A n n a l s o f C linical Laboratory Science, Vol. 2, No. 4 Copyright © 1972, I n s t i t u t e for Clinical Science

Enzyme Defects in the and Their Application to Diagnosis*

ROSCOE O. BRADY, M.D.

Developmental and Metabolic Neurology Branch National Institute of Neurological Diseases and Stroke National Institutes of Health Bethesda, MD 20014

Chronicle methods for detecting fetuses afflicted with The first clinical indication of a heritable each of the nine now known storage diseases sufficiently early in pregnancy for disorder of appeared just precise genetic counseling. This paper is a over 90 years ago with Warren Tay’s de­ brief review of how these developments scription of changes in the macular region came about. Moreover, an effort is made to of the eyes of children.33 Six years later, anticipate further progress in this family Bernard Sachs reported that patients with of genetic diseases. these eye lesions were also severely re­ tarded.28 In time this condition was named Tay-Sachs disease. However, most of the Clinical Manifestations of Lipid pioneering developments in the field of Storage Diseases lipid storage diseases have arisen from in­ The signs and symptoms of patients with tensive investigations in another of these the sphingolipidoses are quite varied and conditions called Gaucher’s disease. The are functions of the nature of the accumu­ first clinical description of patients with lating lipid and the particular organs and this syndrome postdated Tay’s communica­ tissues involved in the storage process tion by only a year.14 The chemical struc­ (table I). The development of the central ture of the stored lipid was first identified nervous system may be impaired in eight in Gaucher’s disease,1 and the nature of of the nine known disorders; the apparent the explicit metabolic derangement was single exception is Fabry’s disease in which elucidated first in this sphingolipodys- neuralgias and are trophy.7’9 Through this demonstration, an frequently encountered. If the underlying unbroken precedent regarding the under­ metabolic abnormality is less severe than a lying metabolic defect in all of the sphingo­ total loss of the requisite , patients lipidoses was established. Much practical may escape cerebral damage. Patients with benefit followed this demonstration, includ­ the so-called “juvenile” and “adult” forms ing (1) accurate diagnostic tests, (2) pro­ of Gaucher’s disease are frequently en­ cedures for identifying heterozygous car­ countered in medical clinics because of riers of the lipidoses, and (3) reliable organomegaly, thrombocytopenia, or patho­ logic fractures of the long bones and verte­ * Presented at the Applied Seminar on the Clin­ brae in whom mental function is perfectly ical Pathology of the , November, 1971. normal. Improved diagnostic procedures 285 286 BRADY

TABLE I

M a j o r S i g n s a n d S y m p t o m s o f t h e S phingolipidoses

Gaucherrs disease Mental retardation (infantile form only), , hip and long bone involvement, Oil-red-O and PAS-positive lipid-laden (Gaucher) cells in bone marrow, increased serum acid phosphatase, mild anemia, and thrombocytopenia

Niemann-Pick disease Generally similar to Gaucher's disease? 30 percent with cherry-red spot in macula; marrow cells (foam cells) stain for both lipid and phosphorus, cachexia

Globoid Mental retardation? almost total absence of myelin, severe (Krabbe's disease) gliosis, and multinucleated "globoid bodies” in white matter

Metachromatic leukodystrophy Mental retardation? psychological disturbances (adult form)? decreased nerve conduction time? nerve biopsy shows yellow- brown droplets when stained with cresyl violet (metachromasia)

Ceramide lactoside lipidosis Slowly progressing CNS impairment? hepatosplenomegaly; macrocytic anemia, leukopenia and thrombocytopenia due to involvement of bone marrow and spleen

Fabry's disease Reddish-purple maculo-papular rash in umbilical, inguinal and scrotal areas? renal impairment? corneal opacities; peripheral neuralgias and abnormalities of EKG

Tay-Sachs disease Mental retardation, amaurosis, cherry-red spot in macula, macrocephaly, neuronal cells distended with "membranous cytoplasmic bodies”

