Review

Mitochondrial disease in adults: recent advances and

future promise
Yi Shiau Ng, Laurence A Bindoff, Gráinne S Gorman, Thomas Klopstock, Cornelia Kornblum, Michelangelo Mancuso, Robert McFarland,
Carolyn M Sue, Anu Suomalainen, Robert W Taylor, David R Thorburn, Doug M Turnbull

Mitochondrial diseases are some of the most common inherited neurometabolic disorders, and major progress has Lancet Neurol 2021; 20: 573–84
been made in our understanding, diagnosis, and treatment of these conditions in the past 5 years. Development of Wellcome Centre for
national mitochondrial disease cohorts and international collaborations has changed our knowledge of the spectrum Mitochondrial Research,
Translational and Clinical
of clinical phenotypes and natural history of mitochondrial diseases. Advances in high-throughput sequencing
Research Institute,
technologies have altered the diagnostic algorithm for mitochondrial diseases by increasingly using a genetics-first Faculty of Medical Sciences,
approach, with more than 350 disease-causing genes identified to date. While the current management strategy for Newcastle University,
mitochondrial disease focuses on surveillance for multisystem involvement and effective symptomatic treatment, Newcastle upon Tyne, UK
(Y S Ng PhD,
new endeavours are underway to find better treatments, including repurposing current drugs, use of novel small
Prof G S Gorman PhD,
molecules, and gene therapies. Developments made in reproductive technology offer women the opportunity to Prof R McFarland PhD,
prevent transmission of DNA-related mitochondrial disease to their children. Prof R W Taylor PhD,
Prof D M Turnbull PhD); NHS
Introduction techniques, such as mitochondrial replacement therapy, Highly Specialised Service for
Rare Mitochondrial Disorders
Inherited mitochondrial diseases are complex neuro­ has provided hope for mothers with mtDNA-related (Y S Ng, Prof G S Gorman,
genetic conditions that present clinical, diagnostic, and disease. Prof R McFarland,
treat­
ment challenges for neurologists. The substantial Prof R W Taylor,

clinical variability and frequent involvement of other Clinical aspects of mitochondrial disease Prof D M Turnbull) and
Directorate of Neurosciences,
tissues means that recognition of potential mitochondrial Since mitochondria are essential organelles in all human Royal Victoria Infirmary
disease might be delayed. Establishing the genetic diag­ cells, mitochondrial disease can affect all organs; involve­ (Y S Ng, Prof G S Gorman),
nosis of mitochondrial disease can be difficult because ment of the nervous system, however, can be substantial. Newcastle upon Tyne Hospitals
NHS Foundation Trust,
mito­chondria are under dual genetic control by the Initially, many mitochondrial diseases were defined as a Newcastle upon Tyne, UK;
nuclear and mitochondrial genomes. For individuals who cluster of clinical syndromic features—eg, mitochondrial Department of Clinical
present with a recognised clinical syndrome or a positive myopathy, encephalopathy, lactic acidosis, and stroke- Medicine, University of Bergen,
family history, genetic diagnosis is straightforward, but for like episodes (MELAS) syndrome and Kearns-Sayre Bergen, Norway
(Prof L A Bindoff PhD);
many people, mitochondrial disease is just part of the syndrome.1 However, only a few individuals show the full Neuro-SysMed, Department of
differential diagnosis of complex neurogenetic disorders. spectrum of signs at presentation. Key to recognising Neurology, Haukeland
Nevertheless, establishing the genetic cause is crucial for patients with mitochondrial disease has been the use of University Hospital, Bergen,
both prog­nosis and genetic counselling, particularly in the deep phenotyping, full characterisation of clinical features, Norway (Prof L A Bindoff);
Department of Neurology,
case of mitochondrial DNA (mtDNA)-related disease, and identification of other organ involve­ment (appendix Friedrich-Baur-Institute, LMU
which presents unique challenges for affected women in pp 2–4). The initial clinical mani­festation of mitochondrial Hospital, Ludwig Maximilians
terms of reproductive options. In past years, even with a disease might be neuro­logical, or not, and can present University, Munich, Germany
diagnosis of mitochondrial disease, few treatment options acutely, subacutely, or evolve slowly. (Prof T Klopstock MD); German
Center for Neurodegenerative
were available and there were no care guidelines. Diseases , Munich, Germany
The aim of this Review is to highlight the substantial Acute and subacute neurological presentations (Prof T Klopstock); Munich
progress made in mitochondrial disease research in the Stroke-like episodes Cluster for Systems Neurology,
Munich, Germany
past few years, showcasing the important aspects for Stroke-like episodes in individuals with mitochondrial
(Prof T Klopstock); Department
neurologists to recognise, diagnose, and manage adult disease are characterised by headache, nausea and vomit­ of Neurology, Neuromuscular
patients with mitochondrial disease. Our understanding ing, encephalopathy, focal-onset seizures, or psychiatric Disease Section
of the clinical spectrum of mitochondrial disease has symptoms before development of a focal neurological (C Kornblum MD), and Centre
for Rare Diseases (C Kornblum),
progressed with development of large patient cohorts and deficit. Epilepsia partialis continua and, rarely, generalised
University Hospital Bonn,
recognition of genotype-specific symptoms. Advances in status epilepticus might also occur during the stroke-like Bonn, Germany; Department of
genetic sequencing technology have led to recognition episode. Headache and visual disturbance are often Clinical and Experimental
that defects in more than 350 genes of both mitochondrial experi­enced during the prodrome. A consensus statement Medicine, Neurological
Institute, University of Pisa,
and nuclear origin might cause mitochondrial disease, proposed that “a mitochondrial stroke-like episode is a
Italy (Prof M Mancuso PhD);
thereby transform­ ing diagnosis. Although no cure for subacute, evolving brain syndrome driven by seizure Department of Neurogenetics,
mito­chon­drial dis­ease currently exists, consensus-based activity in genetically determined mitochondrial disease”.2 Kolling Institute, Faculty of
clinical guide­lines are available to help manage patients Although stroke-like episodes were originally described Medicine and Health,
University of Sydney,
and, for some mitochondrial diseases, potential treatments in individuals younger than 40 years, late-onset presenta­
Sydney, NSW, Australia
are emerg­ing. An increasing number of clinical trials are tion is now recognised.3 Stroke-like episodes are the (Prof C M Sue PhD); Department
investi­
gating new or repurposed small molecules and defining feature of MELAS syndrome, with the pathogenic of Neurology, Royal North
gene therapy. Finally, the development of reproductive m.3243A→G MT-TL1 gene variant accounting for up Shore Hospital, Northern

