BIlNS
Child's Nerv Syst (1992) 8:468-470
9 Springer-Verlag 1992
Biochemical diagnosis of Canavan disease G. Bartalini, M. Margollicci, P. Balestri, M.A. Farnetani, M. Cioni, and A. Fois Istituto di Clinica Pediatrica, Universitfi degli Studi di Siena, Via P.A. Mattioli, 10, 1-53100 Siena, Italy Received September 23, 1991
Abstract. C a n a v a n disease (CD) is a rare a u t o s o m a l recessive disorder characterized by m a c r o c e p h a l y and progressive l e u k o d y s t r o p h y . U p to n o w biopsy or n e c r o p s y were required to define the diagnosis. Recently the disease has been related to N-acetylaspartic aciduria and deficiency o f aspartoacylase, an e n z y m e possibly involved in the myelin synthesis. These biochemical findings have p r o v i d e d a diagnostic m a r k e r for the disease. We report a new case o f infantile C D in which the d e m o n stration o f N-acetylaspartic aciduria and a m a r k e d deficiency o f a s p a r t o a c y l a s e activity confirmed the diagnosis. Key words: C a n a v a n disease - N-acetylaspartic aciduria A s p a r t o a c y l a s e deficiency
C a n a v a n disease (CD) is a rare a u t o s o m a l recessive disorder characterized by degeneration o f cerebral white m a t ter. It has been f o u n d to be m o r e c o m m o n a m o n g Ashkenazi Jews [14]. Three variants o f the disease, congenital, infantile, and late onset, have been reported [I]. I n the m o s t frequent form, the infantile, s y m p t o m s occur within the first few m o n t h s o f life and include failure o f m o t o r and m e n t a l development, progressive spasticity, blindness and optic a t r o p h y , a n d m a c r o c e p h a l y . N e u r a l imaging studies d e m o n s t r a t e symmetrical demyelination a n d l e u k o d y s t r o p h y [10]. T h e m a i n pathological features o f the brain consist o f s p o n g y degeneration. Electron mic r o s c o p y shows vacuoles in the myelin sheaths, swelling o f astrocytes, and elongation o f m i t o c h o n d r i a . U p to n o w brain biopsy or n e c r o p s y has been required to c o n f i r m the diagnosis and differentiate the disease f r o m other types o f m a c r o c e p h a l i c l e u k o d y s t r o p h y (e.g., Alexander disease). H o w e v e r , recent studies have reported increased a m o u n t o f N-acetylaspartic acid in urine, plasma, and C S F and a deficiency o f aspartoacylase activity in fibroblasts f r o m patients in which the diagnosis o f C D h a d previously been c o n f i r m e d by m o r p h o l o g i c a l Correspondence to." A. Fois
e x a m i n a t i o n o f the brain [3, 8]. These observations have p r o v i d e d a biochemical diagnostic m a r k e r for the disease. We describe here a new case o f m a c r o c e p h a l i c l e u k o d y s t r o p h y in which N-acetylaspartic aciduria and aspartoacylase deficiency have been demonstrated.
Case report The patient, a girl, was born at term by cesarean section after an uneventful pregnancy. The birth weight was 3400 g; length and head circumference are not known. Her parents are not related but both families come from the same small village (2500 inhabitants). One older sister is healthy. From the patient's 15th day of life, tremors and progressive muscular rigidity were noted. At 5 months severe neuromotor delay was observed. At that time she did not show any interest in her surroundings and head control was absent. The head circumference at 6 months of age was 45 can (above the 95th percentile). A cranial CT scan at this time revealed demyelination. At the age of 21 months, when she was referred to our hospital, physical examination revealed macrocephaly (head circumference 51 cm), nystagmus, no visceromegaly, axial hypotonia with absent head control, limb hypertonia with brisk deep tendon reflexes, bilateral foot clonus, and Babinski's sign. She had no spontaneous movements, showed very little interest in her surroundings, did not grasp objects, and was not able to fix her eyes; language was absent. At the age of 2 years and 10 months (last observation) her clinical condition had further deteriorated. Her head circumference was 52 cm. Interest in her surroundings was completely lacking and she appeared to be blind. The ophthalmologic examination showed pale discs bilaterally. Cerebral magnetic resonance imaging at 21 months revealed an increased signal in T2-weighted images, indicating diffuse symmetrical demyelination and moderate widening of the lateral and III ventricles. These findings were more evident at 2 years and 10 months. EEG showed alternate slow and fast rhythms and absence of sleep spindles. Blood ammonia, lactate, aminoacids, cortisol, very long chain fatty acids and urinary glycosaminoglycans and oligosaccharides were normal. The cerebrospinal fluid protein level was also normal. Nerve conduction velocity was within the normal limits. The following acid hydrolases on leukocytes and/or fibroblasts were normal: fi-hexosaminidase, galactosylceramidase, arylsulfatase A, fi-galactosidase. The ultrastructural examination of skin biopsy was normal. The study of urinary organic acids revealed an high excretion of N-acetylaspartic acid (1026 mmol/mol creatinine). The aspartoacylase activity test was performed as follows.
