Metab Brain Dis (2014) 29:139–144
DOI 10.1007/s11011-013-9464-5

ORIGINAL PAPER

MERRF/MELAS overlap syndrome due
to the m.3291T>C mutation
Kaiming Liu & Hui Zhao & Kunqian Ji & Chuanzhu Yan

Received: 17 October 2013 / Accepted: 26 November 2013 / Published online: 12 December 2013
# Springer Science+Business Media New York 2013

Abstract We report the case of a 19-year-old Chinese female
harboring the m.3291T>C mutation in the MT-TL1 gene
encoding the mitochondrial transfer RNA for leucine. She
presented with a complex phenotype characterized by progressive cerebellar ataxia, frequent myoclonus seizures, recurrent stroke-like episodes, migraine-like headaches with nausea
and vomiting, and elevated resting lactate blood level. It is
known that the myoclonus epilepsy with ragged-red fibers
(MERRF) is characterized by cerebellar ataxia and myoclonus
epilepsy, while that the mitochondrial encephalopathy, lactic
acidosis, and stroke-like episodes (MELAS) is characterized
by recurrent stroke-like episodes, migraine-like headaches,
and elevated resting lactate blood level. So the patient’s
clinical manifestations suggest the presence of a MERRF/
MELAS overlap syndrome. Muscle biopsy of the patient
showed the presence of numerous scattered ragged-red fibers,
some cytochrome c oxidase-deficient fibers, and several
strongly succinate dehygrogenase-reactive vessels, suggestive
of a mitochondrial disorder. Direct sequencing of the complete
K. Liu : H. Zhao : K. Ji : C. Yan
Laboratory of Neuromuscular Disorders and Department of
Neurology, Qilu Hospital, Shandong University, Jinan 250012,
China
K. Liu
Department of Geriatrics, Qilu Hospital of Shandong University,
Jinan 250012, China
H. Zhao : K. Ji : C. Yan
Key Laboratory for Experimental Teratology of the Ministry of
Education, Brain Science Research Institute, Shandong University,
Jinan 250012, China
C. Yan (*)
Department of Neurology, Qilu Hospital of Shandong University,
Shandong University, No. 107, West Wenhua Road, Jinan 250012,
China
e-mail: chuanzhuyan@163.com

mitochondrial genome of the proband revealed no mutations
other than the T-to-C transition at nucleotide position 3291.
Restriction fragment length polymorphism analysis of the
proband and her family revealed maternal inheritance of the
mutation in a heteroplasmic manner. The analysis of aerobic
respiration and glycolysis demonstrated that the fibroblasts
from the patient had mitochondrial dysfunction. Our results
suggest that the m.3291T>C is pathogenic. This study is the
first to describe the m.3291T>C mutation in association with
the MERRF/MELAS overlap syndrome.
Keywords Point mutation . m.3291T>C(tRNALeu(UUR)) .
MERRF . MELAS . Mitochondrial DNA (mtDNA)

Introduction
Mitochondrial encephalomyopathy is usually caused by point
mutations in mitochondrial genes and is presented as a multisystem disease. Myoclonic epilepsy with ragged-red fibers
(MERRF, OMIM 545000) and mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS, OMIM
540000) are established phenotypes of mitochondrial encephalomyopathy. MERRF is characterized by myoclonic seizures, cerebellar ataxia, myopathy, and ragged-red fibers
(RRFs) on muscle biopsy. Approximately 80 % of MERRF
cases are caused by the A8344G mutation in the transfer RNA
(tRNA) Lys gene (Yoneda et al. 1990). MELAS is characterized by stroke-like episodes, episodic headache and vomiting,
lactic acidosis, and skeletal myopathy; it is commonly caused
by the A3243G mutation in the mitochondrial tRNA
Leu(UUR) gene (Goto et al. 1990).
The correlations between the genotype and clinical phenotype of mitochondrial encephalomyopathy are complex and
imprecise. One type of clinical syndrome can also be associated with different point mutations, whereas the same mtDNA

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point mutations can lead to various clinical phenotypes. The
investigation of the correlations between the genotype and
clinical phenotype is useful to elucidate the pathogenesis and
have implications in the treatment of mitochondrial
encephalomyopathy.
In this study, we report the case of a MERRF/MELAS
overlap syndrome associated with a T-to-C transition at nucleotide position 3291 in the MT-TL1 (OMIM 590050) gene
encoding the mitochondrial tRNA for leucine (UUR). To date,
no other studies have described the association of the MERRF/
MELAS overlap syndrome with the m.3291T>C mutation.

