Title

MERRF/MELAS overlap syndrome: A double pathogenic mutation in mitochondrial tRNA genes

Author(s)

Nakamura, Masakazu; Yabe, Ichiro; Sudo, Akira; Hosoki, Kana; Yaguchi, Hiroaki; Saitoh, Shinji; Sasaki, Hidenao

Citation

Journal of Medical Genetics, 47(10), 659-664
https://doi.org/10.1136/jmg.2009.072058

Issue Date

2010-07-07

Doc URL

http://hdl.handle.net/2115/44728

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This article has been accepted for publication in Journal of Medical Genetics (JMG). The definitive copyedited, typeset
version [insert complete citation information when available] is available online at : http://jmg.bmj.com

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article (author version)

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yabe̲JMG.pdf

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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

M. Nakamura et al, 1

MERRF/MELAS overlap syndrome: A double pathogenic mutation in
mitochondrial tRNA genes

Masakazu Nakamura, MD; Ichiro Yabe, MD; Akira Sudo, MD; Kana Hosoki, MS; Hiroaki
Yaguchi, MD; Shinji Saitoh, MD; and Hidenao Sasaki, MD

Author affiliations: Departments of Neurology (Drs Nakamura, Yabe, Yaguchi, and Sasaki)
and Pediatrics (Dr Sudo, Ms Hosoki, and Dr Saitoh), Hokkaido University Graduate School
of Medicine, Sapporo, Japan.
Dr. Nakamura and Dr. Yabe equally contributed to this work.
Correspondence: Ichiro Yabe, MD, PhD, Department of Neurology, Hokkaido University
Graduate School of Medicine, N15 W7, Kita-ku, Sapporo 060-8638, Japan

M. Nakamura et al, 2

ABSTRACT
Background: Myoclonic epilepsy with ragged-red fibers (MERRF) and mitochondrial
encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) are established phenotypes
of mitochondrial encephalomyopathy. The m.8356T>C transition in the mitochondrial
tRNALys gene is a pathogenic mutations of MERRF. The m.3243A>G transition in the
mitochondrial tRNALeu gene is detected in most MELAS patients. Although previous analyses
of double mutations in mitochondrial DNA (mtDNA) were useful for discussing their nature,
many unsolved questions remain.
Objective: To describe the clinical and genetic features of a family with the above mtDNA
double point mutations, and discuss the role of double mtDNA mutations in diverse clinical
features in the family.
Patients and Methods: The proband was a 23-year-old female with MERRF harboring
m.8356T>C and m.3243A>G transitions in mitochondrial tRNA genes. We assessed her
clinical aspects of her and those of her three relatives, and performed mutation analyses on
their mtDNA.
Results: Phenotypes of the four patients were MERRF, MERRF/MELAS overlap syndrome,
and asymptomatic carrier. We hypothesize that the course of the phenotype of this family
begins with MERRF and is followed by MELAS. This double mutation was heteroplasmic in

M. Nakamura et al, 3

blood of all four patients, but with different rates in each patient, while m.8356T>C appeared
homoplasmic, and m.3243A>G was heteroplasmic in muscle of the two examined cases. No
other mutations were detected in the total mtDNA sequence in this family.
Conclusions: This is the first reported case of a double point mutation in mtDNA, both of
which were heteroplasmic and pathogenic for the established phenotypes.

M. Nakamura et al, 4

The concept of myoclonic epilepsy with ragged-red fibers (MERRF) was first proposed
in 1980, and has been established as one of the phenotypes of mitochondrial
encephalomyopathy [1]. It is maternally inherited and patients express myoclonus, myoclonic
or generalized convulsions, cerebellar ataxia, and myopathy with ragged-red fibers [2]. In
1990, the most common pathogenic mutation, m.8344A>G transition in the mitochondrial
tRNALys gene was reported [3], and report of a second pathogenic mutation, m.8356T>C
transition in the mitochondrial tRNALys gene followed in 1992 [4]. The first report of the
m.8356T>C transition indicated a pure MERRF expression [4], however, subsequent reports
revealed that patients, with or without MERRF symptoms, of the families with this mutation,
frequently

