General overview of Mitochondrial Disease (diagnosis and management) consensus 

Mitochondrial diseases are one of the most common inborn errors of metabolism, with a conservative estimated prevalence of approximately 1:5,000.  Primary mitochondrial diseases are defined as disorders impacting the structure or function of the mitochondria as a result of either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) mutations.

 Establishing a diagnosis often remains challenging, costly, and, at times, invasive.

Patient care standards for primary mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society | Genetics in Medicine 

Definition relating mitochondrial myopathies

Mitochondrial myopathies and neuropathies (neuromyopathies) are heterogeneous group of disorders caused by dysfunction in mitochondria.  Mitochondrial myopathies are progressive muscle conditions caused primarily by the impairment of oxidative phosphorylation (OXPHOS) in the mitochondria.  This causes a deficit in energy production in the form of adenosine triphosphate (ATP), particularly in skeletal muscle.  The diagnosis of mitochondrial myopathy is reliant on the combination of numerous techniques including traditional histochemical, immunohistochemical, and biochemical testing combined with the fast-emerging molecular genetic techniques, namely next-generation sequencing (NGS).

Mitochondrial genetics

Mitochondrial genome is 16.5-kB circular double stranded DNA that does not contain noncoding regions (introns).  In some areas there is overlap of mitochondrial genes.  There is a single promoter site.  The transcription in mitochondria is polycistronic and mitochondrial genes are transcribed as two large RNAs.  These subdivide into 13 mitochondrial mRNA (mt-mRNA) with are translated into 13 polypeptide proteins of the OXPHOS (respiratory chain complexes), 22 mitochondrial tRNA (mt-tRNA), and 2 mitochondrial rRNAs (mt-rRNA).

Primary mutations of the mtDNA include point mutations affecting protein coding regions of the genome and mt-tRNA genes which alter mitochondrial protein synthesis.  They also include single, large-scale mtDNA deletions which can either be inherited or arise sporadically during embryogenesis.  The pathogenicity of mtDNA mutations is further complicated by heteroplasmy (the state in which there is coexistence of both mutant and wild-type mtDNA in a given cell) and the threshold effect (when the proportion of mutant mtDNA exceeds a given limit and causes a biochemical defect in a cell) 

The nDNA encodes 1171 known and 442 predicted mitochondrial proteins (MitoMiner v.Q2 2018), including OXPHOS subunits, assembly factors for OXPHOS complexes and proteins required for mtDNA maintenance.  Mutations in the genes encoding the mtDNA maintenance machinery can lead to either mtDNA depletion or multiple mtDNA deletions.

The mitochondrial genome account for less than 5% of all mitochondrial proteins.  The majority of mitochondrial proteins are encoded by the nuclear genome that are translated in the cytoplasm and subsequently are transported into the mitochondria.  Also, the nucleus regulates the replication of mitochondrial genome.  

Mitochondrial function is under the control of two genomes;  the mitochondrial genome (mtDNA) and the nuclear genome (nDNA); as such, mitochondrial myopathy can be caused by pathogenic genetic variants in either of these genomes.  This dual genetic control also means that mitochondrial disease is transmitted with the following inheritance patterns: maternal (mtDNA), X-linked, autosomal recessive, autosomal dominant.   In addition, some relatively common mitochondrial myopathies occur de novo.

During fertilization, all the mitochondria are contributed by the mother.  There are hundred of mitochondrial present in every cell in the body.  Every mitochondria has several copies of mtDNA.  Mutation is far more common to occur in mtDNA and these are more likely to manifest clinically than mutation in nDNA genes, because of the lack of introns in mtDNA and decreased DNA repair mechanisms in the mitochondrial genome.  mtDNA mutations are randomly distributed in subsequent generation of somatic cells during mitosis and in germ cells during meiosis.   Some cells will have few or no mutant genomes (normal homoplasty), some will have a mix of mutant and normal genomes (heteroplasmy), and some will have only mutant genomes (mutant homoplasty).  Phenotypic expression depends on the burden of mutant genome.  When the number of mitochondria bear sufficient mutated mtDNA, exceeding a certain threshold, mitochondrial function becomes impaired and patients manifest clinical symptoms and signs of disease (threshold effect).  Some of the mutant mitochondria may borrow the mRNAs and tRNAs from neighboring normal mitochondria in a process called complementation.  Thus, there can be a degree of normal translation of mtDNA-encoded proteins even in mitochondria harboring large DNA deletions.  

Different organs have different susceptibility for mitochondrial abnormalities depending on their energy requirements.  CNS has a constant demand for energy and is more severely impacted.  The skeletal muscles have low energy demand at rest, but these demands drastically increase with exercise.  This is the basis for exercise intolerance in many patients with mitochondrial myopathies.  

Primary mutations of mtDNA can only be passed on by the mother.  X-linked inheritance pattern affect men and women equally, while men are generally severely affected.  Based on the degree of heteroplasmy, all the children of an affect mother may be affected to varying degrees.  

95% of mitochondrial proteins are encoded by nDNA and can be inherited in an AD (e.g. CPEO) or AR (e.g. MNGIE) and even X-linked (e.g. some forms of Leigh syndrome) fashion.  KSS is associated with large mtDNA deletions, but is sporadic in nature.  Mutation in nDNA result in syndromes associated with depletion or multiple deletion of mtDNA.  They can demonstrate AD or AR patterns.  They can affect dNTPs (deoxyribonucleoside triphosphates) that serve as the building blocks for mtDNA.  Replication of mtDNA requires POLG1, POLG2, PEO1, DNA2.  Mutations in these genes result in mtDNA depletion and/or multiple mtDNA deletions that are found in various mitochondrial disorders (PEO, MNGIE, SANDO).   POLG related disorders are due to mutations in the nuclear gene POLG and often result in multiple mtDNA deletions of different length.  POLG related disorders may be AR or AD.  A delicate balance of the 4 dNTPs (dATP, dGTP, dCTP, and dTTP) is crucial for mtDNA replication.  Several nDNA encoded enzymes are key in preserving this balance (TK2, DGUOK, SUCLA2, SUCLG1, RRM2B, TYMP, and MPV17).  Mutations in these genes also cause mtDNA depletion resulting in various mitochondrial disorders (mitochondrial depletion myopathy, MNGIE, Navajo neurohepatopathy).  Furthermore, some nDNA genes are responsible for normal mitochondrial dynamics.  For example, fusion of mitochondria is dependent on nDNA encoded profusion GTPases that are located in the mitochondrial membranes and MFN1 and MFN2.

Function of mitochondria:  

• Produces energy in the form of ATP for cellular function by converting fuel substrates (carbohydrates, lipids, and proteins).

• Fatty acids are metabolized into molecules of acetyl CoA within mitochondria.

• Amino acids are converted to pyruvate within mitochondria

• Carbohydrates are metabolized to pyruvate in the cytoplasm and then transported into the mitochondria. 

• Pyruvate from both sources are converted to acetyl CoA.

• Acetyl CoA then enters in the Krebs cycle from which electrons are generated. 

• Electrons derived from the Krebs cycle are shuttled to the respiratory chain and are processed through complexes 1-V to generate ATP molecules.  

The respiratory chain complexes comprise 5 multienzyme complexes (I-V) present in the inner membrane of mitochondria. 

• Complex I (NADH:ubiquinone oxidoreductase) contains 45 polypeptide subunits, 7 are encoded by mtDNA

• Complex II (SDH) contains 4 polypeptide subunits, all encoded by nDNA

• Complex III (ubiquinol–cytochrome c oxidoreductase or bc1 complex) contains 11 polypeptide subunits, 1 encoded by mtDNA

• Complex IV (cytochrome c oxidase) contains 13 subunits, 3 encoded by mtDNA

• Complex V (ATPase synthetase) contains 19 subunits, 2 encoded by mtDNA.

Cytochrome complex I is the first enzyme of the respiratory chain.  It oxidizes NADH, which is generated through the Krebs cycle in the mitochondrial matrix, and uses the two electrons to reduce ubiquinone to ubiquinol.  Ubiquinol is reoxidized by the cytochrome III complex and transfers electrons to reduce molecular oxygen to water at cytochrome complex IV.  The redox energy released during this process is used to transfer protons from the mitochondrial matrix to the periplasmic space that generates proton-motive force across the inner mitochondrial membrane at cytochrome complexes I, III, and IV.  Complex V uses this proton-motive force to produce ATP from ADP and inorganic phosphate.  This entire process constitutes OXPHOS.  Because complex I is the major entry point for electrons to the respiratory chain and is suggested as the rate-limiting step in overall respiration, it plays a central role in energy metabolism. 

Mitochondrial disorders Classification:

• Biochemical defects - metabolic function affected

  • Transport

  • Substrate utilization 

  • Krebs cycle

  • Oxidation/phosphorylation coupling

  • Respiratory chain defects

• Genetic defects

  • mtDNA

  • nDNA affecting directly or indirectly the respiratory chain complex

  • nDNA affecting mtDNA maintenance or mitochondrial dynamics. 

Patients with mitochondrial myopathies have diverse clinical phenotypes, some features may be similar to other myopathies and others are more specific for patients with mitochondrial disease.  In virtually all patients with mitochondrial myopathy, there is potential involvement of other systems which may well be the prominent and life-threatening feature—for example, cardiomyopathy, epilepsy, or stroke-like episodes.  Indeed the most frequent presentation of mitochondrial myopathy is a proximal myopathy in combination with other symptoms which is often the clue to likely mitochondrial involvement.  Therefore, t is essential that they are considered when evaluating all patients.

• Eyes: Ptosis, external ophthalmoplegia, and optic atrophy.

• Nervous system: Seizures, tremor, developmental delay, deafness, strokelike episodes, ataxia, and peripheral neuropathy.

• Heart: Cardiomyopathy.

• Kidneys: Fanconi syndrome, kidney failure, and nephrotic syndrome.

• Liver: Liver failure and hepatic steatosis.

• Pancreas: Diabetes.

• Gastrointestinal: Gut dysmotility, intestinal pseudoobstruction, and chronic diarrhea.

• Skeletal muscle: Muscle weakness, fatigue, exercise intolerance, and cramps.  Respiratory failure due to diaphragmatic dysfunction requiring ventilatory support.

Reproductive Options 

In addition to a wide variety of treatment options, reproductive options are also available.  Patients who have been diagnosed with a pathogenic mutation in either nDNA or mtDNA known to cause mitochondrial disease have a number of reproductive options that aim to reduce the risk of transmitting the disease to their offspring.  One option is oocyte or sperm donation (depending upon which parent is affected) which can prevent the inheritance of mitochondrial disease.  An important consideration for some, however, is that the mother or father will not be genetically related to the offspring. Therefore, an alternative is prenatal diagnosis and preimplantation genetic diagnosis (PGD).  These options have been used successfully to prevent transmission of nDNA and mtDNA mutations associated with mitochondrial disease.  For the latter, the unique features of mtDNA mean that they will not be suitable for women who are homoplasmic for an mtDNA mutation or produce oocytes with high a mutation load.

The most recent reproductive option that may allow some women to have a genetically related child while reducing the risk of mitochondrial disease is mitochondrial donation (also known as mitochondrial replacement therapy). The novel IVF treatment involves transferring the mother’s nDNA from an affected egg into an enucleated egg from an unaffected donor, resulting in an embryo containing nDNA from both parents but mostly wild-type mtDNA from the healthy donor and a much lower risk of mitochondrial disease. The technique can be performed either before or after fertilization.  Before fertilization, maternal spindle transfer (MST) or polar body transfer (PBT) can be used, while in fertilized eggs pronuclear transfer (PNT) can be used [71, 72].  Preclinical research studies have been important in confirming the potential of these techniques to reduce the risk of mitochondrial disease [73–75] and addressing the safety and efficacy of mitochondrial donation before clinical implementation [76].

Mitochondrial donation was approved for clinical use in the UK following an extensive policy process that led to the mitochondrial donation regulations being passed into law in March 2015 [77].  This legislative change allows the Human Fertilizations and Embryology Authority (HFEA) to issue licences to fertility centers who want to offer mitochondrial donation as a novel IVF treatment to reduce the risk of mitochondrial disease.  To ensure strict regulation, license applications must be approved on a case-by-case basis for every patient and will only be considered when certain criteria are fulfilled as outlined by the HFEA. As this option is only applicable for mitochondrial diseases caused by mtDNA mutations, it could potentially benefit approximately 150 women each year in the UK [78].

Fatty Acid Oxidation Disorders

POLG mutations

Mitochondrial Chronic Progressive External Ophthalmoplegia

Chronic progressive external ophthalmoplegia (CPEO) is a common presentation of mitochondrial encephalomyopathies, which can result from alterations in mitochondrial or nuclear DNA.  It is marked by progressive bilateral ptosis and diffuse reduction in ocular motility; later complete external ophthalmoplegia.  It often occurs with other systemic features of mitochondrial dysfunction that can cause significant morbidity and mortality.   It represents a descriptive clinical finding rather than a diagnosis.  When it occurs in isolation, it is often referred to as chronic progressive external ophthalmoplegia (CPEO).   PEO often co-occurs with other symptoms of mitochondrial dysfunction (CPEO plus syndrome) or as one feature of a defined systemic mitochondrial cytopathy.   It can be part of a multisystem disease such as Kearns–Sayre syndrome and is commonly associated with proximal myopathy. 

Patients with PEO may have ptosis and ophthalmoparesis with or without extremity weakness, diplopia, and dysphagia.  They lack pigmentary retinopathy, cardiac conduction defects, endocrinopathy and other systemic manifestations as seen in Kearns Sayre syndrome (KSS).  Some cases of PEO are sporadic and associated with single large deletions of mtDNA, representing a partial expression of KSS.  There are autosomal and recessive forms as well as maternally (mitochondrial) inherited forms of PEO. 

PEO spectrum disorders (e.g., CPEO, CPEO plus syndromes, KSS) 

Clinical Features of CPEO

• The progressive ptosis is typically the most noticeable feature, often leading to the gradual acquisition of a chin-up compensatory head position. 

• Ptosis almost always presents prior to or simultaneous with ophthalmoparesis, although rare cases of PEO without ptosis exist. 

• Ptosis is usually symmetric, it may present in an asymmetric fashion, however. 

• A hallmark exam feature of PEO related ptosis is poor function of the levator palpebrae superioris (LPS) muscle.  Whereas the normal excursion of the superior eyelid from maximal downgaze to upgaze with stabilization of the brow is ≥12 mm, PEO patients often have less than 8–10 mm of function.

• Early on, examination reveals slowed and incomplete, omnidirectional saccades.  Late in the disease course, ophthalmoplegia may become complete. 

• The often-symmetric nature of the disease means that patients do not have diplopia, and reports of manifest strabismus with diplopia in CPEO patients are rare.  

• Retinopathy and optic neuropathy can occur.  Pigmentary retinopathy is a hallmark feature of Kearns-Sayre syndrome (KSS), which includes PEO . 

• CPEO patients (who do not fit the diagnostic criteria of KSS) may also demonstrate pigmentary retinopathy.   Often described as a "salt and pepper" retinopathy, there is a speckled pattern of retinal pigment epithelium (RPE) clumping alternating with areas devoid of normal RPE.  Unlike retinitis pigmentosa demonstrating "bone spicule"-like clumps of RPE in the mid-periphery of the retina, PEO-related RPE changes are most prominent in the macula, peripapillary retina, and the equatorial region. The pigmentary retinopathy only rarely causes severe vision loss, and most patients retain normal or near normal acuity and visual fields. 

• Proximal myopathy, sensory ataxic neuropathy, cardiomyopathy and parkinsonism. 

Differential diagnosis of CPEO

• Myopathic: PEO spectrum disorders (e.g., CPEO, CPEO plus syndromes, KSS), orbital myositis, TED, Grave's disease, Myotonic Dystrophy type 1, and type 2, congenital myopathies, OPMD, OPDM, LGMD with ophthalmoplegia. 

  • HMG-CoA reductase inhibitors (statins) for hyperlipidemia have established systemic myopathic toxicity.  In a 2008 study, 256 reports of ptosis, diplopia, and/or ophthalmoplegia associated with statin use were investigated.  Sixty-two cases of symptom resolution with drug cessation (positive dechallenge) and 14 cases of symptom recurrence with reinitiation of the drug (positive re-challenge) led to a World Health Organization criteria-based conclusion that statin drugs possibly cause ophthalmoparesis.

• Neuropathic:  MFS, MS, A-beta lipoproteinemia, Tolosa-Hunt syndrome, WEBINO, CAPOS (Cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss). CANOMAD (Chronic ataxic neuropathy with ophthalmoplegia, IgM paraprotein, cold agglutinins, and disialosyl antibodies), PSP, HSP, SCA1, 2, 3, 7, 9, 11, 28, Congenital cranial dysinnervation disorders: CFEOM (Congenital fibrosis of the extraocular muscles), Moebius syndrome, and Duane syndrome. Wernicke's encephalopathy.

• NMJ disorderes: MG.  Single-fiber EMG does not contribute diagnostically, since both MG and CPEO have been shown to demonstrate abnormal jitter. CMS, LEMS, and botulism.

• Agerelated levator dehiscence causing ptosis  and sagging eye syndrome (SES) 

• PEO may occur in association with chronic (>10 years) nucleoside reverse transcriptase inhibitor use for human immunodeficiency virus (HIV) 

Laboratory Features in CPEO

Serum CK, lactate, CSF lactate can be normal or elevated.  CSF protein may be increased.  ECG does not demonstrates cardiac conduction defects.  NCS are normal.  EMG is usually normal, although myopathic MUP may be seen in weak extremities.  

MRI: MRI of orbit with contrast can show abnormal bright signal seen within EOMs on T1WI on high-resolution MRI in the absence of orbital or EOM inflammmation, may be clinically useful in distinguishing CPEO from other conditions causing severe ophthalmoplegia.   MRI orbits can show slender EOM (medial and inferior recti), but not in all cases. 

MRI of brain in CPEO: Pyramidal tract hyperintesity as in ALS

Molecular genetics and pathogenesis of CPEO

40% to 70% of PEO have a single large mtDNA deletions similar to KSS.  These cases are sporadic and not genetically inherited. Point mutations have been identified. within various mitochondrial tRNAs (Leu, Ile, Asn, Tryp) genes in several kinships with maternal inheritance of PEO.  

Chronic progressive external ophthalmoplegia (CPEO) can be caused by defects in one of a number of genes within the nuclear genome: DGUOK, DNA2, MGME1, OPA1, POLG, POLG2, RNASEH1, RRM2B, SLC25A4, SPG7, TK2, TWNK, or TYMP.

Inheritance may be primarily autosomal dominant (POLG2, OPA1, DNA2, SLC25A4) or autosomal recessive (DGUOK, MGME1, RNASEH1, TK2, TYMP), while reports of both recessive and dominant inheritance have been documented for a subset of genes (POLG, RRM2B, TWNK, SPG7). 

