European Journal of Paediatric Neurology 1998; 2: 69-76

REVIEW ARTICLE 0

IiJ - D Molecular basis of Canavan disease

REUBEN MATALON,’ KIMBERLEE MICHALS-MATALON

‘Department of Pediatrics; *Primary Care Outpatient Center, University of Texas Medical Branch at Galveston, Galveston, Texas.

Introduction

Canavan disease is a neurodegenerative disorder characterized by spongy degeneration of the white matter of the brain. The brain pathology of the disease was described by Canavan in 1931 in a child thought to have Schilder’s disease.’ In 1949, van Bogaert and Bertrand in their report of three children of Jewish extraction with Canavan disease, recognized that this was a new autosomal recessive disease.* Since then, numerous cases have been reported with prevalence among Jewish indivi- duals.3 The enzyme defect in Canavan disease, aspartoacylase deficiency, was identified by Matalon et al. in 1988.4 The gene for aspartoacylase was cloned in 1993 and mutations causing Canavan disease have since been identified.w Interestingly, only two mutations were found to be the basis for Canavan disease among 98% of individuals of Ashkenazi Jewish ancestry, while the molecular basis of Canavan disease among non-Jewish individuals is caused by a wide range. of mutations. Therefore, because of the limited number of mutations in Ashkenazi Jewish people, screening programmes to identify carriers can be performed on this population.

Clinical course

Babies with Canavan disease appear normal in the first few months of life. They become increasingly irritable, remain hypotonic, and have poor head control. Head lag is a constant finding in Canavan

disease and it is often detected between 3 and 6 months of age. Developmental milestones remain delayed and the head becomes progressively larger, especially after the 6th month of life. The triad of hypotonia, head lag and megalencephaly should suggest Canavan disease, when white matter involvement is suspected.

As children with Canavan disease become older the delay in development becomes obvious, espe- cially in motor and verbal skills. These children, none the less, interact, laugh, smile, reach for objects and can lift their head in the prone position. However, children with Canavan disease do not attain the skills for sitting, standing, walking or talking. As children with Canavan disease get older, the hypotonia gives way to spasticity, similar to that seen in cerebral palsy. Indeed, some children with Canavan disease may carry the diagnosis of cerebral palsy. After the first or second year of life children with Canavan disease may become increasingly irritable, develop feeding difficulties, and sleep disturbances. Although children with Canavan disease develop optic atrophy, they are not blind and can follow objects with their eyes. Figures 1 and 2 show unrelated boys with Canavan disease. Figure 1 shows a 5- year-old boy with macrocephaly, who can still raise his head and follow objects. This child is homo- zygous for mutation Tyr231X the less common Jewish mutation. This mutation is caused by substitution of C to A in position 693 of the aspartoacylase cDNA on exon V. This substitution results in a termination codon in that position. The second child (Fig. 2) also has macrocephaly and seems happy and smiling while looking at the camera, although the head cannot be supported by

Keywords: Canavan disease. N-Acetylaspartic acid. Aspartoacylase. Spongy degeneration of the brain.

Received 8.12.97. Revised 26.2.98. Accepted 27.2.98. Correspondence: Reuben Matalon, Director, Biochemical/Molecular Genetics, Professor, Pediatrics and Human Biological, Chemistry and Genetics, 301 University Boulevard, Galveston, Texas 77555-0359, USA

1090-3798/98/010069+8 $18.00 0 1998 European Paediatric Neurology Society

70 Review article: R Matalon et al.

Fig. 1. (above) A S-year-old with Canavan disease. He is able to raise his head in the prone position. He had increased intracranial pressure at 2 years of age and needed a ventri- culo-peritoneal shunt. He is homozygous for the less common Jewish mutation Tyr231 X.

Fig. 2. (right) A 6-year-old with Canavan disease. He has poor head control but can make eye contact and smile. He cannot support his head and needs a head rest. He is homo- zygous for the prevalent Jewish mutation, Cfu285Ala.

Review article: Canavan disease 71

- OOC.C~.CH.COO- , 7

-0OC.C H1.CH.COO - + CH,.COO-

Hz0 I

HN.CO.CH, NH,

N-Acetybparticacid L-Asperticecid

Fig. 3. Hydrolysis of N-acetylaspartic acid (NAA) by aspartoacylase. The deficiency leads to the accumulation of NAA.

the neck muscles. This child is homozygous for the more common mutation found in Ashkenazi Jewish individuals, a missense mutation on exon VI, (854) which substitutes glutamic acid to alanine in position 285 (Glu285Ala) of aspartoacylase. Some children with Canavan disease develop seizures, although that is not the rule. As feeding difficulties increase, assisted feeding by a nasogas- tric tube or permanent gastrostomy may be needed. Many patients succumb in the first few years of life; however, increasing numbers of patients with Canavan disease are reaching their teens due to improved medical and nursing care.