Generalized Mental retardation, cherry-red spot in macula (50 percent of patients), hepatosplenomegaly, foam cells in bone marrow, rarefaction of all bones and skeletal deformities now available have permitted the conclu­ X-chromosome, and therefore only the fe­ sive identification of patients with Nie- male need be a carrier in order to have a mann-Pick disease also without neuronal male child affected with this disorder. Half involvement. Except for Fabry’s disease of her sons will have the disease, 50 percent and Tay-Sachs disease, hepatosplenomegaly of her daughters will be heterozygous, and is usually observed in patients with a the others will not be involved. In order sphingolipidosis. Large lipid-laden histio­ to produce an offspring afflicted with one cytes are present in the bone marrow and of the other lipid storage diseases, both elsewhere throughout the reticuloendothe­ parents must be heterozygotes. Of four lial system. In Fabry’s disease, there is an children born to such parents on a statis­ accumulation of in the glomeruli tical basis, one child will have the disease, of the , the vascular endothelium, two will be heterozygotes, and one will be and lymphatics. In patients with Tay-Sachs uninvolved. Obviously in these disorders, disease, only the neuronal cells in the both of the sexes are equally affected. and myenteric There are interesting clinical subtleties plexus are extensively involved.34 regarding heterozygotes. Female carriers for Fabry’s disease may have varying de­ Genetics grees of ophthalmological difficulties pri­ All of the lipid storage diseases which marily affecting the cornea. By and large, are known at the present time are trans­ heterozygotes for the other lipidoses are mitted as autosomal recessive traits with symptom-free and some speculation has the exception of Fabry’s disease. The muta­ even appeared regarding the possibility that tion in Fabry’s disease is linked to the survival advantage may be a consequence ENZYME DEFECTS IN THE SPHINGOLIPIDOSES 287 of being heterozygous for Tay-Sachs dis­ lipid component of brain.36 It was there­ ease.31 fore necessary to determine whether gluco­ was synthesized in patients Metabolic Derangements with Gaucher’s disease instead of galacto- At least five biochemical aspects are con­ cerebroside because of faulty galactose me­ stant features of all of the lipid storage tabolism. This possibility was dispelled by diseases: (1) in all of these disorders there Thannhauser in 195035 by his demonstrat­ is an accumulation of a complex lipid in ing that there was no abnormality of galac­ various tissues of the body; (2) a portion tose tolerance in Gaucher patients. We of the structure of all of the involved lipids confirmed Thannhauser’s observation and is similar and it is called (N-fatty set about to try to decide between the acyl ) following two alternatives: (1) the possi­ bility of an overproduction of a normally [CHs—(CH2) 12—CH=CH—CH (OH) minor tissue component, or (2) an impair­ —CH( NHCOR)—CH2OH]; ment of catabolism of the accumulating glycolipid. Experiments were carried out in (3) the rate of synthesis of the stored lipid 1959 dealing with the biosynthesis of cere- in patients with these disorders is com­ brosides in spleen tissue slices obtained parable to that in non-involved humans; from various control human sources and (4) the enzymatic defect in each of the from patients with Gaucher’s disease. diseases is a deficiency of a specific hydro­ These investigations revealed that there lytic enzyme required for the degradation was no increase in the rate of cerebroside of the acculating lipid; and (5) the degree formation in the tissues obtained from of attenuation of enzymatic activity is simi­ Gaucher’s patients.37 This observation led lar in all of the tissues of an affected indi­ to the idea that a deficiency of a catabolic vidual. These important concepts should enzyme was responsible for the accumula­ be kept in mind since they constitute an tion of the lipid. In 1965, it was shown important basis for investigations which conclusively that the activity of glucocere­ have provided impressive advances towards broside /J-glucosidase was drastically dimin­ the understanding and management of the ished in tissue preparations obtained from sphingolipidos es. patients with Gaucher’s disease compared The best documentation of the funda­ with the activity of this enzyme in normal mental biochemical aspects of lipid storage humans.7’9 These experiments provided the diseases has evolved over the years in the foundation for understanding the nature of course of investigations on the etiology of the metabolic abnormality in all of the Gaucher’s disease. The lipid which accu­ presently known lipid storage diseases; viz, mulates in patients with this disease is a deficiency of a specific hydrolytic enzyme called . It consists of a which catalyzes the degradation of the ac­ single molecule of linked by a p- cumulating lipid. Following this initial glycosidic bond to carbon atom 1 of cera­ demonstration, the underlying abnormal mide (figure 1). In order to elucidate the enzymology was soon established for meta- nature of the metabolic abnormality in chromatic leukodystrophy,22 Niemann-Pick Gaucher’s disease, a decision was required disease,8 and Fabry’s disease.5 In time, the which involved at least three separate pos­ specific enzyme deficiency was identified sibilities. The first was the formation of in the remaining lipid storage disease (fig­ an abnormal cerebroside due to an altera­ ure 1). The metabolic lesion in the lipi­ tion of carbohydrate metabolism. It had doses are quite straight-forward with the been known since 1901 that galactocere- exception of Tay-Sachs disease. The miss­ broside (ceramide-galactose) was a major ing enzyme normally catalyzes the cleavage 288 BRADY

