Adult onset motor neuronopathy in the juvenile type of hexosaminidase A and B deficiency

Journal of the Neurological Sciences, 1988, 87:103-119 Elsevier

103

JNS 03050

Adult onset motor neuronopathy in the juvenile type of hexosaminidase A and B deficiency Michael Rubin 1, George Karpati 1, Leonhard S. Wolfe 1, Stirling Carpenter l, Maris H. Klavins 2 and D o n J. Mahuran 2 1Department of Neurology and Neurosurgery, McGill University, and the Montreal Neurological Institute, Montreal, Quebec (Canada), and 2Research Institute, The Hospital for Sick Children, and Department of Clinical Biochemistry, Universityof Toronto, Toronto, Ontario (Canada) (Received 23 November, 1987) (Revised, received 30 May, 1988) (Accepted 31 May, 1988)

SUMMARY

Two sisters presented with progressive muscle cramps, as well as wasting and weakness of the legs with onset after age 20. They also showed intention tremor of the upper extremities and dysarthria starting during the In'st decade. The older patient also had fasciculations; the younger, hyperreflexia. Total plasma beta-hexosaminidase (Hex) activity with 4-methylumbelliferyl-acetyl-glucoszmine as substrate was reduced to 1.4 % and 2.7~o of the control in the 2 patients, respectively. Hex A activity measured by 4-methylumbelliferyl-N-acetylglucosamine-6-O-sulphate as substrate was 9.9~o and 12.8% of the mean control value in the 2 patients, respectively. Hex B activity was undetectable in both patients. Leukocyte total Hex activity was 7-8~o of normal; residual Hex A activity in the 2 patients was 17.89/o and 16.3~o of normal controls, respectively. Fibroblastic residual Hex A activity in the 2 patients was 9.6% and 22~o of normal mean value, respectively. Appendiceal ganglion cells contained membranous cytoplasmic bodies in the younger patient. Thin layer chromatography of the appendiceal extract from one patient (111/2) showed a marked increase of GM 2 ganglioside, and some increase of GM 3 ganglioside. Northern blots performed on fibroblast cell lines from both patients for the demonstration of alpha and beta locus messenger RNA showed no difference between patients and control. These patients have a rare form of adult-onset progressive motor neuron disease presumably due to abnormal beta subunits, causing severe deficiency of both Hex A and Hex B. The phenotypic expresCorrespondence to: Dr. George Karpati, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, H3A 2B4, Canada. 0022-510X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

104 sion of this disease is similar to motor neuron disease due to alpha locus mutations, which suggests that the Hex A deficiency, even though only a partial one, may be the important pathogenic factor.

Key words: Motor neuronopathy; Hexosaminidase deficiency; Northern blot analysis

INTRODUCTION Hexosaminidase A and B (Hex A and Hex B) deficiency typically presents as infantile Sandhoff disease. However, infrequent atypical presentations have been reported, including juvenile or adult onset cerebellar ataxia, and/or motor neuron disease (MND) (Barbeau et al. 1984; Adams and Green 1986; Cashman et al. 1986; Federico et al. 1986; Goldie et al. 1977; Johnson 1981; Johnson et al. 1983; MacLeod et al. 1977; Oonk et al. 1979; Van Hoof et al. 1972). The latter variety is extremely rare, having been described in only 3 patients with involvement of both the upper and lower motor neurons (Federico et al. 1971; Oonk et al. 1979). We describe 2 such additional cases involving a pair of sisters who developed adult-onset, progressive upper and lower MND as a prominent feature of their neurologic illness, which started with tremor during the first decade.

MATERIALS AND METHODS For hexosaminidase (Hex) assays, blood samples were processed as follows: To 10 ml of heparinized blood, 2 ml of a 6% dextran/saline/heparin (6000 units) solution was added. The tubes were gently agitated and the red cells allowed to sediment for 45 rain. The supcrnatant was carefully removed, centrifuged for 10 rain at 3000 rpm and the supernatant discarded. Any remaining red cells in the pellet were removed by adding 5 ml ice-cold distilled water gently mixed and after 45 see 5 ml of 1.8% saline added. The lysed suspension was transferred to another tube and centrifuged at 4 °C to sediment the leukocytes which were then resuspended in saline and either assayed immediately or stored at - 20 °C until assay. Total Hex activity was determined in serum and leukocytes by a fluorometric technique using the artificial substrate of 4-methyl-4-umbdliferyl-2-acetamido-2-deoxyfl-D-glucopyranoside (Kaback et al. 1971). Hex A activity was determined by the heatinactivation method (Kaback et al. 1971), as well as by the use of the substrate 4-methylumbdliferyl-N-acetylglucosamine-6-sulphate or MUGS (Ben-Joseph et al. 1985) obtained from the Research Development Corporation of the Hospital for Sick Children, Toronto, Ont., Canada. A fibroblast cell line was established and Hex activities were assayed as described (Kaback et al. 1971). In addition, Hex A and Hex B were isolated by chromatofocussing

