Molecular Genetics and Metabolism 80 (2003) 74–80

www.elsevier.com/locate/ymgme

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Canavan disease: a monogenic trait with complex genomic interaction

Sankar Surendran, Kimberlee Michals-Matalon, Michael J. Quast, Stephen K. Tyring,Jingna Wei, Ed. L. Ezell, and Reuben Matalon*

Department of Pediatrics, Children�s Hospital, The University of Texas Medical Branch, Galveston, TX 77555-0359, USA

Received 1 July 2003; received in revised form 8 August 2003; accepted 8 August 2003

Abstract

Canavan disease (CD) is an inherited leukodystrophy, caused by aspartoacylase (ASPA) deficiency, and accumulation of

N-acetylaspartic acid (NAA) in the brain. The gene for ASPA has been cloned and more than 40 mutations have been described,

with two founder mutations among Ashkenazi Jewish patients. Screening of Ashkenazi Jews for these two common mutations

revealed a high carrier frequency, approximately 1/40, so that programs for carrier testing are currently in practice. The enzyme

deficiency in CD interferes with the normal hydrolysis of NAA, which results in disruption of myelin and spongy degeneration of the

white matter of the brain. The clinical features of the disease are macrocephaly, head lag, progressive severe mental retardation, and

hypotonia in early life, which later changes to spasticity. A knockout mouse for CD has been generated, and used to study the

pathophysiological basis for CD. Findings from the knockout mouse indicate that this monogenic trait leads to a series of genomic

interaction in the brain. Changes include low levels of glutamate and GABA. Microarray expression analysis showed low level of

expression of GABA-A receptor (GABRA6) and glutamate transporter (EAAT4). The gene Spi2, a gene involved in apoptosis and

cell death, showed high level of expression. Such complexity of gene interaction results in the phenotype, the proteome, with spongy

degeneration of the brain and neurological impairment of the mouse, similar to the human counterpart. Aspartoacylase gene

transfer trial in the mouse brain using adenoassociated virus (AAV) as a vector are encouraging showing improved myelination and

decrease in spongy degeneration in the area of the injection and also beyond that site.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Canavan disease; GABA; EAAT4; Spi2; AAV; Glutamate

Introduction and history

Canavan disease (CD), spongy degeneration of the

brain, is an autosomal recessive disorder. Canavan in

1931, described the histological findings of spongy de-

generation of the white matter of the brain in a patient

thought to have Schilder disease [1]. Canavan disease, as

a specific entity, was recognized in 1949 by van Bogaert

and Betrand [2], who described three Jewish children

with spongy degeneration of the brain. The disease iscaused by aspartoacylase (ASPA) deficiency [3] resulting

accumulation of N-acetylaspartic acid (NAA) in the

brain [3]. The urine of patients with CD contain elevated

levels of NAA, so that urine testing can be diagnostic

and brain biopsy is no longer needed for the diagnosis.

* Corresponding author. Fax: 1-409-772-9595.

E-mail address: rmatalon@utmb.edu (R. Matalon).

1096-7192/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ymgme.2003.08.015

After the discovery of ASPA deficiency in CD, the gene

for ASPA was cloned and mutations that cause CD wereidentified. While two mutations are the molecular basis

of CD among Jewish patients, a wide range of mutations

were found among non-Jewish patients with CD [4–8].

The creation of a knockout mouse for CD [9] affords the

opportunity to investigate the molecular events that lead

to the pathophysiology of CD and also experiment with

gene transfer to the brain.

Clinical course of CD

Infants with CD appear normal during the first fewmonths of life, although careful examination reveals

mild delays, hypotonia, and inadequate visual tracking.

These infants become progressively irritable, and remain

hypotonic with poor head control. Developmental delay

Fig. 1. Diagram of the gene for aspartoacylase with 5 introns and 6

exons. The human ASPA gene spans 39 kb and it is localized in the

short arm of chromosome 17(17p-ter). Mutation E285A mutation in

exon 6 and Y231X in exon 5 are the common Jewish mutations. In

non-Jewish populations, mutations vary.

