OBSERVATION

Revelation of a New Mitochondrial DNA Mutation
(G12147A) in a MELAS/MERFF Phenotype
Mariarosa A. B. Melone, MD; Alessandra Tessa, PhD; Stefania Petrini, PhD; Giacomo Lus, MD;
Simone Sampaolo, MD; Giuseppe di Fede, MD; Filippo M. Santorelli, MD; Roberto Cotrufo, MD

Background: A 26-year-old man presented at onset with

the syndrome of mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) and later
with a phenotype for MELAS and myoclonic epilepsy and
ragged red fiber disease (MELAS/MERRF).
Objective: To identify the possible defects in the mi-

tochondrial genome in blood and muscle samples of the
patient.
Design: Case study of a patient clinically exhibiting
strokelike episodes and then epilepsy with myoclonic features, ataxia, and dementia.
Setting: Research unit of a university hospital.
Main Outcome Measures: Electromyographic, morphologic, and biochemical studies of muscle and molecular analysis of blood and muscle to investigate mitochondrial DNA (mtDNA) size and quantity.

T

From the First Division of
Neurology, Department of
Neurological Sciences, Second
University of Naples, Naples,
Italy (Drs Melone, Lus,
Sampaolo, di Fede, and
Cotrufo); and the Department
of Molecular Medicine, Istituto
di Ricerca e Cura a Carattere
Scientifico (IRCCS)–Bambino
Gesù (Drs Tessa, Petrini, and
Santorelli), Rome, Italy.

Results: Morphologically, we found abnormal mitochondrial proliferation with several cytochrome-c oxidase (COX)–negative fibers in muscle biopsy specimens; the analysis of serial sections showed a decreased
immunoreactivity for the mtDNA-encoded subunits
COXII and, partially, COXI. Biochemically, we found a
partial and isolated COX deficiency. The complete mtDNA
sequence analysis identified 3 sequence changes, 2 of
which were reported polymorphisms. The remaining
change, a G12147A transition in the transfer RNAHis gene,
appeared to be the likely pathogenic mutation.
Conclusions: Our data propose that the G12147A
change, the first mutation in the transfer RNAHis gene associated with an overlapped MELAS/MERFF phenotype, is the cause of the encephalomyopathy in this patient interfering with the overall mitochondrial protein
synthesis.

Arch Neurol. 2004;61:269-272

HE ASSOCIATION BETWEEN

the syndrome of mitochondrial encephalomyopathy,
lactic acidosis, and strokelike episodes (MELAS)
(Mendelian Inheritance in Man 540000)
and the A3243G mutation in the mitochondrial DNA (mtDNA) transfer RNA
(tRNA)Leu(UUR) gene is well established in
all ethnic backgrounds.1 However, clinical and genetic heterogeneity is also recognized. Besides the obligatory signs indicated in the acronym, virtually any
neurological abnormality described in mitochondrial disorders (often in variegated combination) can occur in patients
with typical MELAS.2 Moreover, approximately 20% of A3243G-positive patients
are affected by other syndromes, including progressive external ophthalmoplegia3 and a syndrome combining deafness
and diabetes mellitus.4 Furthermore, not
all typical patients with MELAS carry the
A3243G mutation. Generally, myoclonic

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269

epilepsy and ragged red fiber disease
(MERRF) (Mendelian Inheritance in Man
545000) can be clinically distinguished
from MELAS on the basis of the absence
of strokes.5 Seldom, features of MERRF and
MELAS can be seen during the clinical
course of some patients6-8 and within the
same pedigree.9 Approximately 80% to 90%
of MERRF cases are caused by a heteroplasmic G-to-A point mutation at base pair
8344 in the T␺C loop of the tRNALys gene
(G8344A mutations).10,11
In a molecular investigation of a case
that was negative for A3243G and G8344A,
we identified a novel heteroplasmic mtDNA
mutation (G12147A) in the tRNAHis gene.
The patient exhibited a progression from
MELAS to an overlapped MELAS/MERRF
phenotype.
REPORT OF A CASE

Patient III:1 is a 26-year-old man whose
medical history was significant for psyco-

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METHODS

Figure 1. Axial noncontrast T2-weighted magnetic resonance image in the
proband (III:1).