105 columns and their activities were measured as described (Lowden et al. 1973). Thin layer chromatography of the urine for oligosaccharide screening was performed using silica gel 150 ,~ K5 plates (Chromatographic Specialties, Brockville, Ont., Canada). 35 and 75/~1 of patients' untreated urine were spotted along with 35 #1 of normal control, 35 #1 of a proven infantile Sandhoff sample and 5 #1 of lactose and N-acetylneuraminic acid as standard (McLeod et al. 1977). The plate was run overnight in butanol/acetic acid/H20 mixture (50 : 25 : 25, v/v). The plate was then dried and sprayed with orcinol or resorcinol to stain the neutral sugar or sialic acid containing compounds. Thin layer chromatography of appendiceal tissue Folch extract of patient III-2 and controls for the demonstration of gangliosides was done as described (Parnes et al. 1985). For mRNA analysis, total fibroblast RNA was isolated by guanidinium isothiocyanate extraction followed by CsC1 centrifugation (Chirgwin et al. 1979). RNA hybridization was performed by the technique of Maniadis et al. (1982) using one of three 32p-labeled probe fragments: (i) the complete 1.7 kb insert from pHexB 1, a cDNA clone coding for the pre-beta-chain of hexosamim'dase (O'Dowd et al. 1986), (ii) the complete 1.8 kb insert from pHexA49, a cDNA clone coding for the pre-alpha-chain of hexosaminidase (Korneluk et al. 1986); (iii) the complete 1.9 kb insert from pHUGF, a cDNA clone coding for beta-glucuronidase (Oshima et al. 1987).

CASE REPORTS AND RESULTS The 2 sisters, children of non-consanguinous French Canadian parents, belong to the pedigree shown in Fig. 1 and are identified as patients III-2 and III-5. No other family members were available for examination, but on the basis of the history obtained from the patients, all other family members were considered neurologically normal. Patient III-2 is a 39-year-old woman born al~cr normal gestation and defivery, and was well until age 6 when she developed intention tremor and dysarthria which were mild and nonprogressive. Nevertheless, she completed high school with average grades and subsequently she worked as a waitress. At age 20 she noted progressive limb weakness, cramping and twitching of her muscles which were exacerbated by exercise. At age 39 she had mild to moderate weakness of hip flexors; other muscles were not weak on formal testing, although there was some atrophy of quadriceps and leg muscles (Fig. 2). There was spasticity at the knees and ankles. Tendon reflexes were moderately brisk throughout. She also showed myoclonic and choreiform movements of the right forearm at rest, and dysmetria of the right upper extremity. Plantar responses were flexor. Her overall mental status examination revealed no appreciable abnormalities.

m" ~Z 1

2

3

4

5

6

8

g

10

Fig. 1. Pedigree of family P. Probands are represented in black symbols.

106

Fig. 2. Patient 1II-2. Note proximal muscle atrophy of lower limbs.