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 Review

Sydney Local Health District, Patients with the m.8344A→G variant can have both myo­
St Leonards, NSW, Australia A B C D clonic epilepsy and subcortical myoclonus without any
(Prof C M Sue); Research
Program in Stem Cells
EEG correlates.11,12
and Metabolism,
Faculty of Medicine Metabolic decompensation of brainstem function
(Prof A Suomalainen PhD) and Leigh syndrome is the most common presentation of
Neuroscience Centre, HiLife
(Prof A Suomalainen),
paediatric mitochondrial disease,13 although some children
E F G H present late and those who are mildly affected can survive
University of Helsinki, Helsinki,
Finland; Helsinki University into adulthood.14 Patients with Leigh syndrome can develop
Hospital, HUSlab, Helsinki, subacute brainstem dysfunction when metabolically chal­
Finland (Prof A Suomalainen);
Murdoch Children’s
lenged (eg, during intercurrent infections; figure 1).
Research Institute Features of Leigh syndrome include ophthalmoplegia,
(Prof D R Thorburn PhD), and Figure 1: Neuroimaging features of mitochondrial diseases ataxia, dysphagia, dysarthria, central hypoventilation,
Victorian Clinical Genetics Stroke-like lesions involving the right temporal, occipital (A) and parietal lobes hyper­­somnolence, and autonomic instability. Brainstem
Services (Prof D R Thorburn), (B) with restricted diffusion (not shown) are seen in a 20-year-old man with
Royal Children’s Hospital, m.3243A→G-related mitochondrial myopathy, encephalopathy, lactic acidosis,
syndrome has also been reported in adult patients who
Melbourne, VIC, Australia; and stroke-like episodes syndrome. Severe cerebral atrophy (C) is evident in a initially presented with neuropathy and ataxia related
Department of Paediatrics, 50-year-old man with the m.3243A→G variant who had experienced recurrent to MT-ATP6 pathogenic variants, without a pre-existing
University of Melbourne, stroke-like episodes since his mid-30s. Asymmetrical medullary lesions with diag­nosis of Leigh syndrome.15 Partial recovery of brain­
Melbourne, VIC, Australia restricted diffusion (D) are seen in a 25-year-old woman with the
(Prof D R Thorburn) m.9185T→C MT-ATP6 variant who presented with subacute onset dysphagia,
stem function occurs in some patients who survive the
Correspondence to: central hypoventilation, and type 2 respiratory failure; her initial clinical metabolic crisis.
Prof Doug M Turnbull, Wellcome phenotype was teenage-onset progressive ataxia and neuropathy. Symmetrical
Centre for Mitochondrial T2-hyperintensities in the basal ganglia (E) are shown in a woman in her Subacute visual loss and optic neuropathy
Research, Translational and mid-40s who had childhood-onset Leigh syndrome related to the
m.8993T→C MT-ATP6 variant. Symmetrical leukodystrophy (F) is identified in a
Subacute, painless, and progressive impairment of central
Clinical Research Institute,
Faculty of Medical Sciences, 13-year-old boy with Kearns-Sayre syndrome due to a single, large-scale vision is a typical presenting feature of the mitochon­
Newcastle University, mitochondrial DNA deletion. Asymmetrical reduction in dopamine uptake (G) is drial disease Leber hereditary optic neuropathy (LHON).
Newcastle upon Tyne seen on a dopamine transporter PET scan of a 45-year-old man with recessive Typically, onset of this disorder is unilateral, with the
NE2 4HH, UK POLG variants who presented with chronic progressive external ophthalmoplegia,
ataxia, and parkinsonism. Diffuse lipomatosis (H) is seen in the shoulder girdles
second eye becoming affected within weeks or months.
doug.turnbull@ncl.ac.uk
and cervical region in a 44-year-old woman with the m.8344A→G variant. Male carriers of one of the three primary pathogenic
See Online for appendix
variants (m.3460G→A MT-ND1, m.11778G→A MT-ND4,
to 80% of cases; pathogenic variants in other mtDNA and m.14484T→C MT-ND6) associated with LHON are
genes and in the nuclear gene POLG are also known to more likely to be affected by visual impairment than are
cause stroke-like episodes.2,4 women, and smoking and excess alcohol are trigger factors
On conventional MRI, stroke-like lesions associated for visual loss.16 Rapid deterioration of visual function can
with mitochondrial disease typically do not correspond to raise suspicion for inflammatory optic neuritis initially.
vascular territories and involve cortex and juxtacortical Therefore, early diagnosis of LHON could avoid the initia­
white matter (figure 1). MRI proton spectroscopy shows a tion of long-term immunotherapy, which is unnecessary,
lactate peak both in the affected regions and in ventricular ineffective, and potentially detrimental. Clinical features of
CSF or other apparently unaffected brain areas. Lesions LHON are young age (with peak age of onset in the second
might spread over days, weeks, and even months. Some and third decade of life), male sex, absence of pain with eye
signal abnormalities resolve completely; however, most movements, no relative afferent pupillary deficit, maternal
severe lesions develop into cortical laminar necrosis, family history of blindness, normal MRI and CSF results,
gliosis, and atrophy, potentially leading to progressive and no benefit from steroids. Typical ophthal­mological
physi­cal and cognitive decline. Based on the clinical and findings during the acute phase of LHON include hyper­
MRI features, clinicians should think about a mitochondrial aemia of the optic disc, oedema of the peripapillary retinal
stroke-like episode as a main differential diagnosis to nerve fibre layer, retinal telangiectasia, and absence of
atypical recurrent ischaemic stroke,5 encephalitis with a leakage on fluorescein angiography.17 A clinical study using
nega­tive infectious and autoimmune screen,6 and posterior optical coherence tomography showed that thicken­ing of
reversible encephalopathy syndrome.7 the retinal nerve fibre layer preceded angiopathic changes,
measured by an increase in choroidal thickness during the
Epilepsy acute phase of LHON.18
Epilepsy is a common feature of mitochondrial dis­ ­
ease8,9 with both generalised and focal seizures observed. Chronic neurological presentations
In individuals with m.3243A→G and POLG pathogenic Chronic progressive external ophthalmoplegia (CPEO)
variants, seizure activity leading to development of a CPEO is one of the most common adult clinical presenta­
stroke-like episode is typically refractory or super- tions of mitochondrial disease. Only a small proportion of
refractory to phar­maco­logical treatment, including general patients (<30%)19 report diplopia (which can affect reading
anaesthesia—particularly in POLG-related epilepsy.10 ability, balance, and movement; and an early onset of

