Article Text


Heterogeneous presentation in A3243G mutation in the mitochondrial tRNALeu(UUR) gene


AIMS To clarify the phenotype–genotype relation associated with the A3243G mitochondrial DNA mutation.

METHODS Five unrelated probands harbouring the A3243G mutation but presenting different clinical phenotype were analysed. Probands include Leigh syndrome (LS3243), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS3243), progressive external ophthalmoplegia (PEO3243), and mitochondrial diabetes mellitus (MDM3243). Extensive clinical, histological, biochemical, and molecular genetic studies were performed on five families.

RESULTS All patients showed ragged red fibres (RRF), and focal cytochrome c oxidase (COX) deficiency except for the patient with MDM3243. The mutation load was highest in the proband with LS3243 (>90%), who also presented the highest proportion of RRF (68%) and COX negative fibres (10%), and severe complex I plus IV deficiency. These proportions were lower in the probands with PEO3243 and with MDM3243.

CONCLUSION The most severe clinical phenotype, LS3243, was associated with the highest proportion of the A3243G mutation as well as the most prominent histological and biochemical abnormalities.

  • Leigh syndrome
  • progressive external ophthalmoplegia
  • mitochondrial DM

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An A3243G mutation in the mitochondrial tRNALeu(UUR) gene has been associated with various types of mitochondrial multisystem disorder, such as mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS),1 2 myoclonus epilepsy with ragged red fibres (MERRF),3 MELAS+MERRF,4 maternally inherited progressive external ophthalmoplegia (PEO),5MELAS+PEO,6 mitochondrial diabetes mellitus (MDM),7 hypertrophic cardiomyopathy,8 cluster headache,9 pancreatitis,10 and a subtype of Leigh syndrome (LS).11

Here we report the five unrelated probands having the A3243G mutation in the mitochondrial tRNALeu(UUR) gene, including clinically and pathologically defined LS3243, MELAS3243, PEO3243, and MDM3243. Our LS proband is the second case found to be associated with A3243G mutation. Though the muscle is not the main target of any of the four clinical phenotypes studied here (except, perhaps, PEO), we analysed the variations in the distribution pattern of the A3243G mutation in muscle in order to clarify the phenotype–genotype relation in A3243G disorders.

Patients and methods


Figure 1 shows the pedigrees of LS3243 (A), MELAS3243 (B and C), PEO3243 (D), and MDM3243 (E). Table 1 summarises the clinical findings of all these probands and their family. All probands and their family members gave their informed consent and the study was approved by the local ethics committee.

Figure 1

Pedigrees of five families with A3243G mutation: family A (LS2433); families B and C (MELAS3243); family D (PEO3243); family E (MDM3243). The proportions of mutant mtDNA in peripheral blood lymphocytes (B), hair follicles (H), and muscle (M) are shown. Arrows denote the probands. Solid symbols denote patients having each symptom; shaded symbols denote oligosymptomatic relatives.

Table 1

Patients with A3243G mutation


The proband, subject AIII3 died at 10 years of age. He had been clinically and pathologically described previously as having Leigh disease.12 He was born from non-consanguineous parents, at 39 weeks gestation, with an uneventful delivery. The early developmental milestones were normal. However, no further developmental progress occurred until after 20 months of age. At 38 months old, he was admitted for generalised seizure, generalised ataxia, and diffuse muscle weakness. His height was 89 cm (−2.7 SD below normal), body weight 10.4 kg (−3SD below normal), and his developmental quotient (DQ) was 68. A Levine 2/6 ejection systolic murmur was audible on the fourth left intercostal line. Deep tendon reflexes were diminished. A chest x ray showed an enlargement in the cardiothoracic ratio (68%). An echocardiogram showed hypertrophic cardiomyopathy. On laboratory examination, hepatic transaminase and blood urea nitrogen were within the normal range. The concentrations of lactate and pyruvate were increased to 6.0 mmol/l (normal 0.3–1.3 mmol/l) and 0.17 mmol/l (0.03–0.08 mmol/l) in blood, and 8.45 and 0.26 in CSF, respectively. A muscle biopsy was taken at the age of 3 years 9 months.

His condition remained stable until 9 years of age when he developed slight but increasing impairment in visual acuity. Progressive muscle weakness occurred. At the age of 10 years 3 months, he developed intractable epilepsy characteristic of Lennox–Gastaut syndrome. A computed tomography (CT) scan of the brain revealed notable cortical atrophy, ventricular dilatation, and enlargement of the fourth ventricle. At the age of 10 years 10 months, he underwent an operation for paralytic ileus; 10 days later he developed respiratory insufficiency. He died with complications of respiratory distress and severe infection. An autopsy was performed after receiving informed consent.

The mother (AII2), a 42 year old woman, had been healthy until about 10 years previously, when she first noticed progressive muscle weakening; since then she has also suffered migraine headaches. However, she has never been admitted for any detailed examination.

