Brain & Development 25 (2003) 362–366
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Case report

A case of megalencephalic leukoencephalopathy with subcortical cysts
(van der Knaap disease): molecular genetic study
Harumi Saijoa,*, Harumi Nakayamaa, Takanori Ezoea, Katsuhito Arakia, Sui Sonea,
Hiroshi Hamaguchia, Hisaharu Suzukia, Naohide Shiromab, Naomi Kanazawab, Seiichi Tsujinob,
Yoshito Hirayamaa, Masataka Arimaa
a

b

Tokyo Metropolitan Higashiyamato Medical Center for the Severely Disabled, 3-44-10 Sakuragaoka, Higashiyamato, Tokyo 207-0022, Japan
Department of Inherited Metabolic Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira,
Tokyo 187-8502, Japan
Received 8 July 2002; received in revised form 5 November 2002; accepted 10 December 2002

Abstract
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder characterized by macrocephaly,
deterioration of motor function with ataxia, spasticity and mental decline. It has been revealed that the mutations in the gene, KIAA0027,
were responsible for MLC and the gene was renamed subsequently ‘MLC1’. A 41-year-old Japanese male with MLC, in whom a
homozygous missense mutation, TCG to TTG at codon 93 resulting in S93L, was detected in the MLC1 gene, was described. MRI revealed
marked cerebral atrophy and enlargement of the ventricular system. The subject’s motor function had severely deteriorated, while his
cognitive function had maintained at the level of a 2-year-old for the past 10 years. The mutation in the MLC1 gene of the patient is
considered to be a common mutation responsible for MLC in Japanese patients because the same mutation had been detected in two other
Japanese patients with MLC.
q 2003 Elsevier Science B.V. All rights reserved.
Keywords: Leukoencephalopathy; Megalencephalic leukoencephalopathy with subcortical cysts; Mutation; MLC1 gene

1. Introduction
Megalencephalic leukoencephalopathy with subcortical
cysts (MLC; MIM604004) is a neurological disorder
characterized by a macrocephaly noticed within the first
year of life and a delayed onset of the deterioration of motor
functions with ataxia and spasticity [1]. Recently, Leegwater et al. found that several types of mutation in the gene
(KIAA0027), which had been considered to be one of the
genes responsible for MLC, were definitely responsible for
MLC and they renamed the gene ‘MLC1’ [2].
We followed a 41-year-old male with MLC and a
missense mutation (a substitution of thymine for cytosine at
the nucleotide position of 393, 393C . T) was detected
within the MLC1 gene, resulting in a single amino acid
substitution of leucine for serine at the amino acid position
93 (S93L). We describe here a long survived patient with
* Corresponding author. Tel.: þ81-42-567-0222, x 380; fax: þ 81-42567-0224.

MLC diagnosed not only by clinical manifestations but also
by molecular investigation.

2. Case report
The patient was a 41-year-old Japanese male, the third
child to healthy and non-consanguineous parents. Both his
mother and father originated from the same prefecture. He
was born after 37 weeks’ gestation without any complications weighing 3560 g. His head circumference was 34 cm
at birth. The early motor milestones were normal as follows;
he could control his head by 3 months of age, sit without
support by 7 months of age and started crawling by the age
of 8 months. He subsequently achieved the ability to take a
few steps without support at the age of 15 months. However,
he showed no further progress after that. The macrocephaly
became apparent by the age of 12 months. He started to
speak meaningful words at the age of 14 months and could
speak some sentences at the age of 2, but slowly. After the

0387-7604/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.
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H. Saijo et al. / Brain & Development 25 (2003) 362–366

age of 5, his ability to speak began to deteriorate and
thereafter he could only speak some words. The head
circumference was 55 cm (þ 2.5 S.D.) at the age of 6. He
became completely dependent on a wheelchair at the age of
11. The first seizure, a generalized tonic – clonic convulsion,
occurred when he was 11, but the seizures were kept under
control by the appropriate treatment with antiepileptic
drugs. He could not eat by himself after 10 years of age.
Furthermore, his ability to swallow deteriorated gradually
from the age of 15. He became bed ridden completely when
he was 18 years old. His cognitive function has remained at
the level of a 2-year-old for the past 10 years. Generalized
tonic –clonic convulsion occurs a few times a month. He
needs tube feeding after recurrence of aspiration
pneumonia.
Clinical findings at the age of 41 were as follows. His
height, body weight and head circumference were 166 cm,
43 kg and 61.5 cm, respectively. Eye movement was full in
all directions. The pupils were symmetric and responded
promptly to light stimulation. Horizontal nystagmus
appeared with lateral gaze. Gag reflex was absent. Although
he could rotate his head and flex and extend his bilateral
elbow and shoulder joints, he could not move voluntarily.
No muscle stretch reflexes were seen in the triceps,
brachioradialis, patellar and Achilles, but were detected
weakly in the biceps. The Babinski and Chaddock reflexes
were positive without fanning of the toes. Ankle clonus was
not observed. Intention tremor was not present. Bilateral
optic disks were atrophic. He could understand simple
words and express his intention by either opening his mouth
or closing his eyes.
Routine laboratory investigations were normal. Amino
acid levels were revealed to be normal in both urine and
blood samples. Pyruvate and lactate levels in the blood were