showed

migraine,

stroke-like

episodes,

and

strongly

succinate

dehygrogenase-reactive vessels (SSVs) in muscle pathology, which are typical for another
phenotype of mitochondrial encephalomyopathy; mitochondrial mayopathy, encephalopathy,
lactic acidosis, and stroke-like episodes (MELAS) [5, 6]. It was speculated that the
m.8356T>C transition might occur in patients with some of the expressions of MELAS, such
as migraine, stroke-like episodes, and SSVs; however, the mechanism by which various
expressions typical for two distinct phenotypes might occur in patients with only this point
mutation in mitochondrial DNA (mtDNA) has not been clarified [5, 6].
The concept of MELAS was proposed in 1984, also as one of the phenotypes of

M. Nakamura et al, 5

mitochondrial encephalomyopathy [7]. This entity was maternally inherited and characterized
by stroke-like episodes, episodic headache and vomiting, seizures, dementia, lactic acidosis,
skeletal myopathy, short stature and as being with or without MERRF symptoms [7]. The first
pathogenic mutation, the m.3243A>G transition in the mitochondrial tRNALeu gene, was
reported in 1990 [8]. Moreover, it was revealed that most of the patients with MELAS had
this mutation as a primary cause of the disease [9]. On the other hand, there were several
reports of patients with an m.3243A>G transition, who also expressed features typical of
phenotypes of mitochondrial encephalmyopathy other than MELAS, such as MERRF [10, 11],
MERRF/MELAS overlap syndrome [12, 13], progressive external ophthalmoplegia (PEO),
and MERRF/PEO overlap syndrome [14], suggesting that the m.3243A>G transition was
potentially indicative of a broad expressive spectrum. However, what determined the range of
expression in each patient had not been resolved [10-14].
There are reports of rare families with mitochondrial encephalomyopathy, possessing
double point mutations in mtDNA [15-22]. Some of these consist of two mutations reported to
separately express the same [15, 18, 20, 22] or different [17, 19, 21] distinct phenotypes, and
others consist of one mutation of a well-known pathogenesis and one mutation of unknown
effect [16]. It is speculated that in some families only one of the mutations expresses a
phenotype [16, 17], that each mutation expresses different phenotypes in a given family [19],

M. Nakamura et al, 6

and both mutations act synergistically to express one phenotype in other families [18, 21, 22].
These double mtDNA mutations might be the keys to resolving the mechanisms underlying
the formation of threshold effects. However, these issues have yet to be resolved, presumably
due in part to the various combinations of established phenotypes and mutational expressions
in each of the families mentioned above.
We encountered a family with the double mutation, m.8356T>C and m.3243A>G.
Individual family members expressed MERRF, MERRF/MELAS overlap syndrome, and
other minor complications compatible with symptoms derived from mitochondrial
dysfunctions. Because the various family members express potential phenotypes known to be
caused by these two mtDNA mutations they were considered advantageous for to discussing
the relationship between phenotypes and genetic effects. We had a chance to examine four
members of this family, and tried to clarify the expression and the role of mtDNA point
mutations.

