~~

We studied a patient with a rnitochondrial encephalomyopathy characterized by the presence of all the cardinal features of both myoclonic
epilepsy and ragged-red fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndromes. Muscle biopsy showed ragged-red fibers (RRF). Some RRF
were cytochrome c oxidase (COX)-negative while some others stained
positive for COX. Muscle biochemistry revealed defects of complexes I
and IV of the respiratory chain. Both muscle and blood mitochondrial
DNA from the patient showed the presence of the mutation at nucleotide position 3243 in the tRNALeU("UR)gene and the absence of point
mutations related to MERRF syndrome. The proportions of mutant
mtDNA were 70% in muscle and 30% in blood. The mutation was absent
in blood from all maternal relatives, in hair follicles from the mother,
and in muscle from one sister of the proband. Therefore, there was no
evidence of maternal inheritance. 0 1996 John Wiley 8. Sons, Inc.
Key words: rnitochondrial DNA rnitochondrial myopathy MERRF
MELAS maternal inheritance
MUSCLE 81NERVE 19:187-190 1996

SPORADIC MERRF/MELAS OVERLAP
SYNDROME ASSOCIATED WITH THE
3243 tRNALeU(UUR)
MUTATION OF
MITOCHONDRIAL DNA
YOLANDA CAMPOS, MSc, MIGUEL ANGEL MARTIN, MSc,
GUSTAVO LORENZO, MD, MANUEL APARICIO, MD, ANA CABELLO, MD, and
JOAQUIN ARENAS, PhD

Mitochondria1 encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) and myoclonic epilepsy and ragged-red fibers (MERRF) are two
commonly maternally inherited syndromes within
the diverse clinical spectrum of mitochondrial encephalomyopathie~.~
Most patients with MELAS
harbor a point mutation at nucleotide position (np)
3243 in the tRNALeU(UUR)
gene of mitochondrial
deoxyribonucleic acid (mtDNA).8 Some few have a
distinct point mutation at n p 3271 in the same
transfer ribonucleic acid (tRNA) gene.g About 85%
of patients with MERRF syndrome have a point
mutation at n p 8344 within the tRNALysgene of

From the Centro de Investigaci6n. Hospital 12 de Octubre, Madrid, Spain
(Ms. Campos, Mr. Martin, and Drs. Cabello and Arenas); and Department
of Pediatrics, Hospital Ramon y Cajal, Madrid, Spain (Drs. Lorenzo and
Aparicio)
Acknowledgments: Supported by a grant from FIS number 95/0658, Ministry of Health, Spain. Yolanda Campos was supported by a grant from
Sigma-Tau
Address reprint requests to Joaquin Arenas, Centro de Investigacion,
Hospital 12 de Octubre, Avda de Cordoba Km 5.4, 28041, Madrid, Spain.
Accepted for publication May 19. 1995.
CCC 0148-639)(/96/020187-04
0 1996 John Wiley & Sons, Inc

Overlap

mtDNA.I3 Some others harbor a different point
mutation at position 8356 in the same gene.14
Although these syndromes are clinically very
distinctive, there can be overlap between them in
some patients. Molecular genetics can help classify
such patients. We describe a patient with all the
cardinal features of both MERRF and MELAS syndromes and no evidence of maternal inheritance,
harboring the 3243 np mutation.
PATIENT REPORT

The patient, a 14-year-old boy, began at age 2
years to have generalized tonic-clonic seizures and
multifocal head myoclonus that were difficult to
control. At age 7 years, he presented with transient
cortical blindness, tonic-clonic seizures, and severe
multifocal myoclonus involving the head and
limbs. The electroencephalogram (EEG) showed
slow background, bilateral spike, and spike-andslow wave discharges. A C T scan was normal. Over
the next 3 years, he had several strokelike episodes
accompanied by migrainous headache, cortical
blindness, generalized tonic-clonic seizures, multifocal head myoclonus, and sometimes right hemi-

