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1090-3798/$ - see frodoi:10.1016/j.ejpn.20

�Corresponding autE-mail address: s

Official Journal of the European Paediatric Neurology Society

Case study

Increased level of N-acetylaspartylglutamate (NAAG) in theCSF of a patient with Pelizaeus-Merzbacher-like diseasedue to mutation in the GJA12 gene

Stefano Sartoria,�, Alberto B. Burlinab, Leonardo Salviatic, Eva Trevissonc, Irene Toldoa,Anna Maria Laverdaa, Alessandro P. Burlinad

aDepartment of Pediatrics, University of Padua, Via Giustiniani, 3, 35128 Padova, ItalybMetabolic Unit, Department of Pediatrics, University Hospital, Padua, ItalycClinical Genetics, Department of Pediatrics, University Hospital, Padua, ItalydDepartment of Neuroscience, Neurological Clinic, University Hospital, Padua, Italy

a r t i c l e i n f o

Article history:

Received 25 March 2007

Received in revised form

10 July 2007

Accepted 30 July 2007

Keywords:

GJA12

PLP1

Pelizaeus-Merzbacher disease

Pelizaeus-Merzbacher-like disease

NAAG

nt matter & 2007 Europe07.07.011

hor. Tel.: +39 049 8218094tefano.sartori@unipd.it (

a b s t r a c t

Autosomal recessive Pelizaeus-Merzbacher-like disease 1 (PMLD1) is a hypomyelinating

disorder of the central nervous system (CNS) with virtually identical phenotype to

Pelizaeus-Merzbacher disease (PMD). PMLD1 is caused by mutations in GJA12 gene, PMD

is due to mutations in PLP1 gene.

Elevated levels of N-acetylaspartylglutamate (NAAG), the most abundant peptide

neuromodulator in the human brain, have been recently reported in cerebral spinal fluid

(CSF) of patients with PMD.

Using capillary electrophoresis, we analyzed for the first time, the CSF from a girl with

PMLD1 and detected high concentrations of NAAG.

This finding confirms the hypothesis that NAAG may be involved in myelination-related

processes and can be considered as a useful diagnostic marker not only for patients with

the PLP1 related disorder, but also in those with Pelizaeus-Merzbacher like hypomyelinating

disease due to other defined genetic causes, such as PMLD1.

& 2007 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Autosomal recessive Pelizaeus-Merzbacher-like disease 1

(PMLD1-MIM 608804) is a disorder of myelination character-

ized by diffuse and symmetrical hypomyelination in the

brain, clinically undistinguishable from Pelizaeus-Merzba-

cher disease (PMD-MIM 312080). The phenotypes are virtually

identical: nystagmus, impaired motor development, hypotonia,

progressive spasticity, ataxia, choreoathetotic movements

and development delay.1 The MRI picture is characteristic,

an Paediatric Neurology

; fax: +39 049 8213509.S. Sartori).

showing an uniformly increased intensity of the white matter

signal in T2-weighted images, with inconstant high signal

intensity in the internal capsule, optic radiation and proximal

corona radiata in T1-weighted images. PMD is an X-linked

recessive disorder caused by mutations in the PLP1 gene

encoding the most abundant protein of CNS myelin, while

PMLD1 is an autosomal recessive disorder caused by mutations

in GJA12 that codes for a gap junction protein (also known as

connexin 46.6 or connexin 47) which is highly expressed in

oligodendrocytes.2–4

Society. Published by Elsevier Ltd. All rights reserved.

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N-acetylaspartylglutamate (NAAG) is the most abundant

dipeptide in the mammalian central nervous system (CNS).5 It

is synthesized from N-acetylaspartate (NAA) and glutamate

in neurones and is catabolized in the extracellular space by

astrocyte-membrane bound NAAG dipeptidases.6,7 In vivo

proton magnetic resonance spectroscopy demonstrated that

NAAG concentrations in the human brain are higher in white

matter than those found in gray matter.8 Furthermore, it has

been reported that NAAG levels increase in white matter

during childhood.9

High concentrations of NAAG have been detected in the

cerebral spinal fluid (CSF) of patients with PMD, but also in

patients with a genetically undefined hypomyelinating phe-

notype resembling PMD.10–12

2. Case study

A detailed clinical history and the molecular analysis of the

patient have already been reported elsewhere.4 She is a

5-year-old girl with a clinical, neuroradiological and electro-

physiological picture highly suggestive of PMD. Duplication

and coding region mutations in the PLP1 gene were excluded

by real-time PCR and direct sequencing, whereas she

harbored a homozygous 34 bp deletion within the coding

region of the GJA12 gene [c. 914_947del].

