Journal of the Neurological Sciences 296 (2010) 101–103

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Journal of the Neurological Sciences
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

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MELAS syndrome associated with both A3243G-tRNALeu mutation and multiple
mitochondrial DNA deletions
Sharon Aharoni a,d, Teres A. Traves b,d, Eldad Melamed b,d, Sarit Cohen c,d, Esther Leshinsky Silver c,d,⁎
a

Institute of Pediatric Neurology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel
Department of Neurology, Rabin Medical Center, Beilinson Campus, Petach Tikva, Israel
Molecular Genetics Laboratory, Wolfson Medical Center, Holon, Israel
d
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
b
c

a r t i c l e

i n f o

Article history:
Received 26 January 2010
Received in revised form 29 June 2010
Accepted 30 June 2010
Available online 22 July 2010
Keywords:
MELAS syndrome
mtDNA
Multiple deletions
tRNALeu

a b s t r a c t
The syndrome of mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS) is
characterized clinically by recurrent focal neurological deficits, epilepsy, and short stature. The phenotypic
spectrum is extremely diverse, with multisystemic organ involvement leading to isolated diabetes, deafness,
renal tubulopathy, hypertrophic cardiomyopathy, and retinitis pigmentosa. In 80% of cases, the syndrome is
associated with an AG transmission mutation (A3243G) in the tRNALeu gene of the mitochondrial DNA
(mtDNA).
We describe a woman with a unique combination of the MELAS A3243G mutation and multiple mtDNA
deletions with normal POLG sequence. The patient presented with diabetes mellitus, sensorineural deafness,
short stature, and mental disorientation. All her three children died in early adolescence.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
The mitochondrial encephalomyopathies due to defects in oxidative phosphorylation are genetically and clinically heterogeneous [1].
Mutations in different genes can lead to the same manifestation, and a
single mutation in one gene can lead to a variety of clinical
manifestations [1]. The syndrome of mitochondrial encephalopathy,
lactic acidosis, and stroke-like episodes (MELAS) is characterized by
recurrent focal neurological deficits, epilepsy, and short stature [2–5].
About 80% of patients carry an AG mutation (A3243G) in the tRNALeu
gene of the mitochondrial DNA (mtDNA). The prevalence of the
A3243G mutation is 1 in 6000 in the general population and higher in
specific populations [6]. The syndrome is progressive, and the
phenotypic spectrum is extremely diverse, with multisystemic
organ involvement leading to diabetes, deafness, renal tubulopathy,
hypertrophic cardiomyopathy, and retinitis pigmentosa, alone or in
combination. The clinical variability can be partly explained by tissue
specificity and mutant load, but other factors are probably involved as
well [2–4].
MELAS syndrome has a high morbidity and mortality. The
encephalopathy may lead to severe disability and premature death.
Cardiomyopathy and renal failure may also account for the high
mortality.

⁎ Corresponding author. Wolfson Medical Center, Holon 58100, Israel. Tel./fax: + 972
3 502 8543.
E-mail address: leshinsky@wolfson.health.gov.il (E.L. Silver).
0022-510X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jns.2010.06.029

We describe a unique combination of MELAS A3243G mutation
and multiple mtDNA deletions with normal POLG sequence in an
adult woman, whose all three children died in early adolescence.
2. Case presentation
A 52-year-old woman was admitted for evaluation of confusional
state. Past medical history was remarkable for diabetes mellitus type
2 and hypertension. Sensorineural deafness had been documented
3 years prior to the present admission. General physical examination
demonstrated short stature and normal vital signs. The patient was
alert and oriented in time and place. The neurological examination
revealed no abnormalities other than anomic aphasia. The patient
reported difficulty in finding words and had lost the ability to name
people and objects. Laboratory results were within normal range. At
that stage, no other medical or family history data were revealed
because of the patient's disorientation. Suspecting that a lesion in
the temporal lobe accounted for the language disorder, brain
computer tomography (CT) was performed, which indeed showed
a temporal hypodense area with gyral enhancement. The initial
differential diagnosis included stroke, proliferate or inflammatory
disease, herpes infection, or lymphoma. Cerebrospinal fluid findings
were normal; polymerase chain reaction (PCR) testing for herpes
was negative. Magnetic resonance imaging (MRI) demonstrated
cortical hyperintensity of the temporal and occipital lobes with
restricted diffusion.
Over the next few days, the patient's condition deteriorated. She
became disoriented in time and place and experienced a focal