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 Review

which might result in chronic suppression and amblyopia) other mitochondrial diseases—eg, in m.8344A→G-related
due to the insidious nature of disease progression. or m.3243A→G-related disease.
Depend­ ing on the genetic defect, CPEO can occur in
isolation or with other neurological features and multi­ Other chronic neurological presentations
system involvement. Seronegative myasthenia gravis Cerebellar or sensory ataxia, peripheral neuropathy,
(without AchR and MuSK antibodies) is an important migraine, cognitive impairment, spasticity, and extra­pyra­
differential diagnosis, and patients with CPEO can midal movement disorders including tremor, parkinson­
have minor neuromuscular junction electro­physiological ism, dystonia, and chorea can also be presenting features
abnor­mali­ties.20 Other differ­
ential diagnoses21 include of mitochondrial disease. In some patients, the diagnosis
oculo­pharyngeal mus­ cular dys­
trophy, congenital myas­ of mitochondrial disease is straightforward because of
thenic syndrome, congenital myopathy, and myotonic other classic symptoms, such as a combination of CPEO
dystrophy. and parkinsonism in POLG-related disease, or maternally
inherited diabetes and deafness in m.3243A→G-related
Myopathy disease. In other patients, mitochondrial disease is just
Features that can alert the clinician to mitochondrial one of several diagnostic possibilities. Making a diagnosis
myopathies include fatiguability, myalgia (often exercise- in these patients can be challenging, particularly when
induced), exercise intolerance, and lactic acidaemia. disease onset is late and there is no family history.
Many patients with CPEO also develop myopathy, and Moreover, many aspects of mitochondrial disease are still
some mitochondrial diseases might have concomitant not understood, such as selective tissue involvement and
peri­pheral neuropathy that can be axonal or demyelinat­ the unique patterns of clinical manifestations that occur
ing, including POLG-related disease and mitochondrial with specific gene variants. In such cases, development of
neurogastrointestinal encephalomyopathy.22 Mild bulbar large cohorts and natural history studies should be help­
weakness is not uncommon;23 however, some patients ful, particularly when combined with mechanistic studies.
develop severe dysphagia and are at risk of recurrent
aspiration. Although an elevated creatinine kinase level Non-neurological features
(typically >1000 IU/L) is a recognised laboratory finding Management of patients with mitochondrial disease is
of mitochondrial disease, rhabdomyolysis manifesting complicated by the recognition that multiple organs can
with myoglobinuria is very rare.24 Severe neuromuscular be involved. Moreover, systemic features might be more
weakness (including diaphragmatic weakness) requiring prominent than neurological signs (appendix pp 2–4).
ventilatory support is infrequent in adult mitochondrial
diseases and is typically associated with specific genetic Recent advances in diagnosis and biomarkers
defects such as TK2 deficiency25 and the m.8344A→G Genomics and biomarker development are transforming
mutation. the diagnosis of mitochondrial disease.30,31 Securing a
precise genetic diagnosis is important since knowledge
Sensorineural hearing loss about genetic variants can facilitate the targeting of treat­
To be suggestive of a mitochondrial disorder, hearing ments, help with management of and surveillance for
loss is generally young-onset with other systemic fea­ specific complications, inform counselling for reproduc­
tures associated with a mitochondrial dis­ease,26 and a tive options, and enable patients to be enrolled into
maternal pattern of transmission. Some individuals with appropriate clinical trials. However, access to diagnostic
specific mtDNA pathogenic variants (eg, a homoplasmic tests remains inequitable in some countries.
m.1555A→G variant) develop deafness after exposure to
aminoglycoside antibiotics. The onset of hearing loss is Mitochondrial biomarkers
usually after postlingual develop­ment, indicating that Validated biomarkers now make it possible to avoid
cochlear implantation could be useful as a treatment in invasive muscle biopsies in some cases.32 Two novel
those patients with profound hearing loss.27 biomarkers of mitochondrial dysfunction have been
identified, FGF2133 and GDF15.34 In adults, increased
Optic neuropathy amounts of FGF21 and GDF15 are associated with muscle-
Around 60% of cases of autosomal-dominant optic manifesting mitochondrial translational defects (patho­
atrophy are due to OPA1 genetic defects, which have an genic mt-tRNA variants and mtDNA deletions) and levels
estimated prevalence of 1 in 25 000 people.28 Optic atrophy of the biomarkers correlate with the severity of the
is characterised by a slowly progressive, childhood-onset biochemical defect in muscle.35 Both FGF21 and GDF15
optic neuropathy with moderate-to-severe loss of visual have better sensitivity and specificity for adult mito­
acuity. At least a fifth of patients with OPA1 pathogenic chondrial disease than do other conventional serum
variants develop extra-optic nerve manifestations—eg, biomarkers, such as creatinine kinase and lactate.36,37
hearing impairment, CPEO, neuropathy, cerebellar symp­ Studies have shown that coupled measurement of serum
toms, or parkinsonism.29 Additionally, subclinical optic FGF21 and GDF15 was more sensitive in diagnosing
neuropathy can be an accompanying feature in many mitochondrial disease than were muscle histopathological

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Cytosol low amounts of oxidised nicotinamide adenine dinucleo­

