Near-Identical Segregation of mtDNA Heteroplasmy in Blood, Muscle, Urinary Epithelium, and Hair Follicles in Twins With Optic Atrophy, Ptosis, and Intractable Epilepsy

Importance  Mitochondrial DNA (mtDNA) disorders have emerged as major causes of inherited neurologic disease. Despite being well recognized for more than 2 decades, the clinical presentation continues to broaden. The phenotypic heterogeneity is partly owing to different percentage levels of mutant mtDNA heteroplasmy in different tissues, but the factors influencing this are poorly understood.

Observations  This case report describes monozygotic male twins with ptosis, optic atrophy, and recent-onset intractable myoclonic epilepsy. The assessment of respiratory chain enzyme activities in the muscle from 1 twin revealed a severe and isolated defect involving mitochondrial complex I. Mitochondrial DNA sequencing revealed a pathogenic m.14487T>C MTND6 mutation, which was present at very high levels of heteroplasmy in muscle (84%) and lower levels in blood (15%), urinary epithelium (75%), and buccal mucosa (58%). Of particular interest, his identical twin was found to harbor very similar levels of the m.14487T>C mutation in his blood, urine, buccal mucosa, and hair follicle DNA samples, while the presence of low levels in the mother’s tissues confirmed maternal transmission.

Conclusions and Relevance  It was shown that m14487T>C can also cause the unusual combination of optic atrophy, ptosis, and encephalomyopathy leading to intractable seizures. Near-identical heteroplasmy levels in different tissues in both siblings support a nuclear genetic mechanism controlling the tissue segregation of mtDNA mutations.

Pathogenic mitochondrial DNA (mtDNA) mutations were first described more than 25 years ago, and mtDNA sequencing has been a mainstream diagnostic test for more than a decade. Therefore, it is remarkable that new phenotypes continue to be identified that defy clinical paradigms. Here we describe 2 monozygotic twins with bilateral optic neuropathy and ptosis, preceding a fluctuating leukoencephalopathy with myoclonus and intractable seizures. Despite strong clinical clues, the underlying gene defect was not in the expected place and was only identified through a systematic clinical approach and respiratory chain biochemistry.

Monozygotic male twins were born at 38.5 weeks through spontaneous vaginal delivery, with no perinatal problems. Mild bilateral ptosis was noticed at birth, but they were otherwise well and had normal development. There was no relevant family history.

Twin 1 presented at age 14 years with visual acuity (VA) of 6/9 on the right and 6/24 on the left. His left visual field was restricted peripherally secondary to a previous retinal detachment that was surgically treated. At the age of 18 years, he experienced intermittent tingling in his right hand and twitching movements of his fingers and arm. At the of age 20 years, he had 2 generalized tonic-clonic seizures, followed by persistent jerking of his right upper limb. Brain magnetic resonance image (MRI) findings showed lesions in the postcentral gyrus bilaterally and in the left posteromedial thalamus (Figure 1). An electroencephalogram showed an alpha rhythm of 9 to 11 Hz, with some theta waves posteriorly and multifocal spike-wave discharges predominantly over the left centroparietal region. Repeat imaging showed regression of the lesion in the left thalamus but a new mirror lesion in the right thalamus. Two and a half years after his first scan, there was a new left superior temporal lesion and a new cingulate lesion. Nearly 2 years later, these lesions had resolved but there was a new small (7-mm) lesion in the right posterior frontal region.

On examination at age 23 years, he had poor color vision and VA had deteriorated further to 6/36 (left) and 6/60 (right). He had bilateral optic atrophy with thinning of the retinal nerve fiber layer on optical coherence tomography (Figure 2). Bilateral ptosis was evident, with no ophthalmoparesis. The rest of the cranial nerve examination findings were normal. He had brisk symmetrical reflexes and downgoing plantars. There was continuous myoclonic jerking of his right upper limb.