S. Surendran et al. / Molecular Genetics and Metabolism 80 (2003) 74–80 75

and larger head become noticeable after 6 months ofage. The hypotonia, head lag, and megalencephaly are

common features of CD, and should lead the physician

to consider leukodystrophy [10]. When children with

CD become older, developmental delays such as, motor

and verbal skills become obvious. In spite of the pro-

found delays, children with CD are able to interact,

laugh, smile, reach for objects, and lift their head when

in prone position. Patients do not develop the ability tosit, stand, walk or talk. Children with CD develop optic

atrophy and have difficulty focusing, but are able to

recognize their surroundings. When patients with CD

become older, hypotonia gives way to spasticity. Feed-

ing difficulties increase with age, and feeding by a na-

sogastric tube or permanent gastrostomy will be needed.

With improved nursing and medical care such patients

can reach the second decade of life or beyond that[11,12].

Diagnosis of CD

The diagnosis of CD relies on demonstrating high

levels of NAA in the urine. The NAA in the urine of a

patient with CD is more than 50 times the normal uri-nary level. The mean values of urine NAA in normal

and CD patients were, 23.5� 16.1 (n ¼ 53) and

1440.5� 873.3 (n ¼ 95) lmol/mmol creatinine, respec-

tively [13]. Patients with a slight increase in urine NAA

are often confused with Canavan disease [7]. NAA is

elevated about 3-fold in blood and CSF in patients with

CD. Blood does not have ASPA activity and enzyme

activity in cultured fibroblasts is difficult to interpretbecause the enzyme activity is sensitive to culture con-

ditions. Brain biopsy is no longer needed for the diag-

nosis of CD. Mutation analysis is important to

determine the genotype for purposes of counseling.

Mutation expression needs to be determined because

some mutations are polymorphic [7].

Computed tomography (CT) scan of the head or

magnetic resonance imaging (MRI) of the brain showdiffuse white matter degeneration in CD [14–16].

Nuclear magnetic resonance spectroscopy (MRS) of the

CD brain show increase in the peak of NAA [16–18].

Aspartoacylase gene

Aspartoacylase gene was cloned and localized on theshort arm of chromosome 17 (17p13-ter) [19,20]. The

human aspartoacylase gene spans 30 kb, contains five

introns and six exons coding for 313 aminoacids [19], an

enzyme with a molecular weight of 36 kDa. Southern

blotting of genomic DNA from eukaryotes including

rabbit, chicken, monkey, mouse, dog, cow, and yeasts

show fragments that hybridize with human ASPA

cDNA, suggesting that ASPA is highly conserved duringevolution [20]. The genomic organization of the gene for

ASPA with various mutations is shown in Fig. 1. Some

mutations in exon 6 of the gene shows polymorphism

and the polymorphic mutations do not change the

enzyme activity [21,22]. Therefore expression studies

are important to understand functional significance of

mutations.

There are two mutations, E285A and Y231X thataccount for over 96% of the mutations among Ashke-

nazi Jewish population [13,19]. Carrier frequency for

CD among Ashkenazi Jewish populations has ranged

from 1/37 to 1/60 [23,24]. This high frequency of carriers

means that routine preventive measure using DNA

analysis for the common Jewish mutations needs to be

recommended for Ashkenazi Jews.

In non-Jewish patients the mutations are more var-iable. The most common mutation in non-Jewish pa-

tients is A305E [23]. There have been over 40

mutations identified in various ethnic groups [8,12,25–

28]. Many appear to be sporadic mutations that run in

families. Mutation D114Y was found in a small geo-

graphical region in Norway and D249V mutations was

specific to Norwegian and Swedish population [29] in-

dicating some mutations in the ASPA gene are foundermutations.

Genotype and phenotype correlation

The majority of patients with Canavan disease have

a severe phenotype. The Jewish mutations E285A and

Y231X lead to a severe phenotype. Homozygosity ofthe common non-Jewish mutation A305E has been

reported with both severe and mild CD [25]. The non-

Jewish mutation, D249V, converts a negatively charged

aspartate residue into a hydrophobic valine residue,

resulting in complete loss of ASPA activity. Pheno-

typically patients with D249V have a severe phenotype

76 S. Surendran et al. / Molecular Genetics and Metabolism 80 (2003) 74–80

with nystagmus and irritability at birth [8]. Mutationsthat disrupt the conformation of the active site of

ASPA [26,27] will result in total loss of enzyme activity

and a severe phenotype. Mutation C152W forms a

disulfide bond that disrupts the active site [8]. Thus

severe genotypes often correspond to a severe pheno-

type.