Outer membrane tide (NAD+) in patients with mitochondrial myopathy
Fusion and fission
Inner membrane space machinery were restored by the NAD-booster niacin.46 The potential
(eg, MFN2 and OPA1)
for using blood NAD+ concentrations as a mitochondrial
Matrix
disease biomarker requires further study.
mtDNA
Thymine replication
POLγ
TWNK Genomics
m-AAAs
Inner 22 tRNAs Mitochondrial diseases are the result of a defective oxida­
TYMP

Translation
membrane
13 mRNAs tive phosphorylation system (figure 2), which com­prises
dTTP mtDNA
dNTP 2 rRNAs subunits encoded by both the nuclear and mitochondrial
synthesis dCTP
and salvage
genomes. Mutations in genes encoding subunits of
dATP Transcription ADP + Pi H⁺ ATP
(eg, TK2) this system will lead to mitochondrial disease, as will
dGTP
V
defects in the many other nuclear mitochondrial proteins
NADH H2O
required for normal assembly and activity of the oxidative
NAD⁺ O2
FADH2 FAD
phosphorylation system.1 Nuclear mitochondrial proteins
are also essential for the replication, transcription, and
IV

III e
H⁺

I
II e
CoQ10 Cytochrome c maintenance of mtDNA and defects in these proteins can
H⁺ H⁺ lead to mtDNA depletion or multiple mtDNA deletions
(figure 2).
The genetic characteristics of mtDNA differ from those
Figure 2: Illustration of mitochondrial oxidative phosphorylation system and other pathways that are
commonly implicated in adult mitochondrial disease of nuclear DNA: mtDNA is maternally inherited and
The mitochondrial oxidative phosphorylation system comprises complexes I–V and two mobile electron carriers, present in multiple copies in individual cells. Pathogenic
coenzyme Q₁₀ and cytochrome c. As high-energy electrons are passed along the complexes, protons are pumped out variants can, therefore, affect all copies (homoplasmy) or
of the matrix space, creating an electrochemical membrane potential that is used by ATP synthase (complex V) to
only some (heteroplasmy). In the case of heteroplasmy,
generate ATP. The mitochondrial genome encodes 13 protein subunits, 22 tRNAs, and two rRNAs; there are multiple
copies of mtDNA per cell, ranging from hundreds to thousands depending on the cell type. The replication, the percentage of mtDNA copies affected by a pathogenic
maintenance, transcription, and translation of mtDNA and mtDNA-encoded proteins are dependent on many variant is an important factor in determining whether a
nuclear-encoded proteins. m-AAAs play an important role in the quality control of mitochondrial proteins. Genetic clinical phenotype will manifest (appendix pp 10).
defects in nucleotide synthesis and salvage (eg, TYMP and TK2), mitochondrial DNA replication and maintenance
(eg, catalytic subunit of POLγ and TWNK), fusion and fission machinery (eg, OPA1 and MFN2), and m-AAAs
If there is a high clinical suspicion of mitochondrial
(eg, SPG7) perturb mtDNA integrity and mtDNA copy number, leading to the formation of multiple deletions and disease, such as syndromic presentation and multi­
mtDNA depletion. black stars=amino acid. dA=deoxyadenosine. dC=deoxycytidine. dG=deoxyguanosine. system involvement, genetic testing should be prioritised
dT=thymidine. dATP=deoxyadenosine triphosphate. dCTP=deoxycytidine triphosphate. dGTP=deoxyguanosine (figure 3). Deep clinical phenotyping and an accurate
triphosphate. dNTP=deoxynucleoside triphosphate. dTTP=deoxythymidine triphosphate. m-AAAs=mitochondrial
ATP-dependent proteases. mtDNA=mitochondrial DNA. Pi=phosphate.
family history remain essential when considering if
genomic variants identified by techniques such as whole
abnormalities.38 A meta-analysis of data on the use of exome sequencing (WES) or whole genome sequencing
FGF21 and GDF15 for diagnosis (including five studies (WGS) can account for the clinical presentation. Systematic
of FGF21 and seven studies of GDF15) concluded that enquiries about the clinical histories of other family
FGF21 and GDF15 showed acceptable sensitivity (71% members should include asking about both neurological
for FDF21 and 83% for GDF15) and high specificity and extra-neurological signs and symptoms. Parental
(88% for FDF21 and 92% for GDF15).39 These findings consanguinity strongly sug­ gests an autosomal-recessive
highlight a supportive role for these two biomarkers in the disorder, but there are exceptions such as maternally
diagnosis of mito­ chondrial disease and potentially for inherited mtDNA disease in consanguineous families.48
monitoring of dis­eases such as TK2 deficiency.40 FGF21 is Conversely, an absence of family history should not reduce
of reduced value for non-muscle-related phenotypes of clinical suspicion since de-novo single, large-scale mtDNA
mitochon­drial disease.36 deletions, de-novo mtDNA pathogenic variants,49 and
New fluid biomarkers for mitochondrial disease are de-novo autosomal-dominant and recessive traits are all
emerging, including in amino acid and one-carbon known causes of adult mitochondrial disease.50,51 In clinical
metabolism. Circulating cell-free mtDNA was found to be practice, assessment of late-onset, non-syndromic neuro­
increased in m.3243A→G-related mitochondrial disease logical presentations in adults for a mitochondrial cause
and could be useful for monitoring disease severity,41 and remains a considerable challenge, even after extensive
the amount of serum neurofilament light chain seems to investigations to exclude other conditions.
reflect CNS involvement.42 Tissue-specific stress responses
in the affected organ involving one-carbon cycle, trans- mtDNA genomics
sulphuration, and mTORC1 remain to be established as Approximately two-thirds of cases of adult-onset disease are
specific biomarkers for diagnosis.43,44 Multibiomarkers, in caused by pathogenic mtDNA variants.52 The first step in
the form of metabolomic and proteomic approaches, diagnosing adults is typically mtDNA testing, unless there
show promise30,45 but have not yet advanced to clinical are specific indicators suggesting another gene or mode
implementation. A pilot study showed that systemically of inheritance. For the classic syndromic presentations

