Atypical MELAS
Associated With
Mitochondrial
tRNALys Gene
A8296G Mutation
Ryoichi Sakuta, MD*,
Shiho Honzawa, MD*,
Nobuyuki Murakami, MD*,
Yuichi Goto, MD†, and Toshiro Nagai, MD*

We report on a unique patient with mitochondrial
myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) presenting optic atrophy, cardiomyopathy, and bilateral striatal necrosis before
stoke-like episodes became apparent. Skeletal muscle
total mitochondrial DNA analysis identified a heteroplasmic A to G point mutation in the tRNALys gene at
position 8296. Skeletal muscle pathology revealed typical MELAS findings, including ragged-red fibers cytochrome c oxidase positive strongly succinate dehydrogenase-reactive blood vessels. Recent reports
describe the 8296 mutation identified in patients with
diabetes mellitus or myoclonus epilepsy with raggedred fibers, not MELAS. We conclude that the 8296
mutation is likely to be pathogenic and that it may be
not only a mutation responsible for diabetes mellitus or
myoclonus epilepsy with ragged-red fibers but also for
MELAS. © 2002 by Elsevier Science Inc. All rights
reserved.
Sakuta R, Honzawa S, Murakami N, Goto Y, Nagai T.
Atypical MELAS associated with mitochondrial tRNALys
Gene A8296G mutation. Pediatr Neurol 2002;27:
397– 400.

From the *Department of Pediatrics; Koshigaya Hospital; Dokkyo
University School of Medicine; Saitama, Japan; and the †Department
of Clinical Laboratory; National Center Hospital for Mental, Nervous
and Muscular Disorders; National Center of Neurology and Psychiatry;
Tokyo, Japan.

© 2002 by Elsevier Science Inc. All rights reserved.
PII S0887-8994(02)00456-3 ● 0887-8994/02/$—see front matter

Introduction
Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) are characterized
by recurrent neurologic deficits including seizures, headache, dementia, and psychomotor retardation that initiate
in childhood. Although symptomatic heterogeneity among
MELAS patients has been known, the combination of
chronic progressive external ophthalmoplegia, hearing
loss, and diabetes mellitus is shared by most of them [1].
It is rare to have optic atrophy of early childhood onset
with cardiomyopathy and bilateral striatal necrosis before
stoke-like episodes become apparent.
We performed direct sequencing of all mitochondrial
DNA (mtDNA) and identified the A8296G substitution in
the tRNALys gene. In recent reports the A8296G mutation
has been identified in patients with diabetes mellitus and
myoclonus epilepsy with ragged-red fibers (MERRF).
Herein we report on the first patient with atypical MELAS
associated with the A8296G mutation and its causal link to
the clinical features.
Case Report
A 14-year-old Japanese male was born by normal spontaneous
delivery at 38 weeks of gestation weighing 2,675 gm. His mother, 39
years old, was healthy, but his 65-year-old maternal grandmother had
hearing loss and noninsulin-dependent diabetes mellitus. In his prenatal
and perinatal periods and through early infancy, no abnormal characteristics were observed. At 4 years of age, he temporarily lost central vision
in both eyes without noticeable pain. However, the loss of vision was
transient, lasting for a few days; his visual acuity was limited to
perceiving hand movements in both eyes. The optic disc pallor developed
after a few months, and he was thus diagnosed as having optic atrophy.
His visual acuity was gradually reduced. Hypertrophic cardiomyopathy
was found at a school check-up when he was 6 years of age. However,
he did not develop heart failure. Then he developed persistent fatigue and
weakness of skeletal muscles between 8 and 9 years of age. At that time,
he was noted to have short stature, sensorineural hearing loss, a variable
speech disorder, poor short-term memory, and clumsiness. A computed
tomography brain scan revealed a low-density area in bilateral striatal
lesions. At 14 years of age, he had an episode of recurrent headache with
nausea accompanied by focal motor and generalized epileptic seizures
and lactic acidosis. He was referred to our hospital for further examination, including muscle biopsy, biochemical analysis, and DNA analysis.
On admission, physical examination revealed short stature and body
weight both below the second percentile. His pubertal development was
normal. General examination findings were otherwise unremarkable.
Optic atrophy was observed, but there was no retinal pigmentation. He
had myoclonus with hypertonicity. There was no objective sensory
deficit. Brain magnetic resonance imaging demonstrated diffuse cortical

Communications should be addressed to:
Dr. Sakuta; Department of Pediatrics; Koshigaya Hospital; Dokkyo
University School of Medicine; 2-1-50 Minamikoshigaya; Koshigaya;
343– 8555, Saitama, Japan.
Received February 26, 2002; accepted May 28, 2002.

