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MELAS associated with a mutation in the valine transfer RNA gene of mitochondrial DNA.

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specific. Our findings contribute to the hypothesis that
a still unknown cellular dysfunction may lead to the
selective vulnerability of the astroglial glutamate transport [IO]. In future studies, patients with a biochemically or immunohistochemically defined transport defect should be screened. Furthermore, quantitative
investigations of the transporter mRNA expression will
allow us to distinguish between a transcriptional and
posttranscriptional cause of the reported loss of transporter expression in ALS. Further work is required to
identify a functional role of each of the transporter
variants and their eventual differential expression in
normal and diseased tissue.
This work was supported by a research grant for Drs Ludolph,
Speer, and Meyer from the Deutsche Forschungsgemeinschaft
(grant Lu 336/5-1) and the Universitatsklinikum Charit6 at Humboldt University, Berlin.
We cordially thank Dr N. Cairns at the Medical Research Council’s
Alzheimer’s Disease Brain Bank for supporting the project by providing normal postmortem brain and spinal cord tissue. We are
grateful to Dr G. Kuther at the Medizinische Hochschule Hannover
and Dr A. Weindl at the Technical University, Munich, for providing ALS brain and spinal cord tissue.
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MELAS Associated with a
Mutation in the Valine
Transfer RNA Gene of
Mitochondria1 DNA
R. W. Taylor, PhD,* P. F. Chinnery, MRCP,* F.
Haldane,* A. A. M. Morris, MRCP,* L. A. Bindoff,
MRCP,* 1. Wilson, FRCP,? and D. M. Turnbull, FRCP*
We describe a patient with the mitochondrial myopathy,
encephalopathy, lactic acidosis, and strokelike episodes
(MELAS) phenotype in whom initial investigations in
skeletal muscle failed to show any histochemical or biochemical defect. Subsequent analyis of the mitochondrial
genome identified a new heteroplasmic mutation in the
valine transfer RNA gene, the first described in this region.
Taylor RW, Chinnery PF, Haldane F, Morris
AAM, Bindoff LA, Wilson 1, Turnbull DM.
MELAS associated with a mutation in the valine
transfer RNA gene of mitochondrial DNA.
Ann Neurol 1996;40:459-462
8. Rorhstein JD, Jin L, Dykes-Hoberg M , Kuncl R. Chronic inhibition of glutamate uptake produces a model of slow toxicity.
Proc Natl Acad Sci USA 1993;90:6591-6595
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transport by the brain and spinal cord in ALS. N Engl J Med
10. Rothstein JD, Van Kammen M, Levey AI, et al. Selective loss
of glial glutamate transporter GLT-1 in amyotrophic lateral
sclerosis. Ann Neurol 1995;38:73-84
11. Tandan R. Clinical features and differential diagnosis of classical motor neuron disease. In: Williams AC, ed. Motor neuron
disease. London: Chapman and Hall Medical, 1974:l-27
12. Chomczynski P, Sacchi A. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 1987;162:156-159
Over 80% of cases of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes
(MELAS) are associated with an A-to-G substitution
gene of the mitoat position 3243 in the tRNAhU(UUR)
From the *Department of Neurology, University of Newcastle upon
Tyne, Newcastle upon Tyne, and ?Great Ormond Street Hospital,
London, United Kingdom.
Received Nov 8, 1995, and in revised form Mar 25, 1996. Accepted
for publication Apr 1, 1996.
Address correspondence to Prof Turnbull, Department of Neurology, The Medical School, Framlington Place, Newcastle upon
Tyne, NE2 4HH, United Kngdom.
Copyright 0 1996 by the American Neurological Association 459
the third centile. H e had normal pubertal development and
chondrial genome (mtDNA) [ 1 ,
Skeletal muscle
general examination findings were otherwise unremarkable.
biopsy specimens from these patients usually contain
He had a severe global cognitive impairment but his speech
cytochrome c oxidase-negative fibers and a correwas normal. Cranial nerve examination was unremarkable,
sponding defect in mitochondria1 respiratory chain
he had full visual fields to confrontation, and there was no
function [3].There is, however, ploor correlation beretinal pigmentation. H e had myoclonus with normal petween genotype and phenotype in this condition: The
ripheral tone, a subtle pyramidal weakness in his right arm,
A3234G substitution may be associated with other
and brisk symmetrical tendon reflexes. Plantar responses were
phenotypes [ 4 ] ,and the MELAS syndrome may also
flexor and on formal testing he had mild gait and appendicuoccur in patients with mutations at positions 3251 [ 5 ] ,
lar ataxia. There was no objective sensory deficit.