CONSTELLATION OF METABOLIC DISEASES CHARACTERIZED BY INABILITY TO DEGRADE

Disease Major accumulated Enzyme defect

G a ucher C e r 0-Glucosidase

Ceramide glucoside (glucocerebroside)

Niemann-Pick C e r Sphingomyelinase

Sphingomyelin

K ra b b e Cer ß - Galactosidase

Ceramide galactoside (galactocerebroside)

Metachromatic Leukodystophy Cer Gal Sulfatidase ~oso3- Ceramide galactose-3-sulfate (sullalide)

C er Ceramide Lactoside Lipidosis ^■Galactosidase

Ceramide lactoside

Fabry C e r a-Galactosidase

Ceramide trihexoside

T a y-Sa ch s C e r A

N A cN A GM;

Cer = N-Acyl Sphingosine (Ceramide) Glc=Glucose PChol=Phosphorylcholine Gal=Galactose

NAcNA = N-Acetylneuraminic Acid NAcGal=N-Acetylgalactosamine

F i g u r e 1. Metabolic lesions in the sphingolipidoses. of a terminal hexose molecule or in Nie- determine the catabolic pathway(s) of mann-Pick disease, the phosphorylcholine Tay-Sachs ganglioside. It was anticipated portion of the lipid. However, in Tay-Sachs a number of years ago tha the N-acetyl- disease, the accumulating ganglioside is galactosaminyl portion of this ganglioside branched in the terminal portion and con­ might be cleaved before the molecule of siderable experimentation was required to N-acetylneuraminic acid (reaction l).s ENZYME DEFECTS IN THE SPHINGOLIPIDOSES 289

hexosaminidase 1. Cer-Glc-Gal- (NAcNA) -NAcGal + H20 ------> Cer-Glc-Gal-NAcNA + N-acetylgalactosamine Alternatively, the molecule of N-acetylneuraminic acid (NAcNA) might be cleaved prior to the hydrolysis of N-acetylgalactosamine (reaction 2).