469 Table 1. Aspartoacylase activity in cultured fibroblasts
Aspartoacylase activity (U/rag protein) Patient
0.11
Father Mother Sister Controls (n = 7)
0.86 1.44 1.32 2.91 + 1.83
Residual activity (% of control) 3.8
29.5 49:5 4514 100.0
Materials and methods
Skin fibroblasts from the patient, her parents, her sister, and seven controls were utilized for the enzymatic assay of aspartoacylase. The cells were cultured in MEM 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) containing 10% fetal calf serum, penicillin, streptomycin, and L-glutamine. Aspartoacylase activity was determined according to the procedure described by Matalon et al. [8]. Confluent cells were harvested by scraping, suspended in water, and disrupted by sonication for 3 x 10 s. After centrifugation at 10000 g x 30 min, the supernatant aliquots were incubated for 5 h at 37 ~ with and without the substrate 1.7 mmol/1 N-acetyl-L-aspartic acid in 0.05 Tris-HC1 buffer pH 8. Each incubation mixture contained 350-450 gg protein [7]. The reaction was terminated by boiling for 3 min. Precipitated proteins were removed by centrifugation at 11000 rpm and asparrate content was assayed spectrophotometrically at 340 nm after addition of 2-oxoglutarate (t .4 retool/l), NADH (0.148 nmol/1) and malate dehydrogenase and aspartate aminotransferase in excess. A slight Iinear decrease of adsorbance for conversion of NADH to NAD occurred which was recorded for 5 min. The adsorbance measurements were converted to enzyme activities by using the molar extinction coefficient for NADH 6.3 x 1000.
Results
Aspartoacylase activity in control fibroblasts was 2.9l + 1.83 U/rag protein. In the patient's fibroblasts the enzyme activity was 0.11 U / m g protein, 3.8% of the mean control value. The activity of the same enzyme in the parents' and sister's fibroblasts was less than 50% of the control value (Table 1). Discussion
N-acetylaspartic aciduria was initially described by Kvittingen et al. in 1986 [6] in a 6-year-old b o y with macrocephaly, spasticity, p s y c h o m o t o r retardation, and leukodystrophy. Afterwards this biochemical change was observed in four other patients with similar clinical findings [2, 4]. Matalon et al. [8] and Echenne et al. [3] independently reported the association between N-acetylaspartic aciduria due to aspartoacylase deficiency and C D proven by brain biopsy. This correlation has since been demonstrated in other cases of C D and the biochemical defect is currently considered to be specific to the disease [9, 12, 151. Synthesis of N-acetylaspartic acid has not been found to occur in any organ other than the central nervous system, where it is mainly localized in the neurons [11]. To date, the true functional role of this c o m p o u n d is not
yet understood. A remarkable increase of N-acetylaspartic acid has been observed during brain m a t u r a t i o n in animals, and an increase in the concentration has been also demonstrated in h u m a n brain during early life [5]. This observation has suggested that this substance m a y have a role in the myelination process, and it is possible that a defect in its metabolism determines an abnormal myelinization and spongy degeneration in CD. It has been proposed in fact that N-acetylaspartic acid is an essential; factor for the conversion of lignoceric acid to cerebronic acid, a precursor of ceramide, Which is one Of the major lipid components of myelin [13]. Another mechanism for the pathogenesis of C D m a y be the accumulation :of N-acetylaspartic acid in brain::tissue, result: ing i n m y e l i n damage and subsequent spongy degenera: tion [8]. :: In our case, the clinical presentation, which includes progressive and severe mental retardation with early onset, macrocephaly, and leukodystrophy, is similar to that of the infantile type of CD. The demonstration of increased amounts of N-acetylaspartic acid in the urine and of a marked deficiency of aspartoacylase activity in cultured fibroblasts confirms the diagnostic value of these biochemical analyses in C D and means that the diagnosis can now be made without invasive investigation (i.e., brain biopsy). The levels of aspartoacylase activity in fibroblasts from the parents and sister, consistent with the heterozygote status, confirm the reliability of this method of enzymatic determination for the identification of carriers.