The grandmother (AI4), a 78 year old woman, had developed muscle weakness and easy fatiguability for the past 25 years. However, she declined to undergo any clinical or other further examination.

The proband's elder brother (AIII1) has noticed migraine headache. However, all other relatives presented no symptoms suggesting any mitochondrial disorders.


The proband, MELAS3243 (BII2) was admitted to our hospital at the age of 10 years for generalised muscle weakness, migraine headache, periodic vomiting, and right side hemiparesis. On admission, her height was 121 cm (−2.7 SD below normal), body weight 17 kg (−3SD below normal), and her IQ was 102. She had bilateral sensorineural hearing loss (below 40 db), severe muscle weakness, and muscle atrophy. CT and magnetic resonance imaging (MRI) scans showed cortical atrophy and calcification of the brain stem. She fulfilled the clinical criteria of MELAS. She underwent a muscle biopsy at the age of 13 years.

The elder sister (BII1) and the mother (BI2) have been suffering migraine headaches for 10 years.


The proband, MELAS3243 (CII2) was admitted to our hospital at the age of 23 years for generalised muscle weakness, periodic vomiting, and hemiparesis. On admission, her height was 146 cm (−1.5 SD below normal), body weight 45 kg (−1.3 SD below normal), and her IQ was 106. She had sensorineural hearing loss (below 30 db), severe muscle weakness, and muscle atrophy. CT and MRI scans showed cortical atrophy and calcification in the brain stem. She fulfilled the clinical criteria of MELAS. She underwent a muscle biopsy at the age of 26 years.

The elder brother (CII1) and the mother (CI2) suffered migraine headaches.


The proband, PEO3243 (DIII3) was admitted to our hospital at the age of 42 years for progressive external ophthalmoplegia (PEO) and generalised muscle weakness. His height was 168 cm (mean), body weight 52 kg (−1.3 SD below normal), and his IQ was 98. CT and MRI scans revealed no abnormality. He fulfilled the clinical criteria of maternally transmitted PEO. He underwent a muscle biopsy at the age of 42 years.

Though his maternal relatives (DI4, DII4, DII5, and DIII2) showed PEO, no family member had diabetes, muscle weakness, short stature, or any hearing loss suggesting mitochondrial disorder.


The proband, MDM3243 (EIII3) was admitted to our hospital at the age of 26 years for the control of insulin dependent diabetes mellitus. On admission, he had short stature (−2.0 SD below normal), mild sensorineural hearing loss, and mild muscle weakness. He showed no stroke like episodes and no other symptoms.

His mother had been diagnosed as having diabetes mellitus and had earlier died at the age of 49 years, before the proband was diagnosed. We did not have any opportunity to examine the mother.


Respiratory chain enzyme activities were measured in muscle biopsy specimens from patients with LS3243 (AIII3), MELAS3243 (BII2, CII2), PEO3243 (DIII3), and MDM3243 (EIII3,) and in tissues taken at postmortem examination from patient LS3243(AIII3), according to established procedures.13


Total DNA was extracted from leucocytes, hair follicles, and muscle biopsy specimens. The A3243G mutation was analysed by polymerase chain reaction (PCR)–restriction fragment length polymorphism according to methods described previously.14 Tissues from postmortem examination of LS3243 were also subjected to DNA analysis. The DNA signals were quantified using a BAS 2000II Bioimage Analyser (Fujix Inc., Tokyo, Japan).



There was mild to moderate variation in fibre size in both type 1 and type 2 fibres in all patients examined. Scattered ragged red fibres (RRF) were seen in all probands, except the probands with MDM3243. RRF showed speckled, patchy, or heterogeneous pattern, stronger at the subsarcolemmal regions, while non-RRF showed homogeneous on cytochrome c oxidase (COX) staining. In LS3243, 10% of RRF showed a homogeneously negative pattern on COX. The intramuscular small arteries showed hyperactivity on SDH staining; strongly succinate dehydrogenase reactive blood vessels (SSVs) were seen in all patients, except PEO3243. SSVs were always COX positive. Table 2 summarises the percentages of RRF and of COX negative fibres.

Table 2

Pathological and biochemical analysis of muscle biopsy specimens from LS3243, MELAS3243, and PEO3243


An autopsy was performed four hours after death at the age of 10 years 10 months. The brain was oedematous and increased in softness with leptomeningeal turbidity. Microscopically, there were extensive symmetric necrotic lesions in the brain stem in which the bilateral putamen and thalamus showed necrotic softening. Some symmetrical necrotic legions were noted around the midbrain extending down to the pons, where demyelination, proliferation of capillaries, and gliosis were noted. These findings were consistent with those reported as characteristic lesions of LS. The heart muscle showed increased vacuolation in the myocardium, and also increased fibrosis in the perimysium and endomysium area, showing characteristic features of cardiomyopathy.