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convulsion. Intravenous phenytoin was initiated. The electroencephalogram showed background slowing. New data obtained from the
patient's family revealed that her three children had all died in early
adolescence. The children's medical files revealed developmental delay,
cardiomyopathy, hearing loss, and recurrent stroke-like episodes.
MELAS was the possible diagnosis. More attention was now directed
to the patient's family history of short stature, hearing loss, and
diabetes. Additional work-up revealed the following findings: blood
lactate — normal; vision-evoked potentials — normal; brain auditory
evoked potentials — abnormal. The patient was referred for genetic
testing for the most frequent MELAS mutation. Pending the results,
however, there was a further deterioration in her condition, and to rule
out brain lymphoma, a brain biopsy was performed.
Over the next months, the patient's condition continued to worsen
progressively. Her mental status and cognition decreased, and she had
focal seizures, dysphagia, and recurrent pneumonia. A tracheotomy
was inserted. The patient was discharged to a nursing institution and
died 18 months later of pneumonia and hypotension. Autopsy was not
performed.
Unfortunately, no biological tissue of the children was preserved
for genetic analysis.
3. Molecular analysis
Total DNA was extracted from blood and brain using the Puregene
Kit (Gentra, Minneapolis, USA), according to the manufacturer's
instructions. The A3243G mutation was detected by PCR amplification
with primers corresponding to mtDNA at positions 3160–3550. The
amplified product (400 bp) was digested with ApaI and run on 3%
agarose gel. Mutant is digested to 307 bp + 93 bp, while wild type is
undigested. To evaluate mutant load, the proportions of digested
(mutant) and undigested (wild type) PCR product were determined
using the ImageQuant program (Molecular Dynamics, Sunnyvale, CA)
following ethidium bromide staining.
Southern blot analysis was performed as follows: five-microgram
samples of total DNA were subjected to 24-hour digestion with PvuII
(New England Biolabs, Beverly, MA, USA), as recommended by the
manufacturer, separated by electrophoresis on 0.8% agarose gel, and
transferred to a N+ nylon membrane. The membrane was hybridized
with 100 ng of a 519 bp PCR product of mtDNA (expanding
nucleotides 4981–5500). The probe was labeled by DIG dUTP
incorporation during a PCR procedure using a PCR DIG Probe
Synthesis Kit (Cat. No. 1636090, Roche, Basel, Switzerland). PCR
conditions were at 95 °C for 7 min, (1 cycle), 30 cycles of 95 °C for
30 s, 55 °C for 45 s and 72 °C for 90 s, and a final elongation step at
72 °C for 10 min. Hybridization was performed at 65 °C. The blot was
washed twice with 2 × SSC, 0.2% SDS and once with 1 × SSC, 0.1% SDS.
The hybridized probe was immunodetected with anti-digoxigenin Fab
fragments conjugated to alkaline phosphatase and visualized with a
chemiluminescence substrate (CDP star), according to the manufacturer's instructions (Cat. No. 1363514, DIG Luminescent Detection Kit,
Roche). A DIG-labeled 48-kb ladder was also used for fragment sizing
(#301-9L, Lambda DNA Mono Cut Mix, New England Biolabs, Cat, No.
1836463, Chem-Link Labeling and Detection Kit, Roche), exposed and
read by a phospho imager.
POLG1 sequencing: Genomic DNA was used to amplify the exons
and exon–introns boundaries of POLGα. PCR products were purified by
the Exo-Sap reagent. Sequencing was performed on an ABI sequencer
and results were compared to the wild type sequence using the
Ensemble database.