Acknowledgement. We wish to thank Professor Claude Bachmann, Laboratoire Central de Chimie Clinique, Centre Hospitalier Universitaire Vaudois, Lausanne, for the determination of urinary Nacetylaspartic acid.
References
1. Adachi M, Schneck L, Cara J, Volk BW (1973) Spongy degeneration of the central nervous system (Van Bogaert and Bertrand type; Canavan's disease). Hum Pathol 4:331-347 2. Divry P, Vianey-Liaud C, Gay C, Macabeo V, Rapin F, Echenne B (1988) N-acetylaspartic aciduria: report of three new cases in children with a neurological syndrome associating macrocephaly and leukodystrophy. J Inherited Metab Dis 11: 3073O8 3. Echenne B, Divry P, Vianey-Liaud C (1988) Spongy degeneration of the neuraxis (Canavan-Van Bogaert disease) and Nacetylaspartic aciduria. Neuropediatrics 20:79-81 4. Hagenfeldt L, Bollgren I, Venizelos N (1987) N-acetylaspartic aciduria due to aspartoacylase deficiency - a new aetiology of childhood leukodystrophy. J Inherited Metab Dis 10: 135-141 5. Jacobsen KB (1957) Studies on the role of N-acetylaspartic acid in mammalian brain. J Gen Physiol 43:323-333 6. Kvittingen EA, Guldal G, Borsting S, Skalpe IO, Stokke O, Jellum E (1986) N-acetylaspartic aciduria in a child with a progressive cerebral atrophy. Clin Chim Acta 158:217-227 7. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275 8. Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J (1988) Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease. Am J Med Genet 29:463-471
470 9. Matalon R, Kaul R, Casanova J, Michals K, Johnson A, Rapin I, Gashkoff P, Deanching M (1989) Aspartoacylase deficiency: the enzyme defect in Canavan disease. J Inherited Metab Dis 12:329-331 10. McAdams HP, Geyer CA, Done SL, Deigh D, Mitchell M, Ghaed VN (1989) CT and MR imaging of Canavan disease. Am J Neuroradiol 11:397-399 11. Nadler JV, Cooper JR (1972) N-acetyl-L-asparfic acid.content of human neural tumours and bovine peripheral ner'~ous tissues. J Neurochem 19:313-319 12. Ozand PT, Gascon GG, Dhalla M (1990) Aspartoacylase deficiency and Canavan disease in Saudi Arabia. Am J Med Genet 35:266-268
13. Shigematsu H, Okamura N, Shimeno H, Kishimoto Y, Kan L, Fenselau C (1983) Purification and characterization of the heatstable factors essential for conversion of lignoceric acid to cerebronic acid and glutamic acid: identification of N-acetyl-g-aspartic acid. J Neurochem 40:814-820 14. Ungar M, Goodman RM (1983) Spongy degeneration of the brain in Israel: a retrospective study. Clin Genet 23:23-29 15. Yalaz K, Topcu M, Topalo~lu H, Gurcay O, Ozcan OE, Onol B, Renda Y (1990) N-acetylaspartic aciduria in Canavan disease: another proof in two infants. Neuropediatrics 21: 140-142
Child's Nerv Syst (1992) 8:468-470
9 Springer-Verlag 1992
Biochemical diagnosis of Canavan disease G. Bartalini, M. Margollicci, P. Balestri, M.A. Farnetani, M. Cioni, and A. Fois Istituto di Clinica Pediatrica, Universitfi degli Studi di Siena, Via P.A. Mattioli, 10, 1-53100 Siena, Italy Received September 23, 1991
Abstract. C a n a v a n disease (CD) is a rare a u t o s o m a l recessive disorder characterized by m a c r o c e p h a l y and progressive l e u k o d y s t r o p h y . U p to n o w biopsy or n e c r o p s y were required to define the diagnosis. Recently the disease has been related to N-acetylaspartic aciduria and deficiency o f aspartoacylase, an e n z y m e possibly involved in the myelin synthesis. These biochemical findings have p r o v i d e d a diagnostic m a r k e r for the disease. We report a new case o f infantile C D in which the d e m o n stration o f N-acetylaspartic aciduria and a m a r k e d deficiency o f a s p a r t o a c y l a s e activity confirmed the diagnosis. Key words: C a n a v a n disease - N-acetylaspartic aciduria A s p a r t o a c y l a s e deficiency