neuraminidase 2. Cer-Glc-Gal-(NAcNA)-NAcGal + H20 ------> Cer-Glc-Gal-NAcGal + N-acetylneuraminic acid Actually, both of these reactions have now natal period of life, it may be assumed been demonstrated. Tay-Sachs ganglioside that a major portion of ganglioside catabo­ was specifically labeled in the N-acetylneu- lism in brain proceeds via reaction 1. If raminic acid portion of the molecule.16 An this enzyme is deficient as in Tay-Sachs enzyme was detected in a number of tissues disease, then ganglioside catabolism must which catalyzes the cleavage of this N- occur exclusively via reaction 2 which is acetylneuraminyl moiety from Tay-Sachs a rate-limiting step. Furthermore, this ganglioside.17’20 The activity of this neura­ neuraminidase is inhibited by higher gan- minidase in organs and tissues obtained gliosides,10 and, therefore, the accumula­ from patients with Tay-Sachs disease was tion of ganglioside occurs on an ever- within the range of values obtained with increasing kinetic course. The absence of control human tissue specimens.18 The re­ systemic tissue involvement in the classic sults of this study prompted an investiga­ form of Tay-Sachs disease may be due to tion of the metabolism of Tay-Sachs the fact that the rapidity and quantity of ganglioside labeled in the N-acetylgalac- ganglioside turnover in peripheral tissues tosaminyl portion of the molecule.26 An is much less than that in brain and the enzyme was found in human tissues which combined activity of Tay-Sachs ganglioside catalyzes the cleavage of this portion of neuraminidase and hexosaminidase B are the ganglioside molecule.32 This enzyme is sufficient to prevent the accumulation of lacking in the tissues of patients with Tay- a significant quantity of Tay-Sachs gan­ Sach disease.18*26 Furthermore, there is gen­ glioside in the non-neural tissues. erally an increase in the activity of a residual hexosaminidase isozyme (Hex B) Practical Applications in serum and tissues obtained from Tay- D i a g n o s i s o f H o m o z y g o t e s Sachs patients and this isozyme very effectively catalyzes the hydrolysis of Knowledge of the nature of the specific asialo-Tay-Sachs ganglioside (Cer-Gle-Gal- enzymatic abnormalities in the various NAcGal), a product of reaction 2. lipid storage diseases has provided the These interwoven observations require a basis for developing important aids to practicing clinicians. Assays of lipid hydro­ word of interpretation. Since neuramini­ lase activity in washed leukocyte prepara­ dase (reaction 2) is normal and hexosami­ tions provide for the reliable identification nidase B is active and even increased in of patients with Gaucher’s disease and patients with Tay-Sachs disease, why does Neimann-Pick disease.15 Analyses of this the entire molecule of Tay-Sachs ganglio­ type are also useful for the identification side accumulate? The activity of this par­ of patients with metachromatic leukodys­ ticular neuraminidase is comparatively low trophy.25 More recently, assays with the in brain preparations.20 Since cerebral gan­ fluorogenic substrate 4-methylumbelliferyl- glioside turnover is very rapid in the neo­ /3-D-N-acetylglucosaminide (figure 2) have 290 BRADY

HEXOSAMINIDASE + H20

c h 3

4-METHYLUMBELLIFERYL-2-ACETAMIDO-2-DEOXY-/?-D-GLUCOPYRANOSIDE

Figure 2. Schematic representation of the use of 4-methylumbelliferyl-/3-D-N-acetylglucos- amine as substrate for hexosaminidase assays. been successfully employed to identify as serum (table II), circulating leukocytes Tay-Sachs homozygotes using small sam­ (table III), or cultured skin fibroblasts ples of serum.23’24 Similar tests with various (table IV). Furthermore, as clinical inves­ natural and artificial substrates are now tigators are well aware, there is usually available for the substantive diagnosis of a fairly wide variation of normal values all of the sphingolipidoses. in humans. Thus, on occasion, it may be difficult to decide that a particular indi­ D etection of C arriers vidual is truly a heterozygote. However, The second impressive practical advance for the most part, the level of activity of in this area is the recent development of the enzyme in question in the heterozygotes reliable procedures for the detection of is depressed to between 50 and 70 percent heterozygous carriers of the lipidoses.6 of normal in the leukocytes, cultured fibro­ Identification of heterozygotes is required blasts, and serum obtained from these for accurate genetic counseling. At the individuals. present time, carrier detection is the most subtle and most elusive aspect of the in­ Pre-n a ta l D iagnosis vestigation of genetic diseases because The development and application of there is only a moderate diminution of the these diagnostic tests for homozygotes and activity of the enzyme in question, and heterozygotes has maximum benefit at the that only in a few appropriate sources such present time for the prenatal monitoring ENZYME DEFECTS IN THE SPHINGOLIPIDOSES 291