Aspartoacylase: the enzyme defect - _-----__ - _-- -- - .-~- -.-_ -.-“---.- - .-- - .̂ Canavan disease is caused by a deficiency of the enzyme aspartoacylase. Aspartoacylase is one of two different aminoacylases, I and II. Aminoacy- lase I is not specific for certain amino acids and hydrolyses acetate from all amino acids with the exception of aspartic acid. Aspartoacylase (amino- acylase II) is specific for the hydrolysis of acetate from aspartic acid only. Aspartoacylase hydrolyses N-acetylaspartic acid (NAA) to aspartate and acetate and its deficiency leads to the accumulation of NAA (Fig. 3). Aspartoacylase is abundant in the white matter of the brain and can be traced to the myelin tracts. Aspartoacylase is also found in kidneys, lungs and to a lesser extent in liver and other tissues. It is not present in blood cells, so its deficiency or presence cannot be assayed using blood. Cultured skin fibroblasts would be needed for these purposes.

Antibodies to human aspartoacylase cross-react with brain, aspartoacylase of other species; bovine, rats, mice and monkeys. In the brain aspartoacylase is localized to the white matter, while the substrate for aspartoacylase, NAA, is synthesized only in the brain and only in the grey matter. Aspartoacylase has been purified to homogeneity from different tissues. It is monomeric and the bovine enzyme has similar properties to the human suggesting a high

degree of conservation in these species. So the process that leads to Canavan disease is caused by the deficiency of aspartoacylase in the brain white matter and one can assume that aspartoacylase in the peripheral tissues is for housekeeping pur- poses. The increased levels of NAA in the brain leads to “swelling” or sponginess of the brain and disruption of the white matter. The exact mechan- ism of this process is unclear and the function of NAA and its role in maintaining intact white matter also need to be clarified. Severe neurological problems found in Canavan disease are the result of the derangement of the normal metabolism of NAA.1-3 Human aspartoacylase is similar to other esterases, and it is composed of 313 amino acid residues, with molecular weight of 36 kD. Its functional domains, such as the hydrolytic site(s) and the binding site(s) have not been character- ized.

The gene for aspartoacylase

The molecular basis for Canavan disease is caused by mutations in the gene coding for aspartoacylase, resulting in a deficient enzyme. This gene has been cloned and localized on the short arm of chromo- some 17 (17p13-ter). 5~7 The human aspartoacylase gene spans 39 kb, with 6 exons and 5 introns. The exons vary in size from 94 bases in exon III to 514 in exon VI. The cDNA for aspartoacylase is 1435 bp long coding for 313 amino acids. The aspartoacy- lase gene is conserved among species and the coding sequence of the bovine and human cDNA shows 92% identity. The mouse cDNA shows 86% identity with human. 5t7 Southern blot analysis of genomic DNA from eukaryotic species including monkey, mouse, dog, cow, rabbit, chicken and yeast show fragments that hybridize with the human aspartoacylase cDNA, indicating conserva- tion during evolution. 7 Figure 4 shows the genomic organization of the gene for aspartoacylase based on restriction map of human genomic DNA. The

72 Review article: R Matalon et al.

cDNA position ii s E

Exons I II 111 IV V VI

Y Ikl t,

1 1 1 c Aspll4Gl~ QllS~ 527deilNl

Mutations Gty27Aq WI=8 %I- CyrzlSX Cly274Arg

527dd6bp Qd3lX 827&lCT b@fA* IleluTbr ~16acIm 566dei7bp 87OdeUbp 32ddT 876dellbp

GldESAla PWMScr

,&lEZ!?mr

Fig. 4. Diagram of the gene for aspartoacylase with 5 introns and 6 exons. The gene spans 29 kb of DNA and it is localized on the short arm of chromosome 1 i’(1 i’p-ter). A newly described mutation on exon II, 11143Thr, has been described in a Japanese patient.35 The two mutations in italics are the common Jewish mutations. The mutation underscored with the interrupted line is the common European mutation.

mutation distribution on the various exons of the aspartoacylase gene are shown.

The two predominant Jewish mutations; a mis- sense mutation on exon VI, Glu285Ala, found in 86% of the alleles and the other mutation, a nonsense mutation on exon V, Tyr231X (termina- tion codon) found in 14% of the alleles. These two mutations were found in 98.8% of all Jewish patients with Canavan disease. The third common mutation found in patients with Canavan disease (Ala305Glu) has been reported in 40-48% of non- Jewish patients. 30,s3 Table 1 shows these common mutations in Canavan disease with the residual activity of the enzyme. Less common mutations are listed in Table 2. The residual activity is shown in some of the mutations. As more mutation analysis is performed for patients with Canavan disease the number of individual mutations will increase.