Materials and methods

Metab Brain Dis (2014) 29:139–144

The proband’s mother is 48 years old, and her neurological
examination showed no abnormalities except for short stature
with emaciation.
The proband’s sister is 2 years and 6 months old, who
showed normal development and no neurological abnormalities until now.
Muscle histology and histochemistry
The left bicep muscle of the proband was subjected to muscle
biopsy, and serial frozen sections (10 μm) were stained with
standard hematoxylin-eosin (H&E), modified Gomori
trichrome (MGT), cytochrome c oxidase (COX), and succinate dehydrogenase (SDH) according to established protocols
(Old and Johnson 1989).

Subjects
Cell culture
The proband was a 19-year-old female born from a
nonconsanguineous Chinese couple after a regular pregnancy
and delivery. She had frequent myoclonic seizures and progressive cerebellar ataxia since the age of 14. At age 17, she
had recurrent stroke-like episodes, migraine-like headaches
with nausea and vomiting, homonymous hemianopsia,
hemiparesis, and hemihypesthesia. She had a height of
168 cm and a weight of 38 kg. Neurological examinations
revealed very low visual acuity, constricted visual fields, and
nystagmus, but funduscopic examination showed no abnormalities in the eyeground. External ophthalmoplegia was not
observed. Her deep tendon reflexes were exaggerated and
muscle strength was reduced. The myoclonus could be induced by sudden buckling limbs. She had dysmetria of the
upper and lower limbs with a broad-based gait. She cannot
perform a tandem gait. Both her Babinski sign and meningeal
irritation signs were negative. Neuropsychological testing
showed that she scored 18 on the Mini-Mental State Exam
and 13 on the Montreal Cognitive Assessment, indicating
cognitive impairment. Laboratory examinations revealed
increased levels of resting lactate acid in the blood
(17.1 mmol/L, normal<1.55 mmol/L). Fundus photography
examination revealed no papillary edema or optic nerve atrophy. Electromyography revealed myopathic pattern in all
muscles in the lower limbs and a slight decrease in exerciseinduced conduction velocity in the bilateral tibial nerve. When
the myoclonus epilepsy attacked the patient causing sudden,
quick, small jerks of her arms, the electroencephalogram
(EEG) recorded multiple spike-and-wave complexes (3 Hz
to 4 Hz) (Fig. 1a). Computed tomography (CT) showed
bilateral symmetrical calcification in the region of the basal
ganglia (Fig. 1b). Brain magnetic resonance imaging (MRI)
showed cerebral and cerebellar atrophy (Fig. 1c and d). Twice
stroke-like lesions located in the occipito-parieto-temporal
cortexes of bilateral hemispheres were also detected by MRI
(Fig. 1c and d).

Primary fibroblasts were derived from the skin of the proband.
This experiment was approved by the local ethics committee
and conforms to the Declaration of Helsinki. The cells were
plated in Dulbecco’s modified Eagle’s medium (DMEM,
Hyclone, USA) supplemented with 10 % fetal calf serum
(Hyclone, USA), 100 U/mL penicillin, and 100 μg/mL
streptomycin and then incubated at 37 °C in a humidified
atmosphere of 95 % air and 5 % CO2. The medium was
changed twice a week.
mtDNA mutational analysis
DNA samples were prepared from the proband’s skeletal
muscle, blood, and fibroblasts. DNA samples from the blood
of the proband’s mother and sister were also prepared. Total
DNA was extracted from tissue samples as previously described (Rieder et al. 1998). Direct sequencing of the complete
mitochondrial genome and polymerase chain reaction (PCR)–
restriction fragment length polymorphism (RFLP) analysis
were performed to detect the mtDNA mutation (Rieder et al.
1998). The following sets of primers were used to detect the
m.3291T>C mutation: forward primer, 5′-CACCCAAGAA
CAGGGTTTGT-3′; reverse primer, 5′-AGGAATGCCATT
GCGATTAG-3′. Wild-type mtDNA was cut into two fragments (80 and 74 bp) by the enzyme MluCI. Mutant mtDNA
cannot be digested. The digestion products were analyzed in a
phospho-imager (Molecular Analyst, BioRad, Hercules, CA)
using Image-Quant software (Molecular Dynamics,
Sunnyvale, CA) to assess the mutation load.
Measurement of O2 consumption rate (OCR) and extracellular
acidification rate (ECAR)
OCR and ECAR were measured with an XF24 Extracellular
Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) as

