalized gangliosidosis is The brain is 80 percent water, yet of the dry due to an accumulation of GM1 ganglioside in weight, brain lipids are the major constituent (60 brain and viscera as well as mucopolysaccharide percent and are classified into three main groups: in the latter. Death ensues in the first two years (1) the sterols, primarily cholesterol, (2) the of life. The disease has been confused with Tay- phospholipids, and (3) the . The Sachs disease because of the finding of a cherrv major types of sphingolipids are gangliosides, cer- red spot in the macula and psychomotor retarda- ebrosides, sulfatides, and sphingomyelin. Sphin- tion, with Hurler's disease because of the presence golipids constitute approximately 10 percent of of similar phenotypic abnormalities. Enzymic whole brain lipids and approximately 20 percent studies disclose a pronounced deficiency of beta- of purified myelin lipids. Of interest is the locali- galactosidase which accounts for accumulation of zation of gangliosides in neurons and their virtual the GM1 ganglioside and the mucopolysaccharide. absence in myelin. There is as yet no specific form of therapy but The sphingosine molecule is an essential con- early recognition and diagnosis is important so stituent for the structure of sphingolipids from that accurate genetic counseling can prevent simi- which it derives its name. Sphingosine is synthe- lar births. sized from the condensation and subsequent de- Juvenile GM1 gangliosidosis (or GM1 gangliosi- carboxylation of palmitic acid and serine to form dosis, type 2) has as its onset about age one and an 18-carbon amino-sugar. The three main groups is due to the cerebral but not visceral' accumula- of lipids noted above of cholesterol, phospho- tion of GM1 ganglioside. Death ensues within lipids, and sphingolipids are assembled with pro-

CALIFORNIA -MEDICINE 5 The Western Journal of Medicine tein moieties to form membranes and become the cal identification of the enzymatic defect. These structural boundary of cells and their subcellular "experiments of nature" have provided a bio- organelles. A postulated structure of membranes chemical insight into the complexity of the nerv- is that of a "bimolecular leaflet" with interdigita- ous system. It is anticipated that continued ex- tion of opposing lipid molecules. This concept ploration of these and similar disorders of brain was derived from electron-microscopic and x-ray will yield rational clues to their therapy as well diffraction data. as shed light on the molecular basis of normal Anatomically, gangliosides are associated pri- neural function. marily with neurons, or "ganglion" cells, from which they derive their name. This highly polar REFERENCES lipid plays an important role in neuronal function, 1. Folch-Pi J, Bauer HD (ed): Brain Lipids and Lipo-proteins and but its precise nature is as yet unknown. Subcel- the Leukodystrophy. Amsterdam, Elsevier, 1963 2. Aronson SM, Volk BW (ed): Inborn Errors of Me- lular fractionation of nerve cells has yielded infor- tabolism. New York, Pergamon Press, 1967 3. Stanbury JB et al. (ed): The Metabolic Basis of Inherited Dis- mation that gangliosides are associated primarily ease. New York, Blakiston, 1966, pp 523-634 4. Brady RO: The sphingolipidoses. New Eng J Med 375:312-318, with microsomes and synaptosomes. Gangliosides 1966 are distinct from other sphingolipids by contain- 5. Brocker AC, Farber S: Niemann-Pick disease-A review of 18 patients. Medicine 37:1-45, 1958 ing one or more N-Acetylneuraminic acid (NANA) 6. Schneider PT, Kennedy EP: Sphingomyelinase in normal human spleens from subjects with Niemann-Pick disease. J Lipid Res 8:202, molecules, the biologically most significant mem- 1967 7. Philippart M et al: Niemann-Pick disease. Arch Neurol 20:227- ber of the family of sugars called sialic acid. Ten 238, 1969 separate gangliosides have so far been identified, 8. D'Agostino AN et al: Krabbe's disease. Arch Neurol 8:82-96, 1963 depending on the number of attached NANAS ( 1 9. Austin J: Studies in globoid (Krabbe) leukodystrophy. Arch Neurol 9:207-231, 1963 to 3), their combination, and the number of at- 10. Dunn HG et al: The neuropathy