T a b l e II

H exosaminidase L e v e l s i n H u m a n S e r u m

Source Enzymatic Activity

Hex A Hex B. Total nmoles/ml/hr

Normals8 990 ± 15.0 940 ± 140 1933 ± 256 L Tay-Sachs heterozygotes 548 ± 48 793 ± 79 1340 ± 90 3 Tay Sachs homozygotes 153 ± 32 3716 ± 278 3870 ± 303

Tay-Sachs variant heterozygotes2 558 570 1128 o Tay-Sachs variant homozygotes ** 55

*Too low for accurate assay.

of pregnancies at risk for lipid storage dis­ clinics throughout the world. For maximal eases.4 These determinations are carried accuracy the procedure of choice at the out when there is a positive family history present time is the determination of enzy­ or when both parents have been shown to matic activity in extracts of cultured des­ be carriers (except of course in Fabry’s quamated fetal cells obtained by transab­ disease). In time, as more facile screening dominal amniocentesis. These samples are tests are developed for detecting hetero­ generally obtained around the 14th week zygotes, an ever increasing number of of pregnancy, and a sufficient number of pregnancies will be monitored in medical cells can be grown in tissue culture in a

T a b l e III

D e t e c t i o n o f H eterozygous C a r r i e r s o p G a u c h e r ’s D i s e a s e U s i n g S o n i c a t e d W h i t e B l o o d C e l l P reparations *

Source of Leukocytes Glucocerebroside Hydrolyzed

Nanomoles/mg Percent of protein/hr of Controls

Controls (22) Mean - 6.77 ± 1.67 S.D.

Family 1 Patient (Gaucher's disease) 1.06 16.0 Father 4.51 67.0 Mother 4.25 63.0

Family 2 Patient (Gaucher's disease) 0.44 6.5 Father 3.61 53.0 Mother 3.65 54.0

* Summary of data from reference 6 . Leukocytes from small samples of venous blood were prepared and subjected to a brief period of osmotic shock.15’19 The cells were pelleted by centrifugation at 500 X g for 10 min and resuspended in an aqueous solution of sodium cholate (5 mg per ml, w/v). The suspensions were sonicated at 0° for 15 seconds, kept on ice for 1 hr, and then centrifuged at 25,000 X g for 20 min. The activity in the supernatant solutions was determined at 41° using glucocerebroside-14C as substrate. 292 BRADY

TABLE IV

D e t e c t i o n o f H eterozygous C a r r i e r s o f G a u c h e r ’s D i s e a s e i n E x t r a c t s o f C u l t u r e d S k i n F i b r o b l a s t s *

Source of Cells Glucocerebroside Hydrolyzed

Nanomoles/mg Percent of protein/hr of Controls

Family A Controls (7) Mean » 139.0 24.0 S.D. Propositus 5.0 3.6 Father 89.0 64.0 Mother 74.0 53.0 Sister 61.0 44.0

Family B Controls (5) Mean = 119.0 17.0 S.D. Female Gaucher patient 1 8.9 7.5 Female Gaucher patient 2 6.3 5.3 Obligatory heterozygote 49.0 41.0 Possible heterozygote 41.0 34.0