C a n a v a n disease (CD) is a rare a u t o s o m a l recessive disorder characterized by degeneration o f cerebral white m a t ter. It has been f o u n d to be m o r e c o m m o n a m o n g Ashkenazi Jews [14]. Three variants o f the disease, congenital, infantile, and late onset, have been reported [I]. I n the m o s t frequent form, the infantile, s y m p t o m s occur within the first few m o n t h s o f life and include failure o f m o t o r and m e n t a l development, progressive spasticity, blindness and optic a t r o p h y , a n d m a c r o c e p h a l y . N e u r a l imaging studies d e m o n s t r a t e symmetrical demyelination a n d l e u k o d y s t r o p h y [10]. T h e m a i n pathological features o f the brain consist o f s p o n g y degeneration. Electron mic r o s c o p y shows vacuoles in the myelin sheaths, swelling o f astrocytes, and elongation o f m i t o c h o n d r i a . U p to n o w brain biopsy or n e c r o p s y has been required to c o n f i r m the diagnosis and differentiate the disease f r o m other types o f m a c r o c e p h a l i c l e u k o d y s t r o p h y (e.g., Alexander disease). H o w e v e r , recent studies have reported increased a m o u n t o f N-acetylaspartic acid in urine, plasma, and C S F and a deficiency o f aspartoacylase activity in fibroblasts f r o m patients in which the diagnosis o f C D h a d previously been c o n f i r m e d by m o r p h o l o g i c a l Correspondence to." A. Fois
e x a m i n a t i o n o f the brain [3, 8]. These observations have p r o v i d e d a biochemical diagnostic m a r k e r for the disease. We describe here a new case o f m a c r o c e p h a l i c l e u k o d y s t r o p h y in which N-acetylaspartic aciduria and aspartoacylase deficiency have been demonstrated.
Case report The patient, a girl, was born at term by cesarean section after an uneventful pregnancy. The birth weight was 3400 g; length and head circumference are not known. Her parents are not related but both families come from the same small village (2500 inhabitants). One older sister is healthy. From the patient's 15th day of life, tremors and progressive muscular rigidity were noted. At 5 months severe neuromotor delay was observed. At that time she did not show any interest in her surroundings and head control was absent. The head circumference at 6 months of age was 45 can (above the 95th percentile). A cranial CT scan at this time revealed demyelination. At the age of 21 months, when she was referred to our hospital, physical examination revealed macrocephaly (head circumference 51 cm), nystagmus, no visceromegaly, axial hypotonia with absent head control, limb hypertonia with brisk deep tendon reflexes, bilateral foot clonus, and Babinski's sign. She had no spontaneous movements, showed very little interest in her surroundings, did not grasp objects, and was not able to fix her eyes; language was absent. At the age of 2 years and 10 months (last observation) her clinical condition had further deteriorated. Her head circumference was 52 cm. Interest in her surroundings was completely lacking and she appeared to be blind. The ophthalmologic examination showed pale discs bilaterally. Cerebral magnetic resonance imaging at 21 months revealed an increased signal in T2-weighted images, indicating diffuse symmetrical demyelination and moderate widening of the lateral and III ventricles. These findings were more evident at 2 years and 10 months. EEG showed alternate slow and fast rhythms and absence of sleep spindles. Blood ammonia, lactate, aminoacids, cortisol, very long chain fatty acids and urinary glycosaminoglycans and oligosaccharides were normal. The cerebrospinal fluid protein level was also normal. Nerve conduction velocity was within the normal limits. The following acid hydrolases on leukocytes and/or fibroblasts were normal: fi-hexosaminidase, galactosylceramidase, arylsulfatase A, fi-galactosidase. The ultrastructural examination of skin biopsy was normal. The study of urinary organic acids revealed an high excretion of N-acetylaspartic acid (1026 mmol/mol creatinine). The aspartoacylase activity test was performed as follows.