M. Nakamura et al, 7

PATIENTS and METHODS
Patients
The pedigree of the family we will discuss is shown in Figure 1. We first met patient III-1
(proband). She was introduced to us from another institution, diagnosed with myoclonic
epilepsy. An interview of her and her mother (II-1) revealed that they had a familial history of
mitochondrial encephalomyopathy (II-2; maternal aunt of the proband). We subsequently
discovered that one of our colleagues had examined II-2 and had identified a mtDNA double
point mutation (m.8356T>C and m.3243A>G) by a commercial-based (SRL Inc.) analysis of
common mtDNA mutations, and that her muscle specimen had been preserved in our
institution. Another patient of this family (ΙΙΙ-6; cousin of the proband) was introduced to us
from another institution, also diagnosed with myoclonic epilepsy. Thus, the main subjects of
this study are these four cases; II-1 (mother), II-2 (maternal aunt), III-1 (proband), and III-6
(cousin). This pedigree contains other cases with hearing loss (I-1), hearing loss and mental
retardation (III-5), and sudden death at one year of age (III-7). However, as we did not
encounter these family members, we have no further information about them.
We conducted clinical (physical, neurological, laboratory, and radiological) examinations of
II-1, IIΙ-1, and IIΙ-6 as far as we could. Clinical information of II-2 was gained from medical
records. Histochemical analysis of muscle was conducted for II-2 and IIΙ-1. Genetic analyses

M. Nakamura et al, 8

of mtDNA from blood and / or muscle was conducted in all four cases.

Report of cases
III-1; The proband
A 17 year-old female with bilateral sensorineural deafness developed a generalized clonic
convulsion; she was diagnosed with myoclonic epilepsy one year later. Sodium valproate had
been effective for preventing relapses of epileptic seizures. She was introduced to us at age 23
because her maternal aunt (II-2) was was diagnosed with MELAS. She exhibited a slightly
short stature (154 cm) and definitely low body weight (36 kg). She did not complain of either
episodic headache or nausea. Neurological examinations revealed deafness, slurred speech,
weakness of bilateral quadriceps, and limb and truncal ataxia. By conversation, we were
impressed that although she was somewhat childish for her age, her cognitive function was
preserved. Her visual acuity and field were not disturbed. External ophthalmoplegia was not
observed. Serum chemistry examinations detected no elevations in lactate, creatine kinase,
transaminases, lactate dehydrogenase, amylase, hemoglobin A1c, and creatinine. Blood
hormonal evaluations of pituitary gland (ACTH, TSH, LH, FSH, GH, and prolactin), thyroid
(fT3, fT4), adrenal cortex (cortisol), and genitals (estradiol) were normal. Elevated lactate was

M. Nakamura et al, 9

detected in cerebrospinal fluid (35.3 mg/dL; normal <20.0 mg/dL). A CT scan of the trunk
showed no abnormal forms of the organs in chest and abdomen. Transthoracic ultrasonic
cardiography did not reveal cardiomyopathy. A brain MRI scan showed diffuse mild brain
atrophy without focal lesions. Electroencephalography (EEG) showed bilateral slow waves. A
muscle biopsy of the left rectus femoris revealed fiber size variability, scattered ragged-red
fibers (RRFs), and vessels strongly stained with succinate dehydrogenase (SSVs) (Figure 2A).
Cytochrome c oxidase defects were present in occasional fibers (focal COX deficiency), also
among RRFs (Figure 2B). Most of the SSVs (approximately 80%) possessed COX activity.

II-2; Maternal aunt of III-1
The proband’s maternal aunt had bilateral sensorineural deafness and progressive
myoclonic epilepsy when she was 26 years old. At age 38, she developed stroke-like episodes
with headache, truncal instability, and convulsion; she exhibited emaciation (stature: 157 cm,
body weight 38 kg). Neurological examinations revealed aphasia, right hemianopsia, and
cerebellar ataxia. A brain MRI scan showed a large lesion in the left cerebral hemisphere
(Figure 3). Elevated lactate (34.6 mg/dL; normal <16.0 mg/dL) and impaired glucose
tolerance (data not shown) were detected in serum. A muscle biopsy of the left rectus femoris
showed fiber size variability, RRFs, focal COX deficiency, and SSVs (Figure 2C, E). Some

M. Nakamura et al, 10

SSVs (approximately 40%) lacked COX activity (Figure 2C-F), and occasional RRFs also
lacked COX activities. A stroke-like episode relapsed with disturbed consciousness at age 45,
and the patient died of renal and heart failure one year later.