* Summary of data from reference 6 . The cells were grown under standard tissue culture conditions, harvested, and acetone powders were pre­ pared. The glucocerebrosidase was quantitatively extracted with 0.01 M potassium phosphate buffer solution (pH 6.0) containing 5 mg of sodium cholate per ml. Enzyme activity was assayed with glucocerebroside-uC as substrate. few weeks’ time for enzyme assays. This plement of fetal cells.29 The interpretation technique has been shown to be reliable of such determinations is a bit more un­ for the identificatoh of fetuses afflicted with certain than assays carried out on cultured Fabry’s disease,11 Niemann-Pick disease13 fetal cells. In fact, with this direct tech­ and table V, as well as Gaucher’s disease.30 nique, an erroneous diagnosis was made The prenatal diagnosis of Tay-Sachs dis­ and the pregnancy was terminated in ease has been accomplished by determining which the fetus was heterozygous for Tay- the activity of hexoasminidase A directly Sachs disease.27 Thus, a choice of prenatal on aliquots of amniotic fluid with its com­ diagnostic procedures is available and is

TABLE Y

P r e n a t a l D i a g n o s i s o f N i e m a n n -P i c k D i s e a s e

Source of Cells Enzymatic Activity

(Amniocentesis) Sphingomyelinase Glucocerebrosidase nmoles/mg protein/hr

Control 155.0 128.0

Control 74.0 77.0

At Risk 1.8 128.0

The cultured fetal cells were homogenized in 0.01 M potassium phosphate buffer (pH 6.0) containing 5 mg of sodium cholate per ml. After standing for 1 hr at 0°, the samples were centrifuged at 25,000 X g for 20 min. Enzymatic activity was assayed in aliquots of the supernatant solutions with -I4C and gluco- cerebroside-14C [as control] according to previously published procedures.15 ENZYME DEFECTS IN THE SPHINGOLIPIDOSES 293 at the discretion of the physician caring for lipids is required for such testing for Nie- the case. If monitoring of the pregnancy mann-Pick disease, Krabbe’s disease, and is undertaken too late in gestation to wait ceramide lactoside lipidosis.4’6 An artificial for cell culturing, it may be necessary to substrate has been proposed and will be base the management of the pregnancy on synthesized for an examination of its use­ the less certain direct determination of fulness for identification of patients and hexosaminidase activities on uncultured carriers of Niemann-Pick disease.6 Still amniotic fluid samples. other alternative procedures are under con­ sideration for the facile detection of pa­ Prospectus tients with Krabbe’s disease and ceramide The rapid scientific advances which have lactoside lipidosis.2 occurred in the lipidoses during the past Acknowledgment six years permit a fair degree of extrap­ Thanks are extended to Miss Jane M. Quirk for olation and fairly confident anticipation the determinations listed in table II. of future developments along several lines. References It seems likely that at least one additional lipid storage disease will be identified and 1. A g h i o n , A .: La maladie