363

also normal. The activities of lysosomal enzymes in
leukocytes or fibroblasts were normal. The long fatty
acids were also confirmed to be within the normal range.
The a B crystalline level was normal in cerebrospinal fluid.
Chromosomal analysis revealed the presence of 46 XY
chromosomes.
Auditory evoked potentials in the brainstem revealed a
gradual elongation of the latency time between I and V
waves; 4.59 ms (þ 2.8 S.D.) and 4.86 ms (þ 4.2 S.D.) at the
ages of 33 and 41, respectively. Flash-visual evoked
potentials were normal. In short somatosensory evoked
potentials, cortical responses were absent, while the
peripheral and spinal components were normally observed.
Electroencephalography showed a slow a wave over the
occipital area during waking and a bilateral slow spike and
wave in the central and parietal areas during sleep.
Brain computed tomography (CT) images reveal a
progressive increase in the size of the cysts and the
enlargement of the ventricular system (Fig. 1A,B). The
magnetic resonance imaging (MRI) findings of the brain are
shown in Fig. 2 with explanations (Fig. 2A –D).
Total RNA was extracted from the peripheral leukocytes
of the patient using the Isogen kit (Nippon Gene, Tokyo,
Japan) under informed consent. First-strand cDNA was
synthesized using oligo dT and a Superscript Preamplification System (Life Technologies, Rockville, MD) according
to the manufacturer’s specifications. Then, three DNA
fragments encompassing the entire coding region of the
MLC1 cDNA was amplified by PCR using primers 1 (50 -AC
ACGTGGCTGTACATTCAG-30 ) and 2 (50 -TGGGTTCAG
GACTAGTTTGC-30 ) for fragment 1, primers 3 (5’ACGCCAATGTGATTCCCAAC-30 ) and 4 (50 -AGACGTGAGGCTGCTTATGG-30 ) for fragment 2, and primers 5
(50 -TGCCATTGCCAGTCATGTGG-30 ) and 6 (50 -TTGTG

Fig. 1. Brain CT findings at the ages of 25 (A) and 41 (B). A diffuse low density area is seen in the subcortical area (A and B). Large cysts are also seen in the
frontoparietal area and in the tips of the temporal lobes. A progressive increase in the size of the cysts and enlargement of the ventricular system is noticed.

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H. Saijo et al. / Brain & Development 25 (2003) 362–366

Fig. 2. Brain MRI findings at the age of 41: flair coronal images (A, C) and T2 -weighted axial images (B, D). Abnormal signal intensity is seen in the area of
subcortical white matter (A –D). Large cysts are observed in the bilateral frontoparietal areas and in the tips of the temporal lobes (A). The gyrus expands
generally but not so in occipital lobe (A, B). The subcortical fibers in the occipital lobe are spared slightly (B). In central white matter, there is some sparing of
structures. The corpus callosum is very thin but its intensity is normal. The anterior and partial posterior limbs of the internal capsule, and the periventricular
rim of the occipital white matter are preserved (B). Cortical and central gray matter structures are normal in signal intensity. The caudate nucleus and thalami
shows mildly atrophic changes (B). The cerebellum is atrophic and the central white matter shows abnormal signal intensity (C). The caudal part of the
posterior limb of the internal capsule and pyramidal tracts of the mesencephalon show abnormal intensity (B, D). The pons and medulla oblongata are normal.
Cavum septi pellucidi and vergae are present (B).