PEO (PEOA: PEOA1 due to mutation in POLG1 (polymerase gamma 1 gene);  PEOA1 caused by mutation in ANT1 gene that encodes for adenine nucleotide translocator (ANTI; PEOA3 caused by mutations in twinkle gene (C10orf2). 

ANT is the most abundant mitochondrial protein and transports ATP across the mitochondrial inner membrane, while twinkle and POLG are involved in mtDNA replication.  Less common, PEO has been associated with mutations in POLG2, TK2, OPA1, DGOUK, and RRM2B. 

OPA1 gene mutations can also cause a subset of CPEO plus syndrome with optic atrophy .

Autosomal recessive form of PEO (PEOB) is also commonly caused by mutations in POLG1 gene.  It has been implicated with Alper syndrome, which causes a clinical triad of psychomotor retardation, intractable epilepsy, and liver failure in infants and young children.  Less common mutations have been identified in TK2, DGOUK, RRM2B, MPV17, DNA2, and SPG7 genes. 

https://www.preventiongenetics.com/testInfo?val=Chronic-Progressive-External-Ophthalmoplegia-%28CPEO%2FPEO%29-Panel

Nuclear DNA Gene Mutations/Protein Dysfunction Causing CPEO

DNA Polymerase Subunit Gamma (POLG)

POLG-related disorders affect the nDNA that encodes mtDNA polymerase gamma. The first identified POLG mutation variant was inherited in an autosomal dominant manner; however, it was identified in recessive variants later in other families. CPEO was observed in both autosomal dominant and recessive carriers along with other neurodegenerative disorders associated with POLG that include myoclonic epilepsy myopathy sensory ataxia (MEMSA), childhood myocerebrohepatopathy spectrum (MCHS), Alpers syndrome, Alpers–Huttenlocher syndrome (AHS), and ataxia neuropathy spectrum (ANS) disorder.

The clinical presentation of POLG-related disorders can vary widely and include neurological features such as ataxia, axonal neuropathy, myoclonic epilepsy, and sensorineural hearing loss. Other features are PEO, cataracts, hypogonadism, liver dysfunction, and possible renal manifestations.

The diagnostic approach of POLG-related disorders proposed by Hikmat et al. is simplified and accounts for the age of onset and clinical picture with supportive and definitive investigations. EEG, MRI, muscle biopsy, and laboratory investigations are used as supportive investigations and ordered depending on the clinical presentation. POLG gene sequencing is the definitive investigation.

Twinkle mtDNA Helicase (TWNK)

The TWNK gene, also known as C10orf2 or PEO1, is responsible for encoding TWINKLE, an mtDNA helicase, an enzyme that unwinds DNA temporarily for replication. The dysfunction in TWINKLE is thought to pause or stall mtDNA replication and accumulate many mtDNA deletions over time. Mutations in this gene are usually associated with adult-onset autosomal dominant PEO (adPEO). The clinical presentation can be isolated CPEO or with systemic muscle weakness, dysarthria, dysphagia, and cardiac or neurological involvement. Treatment options for this disease are supportive and focused on alleviating symptoms.

Thymidine Phosphorylase

The TYMP gene encodes thymidine phosphorylase (TP), an enzyme that catalyzes thymidine and deoxyuridine into thymine and uridine, respectively. Defects in this gene cause the accumulation of thymidine and uracil in the blood, resulting in mitochondrial neurogastrointestinal encephalopathy (MNGIE). MNGIE is a rare multisystemic autosomal recessive disorder that typically starts before the second decade of life but can manifest up to the fifth decade. It is characterized by CPEO, cachexia, severe gastrointestinal dysmotility, sensorineural hearing loss, peripheral neuropathy, and leukoencephalopathy. Clinically, diagnosis can be supported by increased plasma levels of thymidine (>3 micromol/L) and deoxyuridine (>5 micromol/L) or a decrease in the buffy coat of TP activity to less than 8% of controls. It is important to note that TYMP is not the only mutation attributed to a MNGIE-like phenotype, as POLG1, RRM2B, and LIG3 mutations were reported with a somewhat similar clinical phenotype in the literature. This can be of therapeutic value since TYMP mutations have ongoing treatment modalities to restore TP activity and target the toxic effects of thymidine and uracil, such as platelet infusion, continuous ambulatory peritoneal dialysis, enzyme replacement therapy, hematopoietic stem cell transplantation, and liver transplantation.

Ribonucleoside–Diphosphate Reductase Subunit M2 B (RRM2B)

Ribonucleotide–diphosphate reductase subunit M2 B is an enzyme encoded by RRM2B, which produces one of the two subunits of ribonucleotide reductase. Ribonucleotide reductase is induced by p53 to produce deoxyribonucleoside diphosphatase, a nucleotide precursor, for DNA repair and mtDNA synthesis in non-proliferating cells.

Defects in this gene can cause mtDNA maintenance defects, either from mtDNA depletion in RRM2B encephalomyopathic mitochondrial DNA maintenance defect (MDMD) and in RRM2B mimicking mitochondrial neurogastrointestinal encephalopathy (MNGIE) or multiple mtDNA deletions in RRM2B adPEO and RRM2B autosomal recessive PEO (arPEO). A common feature in these subtypes is ophthalmoplegia and ptosis; however, the age of onset and other clinical features may differ. For example, RRM2B adPEO usually accompanies bulbar dysfunction, hearing loss, and gastrointestinal motility]. On the other hand, arPEO is a childhood-onset disease that is more severe with associated retinopathy, myopathy, and mood disorders. RRM2B encephalomyopathic MDMD is an infantile-onset severe multisystem disease that usually presents with hypotonia, poor feeding, and failure to thrive. In addition, other manifestations include respiratory failure, renal tubular necrosis, and sensorineural hearing loss. Finally, MNGIE-like RRM2B is a rare phenotype that can occur with cachexia, gastrointestinal dysmotility, and peripheral neuropathy.

Optic Atrophy 1 (OPA-1)

The OPA-1 (optic atrophy 1) gene is a membrane-remodeling protein that regulates mitochondrial dynamics with both energetics and mitochondrial morphology. It was discovered in 2000, along with its association with dominant optic atrophy (DOA). Since then, a broad spectrum of clinical features have been reported in DOA plus syndromes that include Behr syndrome, syndromic parkinsonism, dementia, CPEO, and other neuromuscular features. The clinical features characterizing DOA are bilateral progressive visual loss involving color vision and the central or paracentral visual field with varying severity. Funduscopic examination exhibits optic pallor or atrophy related to retinal ganglionic cell (RGC) layer death. Additional diagnostic modalities for DOA can involve optical coherence tomography, which shows nonspecific retinal nerve fiber layer thinning and abnormal visual evoked potentials due to RGC dysfunction. In a cohort by Romagnoli et al. for using Idebenone as a therapeutic option for OPA1-DOA, patients who underwent the therapy benefited in terms of visual recovery four times more than those who did not. This shows promising results that have yet to be confirmed by future studies [74].

Thymidine Kinase 2 (TK2)

The Thymidine kinase 2 (TK2) gene encodes for an enzyme integral for mtDNA replication and maintenance since it phosphorylates deoxythymidine (dT) and deoxycytidine (dC) into deoxynucleotide triphosphates in the deoxypyrimidine salvage pathway. The clinical picture of TK2 deficiency (TK2d) varies with the age of onset and is categorized into early-onset (≤1 year), childhood-onset (>1 to ≤12 years), and late-onset (>12 years) TK2d.  

Early-onset TK2d is usually a severe myopathic form that is fatal within a year, with early symptoms preceding muscle weakness that include esophageal reflux, vomiting, intestinal dysmotility, and failure to thrive. These patients can also exhibit neurological and extra-skeletal manifestations, which include seizures, cognitive impairment, bilateral optic atrophy, multiple fractures, rigid spine, nephropathy, and cardiomyopathy.

The childhood-onset form of the disease typically has an intermediate to a rapidly progressive phenotype of proximal myopathy with Gowers signs and a dropped head. Some cases can also show CPEO, facial diplegia, dysphagia, and restrictive lung disease, aiding the diagnosis. Extra-skeletal findings may include multiple fractures, cognitive decline, encephalopathy, hearing loss, renal tubulopathy, and arrhythmias.

Patients with late-onset TK2d have the characteristic progressive proximal muscle wasting, with the addition of axial neck flexors and facial weakness. This form usually accompanies CPEO, bulbar weakness, and early respiratory muscle involvement requiring non-invasive ventilation. In some cases, peripheral neuropathy and hearing loss can also occur.

A recent cohort by Domínguez-González et al. demonstrated a characteristic lower limb muscle MRI pattern that can differentiate the condition from other myopathies with similar clinical features.

Therapy using the active substrates dT and dC in TK2d patients has been reported to improve muscle weakness and ambulation, as well as discontinuing mechanical ventilation and gastrostomy in affected children. However, late-onset cases showed minimal benefits, and further studies are needed to establish a clear benefit.

Deoxyguanosine Kinase

Deoxyguanosine kinase (DGK) phosphorylates purine deoxyribonucleosides and contributes to the deoxyribonucleoside salvage pathway in the mitochondrial matrix. Two forms of DGUOK gene deficiency have been described in the literature, with neonatal multisystem disorder being the most common. It is characterized by hepatic and neurological manifestations, including developmental delay, hypotonia, nystagmus, jaundice, cholestasis, and hepatomegaly. The second less severe form is an isolated childhood hepatic disorder. Long-term follow-up of varying cases with this phenotype showed renal involvement, myopathy, and parkinsonism with CPEO, rigidity, and bradykinesia. The most common cause of mortality in both forms is progressive hepatic disease, and a decision on whether a transplant is needed should be as per hepatologist since the topic is under debate.

Ribonuclease H1 (RNase H1)

RNase H1, or ribonuclease H1, is an enzyme encoded by the gene RNASEH1, located in chromosome 17p11.2. This enzyme contributes to mitochondrial dynamics through primer maturation, removal, synthesis of replication primer, and pre-RNA processing in mtDNA replication. In a cohort conducted by Bugiardini E. et al., patients harboring RNASEH1 mutations had characteristic features of CPEO, cerebellar ataxia, and dysphagia, with CPEO being a universal feature in all cases. In contrast, ataxia and dysphagia were concomitantly present in approximately 50% of cases. Other less frequent symptoms were proximal muscle weakness, peripheral neuropathy, and pyramidal signs. This study also concluded that in the presence of POLG-negative ataxia neuropathy spectrum, all patients should be considered for genetic analysis for RNASEH1 mutations since it is the fourth most common cause of adult mendelian PEO with multiple mtDNA deletions in their cohort, following POLG, TWNK and RRM2B. Manini et al. reported similar findings in their case report and compiled data from several reports of patients with the RNASEH1 mutation and noted that some of these frequent findings have been observed in other mitochondrial diseases, such as dysarthria in adults with POLG and TK2 mutations, and cerebellar signs in late-onset RRM2B mutations.

Mitochondrial Genome Maintenance Exonuclease 1 (MGME1)

Mitochondrial genome maintenance exonuclease 1 (MGME1), or Ddk1, is an exclusive mitochondrial DNase responsible for mtDNA maintenance by preferentially cutting single-stranded DNA (ssDNA) flaps and enabling the ligation of new DNA strands. MGME1 affects the turnover of 7S DNA and causes its accumulation when depleted or causes 7S DNA attrition when overexpressed. 7S DNA is a ssDNA arising from the non-coding region and is postulated to contribute to the mtDNA displacement loop (D-loop) as an intermediate of premature termination of mtDNA replication. Patients with this mutation have shown an increase in 7S DNA and a multisystemic phenotype of PEO, muscle wasting, emaciation, and respiratory failure. A similar phenotype was reported recently with skeletal malformations, atrioventricular block, and cerebellar atrophy in magnetic resonance imaging

Adenine Nucleotide Translocator 1 (ANT1)

ANT1-related PEO is associated with adPEO and affects the adenine nucleotide translocase type 1 (ANT1) gene, which encodes the translocator responsible for ADP to ATP exchange in the inner mitochondrial membrane and regulates the mitochondrial permeability transition pore that initiates apoptosis. The complete loss of this gene causes the characteristic clinical phenotype of cardiomyopathy and myopathy. On the other hand, the overexpression of this gene results in cardioprotective features. Other clinical features of this disorder include exercise intolerance, muscle weakness, ptosis, and lactic acidosis. All of the symptoms mentioned assist in suspecting the diagnosis, which can later be confirmed with genetic testing. There is no consensus on the treatment of this disorder. Standard heart failure treatment has been used to manage some cases to tackle cardiomyopathy; however, the results did not show any benefit in halting disease progression. Recent studies suggest that reducing protein leak can be an effective treatment option for aged cardiomyocytes, which may assist in ANT1-related symptoms. Another showed improvement in exercise intolerance using nicotinamide riboside in ANT1-deficient mice.

Mitochondrial Inner Membrane Protein MPV17

MPV17 is an inner mitochondrial non-selective channel that is thought to play a role in mitochondrial maintenance by preventing the formation of reactive oxygen species. The clinical picture of this mutation is similar to DGUOK with an early-onset hepatocerebral phenotype with hypoglycemia, metabolic acidosis, gastrointestinal findings of poor feeding, failure to thrive, and dysmotility. Rare cases of this mutation with a late-onset neuromyopathic phenotype have also been reported. Brain MRI findings may harbor abnormalities in lower brainstem reticular formation, reticulospinal tracts at the cervicocranial junction, and cerebral leukoencephalopathy.

Management of CPEO

• Treatment of CPEO is focused on surgical correction of ptosis.  Surgery is the mainstay treatment and is dependent on LPS function.  Resection of the levator tendon along the superior tarsus is available for mild LPS impairment, while in more severe cases, frontalis suspension procedures with facia lata or silicon are used.

• When strabismus and diplopia occur, prismatic glasses are prescribed to correct small malalignments, and strabismus surgery can be performed to improve the patient’s quality of life.

CPEO case Template: 

CPEO (chronic progressive external ophthalmoplegia), bilateral [H49.43]

The vast majority of CPEO patients have clinically evident weakness of limb muscles or facial muscles.  Late onset CPEO (defined as symptom onset after age 20) suffer from dysphagia.  Manometry in CPEO patients has shown abnormal contractions with decreased amplitude and duration predominately in the proximal esophagus [Domenis DR, Okubo PM, Sobreira C, Dantas RO. Esophageal contractions in patients with chronic progressive external ophthalmoplegia. Dig Dis Sci. 2011;56(8):2343–8.]  Patients with CPEO or KSS also demonstrated cricopharyngeal achalasia on esophageal videofluoroscopy and manometry [Kornblum C, Broicher R, Walther E, et al. Cricopharyngeal achalasia is a common cause of dysphagia in patients with mtDNA deletions. Neurology. 2001;56(10):1409–12.]  

Other associated features include migraine, fatigue, neuropsychiatric disorders, hypothyroidism, diabetes and optic atrophy.  (5 %) [12].  PEO-related syndromes can have overlapping features including CPEO and Kearns-Sayre-syndrome (KSS) and exhibit ataxia, cardiomyopathy, cardiac conduction abnormalities, pigmentary retinopathy, and short stature PEO plus - KSS).  Autosomal dominant PEO is usually associated with mutiple mtDNA deletions and is genetically heterogeneous.  Genes identified in autosomal dominant PEOs include mutation in POLG1, ANT1, twinkle gene (C10orf2). Less common, mutations POLG2, TK2, OPA1, DGOUK, and RRM2B. MPV17, DNA2, and SPG7 genes. 

RECOMMENDATIONS:

ECG and echocardiogram every six to 12 months to monitor cardiac conduction and contractility.  Yearly evaluation by neurologist, audiologist, ophthalmologist, and endocrinologist; referral to other specialists (e.g., gastroenterologist, pulmonologist) based on symptom occurrence.  Significant PEO-associated dysphagia should prompt referral to a gastroenterologist because cricopharyngeal myotomy for achalasia could be beneficial.  Evaluation by neuroophthalmologist and/or retinal specialist for CPEO, pigmentary retinopathy with appropriate surveillance testing (e.g., electroretinography, optical coherence tomography, visual fields.)   Agents/circumstances to avoid: Drugs potentially toxic to mitochondria, including chloramphenicol, aminoglycosides, linezolide, valproic acid, and nucleoside reverse transcriptase inhibitors. Volatile anesthetic hypersensitivity may occur. Avoid prolonged propofol (>30-60 minutes.)  

Patient hand out in CPEO.  

Chronic Progressive Ophthalmoplegia (CPEO) 

What is CPEO? 

Chronic Progressive External Ophthalmoplegia or CPEO is the medical term for a disease that affects the muscles of the eye lids and eye ball. CPEO is progressive, which means it gradually gets worse over time. 

What causes CPEO? 

CPEO is a genetic disease where there is a piece of DNA material missing (deletion) from the mitochondrial DNA. This may be inherited (passed down from one or both parents), but usually this is often caused by a spontaneous DNA mutation (85%). Spontaneous means that the deletion occurs at conception for no known reason, despite parents’ healthy genes and no family history of the disease. The more rare (15%) inherited forms can either be autosomal dominant, meaning that a person with the disease has a 50% risk of passing it on to each child, or maternally inherited where all offspring could be affected. The abnormal gene affects mitochondria, tiny structures inside almost every cell in your body. Their main job is to use the food and oxygen that enter the cells to make energy. Almost all of the energy your body needs for daily life and growth comes from mitochondria. 

What are the characteristics of CPEO? 

Each person responds differently to the disease. The most common characteristics of CPEO are:  

• Droopy eyelids (ptosis). This can make the muscles of the forehead overactive, as they try and do the work of the eyelids.  

• Limited eye movements. As the disease progresses, all eye movements can be lost. The eye can become fixed in the mid-position and the person has to turn his or her head to see in different directions.  

• Hearing loss – especially at high sound frequencies.  

• Mild muscle weakness of the arms and legs.  

• Difficulty with swallowing (dysphagia).  

• Cataracts. 

How do I know I have CPEO? 

Your doctor or specialist can tell if you have CPEO from:  

• Talking with you about your symptoms and family history.  

• Checking for the physical characteristics of CPEO with a complete neurological exam.  

• Reviewing the results of the following tests: 

  • Muscle biopsy  A biopsy is a procedure to take a tiny sample of muscle to be examined under a microscope.  This procedure is done in the clinic and takes about 20 minutes. The sample is usually taken from your thigh muscle (quadriceps). First the area is “frozen” or numbed with a local anesthetic. Then the doctor makes a tiny cut (incision) and removes a sample of muscle with a needle. The incision is closed with a stitch that will need to be removed in a few days. 

  • Blood and urine tests  Several tests may be abnormal in patients with CPEO including amino acids (elevated alanine), lactate, CK activity, and urine organic acids. 

  • Genetic tests  Blood tests to check for mutations in the genetic material (DNA) of your chromosomes and the mitochondria’s own DNA.  CPEO usually requires that the genetic test be done on a muscle biopsy sample.