Pa thophysiology

The exact relationship between increased NAA and spongy degeneration of the brain is unknown. The discovery of the enzyme defect in Canavan disease suggests that the normal metabolism of NAA is important in the maintenance of healthy white matter. Although NAA synthesis occurs in the grey matter, aspartoacylase is found mainly in the white matter.- The different localization of the enzyme and substrate in the brain suggests chemical compartmentation of enzyme and sub- strate. The grey matter of the brain becomes involved as well in the process as the disease progresses. Microscopy of the brain shows spongy degeneration throughout the white matter includ-

ing the subcortical regions. The astrocytes are swollen and electron microscopy shows distorted and elongated mitochondria.“-‘-r3 These histological changes used to be the major diagnostic criteria for Canavan disease.

Diagnosis

Computed tomography (CT) scan of the head or magnetic resonance imaging (MRI) of the brain reveal diffuse white matter degeneration in Cana- van disease.“16 The involvement is primarily in the cerebral hemispheres with less involvement in the cerebellum and brain stem. Early in life the MRI may be interpreted as normal so follow-up evalua- tions may be needed. I6 Figure 3 shows the MRI of a 16-month-old boy with Canavan disease. Nuclear magnetic resonance spectroscopy of the Canavan brain has revealed increased NAA compared with normal brain.l&*’ Increased brain NAA can be used for initial diagnostic purposes.18

The specific test that is confirmatory of the diagnosis of Canavan disease is elevated NAA in the urine or blood. In our experience some increase in NAA may erroneously lead to a misdiagnosis.

Table 1 Common Canavan disease mutations with residual activity of aspartoacylase’

Mutation Residual

activity (%) me Ethnic group

Glu285Ala Tyr231X Ala305Glu

2.5 Missense Jewish 0.0 Nonsense Jewish 0.0 Missense Non-Jewish

Review article: Canavan disease 73

Table 2 Less common Canavan disease mutations among non-Jewish individuals with residual activity of asparatoacylase7~32-35

Mutation

Residual

Activity % TYPe Mutation

Residual

Activity % TYPe

Cysl52Tyr 876de14bp 32delT Ilel6Thr Gly27Arg Asp27Arg Aspll4Glu Gly123Glu Arg168Glu Cysl52Arg

0.16 Missense 0.0 Deletion NE Deletion 0.38 Missense 3.07 Missense 3.07 Missense 0.35 Missense

26.90 Missense 0.0 Missense 0.0 Missense

Cys218X Phe295Ser Gly274Arg 827delGT 870de14 566de17 527de16 527de1108 Ile143Thr

NE NE NE NE NE NE NE NE NE

Nonsense Missense Missense Deletion Deletion Deletion Deletion Deletion Missense

The concentration of NAA in Canavan disease needs to be more than 5- to lo-fold the normal base line.lg Cultured skin fibroblasts can be used to manifest the enzyme deficiency, but the urinary determination of increased NAA is sufficient to make the diagnosis. Brain biopsy which used to be the major confirmatory procedure for the diagnosis of Canavan disease is no longer necessary.

Nuclear magnetic resonance (NMR) spectro- scopy of the brain can be used as a first step screen for NAA when MRI is being performed. Usually NAA levels are increased in patients with Canavan disease. However, NAA is not uniformly increased in all patients and in some patients with other leukodystrophies NAA may seem increased. Figure 6 shows the NMR spectroscopy of a patient with Canavan disease. One cm3 voxel taken in the deep white matter of the frontal lobe shows increase in NAA while the one taken in the medulla of the same child shows no increase.

Once the diagnosis of Canavan disease is reached, DNA mutation studies can be performed on the proband and family members for counsel@ and preventive measures.

Differential diagnosis

Fig. 5. A T2-weighted magnetic resonance image of the brain of a 16-month-old boy with Canavan disease, showing diffuse signal changes in the white matter. It involves the subcortical white matter, the posterior fossa and the internal and external capsules.

The macrocephaly, characteristic of Canavan dis- ease, can be seen in Alexander disease, Tay-Sachs and other neurodegenerative diseases. Recently 3-OH glutaric acidaemia has been added to the list of diseases with macrocephaly and white matter involvement.20 Another newly recognized autoso- ma1 recessive megalencephaly with vacuolating leukoencephalopathy and a mild clinical course has been reported and needs to be differentiated from Canavan disease.2*-23 This is a distinct entity

and seems to be common in India. Autosomal dominant megalencephaly can also be confused with Canavan disease. These individuals are only slightly mentally impaired.