Patient III-5, 36 years old, was also born after normal gestation and delivery. She was well until age 5, when she also developed intention tremor and dysarthria. In addition, she was a "slow learner" but completed grade 10. Subsequently, she worked as a sales clerk in a furniture store and got married. At age 22, she noted progressive leg weakness, muscle cramps and twitching. At age 32, she had the first of 3 episodes of acute paranoid psychosis treated successfully with phenothiazines. At age 34 and 36 two similar episodes occurred; both responded well to phenothiazines. At age 36, she showed a normal mental status except for mildly impaired recent and remote memory. There was marked muscle atrophy of the lower limbs both proximally and distally (Fig. 3). However, on formal testing of muscle strength, she had no appreciable weakness, but she could not get up from a deep-seated position or walk on tiptoes. She showed spasticity at the major limb joints, and moderately increased symmetrical tendon reflexes in arms and legs. Plantar responses were flexor. Gait was broad-based and somewhat spastic. C o m p l e t e l a b o r a t o r y e v a l u a t i o n w a s p o s s i b l e o n l y o n p a t i e n t 111-5. T h e following tests w e r e n o r m a l : c o m p l e t e b l o o d c o u n t , S M A C - 1 6 (including c r e a t i n e k i n a s e activity), urinalysis, c h e s t X-ray, E C G , skull r a d i o g r a p h , E E G , s e r o l o g y by v e n e r e a l d i s e a s e r e s e a r c h l a b o r a t o r y test ( V D R L ) . E l e c t r o m y o g r a p h y s h o w e d a n e u r o p a t h i c m o t o r unit

107

Fig. 3. Patient III-5. Note atrophy of lower leg muscles. profile in several muscles. Motor and sensory nerve conduction velocities were within normal range. Right gastrocnemius muscle biopsy showed denervation atrophy with evidence of reinnervation. Right sural nerve biopsy was normal. No abnormal inclusions in Schwann cells were noted. In both sisters, assays showed severe reduction of total Hex activity in plasma, in cultured fibroblasts and in leukoeytes. Hex activities in the patients and members of the patients' pedigree are shown in Table 1. When Hex A and Hex B were isolated by a chromatofocussing column and their activities were measured using the synthetic substrate (Kaback et al. 1971; Lowden et al. 1973), residual Hex A activity in fibroblasts was 22 ~ and 11 ~o of normal, in the sisters 111-2 and 111-5, respectively. By using a specific substrate (MUGS), for Hex A activity, residual enzyme activity ranged from 9.6 to 22~o of the normal mean values in sera, leukocytes and fibroblasts (Table 2). Hex activities suggested that 3 of the patients' siblings (111-4, 11I-8 and III-10) and the son of patient 111-2 (IV-l) are heterozygotes.

1180_+870 560-3120

Normal subjects Mean (n = 40) +SD Range 62.6+ 1 0 . 2 51-75

100 100 72.7 75 61.1 81.2 74.8 73.2

37.4_+9.6 25-49

0 0 27.3 25 38.9 18.8 25.2 26.8

2400_+ 1120 804-3290

160.6 167.5 1380.3 761.1 762.2 1341.6 885 1571.7 602.6 842.1 1259.9

70.1 + 8.6 57-85

97.3 96.4 71 74.7 75.4 60.7 73.8 64.3 72.2 76.5 74.3

% Hex A

29.9+ 7.8 15-43

2.7 3.6 29.0 25.3 24.6 39.3 26.2 35.7 27.8 23.5 25.7

% Hex B

3430+ 1800 2600-12,000

128 55

Total (nmol/mg protein/h)

65.3 + 10.1 68-87

100 c 100 ¢

% Hex A

Fibroblast cell lines

34.7+4.3 13-39

0 0

~o Hex B

a Hexosaminidase A determined by heat inactivation method at 52 °C for 2 and 3 h. b Refers to members shown in Fig. 1. c Residual Hex A activity (22% and 11% of normal in patient Ill-2 and III-5, respectively) was present when the isoenzymes were first separated by chromatofocussing column and then assayed. Residual Hex A activity was also measured by the 4-methylumbelliferyl-N-aeetylglucosamine-6-sulphate substrate and the results are shown in Table 2. Hex A = hexosaminidase A; Hex B = hexosaminidase B.

1.6 3.2 1056 600 1068 588 776 1032

1II-2b III-5 11-12 I1-13 11I-4 111-8 III-10 III-12 III- 13 IV- 1 IV-2

Total (nmol/mg protein/h)

~o Hex B

Total (nmol/ml/h)

% Hex A

Leukocytes

Plasma

H E X O S A M I N I D A S E ACTIVITIES IN PROBANDS A N D MEMBERS OF FAMILY pa

TABLE 1

Go

109 TABLE 2 H E X O S A M I N I D A S E A ACTIVITIES IN P R O B A N D S A N D M E M B E R S O F F A M I L Y pa Plasma

Leukocytes

Fibroblast cell lines

III-2 b III-5 II-12 II-13 11I-4 111-8 III-10 Ili-12 III-13 IV-1 IV-2

17 22 132 97 109 92 109 136

59 54 137 142 137 134 67 151 142 253 125

73 173 -

Mean of normal subjects

189 + 35 (115-273) (n = 27)

379 + 93 (279-464) (n = 6)

767 + 51 (691-801) (n = 4)

a Hex A activity was determined by using 4-methylumbelliferyl-N-acetyl-glucosamine-6-sulphate as substrate (Ben-Joseph et al. 1985) and expressed as nmol/ml of plasma/h or nmol/mg of protein/h. b Refers to members shown in Fig. 1.