CGTTTCCATGCTTGG-30 ) for fragment 3 with Taq
polymerase (Nippon Gene). The PCR products were
directly sequenced using the DYEnamic ET Terminator
Cycle Sequencing kit (Amersham Pharmacia Biotech Inc,
Piscataway, NJ) and using an ABI Prism 310 Genetic
Analyzer (Applied Biosystems, Foster City, CA) according
to manufacturer’s instructions. Sequencing of the MLC1
cDNA from the patient revealed the presence of a 393C . T
mutation, resulting in a missense mutation of S93L (TCG to
TTG at codon 93) (Fig. 3A). Genomic DNA was isolated
from peripheral leukocytes of the patient and a control
individual. A genomic fragment containing exon 4 was
amplified by PCR with intronic primers 7 (50 -TTCAGATCCTTCTGGAAGCG-30 ) and 8 (50 -ACACTGTCTGTCAGCCCCTC-30 ), and sequenced by the same method as
described above. The same nucleotide substitution was also
detected by sequencing the genomic PCR fragment of the
patient. The PCR fragments from the patient and a control

were digested with the restriction endonuclease Sty I (New
England Biolabs, Inc., Beverly, MA), and analyzed using
electrophoresis in 3.0% NuSieve agarose (BMA, Rockland,
ME). This PCR-restriction fragment length polymorphism
confirmed the presence of the 393C . T mutation and
showed that the patient was homozygous for the mutation
(Fig. 3B,C). Because of this nucleotide substitution,
393C . T, the PCR product (210 bp) was cleaved into
two segments (106 and 104 bp), whereas the wild-type PCR
product was not cleaved (Fig. 3B,C).

3. Discussion
The formation of cysts was observed in the frontoparietal
and temporal lesions in the patient. On the other hand, there
is some sparing of structures in central white matter
including the corpus callosum, the anterior limb of the

H. Saijo et al. / Brain & Development 25 (2003) 362–366

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Fig. 3. Genetic study by sequencing the MLC1 cDNA (A) and RFLP analysis (B, C). A C-to-T transition at the nucleotide position 393 was detected (A, arrow).
A Sty I site was generated by this mutation (A). Schematic representation of segments of the mutant PCR fragment and of the wild-type PCR fragment digested
with Sty I (B). The genomic PCR products digested with Sty I were visualized at the position of around 100 bp in the lane of patient, whereas those of control
were at position 210 bp (C).

internal capsule, the periventricular rim of the occipital
white matter, partially the posterior limb of the internal
capsule and some of the subcortical fibers in the occipital
lobes (Figs. 1 and 2). The same findings have also been
reported in the patients with MLC [1,3,4,8,9]. Barkovich
et al. reported that myelination in frontoparietal and
temporal areas starts in the later stage of brain development
whereas myelination in basal ganglia, brian stem, corpus
callosum and subcortical white matter of occipital lobe
starts in the earlier stage [5]. We speculate that the
pathogenesis of MLC might associate with the stages of
myelination because cyst formation occurs in the areas
where myelination starts in the later stage of brain
development, while the areas where myelination starts in
the earlier stage are spared.
In other forms of leukoencephalopathy with megalencephaly such as Canavan disease and Alexander disease, there
are some changes in the size and/or intensity of basal
ganglia in addition to diffuse white matter abnormalities [6,
7], but not in cases of MLC [1,3,4,8,9]. Although there were
mildly atrophic changes in the basal ganglia and thalami in
the patient, the signal intensity was normal (Fig. 2A,B).
Furthermore, the cerebral atrophy and enlargement of the
ventricular system were marked.
To our knowledge, this case is the oldest patient with

MLC yet reported. Therefore, this report on the long clinical
course of MLC is of value. The clinical course during the
first decade of this patient was similar to that of young
patients previously reported [1,3,4,8,9]. However, motor
deterioration advanced more severely after the first decade
in this patient. Furthermore, he presented severe motor
deterioration at the age of 41, whereas his cognitive function
had remained at the level of a 2-year-old for the past 10
years.
van der Knaap and her colleagues described that vacuole
formation was only present within the outermost lamellae of
the myelin sheath, sparing the inner part in MLC [10]. They
speculated these features explain why the disease is
discrepantly mild in their early childhood [10]. But this
patient had shown severe motor deterioration since around
18 years old, and cerebral atrophy progressed. Therefore, we
suspect that more severe vacuole formations and subsequent
neuronal loss progress gradually and constantly in patients
with MLC.
The MLC1 gene encodes a putative membrane protein
with eight predicted transmembrane domains [2]. This
protein is considered to be important for the maintenance
and/or compaction of the outermost lamellae of the myelin
sheath.
A missense mutation, 393C . T (S93L), was detected in

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H. Saijo et al. / Brain & Development 25 (2003) 362–366

this patient and the patient was homozygous for the
mutation (Fig. 3). The same mutation was detected in two
other Japanese patients with MLC [2]. Therefore, this
mutation is thought to be one of the common types of
mutation in Japanese patients with MLC, although further
study is needed.
In summary, the clinical course in a Japanese 41-year-old
patient with MLC was presented, and the responsible
mutation in the MLC1 gene was identified.

Acknowledgements
We thank Dr Y. Ochiai, Tokyo Metropolitan Kita
Medical Center, Kita-ku, Tokyo, Japan, for his excellent
assistance in this study.

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