Prevention and prenatal diagnosis

Carrier detection and genetic counseling are impor-

tant to prevent CD. These approaches are now being

promoted for the Jewish population using DNA sam-

ples to determine the common Jewish mutations for

CD. When both parents are carriers and their muta-

tions are known they are informative for prenatal di-agnosis [30,31]. Prenatal diagnosis based on mutation

analysis should also include the study of other DNA

markers to avoid possible maternal cell contamination

[31]. In non-informative families, the biochemical assay

for NAA in amniotic fluid should be offered and pro-

gressive increase of NAA (5- to 10-fold) in the amni-

otic fluid can be used for prenatal diagnosis of CD

[32,33].

Fig. 3. Subcortical spongy changes in the white matter of the brain in a

patient with CD.

Pathology of CD brain

Aspartoacylase, the enzyme deficient in Canavan

disease, hydrolyzes NAA to acetate and aspartate

(Fig. 2). Aspartoacylase is abundant in the white

matter of the brain, kidney and to a lesser extent inliver and other tissues. Aspartoacylase activity is lo-

calized in the white matter and NAA is synthesized in

the gray matter of the brain [9,34] and this compart-

mentation of substrate and enzyme in different regions

of the brain require a mechanism to transport NAA to

the site of the enzyme, the oligodendrocytes for its

normal metabolism [35]. The discovery of the enzyme

defect in CD indicates that normal metabolism ofNAA is important for the synthesis and maintenance

of healthy white matter. The level of NAA in the hu-

man brain is 8-mmol/g tissue [10]. The swollen astro-

cytes from the brain of a child with CD are shown in

Fig. 3. The increased levels of NAA in CD lead to

swelling or sponginess of the brain. The osmolite role

ASPA-OOC. CH2. CH. COO- + H2O -OOC. CH2.CH.COO- + CH3. COO-

HN.CO.CH3N-Acetylaspartic acid L-Aspartic acid Acetate

Fig. 2. Aspartoacylase (ASPA) hydrolyzes N-acetylaspartic acid

(NAA) to acetate and aspartic acid. Deficiency of ASPA leads to

accumulation of NAA.

of NAA and its lack of hydrolysis in CD lead to water

accumulation in the brain [36,37]. The mitochon-dria also gets distorted and elongated in CD brain

[13,38,39] suggesting that energy metabolism may be

affected.

Knock-out mouse for CD

The mouse 129/SvJ ASPA gene was cloned in ourlaboratory. The ASPA coding sequence in mouse is

approximately 86% identical to the human ASPA

cDNA sequence in the ORF region [20]. The longest

uninterrupted ORF in the cDNA is 936 bases that

predicted 312 aminoacids residues of mouse ASPA

protein, while 313 aminoacids are observed in human

ASPA protein. Deletion of 10 bp from exon 4 of the

mouse ASPA cDNA was accomplished and followedby Cre-mediated recombination. These experiments

resulted in a knockout mouse for Canavan disease

[9].

Spongy degeneration observed by MRI and peaks

of NAA in the CD mouse brain analyzed using MRS

Fig. 4. The proton spectra of brain extracts of (A) Canavan and (B) wild type mice. The peak areas are creatine, NAA, glutamate, and GABA. In the

wild type brain, GABA and glutamate levels are higher than Canavan mice. The NAA level is high in the Canavan mouse brain.

S. Surendran et al. / Molecular Genetics and Metabolism 80 (2003) 74–80 77

are shown in Fig. 4. This type of signal intensity and

elevated NAA is similar to the white matter changes

observed in patients with CD. The vacuolation of the

white matter in the deep cortex and white matter

bundles in the corpus striatum was found in the CDmouse [9] and can be observed in patients with CD.

Vacuolation in the brain of CD mouse is shown in

Fig. 5. Urine NAA is approximately 10-fold higher in

CD mice compared to the wild type [9]. The

knockout mouse for CD showed higher bone mineral

loss compared to the wild type of similar age [40].

This is probably associated with the muscle weakness

observed in the mouse with CD.Analysis of CD mouse brain revealed abnormal

expressions of serine proteinase inhibitor (Spi2), the

GABA-A receptor-GABRA6, neurogenic differentia-

tion factor, genes involved in inflammatory reaction

and cell death and the glutamate transporter, EAAT4.