Twin 2 presented at age 21 years with intermittent numbness and tingling affecting his right hand. At the time, brain MRI, electroencephalography, and nerve conduction study findings were normal. He had his first generalized tonic-clonic seizure at the age of 23 years, then experienced episodes of right hemiparesthesia as he was going into sleep or as he was waking. He subsequently developed visual symptoms, which consisted of seeing red dots and flashing blotches and eventually obscured central vision. He had reduced VA (6/9 bilaterally). Visual evoked potentials and electroretinography findings were consistent with a bilateral optic neuropathy secondary to retinal ganglion cell dysfunction. At the age of 24 years, he suffered clusters of partial seizures affecting his right hand about once per month. Brain MRI showed 2 small, predominantly cortical lesions on fluid-attenuated inversion recovery imaging in the right superior temporal gyrus and posterior frontal region. Six months later, 2 new lesions had appeared in the postcentral gyrus on either side. On examination at age 26 years, his VA was 6/9 (left) and 6/18 (right). He had markedly reduced color vision in both eyes and bilateral dense central scotomas. Fundal examination revealed bilateral optic atrophy. Optical coherence tomographic imaging confirmed significant thinning of the peripapillary retinal nerve fiber layer bilaterally (eFigure in Supplement). He had bilateral ptosis with symmetrical bilateral restriction of eye movements and intermittent jerky movements of his right arm, but neurological examination of the limbs was otherwise normal.

Given the familial optic atrophy, twin 1 was initially tested for common mtDNA mutations. Selective sequencing of blood mtDNA was negative for the m.11778A>G, m.3460A>G, and m.14484T>C Leber hereditary optic neuropathy (LHON) mutations. Given the fluctuating leukoencephalopathy, m.3243A>G and POLG1 were also sequenced, and the familial ptosis prompted m.8344A>G, PEO1, ANT1, RRM2B, and POLG2 sequencing, all of which were normal. He subsequently underwent a muscle biopsy, which showed no histochemical evidence of mitochondrial dysfunction. However, measurement of the individual respiratory chain enzyme activities revealed an isolated deficiency in mitochondrial complex I (21% of controls), with normal activities of complexes II, III, and IV. Sequencing the entire mitochondrial genome in muscle DNA revealed the m.14487T>C (p.Met63Val) MTND6 gene mutation, a recognized cause of isolated complex I deficiency.^1-9 Quantitative pyrosequencing showed the mutation to be present at levels of 84% in skeletal muscle, 15% in blood, 75% in urinary epithelium, 58% in buccal mucosa, and 86% in hair follicles. His identical twin harbored the similar mutation at levels of 17% in blood, 71% in urine, 57% in buccal mucosa, and 74% in hair follicles, while their unaffected mother had significantly lower mutation levels (5% in blood, 25% in urine, 30% in buccal mucosa, and 34% in hair follicles).

The pathogenicity of the m.14487T>C mutation has been previously confirmed because (1) it has never been reported as a polymorphism in more than 10 000 control subjects.¹⁰ (2) It is predicted to cause an amino acid substitution of a highly conserved methionine to valine (p.Met63Val) in the ND6 protein associated with an isolated complex I defect. (3) Transmitochondrial cybrid studies have demonstrated that the complex I defect correlates with the mutant heteroplasmy levels.^7,9 (4) The mutation is heteroplasmic and segregates with a clinical phenotype. (5) It is a recurrent cause of mitochondrial disease, associated with several clinical presentations including severe infantile and childhood-onset Leigh syndrome, Leigh-like encephalopathy^1-8 and progressive dystonia,⁹ and juvenile-onset Leigh syndrome with optic atrophy, ataxia, and epilepsy.⁴ Low levels of m.14487T>C mutation heteroplasmy were associated with a variety of phenotypes in a Belgian family that included subclinical LHON, migraine with aura, bilateral sensorineural hearing loss, or type II diabetes mellitus.¹