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141

Fig. 1 EEG and neuroradiologic
findings in the proband. a When
the myoclonus epilepsy attacked
the patient causing sudden, quick,
small jerks of her arms, the
electroencephalogram (EEG)
recorded multiple spike-andwave complexes (3 Hz to 4 Hz). b
CT showed bilateral symmetrical
calcification in the region of the
basal ganglia. c–d Twice strokelike lesions located in the
occipito-parieto-temporal
cortexes of bilateral hemispheres
were detected by FLAIR images
of brain MRI, which also showed
cerebral and cerebellar atrophy.
The brain edema in the second
stroke-like episode (d) is more
extensive than that in the first
stroke-like episode (c). L left

previously described (Invernizzi et al. 2012). Fibroblasts were
seeded into 24-well Seahorse XF24 plates at a density of 20×
103 cells/well in 200 μL of DMEM. The basal OCR of the cells
was measured in the absence of an uncoupler for six cycles.
The mitochondrial inhibitors oligomycin (1 μM) and mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (0.7 μM) were injected into the wells before
measuring the OCR. ECAR was measured by determining the
change in extracellular pH over time. OCR and ECAR were
normalized to the cell number.
Western blot analysis
We used Western blots to estimate the content of respiratory
complexes in the proband’s muscle (Luo et al. 2013; Patel et al.
2009). Mitochondrial proteins (15 μg/lane) were resolved
electrophoretically in SDS-polyacrylamide gels (Bio-Rad,
Hercules, CA, USA) and were transferred to polyvinylidene
difluoride membranes (Immobilon; Millipore, Bedford, MA,
USA). Membranes were blocked and incubated overnight with
primary antibodies (Invitrogen, Carlsbad, CA, USA) against
the following subunits of mitochondrial respiratory complexes:
OxPhos Complex I subunit NDUFS3, OxPhos Complex II
30 kDa subunit, OxPhos Complex II 70 kDa subunit, OxPhos
Complex III subunit core 1, OxPhos Complex IV subunit I,

OxPhos Complex V (F1F0-ATPase subunit a). After the membranes were washed, they were incubated for 1 h with secondary antibody and were washed again. Western blots were
developed using chemiluminescent HRP substrate. Density of
the resultant bands was quantified using Image-Quant software
(Molecular Dynamics, Sunnyvale, CA, USA).
Statistical analysis
All the data were presented as the mean ± standard deviation.
Student’s t test was performed to compare the two experimental groups. A statistically significant difference was defined as
p <0.05. All the procedures were performed using SPSS 19
software (SPSS Inc., Chicago, IL, USA).

Results
Histological and histochemical analyses
H&E-stained muscle biopsies showed a moderate variation in
fibre size (Fig. 2a). Numerous scattered RRFs (approximately
10 %) (Fig. 2b) were observed with MGT stain. Several
strongly succinate dehygrogenase-reactive vessels (SSVs)
were scattered on SDH stain (Fig. 2c). Staining for COX

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Metab Brain Dis (2014) 29:139–144