III-6; Cousin of III-1
A female cousin of the proband developed slurred speech, dysmetria of all four extremities,
and truncal instability at age 18. After a few months, myoclonic jerks of the upper extremities
often appeared, followed by generalized clonic convulsions. Her stature and body weight
were in the normal range. She had neither deafness nor a history of episodic headache and
nausea. Neurological findings revealed a slight cerebellar ataxia of the trunk and limbs,
transient myoclonus of the upper extremities, and an episode of generalized convulsion. She
appeared childish during conversation, but intellectual ability was normal. Her visual acuity
and field were intact. External ophthalmoplegia was not observed. Serum creatine kinase,
transaminases, lactate dehydrogenase, amylase, hemoglobin A1c, and creatinine were within
normal limits, but lactate was elevated (16.4 mg/dL; normal <16.0 mg/dL). Transthoracic
ultrasonic cardiography indicated normal wall motion of the heart. A brain MRI showed no
definite abnormalities. She showed left dominant spikes and waves on an EEG and was
diagnosed with myoclonic epilepsy at age 19. For about one and a half years, myoclonic

M. Nakamura et al, 11

epilepsy was well controlled with carbamazepine; however, at age 21 the frequency of the
epileptic seizures increased. Despite changes to, or additions of, other antiepileptic drugs
(phenytoin, zonisamide, topiramate, sodium valproate), control of seizures was difficult, and
she required intensive care with mechanical ventilation twice during status epilepticus (SE).
Moreover, in the course of treatment for the second SE, abdominal pain and elevated serum
amylase and lipase occurred. A CT scan and an ultrasonogram detected no abnormal changes
in the pancreas. Following treatment with camostat mesilate, ursodeoxycholic acid, and
ulinastatin, the symptoms were relieved and serum pancreatic enzymes were normalized in
one month.

II-1; Mother of III-1
The proband’s mother, at age 51, was asymptomatic.

mtDNA mutation analysis
The examination of mtDNA mutations was first conducted in II-2 at another institution. She
appeared to express the features of both MERRF and MELAS. Therefore, two established
pathogenic mutations in mtDNA found in these phenotypes, m.8356T>C and m.3243G>A,
were selected and screened for using commercial-based analyses of blood (SRL Inc.); both

M. Nakamura et al, 12

point mutations were found. Therefore, we also screened for these mtDNA mutations in blood
and muscle of III-1, and in the preserved muscle specimen of II-2. The m.8356T>C and
m.3243A>G mutations were detected by PCR-restriction fragment length polymorphism
analysis. Genomic DNA was extracted from blood and muscle specimens and amplified by
PCR. PCR primers for m.3243A>G were adopted from Goto et al. [8], while mismatch
primers to detect m.8356T>C substitution were adopted from Silvesti et al [4]. PCR products
were digested with ApaI and DraI at positions 3243 and 8356, respectively. Plasmids
containing only wild type or mutant mtDNA were constructed and used as controls. PCR
products were also sequenced to confirm the base substitutions. Blood samples of two other
family members whom we had chances to examine (II-1 and III-6) were also analyzed with
the same procedure. Moreover, whole mtDNA genome analyses in blood and muscle of III-1,
and in blood of II-1 were conducted（mitoSEQr resequencing system, for resequencing the
entire mitochondrial genome with 46 RSAs. Applied Biosystems, USA).

RESULTS
Symptoms and phenotypes
The phenotypes of the proband (III-1) and cousin (III-6) were MERRF, the aunt (II-2) was
MERRF/MELAS overlap syndrome, and the mother (II-1) was an asymptomatic carrier.

M. Nakamura et al, 13

Three other members (I-1, III-5, and III-7) also had some symptoms that could not be
categorized in distinct entities. Of the three patients with MERRF (III-1 and III-6) and
MERRF/MELAS overlap syndrome (II-2), two (II-2 and III-1) showed short stature and low
body weight with bilateral sensorineural deafness from childhood. All three patients suffered
from myoclonic epilepsy from the late teens to the mid twenties. Patient II-2 subsequently
exhibited episodic headache in her mid thirties, with a stroke-like episode that repeatedly
occurred afterwards. The other two patients (III-1 and III-6) had not reached their thirties
when this study was conducted.