 How is CPEO treated? 

There is no cure for CPEO, but treatment can control your symptoms. Special glasses with fine metal bars (ptosis props) can help to lift up drooping eyelids. If these are unacceptable, and the ptosis is severe, surgery may be done to lift the eyelids. 

Some patients find that hearing aids may improve hearing. If hearing loss is severe, a special surgical procedure (cochlear implant) may be needed. 

Muscle weakness is best prevented and treated with resistance exercise.

A supplement of Creatine Monohydrate can also help improve strength in conjunction with exercise. The amount you should take is based on your weight. You can add it to a glass of juice, milk, or a smoothie – or sprinkle it on cereal, applesauce, or yogurt. We have studied the effects of anti-oxidants such as alpha lipoic acid, coenzyme Q10, and vitamin E in a large group of individuals with various types of mitochondrial diseases. There was evidence of lower oxidative stress, but these effects were less apparent in individuals with CPEO. If you smoke, we strongly recommend that you quit, because the mitochondria are very sensitive to smoke. There is some evidence that the anti-oxidants discussed above may actually INCREASE cancer risks in smokers, so we strongly advise against taking alpha lipoic acid, coenzyme Q10, and vitamin E if you smoke. 

A Speech-Language Pathologist can help you with swallowing problems by ordering a swallowing study. 

Your treatment plan may include special exercises and following a diet with thickened foods.

How will having CPEO affect my life? 

CPEO progresses slowly, and you can expect the symptoms to gradually worsen over time. It is rare, but possible that you may pass this disease on to your children. If you want to have children, you can meet with a Genetic Counsellor for more information and advice. 

MERRF (myoclonic epilepsy and ragged red fibers)

• Myoclonus, generalized seizures (myoclonic and tonic-clonic), ataxia, dementia, SNHL, optic atrophy, and progressive muscular weakness developing in childhood or adult life.  The myoclonus is stimulus sensitive, but can be present at rest.  The seizures may be photosensitive.  Patients are often misdiagnosed as having juvenile myoclonic epilepsy (JME). 

• Muscle weakness and atrophy can be generalized, but there is a predilection for involvement of proximal arm and leg muscles.  

• Generalized sensorimotor PN and pes cavus deformities may be appreciated. 

• MERFF unlike KSS does not present with ptosis, ophthalmoparesis, and pigmentary retinopathy.  

• CM with conduction block or heart failure may also be seen in MERRF, in cases that present early.  

• MERFF can also be complicated with ventilatory muscle weakness and associated with life-threatening hypoventilation in the setting of surgery, sedation, or intercurrent infection.  

• Some patients present with multiple symmetric lipomatosis. 

Laboratory features:

• CK is normal or mildly elevated. 

• Serum lactate is normal or elevated. 

• EEG: generalized slowing of background activity and burst of spikes and slow waves.  

• MRI or CT of brain reveals cerebral and cerebellar atrophy. 

• EDX:

  • NCS may demonstrate reduced SNAP amplitudes - axonopathy.  

  • EMG is usually normal except for myopathic motor units seen in weak muscles. 

Histopathology:

• RRF, COX-negative and increased SDH staining. 

• Neuronal loss and gliosis of dentate nuclei, GP, red nuclei, substantia nigra, inferior olivary nuclei, optic nerves, and cerebellar cortex.  Demyelination and gliosis of corticospinal and spinothalamic tracts and posterior columns - autopsy. 

Molecular genetics and pathogenesis:

• Maternally inherited.  80% caused by point mutations at nucleotide position 8344 of the mitochondrial genome that results in an A to G transition in the tRNA gene (MTTK).  There is phenotypic pleiotropy with this gene as patients with PEO, Leigh syndrome, or multiple symmetric lipomatosis can present with this specific mutation.

• Mutations in other locations of tRNA gene (positions 8356, 8366) and mutations in tRNA (MTTL1) can also present with MERRF.  The latter mutation can be seen in MELAS. MTTH, MTTF and MTTS1 are also associated with MERRF.

• MERRF phenotype can also be seen in multiple mtDNA deletions caused by mutations in POLG1 and MTND5.

Treatment:

• No specific treatment

• Treat myoclonus with clonazepam and seizures with AEDs. 

• Creatine monohydrate 5-10 g/day may be tried as a slight benefit was reported ; although evidence is weak. 

• Special care in patients with mitochondrial myopathies is needed as they can develop marked alveolar hypoventilation in response to sedating medications and anesthetic agents.  

MELAS (Mitochondrial encephalomyopathy lactic acidosis and stroke like episodes)

• MELAS syndrome, which is known by the acronym of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, is a progressive neurodegenerative disorder characterized by acute neurological episodes resembling strokes associated with lactic acidosis and mitochondrial myopathy.

• This syndrome, most commonly caused by an A-to-G RNA transfer  mutation (Leu (UUR)) in the most commonly m.3243A>G mutation.  It is typified by characteristic neurological manifestations including seizures, encephalopathy, and strokelike episodes, as well as other frequent secondary manifestations including short stature, cognitive impairment, migraines, depression, cardiomyopathy, cardiac conduction defects, and diabetes mellitus. 

• Less commonly, it can present with gastrointestinal manifestations that include gastric perforation, ischemic colitis, segmental ileal paralysis, pseudo-obstruction, or megacolon.

• Muscle weakness, high lactate levels in serum or CSF, and stroke-like episodes.  

• Three almost invariant criteria of MELAS, namely, (1) strokelike episode before age 40 years; (2) encephalopathy characterized by seizures, dementia, or both; and (3) lactic acidosis, ragged-red fibers, or both.  Lactic acidosis, either measured in serum or cerebrospinal fluid (CSF), was a near universal finding, occurring in 94%, as were seizures 96%, and stroke like events, 99%. 

Pathophysiology:

The exact pathogenic mechanism for these episodes is yet to be determined; however, three theories have been postulated. The first is insufficient energy due to mitochondrial dysfunction, supported by the increase in lactate peaks and decreased N-acetyl aspartate peaks of the occipital regions in brain magnetic resonance spectroscopy (MRS). The second is nitric oxide (NO) deficiency, which usually regulates oxygenation and blood flow. This hypothesis is supported by a reduction in NO metabolites during acute attacks and an increase in NO synthase inhibitors in the COX-negative fibers of MELAS patients. The third theory is mitochondrial angiopathy, an accumulation of mitochondria in the smooth muscle cells and endothelial cells of small cerebral arteries leading to the narrowing of the lumen of blood vessels and reducing perfusion. MRI findings of patients with stroke like episodes exhibit stroke-like lesions (SLLs) that are usually differentiated from other pathologies by initially observing cortical and deep white matter lesions, in addition to occipital and parietal lobe lesions or lesions not confined to arterial territories. PWI/ASL can also show hyperperfused lesions, and MRS exhibits lactate peaks. Another distinctive finding in neuroimaging was reported in some cases of MELAS as cerebellar lesions SLL 

Endocrine manifestations:

• Diabetes mellitus is firmly established as association with MELAS.  Dysregulation of hepatic gluconeogenesis in the context of lactic acidosis may be another contributing factor. Mitochondrial dysfunction may lead to increased lactate flux into the liver, fueling gluconeogenesis.  Failure of insulin secretion by pancreatic β cells is the more widely presumed underlying mechanism, possibly due to energy failure and dependence of these cells on ATP driven sodium–potassium pumps.

• Growth failure.  Another widely recognized systemic manifestation of MELAS is growth failure. Individuals with MELAS are typically of short stature relative to other (unaffected) family members. This is often, together with sensorineural hearing impairment, migraine headaches, learning difficulties, and exercise intolerance.

Cardiac manifestations:

• Because of the high energy requirements of cardiac muscle, cardiac disease features prominently among the systemic manifestations of MELAS and other mitochondrial cytopathies. Cardiomyopathy was an early recognized manifestation of mitochondrial disease and has featured particularly prominently in the systemic pathology of patients harboring mtDNA mutations associated with MELAS. Both dilated and hypertrophic cardiomyopathies have been well described in association with MELAS syndrome, although the more typically reported pathology is a non-obstructive,concentric hypertrophy.  Cardiomyopathy is now a firmly entrenched and accepted complication of MELAS. Conduction defects, including Wolff–Parkinson–White (WPW) syndrome, have also been reported commonly in association with MELAS syndrome.

Myopathy:

• Myopathy has long been recognized as a major and common feature of mitochondrial cytopathy and has been well described in the context of MELAS and many other mitochondrial diseases.  Exercise intolerance is an often cited complaint in patients with the m.3243A>G mutation, with or without full-fledged MELAS syndrome.  There is an inverse correlation between mutation load and both maximal oxygen uptake and maximal workload/  Moreover, there appeared to be a level of mutation burden (50%), above which all patients showed ragged-red fibers suggestive of mitochondrial disease.

Renal disease:

• While less common than cardiac and endocrine abnormalities, renal disease has also been reported as a manifestation of MELAS causing mtDNA mutations and other mitochondrial diseases.  The most commonly reported kidney diseases associated with MELAS are proximal tubulopathy as de Toni–Debr´e–Fanconi syndrome, nephrotic range proteinuria, and focal segmental glomerulosclerosis (FSGS).  Other renal manifestations reported in the context of mitochondrial disease include Bartter-like syndrome, hypercalciuria, and tubulointerstitial nephritis.

• De Toni-Debr'e-Fanconi syndrome is characterized by an impairment of proximal tubular reabsorption, leading to urinary losses of amino acids, glucose, proteins phosphate, uric acid, bicarbonate, potassium, and water. This disorder is thought to stem from dysfunction of the active, ATP-dependent sodium–potassium pumps that generate the electrical gradient necessary to drive reabsorption of these electrolytes.

• Glomerular disease with nephritic syndrome has been reported in association with MELAS and the m.3243A>G mutation. Although the mechanism of this process remains elusive, FSGS does not appear to be immunologic in origin on the basis of histopathology.  Steroid therapy was ineffective in all four patients.

Gastrointestinal dysfunction:

• Constipation and gastric discomfort, hepatopathy.

• Recurrent, sometimes triggerable, vomiting; recurrent pancreatitis; gastrointestinal dysmotility; gastroparesis; progressive intestinal pseudo-obstruction; and malabsorption with progressive malnutrition.

• Other mitochondrial syndromes with significant gastrointestinal manifestations include MERRF, Pearson syndrome, Wolfram syndrome, and Leigh disease. Mitochondrial neurogastrointestinal encephalopathy is a chronic and progressive disease caused by loss-of function mutations in the gene encoding thymidine phosphorylase, which typically presents in the second or third decade with progressive external ophthalmoplegia, severe gastrointestinal dysmotility, cachexia, and peripheral neuropathy.

• MELAS or the m.3243A>G mutation has been associated with gastrointestinal complications such as diarrhea and constipation leading to intestinal pseudo-obstruction,gastric dysmotility, and cyclic vomiting. 

• Moreover, clinicians ought to consider the risk of exacerbating underlying hepatic dysfunction, particularly when they choose seizure prophylaxis. Valproic acid, a drug with well-known and demonstrated antiepileptic effects, should be particularly avoided in these individuals. Valproic acid, a drug with well-known and demonstrated antiepileptic effects, should be particularly avoided in these individuals. In addition to the potential for hepatotoxicity, there is evidence that this medication can have deleterious effects on mitochondrial structure, altering the mitochondrial membrane by inducing paroxysmal depolarization shifts. Clinically, this may manifest as a worsening of seizure control in MELAS patients when they are switched to valproic acid from another antiepileptic medication.  Valproic acid may trigger seizures in patients with mitochondrial disorders by impairing the proton pumping activity of complex IV, cytochrome c oxidase.  Although not formally demonstrated, the additional effect of valproic acid on mitochondrial functionmay exacerbate an already fragile and tenuous oxidative phosphorylation system, further worsening pathology. This medication should therefore be avoided in patients with MELAS or suspected to have another mitochondrial disease.

• Alpers, a mitochondrial depletion syndrome resulting from mutations in the POLG-1 (polymerase gamma 1) gene, presents specifically with hepatic dysfunction and seizures, along with developmental delay, hypotonia, ataxia, and cortical blindness.

Dermatological manifestations:

• Vitilgio, pigmentary changes, scaly pruritus rash with diffuse erythema and reticular pigmentosa. 

Pulmonary manifestations:

• Very rare.

• Pulmonary HTN may be reported. 

Clinical features:

• Neurological/neuropsychiatric: Headache, sensory loss, depression. memory problems 

• Visceral: Exercise intolerance, hearing loss, short stature, growth failure, diabetes, heart disease, and gastrointestinal disturbance.

• Clinically, cases are often marked by episodes of at least partially reversible aphasia, hemianopsia, and cortical blindness with eventual progressive accumulation of neurological deficits and dementia.  Recurrent episodes of migraine-type headaches with nausea and vomiting.  These are not long-lasting episodes and most recover.   These episodes may be provoked by exercise or intercurrent infection.  Progressive dementia may occur.

• The term strokelike episodes was coined to stress the nonischemic origin of these events.The affected areas do not correspond to classical vascular distributions but rather have an irregular distribution, more consistent with a metabolic or small-vessel etiology 

• Cortical involvement can be of any size or apparent severity but typically presents in an asymmetric pattern that affects predominantly the temporal, parietal, and occipital lobes and is often restricted to the cortex, with relative sparing of the deep white matter 

• Rare individuals with the most common A3243G mutation in the MTTL1 gene manifest with only diabetes mellitus and/or deafness

• Many other neuropsychiatric manifestations are seen often in this disorder. Although less specific, sensorineural hearing loss, migraine headaches, peripheral neuropathy, depression and other psychiatric disorders, and learning disabilities are commonly seen in MELAS, not just in fully symptomatic patients but also in oligosymptomatic family members who do not manifest the full phenotype. 

• Dementia, reflective both of the accumulation of cortical injury and of the underlying neuronal dysfunction, is an important and common feature of this syndrome. Cognitive impairments, including language, perception, and memory function, have been widely recognized complications of mitochondrial diseases since the early descriptions of these disorders. 

• MELAS patients, in particular, tended to perform the worst on batteries of tests that examined memory, orientation, nonverbal intelligence, working memory, verbal fluency, visuomotor skills, processing speed, and attention. 

• Deficits of executive function, thought suggestive of frontal lobe pathology, are widely seen despite a relative radiographic sparing of this region, with minimal pathology evident on magnetic resonance imaging (MRI). This finding suggests that there may be a more diffuse underlying neurodegenerative process, which is punctuated by strokelike episodes that affect predominantly posterior regions of the brain. 

• A mild, insidiously progressive sensorineural hearing loss is also commonly seen in this disorder and is often an early clinical manifestation. Overall, hearing impairment, with a frequency of at least 1/1000 at birth and greater than 50% by age 80 years, is probably the most common sensory handicap. A seemingly rare cause of lingual hearing loss, mtDNA mutations may underlie up to 20% of inherited cases of postlingual hearing loss. Among the white population, at least 5% of post-lingual, non-syndromic hearing impairment is caused by known mtDNA mutations, representing the second most frequent cause of hearing impairment after the 35delG mutation in the GJB2 gene encoding connexin. MELAS is one of several neuromuscular syndromes, including MERRF,,NARP, Wolfram syndrome, and Pearson syndrome, strongly associated with sensorineural hearing loss.  The m.3243A>G mutation responsible for more than 80% of MELAS cases is also commonly associated with the syndrome of maternally inherited diabetes and deafness, which probably reflects a milder (oligosymptomatic) expression on the continuum of MELAS.

• Although clinical studies did not note it as a complication in the initial clinical descriptions of MELAS, they did report a prevalence of neuropathy in MELAS of up to 22%.  The neuropathy, when present, is usually chronic and progressive, with mild sensory complaints and loss of proprioception and vibration in a “stocking–glove” distribution. The distal lower limbs are usually affected first.  A study done by Kaufmann et al., describing the clinical and neurophysiological characteristics of a cohort of 30 patients with MELAS and the m.3243A>G mutation enrolled in a clinical trial, noted that almost all (29 of 30) had abnormal findings suggestive of peripheral neuropathy on neurological examination, including abnormalities on reflex testing, sensory examination, distal muscle strength testing, and gait. Moreover, 23 (77%) of the 30 patients had abnormal nerve conduction measures, typically of an axonal (12/23) or mixed (7/23) nature.

• Visceral Manifestations and Oligosymptomatic Carriers: Fully symptomatic MELAS syndrome probably represents only the merest fraction of patients manifesting mitochondrial disease due to MELAS-causing mutations.  In fact, there is a growing literature reporting the multifaceted presentation of patients harboring mtDNA mutations associated with MELAS. These so-called oligosymptomatic patients, who manifest symptoms of a mitochondrial cytopathy but who do not manifest the full-fledged syndrome and clinical phenotype probably represent almost all cases.

• The diagnosis is based on the clinical presentation and brain imaging which may reveal numerous hyperintense T2 lesions in cerebral white and grey matter, while CT scan shows cerebral atrophy and basal ganglia calcifications. Lactic acidosis is common. Muscle biopsy usually shows abnormal mitochondrial proliferation in ragged-red fibers and cytochrome c oxidase-deficient fibers.  Analysis of muscle respiratory chain activities may reveal complex I deficiency or a combined deficiency of complexes I and IV.)  The MELAS mutation should be better studied in muscle than in blood because of the different load of mutated mtDNA.

• Heteroplasmy (coexistence of mutant DNA with wild-type mtDNA) is above the threshold levels of 80% in muscle and complicates genetic counseling and prenatal diagnosis.

Laboratory features: 

• CK is normal or elevated. 

• serum lactate and pyruvate 

• Serum uric acid

• Serum and CSF lactate is elevated.

  • Elevated lactate, although the sine qua non of most (though not all) mitochondrial diseases, and MELAS in particular, is a nonspecific marker of metabolic derangement of any cause. Among the myriad etiologies of lactic acidosis not reflective of derangement of oxidative phosphorylation are organic acidurias and aminoacidopathies, defects of the citric acid (Krebs) cycle, pyruvate dehydrogenase deficiency, and of course laboratory error caused by improper handling of the serum specimen. Demonstration of an elevated lactate level in the cerebrospinal fluid provides another level of suspicion regarding this diagnostic possibility spurring further diagnostic evaluation.

• EEG may show epileptiform activity

• EDX:

• NCS is normal

• EMG may reveal myopathic MUAPs. 

• MRI of brain reveal cortical atrophy and high T2 signal and FLAIR abnormalities in the cerebral cortex, BG, and thalamus. 

• MRS of acute cortical lesions reveal severely elevated lactate levels and reduced concentrations of NAA, glutamate, and myo-inositol.  

• MRS of skeletal muscle may show reduced phosphocreatine level, and elevated concentration of inorganic phosphate and free adenosine-5'-diphosphate and an abnormally low phosphorylation potential. 