469 Table 1. Aspartoacylase activity in cultured fibroblasts
Aspartoacylase activity (U/rag protein) Patient
0.11
Father Mother Sister Controls (n = 7)
0.86 1.44 1.32 2.91 + 1.83
Residual activity (% of control) 3.8
29.5 49:5 4514 100.0
Materials and methods
Skin fibroblasts from the patient, her parents, her sister, and seven controls were utilized for the enzymatic assay of aspartoacylase. The cells were cultured in MEM 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) containing 10% fetal calf serum, penicillin, streptomycin, and L-glutamine. Aspartoacylase activity was determined according to the procedure described by Matalon et al. [8]. Confluent cells were harvested by scraping, suspended in water, and disrupted by sonication for 3 x 10 s. After centrifugation at 10000 g x 30 min, the supernatant aliquots were incubated for 5 h at 37 ~ with and without the substrate 1.7 mmol/1 N-acetyl-L-aspartic acid in 0.05 Tris-HC1 buffer pH 8. Each incubation mixture contained 350-450 gg protein [7]. The reaction was terminated by boiling for 3 min. Precipitated proteins were removed by centrifugation at 11000 rpm and asparrate content was assayed spectrophotometrically at 340 nm after addition of 2-oxoglutarate (t .4 retool/l), NADH (0.148 nmol/1) and malate dehydrogenase and aspartate aminotransferase in excess. A slight Iinear decrease of adsorbance for conversion of NADH to NAD occurred which was recorded for 5 min. The adsorbance measurements were converted to enzyme activities by using the molar extinction coefficient for NADH 6.3 x 1000.
Results
Aspartoacylase activity in control fibroblasts was 2.9l + 1.83 U/rag protein. In the patient's fibroblasts the enzyme activity was 0.11 U / m g protein, 3.8% of the mean control value. The activity of the same enzyme in the parents' and sister's fibroblasts was less than 50% of the control value (Table 1). Discussion
N-acetylaspartic aciduria was initially described by Kvittingen et al. in 1986 [6] in a 6-year-old b o y with macrocephaly, spasticity, p s y c h o m o t o r retardation, and leukodystrophy. Afterwards this biochemical change was observed in four other patients with similar clinical findings [2, 4]. Matalon et al. [8] and Echenne et al. [3] independently reported the association between N-acetylaspartic aciduria due to aspartoacylase deficiency and C D proven by brain biopsy. This correlation has since been demonstrated in other cases of C D and the biochemical defect is currently considered to be specific to the disease [9, 12, 151. Synthesis of N-acetylaspartic acid has not been found to occur in any organ other than the central nervous system, where it is mainly localized in the neurons [11]. To date, the true functional role of this c o m p o u n d is not
yet understood. A remarkable increase of N-acetylaspartic acid has been observed during brain m a t u r a t i o n in animals, and an increase in the concentration has been also demonstrated in h u m a n brain during early life [5]. This observation has suggested that this substance m a y have a role in the myelination process, and it is possible that a defect in its metabolism determines an abnormal myelinization and spongy degeneration in CD. It has been proposed in fact that N-acetylaspartic acid is an essential; factor for the conversion of lignoceric acid to cerebronic acid, a precursor of ceramide, Which is one Of the major lipid components of myelin [13]. Another mechanism for the pathogenesis of C D m a y be the accumulation :of N-acetylaspartic acid in brain::tissue, result: ing i n m y e l i n damage and subsequent spongy degenera: tion [8]. :: In our case, the clinical presentation, which includes progressive and severe mental retardation with early onset, macrocephaly, and leukodystrophy, is similar to that of the infantile type of CD. The demonstration of increased amounts of N-acetylaspartic acid in the urine and of a marked deficiency of aspartoacylase activity in cultured fibroblasts confirms the diagnostic value of these biochemical analyses in C D and means that the diagnosis can now be made without invasive investigation (i.e., brain biopsy). The levels of aspartoacylase activity in fibroblasts from the parents and sister, consistent with the heterozygote status, confirm the reliability of this method of enzymatic determination for the identification of carriers.