Blood and cerebrospinal fluid analysis
Of the three patients examined (II-2, III-1 and III-6), two showed elevated lactate (II-2 and
III-6) in serum and one (II-2) had impaired glucose tolerance; at the same time, other items in
serum chemistry, involving creatine kinase, were within normal limits in all three patients. No
hormonal abnormalities except short stature were found in blood of III-1. Patient III-1 showed
high levels of lactate in cerebrospinal fluid (CSF was not examined in the other patients).

Radiological examinations and EEG findings
Of the three patients examined, brain MRIs in two of them (II-2 and III-1) showed diffuse

M. Nakamura et al, 14

brain atrophy, with stroke-like lesions in II-2, while patient III-6 exhibited a normal brain
MRI. Transthoracic ultrasonic cardiographies demonstrated normal cardiac wall motion in the
two patients examined (III-1 and III-6). Thoracic and abdominal CT scans detected no
abnormalities in III-1. Two patients (III-1 and III-6) underwent EEG. III-1 showed generalized
slow waves, and III-6 exhibited focal spike-and-waves.

Muscle pathology
Muscle biopsies were conducted in two cases (II-2 and III-1). II-2 had already expressed
stroke-like episodes at the time this procedure was conducted. The two patients had similar
morphological findings: fiber size variability, RRFs, focal COX deficiency, SSVs, and a
mosaic distribution of COX activities in both RRFs and SSVs (Figure 2). The proportion of
the SSVs with COX deficiencies was higher in III-1 than in II-2.

Genetic analysis
Results of genetic analyses are shown in Figure 4. Genetic analysis of mtDNA in III-1
revealed heteroplasmic m.8356T>C and m.3243A>G mutations in blood. This double mtDNA
mutation was also detected and was heteroplasmic in the blood of II-1 and III-6, although the
proportions of both mutations were clearly less in asymptomatic II-1. In muscle of III-1 the

M. Nakamura et al, 15

proportion of m.3243A>G was higher than that in blood and m.8356T>C appeared to be
homoplasmic. In muscle of II-2, m.3243A>G was heteroplasmic and m.8356T>C also
appeared to be homoplasmic. Screening of the entire mtDNA genomes from blood and muscle
of III-1, and from blood of II-1 revealed no mutations other than the m.8356T>C and
m.3243G>A transitions.

Discussion
All four patients that we encountered carried the double point mutations
m.8356T>C and m.3243A>G in mitochondrial tRNA genes. Both mutations were
heteroplasmic in blood, while m.8356T>C appeared to be homoplasmic in muscle. The
proportion of m.3243A>G transitions was higher in the muscle of III-1, and seemed to be
associated with clinical severity because all symptomatic patients had significant proportions
of the mutation but the asymptomatic patient II-1 carried the lowest mutational load. The
m.8356T>C mutation appeared to be homoplasmic in muscle of III-1 and II-2, although a low
level of heteroplasmy cannot be excluded due to the limitation of our current
semi-quantitative PCR analysis. Nevertheless, it is fair to say that the m.8356T>C transition
was higher in muscle than in blood, at least in III-1. The proportion of m.8356T>C transitions
also appeared to be associated with clinical severity.

M. Nakamura et al, 16

The four patients showed phenotypes of MERRF (III-1 and III-6), MERRF/MELAS
overlap syndrome (II-2), and of an asymptomatic carrier (II-1). There were several other
symptomatic family members (I-1, III-5, and III-7). We suggest that the typical course of
disease in this family is as follows: the onset is characterized by short stature with emaciation
and bilateral sensorineural deafness in childhood followed by cerebellar ataxia and myoclonic
epilepsy; MERRF expression follows in young adulthood. By middle age the disease
progression is characterized by the addition of migraine, vomiting, and stroke-like episodes,
symptoms of MELAS expression, that indicates completion of the MERRF/MELAS overlap
syndrome.