of Krabbe's infantile cerebral tached hexoses. Over 90 percent of the ganglio- sclerosis. Brain 92:329-344, 1969 11. Bachhawat BK et al: A cerebroside sulphotransferase deficiency sides are accounted for by four major ganglio- in a human disorder of myelin. Biochem J 104:15c, 1967 12. Suzuki K, Suzuki Y: Globoid cell leucodystrophy (Krabbe's dis- sides: one monosialoganglioside (different from ease)-Deficiency of galactocerebroside -,/-galactocerebrosidase. Proc Nat Acad Sci, in press the Tay-Sachs ganglioside which lacks a terminal 13. Philippart M, Rosenstein S, Menkes JH: Isolation and charac- terization of the main splenic in normal organ and in galactose), two disialogangliosides, and one tri- Gaucher's disease Evidence for the site of metabolic block. J Neuro- sikloganglioside. The complex nature of ganglio- path Exp Neurol 24:290-303, 1965 14. Suomi WD, Agranoff BW: Lipids of the spleen in Gaucher's sides is suggested by their dependence upon pro- disease. J Lipid Res 6:211-219, 1965 15. Tuchman LR et al: Studies on the nature of the increased acid tein synthesis, since ganglioside synthesis is phosphatase in Gaucher's disease. Amer J Med 27:957, 1959 by puromycin, an inhibitor of protein 16. Austin JH: Metachromatic form of diffuse cerebral sclerosis.- inhibited 2. Diagnosis during life by isolation of metachromatic lipids from synthesis. urine. Neurology 7:716-723, 1957 17. Svennerholm L et al: Quantitative isolation of brain sulfatides. Although sphingomyelin contains the word J Lipid Res 3:483, 1962 18. O'Brien JS: A molecular defect of myelination. Biochem Bio- "myelin," it is not found exclusively nor abun- phys Res Commun 15:484-490, 1964 in 19. Jatzkewitz H, Mehl E: Cerebroside-sulphatase and aryl-sulpha- dantly myelin and, therefore, is a misnomer. tase.-A deficiency in metachromatic leukodystrophy. J Neurochem Cerebroside, on the other hand, is found in rela- 16:19-28, 1969 20. Clausen J: The effect of vitamin A deficiency on myelination in tive abundance in myelin and is frequently con- the central nervous system of the rat. Europ J Biochem 7:575-582, 1969 sidered a "marker" of myelin lipids. 21. Moser HW, Moser AB, McKhann, GM: The dynamics of a lipidosis. Arch Neurol 17:494-511, 1967 Normally, myelin contains three to four times 22. Svennerholm L: The chemical structure of normal human brain and Tay-Sachs gangliosides. Biochem Biophys Res Commun 9:436- as much cerebroside as sulfatide. Sulfatide is a 441, 1962 sulfate ester of cerebroside and is synthesized by 23. Aronson SM, Perle G et al: Biochemical identification of the carrier state in Tay-Sachs' disease. Proc Soc Exp Biol Med 111:664- the addition of sulfate to cerebroside from the 667, 1962 24. Korey SE et al: Studies in Tay-Sachs disease I(A) Methods activated sulfate, phosphoadenosine-phosphosul- (1) Biochemical (2) Electron microscopic (B) Clinical and pathologic descriptions. J Neuropath Exp Neurol 22:2-9 and 10-17, 1963 fate (PAPAS), by the cytoplasmic enzyme, cer- 25. Okada S, O'Brien JS: Tay-Sachs disease Generalized absence of a Beta-D-N-acethylhexosaminidase component. Science 165:698- ebroside sulfotransferase. 700, 1969 This brief review has summarized the current 26. Okada S, O'Brien JS: Tay-Sachs disease-Absence of hexosam- inidase isoenzyme. Amer J Hum Genet 21:533, Sep 1969 knowledge of the biochemical abnormalities in 27. Schneck L, Volk BW, Saifier A: The gangliosidoses. Amer J Med 46:245-263, 1969 the sphingolipidoses. Elucidation of this hetero- 28. Nadler HL, Gerbie AB: Amniocentesis in the intrauterine de- geneous group of rare diseases of the central nerv- tection of genetic disorders. New Eng J Med 282:596-599, 1970 29. O'Brien JS: Generalized gangliosidosis. J Ped 75:167-186, 1969 ous system has followed the traditional sequence 30. Landing BH et al: Familial neurovisceral lipidosis. Amer J Dis Child 108:503-522, 1964 of clinical characterization, pathological verifica- 31. Suzuki K et al: Morphological and biochemical studies on a case of s5:stemic late infantile lipidosis (generalized gangliosidosis). J Neu- tion, chemical analysis, and finally the biochemi- ropath Exp Neurol 27:15-38, 1968

6 APRIL 1971 * 14 * 4