Fig. 4. Thin layer chromatogram of urine stained with resorcinol for oligosaccharides. Lane A is normal control. Lane C is from a child with infantile Sandhoffdisease and shows two prominent spots representing hexa- (small arrows) and heptasaccharides (large arrows) that are not present in the normal control. Lane B is from patient III-2; lane D from patient III-5. Arrows indicate presence of the hexa- and heptasaccharides, though less prominent than in infantile Sandhoff patient. Resorcinoi staining was negative indicating absence of sialic acid. Lane E is lactose standard.

110

Fig. 5. Conspicuous storage deposits fill the cytoplasm of most of the neurons of Auerbach's plexus in the appendix of patient 111-5. Paraphenylene diamine, phase optics x 540.

Fig. 6. This electron mlcrograph shows a membranous cytoplasmic body from a neuron in Auerbach's plexus of patient 111-5. x 50000.

111 Urine thin-layer chromatography (TLC) stained for oligosaccharides indicated the abnormal presence of hexa- and heptasaccharides in both patients (Fig. 4); the identity of these oligosaccharides was confirmed by standards isolated from urine of classical Sandhoff patients (Ng Ying Kin and Wolfe 1978, 1980). Diagnostic biopsy of the appendix performed on patient 111-5 revealed that many neurons of the myenteric plexus were distended and contained osmiophilic bodies (Fig. 5). Electron microscopy showed that these neurons contained numerous typical membranous cytoplasmic bodies (MCB) (Fig. 6). Thin layer chromatography revealed a marked excess of GM2 ganglioside and a mild excess of G M 3 ganglioside in the appendix of patient II1-5 (Fig. 7). The gangliosides were identified by the mobility (Rf) of standards according to the method of Ledeen and Yu (1978). Northern blots performed on fibroblast cell lines from both patients for the alpha and beta locus message using cDNA probes are shown in Fig. 8. There was no difference between the patients and control.

GM3 GM2 GM1

TS EP PA Normal Gangl. Brain Appx. Appx. Appx. Std. Fig. 7. Thin layer chromatographof appendiceal Folch extracts for the demonstration of GM~, GM2 and GM3gangliosides.In patient III-5 (EP) there is a marked increaseofGM2 ganglioside and a mild increase of GM3 ganghoside in relation to normal control. A Tay-Sachs brain extract (TS) and the appendix of an adult onset Hex A deficiencypatient (PA) are also shown. The fight lane contains the ganglioside standards.

112

13-chain 1 2 3

1

2- 3

28s~

13- G ~ r o n i d a s e

28S

18s

Fig. 8. Northern blot for beta locus messenger RNA (upper left panel) and alpha locus messenger RNA (upper fight panel). Lane 1 is normal control; lane 2 is from patient III-2; lane 3 from patient III-5. Lower panels are corresponding Northern blots for bcta-glucuronidase messenger RNA, used for validation of the hybridization technique.

DISCUSSION

Clinical picture The two patients described in this report represent a rare phenotypic variant of Hex A and B deficiency in which adult-onset upper and lower motor neuron involvement accompanied by muscle cramps is a prominent feature. Although the onsot of neurological symptoms and signs was in the first decade, upper and lower motor neuron involvement did not become apparent until adult age.