Histopathology:

• Similar to MERRF but with more variability in COX staining.  Arterioles are also strongly SDH reactive and an increased number of mitochondria are found in the muscle wall of the arterioles.  

• Mitochondrial enzyme analysis of muscle tissue reveals reduced activities of complexes I, III, IV, and V. 

Molecular Genetics and Pathogenesis:

• Maternally inheritied.  70% of cases are caused by mtDNA mutation, an A to G substitution, at nucleotide position 3243 in the gene MTTL1 encoding tRNA.  Mutations have been also identified at positions 3252, 3260, 3271, 3291 in the tRNA gene as well as in MTTV, MTTK, MTTF, MTTS1, MTND1, MTND4, MTND5, and MTCYB. 

• One specific substitution, the m.3243A>G (A-to-G substitution at nucleotide 3243), is responsible for 80% of cases, whereas another tRNA variation, the m.3271T>C (T-to-C substitution at nucleotide 3271), accounts for the remaining cases. 

Treatment: 

• No specific therapy other than treatment for seizures and myoclonus. 

• Creatine monohydrate 5-10 g/day modestly improved in a few patients with MELAS.  

• Coenzyme Q10 and its analog )idebenone, creatine monohydrate, and arginine)

• L-arginine ?

• Avoid valproic acid (an antiepileptic drug reported to provoke stroke-like episodes).

• MELAS syndrome is a mitochondrial inherited genetic disorder that is significantly impacted by a deficiency in nitric oxide. Administering nitric oxide precursors, such as arginine and citrulline, may increase nitric oxide availability and reduce the effects of nitric oxide deficiency. During an acute stroke-like episode, clinicians may administer arginine to reduce brain damage due to impaired vasodilation in intracerebral arteries caused by nitric oxide depletion.  A systematic review by Argudo et al. concluded that the studies conducted showed promising results in managing SLEs. 

  • Acute phase management consists of giving an intravenous dose of 500 mg/kg/day or 10 g/m2 in 24 h for 3–5 days. Whereas chronically, 150–300 mg/kg/day (maximum of 500 mg) is used instead. A study conducted by Pek et al. using induced pluripotent stem cell-derived endothelial cells vouched for edaravone, a potent antioxidant, to be used for improving the vascular function in MELAS since it scavenges ROS and inhibits the inflammatory response in cerebrovascular diseases, which L-arginine and citrulline do not tackle. For treating epilepsy, levetiracetam is considered to be the first-line anticonvulsant in mitochondrial encephalomyopathy due to the mitochondrial toxicity of other anticonvulsant agents 

• Arginine (R-gene 10): Patients experiencing an acute stroke-like event should receive a bolus of intravenous L-arginine (500 mg/kg for children or 10 g/m2 body surface area for adults) within 3 hours of symptom onset followed by the administration of similar dosage of intravenous arginine as continuous infusion over 24 hours for the next 3 to 5 days. Once an individual with MELAS has the first stroke-like episode, arginine should be administered prophylactically to reduce the risk of recurrent stroke-like episodes. A daily dose of 150 to 300 mg/kg/d oral arginine in 3 divided doses is recommended 

• Citrullline: Interestingly, citrulline supplementation was found to induce a greater increase in the NO synthesis rate than that associated with arginine supplementation, indicating that citrulline is a more effective NO precursor than arginine 

• Arginine for the Treatment of Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-Like Episodes: A Systematic Review - PMC 

• Patients with MELAS may experience hypocitrullinemia. Researchers have observed that short-term supplementation with citrulline enhances nitric oxide production more than arginine. A substantial de novo arginine synthesis occurs in response to citrulline supplementation. Consequently, in addition to arginine, citrulline administration holds therapeutic potential for MELAS. However, controlled studies assessing the effects of citrulline supplementation on the clinical aspects of MELAS are required to establish its use as a therapeutic.

• Other treatment options for MELAS include coenzyme Q10, menadione or vitamin K3, phylloquinone or vitamin K1, and ascorbate, which are used to donate electrons to cytochrome c. Furthermore, several case reports suggest improvement with riboflavin, dichloroacetate, sodium succinate, and creatinine monohydrate.

• Vitamins such as coenzyme Q10 or L-carnitine are believed to aid in boosting energy production by mitochondria and may potentially decelerate the progression of the disease. Ongoing phase I and II trials of Idebenone, a synthetic coenzyme Q10, are being conducted for MELAS, showing promise in improving neurological function in other mitochondrial disorders (Scaglia, ClinicalTrials.gov Identifier: NCT00887562).

• Seizures in patients with MELAS syndrome may be refractory to treatment. Notably, valproate is not an appropriate treatment for patients with MELAS syndrome. Many reports exist of valproate aggravating encephalopathy and seizures in patients with MELAS syndrome. Numerous reports document valproate exacerbating encephalopathy and seizures in individuals with MELAS syndrome. The primary mechanism of valproate toxicity involves interference with mitochondrial β-oxidation or direct mitochondrial toxicity, explaining the frequently observed elevated ammonia levels in patients taking valproate 

• Levocarnitine: 1000 mg 

• Ubiquinol: 50 mg

• Citrulline: 3,500 mg (50 mg/kg/dose)

• Arginine: 150 - 300 mg/kg/day in 3 divided dosese. 

• Riboflavin: 50 mg

• Creatine: 1.75 mg 

Prognosis:

• MELAS syndrome, once stroke or epilepsy presents, might have fast deterioration with dementia-like syndrome and death.

• Prognosis is poor. Patients may die during one stroke-like episode, and, along with recurrent episodes, they often develop mental deterioration, loss of vision and hearing, as well as severe myopathy, potentially leading to loss of autonomy.

Radiographic Features of MELAS

Strokelike episodes, ischemic events not adhering to vascular territories can be demonstrated using diffusion-weighted imaging (DWI) sequences on MRI. In combination with other techniques such as MRS, MRI is a powerful tool in the diagnostic evaluation of suspected cases of MELAS. The MRI of patients with MELAS typically demonstrates asymmetric lesions of the occipital and parietal lobes that mimic ischemia, except that they usually do not respect vascular territories and are often restricted to the cortex with relative sparing of deep white matter. MR angiography is typically normal.  Characteristic is a fluctuation of lesions over time.  Other nonspecific features generally suggestive of a metabolic disorder include basal ganglia findings or periventricular white matter abnormalities with preservation of the remaining deep white matter. MR spectroscopy is a frequency analysis of the MR signal that permits a unique in vivo evaluation of brain metabolism. Using this technique, chemical species can be identified on the basis of their chemical shift value, which is recorded in parts per million so that spectra from different fields can be compared. The most common abnormalities in MELAS seen with MR spectroscopy include a N-acetyl aspartate signal decrease and the accumulation of lactate. There is a general consensus that a lactate peak represents a sensitive metabolic marker of disease.  Moreover, there appears to be a strong correlation between high ventricular lactate as measured using MRS and the degree of neuropsychological and neurologic impairment.  Ventricular CSF may be the simplest and most sensitive site for screening by spectroscopy. With CSF lactate greater than 4.0 mmol/L, the lactate peak may be easily detected88,89 and therefore serve as a good confirmatory test for elevated lactate within the CNS.

Patient Hand-out on MELAS

Primary mitochondrial disorders (PMDs) are a heterogeneous group of disorders characterized by impaired mitochondrial structure and/or function due to mutations in nuclear or mitochondrial DNA. Primary mitochondrial disorders are the most common inborn errors of metabolism, and have a prevalence of about 1:5000. We reviewed that mitochondria are components of body cells and are responsible for the production of "energy" for the body. The metabolic pathway most affected in PMDs is the mitochondrial electron transport chain (ETC), which is the most important pathway for producing energy in the form of adenosine 5’-triphosphate (ATP). The ETC, found in the inner membrane of the mitochondria, consists of four protein complexes commonly referred to as complexes I, II, III, and IV, along with two mobile electron carriers (ubiquinone or coenzyme Q [CoQ] and cytochrome c) and the ATP synthase complex. ETC dysfunction reduces ATP production, increases anaerobic metabolism (which can lead to elevated lactate concentrations in the blood [lactic acidosis] and/or cerebrospinal fluid [CSF]), and increases free radical production, which can lead to oxidative stress and additional cellular damage and dysfunction.

Tissues that have high energy requirements are the most affected in PMDs. These tissues include the central nervous system, peripheral nerves, eyes, ears, skeletal and cardiac muscles, kidneys, endocrine organs, and gastrointestinal tract. Patients typically present with multisystem or organ-specific disorders. The clinical features of PMDs vary, due to differing levels of heteroplasmy. The number of mitochondria that carry the gene mutation (called mutational load) can vary between individuals in the same family and even between various body tissues within the same individual (called tissue distribution). We refer to the mix of mutated and normal mitochondria as heteroplasmy. The extent to which an individual is affected by symptoms of a mitochondrial condition can be dependent on this heteroplasmy and overall tissue distribution. Central neurological features include encephalopathy, seizures, stroke-like episodes, dementia, ophthalmoplegia, and hearing deficits. Peripheral neurological features include myopathy (signs include fatigue and exercise intolerance), ataxia, and peripheral neuropathy.

MELAS syndrome is passed on in families via mitochondrial inheritance. We reviewed that mitochondria are components of body cells and are responsible for the production of "energy" for the body. We inherit all mitochondria from our mother and thus, if a woman carries a mitochondrial gene mutation, all of her children will inherit that mutation and will be at risk for the features associated with that mitochondrial condition. It is important to note, however, that the number of mitochondria that carry the gene mutation (called mutational load) can vary between individuals in the same family and even between various body tissues within the same individual (called tissue distribution). We refer to the mix of mutated and normal mitochondria as heteroplasmy. The extent to which an individual is affected by symptoms of a mitochondrial condition can be dependent on this heteroplasmy and overall tissue distribution.

Primary mitochondrial disorders have no cure. Consensus-based recommendations for standardized treatment and preventive health care of patients with PMDs have been published. Though there is no cure, certain dietary supplements can be of value in treating PMDs because, as nutrients and metabolic cofactors, they help increase energy production, bypass a cellular defect (such as deficiency in activity of complexes I, II, or III in the ETC), or remove toxic metabolites. However, while in theory, these supplements should be of benefit, in practice (and from published clinical trials), there is no concrete evidence that these supplements are actually efficacious. The most commonly used dietary supplement ingredients include antioxidants, electron donors and acceptors, compounds that can be used as alternative energy sources, and compounds that can conjugate or bind mitochondrial toxins.

 From a surveillance standpoint, we would recommend the following to be considered:

1. EKG and echocardiogram annually (to monitor cardiac conduction and contractility, and to assess for cardiomyopathy).  Cardiac MRI

2. MRI, MRS, DEXA. swallow evaluaiton

3. Annual audiology evaluation (to detect hearing loss)

4. Annual ophthalmology evaluation (to screen for ptosis, optic atrophy, pigmentary retinopathy, ophthalmoplegia, and/or vision deficits), OCT, ERG

5. Psychiatry

6. PSG, PFT

7. Annual labs: CBC with differential, CMP, Vit D, iron, ferritin, tranferrin, HbA1c, TSH, iPTH, cortisol, ACTH, gonadotrophins, amylase, lipase, stool elastase, 

8. Neurological follow-up determined based on signs/symptoms

9. Anesthesia can potentially aggravate respiratory symptoms and precipitate respiratory failure; thus, careful consideration should be given to its use and to monitoring the individual prior to, during, and after its use [Shear & Tobias 2004, Niezgoda & Morgan 2013].

10. Additionally, check a set of metabolic labs, including plasma amino acids, STAT lactate, CBC, CMP, urine organic acids to determine metabolic stability.  

11. Avoid Valproic acid.  Can result in rrreversible liver failure and onset of hepatoencephalopathy, especially in POLG-related disorders; worsening of seizures. 

 References:

1. https://ods.od.nih.gov/factsheets/PrimaryMitochondrialDisorders-HealthProfessional/

2. Parikh S, Goldstein A, Koenig MK, Scaglia F, Enns GM, Saneto R, et al. Diagnosis and management of mitochondrial disease: A consensus statement from the Mitochondrial Medicine Society. Genet Med 2014.

3. Haas RH, Parikh S, Falk MJ, Saneto RP, Wolf NI, Darin N, et al. Mitochondrial disease: a practical approach for primary care physicians. Pediatrics 2007;120:1326-33.

4. Niyazov DM, Kahler SG, Frye RE. Primary mitochondrial disease and secondary mitochondrial dysfunction: Importance of distinction for diagnosis and treatment. Mol Syndromol 2016;7:122-37.

5. Goldstein A, Wolfe LA. The elusive magic pill: Finding effective therapies for mitochondrial disorders. Neurotherapeutics 2013;10:320-8.

6. Pfeffer G, Majamaa K, Turnbull DM, Thorburn D, Chinnery PF. Treatment for mitochondrial disorders. Cochrane Database Syst Rev 2012;4:CD004426.

7. Parikh S, Goldstein A, Karaa A, Koenig MK, Anselm I, Brunel-Guitton C, et al. Patient care standards for primary mitochondrial disease: A consensus statement from the Mitochondrial Medicine Society. Genet Med 2017;19.

8. Marriage B, Clandinin MT, Glerum DM. Nutritional cofactor treatment in mitochondrial disorders. J Am Diet Assoc 2003;103:1029-38.

9. Tarnopolsky MA, Raha S. Mitochondrial myopathies: Diagnosis, exercise intolerance, and treatment options. Med Sci Sports Exerc 2005;37:2086-93.

10. Barcelos I et al. Mitochondrial medicine therapies: rationale, evidence, and dosing guidelines. Curr Op Pediatr. 2020 Dec;32(6):707-718. doi: 10.1097/MOP.0000000000000954 

Migraine in MELAS

Mitochondrial Disorder Aggravated by Metoprolol - PMC 

Patient care standards for primary mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society

Ilness management:

1. Specific decisions about patient management including hospitalization require clinical judgment and should be case-specific. Decisions should reflect the individual patient’s presentation as well as an understanding of the etiology for the acute decompensation and the pathophysiology of the underlying mitochondrial disorder.

2. Patients with a mitochondrial disease should carry an emergency care plan that details their underlying disorder and provides management recommendations.

3. Patients with a mitochondrial disease should consider wearing a medical alert bracelet when appropriate depending on their clinical symptomology.

4. Mitochondrial patients should take precautions to prevent entering catabolism, especially when exposed to medical stressors, including avoiding prolonged fasting and receiving dextrose-containing IV fluids before, during, and after procedures and surgeries. (Dextrose should not be provided or provided in limited quantity as indicated by clinical status in suspected or confirmed disorders of pyruvate metabolism, if the patient is on a ketogenic diet, or the patient has had a previous adverse response to high glucose delivery.)

5. Evaluation of a mitochondrial patient in the acute setting should include evaluation of routine chemistries, glucose, transaminases, and lactate; all other testing is as clinically indicated, although one must keep in mind the potential for cardiac and neurologic decompensations in these patients.

6. Treatment during acute decompensation should include dextrose-containing IV fluids, stopping exposure to potentially toxic medications, and correction of any metabolic derangements. (Note: dextrose should be provided only in limited in quantity or not at all, as indicated by clinical status in suspected or confirmed disorders of pyruvate metabolism, if the patient is on a ketogenic diet, or the patient has had an adverse response to high glucose delivery.) IV fluid rate should be based on the clinical situation. Outpatient mitochondrial therapies should be continued when possible.

7. Lipids can be used when needed in mitochondrial patients, even in the presence of secondary fatty acid oxidation dysfunction.

8. The following medications should be avoided in patients with mitochondrial disease when possible and, if given, they should be used with caution: valproic acid; statins; metformin; high-dose acetaminophen; and selected antibiotics, including aminoglycosides, linezolid, tetracycline, azithromycin, and erythromycin.

9. Repeat neuroimaging should be considered in any mitochondrial patient with an acute change in neurologic status.

Anesthesia and surgical management:

1. Patients with mitochondrial diseases are at an increased risk of anesthesia-related complications.

2. Preoperative preparation of patients with mitochondrial disease is crucial to their perioperative outcome. Patients should minimize preoperative fasting and have glucose added to their perioperative IV fluids, unless they are on a ketogenic diet or have been demonstrated to have adverse reaction to higher glucose intake.

3. Caution must be used with volatile anesthetics because mitochondrial patients may potentially be hypersensitive.

4. Caution must be used with muscle relaxants in those mitochondrial patients with a preexisting myopathy or decreased respiratory drive.

5. Mitochondrial patients may be at a higher risk for propofol infusion syndrome and propofol use should be avoided or limited to short procedures.

6. One should consider slow titration and adjustment of volatile and parenteral anesthetics to minimize hemodynamic changes in mitochondrial patients.

7. Local anesthetics are generally well tolerated in patients with mitochondrial defect.

8. There is no clear established link between malignant hyperthermia and mitochondrial disease.

Stroke management:

1. Strokelike episodes in primary mitochondrial disease typically have correlating visible magnetic resonance imaging abnormalities.

2. IV arginine hydrochloride should be administered urgently in the acute setting of a strokelike episode associated with the MELAS m.3243A>G mutation in the MTTL1 gene and considered in a stroke-like episode associated with other primary mitochondrial cytopathies as other etiologies are being excluded. Patients should be reassessed after 3 days of continuous IV therapy.

3. The use of daily oral arginine supplementation to prevent strokes should be considered in MELAS syndrome.

4. The role of monitoring plasma arginine and citrulline levels and oral citrulline supplementation in the treatment of MELAS requires further research.

5. MELAS patients with chronic pseudo-obstruction (CIPO) should receive closer neurologic follow-up as the symptom may represent a harbinger of stroke. 

   • CIPO is characterized by more than 6 months of severe symptoms of intestinal obstruction such as abdominal distention and pain, nausea, and vomiting with clinical/radiographic evidence of dilated bowel in the absence of mechanical obstruction. 

KSS (Kearns-Sayre Syndrome)

• Kearns–Sayre syndrome is a syndrome of CPEO and pigmentary retinopathy, with onset before the age of 20 years as well as one of the following features: a complete heart block, cerebellar ataxia, dementia, deafness, short stature, endocrine abnormalities, and cerebrospinal fluid (CSF) protein of more than 100 mg/dL. If the diagnostic criteria are not met, the patient is termed “CPEO plus” or “KSS-minus”. 

• When a patient presents with CPEO before the age of 20, they should be evaluated with mtDNA sequencing followed by regular ophthalmologic assessments and screening for systemic signs and symptoms. A muscle biopsy can be performed to look for the ragged red fibers. The fundoscopic examination reveals pigmentary retinopathy that should be distinguished from retinitis pigmentosa since they might share similar symptoms like mildly reduced night vision and visual acuity. Retinitis pigmentosa typically affects the peripheral or the mid-peripheral retina with a bone spicule pattern, whereas KSS affects the posterior retina with a salt and pepper pattern.