Sakuta et al: Atypical MELAS with A8296G Mutation 397

DNA Analyses
Total DNA was extracted from a part of the muscle biopsy specimen.
DNA sample was analyzed for the mutations previously identified in
MELAS or MERRF, as described [2-6]. For sequencing analysis, 12 sets
of primers were used to amplify the DNA fragments covering all of the
mitochondrial tRNA regions. Polymerase chain reaction products were
purified and their nucleotide sequences determined by an ABI 377
Automatic Sequencer (Perkin Elmer Biosystem, Foster City, CA) using
the DNA Sequencing Kit and the Dye-Terminator Protocol (Perkin Elmer
Biosystem, Foster City, CA).
Based on the sequencing result a simple screening method was devised
to detect the A8296G mutation. This method utilizes an AciI restriction
site that is only present in the mutated allele. The mtDNA fragment
surrounding the tRNALys was amplified by polymerase chain reaction
using a sense primer, 5⬘;-AACCAAACCACTTTCACCGCT-3⬘; (8123–
8143), and an antisense primer, 5⬘;-CATACGGTAGTATTTAGTTGG3⬘; (8391– 8371). The polymerase chain reaction was performed in a total
volume of 5 ␮L containing 5 ng of genomic DNA, 50 nM of two primers,
62.5 ␮M of dNTP, 50 mM KCl, 10 mM Tris-HCl, 2.0 mM MgCl2, and
0.25 U of the Taq DNA polymerase. The polymerase chain reaction was
performed for 30 cycles consisting of 15 seconds denaturation at 94°C,
15 seconds annealing at 50°C, and 30 seconds primer extension at 72°C
in a Thermal Cycler (Perkin-Elmer Biosystem, Foster City, CA).

Figure 1. Axial T1-weighted brain magnetic resonance image (TR ⫽
400 ms, TE ⫽ 15 ms) is presented. Note the diffuse cortical atrophy and
bilaterally symmetric lesions (low-intensity area) of the putamen and
caudate nucleus.

atrophy and bilaterally symmetric lesions at the putamen and caudate
nucleus (Fig 1). Echocardiography revealed hypertrophy of the anterolateral and posterior walls of the left ventricle (Fig 2). We treated the
patient with cytochrome c and dichloroacetate for mitochondrial dysfunction, without significant effects.
Informed consent from the patient and patient’s mother was obtained
for all investigations.

Muscle Biopsy
We obtained biopsied muscle from the left biceps brachii muscle.
Serial frozen sections were stained with hematoxylin and eosin, modified
Gomori trichrome, succinate dehydrogenase (SDH), cytochrome c oxidase (COX), and a battery of histochemical methods.

Figure 2. Echocardiography reveals hypertrophy of the anterolateral
and posterior walls of the left ventricle at 14 years of age (parasternal
long-axis view).

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Results
Biochemical and Histologic Characteristics
Routine hematologic and biologic investigations were
indistinguishable from normal. Glucose tolerance test was
normal. However, lactate levels of fasting blood and
cerebrospinal fluid were elevated to 40 mg/dL and 80
mg/dL (normal range ⫽ 3.3–14.9 mg/dL), respectively.
The muscle fibers varied in size from 20 to 80 ␮m in
diameter. Some ragged-red fibers (RRFs) and strongly
SDH-reactive blood vessels (SSVs) were recognized. All
the muscle fibers were COX positive. RRFs and SSVs
demonstrated increased levels of COX activity (Fig 3).
Enzymes involved in mitochondrial respiratory chain were
not analyzed.
DNA Analyses
The DNA sample was screened for the mutations in
mitochondrial tRNA genes that were previously found to
be associated with MELAS or MERRF. They included the
3243, 3271, 3291 mutation of the tRNALeu(UUR) gene, and
the 8344 and 8363 mutation of the tRNALys gene [2-6].
However, none of these were detected. Sequence analysis
identified a number of nucleotide substitutions, A1438G,
T2626C, A2706G, C2772 torr, G3423 torr, A4769G,
A4958G, G4985A, C7028 torr, A8296G, A8701G,
A8860G, T9540 torr, T11335C, C12705 torr, and
G14365C, in comparison to the Cambridge sequence.
With the exception of the A8296G mutation, these substitutions have been viewed as polymorphisms without
negative effects on function. Cleavage of the 268-bp
polymerase chain reaction product containing the mutated
region by AciI revealed a heteroplasmic proportion (Fig 4).
With this simple and specific screening method, the

Figure 3. Frozen cross-sections are illustrated with cytochrome c
oxidase (COX) stain. There was a mild variation in fiber size. Some
ragged-red fibers (RRFs)(多) and strongly SDH-reactive blood vessels
(SSVs)(4) were recognized. All the muscle fibers were COX positive.
RRFs and SSVs demonstrated increased levels of COX activity (bar ⫽ 50
␮m).