Muscle pathology is compatible with MELAS before and after phenotypic

expression of MELAS [9, 23]. However, a quantitative association between SSVs with COX
deficiencies and expression of MELAS was not found in this study. This might mean that
these family members gain the potential to express MELAS from the mutation loads in their
primary phases. The characteristic brain MRI and EEG findings were not observed in this
family. Neither external ophthalmoplegia nor cardiomyopathy were detected.
We presume that these mutations are pathogenic for the observed clinical
phenotypes based on the following criteria; i) m.8356T>C and m.3243A>G transitions are
pathogenic for MERRF [5, 6] and MELAS [8], respectively; ii) both mutations were
heteroplasmic in blood of all four cases; iii) no mutations other than m.8356T>C and

M. Nakamura et al, 17

m.3243A>G were detected in total sequences of mtDNA from blood of II-1 or from blood and
muscle of IIΙ-1; iv) other symptomatic members of the pedigree had symptoms indicative of
mitochondrial dysfunction [2].
Patients with the m.8356T>C transition frequently express both MERRF and
MELAS [5, 6]. Moreover, some patients with the m.3243A>G transition exhibit MERRF [10,
11] or MERRF/MELAS overlap syndrome [12, 13]. As we did not quantitatively evaluate
these mutations or clarify their thresholds, we were unable to determine whether both
mutations simultaneously affected the observed clinical phenotypes.
In ΙΙ-2, with MERRF/MELAS overlap syndrome, MERRF preceded MELAS. In the
next generation, the proband (III-1) expressed MERRF without MELAS. However, her
muscle pathology showed RRFs and SSVs with COX activities, which are more characteristic
of MELAS than MERRF [23], and SSVs might be a predictor of stroke-like episodes [24] and
additional MELAS expression. This finding leads us to speculate that the m.3243A>G
transition might be pathogenic for expressing MELAS. Because the proportion of another
mtDNA point mutation in muscle (m.12320A>G) was shown to increase over time [25], we
hypothesize that the possibly increasing m.8356T>C transitions in our family might first
overcome the threshold for the biochemical and symptomatic expressions of MERRF, and the
increasing m.3243A>G transitions might then finally reach the threshold of symptomatic

M. Nakamura et al, 18

MELAS expression. It is presumed that neither mutation had reached its threshold in the
asymptomatic family members. However, to demonstrate the pathogenicity of the
m.3243A>G transition in the expression of MELAS, single muscle fiber studies and more
precise mtDNA species analyses are required. Without these data, it remains unknown
whether a stroke-like episode of patient II-2 may be ascribed to m.3243A>G, because the
m.8356T>C mutation itself can lead to the expression of MELAS/MERRF overlap syndrome
[5, 6]. Among further studies that we are planning, the screening for m.3243A>G transitions
in urinary sediment samples will also be informative for determining clinical severity. Data
have shown that assessment of mtDNA mutational load in urinary cells correlates closely with
that observed in skeletal muscle [26, 27], and is also a reliable measure of clinical severity
[28].
Most of the reported cases of patients with more than one mtDNA mutation carried
double point mutations [15-22]; however, cases with two heteroplasmic mtDNA point
mutations were observed by Bidooki et al. in only one family with m.10010T>C and
m.5656A>G transitions in the tRNAGly gene and in the non-coding region between the
tRNAAsn and tRNAAln genes, respectively [16]. The former mutation was pathogenic, while
the latter was silent, and the associated phenotypes have not been generally established even
in the former [16]. By contrast, both of the mutations in our family are known to express

M. Nakamura et al, 19

established phenotypes. The difference between this work and the 1997 publication by
Bidooki et al [16]. is that in the current study, there are potentially two pathogenic mtDNA
mutations, rather than a pathogenic mutation and a SNP. However, the causal effects must be
investigated experimentally.
There are, as yet, no reports of double mtDNA mutations in which no evolutionary
associations with one another were shown [15-22]. Thus, we presume that some of the double
mutations in our family were in the same mitochondria and some occurred as single mutations
in distinct mitochondria. If further studies demonstrate this condition (i.e., the recombination
of mtDNA [16] and one mutation’s effect arising along with another in mtDNA [29]), single
muscle fiber analysis and quantitative evaluations of the heteroplasmic rate of both mutations
in each tissue might provide more profound insights into mtDNA mutations.