pathological disturbances (hysterical and paranoid personality), left sensorineural hearing loss, and endocrine
abnormalities (hypogonadotropic hypogonadism). At 20
years of age, he experienced sudden severe migrainous
headache and vomiting. These episodes were followed
by left lateral homonymous hemianopia and left hemiparesis, complex partial seizure, and stupor for a few days.
Results of a neurological examination showed left hemiparesis, hypoesthesia, and lateral homonymous hemianopia slowly and incompletely remitting in 3 months.
At 22 years of age, he had 2 seizures in 2 consecutive days,
without modifications of his neurological status. A third
episode of his disease occurred at 23 years of age, characterized by sudden cortical blindness, without recovery. At 25 years of age, he was admitted to the hospital
because of the appearance of myoclonus and ataxia, which
emphasized a progression to a MERRF phenotype. A brain
magnetic resonance image (Figure 1) disclosed T2weighted hyperintense signals in the right posterior temporal lobe and bilaterally in the occipital lobe. Moreover, as a sequela of previous episodes, small areas of
abnormal signal intensity were noticed in the left temporal lobe, right thalamus, and periaqueductal gray matter. Blood and cerebrospinal fluid lactate levels and serum creatine kinase activity were abnormally increased.
The proband’s mother (II:1) had died after an extended strokelike attack at 36 years of age. Her medical
records showed recurrent strokelike episodes of hemiparesis and hemianopia, often associated with severe migrainous headache, that began in adolescence.
A total of 11 relatives underwent direct examination and were deemed free of signs and symptoms often
associated with mtDNA disorders. They underwent sampling with informed consent.
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270

Two muscle biopsy specimens from the proband, taken at 20 and
23 years of age, underwent histological and histochemical processing. Also, we performed immunohistochemical incubations with the primary antibodies slow myosin (Ylem Co, Rome,
Italy) and cytochrome-c oxidase (COX) subunits I, II, IV, and
VIc (Molecular Probes, Inc, Eugene, Ore) and detected them with
a streptavidin-biotin method described elsewhere.12 Respiratory chain complexes were measured in a post–600-revolution
supernatant and expressed relative to the activity of the matrix
marker enzyme citrate synthase. The size and quantity of mtDNA
were investigated by means of Southern blotting in skeletal muscle.
We used a procedure described elsewhere1 to screen for the common A3243G MELAS and G8344A MERRF mutations. The entire mtDNA coding region was amplified in 13 overlapping amplicons and sequenced directly with BigDye chemistry (Applied
Biosystems, Foster City, Calif). Using suitable oligonucleotide
primers and the endonuclease Tsp509I, we screened for the
G12147A mutation in tissues from the proband, his relatives, and
control subjects. To assess the abundance of mutated genomes,
radiolabed amplicons were analyzed by means of ImageQuant
software (Molecular Dynamics, Eugene, Ore).
Single-muscle fibers were isolated from cryostat cross section after succinate dehydrogenase staining in the proband. Staining with COXIV was also performed in some experiments. Abnormal fibers were considered those showing unusual
accumulation of mitochondria or pale to absent COX staining. Selected normal and abnormal muscle fibers were processed using a modification of the laser capture microdissection technique (PixArthurus, MWG Biotech, Ebersberg,
Germany) that allows the selective sampling of tissue from histological sections,13 following the manufacturer’s suggested procedures. The relative proportion of wild-type and mutant mtDNA
genomes was determined as described above. We used the nonparametric Mann-Whitney test to compare the levels of mutated genomes in normal and abnormal muscle fibers.
RESULTS

Morphologically, we found abnormal mitochondrial proliferation by means of succinate dehydrogenase staining, with several COX-negative fibers in the muscle biopsy specimens (data not shown). Fibers with strong
succinate dehydrogenase staining represented about 15%
of the muscle fibers. In addition, we found vessels that
were strongly reactive for succinate dehydrogenase. The
mosaic pattern of the COX activity suggested an mtDNA
origin of the disease. This finding was further corroborated by the analysis of serial sections, which showed a
decreased immunoreactivity for the mtDNA-encoded subunits COXII and, partially, COXI (Figure 2A). The
nuclear DNA–encoded subunits COXIV and COXVIc
were virtually normal. Biochemically, we found a partial and isolated COX deficiency (residual activity was
50% normal) in the patient’s muscle homogenate and increased citrate synthase activity indicating mitochondrial proliferation. When compared with the human reference sequence, complete mtDNA sequence analysis
identified 3 sequence changes, 2 of which were reported polymorphisms. The remaining change, a
G12147A transition in the tRNAHis gene (Figure 2B), appeared to be the likely pathogenic mutation. The mutation was present in 81% of the muscle mtDNA molecules and in 25% of the mtDNA from the patient’s blood,
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but it was absent in blood, hair roots, and urinary sediment from 11 healthy maternal relatives (Figure 2C).
Single-muscle fiber analysis showed more abundant mutated genomes in abnormally appearing muscle fibers
(n=22) than in normal fibers (n=15). The mean±SD percentage of mutant mtDNA was 90% ± 2% in the former
group and 58%±6% in the latter, and this difference was
significant (P = .007). In the 12 ragged red–COXnegative fibers studied, the average of mutated genomes
was even more abundant (94%).