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such as MELAS syndrome and LHON, screening for required, alternative approaches such as quantitative
common pathogenic mtDNA variants (eg, m.3243A→G in PCR should be used. Pathological mtDNA variants can
MELAS or three primary MT-ND gene variants in LHON), be detected robustly from WGS data and, with lower
will still have a moderate-to-high diagnostic yield (figure 3). sensitivity, from WES data.30,54
Testing sometimes proceeds straight to amplification of a Testing for pathogenic mtDNA variants in blood alone
full-length mtDNA amplicon, or overlapping amplicons, can yield false-negative diagnoses. Additional use of non-
for massively parallel sequencing.30,53 The high depth of invasively obtained samples (eg, urinary sediment, buccal
coverage with such sequencing provides more sensitive swab) can improve the sensitivity of diagnostic mtDNA
detection of single nucleotide mtDNA variants than with testing and familial screening, although the age-corrected
previous methods such as Sanger sequencing, and accurate level of mutant mtDNA hetero­plasmy in blood for the
quantitation of mutant load. Large-scale mtDNA rearrange­ common m.3243A→G variant has a stronger correla­
ments are also detected, but if accurate quantitation is tion with disease burden and progression than in urine.55

A Suspected acute presentations of mitochondrial disease in adults*

Stroke-like episodes or focal-onset status Brainstem syndrometh with or without basal Subacute optic neuropathy (LHON)
epilepticus (MELAS) ganglia changes (Leigh syndrome)

m.3243A>G and POLG† Common LHON variants (blood)‡

negative Full mtDNA sequencing (blood or urine) with or negative

without WES or WGS§

negative

Novel mtDNA variant or variant of undefined Discussion with mitochondrial specialist service
significance

Familial segregation studies (blood and urine)

and muscle biopsy

B Suspected slowly progressive neurological presentations of mitochondrial disease in adults¶||

Ataxia with Myopathy or exercise intolerance†† Neuropathy Progressive

other systemic optic
features** neuropathy

With other With CPEO Without With other Prominent OPA1§§

systemic CPEO‡‡ systemic gastrointes-
features features tinal symptoms

TYMP

negative
Full mtDNA (blood) or POLG gene Single mtDNA negative Muscle Full mtDNA
sequencing deletion biopsy|||| sequencing
negative
(urine)¶¶ (blood)
Mendellian inheritance

negative

Full mtDNA mtDNA

sequencing deletions OR
(blood) mtDNA
depletion
negative negative
negative
negative WES or
WGS***

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If mtDNA screening is uninformative in blood, testing of advisable to expand the gene list for reanalysis of
muscle might be needed to fully exclude mtDNA-related undiagnosed cases. Early WES analyses were designed
disease.56 primarily to detect single nucleotide variants and short
insertions or deletions (indels), and the limitations of
Nuclear DNA genomics these analyses for identifying repeat-expansion disorders
Pathogenic variants in more than 300 nuclear genes such as Huntington’s disease, Friedreich’s ataxia, and
cause mitochondrial disease.50,57 New gene and pathway spinocerebellar ataxias have been highlighted. However,
discoveries and their link to mitochondrial disease will new bioinformatic approaches have been developed to
continue, as more than 1100 mitochondrial proteins have address such challenges.59
been identified to date.58 In many laboratories, unbiased Interpretation of genomic sequencing results has been
WES and WGS data have replaced the testing of single greatly improved by algorithms that prioritise the likeli­
genes. The improved detection of copy number variants hood of rare variants being pathogenic and by widespread
and mtDNA sequences, and the ability to investigate adoption of the American College of Medical Genetics
non-coding regions in the genome, means that the criteria for variant classification.60 Interpretation of data is
diagnostic yield of WGS is usually higher than with WES. usually straightforward when patients are found to have
A key consideration in WES or WGS is selection of the pathogenic or probably pathogenic variants, consistent
gene list to be analysed and whether to use validated and with their phenotype. However, variants of uncertain
curated gene panels (ie, a clinical exome). International impor­tance, without sufficient evidence to determine if
practices differ regarding selection of a narrow phenotype- they are pathogenic or benign, are common. Some of these
defined gene list or a mendeliome containing all genes variants require further functional testing or other studies
causing inherited disease. Standard­isation of gene lists to determine causality. Secondary findings of patho­genic
occurs through the use of curated consensus panels such variants in genes unrelated to the primary medical reason
For PanelApp see https:// as PanelApp. Phenotypic mimicry (eg, Charcot-Marie- for testing are found in up to 4% of patients.61,62 Finally,
panelapp.genomicsengland. Tooth neuro­pathy, genetic myopathy, or spinocerebellar up to 5% of patients with inherited conditions have
co.uk/panels/
ataxia)20 means that if a relatively narrow mitochondrial phenotypes result­ing from pathogenic variants in two or
gene list is used for initial variant analysis, it is usually more differ­ent genes.63 The quality of curation of genomic
variants is higher when ratified by a multi­ disciplinary
team com­ prising clinicians and clinical scientists with
expertise in the conditions being investi­gated. For adults
Figure 3: Algorithms for diagnosis of mitochondrial disease with ambiguous genetic results, or for whom no likely
(A) Diagnostic algorithm for suspected acute presentations of adult mitochondrial genetic diagnosis is achieved, func­tional testing is a follow-
disease. (B) Diagnostic algorithm for suspected slowly progressive neurological
presentations of adult mitochondrial disease. MELAS=mitochondrial myopathy,
up approach, most usually entailing muscle biopsy.
encephalopathy, lactic acidosis, and stroke-like episodes. LHON=Leber hereditary
optic neuropathy. mtDNA=mitochondrial DNA. WES=whole exome sequencing. Biochemical assessment of mitochondrial function
WGS=whole genome sequencing. CPEO=chronic progressive external Traditionally, investigation of suspected mitochondrial
ophthalmoplegia. *Evaluation for multisystem involvement such as sensorineural
hearing loss, diabetes, cardiac abnormality, gastrointestinal, renal, or hepatic
disease focused on detecting evidence of biochemical
involvement. †m.3243A→G and POLG pathogenic variants account for >80% of dysfunc­tion in muscle tissue, including the demonstration
cases of mitochondrial stroke-like episodes. ‡m.3460G→A, m.11778G→A, of COX-deficient fibres and measurements of oxidative
and m.14484T→C pathogenic variants account for >90% of cases of LHON. phosphorylation system enzyme activities. Some biopsy
§Consider other acquired or genetic mimics of mitochondrial disease. ¶Evaluation
for multisystem involvement. Ascertainment of family history. ||If myopathy findings, such as focal COX deficiency, have imperfect
forms part of the overall clinical phenotype, and plasma GDF15 and FGF21 levels sensitivity and specificity for mitochondrial disease since
are within the normal range, the probability of mitochondrial disease is low. defects in COX accumulate with age due to somatic
**If CPEO is also evident, follow the myopathy pathway. Isolated cerebellar mtDNA deletions. Muscle biopsy is typically used after
syndrome is uncommon in primary mitochondrial disease. ††Persistent creatinine
kinase concentration >1000 U/L is rare in mitochondrial disease. ‡‡Neurologists inconclusive genomic testing, and analysis of muscle
face considerable challenges when investigating late-onset, isolated, and tissue64 can provide invaluable information for interpreting
progressive myopathy in adults. Many differential diagnoses are possible, the pathogenicity of novel genetic variants; techniques
including both acquired and genetic causes, and muscle biopsy is important to
such as imaging mass cytometry offer the potential to study
identify the correct diagnosis. MRI of pelvis and lower limbs show a good
diagnostic value for specific genetic muscle diseases; however, no clear pattern of mitochondrial protein signatures at single cell resolution.65
muscle involvement has been identified in primary mitochondrial disease to date. Although many genetic variants can be studied in blood or
§§Account for at least 60% of dominant optic atrophy cases. ¶¶Accounting for non-invasive tissues, muscle-restricted mtDNA variants
30–50% of CPEO cases, the ability to detect a single, large-scale mtDNA deletion in
can only be diagnosed by analysis of muscle.32 Furthermore,
urothelial cells has been reported in patients who previously had muscle biopsy.47
||||Histopathological evidence of mitochondrial dysfunction can be observed in a in single, large-scale mtDNA rearrangements, disease
muscle biopsy, including COX-deficient and other oxidative phosphorylation onset and progression can be predicted by identifying
complex-deficient fibres, or mitochondrial subsarcolemmal accumulation the deletion size and mutant load within muscle tissue.66
(ragged-red fibres). However, these changes, usually occurring in only occasional
Finally, muscle biopsy helps confirm other causes (eg,
fibres, can be observed in non-primary mitochondrial disorders. ***Identification
of variants of undefined significance through WES or WGS might require further inflammatory myositis, inclusion body myositis, and myo­
functional characterisation by muscle biopsy to prove pathogenicity. fibrillar myopathies) and is a source of tissue for RNA