Acknowledgements: We thank all patients for their active cooperation. This work was
supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education,
Science, Sports, and Culture, Japan, and by a grant from the Uehara Foundation.

M. Nakamura et al, 20

Patient consent: We obtained written consent from the patients themselves or their family
members for publication of data and images of our subjects in print and online.

Competing Interest: None declared.

The Corresponding Author has the right to grant on behalf of all authors and does grant on
behalf of all authors, an exclusive license (or non exclusive for government employees) on a
worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be
published in JMG and any other BMJPGL products and sublicenses such use and exploit all
subsidiary rights, as set out in our license (http://group.bmj.com/products/journals/
instructions-for-authors/licence-forms).

M. Nakamura et al, 21

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M. Nakamura et al, 26

Figure Legends
Figure 1
Proband’s family pedigree. ○= female; □ = male;

/ = deceased. Solid symbols are

symptomatic members. The arrow shows the proband. * Patients we encountered.

Figure 2
Pathology observed in rectus femoris muscle biopsies in III-1 (A and B) and II-2 (C, D, E and
F). A & B, C & D and E & F are pairs of adjacent sections stained as indicated. A; Scattered
ragged-red fibers (RRFs). Modified Gomori trichrome stain, ×400. B; Occasional RRFs with
(arrow heads) or without (arrow) cytochrome c oxidase (COX) activities. COX stain, ×400. C;
Strongly succinate dehydrogenase-reactive blood vessels (SSVs) (arrow). Succinate
dehydrogenase (SDH) stain, ×400. D; SSVs with COX activities (arrow). COX stain, ×400. E;
SSVs (arrow heads). SDH stain, ×100. F; SSVs without COX activities (arrow heads) and
with mild COX activities (arrow). COX stain, ×100.

Figure 3
Brain MRI of II-2 during acute phase. FLAIR images detected a hyperintense lesion
extending from the left occipito-parieto-temporal lobe at 38 years. R: right.

M. Nakamura et al, 27

Figure 4
Restriction fragment length polymorphism analysis for the m.3243A>G and m.8356T>C
mutations. Upper panel demonstrates variable heteroplasmy level of the m.3243A>G
mutation in the pedigree. Uncut 294 bp band represents the wild type (3243A) while the 162
bp and 132 bp bands represent the mutant (3243G). Ratio of the mutant in muscle is
significantly higher than in blood of III-1.

Lower panel also shows variable heteroplasmy

level of the m.8356T>C mutation. Uncut 141 bp band represents the mutant (8356C) while
the 119 bp and 22 bp bands (only the 119 bp band is shown) represent the wild-type (8356T).
The mutation rate in muscle is higher than in blood and appears to be homoplasmic in III-1
and II-2.

WT and Mutant represent cloned plasmid DNAs containing only wild type and

mutation sequences, respectively.

Figure 1

Proband’s family pedigree

Ⅰ

1

Ⅱ

*
1

*
2

2

*
6

*

Ⅲ 1

3

4

5

3

7

4

8

5

A

B

C

D

E

F

M. Nakamura et al, 1

Figure 3

Brain MRI of II-2 during acute phase

Figure 4

Restriction fragment length polymorphism analysis for the m.3243A>G and

m.8356T>C mutations

Blood
[bp]
294
162
132
141
119

III-1

II-1

Muscle
III-6

III-1

II-2

Control
WT

Mutant