113 Of the 9 reports on atypical Hex A and B deficient patients, 6 presented in childhood with clinically prominent motor neuron involvement (Table 3). Only 3 reports deal with adult-onset motor neuronopathy associated with Hex A and B deficiency (Table 4). These reports include a total of 6 patients, of which 3 had combined upper and lower motor neuron disease, 2 had only lower motor neuron disease, and 1 had only minimally evident upper motor neuron involvement. Our 2 cases can be added to those with both upper and lower motor neuronopathy. In addition to motor neuronopathy, other CNS manifestations are usually present, similar to those seen with Hex A deficiency, and these should trigger suspicion for the possibility of underlying Hex deficiency (Parries et al. 1985; Harding et al. 1987). These features include psychotic episodes, cerebellar deficits, and movement disorders. Psychotic episodes have previously been noted in Hex A but not in Hex A and B deficiency (Navon et al. 1986; Parries et al. 1985). Our patient 111-5 developed recurrent acute onset psychotic episodes early in her fourth decade. Cerebellar manifestations have been the most frequent presentation of adult onset Hex A and B deficiency (Johnson 1981, 1983; Willner et al. 1981); patient 111-5 had a broad-based gait, 111-2 had fight arm dysmetria and both patients had intention tremor. Movement disorders such as tremor, dystonia and choreiform jerks have been reported in Hex A deficiency (Menkes et al. 1971; Meek et al. 1984; Parnes et al. 1985; Suzuki et al. 1970). Patient 111-2 was minimally affected with choreiform movements of the right forearm. Thus, the similarity of the phenotypic presentation in Hex A and B deficiency to that ofHex A suggests that in this disease, deficiency ofHex A is probably the significant pathogenic factor.

Hexosaminidase genetics Hexosaminidase is a lysosomal enzyme involved in the degradation of several natural substances including GM 2 ganglioside, asialo GM 2 ganglioside GA2, globoside and oligosaccharides (Bach and Suzuki 1975; Seyama and Yamakawa 1974; Tsay and Dawson 1976). The full spectrum of Hex activity requires the presence of at least 3 gene loci (activator, alpha and beta) that code for an activator protein, alpha subunits and beta subunits, respectively (Beutler 1979). Classic Tay-Sachs disease involves a mutation at the alpha locus that leads to impaired alpha subunit formation. This leads to deficiency of both Hex A and Hex S, whereas Hex B, inasmuch as it contains no alpha subunits, has normal activity. Sandhoff disease represents a mutation of the beta locus interfering with beta subunit formation and thus causing deficient activity of both Hex A and Hex B. Hex S would be unaffected (Johnson 1983). Thus, our patients who were severely deficient in both Hex A and Hex B would appear to have impaired beta subunit formation that may be due to an abnormality of the beta locus gene on chromosome 5. Alternatively, an abnormality may be present anywhere along the posttranscription pathway involved in beta subunit formation. In order to address the possibility that our patients had a mutation at the beta locus gene, a Northern blot analysis of the beta locus messenger RNA (mRNA) was performed. During formation of the beta subtmit, the beta locus is fwst transcribed into a pre beta-polypeptide mRNA (Hasilik and Neufeld 1980; Tsui

1

1

1

1 3

1

Van Hoof et al. (1972)

MacLeod et al. (1977)

Goldie et al. (1977)

Johnson et al. (1983)

Adams et al. (1986)

C a s h m a n et al. (1986)

7

2,2,3

2

5

5

1.5

Age of onset (years) of neurological signs

U M N = upper motor neuron; L M N = lower motor neuron.

No. of cases

Authors

10

2-4

2

6

8

3

Age of onset (years) of motor involvement

Dystonia Dysmetria Dementia Tremor Seizures Psychomotor retardation Tremor Peripheral neuropathy

UMN + LMN U M N in 2 U M N + L M N in 1

UMN + LMN

Ataxia Dysmetria Dementia

Myoclonus Akinetic attacks Startle reaction

Other neurologic involvement

UMN + LMN

UMN

UMN ( + ? LMN)

Type of motor involvement

CASES OF H E X A A N D B D E F I C I E N C Y A S S O C I A T E D W I T H P R O G R E S S I V E M O T O R N E U R O N O P A T H Y ( C H I L D H O O D O N S E T )

TABLE 3

2

2

2

Oonk et al. (1979)

Barbeau et al.(1984)

Fedefico et al.(1986)

37,20

6

20

Age of onset (years) of neurological sings

U M N = upper motor neuron; L M N = lower motor neuron.