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 Review

sequencing, which can lead to a genetic diagnosis in up led to idebenone becoming the first licensed drug in
to 40% of patients who are not diagnosed by WES.67 Europe for treatment of visual impairment in patients
with LHON. An inter­ national expert group reached
Natural history studies and patient registries consensus that idebenone was indicated in the subacute
In past years, sparse availability of longitudinal and natural and dynamic stages of LHON (ie, in the first year after
history data for mitochondrial disease created consider­ onset) but evidence was considered insufficient to approve
able barriers to establishing clear and comprehensive the drug for treatment in the chronic stage. In patients
guidance on medical management, and hampered treated with idebenone, clinically relevant recovery can
develop­­ment of clinical trials and patient-centred clinically occur after 24 months of treatment or, rarely, after an even
relevant outcome measures. Publications describing single longer time. Accord­ingly, idebenone treat­ment should be
cases of mitochondrial disease, or retrospective case series maintained for more than 24 months before judging a
of specific genotypes, often report patients with severe patient a non-responder.92 No evidence is available to
and so-called classic clinical manifestations and reflect, support the use of idebenone in asymptomatic carriers of
therefore, a bias towards severe disease. A greater under­ pathogenic LHON variants.
standing of the spectrum of clinical phenotypes of No randomised controlled trials have been done of
mitochondrial disease and disease severity has been treatments for stroke-like episodes. Use of L-arginine
provided by cohort-based, observational studies that used for this neurological presentation remains controver­
standardised methods of data collection.11,68,69 Substantial sial.2 A longitudinal open-label study in patients with
progress has been made in describing the nature and m.3243A→G-related MELAS syndrome did not attain the
frequency of clinical features of patient cohorts, including predefined primary outcome measure (“the improvement
m.3243A→G,69–71 m.8344A→G,11 MTA-TP6,15,72 single large- rates of headache and nausea or vomiting at 2 h after
scale mtDNA deletion,66,73 POLG,74 TK2,75 TYMP,76 and completion of the initial intravenous administration”),
Leigh syndrome.77 Moreover, establishing these cohorts and L-arginine did not prevent the development of
has facilitated investigation of a phenotype first approach, neurodegeneration.93 A prospective, multicentre, random­
furthering our understanding of the diverse genetic causes ised controlled trial would be required to evaluate the
of specific clinical features of mitochondrial diseases, efficacy of L-arginine to reduce the recurrence of stroke-
such as extrapyramidal movement disorders,78 epilepsy,8,9 like episodes. In an open-label study, taurine supple­
myoclonus,79 CPEO,80,81 pregnancy-related com­pli­cations,82 mentation reduced the frequency of stroke-like episodes
and gastrointestinal symptoms.83 Additionally, our under­ in ten patients with MELAS syndrome.94 However, the
standing of very rare but clinically important events, absence of a control arm in the study questions whether
such as sudden unexpected death in patients with the the change in the number of stroke-like episodes was due
m.3243A→G variant,84 has been augmented by systematic to the efficacy of taurine, a placebo effect, or simply
assessment and follow-up afforded by recruit­ment to these reflected the natural history of stroke-like episodes, which
cohort studies. These natural history studies have provided are paroxysmal and unpredictable. European consensus-
insight into different stages of disease and risk stratifica­ based guidance recommends an urgent administration
tion, and surveillance for complications is now routine of intravenous anti-epileptic drugs for all stroke-like
for common mtDNA-related diseases. Studies of patient episodes.2 Valproate should not be used in patients with
cohorts have also provided insights into new disease POLG-related disease, because this drug might precipitate
mechanisms caused by mitochondrial dysfunction—eg, liver failure.87 However, valproate can be useful for
the leukodystrophy observed on MRI in patients with myoclonic epilepsy in other mitochondrial diseases.
defects in several mt-tRNA synthetases (eg, AARS2, Young adults with pathogenic variants in mtDNA-
DARS2, and EARS2) that lead to impaired translation encoded complex I subunits95 or MTA-TP615 are at risk
of mitochondrial protein85 indicates a role for mito­ of developing subacute brainstem dysfunction. Prompt
chon­dria in myelin metabolism. Furthermore, establish­ recognition is crucial, and critical care support might be
ment of patient cohorts has been essential for development required in some cases.
of consensus-based international guidelines for the care of With few treatments specific for neurological presenta­
patients with mitochondrial disease,2,86–88 development tions of mitochondrial diseases, symptomatic manage­
of clinical phenotype and genomic resources dedicated to ment of chronic neurological symptoms is very important.
mitochondrial disease (including mtDNA variant data in For example, discussing management with an expert
gnomAD and GENOMIT) and selection of outcome oculo­plastic surgeon, who has experience in mitochondrial For gnomAD see https://
measures for clinical trials.89,90 disease, can result in substantial improve­ments in quality gnomad.broadinstitute.org/
of life for patients with CPEO. Exercise is encouraged For GENOMIT see
Treatment because of general health benefits (including cardiovascular https://genomit.eu/