3252 [d], 3271 [7],and 3291 [S] of the tRNALeU(UUR)
gene, and position 9957 [9] of the cytochrome oxidase
CLINICAL INVESTIGATIONS. Routine hematological and
subunit I11 (COX 111) gene.
biochemical investigations were unremarkable. A fasting
We describe a patient with the MELAS phenotype
blood lactate level was within the normal range but cerebrowho did not have histocytochemical abnormalities or a
spinal fluid (CSF) analysis revealed an elevated lactate level
of 9.23 mmol/liter and a moderately elevated protein conbiochemical respiratory chain defeci in skeletal muscle.
centration of 0.55 gmlliter. An EEG was normal, but T2Subsequent analysis of mtDNA revealed the wild-type
MRI revealed multiple areas of high signal intensity
sequence at the site of the recognized mutations associin
lobes, the left frontal lobe, and the left
ated with MELAS. Direct sequencing of all 22 mitoparietal lobe. These areas extended from the cortex to the
chondrial tRNA genes identified a single heteroplasmic
subcortical white matter and were consistent with infarction.
pathological base substitution in the tRNA valine gene.
INVESTIGATIONS. Morphological and histochemical fea-
Case History and Methods
Cuse Histov
A 16-year-old boy developed anorexia and weight loss from
the age of 9 years. Prior to that time he had been fit and well,
had achieved normal developmental milestones, and reported
average academic and sporting performance at school. He
developed persistent headaches between the ages of 9 and
10, noted intermittent involuntary twitching of both arms,
and subsequently had a tonic-clonic generalized seizure without provocation that lasted for 7 hours. Despite a full neurological recovery, he subsequently experienced intermittent visual hallucinations and a behavioral disturbance. An
electroencephalogram (EEG) was nornial at the age of 10'/2
years, but a cranial computed tomogram (CT) revealed a
low-attenuation lesion in the left occipital region. Biopsy was
performed based on suspicion of malignancy, but histological
findings were consistent with cerebral infarction.
From the age of 11 he experienced five episodes of transient right-sided weakness and on one (occasionhe lost vision
in both eyes for 12 days. Headache remained a prominent
feature but did not appear to have a temporal relationship
to these symptoms. He had no further episodes of disturbed
consciousness, but was noted to have a variable speech disorder, poor short-term memory, and clumsiness. Nocturnal enuresis became a problem and he developed progressive learning difficulties between the ages of 12 and 16. Magnetic
resonance imaging (MRI) of the brain, including MRI angiography and spectroscopy, revealed lesions consistent with
multiple cerebral infarcts, and he was maintained on anticonvulsant medication (clobazam 10 mg twice a day, carbamazepine 400 mg twice a day) and aspirin (300 mg daily).
His parents and 18-year-old sister had no relevant medical
history, but one of his maternal uncles was reported to be
deaf in one ear. Two of his cousins (hoth male and born to
separate maternal aunts) also had uniIatera1 deafness and a
female cousin had insulin-treated diabetes mellitus. There
was no consanguinity.
Examination revealed short stature and body weight below
460 Annals of Neurology
Vol 40
No 3
September 1996
tures of a muscle specimen obtained by routine needle biopsy
were within normal limits. There were no ragged-red or cytochrome c oxidase-negative fibers. Biochemical studies revealed normal activities of the individual respiratory chain
complexes I, 11, 111, and IV (results not shown).
Moleculur Genetic Investigutions
Total DNA was extracted from both skeletal muscle, blood,
and formalin-fixed brain. Southern analysis was performed,
and the sample screened for the 3243 mutation. The 22
tRNA genes were then sequenced directly [lo]. The identified mutation created a restriction site for the restriction endonuclease Mbo I1 in a polymerase chain reaction (PCR)amplified fragment containing the valine tRNA gene, which
was used to screen for the novel mutation. The level of mutant mtDNA w a s quantified by the last cycle hot-PCR technique (forward primer position 461 to 477,reverse primer
position 1790 to 1773, 30 cycles of PCR with [a-'*P]deoxycytidine triphosphate added for the last cycle). Mbo 11-digested products were run out on a 4% nondenaturing polyacrylamide gel and quantified using a PhosphorImager
(Molecular Dynamics).