•  It is essential to perform an electrocardiogram on these patients to rule out a complete heart block. Endocrine abnormalities affecting the adrenals, parathyroid, and hypothalamus can present with diabetes mellitus, growth hormone deficiency, and short stature. Orbicularis oculi muscle weakness can impair eyelid closure, and frontalis weakness can affect eyelid elevation. Dysphagia is a rare presentation of KSS and may result from upper esophageal sphincter dysfunction and reduced peristalsis in the pharynx and upper esophagus, as observed in a manometric study 

Laboratory features: 

• Serum CK is normal

• Serum lactate and pyruvate may be elevated. 

• CSF protein may be increased. 

• ECG reveals conduction defects. 

• EDX: normal to axonal sensory or sensorimotor polyneuropathy.  EMG may show normal insertional and spontaneous activity, but may reveal myopathic MUAPs in weak muscles.  

Histopathology

• RRF, little variability in COX unlike MERRF and MELAS. 

• Spongy degeneration of cerebral white matter - autopsy. 

Molecular genetics and pathogenesis

• Single large mtDNA deletions (1.3 to 8.8 kB) can be demonstrated in most patients with KSS.

• More than 150 different mtDNA deletions have been associated with KSS.  A deletion of 4977 base pairs known as m.8470_13446del4977 is encountered most frequently. The same mutation and numerous other types of deletions of varying length have been identified in Pearson syndrome (sideroblastic anemia and exocrine pancreas dysfunction) and progressive external ophthalmoplegia (PEO).

Management of KSS:

No definitive treatment option is available for KSS. Symptomatic treatment includes correction of CPEO, treating heart blocks with pacemakers with a long-term cardiology follow-up, correction of endocrine abnormalities, and cochlear implants in cases of hearing loss. 

Autosomal recessive cardiomyopathy and ophthalmoplegia (ARCO). 

Mitochondrial depletion myopathy - TK2, DGOUK

• Some forms of PEO, MNGIE, MIRAS, OA1, and Navajo neurohepatopathy are associated with MDS.

• TYMP, POLG1, POLG2, twinkle (C10orf2), MPV17, RRM2B, OPA1, SUCLG1, SUCLA2.

POLG1 related mitochondrial disorders: PEO1 - AR, MGNIE, SANDO, MIRAS, MERRF.

MGNIE (Mitochondrial neurogastrointestinal encephalomyopathy)

• AR mitochondrial disorder due to mutations in TYMP, the gene encoding thymidine phosphorylase enzyme.  The activity of thymidine phosphorylase enzyme is reduced resulting in elevated plasma concentrations of thymidine.

• POLG1 mutations may cause MNGIE-like syndrome without leukoencephalopathy and normal thymidine plasma levels

• Sensorimotor PN, leukoencephalopathy on MRI of brain, RRF on muscle biopsy, and chronic intestinal pseudo-obstruction. 

• Onset <20 years of age

• Progressive with severe disability or death by 3rd or 4th decade of life. 

• Distal greater than proximal muscle weakness and atrophy, stocking-glove distribution of sensory loss, and reduced MSR throughout.  

• Ptosis and EOM weakness. 

• Despite of leukoencephalopathy on MRI, most affected patients have little in the way of CNS symptoms or signs.  Some have mental retardation.  Others have retinitis pigmentosa, SNHL, facial weakness, hoarseness, or dysarthria.  

Laboratory features:

• Serum CK can be normal or mildly elevated. 

• Serum lactate, pyruvate, and CSF protein is elevated. 

• Thymidine phosphorylase activity is decreased (<10%) in leukocytes and platelets.  Thymidine levels are increased in plasma and deoxyuridine is increased in serum and plasma in those cases caused by mutation in the TYMP gene that encodes for thymidine phosphorylase. 

  • Plasma thymidine levels greater than 3 umol/L and deoxyuridine concentration greater than 5 umol/L can be sufficient to confirm the diagnosis.

• MRI of brain shows leukoencephalopathy in cerebral and cerebellar white matter. 

• Dilatation and dysmotility of esophagus, stomach, and small intestine.  

• ECG has shown conduction defects but many remain asymptomatic. 

• EDX:

  • Motor and sensory NCV may be slow to within the demyelination range, while F-wave latencies are usually markedly prolonged. 

  • Other cases are more suggestive of axonopathy with reduced SNAP and CMAP amplitudes.  

  • EMG shows fibrillation potentials and psw.  Recruitment of MUAPs can be decreased in distal muscles.  Quantitative EMG of proximal muscle show myopathic motor units.  

SANDO (sensory ataxia, neuropathy, dysarthria/dysphagia, and ophthalmoplegia)

•  POLG1, C10orf2

MIRAS (mitochondrial recessive ataxic syndrome) - POLG1

NARP (neuropathy ataxia and retinitis pigmentosa)

Navajo Neurohepatopathy

Optic atrophy 1 (OA1) syndrome

Focal Mitochondrial depletion

Mitochondrial myopathies associated with exercise intolerance/recurrent myoglobinuria

CMT2A, CMT2K, CMT4.  

Pearson Syndrome

Pearson syndrome (PS), also known as Pearson marrow–pancreas syndrome, is a rare fatal multisystemic mitochondrial disease due to deletions in mtDNA, and it typically affects infants. Ophthalmologic manifestations include corneal endothelial dysfunction, ptosis, CPEO, and mild peripheral pigmentary retinopathy. It is also characterized by refractory sideroblastic anemia, lactic acidosis, and exocrine pancreatic dysfunction. It can also present with vacuolization of hematopoietic precursors, pancytopenia, failure to thrive, diarrhea, hypospadias, cleft lip palate, diabetes mellitus, renal tubular dysfunction, hepatic failure, enteropathy, and rashes. Cardiac manifestations, such as bundle branch blocks and supraventricular tachycardia, have been reported; however, cardiac involvement is not yet a part of the major criterion of the disease/  

Usually, premature death at three years of age occurs due to infection from neutropenia or metabolic crisis. Thus, early diagnosis is essential in improving the poor prognosis for these patients. The diagnosis of Pearson syndrome is challenging due to the atypical presentation in infancy. It can be confirmed via mtDNA sequencing and observing multiple deletions of varying lengths. Interestingly, these single large-scale mtDNA deletions can also be found in young patients with CPEO and KSS. They, therefore, form a continuous spectrum of diseases termed “mtDNA deletion syndromes”, supported by reports of a KSS-like phenotype in PS survivors.

Treatment for Pearson syndrome is supportive and may include blood transfusions, iron chelating therapy, pancreatic replacement therapy, and prompt detection and management of cardiac dysfunction. Bone marrow transplant has been tested and, unfortunately, yielded poor outcomes 

Leigh Syndrome

Adult onset Leigh

Clinical Diagnosis and Treatment of Leigh Syndrome Based on SURF1: Genotype and Phenotype - PMC (nih.gov) 

https://www.invitae.com/us/providers/test-catalog/test-98002

https://www.ncbi.nlm.nih.gov/books/NBK1173/

https://www.sciencedirect.com/science/article/pii/B9780128217511000154?via%3Dihub

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11177993/

Leigh syndrome, or infantile subacute necrotizing encephalopathy, is a severe early-onset progressive neurodegenerative disorder, characterized by symmetrical necrotic lesions in one or more areas of the central nervous system, including the brainstem, thalamus, basal ganglia, cerebellum, and spinal cord. The lesions include demyelination, gliosis, necrosis, spongiosis, and capillary proliferation.

It is a fatal, progressive neurodegenerative disease that typically manifests in infants and young children of 3 months to 2 years of age. It can be caused by multiple mtDNA deletions as well as nDNA defects in more than 75 different monogenic causes, most commonly by the SURF1 variant.  

Leigh syndrome is commonly associated with systemic deficiency of cytochrome C oxidase (COX), but it may result from a deficiency of any of the five different mitochondrial respiratory chain complexes.  It is genetically heterogeneous and causative mutations have been identified in both nuclear- and mitochondrial-encoded genes, including mitochondrial respiratory chain complexes I, II, III, IV, and V, which are involved in oxidative phosphorylation and the generation of ATP, and components of the pyruvate dehydrogenase complex (sexlinked (pyruvate dehydrogenase E1 gene mutations).  Mutations in the SURF1 gene, encoding the surfeit-1 protein involved in the COX complex's biogenesis, are responsible for most of the COX-deficient cases of Leigh syndrome.  The SURF 1 gene is located on chromosome locus 9q34.2  and has autosomal recessive inheritance.

COX deficiency is the most common biochemical abnormality in Leigh syndrome.  However, COX defect can be caused by both mutations in mtDNA-encoded and nuclear DNA-encoded genes. In addition, mutations in the SURF-1, SCO1, SCO2, and COX10 genes have been identified.  The role of SURF1 is to allow a proper COX complex assembly

Prevalence of 1:100,000 to 1:140,000 births.  

 Over 50% of cases are present in the 1st year of life, usually before 6 months of age.  Late-onset varieties are rare and only a few cases were reported all over the world

Clinical symptoms depend on which areas of the central nervous system are involved.  It includes developmental delay, hypotonia, respiratory dysfunction, epilepsy, reduced feeding, and weakness. The ocular features of LS include nystagmus, ptosis, ophthalmoplegia, strabismus, pigmentary retinopathy, and optic atrophy. Common cardiac abnormalities are hypertrophic or dilated cardiomyopathy and conduction defects such as Wolff–Parkinson–White syndrome. 

Case Report 1: 

A 37-year-old female presented with protracted pain in the abdomen and vomiting for 3 months; followed by dizziness, headache, and diplopia for 15 days.  There was no fever, seizures, limb weakness, or sensory symptoms.  Personal and family history was unremarkable.  General and other systemic examinations were normal.  Neurological examination revealed bilateral horizontal gaze palsy with gait ataxia.  The rest of the examination was normal.  MRI of the brain showed dorsal brain stem (midbrain and pons) T2/fluid-attenuated inversion recovery (FLAIR) hyperintensities.  Given recurrent vomiting, and clinical and MRI pictures, a diagnosis of Wernicke’s encephalopathy was made and she was treated with parenteral thiamine.  There was marked improvement and she was discharged.  A few days later she presented with diplopia, increased swaying while walking, bulbar palsy, breathlessness, and shock.  Arterial blood gas (ABG) analysis showed severe metabolic acidosis.  Fasting serum lactate was elevated (8 mmol/L; normal: 0.8-2.4 mmol/L).  MRI revealed increased brainstem hyperintensities with MR spectroscopy (MRS) of the lesion showing peak lactate.  Cerebrospinal fluid (CSF) lactate was also elevated (4.4 mmol/L; normal: 1.1-2.3 mmol/L).  The hemogram and renal and liver function tests were normal.  CSF cytology and biochemistry were normal.  Further, the metabolic workup revealed normal serum copper, ceruloplasmin, and urine copper levels.  Serum aquaporin antibodies were negative.  A provisional diagnosis of adult-onset LD was considered and the patient was treated with a mitochondrial cocktail (intravenous thiamine, coenzyme-Q, riboflavin, L-carnitine, and L-arginine) along with ventilator support for respiratory failure.  The patient improved dramatically in neurological symptoms and was slowly weaned from the ventilator.  Muscle biopsy revealed reduced COX and COX-succinate dehydrogenase (SDH) activity without any evidence of ragged red fibers.  Muscle biopsy where muscle deoxyribonucleic acid (DNA) was extracted and sequential analysis for complete mitochondrial genome was done; no pathogenic mutations were observed, however, nuclear part of mitochondrial DNA (mtDNA) of blood was not analyzed.  A repeat MRI after 3 months revealed a complete disappearance of the hyperintensities.  A diagnosis of adult-onset LS was made based on classical radiological appearance, biochemical and histochemical evidence, and excellent response to mitochondrial cocktail.  At the 1-year follow-up, she was asymptomatic neurologically but had moderate obstructive sleep apnea with an apnea-hypopnea index of 20/h on overnight polysomnography.  Cardiac evaluation which included a two-dimensional (2D) echocardiogram, treadmill test, and 24-hHolter monitoring was normal.

Adult LD was defined as patients who survived longer than 18 years.  It is rare and its clinical manifestations were different from those of children.  These cases are divided into those that fulfilled the Rahman et al., criteria (Rahman’s criteria group (RCG)) and those that were diagnosed with the identification of genetic abnormality (laboratory-diagnosed group (LDG)).  Adult-onset LD tends to have less incidence of developmental delay, COX deficiency, serum lactate elevation, and basal ganglia lesions.  In contrast, they have cranial nerve disturbance, pyramidal signs, and cerebellar dysfunction. 

The diagnostic criteria by Rahman et al 1996 for LS are as follows: 

1. Progressive neurologic disease with motor and intellectual developmental delay

2.  Signs and symptoms of brain stem and/or basal ganglia dysfunction

3. Elevated lactate levels in the blood and/ or CSF; and 

4. One or more of the following: 

a.  Characteristic features of LS on neuroimaging (symmetric hyperintense lesions in basal ganglia and/ or brainstem in T2 sequence), 

b.  Typical neuropathological changes at postmortem examination, and 

c.  Typical neuropathology in a similarly affected sibling. 

The criteria proposed by Sakushima et al., 2011 are: 

1. History of cryptogenic thrive failure or signs of mental retardation, pyramidal signs, cerebellar disturbances, ophthalmoplegia, deafness, dysarthria, or other neurological symptoms are present; and 

2.  Bilateral basal ganglia lesions or brainstem lesions with serum or CSF lactate elevation are present (lactate stress test (LST) should be considered when resting lactate levels are normal);

3. Mitochondrial abnormalities are present in muscle pathology or in biochemical analyses, or known LD gene mutations are present; and 

4. Metabolic disorders, toxins, infection, multiple sclerosis, and Wernicke’s encephalopathy can be excluded. 

Our patient fulfilled the later criteria as she did not have a history of failure to thrive or motor/ intellectual delay as required in Rahman et al., criteria. Rather she had recurrent vomiting and brainstem signs.

Most of the reported cases excluded infections, autoimmune diseases, and toxins as in our case.  Some cases were first misdiagnosed as multiple sclerosis and few are indistinguishable from Wernicke’s encephalopathy as occurred in our patient during the first admission.  Our patient satisfied the clinical, radiological, and biochemical criteria for LS.  Muscle histochemistry reinforced the diagnosis.  Also, striking imaging findings described previously with LS were found in our patient as well, moreover, excellent response with complete resolution of both MRI and clinical signs and symptoms to mitochondrial cocktail confirmed our patient as a case of LS.  The excellent response to treatment in our patient is similar to the case reported by Goldenberg et al., where there was a partial deficiency of COX.  Recent literature also supported the frequent occurrence of sleep apnea syndrome and abdominal symptoms in patients with mitochondrial diseases as occurred in our patient.  The major mutations known to occur in LD patients are T8993C, T8993G, T10191C, G13513A, A8344G, and A3243G in mitochondrial genes, and SURF1 in the nuclear genome.  Currently, there are 24 known mutations in mitochondrial genes and 21 in nuclear genes. The underlying genetic etiology could not be ascertained in our patient. Recent studies have shown that recognized mtDNA mutations only account for a small proportion of cases of mitochondrial disease.  In addition, nuclear DNA mutations account for a substantial number of mitochondrial disorders which could not be done in our patient due to unavailability.  Moreover, mitochondrial respiratory chain analysis in muscle or fibroblasts could not be done in our patient.  Adult-onset LD is extremely rare and requires a high index of suspicion.

The following characteristic features of adult LD for early diagnosis:

• History of cryptogenic thrive failure or signs of mental retardation, pyramidal signs, cerebellar disturbances, ophthalmoplegia, deafness, dysarthria, or other neurological symptoms are present. 

• LS is suspected through the hallmarks of the disease along with findings suggestive of brainstem dysfunction in addition to T2 weighted brain MRI lesions and accessory laboratory findings.

  • Bilateral basal ganglia lesions or brainstem lesions.

  • Brain MRI findings typically show bilateral symmetrical supra-tentorial (basal ganglia, thalamus, and sub-thalamus) and/or infra-tentorial (brainstem and dentate nuclei) lesions.

  • Serum or CSF lactate elevation are present. (LS should be considered when resting lactate levels are normal) 

• Mitochondrial abnormalities are present in muscle pathology or in biochemical analyses, or known LS gene mutations are present. 

• Metabolic disorders, toxins, infection, multiple sclerosis, and Wernicke encephalopathy can be excluded

• A study by Ardissone et al. presented a predominating basal ganglia involvement of 90.2%. They also showed that both supra and infra-tentorial involvement is dominant in cases of both mtDNA (74%) and -nDNA (67%) variants, while isolated infra-tentorial variants are rare. Extensive research is being conducted to find genetic correlations with MRI findings of LS. For example, a retrospective cohort found significant associations between the SURF1 variant and inferior olivary nuclei lesions.

• Abnormal laboratory findings may yield elevated blood, urine, and CSF lactate levels. 

• Additional deficiencies may be observed in respiratory chain complexes through enzyme assays and pyruvate dehydrogenase complex. However, these laboratory findings are not consistently present. Therefore, confirmatory tests with genetic assays are required for a definitive diagnosis and the identification of specific variants of LS 

Case Report 2: 

A 26-year-old woman presented with acute encephalopathy after spastic paraplegia.  On her first admission, she exhibited bilateral basal ganglia lesions on magnetic resonance images and normal serum lactate levels. On the second admission, she had acute encephalopathy with lactic acidosis and bilateral basal ganglia and brainstem lesions.  A muscle biopsy revealed cytochrome c oxidase deficiency and a diagnosis of adult LD was made.  Despite treatment in the intensive care unit, she died 9 days after admission.  

Conclusions:  A review of the literature describing adult LD revealed that developmental delay, COX deficiency, serum lactate elevation, and basal ganglia lesions occurred less frequently than they did in children with LD.  Cranial nerve disturbance, pyramidal signs, and cerebellar dysfunction were the primary symptoms in adult LD.  Thus, the many differences between childhood and adult LD may help diagnose adult LD

Work-up:  MRI of brain w/wo contrast. MRS - raised lactate peak.  Muscle bx: COX negative fibers, SDH, no RRF.  Genetic testing: Invitae (98002): SURF1, SCO1, SCO2, COX10.  Serum thiamine, lactate, pyruvate, LST, LFTs, CMP, CBC with diff, CSF lactate, Sr. Cu, ceruloplasmin, urinne copper, AQP4.  Muscle bx with DNA extracted for mitochondrial genome. 

Lactate Stress Test (LST)

https://exrx.net/Calculators/CycleMETs

After a rest of 10 minutes immediately before the test, lactate was determined for the first time (R1). 

Afterward, subjects were told to cycle continuously at 30W on a paddle-rate independent electronic bicycle ergometer (E980, Tunturi, Piispanristi, Finland) for 15 minutes. 

During this exercise, lactate was determined 5, 10, and 15 minutes after starting cycling. 

Finally, lactate was determined 15 minutes after finishing cycling (R2).