Attenuation of the white matter on MRI or CT scans should exclude Tay-Sachs disease, particu- larly when it is universal. The U fibres are typically involved with Canavan disease. Spongy degenera- tion of the brain can also occur with viral

74 Review article: R Matalon et al.

Fig. 6. Proton nuclear magnetic resonance spectroscopy for the measurements of NAA in the brain of a patient with Canavan disease. The upper panel shows NAA (at 2.00) which is elevated. The voxel is 1 cm3 taken in the deep white matter of the frontal lobe. The lower panel shows the same measurements taken in the medulla of the same patient, which shows normal concentration of NAA.

infections, mitochondrial diseases and other meta- bolic diseases.24-2* The unique feature of Canavan disease is the increased urinary excretion of NAA.2g

Epidemiology

Canavan disease is pan-ethnic. It is more prevalent among Ashkenazi Jews of Eastern European extraction. There are two specific mutations common among Jews. The predominant mutation among Jewish individuals is a missense mutation (Glu285Ala) with substitution of glutamic acid to alanine. A nonsense mutation where the codon for tyrosine is substituted by a termination codon (Tyr231X), is also prevalent among Jews (Table 1).30 Screening of healthy Jews reveals that l/37-1/40 is a carrier of one of these two mutations.@’ The incidence of the disease in this population is estimated at l/6000. This is a rather high incidence and now there are programmes to screen for carriers for Canavan disease among healthy Jewish individuals for prevention purposes. These programmes are similar to the screening pro- grammes for Tay-Sachs disease.

In non-Jewish patients the mutations are differ- ent and more diverse. Canavan disease has been reported among individuals of European, Middle

Eastern, Turkish, Gypsies and African American ancestry.32-35 The most common Canavan mutation (about 40-48%) in non-Jewish patients of European ancestry is Ala305Glu which is a missense muta- tion in exon VI, substituting alanine to glutamic acid (Table l).30 Many of the other mutations in the non-Jewish patients often occur only in one family or in few patients (Table 2).32-35 Mutation Ilel43Thr has recently been reported in a Japanese patient.34

Genotype-phenotype correlation

Experience with Jewish patients with Canavan disease with the two predominant mutations is rather extensive. Over 96% of Jewish patients are homozygous either for mutation Glu285Ala or Tyr231X. While the residual activity of aspartoacy- lase of mutation Tyr231X is not detectable, children cannot be clinically distinguished from those with mutation Glu285Ala with some residual enzyme activity (Table 1). These patients seem to have a similar course. Life expectancy has been variable in patients with Canavan disease with the same genotype. We had a family with genotypically identical siblings; one died aged 1 year and the other aged 30 years. The experience with non- Jewish patients is similar. Patients homozygous for the most common mutation Ala305Glu, with no

Review article: Canavan disease

residual enzyme activity, have a clinical course similar to the two Jewish mutations. A patient heterozygote for mutation Gly123Glu, a mild mutation (Table 2) was phenotypically a severe Canavan patient, when the mutation on the other allele was Ala305Glu. Generally, it is anticipated that a double heterozygote, as in this case, should be phenotypically mild with residual aspartoacy- lase activity of 26.9%. There is a need for more patients to be genotyped and more mutations need measurement of residual aspartoacylase activity so a clearer correlation could be established.

Treatment

There is no proven method of therapy other than symptomatic. Seizures need to be controlled. Children with Canavan disease may need naso- gastric feedings or feeding gastrotomy. Gene therapy on two children with Canavan disease has been done.36 There is no detailed report on these children, but the trial seemed to be ineffec- tive. More recently a trial with acetazolamide to reduce white matter water concentration and NAA was tried for a period of 5 months. Acetazolamide was helpful in reducing the intracranial pressure, but did not reduce water concentration or NAA levels.37

Prevention

If both parents are carriers, the risk of an affected baby is 1:4. Carrier determination and preventive counselling can be attained using DNA analysis.38 The high carrier rate observed in the Ashkenazi Jewish population warrants screening similar to the screening for carriers in Tay-Sachs disease. Carrier testing for Canavan disease, however, requires DNA analysis since the enzyme is not detectable in the blood. In a couple with informa- tive DNA, prenatal diagnosis could be offered using DNA analysis. Other methods of prenatal diagnosis include determination of NAA in amnio- tic fluid which should be increased in an affected pregnancy.3+r0

Animal models

There have been reports of naturally occurring animals with spongy degeneration and macro-

75

cephaly. These include a mouse, silver fox and a dog.41d However, these reports were prior to the discovery of aspartoacylase deficiency in Canavan disease. So, there is no biochemical confirmation and such animals are not in existence.

We are currently engaged in producing a knock out mouse model for Canavan disease. Such a model will be of help in understanding the relationship of the pathophysiology of Canavan disease and formulating possible therapeutic mod- alities.

Conclusion

More studies are needed to elucidate the patho- physiology of Canavan disease and how the non- hydrolysed NAA leads to spongy degeneration. The creation of an animal model would be helpful in the understanding of the disease and the formulation of gene therapy.

References

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