The twins illustrated several important clinical points. First, the combination of optic atrophy and ptosis/progressive external ophthalmoplegia is not typical for a mtDNA disorder. It is usually seen in patients with a Mendelian disorder due to mutations in OPA1. Second, a fluctuating encephalopathy with intractable epilepsy is usually seen in patients with an intramitochondrial protein translation, commonly due to either a mtDNA mutation affecting a transfer RNA gene or to mutations in POLG where secondary mtDNA deletions contribute to the phenotype. A mtDNA protein-coding mutation, such as the one found in the twins we describe here, would be most unusual in both clinical contexts. Although additional clinical features have been described in patients with optic atrophy due to the 3 common LHON mutations in MTND1, MTND4, and MTND6, including psychiatric disturbances, spastic dystonia, ataxia, and juvenile-onset encephalopathy, these extraocular manifestations are individually rare. The complex phenotype displayed by the 2 monozygotic twins in this report have not been described before, adding to the spectrum of LHON plus syndromes (reviewed in the article by Yu-Wai-Man et al¹¹). Likewise, mutations in MTND5 have been associated with mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (MELAS) or with overlap syndromes of MELAS and Leigh syndrome, MELAS and LHON, and MELAS and myoclonic epilepsy and ragged red fibers.¹² Finally, the imaging findings were unusual for a complex I defect, particularly when due to a mtDNA mutation.^13,14

Finally, it is striking that both twins had almost identical heteroplasmy levels in 4 tissues, which were different to their mother. Genetic analysis with 16 highly informative microsatellite markers confirmed that the twins were identical, and thus arose from the same oocyte. Therefore, it is highly likely that they both inherited the same mutation load from their mother. The near-identical levels in other tissues strongly suggest that tissue-specific segregation of mtDNA levels is tightly regulated, and identical levels in identical twins is in keeping with recent animal studies implicating a nuclear-genetic control mechanism.¹⁵ These findings contrast with identical twins harboring a mtDNA deletion,¹⁶ where strikingly different levels of mtDNA heteroplasmy could have arisen through unequal partitioning of genomes during development. This does not appear to be the case for single nucleotide variants based on a recent study in twins.¹⁷

These findings stress the importance of taking a systematic, algorithmic-based approach when investigating suspected mitochondrial disease. Even in this era of high-throughput genomics, a muscle biopsy and subsequent biochemical analysis should be considered in patients who do not harbor a common mutation in mtDNA or a relevant nuclear gene because these well-established clinical investigations may lead directly to the causal gene.

Corresponding Author: Patrick F. Chinnery, FRCP, FMedSci, University of Newcastle Upon Tyne, Institute of Genetic Medicine, Central Parkway, New Castle upon Tyne, Tyne and Wear NE1 3BZ (p.f.chinnery@ncl.ac.uk).

Accepted for Publication: July 3, 2013.

Published Online: October 14, 2013. doi:10.1001/jamaneurol.2013.4111.

Author Contributions: Prof Chinnery had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Manford, Taylor, Chinnery.

Acquisition of data: Spyropoulos, Manford, Alston, Yu-Wai-Man, He, Chinnery.

Analysis and interpretation of data: Spyropoulos, Manford, Horvath, Yu-Wai-Man, Taylor, Chinnery.

Drafting of the manuscript: Spyropoulos, Manford, Chinnery.

Critical revision of the manuscript for important intellectual content: Horvath, Alston, Yu-Wai-Man, He, Taylor, Chinnery.

Obtained funding: Chinnery.

Administrative, technical, or material support: Manford, Taylor, Chinnery.

Study supervision: Manford, Taylor, Chinnery.

Conflict of Interest Disclosures: None reported.

Funding/Support: The Wellcome Trust sponsored this study.

Role of the Sponsor: The Wellcome Trust had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We are grateful to Catherine Zhang for her help collecting some of the clinical data.