While glutamate, EAAT4, c-amino butyric acid

(GABA) and GABRA6 levels were down regulated inthe CD mouse brain, Spi2 level was increased [41]

(Table 1). The peaks of glutamate and GABA are

shown in Fig. 4. The abnormal expression of these

genes in the cerebellum of the brain, may be respon-

sible for hypotonia and muscle weakness observed in

CD [41]. These observations need to be studied in

humans with CD. Aspartate aminotransferase was

also lower in the CD mouse brain [40]. These studiessuggesting involvement of multiple genomic interac-

tions in the pathophysiology observed in CD.

Fig. 5. Spongy degeneration in the subcortex of the CD mouse brain.

Vacuoles are seen in the CD mouse brain, while no vacuoles are found

in the wild type.

78 S. Surendran et al. / Molecular Genetics and Metabolism 80 (2003) 74–80

The brain pathology in CD is more complex than just

NAA accumulation. Thus the creation of the CD mouse

should give insight in the changes of the brain resulting

in the CD phenotype.

Table 1

Microarray expression and quantitative analysis in the brain of knockout m

GenBank Accession No. Fold Genes

(A) Microarray expression analysis showing abnormal gene expression

Cell growth/signal transduction genes

AJ222970 119.1# GABRA6

U28068 7.0# Neurogenic diff

factor mRNA

D83262 9.7# Glutamate tran

EAAT4 mRNA

D10210 16.0# DD-Aminoacid o

mRNA

Cell death and inflammatory genes

M64086 29.8" Spi2 proteinas

mRNA

Y13089 4.4" Caspase-11 mR

L28095 3.8" IL-1b converti

(B) Quantitative analysis showing abnormal levels of glutamate, GABA,

Real-time RT-PCR

GABRA6 Spi2

Knockout mouse >2800-fold# 6-fold"Brain (<10 copies/lg RNA)

Arrows shows: ", higher; #, lower in the knockout mouse brain.

Gene therapy

Gene therapy for CD was first carried out with

plasmid containing ASPA gene prepared by incorpo-

rating 145 bp inverted terminal repeats (ITRs) from

AAV and the LPD/pAAV-ASPA complex was injected

in the ventricles of two patients with CD [42]. Even after

a 1-year period, efficacy of the treatment was retained in

one patient by reducing NAA level to normal range. TheMRI study on the patient suggested new myelination of

the corpus callosum as well as basal ganglia and the

posterior limb of the internal capsule after 9 months of

treatment [42]. The other treated patient showed normal

level of NAA in the occipital lobe for 9 months. How-

ever, improvement of myelination was not observed

after 9 months [42]. Currently, there is a protocol for the

use of rAAV2-ASPA to treat with patients with CD [43].The knockout mouse for CD is being used for ex-

perimentation with gene transfer. The rAAV2-ASPA

was used for injection into the striatum and thalamus of

the brain of the knock-out CD mouse and the efficacy

was studied until 5 months period after treatment. The

mouse showed less sponginess and reduction in the el-

evation of NAA beyond the injected site as examined by

MRI/MRS [44,45]. The ASPA activity increased afterAAV mediated ASPA gene transfer and activity re-

mained even 5 months after injection while the site of

rAAV2-GFP injected mice did not result in any change

in sponginess or ASPA activity [44,45]. Although the

improvement of brain histology extends beyond the site

of injection, remote areas such as cerebellum are not

affected by rAAV-ASPA.

ouse for CD

erentiation

sporter

xidase

e inhibitor

NA

ng enzyme

and Spi2

Biochemical assay

of glutamate

NMR spectra

of GABA

5-fold# 67%#

S. Surendran et al. / Molecular Genetics and Metabolism 80 (2003) 74–80 79

Conclusion

Since the discovery of basic defect for CD, substantial

studies have been made in the identification of the gene

for ASPA and mutations that lead to CD. Thus there is

a clear molecular mechanism for diagnosis and preven-

tion. Studies at the molecular level using the knockout

mouse reveal information about the complexity of this

monogenic trait and the resulting phenotype. Genetransfer in the knockout mouse is an important step for

human gene therapy of CD.

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