Fig. 2 Pathology observed in left
bicep muscle biopsies in the
proband. a, b, c and d are pairs of
adjacent sections stained as
indicated. a H&E stain showed a
moderate variation in fibre size. b
Numerous scattered RRFs were
observed with MGT stain. c SDH
stain showed SSVs (arrowhead).
d COX stain showed scattered
COX-deficient fibers, occasional
RRFs with (the black arrows) or
without (the white arrows) COX
activities, and the SSVs with
COX activities (arrowhead)

activity revealed the presence of scattered COX-deficient
fibers, and showed that some RRFs (approximately 35 %)
and most of the SSVs (approximately 85 %) possessed COX
activity (Fig. 2d). These results are suggestive of mitochondrial disease.
mtDNA mutation analysis
Direct sequencing of the complete mitochondrial genome
from blood and muscle of the proband revealed no mutations
other than the T-to-C transition at nucleotide position 3291 in
the MT-TL1 gene (Fig. 3a). PCR-RFLP revealed the

heteroplasmy of m.3291T>C mutation in the proband, which
were at a level of 93 % in muscle sample, 67 % in blood, and
62 % in fibroblasts (Fig. 3b). This mtDNA mutation was also
detected and was heteroplasmic in the blood of her mother and
sister, the proportions of the mutations in her mother (46 %)
and her sister (50 %) were clearly less than those in the
proband (Fig. 3c).
Functional measurements of mitochondrial activity
in fibroblasts
OCR and ECAR values are indicators of aerobic respiration
and glycolysis (Invernizzi et al. 2012). Both OCR and ECAR
values were normalized to the cell number. The results demonstrated that the basal (p <0.05, Fig. 4a) and maximum (p <
0.01, Fig. 4b) oxygen consumption of the fibroblasts from the
proband was significantly lower than those of the control
fibroblasts OCR defects can be associated with compensatory
stimulation of ECAR, which reflects an increased utilization
of the glycolysis pathway. Coincident with this variation in
OCR, the fibroblasts from the proband were found to generate
more lactic acid than the control fibroblasts (p <0.01, Fig. 4c).
These results suggest that the fibroblasts from the proband
have mitochondrial dysfunction.
Content of mitochondrial respiratory complexes

Fig. 3 mtDNA mutation analysis. a Direct sequencing of the complete
mitochondrial genome from blood and muscle of the proband revealed no
mutations other than the m.3291T>C mutation. b RFLP analysis showed
the variable heteroplasmy level of the m.3291T>C mutation in the pedigree. Uncut 154 bp band represents the wild type (3291 T) while the 80/
74 bp band represent the mutant (3291C). Ratio of the mutant in muscle is
significantly higher than those in blood and fibroblasts of the proband.
The mutation rate in fibroblasts of the proband is higher than those of her
mother and her sister. DNA samples are from muscle (M), blood (B), and
fibroblasts (F)

Consistent with the mitochondrial dysfunction of the fibroblasts, Western blot demonstrated that the contents of
complexes I and IV were significantly lower in the proband’s
muscle mitochondria compared with the normal control
group: 24.9 %±3.4 % and 14.8 %±5.3 % of the contents
measured in the normal muscle mitochondria, respectively
(p <0.01, Fig. 5). While the contents of complexes II,

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143

Fig. 4 Analysis of the aerobic
capacity and glycolysis in
fibroblasts from the proband. Both
the baseline OCR (a) and maximal
OCR (b) of the fibroblasts from the
proband was significantly lower
than those of the control fibroblasts.
(n =6). c Relative ECAR showed
that the fibroblasts from the
proband generated more lactic acid
than the normal fibroblasts. (n =6).
*p <0.05 compared with the
control group, **p <0.01
compared with the control group

III, and V was not significantly different with the
normal control group.

Discussion
This patient’s clinical manifestations together with the presence of cognitive impairment on neuropsychological testing,
basal ganglia calcification on brain CT, brain atrophy and
stroke-like lesions on brain MRI, multiple spike-and-wave
complexes on EEG, RRFs, SSVs and COX-deficient fibres
on muscle biopsy, and elevated resting lactate blood level,
suggest the presence of a MERRF/MELAS overlap syndrome. The MERRF syndrome is characterized by progressive cerebellar ataxia and frequent myoclonus epilepsy, and
the EEG finding of multiple spike-and-wave complexes is the
characteristic EEG pattern of myoclonus epilepsy. By contrast, the MELAS syndrome is characterized by recurrent
stroke-like episodes, migraine-like headaches with nausea
and vomiting, homonymous hemianopsia, hemiparesis, and
hemihypesthesia.
The MERRF/MELAS overlap syndrome has been reported
in seven sporadic cases, which were associated with four
mtDNA point mutations, namely, m.8356T>C (Nakamura
et al. 2010; Sano et al. 1996; Serra et al. 1996; Zeviani et al.
1993), m.3243A>G (Campos et al. 1996; Nakamura et al.