We analyzed a CSF sample that had been previously

collected and stored at �80 1C during an initial work-up

performed—after obtaining parental informed consent—

before the molecular diagnosis had been established. The

amount of NAAG in the CSF was measured using a capillary

electrophoresis system—Beckman Coulter P/ACE MDQ, accord-

ing to Burlina et al. (1999).11 Our detection limit for NAAG was

0.66mmol/L, at a signal-to-noise ratio of approximately 10.

N-acetylaspartylglutamate concentration in the CSF of the

patient was 50mmol/L (normal value 0.73–12.68 mmol/L),

similar to the values found in PMD,10 whereas the NAA level

was normal.

3. Discussion

In 2000, using capillary electrophoresis, Burlina et al.10

reported the presence of high levels of NAAG, with normal

NAA values, in the urine and CSF of patients with PMD.

Subsequently, applying in vitro magnetic resonance spectro-

scopy, Wolf et al.12 found strongly elevated NAAG in CSF of

two unrelated girls with a hypomyelinating disorder resem-

bling severe PMD, but without PLP1 mutations. More recently,

using capillary electrophoresis, high concentrations of NAAG

and normal NAA levels were detected by Burlina et al.11 in the

CSF of seven patients with a phenotype virtually identical to

PMD, but without PLP1 mutations. Conversely, an elevation of

NAAG in the CSF has never been reported in any other

leukodistrophy,11 except for Canavan disease,13 where this

finding is likely to be a consequence of the NAA mass action

in the biosynthetic NAA–NAAG pathway, as recently demon-

strated in the human neuroblastoma cell line where the

addition of unlabeled NAA to the culture media resulted in a

dose-dependent increase in NAAG synthesis from radiola-

beled precursors.6

The increase of NAAG in the CSF in these patients does not

have a clear explanation yet. Even though it has been

hypothesized that the occurrence of axonal degeneration

and neuronal loss might underlie the late-onset, progressive

neurological deterioration of patients with PLP1 mutation,14,15

it also possible the high level of NAAG is not secondary to

neuronal damage or loss, since NAA values are still normal in

the patients described by Burlina and Wolf. The increase in

NAAG could rather be explained by a reduced function of the

NAAG-degrading enzyme on the astrocyte membranes,

secondary to the abnormal or severely reduced myelination.12

Our case is, to our knowledge, the first report of a patient

with a genetically defined Pelizaeus-Merzbacher-like disease,

caused by a mutation in the GJA12 gene, showing increased

levels of NAAG.

This finding confirms that the phenotype of GJA12-mutant

patients is indistinguishable to PMD, not only for the clinical,

electrophysiological and neuroradiological aspects but

also for some biochemical features, namely NAA and NAAG

levels.

Such phenotypic similarity allows us to hypothesize that, in

spite of genetic heterogeneity, a common pathogenic process

leads to the arrest of myelination in these diseases, support-

ing the idea that connexin 47 and PLP1 are part of a well-

orchestrated myelinogenic program.3,16 The disruption of this

program could alter the tricellular metabolic cycle (which

includes NAA, NAAG and glutamate) operating between

neurones, oligodendrocytes and atrocytes, causing elevated

NAAG elevation, subsequently detectable in the CSF of these

patients.12,17 Interestingly, the recently reported possibility of

in vivo differentiation of NAA from NAAG by in vivo magnetic

resonance spectroscopy at 3 T,18 will help to better under-

stand the relationship between the biochemical findings and

the molecular aspects and the role of PLP1, GJA12 and other

genes in the myelination process.

Further, although the link between altered NAAG and

dysmyelination remains to be fully elucidated, our finding

provides additional evidence for NAA and NAAG as having

roles in axonal physiology, axon–glial interactions, and the

production and maintenance of the myelin sheath, both in

normal myelinogenesis and in dysmyelination, as pointed out

recently by Moffet et al.19

In conclusion, we believe that the measurement of NAAG in

the CSF can become a useful biochemical marker for the

investigation of hypomyelinating disorders. It can address

more precisely towards the diagnosis not only of PMD but also

of Pelizaeus-Merzbacher like hypomyelinating disease due to

other defined genetic causes, such as GJA12-related PMLD

(PMLD1).

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