A8296G mutation was not detected in 100 normal control
DNA samples.
Discussion
The diagnosis of MELAS was determined for this
patient based on the following: (1) stroke-like episodes
with recurrent headache and vomiting, (2) high lactate
level presenting both in blood and cerebrospinal fluid, (3)
muscle biopsy findings including RRFs and COX-positive
SSV. The presence of COX-positive SSV is a characteristic vascular finding for MELAS. In MERRF, SSVs also
appear in muscle specimens, but COX activity in SSVs is
almost negative [1].

Figure 4. The A8296G mutation and normal alleles of the tRNALys gene
detected by polymerase chain reaction followed by AciI digest are
depicted. The DNA fragments were stained with ethidium bromide and
visualized using an ultraviolet light transilluminator. The polymerase
chain reaction products from the mutant mtDNA of the patient (lane P)
and wild type of mtDNA in control subjects (lane 1-9) were fractionated
by electrophoresis through a 3% Nusieve agarose gel. The larger DNA
fragment of 268-bp in size is derived from the nonmutated allele. The
mutated allele contains an AciI site and thus generates two smaller
fragments (173 and 95 bp) by an AciI cleavage. The proportion of the
two alleles in the patient DNA sample indicates a characteristic of a
heteroplasmic mutation. M lane is DNA size marker.

The current patient is unusual in that the symptoms are
accompanied by optic atrophy, cardiomyopathy and bilateral striatal necrosis [7], which appeared in early childhood before the onset of stoke-like episodes. Ophthalmologic impairments usually observed in MELAS are
pigmentary retinopathy, ophthalmoplegia, and ptosis [8].
However, optic atrophy observed in this patient has been
rarely reported in association with MELAS [9] and thus
makes this patient unique. Pulkes et al. reported on a
patient having combined syndromes of Leber’s hereditary
optic neuropathy (LHON) and MELAS [10]. A recent
report described a family in which the affected members
had late-onset optic atrophy, ataxia, and myopathy associated with a mutation of a complex II gene. These patients
did not have stroke-like episodes [11]. LHON cause of
permanent blindness in otherwise healthy young adults is
more common in men. LHON is mostly associated with
mtDNA point mutation in the mitochondrial complex I
subunit genes ND1, ND4, and ND6 [12]. Our patient
developed blindness in his early childhood; however the
loss was only temporary. Because of the disparity of
symptoms, our patient was not diagnosed as LHON.
None of the mutations previously associated with
MELAS were detected in our patient, and sequencing of
all mtDNA was necessary to identify any potentially
causal mtDNA mutation. Extensive sequence analysis of
mtDNA identified only one nucleotide substitution,
A8296G, in the tRNALys gene of a MELAS patient who
presented with an unusual combination of symptoms.
More than 80% of MELAS patients are found to have a
single nucleotide substitution at position 3243 from A to G
(A3243G mutation) in the tRNALeu(UUR) gene of the
mitochondrial genome [1]. Mutations at position 3251,
3256, 3271, and 3291 of the tRNALeu(UUR) gene may also
be responsible for various forms of MELAS [3,4]. Thus
the tRNALeu(UUR) gene may be a genetic hot spot with a
specific link to MELAS.
In contrast, many of the mitochondrial DNA mutations
in tRNALys are associated with MERRF [5,6]. Its unique
link to and the atypical nature of clinical features imply
that the mutation is pathogenic. This notion is supported
by the following three additional factors: (1) the mutation
is heteroplasmic, (2) the mutated site is located in the
aminoacyl acceptor stem of tRNALys, in which the wild
type of nucleotide adenine is highly conserved between a
diverse range of species, and (3) the mutation is not
present in 100 healthy control subjects.
The A8296G mutation may be a part of the complex
cause of diabetes, and 1% of diabetic patients have this
particular mutation [13]. Moreover, a recent report documented a double mutation, A8296G and G8363A, being
associated with myoclonus epilepsy with MERRF [14]. In
this context, because the 8296 mutation was nearly homoplasmic, the authors concluded that G8363A mutation
is pathogenic; the co-occurrence of the 8296 mutation is of
unclear significance and is likely to be a rare polymorphism. However, this 8296 mutation alters the double

Sakuta et al: Atypical MELAS with A8296G Mutation 399

helix of the stem and secondary structure of tRNA. This
effect is the impairment of the activities of aminoacyltRNA synthase and peptidil-tRNA hydrolase, which play a
central role in the mitochondrial translation process. Thus
this study reports the first patient of MELAS associated
with the A8296G mutation.
We conclude that the A8296G mutation is likely to be
pathogenic and may have a causal link not only to diabetes
mellitus and MERRF but also to MELAS.

We express our thanks to Dr. Ikuya Nonaka (National Center Hospital for
Mental, Nervous and Muscular Disorders), Dr. Kyoko Koishi (University
of Otago, Dunedin), and Dr. Kazuo Obata (Dokkyo University School of
Medicine, Saitama) for their helpful suggestions and advice.

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