Current treatment and surveillance for systemic fitness) and scant evidence of adverse effects.96,97
complications Some clinical problems, including sensorineural hearing
Findings of a randomised controlled study, and data sub­ loss, cardiac involvement, and gut dysmotility, are encoun­
se­quently derived from an expanded access programme,91,92 tered with multiple genetic defects; effective treatments are

www.thelancet.com/neurology Vol 20 July 2021 579

 Review

available at least for sensorineural hearing loss and

cardiac involvement. By contrast, the risk of complications
A
such as diabetes, renal tubulopathy, hep­atic involvement,
Mitochondria Nucleus
Normal
and sideroblastic anaemia seem more genotype-specific
Mutated Primordial (appendix pp 2–4). Therefore, development of personal­
germ cell ised strategies based on knowledge of the natural histories
of different genetic defects is necessary. In view of the
multisystem involve­ment seen in many patients with mito­
chondrial disease, multidisciplinary clinics pro­vid­ing advice
about non-neurological involve­ment (eg, diabetes, bowel
dysfunction, and cardiac disease), as well as managing the
Primary oocyte neurological features, are crucial for optimal care.
No trial evidence supports routine dietary supple­
mentation in adult patients with mitochondrial dis­
ease.1 However, specific mitochondrial diseases—such as
primary coenzyme Q₁₀ deficiency,98 primary disorders
Mature oocyte
of vitamin cofactor metabolism (eg, thiamine, biotin,
and ribo­ flavin), and multiple acyl-CoA dehydrogenase
Fertilisation deficiency—are likely to benefit from supplementation.99
The long-term outcomes of these rare conditions after
appropriate supplementation are currently unknown.

Increase in disease severity Experimental treatments

Several experimental treatments are in development for
Environmental and other genetic factors mitochondrial diseases.100 The main approaches entail
small molecules (including repurposed drugs) to correct
B the biochemical abnormality, metabolic rewiring or bypass,
Fertilised oocyte or gene therapy to target the mitochondrial genome.
from woman with
mtDNA mutations Zygote Some small-molecule approaches have led to clinical trials
for the treatment of specific mitochondrial diseases.100,101
An area of increasing interest includes disorders
associated with abnormalities of mitochondrial nucleoside
metabolism leading to mtDNA depletion or multiple
Pronuclear
Oocyte Pronuclei replacement mtDNA deletions. For example, in mitochondrial neuro­
gastrointestinal encephalomyopathy due to thymidine
Fertilised oocyte phos­phorylase deficiency, therapeutic approaches include
from unaffected
woman
allogeneic stem-cell transplants, orthotopic liver trans­
planta­tion, and enzyme replacement therapy;102 in a
retrospec­tive study of thymidine kinase deficiency, deoxy­
nucleoside therapy led to clinical improvement.103
Other approaches to treatment of mitochondrial dis­
ease include augmentation of mitochondrial biogenesis,
Removal of
pronuclei
restoration of the cellular NAD+ to NADH ratio, increasing
mitophagy, metabolic reprogramming, or manipulation
Figure 4: Mitochondrial heteroplasmy and pronuclear transfer of oxidative stress.100 A pilot study using niacin, a form
(A) The mitochondrial bottleneck explains how there can be extreme divergence of vitamin B3, showed that it restored low NAD+ amounts
in the heteroplasmy between offspring. There is a genetic bottleneck
and improved muscle strength, performance, and metabo­
(the restriction in the number of mitochondrial genomes repopulating the
female germ line) during development that results in different levels of lome in a small group of patients with mitochondrial
heteroplasmy in each individual oocyte. This is a major problem when providing myopathy.46 These encouraging results need confirmation
genetic counselling for mothers with heteroplasmic variants since but suggest that targeting the NAD+ to NADH ratio might
the heteroplasmy level will affect the clinical outcome in the offspring.
be an important factor in some patients with mitochondrial
(B) Mitochondrial donation (mitochondrial replacement therapy) involves the
transfer of the nDNA from an oocyte or zygote from a woman with a pathogenic diseases.
mtDNA variant into an enucleated, recipient donor oocyte or zygote. Pronuclear Genetic manipulation techniques could be useful for
transfer is an in-vitro fertilisation technique that transfers the pronuclei treatment of mitochondrial diseases. Although techniques
(containing the nDNA) immediately after fertilisation from the patient zygote
to the donor zygote. The embryo has the nDNA of the woman with the mtDNA
such as CRISPR–Cas9 are already established for nuclear
variant (and that of the father) but the mtDNA of the donor woman. disorders, correction of defects in the mitochondrial
nDNA=nuclear DNA. mtDNA=mitochondrial DNA. genome is difficult due to the impermeability of mito­