Fig. 1. Brain biopsy showing perivascular mononuclear infiltrates involving macrophages, lymphocytes and eosinophils (arrows).

tissue were noted. In addition there were multiple small vessels with
endothelial swelling (Fig. 1).
Molecular analysis of the A3243G mutation revealed a 10% mutant
load in the blood and a 60% load in the brain (Fig. 2).
Southern blot analysis of the DNA extracted from blood and brain
revealed multiple mtDNA deletions, restricted to the brain (Fig. 3).
POLG1 revealed normal sequence.

5. Discussion
We describe a patient with MELAS mutation and multiple mtDNA
deletions. The patient presented with typical MELAS symptoms:
diabetes mellitus, sensorineural deafness, short stature, and mental
disorientation. The A3243G mutation in the tRNALeu of the mtDNA
was confirmed. However, because of the severe clinical manifestations and the family history, revealed later during hospitalization, of
the early death of all three of the patient's children, further
investigation was conducted. Southern blot analysis revealed multiple
deletions in the mtDNA, mainly in the brain.
The MELAS A3243G mutation disrupts the tertiary interaction
between the tRNALeu and the codon, resulting in improper tRNA
processing and, consequently, a defect in mtDNA-encoded protein
translation [7–11]. According to one explanation, synthesis of the
protein complexes controlling oxidative phosphorylation is inhibited

4. Histologic and molecular results
Brain tissue biopsy including grey and white matter showed
perivascular mononuclear infiltrates involving macrophages, lymphocytes, and eosinophils. Brain edema and damage of normal brain

Fig. 2. Restriction digest analysis for the A3243G mtDNA mutation. PCR product
encompassing the tRNALeu gene was digested with ApaI before running on 3% agarose
gel. M-100 bp ladder. (1) Brain. (2) Blood. (3) Positive control. (4) Normal control.
Mutant is digested to 307 bp + 93 bp, while wild type is undigested.

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103

In the present patient, the deleterious effect of the high load of the
mtDNA A3243G mutation in the brain combined with the multiple
mtDNA deletions could explain her rapid mental deterioration and the
severe presentation that led to the death of all three of her children. It
would be interesting to check other MELAS patients for mtDNA
deletions.
To the best of our knowledge, there is no report of a combination of
A3243G mutation and multiple deletions in MELAS syndrome.

References

Fig. 3. Southern blot analysis of PvuII digested mtDNA of the patient's blood (1) and
brain (2).

and oxidative damage occurs [12]. These events may cause secondary
instability of the mtDNA downstream to the primary pathological
mutation [13]. Support was provided by the finding that leukocytes
harboring the A3243G mutation have lower levels of mtDNA [13,14].
An alternative explanation is based on reports that cells with the 3243
mutation are associated with downregulation of ubiquitin-mediated
protein degradation and ribosomal proteins [7,15,16]. This reduced
expression is reflected at the level of the cytosolic proteins, which can
have an impact on the mtDNA synthesis machinery.
There is one report of a patient with typical phenotypic MELAS
who did not have the A3243G mutation but rather a defect in the
POLG gene that led to multiple deletions [16]. By contrast, Bosbach
et al. [17] reported on 22 patients with chronic progressive external
ophthalmoplegia and Kearns–Sayre syndrome, which is usually
characterized by mtDNA deletions, who had the A3243G mutation.
Our patient had a normal sequence of POLG1, ruling out multiple
mtDNA deletions due to POLG mutation.
The mental deterioration in our patient was dramatically progressive. Mental disorders (including dementia, atypical psychosis, depression, schizophrenia-like psychosis and mania) are well documented in
mitochondrial diseases in general and in MELAS in particular [18–20].
Psychotic manifestations may even be the presenting symptoms of
MELAS [18].One of the proposed mechanisms is reduced glutamate
transport, demonstrated in 3243 hybrid cell lines [21].
Mancuso et al. [22] described an Italian family with multiple
deletions in the mtDNA in muscle in which bipolar disorder,
schizophrenia, and depression recurred over several generations in
an autosomal dominant pattern, in the absence of other major signs of
mitochondrial dysfunction.

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