Fig. 5 Western blot analysis of mitochondrial respiratory chain complexes
I, II, III, IV, and V. a Representative blots from six different experiments. b
Semi-quantified statistics. The semi-quantified statistics for Western blot

2010), m.13042G>A (Naini et al. 2005), and m.7512T>C
(Nakamura et al. 1995). Previous studies have associated
heteroplasmic m.3291T>C mutation with MELAS (Goto
et al. 1990), isolated mild myopathy (Uziel et al. 2000), dementia with hearing loss (Salsano et al. 2011), MERRF and
Kearns–Sayre overlap syndrome (Emmanuele et al. 2011),
cerebellar ataxia with ophthalmoparesis, hearing loss and myopathy (Sunami et al. 2011), and deafness and lipomas (Yarham
et al. 2013). In this family, PCR–RFLP analysis detected the
m.3291T>C mutation in the MT-TL1 gene, showing maternal
inheritance of the mutation in a heteroplasmic manner.
The asymptomatic mother and sister had the m.3291T>C
mutation in their blood but in a significantly lower percentage
compared with the proband. These findings indicate that the
degree of heteroplasmy is positively correlated with the clinical manifestations. Meanwhile, the amount of mutations
varied in the proband’s different tissues. That is, the amount
of mutations was higher in the muscle and lower in the
lymphocytes and fibroblasts. This result is consistent with
previous studies, Uziel et al. (Uziel et al. 2000) considered
m.3291T>C as a pathogenic mutation with higher amount in
muscles than in lymphocytes and fibroblasts. This result can
be attributed to the fact that the negative selection for defective
mitochondria is possible for rapid turnover cells, such as
lymphocytes and fibroblasts, but difficult for highly differentiated, postmitotic syncytial muscle cells.

was analyzed and normalized with porin. (n =6). **p <0.01 compared with
the control group

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Furthermore, we performed OCR and ECAR measurements to indicate aerobic respiration and glycolysis of the
patient’s fibroblasts. OCR and ECAR measurements are considerably more sensitive than spectrophotometric assays in
reflecting mitochondrial function and activity, which revealed
that mitochondrial function and activity of fibroblasts harboring m.3291T>C mutation were significantly worse than those
of the normal control. Consistent with the aerobic respiration
capacity of the fibroblasts, Western blot demonstrated that the
contents of complexes I and IV were significantly lower in the
patient’s muscle mitochondria. Therefore, these results also
suggest that m.3291T>C is pathogenic.
The m.3291T>C mutation affects a nucleotide in the T-=-C
loop of the tRNALeu(UUR) cloverleaf, which might inhibit
aminoacylation of the wild-type tRNA (Hao et al. 2004), and
also shows partial deficiency of the normal taurine modification
at the wobble position (Kirino et al. 2005). However, some
scholars had questioned the pathogenicity of m.3291T>C mutation because it only shows moderate conservation according
to the consensus panel of species (Ding and Leng 2012).
Yarham et al. (Yarham et al. 2013) have recently proven
through single-fiber studies that the m.3291T>C mutation is
segregated with biochemical deficiency, by which they confirmed the pathogenicity of the m.3291T>C mutation in human
mitochondrial disease.
This study is the first to describe the rare MT-TL1
m.3291T>C mutation in association with the MERRF/
MELAS overlap syndrome, further expanding the clinical
spectrum associated with the m.3291T>C mutation.
Moreover, this case further supports the pathogenicity
of the previously reported m.3291T>C mutation in the
mitochondrial disease.
Acknowledgments This study was supported by the following funding
sources: the National Nature Science Foundation of China (Grant No.
81171182); the National Natural Science Foundation for Young Scholars
of China (Grant No. 81101157); and the Shandong Provincial Natural
Science Foundation, China (Grant No. 2009ZRB01893).
Conflict of interest The authors declare no conflicts of interest.

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