580 www.thelancet.com/neurology Vol 20 July 2021

 Review

chondrial membranes. One approach—called allotopic

expression—delivers wild-type copies of mtDNA genes Search strategy and selection criteria
using viral vectors that remain in the cytosol. Transport of We searched PubMed for articles published in English from
the protein into the mitochondria is facilitated by addition Jan 1, 2010, to Nov 1, 2020, using the search terms
of a mitochondrial targeting signal. An example of this “mitochondrial disease OR disorder”, “neurological feature”,
technique is allotopic AAV2–ND4 gene therapy for patients “MELAS”, “CPEO”, “myopathy”, “biomarker”, “genetic”,
with LHON with the m.11778G→A variant.104 Considerable “treatment”, “clinical trial”, AND “reproductive option”.
progress has been made with development of TALENs or We further examined the reference lists from relevant articles.
zinc finger nucleases that cross the mitochondrial mem­ The references were chosen based on their originality and
brane and directly target mutated mtDNA for degradation relevance to the theme of this Review.
in experimental models.105 Another advance is the discovery
of an enzyme that can precisely edit mtDNA, although the
success of edited bases was very low.106 For women with heteroplasmic mtDNA variants, the
situation with transmission is more complex due to a so-
Reproductive options called genetic bottleneck during development.111 Rapid
Although considerable progress has been made in the intergenerational shifts in the mtDNA heteroplasmy are
treatment of mitochondrial diseases, many of these evident in human pedigrees transmitting pathogenic
conditions remain severe and life-threatening, with high mtDNA variants due to the restriction in the number of
morbidity. Advances in genomics mean that most patients mitochondrial genomes repopulating the female germ
with mitochondrial disease now have a genetic diagnosis. line (the mtDNA bottleneck). Since the range of hetero­
Therefore, it is important that couples considering a plasmy is a major risk factor in developing symptoms,
family should be offered reproductive advice, which offspring of a mildly affected women can have either no
should be focused on the specific nuclear or mitochondrial signs or symptoms or very severe disease (figure 4).
genetic defect. For families with mtDNA pathogenic variants, various
For patients with pathogenic nuclear gene variants, reproductive options are available depending on the
reproductive advice will depend on the inheritance specific genetic abnormality and whether it is homoplasmic
pattern and the availability of IVF techniques such as or heteroplasmic.86 For heteroplasmic mtDNA variants,
PGD. Nuclear mitochondrial diseases typically present in options such as prenatal diagnosis or preimplantation
childhood, although several disorders present across the genetic diagnosis might be appropriate if available. How­
age spectrum (eg, POLG and other genes involved in ever, there could be difficult decisions about embryo
mtDNA maintenance). Reproductive options include, selection or continuation of a pregnancy in terms of a safe
where permitted, prenatal testing and preimplantation level of heteroplasmy. For women with high levels of
genetic diagnosis. For some diseases in which the mutant heteroplasmy or a high frequency of homoplasmic vari­
allele is present at high frequency within a population—eg, ants, mitochondrial donation (mitochondrial replacement
in consanguineous societies or geographically isolated therapy)112 is now an option in the UK in a highly regulated
communities—screening for common pathogenic alleles environment crucial for any new in-vitro fertilisation
might be justified. technique (figure 4).113
For patients with mtDNA pathogenic variants, provision
of reproductive advice is more complex than for other Conclusions and future directions
types of mutation. For example, even the most common Major advances in the past 5 years in our under­standing
sporadic mtDNA pathogenic variant—a single, large- of and ability to diagnose and prevent mito­ chondrial
scale mtDNA deletion—is transmitted on rare occasions. disease give rise to cautious optimism for improving
Therefore, reproductive options for women with mtDNA the lives of patients with mitochondrial disease. Robust
pathogenic variants should be discussed at centres that guidelines are in use for the management of patients,114 For more on guidelines see
specialise in mitochondrial genetics.107 Despite some and targeted treatments now exist for some patient https://www.newcastle-
mitochondria.com/wp-content/
reports suggesting that there is biparental transmission of popula­tions. Moreover, developments in in-vitro fertilisa­
cache/all/guidelines/index.html
mtDNA,108 other studies have shown that this is most tion mean new reproductive options are available for
likely not the case.109,110 All reported cases of transmission families with mitochondrial disease.
of mtDNA pathogenic variants have been maternally Nevertheless, this progress highlights the need to focus
inherited. For women carrying homo­ plasmic mtDNA our future research. Elucidation of disease mechanisms is
pathogenic variants, the advice might seem simple: they necessary to understand why specific patients have tissue-
will transmit the variant to their offspring. However, specific manifestations of mitochondrial disease. New or
transmission can be complicated by variable penetrance repurposed drugs are needed, and recognition is required
due to either nuclear genetic or environmental factors. For that some approaches—such a gene therapy—might be
example, in families that carry one of the three common genotype-specific. Development of new treatments will
pathogenic variants for LHON, men are more at risk of require not only cohorts of patients for clinical trials but
developing visual loss than are women. also validated outcome measures that are pertinent to

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 Review

mitochondrial diseases. Provision of cascade family tracing Sigrid Jusélius Foundation, Jane and Aatos Erkko foundation, and
will be helpful, since early diagnosis is important for the University of Helsinki.
success of disease-modifying treatments and valuable if References
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GSG reports grants from the Wellcome Trust, the UK Medical Research
6 Yokota Y, Hara M, Akimoto T, et al. Late-onset MELAS syndrome
Council (MRC), and Lily Foundation, during the conduct of the study;
with mtDNA 14453G→A mutation masquerading as an acute
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Neurovive, and AMO Pharma, outside the submitted work. TK reports
7 Ng YS, Gorman GS, Turnbull DM, Martikainen MH.
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GmbH; and grants from Gensight Biologics and Stealth Biotherapeutics, Lancet Neurol 2015; 14: 1073.
outside the submitted work. CK reports personal fees from Santhera, 8 Whittaker RG, Devine HE, Gorman GS, et al. Epilepsy in adults
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AS reports grants from the Jane and Aatos Erkko Foundation and Business 10 Anagnostou ME, Ng YS, Taylor RW, McFarland R. Epilepsy due to
Finland; and personal fees from Khondrion, outside the submitted work; mutations in the mitochondrial polymerase gamma (POLG) gene:
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the US Department of Defense, during the conduct of the study. of the 8344A→G mtDNA “MERRF” mutation. Neurology 2013;
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