Lactate was measured using the commercially available Ektachrome Clinical Chemistry Slide (LAC, Kodak, Rochester-N.Y., USA).

The LST was interpreted as abnormal if more than two of the five individual lactate values exceeded the corresponding cut-off values. 

Complex I Deficiency | UMDF 

Summary

Mitochondrial myopathies are progressive myopathies caused by the impairment of oxidative phosphorylation (OXPHOS). Alongside the traditional histochemical, immunohistochemical, and biochemical assays, the diagnosis of mitochondrial myopathies has been revolutionized by the introduction of NGS. NGS allows for a high-throughput screening of mtDNA and nDNA. However, there are currently no effective or disease-modifying treatments available for mitochondrial myopathies to halt the progression of the disease. Instead, existing therapeutic options have been focusing on the symptomatic management of disease manifestations. The development of large cohorts of patients with mitochondrial disease is enabling extensive studies to investigate the therapeutic effects of a variety of compounds shown to be of potential value in animal models. New molecular strategies, namely mtZFNs and mtTALENs, that cause beneficial heteroplasmic shifts in cell lines harboring varying pathogenic mtDNA mutations offer hope for the future. Moreover, recent developments in the reproductive options for patients with mitochondrial myopathies mean that for most families, the possibility of preventing transmission of the mutation to the next generation is now possible. 

MayBeMito | Identifying & Diagnosing Mitochondrial Disease 

UMDF | Mitochondrial Disease Education & Research 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506718/

Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society (nih.gov) 

Clinical approach to mitochondrial diseases:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10605124/

Mitochondrial cocktail:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067597/

Variants in Human ATP Synthase Mitochondrial Genes: Biochemical Dysfunctions, Associated Diseases, and Therapies - PMC (nih.gov) 

[mt] ATP synthase:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278611/

https://www.mitoaction.org/wp-content/uploads/2021/01/Mito-New-Patient-Kit-Updated-3_10_23.pdf

Structure of human ATP synthase:  https://www.sciencedirect.com/science/article/pii/S1097276523003246

[mt] ATP synthase disorders:  https://www.sciencedirect.com/science/article/pii/S0167488908002395

Key: https://pubmed.ncbi.nlm.nih.gov/11735378/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307938/

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum.  These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline.

A subgroup of maternally inherited clinical syndromes has been termed by the ‘striatal necrosis syndromes’, and interestingly, they are all caused by mutations in a single gene, subunit 6 of complex V, or ATP synthase.  One disorder, neuropathy, ataxia, and retinitis pigmentosa(NARP), is characterized by retinitis pigmentosa, proximal neurogenic muscle weakness, seizures, dementia, ataxia, developmental delay, and sensory neuropathy.  NARP is most commonly associated with a T→G transversion at nt-8993 in the ATPase 6 gene, converting leucine at amino acid position 156 to arginine.  Patients are always heteroplasmic but typically harbor between 70% and 90% mutated mtDNAs in examined tissues.  One of the typical characteristics of mitochondrial disease is that there is a ‘threshold effect’. In other words, the phenotypic expression of a pathogenic mtDNA mutation will be evident only if the number of mutant mtDNAs exceeds a certain threshold. The threshold varies among tissues, depending upon their constitutive and acute requirements for oxidative energy.  The T8993G mutation that causes NARP provides an interesting illustration of this phenomenon: when the proportion of the mutation exceeds 90–95%, a clinically different and far more severe disorder, called maternally inherited Leigh syndrome, or MILS, usually (but not always) ensues.  Leigh syndrome is a severe and fatal encephalopathy of infancy that is also inherited as a recessive Mendelian trait, most frequently associated with deficiencies in nucleus-encoded subunits of complex I, complex IV, and pyruvate dehydrogenase.  The specific role of ATPase 6, and of the selective impairment of complex V, in NARP and MILS is unknown. An intriguing aspect of the NARP/MILS phenotype is that a different mutation also located at position 8993 —  T→C transition, converting Leu-156 to Pro — has also been found associated with NARP at low mutant loads, and with MILS at high mutant loads.  The NARP/MILS presentations have also been identified with two other mutations, both at another codon in the ATPase 6 gene, at nt-9176: a T→G transversion converts Leu-217 to Arg,14 whereas a T→C transition converts Leu-217 to Pro.   As with the nt-8993 mutations, low mutant loads (∼70%) cause the ‘milder’ syndrome [i.e. NARP and a related disorder called familial bilateral striated necrosis (FBSN)], whereas high mutant loads cause MILS.  In both sets of mutations, the T→G (i.e. Leu→Arg) mutation is clinically far more severe than the T→C (i.e. Leu→Pro) mutation. Finally, a single case of FBSN was reported in which the patient harbored a T→C transition at nt-8851, converting Trp-109 to Arg.19

MAT

https://www.science.org/doi/10.1126/scitranslmed.abo3724?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed

https://www.neurotherapeuticsjournal.org/article/S1878-7479(23)02027-5/pdf

https://onlinelibrary.wiley.com/doi/10.1111/joim.13054

Phenotypic manifestations of primary mitochondrial disorders:

Central and peripheral neurological symptoms:  Encephalopathy, epilepsy, ataxia, strokelike episodes, migraine, cortical blindness, dystonia, tremors, parkinsonism, developmental delay/regression, cognitive impairment, and peripheral neuropathy.

Visual and hearing symptoms: Visual loss, progressive external ophthalmoplegia, ptosis, optic atrophy, retinitis pigmentosa, sensorineural hearing loss.

Skeletal and cardiac symptoms: Myopathy, exercise intolerance, myoclonus, arrhythmia, cardiac myopathy, cardiac conduction defects.

Endocrine and reproductive symptoms: Diabetes mellitus, gestational diabetes, lactic acidosis, short stature, hypoparathyroidism hypothyroidism, adrenal insufficiency, hypogonadotrophic hypogonadism, infertility.

Gastrointestinal and renal symptoms: Gastroparesis, gastrointestinal dysmotility, dysphagia, cyclic vomiting, gastroesophageal sphincter dysfunction, pancreatitis, hepatopathy, nephropathy, Fanconi syndrome.

EARS2

Expanding the Clinical and Magnetic Resonance Spectrum of Leukoencephalopathy with Thalamus and Brainstem Involvement and High Lactate (LTBL) in a Patient Harboring a Novel EARS2 Mutation - PMC 

Early-Onset Mild Type Leukoencephalopathy Caused by a Homozygous EARS2 Mutation - PMC 

Diagnostic work-up

The diagnosis of mitochondrial myopathies involves a multidisciplinary approach.  History and physical examination are crucial for recognizing that mitochondrial myopathy is a potential diagnosis but also to suggest the most appropriate diagnostic studies.  The diagnostic investigations include histological and immunohistochemical studies, enzymatic analysis of the OXPHOS complexes, and the genetic analysis of the mtDNA.  Additionally, if a nuclear genetic diagnosis is suspected, a targeted nDNA sequencing approach may be used.  If no pathogenic mutation is identified, whole genome and whole exome screens are now commonly used to search for potential genetic diagnoses, with new disease genes constantly being identified. By integrating the information from these diagnostic tests, it allows for a diagnosis in the majority of patients. 

Labs: CK, lactic acid, pyruvate in serum.  CSF lactate, urine amino acids.  Bicycle ergometry, low levels of workload, forearm exercise test.

Lactate and pyruvate ratio

Lactate is a product of anaerobic glucose metabolism which accumulates when the metabolism is impaired, causing a shift in the oxidized-to-reduced NAD+/NADH ratio within the mitochondria—indicated by the blood lactate–pyruvate ratio. The determination of lactate concentrations at rest or following exercise has become a diagnostic tool for mitochondrial myopathy.  However, many patients do present with consistently normal lactate–pyruvate ratio especially in adults. 

The blood lactate to pyruvate (L:P) ratio is used to distinguish between pyruvate dehydrogenase deficiency and other causes of congenital lactic acidosis.  In conjunction with elevated lactate, an L:P ratio greater than 30 suggests inherited disorders of the respiratory chain complex or tricarboxylic acid cycle disorders.  In conjunction with an elevated lactate, an L:P ratio of less than 25 suggests a defect in pyruvate metabolism.  An artificially high L:P ratio can be observed in acutely ill individuals.  Abnormal concentrations of lactate, pyruvate, and the L:P ratio are not diagnostic for any single disorder and must be interpreted in the context of the individual's clinical presentation and other laboratory studies. 

Exercise Test.  The use of exercise testing, usually by bicycle or treadmill, is used for research and as a clinical diagnostic test for mitochondrial myopathy. Exercise intolerance during clinical observation can be demonstrated through taking venous blood sampling both during and after exercise. The test highlights a potential increase in concentration of muscle metabolites (lactate and pyruvate) in venous blood supply (measured as systemic arteriovenous oxygen (a-vO2)) during post-exercise recovery and a slow clearance of the accumulated plasma lactate.  The aerobic forearm test is also used as a screening tool for mitochondrial myopathy, coupled with the venous oxygen saturation measurements.  In a patient with mitochondrial myopathy, the combined test will reveal a decrease in oxygen desaturation in the venous blood as mitochondrial dysfunction in skeletal muscle results in its inability to extract oxygen from blood.

• Forearm exercise test can be performed instead of bicycle ergometry testing.  The patient is instructed to open and close their hand (about once every 2 seconds at 40% of maximal voluntary contraction for 3 minutes).  Use dynamometer to assess maximal and 40% maximal contraction prior to the exercise.  A butterfly needle is place in the antecubital fossa and venous oxygen and lactate can be measured at baseline and each minute during and immediately following exercise.  Patients with mitochondrial myopathies and exercise intolerance often demonstrate excessive and prolonged lactate production and paradoxically increased venous oxygen saturation.  The range of elevated venous PO2 during forearm exercise in mitochondrial myopathy patients (32-82 mm Hg) correlates closely with the severity of oxidative impairment as assessed during cycle exercise.  The forearm exercise test is an easy screening test that sensitively detects impaired oxygen use and accurately assesses the severity of oxidative-impairment in patients with mitochondrial myopathy and exercise intolerance.  

Fibroblast Growth Factor 21:  Fibroblast growth factor 21 (FGF-21), which has a regulatory role in lipid metabolism, can also be used as a blood serum biomarker for mitochondrial diseases.  FGF-21 levels are seen to be raised in patients who have muscle involvement, and thus, in cases with suspected mitochondrial myopathies, the noninvasive measurement of the growth factor acts as a helpful and sensitive first-line investigation in the diagnostic process. Furthermore, FGF-21 specificity for mitochondrial disease has been determined to be above 90% 

Growth Differentiation Factor 15:   GDF-15, which is a member of the transforming growth factor β superfamily, is shown to be significantly higher in the serum of patients with mitochondrial disease . The serum marker has been seen to be associated with disease severitty. Serum biomarkers of growth differentiation factor 15 (GDF-15) and fibroblast growth factor 21 (FGF-21) were proposed to aid in decision making as they increase in metabolic diseases with oxidative stress and inflammation; however, they cannot be used as a diagnostic tool for mitochondrial disorders as they increase in a variety of other non-mitochondrial diseases. Additionally, GDF-15 was noted to be the most useful first-line test for mitochondrial respiratory chain deficiency, with a superior diagnostic sensitivity and odds ratio compared to FGF-21

EDx: NARP is axonal neuropathy.  MNGIE is demyelinating in nature.  EMG

ECG, EEG, MRI of brain, MRS of brain.  MRI of skeletal muscle.  MRS of skeletal muscle with P and H compounds permits the analysis of ATP, creatine phosphate, inorganic phosphate, and pH in muscle and brain.  In mitochondrial disorders there is a rapid fall in levels of creatine phosphate and an abnormal accumulation of inorganic phosphates in tissues with exercise.   In addition, there is delay in recovery of phosphocreatine levels to normal after exercise.  

Muscle Biopsy:  Skeletal muscle is commonly affected in mitochondrial disease and is the most frequently biopsied tissue, although the increasing use of genetic tests is likely to reduce the need for muscle biopsies in the future.  Patients with mitochondrial myopathy may show histochemical alterations in their skeletal muscle which indicate mitochondrial dysfunction, although in some patients, the muscle biopsy can appear normal.  These mitochondrial abnormalities can be identified using several routine histological and immunological studies.  One example detected in some patients is the staining of skeletal muscle cryosections with the modified Gomori Trichrome, highlighting the presence of ragged-red fibers (RRF).  These fibers can also be detected using succinate dehydrogenase (SDH, complex II) histochemistry, which detects the mitochondrial aggregates in the subsarcolemmal region of the fiber due to mitochondrial proliferation that occurs as a result of mitochondrial OXPHOS dysfunction.   Another important diagnostic feature of mitochondrial myopathy is the presence of cytochrome c oxidase (COX, complex IV)-negative fibers as detected by the sequential COX/SDH histochemistry.  A mosaic pattern is commonly observed with COX-negative fibers appearing blue, among the normal COX-positive fibers which appear brown.  Furthermore, COX deficiency is segmental along the length of the muscle fiber.  The mosaic pattern is due to different levels of mutational heteroplasmy with high mutation load leading to respiratory chain deficiency. Mutation load is known to increase in cells throughout life, a process termed clonal expansion, which means that healthy aged individuals accumulate a low frequency of COX-negative fibers.  However, a suggestive diagnosis of mitochondrial myopathy is only made when individuals harbor COX-negative fibers at a frequency of > 5%.  While COX/SDH fails to provide information on complex I deficiency, a more recently established quadruple immunofluorescent assay has been shown to effectively identify isolated complex I downregulation in skeletal muscle biopsies of patients for diagnostic purposes.  These patients had a confirmed genetic diagnosis in a nDNA-encoded subunits of complex I or assembly factors, or mutations in the mtDNA complex I subunits, with some patients having mitochondrial myopathy.  Other pathological features which may be seen in skeletal muscle are nonspecific.  These include neurogenic atrophy, internal nuclei, abnormal variation in fiber size, and accumulations of glycogen or lipid.

• RRF on modified Gomori Trichome Stain.  An increase number of lipid droplets is also present within the abnormal muscle fibers.

• Oxidative stains (NADH, SDH, and COX).

  • Ragged blue fibers on NADH and SDH

  • Mitochondrial myopathies which are not associated with mt-tRNA mutations have no RRF and normal SDH and NADH staining. 

  • COX stain   fibers that have subunits encoded by mtDNA.  It can also highlight the subsarcolemmal accumulations of mitochondria.  

  • COX staining in combination of SDH is valuable.  When SDH intensely stain muscle fibers which are not stained by COX, it implies abnormality in mtDNA.  This is because SDH component of complex II is entirely coded by nDNA, while 3 of 13 subunits of complex IV (COX) are encoded by mtDNA. 

  • EM shows increased number of normal-appearing mitochondria, enlarged mitochondria with abnormal cristae, and mitochondria with paracrystalline inclusions. 

  • Summary:

    • Muscle biopsy are used in some cases of unclear genetic testing or phenotype with the typical findings of ragged red fibers with trichome histological staining, which represent excess mitochondrial proliferation, and cytochrome c oxidase (COX) negative fibers in COX and succinate dehydrogenase stains.

Biochemical Studies:  Spectrophotometric evaluation of individual respiratory chain complex activities is an important approach to the biochemical investigation and diagnosis of mitochondrial myopathies.  It can be performed in either fresh or frozen muscle homogenate; however, the latter is more common in diagnostic centers due to cross-continental or national referral of patients.  Each complex can be analyzed in isolation following the oxidation/reduction of specific substrates or substrate analogs.  The spectrophotometric enzyme assays are as follows:  NADH:ubiquinone oxidoreductase for complex I, succinate:cytochrome c oxidase oxidoreductase for complex II, ubiquinol cytochrome c oxidoreductase for complex III, cytochrome c oxidase for complex IV. The measurement of complex V (oligomycin-sensitive ATP synthase) is more challenging and requires the use of fresh material and is often measured in cultured skin fibroblasts.  Bernier et al. recommended 20–30% of normal complex activities as a criterion for the diagnosis of mitochondrial myopathies.  However, a normal respiratory chain enzyme activity does not exclude the diagnosis of mitochondrial myopathies as a small percentage of respiratory chain deficiency cells may not be detectable by enzyme measurements on tissue homogenates.  Another biochemical study which proves to be a helpful step in the diagnosis of mitochondrial myopathy is the blue native acrylamide page (BN-PAGE) assay, which is used to assess the relative abundance of fully assembled respiratory chain enzyme complexes.  Similar to the spectrophotometric assay, deficiency of the complexes can be seen as an isolated single complex or a combined deficiency.

Molecular Genetics Studies:  While histochemical and biochemical studies pave the way for appropriate molecular genetic testing, the diagnostic procedure in mitochondrial diseases has shifted towards the “genetic first approach” because of the advances made with next-generation sequencing (NGS).  The application of NGS techniques including targeted multi-gene panels of candidate genes, unbiased exome sequencing and whole genome sequencing, has revolutionized the diagnostic approach, replacing the sequential method of sequencing candidate genes through Sanger sequencing as the first diagnostic approach. The high-throughput analysis of many genes has led to an increase in the pace of diagnosis and reducing costs.  Moreover, unlike Sanger sequencing, NGS can be used for determination of mtDNA heteroplasmy and deletions, although real-time PCR, pyrosequencing, and long-range PCR are still commonly used. The identification of a causative molecular defect facilitates a diagnosis of mitochondrial myopathy in the patient and family members, permitting disease management, genetic counselling, and a variety of reproductive options.

Molecular Studies to Identify Causative Genes Mutations causing mitochondrial myopathy can be found in either the mtDNA or nDNA.  For mitochondrial disease in general, pathogenic mutations have been reported in all 37 mtDNA genes and more than 254 nDNA-encoded genes.  A search of OMIM, ClinVar, and MitoMap yielded 31 mtDNA genes and 29 nDNA genes that are associated with mitochondrial myopathy at time of writing.  However, with new disease genes being identified at a rapid rate, this is likely to be an underestimate and the muscle is often involved in many of the more systemic mitochondrial diseases.

When clinical history shows evidence of a maternally inherited disorder, or a typical mitochondrial myopathy clinical phenotype, the entire mitochondrial genome is typically analyzed.  NGS allows for deep coverage across mtDNA and thus detection of low levels of heteroplasmy, point mutations, and breakpoints of single, large-scale mtDNA deletions.  Analysis can be undertaken from mtDNA extracted from blood, skin biopsy, or muscle.  However, possible mitotic segregation or indeed a loss of mtDNA mutation from mitotic tissues means that if an mtDNA mutation is strongly suspected, then analysis of muscle mtDNA is recommended.

When the compilation of various information from the clinical history and histochemical and biochemical tests indicate that the pathogenic mutation is located in the nDNA, whole exome sequencing (WES), whole genome sequencing (WGS) or a targeted multigene panel of candidate genes can be employed to identify the causative gene using DNA extracted from blood.  Due to the large number of nuclear-encoded mitochondrial genes the benefits of NGS are obvious.

