SHORT REPORT

Key words: mitochondria1 DNA tRNA myopathy maternal
MUSCLE 81 NERVE 19:1603-1604 1996

THE 3260 MUTATION IN
MITOCHONDRIAL DNA CAN CAUSE
MITOCHONDRIAL MYOPATHY,
ENCEPHALOPATHY, LACTIC
ACIDOSIS, AND STROKELIKE
EPISODES (MELAS)
ICHIZO NISHINO, MD, MlKlO KOMATSU, MD, SOlCHl KODAMA, MD,
SATOSHI HORAI, MD, IKUVA NONAKA, MD, and YU-ICHI GOTO, MD

Maternally inherited myopathy and cardiomyopathy
(MIMyCa) is one of the phenotypic subgroups of
mitochondrial diseases. There are two reports of European families with MyMICa associated with an Ato-G transition at nucleotide (nt) 3260 in the mitochondrial tRNAkU(UUR)
We have identified
the 3260 mutation in a Japanese family with mitochondrial myopathy, encephalopathy, lactic acidosis,
and strokelike episodes (MELAS) accompanied by
myoclonic epilepsy.
PATIENTS

The female proband had suffered from frequent episodes of headaches, vomiting, and loss of consciousness beginning at the age of 6 years. Electroencephalography during the attacks showed spike-and-wave
complexeswith afocus in the right occipital area, and,
initially, a diagnosis of complex partial seizure was
made. At the age of 9 years, she began to experience
attacks of myoclonic epilepsy occasionally accompanied by numbness of the left upper extremity and/or
transient blindness in her left visual field. Scintillating

scotomas often preceded the headaches. Serum lactate were abnormally high (up to 90.7 mg/dL; normal
range 3.3-14.9 mg/dL) on several occasions. Lactate
in cerebral spinal fluid, determined once during a period without strokelike episode, was also high (52.2
mg/dL; normal range 3.3-14.9 mg/dL). Muscle biopsy, performed at the age of 10years, showed moderate variation in fiber size with type 2B fiber deficiency,
numerous ragged-red fibers (W),
many strongly succinate dehydrogenase-reactive blood vessels (SSV),6
and focal cytochrome c oxidase (COX) deficiency
(Fig. 1). Both RRF and SSV showed a wide range of
COX activitiesvaryingfrom decreased to increased activities, findings which are typical of MELAS5 Brain
magnetic resonance imaging (MRI) performed at the
age of 14years revealed high T2signal area extending
over the right occipital cortex. A series of chest x-rays,
electrocardiograms, and echocardiograms did not
show any evidence of cardiac involvement. Other family members including her maternal groundmother,
mother, and two elder siblings were asymptomatic.
METHODS AND RESULTS

From the Department of Ultrastructural Research, National Institute of
Neuroscience, National Center of Neurology and Psychiatry (NCNP),
Tokyo, Japan (Drs. Nishino, Nonaka. and Goto); Department of Laboratory
Medicine, National Center Hospital for Mental, Nervous and Muscular
Disorders, NCNP, Tokyo, Japan (Dr. Nonaka); Himeji Red Cross Hospital,
Hyogo, Japan (Drs. Komatsu and Kodama); and Department of Human
Genetics, National Institute of Genetics, Mishima, Japan (Dr. Horai).
Acknowledgment: The authors thank Dr. S. M. Sumi, University of Washington, for his helpful comments on the manuscript.
Address reprint requests to lchizo Nishino, MD, Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan.
Accepted for publication April 22, 1996
CCC 0148-639X/96/121603-02
0 1996 John Wiley & Sons, Inc

Short Reports

Total DNA was extracted from muscle by a conventional method. We directly sequenced a series of polymerase chain reaction (PCR)-amplified fragments
encompassing all the mitochondrial tRNA genomes using an automated sequencer (model 377,Applied Biosystems, Perkin Elmer, Foster City, CA,USA). Thirty
sequence primers were set for both L and H strands.
The datawere compared with the reference sequence’
and only one substitution was found at nt 3260.
Restriction fragment length polymorphism
(RFLP) analysis using the previously reported mismatch primer that creates the XmnI recognition site”
showed heteroplasmy not only in the proband but
also in the mother and the siblings. To estimate the

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proband had SSVwith a variable range of COX activities in the muscle biopsy, whereas SSV were not reported in the two MIMyCa families. SSV are often
seen in MELAS, and especially SSVwithvariable COX
activity are typical for M E W . ' It has been reported
that SSV contain increased numbers of mutant
mtDNA, suggesting that SSV are strongly related to
stroke/strokelike episode. Thus, we believe that the
damaged blood vessels (SSV) may be crucial for this
phenotypic difference.
Is the MELAS phenotype of the 3260 mutation specific to the Japanese? The mitochondrial
tRNA""(uUR)gene is a hot spot for the pathogenic
mutations including the 3243,' 3252,8 3271 ,' and
3291,4all of which have been associated with MELAS.
On the other hand, cardiomyopathy is a relatively
frequent complication of mitochondrial encephalomyopathies including MELAS. Thus, we could consider both MELAS and MIMyCa a5 part of a spectrum
of phenotypic expressions of the 3260 mutation.
Although further studies are needed to elucidate
the mechanism for the variable phenotypic expression, our data confirm that the 3260 mutation is a
genetic cause of MELAS as well as MIMyCa.
REFERENCES

FIGURE 1. Muscle biopsy of the proband. Serial sections were
stained with succinate dehydrogenase (SDH) (A) and cytochrome
coxidase (COX) (B). On SDH, SSV (arrows) and RRF (asterisks)
are highlighted. They showed normal to increased COX activities.
(Original magnification x250)

proportion of the mutant mitochondrial DNA, we
performed a "last cycle cold PCR"/RFLP method
which allowed us to avoid errors due to heteroduplex
formation during PCR. In brief, we added R110dUTPs (Applied Biosystems) after the conventional
PCR and performed one additional cycle of 94°C for
5 min, 45°C for 1 min, and 72°C for 12 min. The
amplified fragments were digested with Xmn I overnight and analyzed in an automated sequencer. The
proportions of the mutant mtDNA were estimated as
follows: the mother 29.4% in blood (B); the proband
87.1% in muscle, 51.0% (B); the first brother 31.6%
(B); and the second brother 44.1% (B).
DISCUSSION

An earlier study of rho' transformants derived from
a family with MIMyCa' strongly suggested that the
3260 mutation was the genetic cause. Similarly the
fact that our patient had a higher proportion of mutant mtDNA than asymptomatic family members supports the idea that the 3260 mutation plays an important role in the pathogenesis of MELAS in our family.
Why does the same mutation cause M E W in
our family and MIMyCa in European families? The

1604

Short Reports

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