Gaucher dans l’en- fance. These de Paris, 1934. the metabolic lesion conclusively docu­ 2. B r a d y , R. O.: Genetic counseling. Med. Ann. mented. This putative disorder will prob­ D. C. 40:430-435, 1971. ably involve the accumulation of fucogly- 3. B r a d y , R. O.: The sphingolipidoses. New Engl. J. Med. 275:312-318, 1966. colipids and perhaps glycoproteins as well.12 4. B r a d y , R. O.: Prenatal diagnosis of lipid One is also tempted to speculate on the storage diseases. Clin. Chem. 16:811-815, possibility of other potential disorders of 1970. 5. B r a d y , R. O., G a l , A. E., B r a d l e y , R. M., sphingolipid metabolism. A prime candi­ M a r t e n s s o n , E., W a r s h a w , A. L., a n d date would seem to be a deficiency of gan- L a s t e r , L,: Enzymatic defect in Fabry’s glioside neuraminidase. However, a meta­ disease ceramide-trihexosidase deficiency. New Eng. J. Med. 276:1163-1167, 1967. bolic abnormality such as this may well be 6 . B r a d y , R. O., J o h n s o n , W. G., a n d U h l e n - lethal in utero. Other potential lipid storage d o r f , B . W.: Identification of heterozygous diseases might involve a deficiency of cera- carriers of lipid storage diseases. Current status and clinical applications. Amer. J . Med. midase, the enzyme which catalyzes the 51:423-431, 1971. cleavage of fatty acid from ceramide. Other 7. B r a d y , R. O ., K a n f e r , J. N., B r a d l e y , R. M., deficiencies might devolve from a de­ a n d S h a p i r o , D.: Demonstration of a de­ ficiency of glucocerebroside-cleaving enzyme ficiency of sphingosine kinase or sphingo- in Gaucher’s disease. J. Clin. Invest. 45:1112— sylphosphate aldolase, directly in­ 1115, 1966. volved in the disposal of the end product 8 . B r a d y , R. O., K a n f e r , J. N., M o c k , M . B ., a n d F r e d r i c k s o n , D. S.: The metabolism of of sphingolipid catabolism. Cases such as sphingomyelin. II. Evidence of an enzymatic this must be extremely rare; otherwise one deficiency in Niemann-Pick disease. Proc. Nat. would have anticipated their discovery by Acad. Sci. 55:366-369, 1966. 9. B r a d y , R. O., K a n f e r , J. N., a n d S h a p i r o , this time. D.: Metabolism of . II. Evi­ Concerning two other important clinical dence of an enzymatic deficiency in Gaucher’s aspects of the sphingolipidoses, diagnostic disease. Biochem. Biophys. Res. Commun. 18:221-225, 1965. skill will be improved and, in particular, 10. B r a d y , R. O. a n d K o l o d n y , E. H.: Disorders reliable procedures based on artificial sub­ of ganglioside metabolism. Progress in Medical strates for the identification of homozy­ Genetics, Vol. VIII, Steinberg, A. G. and Beam, A. G., eds., Grune and Stratton, New gotes and heterozygotes for all of these York, pp. 225-241, 1972. diseases will eventually be developed. At 11. B r a d y , R. O., U h l e n d o r f , B . W., a n d J a c o b ­ s o n , C. B .: Fabry’s disease: antenatal detec­ the present, the use of labeled authentic tion. Science 172:174-175, 1971, 294 BRADY