In the case of novel mutations that have not been previously reported, functional tests must be undertaken to confirm the pathogenicity of the defect.  These typically include looking at pathogenicity predictions based on protein structure and “rescue” experiments where patient cells are transfected with a wild-type copy of the suspected gene and this is shown to rescue the disease phenotype.

Additional Molecular Studies of mtDNA.  For some patients with mitochondrial myopathy, it is helpful to determine mtDNA copy number in muscle tissue using real-time PCR.  If the mtDNA content is depleted, it indicates that the defect has occurred in a nuclear gene responsible for mtDNA replication and/or maintenance of the deoxynucleotide pools and thus may be helpful for targeted nuclear genetic testing if not already completed.  

Real-time PCR and long-range PCR may be used if a single, large-scale mtDNA deletion or multiple mtDNA deletions are suspected.  Single, large-scale mtDNA deletions are most reliably detected in muscle and through determining the size of deletion and heteroplasmy provides important information as regards to disease severity and progression.  In comparison, the presence of multiple mtDNA deletions indicates a genetic defect in a nuclear gene involved in mtDNA maintenance and provides guidance for nDNA sequencing.

My Lab work-up

I usually check CK, serum lactate, pyruvate, total and free plasma carnitine, acylcarnitine/free carnitine ratio, amino acids, urine organic acids.  GDF-15 (growth differentiating factor – 15 from Mayo).  EMG/NCS, ophthalmologic evaluation, GI (if MNGIE suspected), PFT, ECG, echocardiogram.  In my opinion EMG, genetics and muscle biopsy have been helpful. 

I explain to patients  that the investigation for mitochondrial disorder requires a structured work-up which will begin with a screening test.  These may include biochemical testing in blood, urine, and if needed spinal fluid.  Based on the results of the screening test, I will consider further testing which may include bicycle ergometry, PFT, CPET, genetic testing, histopathology (tissue biopsy) and neuroimaging.

The level of variant heteroplasmy may differ among tissue so that mtDNA variants may be detected in some tissues, but not others.  Therefore it is usually best to test and involved tissue, such as muscle or liver.  If clinically indicated for sequence analysis and deletion testing of the mitochondrial genome can be repeated on a muscle biopsy (approximately 50 mg).

MITOP - Overview: Mitochondrial Full Genome Analysis, Next-Generation Sequencing (NGS), Varies (mayocliniclabs.com) 

GeneDx: https://providers.genedx.com/tests/detail/combined-mito-genome-plus-mito-focused-nuclear-gene-panel-736

https://mnglabs.labcorp.com/tests/620029/mngenome-trio-sequencing

https://www.mayocliniclabs.com/test-catalog/Overview/617103

https://mnglabs.labcorp.com/tests/NGS301/comprehensive-cellular-energetics-defects-ngs-panel-and-copy-number-analysis-mtdna

Invitae Nuclear Mitochondrial Disorders Panel | Test catalog | Invitae 

Baylor Genetics:

Mitochondrial electron transport chain disorders may be caused by molecular defects in nuclear or mitochondrial genes. 

• Comprehensive sequence analysis of the entire mitochondrial genome (MitoNGS, test code 2055) or selected nuclear genes responsible for the biogenesis and function of mitochondria is available. 

• Sequence analysis by massively parallel sequencing technology of a group of nuclear genes responsible for mtDNA depletion and/or multiple deletions is also clinically available (MitomeNGS, test code 2130). 

• Also, comprehensive Mitome200plus NGS based testing (test code 2085) is available which includes >162 nuclear genes plus the comprehensive mitochondrial genome analysis. 

Diagnosis

Biochemical testing in blood, urine, and spinal fluid

Most diagnostic algorithms recommend the evaluation of selected mitochondrial biomarkers in blood, urine, and spinal fluid.  These typically include measurements of lactate and pyruvate in plasma and cerebrospinal fluid (CSF), plasma, urine, and CSF amino acids, plasma acylcarnitines, and urine organic acids.

Lactate elevation occurs because the flux through glycolysis overwhelms pyruvate utilization in the mitochondria. Errors in sample collection and handling often limit its usefulness.  Venous plasma lactate levels can be spuriously elevated if a tourniquet is applied during the collection and/or if a child is struggling during the sampling.  Markedly elevated plasma lactate (>3 mmol/l), in a properly collected sample, suggests the presence of mitochondrial dysfunction, which can be due either to primary mitochondrial disease or, secondarily, to organic acidemias, other inborn errors of metabolism, toxins, tissue ischemia, and certain other diseases.

Several studies have shown that in patients with primary mitochondrial disease, truly elevated lactate levels have sensitivity between 34 and 62% and specificity between 83 and  100%.   The blood lactate/pyruvate ratio is most reliable in differentiating electron transport chain (ETC) disease from disorders of pyruvate metabolism, but only when lactate levels are high. The sensitivity of this ratio is 31%, with a specificity of 100%.

Elevated CSF lactate can be a helpful marker of mitochondrial disease in patients with associated neurologic symptoms.  Collection artifacts are less of a problem, although a variety of brain disorders, status epilepticus in particular, can transiently increase CSF lactate.  Surprisingly, urine lactate correlates less well with the presence of mitochondrial disease.

Pyruvate elevation is a useful biomarker for defects in the enzymes closely related to pyruvate metabolism, specifically pyruvate dehydrogenase and pyruvate carboxylase.   Blood pyruvate levels are also plagued by errors in collection and handling; furthermore, pyruvate is a very unstable compound.  A single study has shown a sensitivity of 75% and specificity of 87.2% in patients with primary mitochondrial disease.

Quantitative amino acid analysis in blood or spinal fluid is commonly obtained when evaluating a patient with possible mitochondrial disease. Elevations in several amino acids occur due to the altered redox state created by respiratory chain dysfunction including alanine, glycine, proline, and threonine. The exact sensitivity and specificity of alanine or other amino acid elevations in patients with primary mitochondrial disease are not yet known. Elevations may be present in either blood or spinal fluid, and notable findings may only occur during times of clinical worsening. Urine amino acids are most commonly used to assess for mitochondrial disease–associated renal tubulopathy.

Carnitine serves as a mitochondrial shuttle for free fatty acids and a key acceptor of potentially toxic coenzyme A esters. It permits the restoration of intramitochondrial coenzyme A and the removal of esterified intermediates. Quantification of blood total and free carnitine levels, along with acylcarnitine profiling, permits the identification of primary or secondary fatty-acid oxidation defects, as well as some primary amino and organic acidemias. Although acylcarnitine testing is suggested in a variety of mitochondrial reviews, there is limited background literature to clearly support this recommendation. This testing is typically recommended because of the association of a potential secondary disturbance of fatty-acid oxidation in patients with mitochondrial disease and certain mitochondrial phenotypes overlapping other inborn errors of metabolism for which acylcarnitine analysis is diagnostic.

Urinary organic acids often show changes in mitochondrial disease patients. Elevations of malate and fumarate were noted to best correlate with mitochondrial disease in a retrospective analysis of samples from 67 mitochondrial disease patients compared with 21 patients with organic acidemias; other citric acid cycle intermediates and lactate correlated poorly.  Mild-to-moderate 3-methylglutaconic acid (3MG) elevation, dicarboxylic aciduria, 2-oxoadipic aciduria, 2-aminoadipic aciduria, and methylmalonic aciduria can all be seen in certain primary mitochondrial diseases. Although urine organic acid may detect 3MG elevations, specific quantification of 3MG in blood and urine is more reliable, especially when 3MG levels are not markedly elevated.

Elevated creatine phosphokinase and uric acid are common in acute rhabdomyolysis in patients with fatty-acid oxidation disorders, and the elevations are caused by nucleic acid and nucleotide catabolism.  Although not extensively studied in primary mitochondrial disorders, patients with primary mitochondrial diseases may have muscle disease (especially with cytochrome b disease and thymidine kinase 2 deficiency), and elevations can also occur with primary or secondary fatty-acid oxidation disorders. Hematologic abnormalities can be detected with a complete blood count. Aplastic, megaloblastic, and sideroblastic anemias, leukopenia, thrombocytopenia, and pancytopenia have been reported in some primary mitochondrial diseases. Multiple primary mitochondrial diseases are associated with liver pathology based on mtDNA depletion and/or general liver dysfunction, and transaminases and albumin levels may help in diagnosis. New biomarkers of mitochondrial disease such as FGF21 and reduced glutathione await validation.

Cerebral folate deficiency is seen in a wide variety of neurologic and metabolic disorders including mitochondrial disease and is diagnosed via measurements of 5-methyltetrahydrofolate in CSF. Cerebral folate deficiency was initially identified in mitochondrial disease in patients with Kearns–Sayre syndrome (KSS). More recent case series in patients with KSS have further confirmed this finding. Cerebral folate deficiency has been identified in patients with mtDNA deletions, POLG disease, and biochemically diagnosed complex I deficiency. A primary cerebral folate disorder also exists, often due to mutations in the folate receptor 1 (FOLR1) gene encoding folate receptor alpha.

Consensus recommendations for testing blood, urine, and spinal fluid

1. The initial evaluation in blood for mitochondrial disease should include complete blood count, creatine phosphokinase, transaminases, albumin, lactate and pyruvate, amino acids, and acylcarnitines, along with quantitative or qualitative urinary organic acids. Caution must be taken to ensure that specimens are collected appropriately, especially for lactate and pyruvate measurements.

2. Postprandial lactate levels are more sensitive than fasting specimens and are preferred when possible. Caution must be taken to not overinterpret small elevations in postprandial lactate.

3. The lactate/pyruvate ratio in blood or CSF is of value only when the lactate level is elevated.

4. Quantitative 3MG measurements in plasma and urine should be obtained when possible in addition to urine organic acids in patients being evaluated for mitochondrial disease.

5. Creatine phosphokinase and uric acid should be assessed in patients with muscle symptoms who are suspected of having mitochondrial diseases.

6. Urine amino acid analysis should be obtained in the evaluation of mitochondrial tubulopathy.

7. When CSF is obtained, it should be sent for lactate, pyruvate, amino acid, and 5-methyltetrahydrofolate measurements.

8. Further research is needed regarding other biomarkers such as FGF21, glutathione, and CSF neopterin.

DNA testing

Primary mitochondrial disorders are caused by mutations in the maternally inherited mtDNA or one of many nDNA genes.  mtDNA genome sequencing and heteroplasmy analysis can now effectively be performed in blood, although it may be necessary to test other tissues in affected organs. Newer testing methodology allows for more accurate detection of low heteroplasmy in blood down to 5–10% and 1–2%.  Overall, the advent of newer technologies that rely on massive parallel or next-generation sequencing (NGS) methodologies have emerged as the new gold standard methodology for mtDNA genome sequencing because they allow significantly improved reliability and sensitivity of mtDNA genome analyses for point mutations, low-level heteroplasmy, and deletions, thereby providing a single test to accurately diagnose mtDNA disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome in blood, urine, or tissue, depending on symptom presentation and sample availability. Identification of a causative mitochondrial disease mutation allows for families to end their diagnostic odyssey and receive appropriate genetic counseling, carrier testing, and selective prenatal diagnosis.

It may be necessary to preferentially test other tissues as part of the diagnostic evaluation of a patient for a suspected mitochondrial disorder. Urine is increasingly recognized as a useful specimen for mtDNA genome analysis, given the high content of mtDNA in renal epithelial cells. This finding particularly applies to MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome and its most common mutation m.3243 A>G in MTTL1.  Skeletal muscle or liver are preferred tissue sources for mtDNA genome sequencing when available, given their high mtDNA content, reliance on mitochondrial respiration, and the possibility that they may harbor a tissue-specific mtDNA mutation that is simply not present in the blood.

The mtDNA deletion and duplication syndromes manifest along a spectrum of three phenotypic presentations: KSS, chronic progressive external ophthalmoplegia, and Pearson syndrome. The most commonly used methods for the detection of mtDNA deletions previously included Southern blot and long-range (deletion-specific) polymerase chain reaction analysis. However, Southern blot analysis lacks sufficient sensitivity to detect low levels of heteroplasmic deletions. In contrast, array comparative genome hybridization detects deletions and also estimates the deletion breakpoints and deletion heteroplasmy.  All of these methodologies are being replaced by NGS of the entire mitochondrial genome, which provides sufficiently deep coverage uniformly across the mtDNA genome to sensitively detect and characterize either single or multiple deletions. Deletions and duplications may only be detected in muscle or liver in many patients.

The mtDNA depletion syndromes are a genetically and clinically heterogeneous group of disorders characterized by a significant reduction in mtDNA copy number in affected tissues. Abnormalities in mtDNA biogenesis or maintenance underlie the pathophysiology of this class of mitochondrial disorders. They typically result from nDNA mutations in genes that function in mitochondrial deoxynucleotide synthesis or in mtDNA replication. Less frequently, mtDNA depletion can be caused by germline deletions/duplications of mtDNA segments.  Diagnosis therefore requires quantification of mtDNA content, typically in affected tissue, with identification of a significant decrease below the mean of normal age, gender, and tissue-specific control when normalized to nDNA tissue content.  mtDNA content is not assessed by NGS of the mtDNA genome and must be assayed by a separate quantitative real-time polymerase chain reaction.

More than 1,400 nuclear genes are either directly or indirectly involved in mitochondrial function. In addition to single-gene testing, there are many diagnostic laboratories that offer next-generation sequencing-based panels of multiple genes. Some companies offer panels with a small number of targeted genes, varying from a few to a dozen or so per mitochondrial disease phenotype (e.g., for mitochondrial depletion syndrome). Larger panels of more than 100, 400, or 1,000 nuclear genes are also available. Whole-exome sequencing became clinically available in 2011, and it is an increasingly common diagnostic tool utilized in patients with suspected mitochondrial disease. Numerous research reports describe the detection of novel pathogenic mutations in nuclear mitochondrial genes by whole-exome sequencing, but no clear evidence-based practice recommendation has been established related to the use of single-gene sequencing, nuclear gene panels, or wholeexome sequencing for diagnostic purposes in mitochondrial disease patients in clinical practice.

Consensus recommendations for DNA testing

1. Massively parallel sequencing/NGS of the mtDNA genome is the preferred methodology when testing mtDNA and should be performed in cases of suspected mitochondrial disease instead of testing for a limited number of pathogenic point mutations.

2. Patients with a strong likelihood of mitochondrial disease because of a mtDNA mutation and negative testing in blood, should have mtDNA assessed in another tissue to avoid the possibility of missing tissue-specific mutations or low levels of heteroplasmy in blood; tissue-based testing also helps assess the risk of other organ involvement and heterogeneity in family members and to guide genetic counseling.

3. Heteroplasmy analysis in urine can selectively be more informative and accurate than testing in blood alone, especially in cases of MELAS due to the common m. 3243A>G mutation.

4. mtDNA deletion and duplication testing should be performed in cases of suspected mitochondrial disease via NGS of the mtDNA genome, especially in all patients undergoing a diagnostic tissue biopsy.

   1. If a single small deletion is identified using polymerase chain reaction-based analysis, then one should be cautious in associating these findings with a primary mitochondrial disorder.

   2. When multiple mtDNA deletions are noted, sequencing of nuclear genes involved in mtDNA biosynthesis is recommended.

5. When a tissue specimen is obtained for mitochondrial studies, mtDNA content (copy number) testing via real-time quantitative polymerase chain reaction should strongly be considered for mtDNA depletion analysis because mtDNA depletion may not be detected in blood. 

   1. mtDNA proliferation is a nonspecific compensatory finding that can be seen in primary mitochondrial disease, secondary mitochondrial dysfunction, myopathy, hypotonia, and as a by-product of regular, intense exercise.

6. When considering nuclear gene testing in patients with likely primary mitochondrial disease, NGS methodologies providing complete coverage of known mitochondrial disease genes is preferred. Single-gene testing should usually be avoided because mutations in different genes can produce the same phenotype. If no known mutation is identified via known NGS gene panels, then wholeexome sequencing should be considered.

Pathology and biochemical testing of tissue

Patholog: A tissue biopsy, typically muscle, has often been thought of as the gold standard for mitochondrial diagnosis, although the test is affected by concerns of limited sensitivity and specificity. Tissue is typically sent for a variety of histological, biochemical, and genetic studies. With newer molecular testing, there is less of a need to rely primarily on biochemical testing of tissue for diagnosis, although selectively testing tissue remains a very informative procedure, especially for a clinically heterogeneous condition such as mitochondrial disease. Tissue testing allows for detection of mtDNA mutations with tissue specificity or low-level heteroplasmy and quantification of mtDNA content (copy number), directs appropriate molecular studies ensuring that genes of highest interest are covered, and helps validate the pathogenicity of variants of unknown significance found in molecular tests. In patients with a myopathy, certain other neuromuscular diseases can be excluded by a muscle biopsy. There is debate about whether patients need an open biopsy to preserve histology and perform all the necessary testing. A few centers are experienced in performing needle biopsies for mitochondrial testing. Because of potential injury to the mitochondria and the risk of artifactual abnormalities, open mitochondrial tissue biopsies require a different technique than routine biopsies that a surgeon may perform; these considerations are reviewed :

Tissue collection and processing instructions for mitochondrial tissue biopsies

• The vastus lateralis muscle should typically be biopsied because most laboratories standardize their assays to results from this muscle tissue

• Avoid muscles that have experienced electromyogram manipulation or severe wasting

• Avoid infiltrating the muscle with lidocaine or use of isometric muscle clamps

• Never use electrocautery in the biopsy procedure until after the specimen has been removed

• Muscle should not be collected in fragments or from subfascial and myotendinous areas

• Similar cautions need to be followed when liver samples are obtained 

• Tissue obtained during a biopsy should be quickly snap-frozen in liquid nitrogen, and a piece should be fixed in glutaraldehyde for electron microscopy; another specimen is needed if it is being used for polarographic evaluation

• Proper storage of specimens along with close coordination between pathology and the practitioner collecting the sample are essential to avoid degradation of biochemical enzyme activity

Muscle histology routinely includes hematoxylin and eosin (H&E), Gomori trichrome (for ragged red fibers), SDH (for SDH-rich or ragged blue fibers), NADH-TR (NADH tetrazolium reductase), COX (for COX-negative fibers), and combined SDH/COX staining (especially good for COX intermediate fibers).  Other standard stains per the institution’s pathology department should be routinely utilized to explore other myopathies in the differential that can be diagnosed (i.e., glycogen, lipid staining). Electron microscopy (EM) examines the mitochondria for inclusions and ultrastructural abnormalities. Pediatric patients are less likely to have histopathological abnormalities, and irregularities may only be noted on muscle EM, although normal results can also be seen.  Hepatic dysfunction due to mitochondrial disease is mostly seen in pediatric patients. A liver biopsy can show selective histologic and ultrastructural features of mitochondrial hepatopathies, such as steatosis, cholestasis, disrupted architecture, and cytoplasmic crowding by atypical mitochondria with swollen cristae. Ultrastructural evaluation should be performed routinely in unexplained cholestasis, especially when accompanied by steatosis and hepatocyte hypereosinophilia.