MUSCLE & NERVE

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187

paresis. A CT scan among episodes showed a cerebral infarction.
On examination at age 10 he had slow, dysarthric speech. Motor examination showed thin muscle bulk, and slight weakness of the proximal muscles of the arms and legs. There were persistent,
intermittent generalized myoclonic jerks, mild intention tremor, and dysdiadochokinesia. He had
normal ocular movements and his fundoscopic examination was normal. He had truncal and limb
ataxia. Tendon reflexes were hypoactive and gait
was unsteady. There was no evidence of dementia
but psychomotor delay was apparent. He had normal stature, and bilateral pes cavus, but no spasticity. Hearing was normal.
Laboratory studies revealed a serum CK of 350
U/L (normal < 150) and an elevated serum venous
lactate of 4.2 mmol/L (normal < 2.2). Other laboratory tests were normal. Electromyography
(EMG) was myopathic. Nerve conduction velocities
were diminished. Electrocardiogram was normal.
The EEG findings were similar to those indicated
above. A CT scan showed an occipital infarction.
Over the next 8 months the clinical situation worsened and the patient developed pneumonia with a
fatal outcome.
None of his maternal relatives (including the
grandmother) had any of the following: short stature, hearing loss, migraine, seizures, or diabetes
(Fig. 1). There was no history of consanguinity.
One of the sisters of the proband, although asymptomatic, voluntarily underwent a muscle biopsy for
biochemical, morphological, and genetical analysis.
METHODS

Muscle biopsies were taken from rectus femoris,
frozen, and stored in liquid nitrogen until analysis.
Serial frozen sections of muscle were stained with
NADH-tetrazolium reductase, succinate dehydrogenase (SDH), and cytochrome c oxidase (COX) as
described.6 T h e activities of NADH dehydrogenase (complex I), rotenone-sensitive NADH cytochrome c reductase (complexes I and 111),succinate dehydrogenase (complex 11), succinate
cytochrome c reductase (complexes I1 and III), cytochrome c oxidase (complex IV), and citrate synthase were measured in muscle homogenates as
d e ~ c r i b e dT
. ~h e activity of each complex was normalized to that of citrate synthase for correcting to
mitochondria1 volume. T o detect point mutations
associated with MERRF syndrome (at positions
8344 and 8356 within the tRNALysgene) we used
methods described e l ~ e w h e r e . ' ~ .To
' ~ detect the
3243 point mutation we used the following forward and reverse oligonucleotide primers: 5'188

Overlap

133)-3'
(3116)-CCTCCCTGTACGAAAGGA-(3
and 5'-(3353)-GCGATTAGAATGGGTACAATG(3332)-3' to amplify, by polymerase chain reaction
(PCR), a DNA fragment spanning the putative region. Numbering in parenthesis indicates mtDNA
base pairs (bp) positions according to the Cambridge sequence. Amplification was made for 25
cycles with 1 min of denaturation at 94"C, 1 min of
annealing at 55"C, and 45 s of extension at 72°C.
The 237-bp fragment was digested with the restriction enzyme Apa 1.' T h e mutant mtDNA was
cleaved into two smaller fragments of 127 bp and
110 bp, while the wild-type was not. The products
were analyzed by electrophoresis on a 1.8%
agarose gel and visualized by ethidium bromide
staining. Both for quantitative studies and for increasing the sensitivity to detect the mutation,
ApaI-digested mtDNA was analyzed after agarose
gel (0.8%) electrophoresis, Southern blotting, and
hybridization to a fragment of mtDNA spanning
bp 170-1768 labeled with digoxigenin, as described.8 This probe detects normal mtDNA fragments of 1.6 and 3.0 kb. In the presence of the
ApaI site gain, introduced by the 3243-bp mutation, the larger one is cut into two fragments of 1.2
and 1.8 kilobases (kb). Proportions of mutant (1.8
kb) and normal (3.0 kb) mtDNA fragment were
estimated by densitometry.
RESULTS

Muscle histochemistry showed ragged-red fibers
(RRF). Although part of the RRF had no COX
activity, some others stained positive for COX.
Muscle biochemistry revealed combined defects of
complexes I (30% of mean control) and IV (25% of
mean control) of the respiratory chain. Biochemical and morphological analysis of muscle biopsy
from one of the sisters of the proband were normal.
Genetic analysis of both muscle and blood from
the patient showed the presence of the 3243 mutation in the tRNALeu(lJUR)
gene (Fig. 2). In con-

t

I

rI

Ii

I
I

0

FIGURE 1. Family pedigree of sporadic MERRF/MELAS overlap syndrome. Solid symbol denotes the proband. Open symbols indicate asymptomatic relatives.