12. D u r a n d , P., B o r r o n e , C ., a n d D e l l a C e l l a , 24. O k a d a , S. a n d O ’B r i e n , J. S.: Tay-Sachs G.: Fucosidosis. J. Pediat. 75:665-674, 1969. disease: generalized absence of a beta-D-N- 13. E p s t e i n , C. J., B r a d y , R. O., S c h n e i d e r , acetylhexosaminidase component. Science 165: E . L., B r a d l e y , R. M., a n d S h a p i r o , D.: 698-700, 1969. In utero diagnosis of Niemann-Pick disease. 25. P e r c y , A. K. a n d B r a d y , R. O.: Metachro­ Amer. J. Hum. Genet. 23:533-535, 1971. matic leukodystrophy: diagnosis with samples 14. G a u c h e r , C. P. E.: De l’epithélioma primitif of venous blood. Science 161:594-595, 1968. de la rate. Thèse de Paris, 1882. 26. Q u i r k , J. M., T a l l m a n , J. F., a n d B r a d y , 15. K a m p i n e , J. P., B r a d y , R. O., K a n f e r , R. O.: The preparation of trihexosyl- and J. N., F e l d , M., a n d S h a p i r o , D.: Diagnosis tetrahexosyl specifically labeled in of Gaucher’s disease and Niemann-Pick dis­ the N-acetylgalactosaminyl moiety. J. Labeled ease with small samples of venous blood. Compounds, in press. Science 155:86-88, 1967. 27. R a t t a z z i , M.: Communicated at the Con­ 16. K o l o d n y , E. H., B r a d y , R. O., Q u i r k , J. M., ference on Antenatal Diagnosis, Chicago, IL, a n d K a n f e r , J. N. : Preparation of Tay-Sachs June 12, 1970. ganglioside labeled in the sialic acid moiety. 28. S a c h s , B.: On arrested cerebral development J. Lipid Res. 11:144-149, 1970. with special reference to its cortial pathology. 17. K o l o d n y , E. H., B r a d y , R. O., Q u i r k , J. M., J. Nerv. Ment. Dis. 14:541-553, 1887. a n d K a n f e r , J. N.: Studies on the metabolism 29. S c h n e c k , L ., V a l e n t i , C., A m s t e r d a m , D., of Tay-Sachs ganglioside. Fed. Proc. 28:596, F r i e d l a n d , J., A jd a c h i, M., a n d V o l k , B. W.: 1969. Prenatal diagnosis of Tay-Sachs disease. Lancet 18. K o l o d n y , E. H., B r a d y , R. O., a n d V o l k , 1:582-583, 1970. B. W.: Demonstration of an alteration of 30. S c h n e i d e r , E. L., E l l i s , W. G., B r a d y , R. O., ganglioside metabolism in Tay-Sachs disease. a n d E p s t e i n , C. J.: Pediatric Res., in press. Biochem. Biophys. Res. Commun. 37:526-531, 31. S h a w , R. F. a n d S m i t h , A. P.: Is Tay-Sachs 1969. disease increasing? Nature 224:1214-1215, 19. K a m p i n e , J. P., B r a d y , R. O., Y a n k e e , R. A., 1969. K a n f e r , J. N., S h a p i r o , D., a n d G a l , A. E.: 32. T a l l m a n , J . F., J o h n s o n , W. G., a n d B r a d y , Sphingolipid metabolism in leukemic leuko­ R. O.: Enzymatic hydrolysis of Tay-Sachs cytes. Cancer Res. 27:1312-1315, 1967. ganglioside in isolated from normal 20. K o l o d n y , E. H., K a n f e r , J. N., Q u i r k , J. M., and Tay-Sachs brain tissue. J . Clin. Invest., a n d B r a d y , R. O.: Properties of a particle- in press. bound enzyme from rat intestine that cleaves 33. T a y , W.: Symmetrical changes in the region sialic acid from Tay-Sachs ganglioside. J. Biol. of the yellow spot in each eye of an infant. Chem. 246:1426-1431, 1971. Trans. Ophthal. Soc. U.K. 1:55—57, 1881. 21. L o e b , H . , Tondeur, M ., J o n n i a u x , G., 34. T e r r y , R. D. a n d W e i s s , M.: Studies in Tay- M o c k e l -P o h l , S ., a n d V a m o s -H u r w i t z , E . : Biochemical and ultrastructural studies in a Sachs disease. II. Ultrastructure of the cere­ case of mucopolysaccharidosis “F” (Fucosi­ brum. J. Neuropath. Exp. Neurol. 22:18-55, dosis). Helv. Paediat. Acta 5:519-537, 1969. 1963. 22. M e h l , E. a n d J a t z k e w i t z , H . : Evidence for 35. T h a n n h a u s e r , S. J.: Lipidoses: Diseases of a genetic block in metachromatic leuko­ Cellular Lipid Metabolism, H. A. Christian, dystrophy (M L). Biochem. Biophys. Res. H. A., ed., Oxford Press, New York, 1950. Commun. 19:407-411, 1965. 36. T h u d i c u m , J. L. W.: Die chemische Konstitu­ 23. O ’B r i e n , J. S., O k a d a , S., C h e n , A., a n d tion des Gehirns des Menschen und der Tiere, F e l l e r u p , D. L.: Tay-Sachs disease. Detec­ Pietziker, Tubingen, 1901. tion of heterozygotes and homozygotes by 37. T r a m s , E. G. a n d B r a d y , R. O.: Cerebro- serum hexosaminidase assay. New Eng. J. Med. side synthesis in Gaucher’s disease. J. Clin. 283:15-20, 1970. Invest. 39:1546-1550, 1960.