No. of cases

Author

37,20

22

20

Age of onset (years) of motor involvement

UMN + LMN

LMN

UMN; UMN + LMN

Type of motor involvement

CASES OF H E X A A N D B D E F I C I E N C Y A S S O C I A T E D W I T H P R O G R E S S I V E M O T O R N E U R O N O P A T H Y ( A D U L T O N S E T )

TABLE 4

Autonomic abnormalities Ataxia Tremor Paresthesiae

Tremor Ataxia Dystonia Myoclonus

"Choreic unrest" Ataxia Dysmetria

Other neurologic involvement

116 et al. 1983). This mRNA is translated into the pre-beta-polypeptide which is subsequently cleaved into a beta a and a betab by disulfide bonds to form the ultimate beta subunit (Mahuran and Lowden 1980; Mahuran et al. 1982). In the Northern blot analysis, a complementary DNA (eDNA) probe for the pre-beta-polypeptide mRNA is used to hybridize with any pre-beta-polypeptide message that is present (O'Dowd et al. 1985). As shown in Fig. 8, the Northern blot result was no different in our two patients from that of normal controls, indicating that a major deletion was not present at the beta locus. It is still possible that a point mutation that is not detectable by hybridization may exist or, that an abnormality in beta subunit synthesis or processing is located further along the transcription-translation pathway.

The enzyme defect Our patients had severe Hex A and Hex B deficiency in serum, leukocytes and fibroblasts. While the heat inactivation method of assay revealed no appreciable Hex A or Hex B activity, using the more precise technique of chromatofocussing column elution, or a specific substrate (MUGS) for enzyme assays, 9-22% residual Hex A activity was present in plasma, leukocytes and fibroblasts. The residual Hex A activity could imply that the presumably intact subunit in Hex A is capable of producing some catalytic activity despite the deficient or defective subunits. Deficiency ofHex A and Hex B causes characteristic biochemical and pathologic abnormalities which are demonstrated in our patients. Gangliosides, glycosphingolipids which contain sialic acid in their oligosaceharide chain, are found in most cell types in the body but have their highest concentration in the brain, where they are located primarily in membranes of nerve endings (Norton and Podluso 1971). Some appear to originate in the cell body and are transported to the nerve endings (Forman and Ledeen 1972). Gangliosides are catabolized by the stepwise removal of sugar molecules from the nonreducing end of the oligosaccharide chain by several exohydrolases (Ledeen and Yu 1973; Ohman et al. 1970; Vaes 1973). If even one of these enzymes is deficient, a block occurs at that point in the pathway and accumulation of proximal metabolites results. Hex A and Hex B are two important exohydrolases and their absence produces neuronal lipid storage secondary to abnormal accumulation ofganglioside GM 2, as well as storage and spillage into the urine of structurally related oligosaccharides (Strecker and Montreuil 1971, 1979). The GM 2 ganglioside accumulation produces MCB, which are found in cortical, cerebellar, spinal and autonomic neurons. They are not specific for Hex deficiency states but are seen in other ganglioside storage diseases. The oligosaceharides that accumulate and spill over into the urine derive from degradation of glycoproteins because of Hex B deficiency (Ng Ying Kim and Wolfe 1980; O'Brien 1983). In Tay-Saehs disease, where there is sufficient Hex B present, they do not accumulate and, therefore, are not found in the urine. The diagnosis An abnormality of beta subunit formation causing Hex A and B deficiency is a rare but definite cause of adult onset motor neuronopathy. Suspicion of this possibility

117

should arise if the patient also shows other neurologic abnormalities not usually associated with idiopathic motor neuron disease. These include tremor, dysmetria, dementia, and psychosis. The diagnosis can be verified by Hex enzyme assay on serum, leukocytes and fibroblasts. The overall clinical picture of a suspected beta locus mutation is similar to that of alpha locus mutation (Parnes et al. 1985). Inasmuch as the common denominator between these two possibilities is deficiency of Hex A, this suggests that Hex A deficiency is probably the important pathogenic factor in both instances. The heterozygous or carder state of late-onset Hex A and B deficiency deserves comment. Hex A and B deficiency is an autosomal recessive disorder and thus, the parents of our patients ought to be heterozygous carders of the gene defect. The Hex A and Hex B activity in 2 of the patients' siblings and in one of the patients' sons also appears to be in the heterozygote range for Tay-Sachs disease.

ACKNOWLEDGEMENTS

Supported in part by the Medical Research Council of Canada, the Muscular Dystrophy Association of Canada and The Killam Chair Fund of the Montreal Neurological Institute (G.K.) and by a special grant from the Medical Research Council of Canada to L.S.W., an MRC Career Investigator, and by MRC Program Grant PG-4 to D.J.M.

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