MUSCLE 13NERVE

February 1996

FIGURE 2. Digestion profiles of PCR fragments amplified
with a set of primers for tRNALeU(UUR)
and electrophoresed
through a 1.8% agarose gel. The 237-bp fragment remained
unchanged after digestion with Apal in tissues from asyrnptomatic relatives (lanes 3-6) and generated two additional
fragments (127 and 110 bp) in tissues from the patient (lanes
1 and 2). Lane 1: patient blood; lane 2: patient muscle; lane
3: 11-2 muscle; lane 4: 1-2 blood; lane 5: 11-3 blood; lane 6: 11-4
blood. Blood from 11-2 is not shown. The mutation was also
absent in hair follicles from the mother (not shown).

trast, point mutations at positions 8344 and 8356
within the tRNALysgene were absent. The mutation was heteroplasmic, and represented 70% of
total mtDNA in muscle and 30% in blood. It wa.s
absent in both blood and hair follicles from the
mother, in blood from three sisters, and in muscle
from one sister (Fig. 2).
DISCUSSION

We studied a patient characterized by the presence
of all the cardinal features of both MERRF and
MELAS syndromes. He had all the obligatory findings of MERRF syndrome: myoclonus, ataxia,
muscle weakness, and RRF. I n addition, he had
lactic acidosis, migrainous headache, tonic-clonic
seizures and repetitive strokelike episodes, which
are the usual features of patients with MELAS syndrome. Biochemical analysis showed combined defects of the respiratory chain complexes, as is usual
in patients with point mutations in tRNA genes4
Muscle histochemistry revealed a similar pattern to
that observed in MELAS patients," i.e., some RRF
were COX-negative while others stained normally
for COX. Molecular genetic analysis showed the
absence of the two point mutations characteristic
of MERRF syndrome and the presence of the
"MELAS point mutation."
The development of strokelike episodes in patients with MERRF syndrome has been previously
documented.**' MtDNA analysis has been done on
3 patients,",'* showing the n p 8344 mutation.
Moreover, Zeviani et al. l 6 documented a MERRF/
MELAS overlap patient associated with the n p
8356 mutation.
On the other hand, Ciafaloni et al.3 found the
Overlap

n p 3243 mutation in 2 patients with MELAS plus
myoclonus and ataxia, 1 of them being similar in
age at onset to ours. In both patients, family history was consistent with material inheritance.
By contrast, our patient showed no evidence of
maternal inheritance, because the mutation was
absent in blood from all his maternal relatives, and
more importantly, in muscle from one of his sisters
as well as in hair follicles from the mother. It is,
therefore, plausible that the mutation might have
arisen in the germline cells of the proband's
mother. If these cells contained a low number of
copies of mutant mtDNA, the founder effect,' by
which only a low proportion of the thousands of
mitochondria of oocytes are randomly transmitted
to zygotes, might explain why the mutation was
present in only one of her offspring. However, the
absence of detectable mutant mtDNA in the tissues
analyzed from the patient's mother does not exclude its presence in the postmitotic cells, such as
muscle, where point mutations are more likely to
be fixed. Moreover, a selection against mutant
mtDNAs species or a fall in the proportion of mutant mtDNA with age might account for why some
subjects do not have detectable amounts of the mutation in blood. Consequently, we suggest that the
mutation was in fact inherited from the mother
rather than representing a fresh mutation.
Ciafaloni et al.3 reported good correlation between proportion of mutant genomes in muscle
and disease severity in patients with MELAS harboring the np 3243 mutation. Our patient had remarkable clinical severity, as demonstrated by the
downhill evolutive course and fatal outcome. Yet
the proportion of mutant genomes was low compared with those found by Ciafaloni et al.3 This
lack of correlation may result from a skewed distribution for the mutation in different tissues, the
highest proportions of mutant mtDNA being in
certain areas of the brain rather than in muscle. It
is still unknown why the same mutation can cause
a broad spectrum of clinical phenotypes and, conversely, how distinct mutations can result in the
same clinical phenotype. Different degrees of heteroplasmy for the mutation in each tissue or organ
and tissue dependence on oxidative metabolism
(threshold effect) alone cannot explain this variability. Further mitochondria1 or nuclear factors,
or both, are to be involved in the pathogenetic
mechanisms.
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