Acknowledgement. We wish to thank Professor Claude Bachmann, Laboratoire Central de Chimie Clinique, Centre Hospitalier Universitaire Vaudois, Lausanne, for the determination of urinary Nacetylaspartic acid.
References
1. Adachi M, Schneck L, Cara J, Volk BW (1973) Spongy degeneration of the central nervous system (Van Bogaert and Bertrand type; Canavan's disease). Hum Pathol 4:331-347 2. Divry P, Vianey-Liaud C, Gay C, Macabeo V, Rapin F, Echenne B (1988) N-acetylaspartic aciduria: report of three new cases in children with a neurological syndrome associating macrocephaly and leukodystrophy. J Inherited Metab Dis 11: 3073O8 3. Echenne B, Divry P, Vianey-Liaud C (1988) Spongy degeneration of the neuraxis (Canavan-Van Bogaert disease) and Nacetylaspartic aciduria. Neuropediatrics 20:79-81 4. Hagenfeldt L, Bollgren I, Venizelos N (1987) N-acetylaspartic aciduria due to aspartoacylase deficiency - a new aetiology of childhood leukodystrophy. J Inherited Metab Dis 10: 135-141 5. Jacobsen KB (1957) Studies on the role of N-acetylaspartic acid in mammalian brain. J Gen Physiol 43:323-333 6. Kvittingen EA, Guldal G, Borsting S, Skalpe IO, Stokke O, Jellum E (1986) N-acetylaspartic aciduria in a child with a progressive cerebral atrophy. Clin Chim Acta 158:217-227 7. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275 8. Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J (1988) Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease. Am J Med Genet 29:463-471
470 9. Matalon R, Kaul R, Casanova J, Michals K, Johnson A, Rapin I, Gashkoff P, Deanching M (1989) Aspartoacylase deficiency: the enzyme defect in Canavan disease. J Inherited Metab Dis 12:329-331 10. McAdams HP, Geyer CA, Done SL, Deigh D, Mitchell M, Ghaed VN (1989) CT and MR imaging of Canavan disease. Am J Neuroradiol 11:397-399 11. Nadler JV, Cooper JR (1972) N-acetyl-L-asparfic acid.content of human neural tumours and bovine peripheral ner'~ous tissues. J Neurochem 19:313-319 12. Ozand PT, Gascon GG, Dhalla M (1990) Aspartoacylase deficiency and Canavan disease in Saudi Arabia. Am J Med Genet 35:266-268
13. Shigematsu H, Okamura N, Shimeno H, Kishimoto Y, Kan L, Fenselau C (1983) Purification and characterization of the heatstable factors essential for conversion of lignoceric acid to cerebronic acid and glutamic acid: identification of N-acetyl-g-aspartic acid. J Neurochem 40:814-820 14. Ungar M, Goodman RM (1983) Spongy degeneration of the brain in Israel: a retrospective study. Clin Genet 23:23-29 15. Yalaz K, Topcu M, Topalo~lu H, Gurcay O, Ozcan OE, Onol B, Renda Y (1990) N-acetylaspartic aciduria in Canavan disease: another proof in two infants. Neuropediatrics 21: 140-142