Figure 1 shows the percentage of the A3243G mutation in the muscle of the proband, and also percentages in the leucocytes and/or hair follicles of the family members. Maternal transmission of A3243G mutation was shown in all five families studied, except for MDM3243. In the MDM3243 family, we could not detect the mutation in the relatives.


Table 2 summarises results from the biochemical analysis of the muscle biopsy specimens from each of the probands. Enzyme activities of the respiratory chain complexes IV and I were greatly decreased in all probands, except in PEO3243 and in MDM3243whose activities remained normal. The concentrations of cytochrome a+a3 were decreased in LS3243 and MELAS3243, consistent with the decreased activities of COX. In the tissues from postmortem examination of LS3243(AIII3), all respiratory chain enzyme activities were considerably decreased, except for the activity of complex II which remained normal (data not shown).


The percentage of the A3243G mutation determined in the tissues from postmortem examination, including muscle, heart, liver, kidney, and the cerebrum was 92, 91, 90, 97, and 91%, respectively.


We have performed a comparative study of the clinical symptoms, morphology, and mitochondrial genetics of LS3243, MELAS3243, PEO3243, and MDM3243 in order to understand the phenotype–genotype relation in these four disorders associated with the A3243G mutation. We have found a direct correlation between the percentage mutation and an impairment in the respiratory chain function of the muscle tissues. LS3243harboured the highest percentage of A3243G mutation among these four phenotypes and showed the most severe impairment of respiratory dysfunction. The most striking morphological difference among the muscle specimens from these four phenotypes was the higher percentage of RRF (68% of total muscle fibres), and COX negative RRF in LS3243 (10% of total muscle fibres) than that in MELAS3243 (average 0.15%) and PEO3243 (0.2%). Unlike in LS3243, LS caused by COX deficiency or pyruvate dehydrogenase complex deficiency has usually not been associated with RRF. The reduction in COX activity in muscle fibre of the MELAS patient15 appeared to be less severe than that described in MERRF16 or Kearns–Sayre syndrome (KSS)/PEO17 caused by different mtDNA mutations. In MERRF and KSS/PEO, RRF were consistently COX negative, while in MELAS patients, RRF often showed positive COX staining. Our findings were consistent with those of Petruzzella et al 18 who found that even though PEO3243muscle contained less mutant mtDNA overall, these were distributed more heterogeneously in such a way that many fibres had amounts of mutant genomes sufficiently high to cause COX deficiency. In our LS3243, the percentage of mutant in crude homogenate was so high that the percentage mutation in RRF may be much higher than the threshold effect of COX negative change. This may be clarified by single muscle fibre PCR analysis. As SSVs showed positive COX activities, the COX may be not the primary enzyme defect in A3243G mutation. When the percentage mutation is lower than threshold (<95%), the extensive mitochondrial proliferation is able to compensate for the local needs of oxidative phosphorylation in an affected muscle domain. According to the pedigree of these four clinical phenotypes, a striking inherited difference in the heteroplasmic condition in the family members between LS3243 and the others was the higher percentage of A3243G mutation in LS3243. In LS3243, though the percentages of mutant mtDNA from the mother (AII2) and from the grandmother (AI4) were each less than 15% in blood and hair follicle, all the sisters including the proband had a rather higher percentage of mutant mtDNA in muscle, blood, and hair follicle. As the percentages of mutant mtDNA in the autopsy tissues of the proband (AIII3) were all high (>92%) and homogeneously distributed, we have hypothesised that the proportion of mutant genome in each of the fertilised eggs from the mother of LS3243 was much more significant than the other clinical features.

The pathogenic mechanism by which an individual having the A3243G mutation in the tRNALeu(UUR) gene leads to LS and cardiomyopathy, while in others leads to MELAS3243, PEO3243, or the MDM3243 phenotype, remains to be elucidated. Heterogeneous clinical expression of the individual pathogenic mtDNA mutation has been reported; the T8993G mutation in ATPase 6 was associated with neuropathy, ataxia, and retinitis pigmentosa (NARP)19 at moderately high heteroplasmy (about 70%), and extremely high maternally inherited LS (>95%),20 while the A8344G mutation in the mitochondrial tRNALys gene was associated with MERRF,21 and with LS22 in extremely high amounts.

The findings reported here of the different concentrations and different spatial distributions of a single mtDNA point mutation extend this concept to the cellular level, and probably have relevance to other mitochondrial diseases. Moreover, our analyses have suggested that the LS3243 with cardiomyopathy phenotype was the most severe outcome in the clinical spectrum associated with this point mutation of A3243G in the mitochondrial tRNALeu(UUR) gene.


The authors thank Dr Masanori Nakagawa (University of Kagoshima, Japan) for his initial analysis of LS3243. This work was supported in part by grants 11670805 from the Ministry of Culture and Education, and by a grant from the Ishibashi Research Foundation in Japan.


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