A

B
G T

(REPRINTED) ARCH NEUROL / VOL 61, FEB 2004
271

T T T G G T
210

T A A A C T A T A T T T A C A A
220
230

Control

G T

COMMENT

The combination of the pathologic history of the mother,
clinical data, and biochemical and morphologic impairment of oxidative metabolism strongly indicated a defect
in the mitochondrial genome in this patient. The G12147A
transition in the tRNAHis gene was considered potentially
pathogenic for indirect reasons. First, it was heteroplasmic and detectable at higher levels in skeletal muscle than
in circulating lymphocytes. This is a well-recognized phenomenon for many pathogenic mtDNA point mutations.
Second, the mutation was not identified in 100 healthy subjects or in 35 disease controls, and it has not been reported previously as a common polymorphic variant.14
Third, the involved nucleotide within the dihydrouridine
arm of the tRNA displays high phylogenetic conservation
through evolution. The computer-generated model of the
mutant tRNA using the MFOLD program (available at http:
//www.bioinfo.rpi.edu/applications/mfold) reveals that the
novel change is likely to perturb secondary and tertiary
structure. As shown for another pathogenic mtDNA
change,15 mutations in the dihydrouridine arm might well
cause mitochondrial dysfunction by impairing mitochondrial protein synthesis. For instance, this seems pertinent
for COXIII, the richest polypeptide in histidine residues,14 eventually accounting for the observed COX defect in skeletal muscle. Finally, the deleterious potential of
the mutation was also shown by the highly significant correlation between the morphologic defect and mutation load
in single-muscle fibers as shown by laser capture microdissection–polymerase chain reaction analyses.
Our data propose that the G12147A change, the first
mutation in the tRNA His gene associated with the

Patient III:1

COXI

Control

COXII

Figure 2. A, Results of immunohistochemistry in serial muscle sections from a
control subject and the proband (patient III:1) using antibodies against
cytochrome-c oxidase subunit I (COXI) and COXII (bar indicates 60 mm). In
serial sections, a COX-negative muscle fiber is indicated by an arrowhead. B,
Electropherogram of the mitochondrial DNA (mtDNA) transfer RNA (tRNA)His
gene flanking the novel G12147A mutation. The sequence of DNA from a healthy
control and patient III:1 are presented. Sequencing was performed by means of
an antisense primer. The arrow indicates the novel mutation. C, Autoradiogram
of the restriction-fragment length polymorphism analysis used to quantitate
mutant mtDNA. A 470–base pair (bp) polymerase chain reaction fragment
flanking the tRNAHis gene was amplified using suitable oligonucleotide primers.
The amplicon was cleaved with the endonuclease Tsp509 I and electrophoresed
on a 12% polyacrylamide gel, and the dried gel was exposed to an x-ray film. The
G12147A mutation introduces an additional Tsp509 I site of cleavage in the
389-bp fragment (wild-type [wt]) generating 2 additional fragments sized 163
and 226 bp (mutation [mut]). Lanes 1 to 9 are subjects III:1, III:2, III:3, III:4, II:2,
II:3, I:1, I:2, and I:3 in the superimposed pedigree. Lanes 10 and 11 are from a
healthy control and a patient with the A3243G mtDNA mutation, respectively. The
DNA marker size is also indicated. No sample could be tested from the deceased
mother (II:1). The solid symbols in the pedigree show clinically affected family
members; open symbols, healthy members; squares, male members; circles,
female members; slash, deceased; and arrow, proband.

T T T G G T
220

T A A A T T A T A T T T A C A A
230
240

Patient III:1

C
I

1

2

3

II
1

2

3

4 5

III
1 2
1

3

4

2

3

4

Wt
Mut

5

6

7

8

9

10

11

400 bp
300 bp
200 bp
100 bp

MELAS/MERRF phenotype, is the cause of the encephalomyopathy in this patient. Its pathogenicity stems from
alteration of a highly conserved nucleotide, perturbation of the tRNA secondary structure, and good correlation with the morphologic alterations in the muscle biopsy specimens. Although it is possible that the novel
change interferes, reducing particularly COX peptide levels, the exact mechanisms underlying the clinical presentation of the patient remain to be shown.
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Accepted for publication September 12, 2003.
Author contributions: Study concept and design (Drs
Melone and Santorelli); acquisition of data (Drs Melone,
Tessa, Petrini, Lus, Sampaolo, di Fede, and Santorelli);
analysis and interpretation of data (Drs Melone, Tessa, Petrini, Santorelli, and Cotrufo); drafting of the manuscript
(Drs Melone, Tessa, Petrini, Lus, di Fede, and Santorelli); critical revision of the manuscript for important intellectual content (Drs Melone, Lus, Sampaolo, Santorelli, and Cotrufo); obtained funding (Drs Lus and
Sampaolo); administrative, technical, and material support (Dr Santorelli); study supervision (Drs Melone, Santorelli, and Cotrufo).
Work in the Filippo Maria Santorelli Laboratory, Department of Molecular Medicine, IRCCS Bambino Gesù,
Rome, Italy, was supported by grants from the Italian Ministry of Health, Rome.
Corresponding author and reprints: Mariarosa A. B.
Melone, MD, First Division of Neurology, Department of
Neurological Sciences, Second University of Naples, School
of Medicine, Policlinico Universitario Federico II, Edificio10, Via Sergio Pansini, 5, 80131 Naples, Italy (e-mail:
marina.melone@unina2.it).
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