No rearrangements were observed on Southern analysis
and the 3243 mutation was not identified. Direct sequencing of all 22 tRNAs revealed only one base pair
that differed from the published sequence [ I l l , a Gto-A substitution at position 1642 within the valine
tRNA gene (Fig 1 ) . The mutation was heteroplasmic
with 94% mutant DNA in muscle and 54% in blood
(Fig 2), and 95% in occipital cortex. Restriction digest
analysis also identified the mutation in blood taken
from the patient's mother and the level of mutant
DNA in this sample was 11%. Further restriction en-
Fig 2. Autoradiograph of Mbo II digest indicating the relative amounts of mutant and wild-type mtDNA. Lanes 1 and
5 = control muscle; 2 = blood from patient? mother; 3 =
patient ? blood; 4 = patient ? muscle.
zyme analysis did not detect the mutation in either 49
unrelated control subjects or 52 patients with mitochondrial disorders.
Fig 1. Sequencing chromatograms of a reverse complement
region of the tRNA ualine gene f i r (A) a control subject and
(B) the patient, ilhtrating a heteroplasmic point mutation at
position 1642 (arrowed).
The patient we have described illustrates the genetic
and phenotypic heterogeneity of the MELAS syndrome. Although our patient developed a progressive
encephalopathic illness associated with strokelike episodes leading to epilepsy and dementia, he did not have
a myopathy, nor did he have cytochrome c oxidasenegative fibers in the muscle biopsy specimen. Despite
94% mutant mtDNA, mitochondria isolated from the
muscle biopsy specimen showed normal respiratory
chain function. None of the mutations previously associated with MELAS were detected and sequencing of
all of the mtDNA tRNA genes was necessary to identify the valine tRNA mutation. To date this is the first
significant mutation to have been described in this
tRNA gene.
We believe the single-base substitution at position
1642 has pathological significance on four accounts.
Firstly, there was no evidence of any rearrangement or
other point mutations in our patient’s mitochondria1
Brief Communication: Taylor et al: MELAS due to mtDNA tRNA Valine Mutation
tRNA genes. In particular, nucleotides 3243, 325 1,
3252, 3271, and 9957 were identical to the published
wild-type mtDNA sequence [ 1 I]. Secondly, the mutation was heteroplasmic in the mtDNA extracted from
skeletal muscle, blood, and brain of the affected individual and was at the highest level in the worst affected
tissue. It was not present in muscle from 49 normal
control subjects and 52 patients with mitochondrial
disorders. Thirdly, the point mutation occurs in a region that shares significant homology with corresponding mtDNA sequences from other species [12]. Finally,
it is likely that the base substitution would alter the
synthesis of respiratory chain polypeptides within affected mitochondria. According to the accepted cloverleaf secondary structure of tRNAs, the mtDNA mutation we identified would result in a single-base
substitution in the anticodon stem o f the valine tRNA,
thus altering tertiary structure and function. Probable
pathogenic mutations in the anticodon stem have also
been described in other patients [7, 131, including a
base substitution at an analogous position to this valine
tRNA mutation [14].
This report highlights the difficulties in diagnosing
mitochondrial disorders and illustrates that patients
may have pathological mutations in genes not previously linked with a specific phenotype. Moreover, despite high levels of mutant mtDI\JA in muscle, this
patient did not have a myopathy, nor did he have any
of the morphological, histochemical, or biochemical
features of respiratory chain dysfunction. The strong
clinical evidence, coupled with the elevated CSF lactate
values, motivated our search for a genetic cause, leading
us to a mutation in a tRNA gene that has not been
previously linked to disease.
Financial supporr from the Wellconie Trust and the Muscular Dystrophy Group of Great Britain is acknowledged.
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462 Annals of Neurology Vol 40 No 3 September 1996
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associates, mutation, rna, transfer, dna, melas, genes, mitochondria, valine
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