Consensus recommendations for pathology testing

1. Muscle (and/or liver) biopsies should be performed in the routine analysis for mitochondrial disease when the diagnosis cannot be confirmed with DNA testing.

2. When performing a muscle biopsy, an open biopsy is preferred in the routine analysis for mitochondrial disease, except when the center performing the biopsy is experienced in obtaining an adequate quality and quantity of tissue via a percutaneous biopsy..

3. The vastus lateralis is the preferred site for a muscle biopsy in the evaluation of mitochondrial disease due to this site having been used by most laboratories to establish reference ranges.

4. COX, SDH, NADH-TR, and the combined SDH/COX stain along with EM should be obtained in the routine analysis of tissue for mitochondrial disease; EM is strongly recommended in pediatric patients receiving a tissue biopsy because histological findings are often limited.

5. Mitochondrial hepatopathy may have characteristic findings on liver biopsy histology.

6. When possible, extra tissue should be frozen to allow for additional testing:

Points to consider regarding mitochondrial biochemical testing in tissue

• Assays of spectrophotometric quantification of OXPHOS enzyme activities differ across various laboratories and make interlaboratory comparison of test results highly variable.

• The use of simultaneous control samples may help validate most ETC test results in muscle

• There is little to no margin between patient ranges and control ranges

• Enzyme activities around the lowest reference value cannot always be “absolutely” normal or abnormal

• Electron transport enzyme activity measures are secondarily affected by many factors, with physical inactivity being relevant to most mitochondrial patients

• ETC analysis may be completely normal in any tissue tested early in the disease process, especially in mitochondrial DNA depletion and deletion syndromes

• Interpretation of mitochondrial biochemical testing results is aided by utilizing established diagnostic criteria to avoid mitochondrial dysfunction being identified in a subjective fashion and inter-physician variability in diagnoses provided  

Consensus recommendations for biochemical testing in tissue

1. Biochemical testing in tissue does not always differentiate between primary mitochondrial disease and secondary mitochondrial dysfunction.

2. When obtaining a biopsy in the evaluation of mitochondrial disease, ETC enzymology (spectrophotometry) of complex I–IV activities in snap frozen tissue or freshly isolated mitochondria should be obtained. The affected tissue should be biopsied when possible. The analysis of isolated complex III should be performed when possible because analysis of complex II/III and I/III alone may not be sufficient.

3. ETC results should be interpreted with the use of internal controls (within an assay) and normalized to marker enzymes (such as citrate synthase and/or complex II) to increase the diagnostic reliability of the results.

4. Fresh tissue analysis can allow functional oxidative phosphorylation/ oxymetric measurements of oxygen consumption and adenosine triphosphate production of all five ETC complexes, and it can be sufficient to diagnose mitochondrial dysfunction. These tests are not available in all centers; therefore, they are not considered essential but should be considered in the diagnosis of mitochondrial disease.

5. In some centers, various techniques of evaluating isolated mitochondria, permeabilized myofibers, immunoblot assays, and radiolabeled assays may enhance detection of ETC abnormalities. However, as stand-alone tests they need validation.

6. When interpreting ETC results, one should use published diagnostic criteria. One should cautiously interpret the relevance of ETC enzyme activity above 20% of the control mean. Caution should also be used in providing a primary mitochondrial disease diagnosis based on biochemical abnormalities from tissue testing alone.

7. The findings of significantly reduced ETC components or reduced enzyme activity from isolated components can give supplementary information in evaluating a patient with possible mitochondrial disease.

8. Tissue analysis of ETC complex enzyme activities may be falsely normal depending on a variety of factors, including the timing of the assay and use of less affected tissue. Therefore, ETC findings should not be used as the sole criterion for excluding mitochondrial dysfunction.

9. Muscle CoQ10 levels are necessary to determine primary CoQ10 synthesis defects, especially when genetic studies are not diagnostic. Leukocyte CoQ10 levels are inadequate to determine primary CoQ10 synthesis disorders. Reduced CoQ10 levels in muscle can be seen in other conditions.

10. Fibroblast ETC assays can help identify mitochondrial dysfunction in some cases, although testing can lead to false-negative results.

11. Buccal swab analysis should not be a first line for mitochondrial testing; additional comparisons of buccal swab ETC results with muscle ETC activity and genetically confirmed patients are needed.

Neuroimaging

Neuroimaging in the form of computed tomography and magnetic resonance imaging of the brain has been used to assist in the diagnosis of mitochondrial disorders.  Some diagnostic criteria protocols include neuroimaging but some do not.  Depending on the type of mitochondrial disorder and type of central nervous system involvement, neuroimaging may or may not show structural alterations. Stroke-like lesions in a nonvascular distribution, diffuse white matter disease, and bilateral involvement of deep gray matter nuclei in the basal ganglia, mid-brain, or brainstem are all known classic findings in syndromic mitochondrial disease. These “classical” changes are selectively also observed in nonsyndromic mitochondrial diseases and other metabolic disorders as well. Thus, they are neither sensitive nor specific enough to allow for a primary mitochondrial disease diagnosis without the presence of other abnormalities. Certain mitochondrial disorders such as KSS and myoclonic epilepsy with ragged red fibers (MERRF) are known to also have other neuroimaging abnormalities such as nonspecific white matter lesions; these findings are not sensitive enough to be considered part of the syndrome’s diagnostic criteria. More florid white matter abnormalities are seen in mitochondrial neuro-gastrointestinal encephalopathy syndrome (MNGIE), Leigh syndrome, and mitochondrial disorders due to defects in the aminoacyl-tRNA synthetases. In addition to qualitative changes, there are quantitative changes that can be seen in specific acquisition sequences, proton magnetic resonance imaging (MRS), and diffusion tensor imaging. MRS provides a semiquantitative estimate of brain metabolites, including lactate, creatine, and N-acetyl aspartate in a single- or multi-voxel distribution. Diffusion tensor imaging detects and quantifies major white matter tracts. MRS and diffusion tensor imaging changes may be found in classic mitochondrial syndromes as well as nonsyndromic patients, but they are not specific to mitochondrial disorders and can be seen in a variety of other metabolic or other brain parenchymal disorders.

Consensus recommendations for neuroimaging

1. When the central nervous system is involved, brain magnetic resonance imaging should be performed in the evaluation of a patient suspected of having a mitochondrial disease. MRS findings of elevated lactate within brain parenchyma are useful as well. Neuroimaging cannot by itself be the absolute criterion for disease confirmation.

2. Neuroimaging can be useful in following the progression of mitochondrial neurologic disease.

3. Further research is needed regarding the role of MRS and diffusion tensor imaging in helping follow the course of mitochondrial disease.

Genettic First Approach (Dr. Salman Bhai).

A “genetics first” approach can reduce diagnostic delay and improve management, where the diagnostic pathway can be an invasive or noninvasive combination of targeted or comprehensive molecular testing. Prior to ordering these tests, clinicians must consider the ambiguities and nuances of various testing modalities during the work-up for mitochondrial diseases. Therefore, due to the diagnostic challenges associated with primary mitochondrial diseases, diagnosis should be made in the context of clinical and molecular data, potentially supplemented with histochemical and biochemical evidence. Confirmation of a diagnosis leads to improvements in the management of the disease, decreases unnecessary testing, informs reproductive planning, and improves research pipelines 

Dropped Head Presentation of Mitochondrial Myopathy Fazal Rahim, MD, DevanshiGupta, MD, Tulio E. Bertorini, MD, and MarkS. LeDoux, MD,PhD 

Management/Treatment

There are currently no effective or disease-modifying treatments available for the vast majority of patients with mitochondrial myopathies.  Existing therapeutic options focus on the symptomatic management of disease manifestations.  

Must Read:  https://www.nature.com/articles/s41392-024-01839-8

The management is tailored to the individual patient according to their specific needs and requires the input of many different healthcare professionals such as neurologists, endocrinologists, cardiologists, dieticians, speech and language therapists, and physiotherapists. 

Treatment guidelines are in place to help provide for the management of patients in a clinical setting (for further information, see the guidelines available at: http://www.newcastle-mitochondria.com/clinical-professional-home-page/clinical-publications/clinical-guidelines/).

An increasing number of clinical trials, usually designed to be double blinded and placebo controlled, have investigated the therapeutic effects of various vitamins, cofactors, and nutritional supplements, though often these trials have failed to show definitive beneficial primary and secondary outcomes (referred to in this review). Moreover, new molecular and cellular strategies are being proposed that act on a molecular or cellular level, for example restriction endonucleases technologies.

Mitochondrial cocktail:  Co-Q10, 200 mg p.o. twice daily, folic acid 1 mg p.o. daily, riboflavin 400 mg p.o. daily, thiamine to 50 mg p.o. daily, vitamin D 200 units p.o. daily.  Ubiquinone and Ni cotinamide riboside (mitochondrial myopathy) may also be added.

New molecular strategies, such as  mitochondrially targeted nuclease technology demonstrates programmable nuclease-based technology that clinical manipulaties mtDNA heteroplasmy may be on the horizon.  Recent developments in  mtZFNs  (mitochondrial zinc-finger protein nuclease) and mtTALENs (mitochondrial transcription-activator-like effector nuclease) technologies that can potentially cause beneficial heteroplasmic shifts in cell lines harboring varying pathogenic mtDNA mutations offer hope for the future.  These technologies act on a molecular level to manipulate the mitochondrial genome and viewed as therapeutic options for mitochondrial diseases. This manipulation of mtDNA is achieved with the use of restriction endonucleases, engineered specifically for the mitochondria. The approaches are designed to recognize and bind to specific mtDNA sequences, induce a double-strand break, and initiate targeted degradation of the mutant mtDNA in a heteroplasmic population.  One such approach involves the use of mitochondrially targeted zinc finger nucleases (mtZFN), a heterodimer nuclease consisting of a DNA binding site (tandem repeat of zinc fingers binding three bases each), and a DNA cleavage domain (a Fok1 endonuclease).  A further development to mtZFN is mitochondrially targeted transcription activator like effectors (TALE) fused with a Fok1 nuclease (together abbreviated to mtTALENs).  Studies have shown the ability of both mtZFNs and mtTALENs to selectively eliminate mutant mtDNA in cell lines harboring a number of pathogenic mutations, including the mtDNA point mutations m.8993T > G (associated with neuropathy, ataxia, and retinitis pigmentosa (NARP)), m.8344A > G (associated with myoclonic epilepsy with ragged-red fibers (MERRF)), m.13513A > G MT-ND5 mutation (associated with MELAS and Leigh syndrome (LS)) and also the “common deletion” m.8483-1345del4977 (associated with CPEO and Kearns–Sayre syndrome (KSS)).  These studies showed that through the targeted elimination of mutated mtDNA, a beneficial shift in heteroplasmy and improvement in the biochemical deficit can be achieved.  However, despite the encouraging findings, these techniques are limited by their lack of efficacy in the delivery mechanism to affected tissues.  Once this issue has been resolved, gene-editing techniques could offer a potential treatment option for patients with primary mutations in the mitochondrial genome. 

Ubiquinone and Ubiquinone Analog CoQ10, an electron transport chain component involved in shuttling electrons from complex I and complex II to complex III via the quinone pool, has been shown to be an effective treatment option in patients who harbor a rare congenital CoQ10 deficiency.  The compound has been evaluated in a phase 2 clinical trial which included adult patients with either myoclonic epilepsy, lactic acidosis, and stroke-like episodes (MELAS), Leber’s hereditary optic neuropathy (LHON), or CPEO.  The study reported minor improvements in the aerobic capacity and post-exercise lactate levels, but there were no improvement in clinical measures such as strength or resting lactate.  A phase 3 trial has also been completed which aimed to show that oral CoQ10 was a safe effective treatment for children with inherited mitochondrial disease caused by defects in specific respiratory chain complexes or mtDNA mutations (Clinical Trial identifier: NCT00432744).  No results have been reported as of yet.

Idebenone, a synthetic quinone analog of CoQ10, may have the potential to restore cellular ATP generation.  It also acts as an antioxidant by protecting the lipid membrane and mitochondria from oxidative damage.  Idebenone was used in a randomized controlled trial (Clinical Trial identifier: NCT00747487) which showed an improvement in secondary outcome measures in LHON patients with discordant visual acuities.  Following this, LHON became the first mitochondrial disease for which a treatment has been approved by the European Medicine Agency.  Idebenone was also investigated in a phase 2 clinical trial (Clinical Trial identifier: NCT00887562) at two different doses of either 900 mg or 2400 mg in MELAS patients.  On the completion of the trial, the results reached no statistical significance. 

Nicotinamide riboside (NR), a form of vitamin B3 and a natural precursor of NAD+, has been shown to be a promising treatment strategy for mitochondrial myopathy.  NR increases the levels of NAD+ which has been shown to induce mitochondrial biogenesis, thus increasing oxidative ATP production capacity.  A study by Khan et al. , investigated the effect of NR using Deletor mice which have a mutation in the mtDNA Twinkle (TWNK) helicase.  Multiple mtDNA deletions accumulate in the skeletal muscle from these mice causing COX-negative fibers and eventually myopathy after the age of 12 months.  The study administered NR at a dose of 400 mg/kg/day to all control, presymptomatic, and postsymptomatic mice for a period of 4 months, a regime which had already proven to increase levels of NAD+ in skeletal muscle of wild-type mice.  The findings showed that NR prevented both the development and progression of mitochondrial myopathy.  The treatment also resulted in a significant induction of mitochondrial biogenesis and oxidative metabolism.  The increased biogenesis resulted in a delayed development of morphological hallmarks, ultrastructural abnormalities, and the prevention of mtDNA deletion accumulation in skeletal muscle.  A more recent clinical trial is underway investigating NR supplementation in patients with mitochondrial disease to induce mitochondrial biogenesis (Clinical Trial identifier: NCT03432871) but results are yet to be confirmed at the completion of the trial.  Further research is also underway to improve the uptake of NR into patient blood stream by providing patients with a potential modified release oral supplement or intravenous version of NR. 

A NAD+ precursor compound named acipimox has also been tested in an interventional clinical trial study which recruited patients with type 2 diabetes mellitus (Clinical Trial identifier: NCT00943059).  Findings from the trial showed that a 2-week treatment with 250 mg of acipimox three times daily resulted in an increase in skeletal muscle mitochondrial oxidative capacity and an improvement in the ATP production.  Though the researchers believe further insight need to be given into the safety and efficacy of the compound as a potential treatment option.  An upcoming trial for acipimox is soon to start in Newcastle, UK. 

Bezafibrate is a pan-PPAR agonist that stimulates PGC1α, a coactivator responsible for the induction of mitochondrial biogenesis through its interaction with a number of transcription factors.  Preclinical studies undertaken on various mouse models have reported varied findings as to whether bezafibrate could be a potential therapeutic strategy for mitochondrial myopathies.  One study on Deletor mice showed that bezafibrate treatment, starting at the time of disease manifestation (12 months of age), resulted in a milder progression of myopathy and reduced COX-negative fibers and mtDNA deletions in skeletal muscle.  In contrast, studies using the Surf1-KO mice (a model of early-onset partial COX-deficiency) and the COX15−/− mice showed no change in COX-activity, percentage COX-deficient fibers, and, importantly, no induction of mitochondrial biogenesis.  Rather, the studies showed a suppression with no increase in the PPARs or PGC1α expression following treatment.  However, both studies did highlight the adverse effects of the drug on mice, mainly severe lipid metabolism side effects and hepatomegaly.  Despite these findings, two clinical trials are currently ongoing for bezafibrate treatment.  The first is a randomized placebo-controlled trial aiming to assess the safety and efficacy of bezafibrate in mitochondrial myopathy patients (EudraCT number: 2012-002692-34). The trial is administrating bezafibrate orally at a dose of 200 mg in patients aged 2 to 50 years.  The second trial, which is a phase 2, open labelled feasibility study is aiming to provide proof of the effects of bezafibrate on six patients with the m.3243A > G mutation (Clinical Trial number: NCT02398201).  The results of these clinical trials are not yet published. 

RTA-408 is a synthetic triterpenoid compound and a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), working to increase the cellular antioxidant response.  Nrf2 is a transcriptional target of PGC1α and Nrf2 which promotes mitochondrial biogenesis.  Therefore, RTA-408 has the potential to improve muscle function, oxidative phosphorylation, antioxidant capacity, and mitochondrial efficiency in patients with mitochondrial myopathy.  The drug form of RTA-408 is called omaveloxolone which has been investigated in a recently completed phase 2, clinical trial (in a total of 53 patients with mitochondrial myopathy (Clinical Trial number: NCT02255422—known as the MOTOR trial); however, the results are not yet published. 

Ketogenic diets, which consist of a low carbohydrate and high lipid content which helps the lipid utilization by the mitochondria, have been proposed as a possible treatment for mitochondrial myopathies.  The diet was found to simulate mitochondrial oxidative metabolism in Deletor mice, reducing the amount of COX-negative fibers, preventing mitochondrial ultrastructural abnormalities in the skeletal muscle and inducing mitochondrial biogenesis.  Another preclinical trial found that a ketogenic culture medium killed cybrid cell lines carrying mtDNA mutation derived from a heteroplasmic patient with a single, large-scale mtDNA deletion.  A reduction in the mtDNA deletion load was detected in the heteroplasmic cells line indicative of a heteroplasmic shift.  Furthermore, Ahola et al.  tested a ketogenic diet called the modified Atkins Diet (mAD) in five patients with mitochondrial myopathy with either single or multiple mtDNA deletions.  Results of the pilot study showed no induction of mitochondrial biogenesis.  A 2-year follow-up of the patients revealed an improvement in muscle strength, showing a potential activation of muscle regeneration.  Despite the positive outcomes, further work is warranted to determine whether ketogenic diets can have a therapeutic effect on patients with mitochondrial myopathy. 

Targeting the Regulation of Lipid Dynamics Elamipretide (previously Bendavia), is a member of the Szeto-schiller (SS) family.  The drug targets the mitochondrial intermembrane lipid cardiolipin, working to stabilize the lipid structure.  Promising findings from preclinical trials have shown that the compound led to an increase in the OXPHOS efficiency through a reduction of ROS generation and so ultimately an increase in ATP production.  The drug has been assessed in mitochondrial myopathy patients (Clinical Trial identifier: NCT02367014, known as MMPOWER) and recently published data from the trial showed that patients had improved exercise intolerance and walking distance after the administration of the drug at the highest dose.  These findings have led to an ongoing extension trial (Clinical Trial identifier: NCT02976038, MMPOWER2). 

Nutritional Supplementations.  Another small molecule being investigated for treatment of mitochondrial myopathy is KH176, a vitamin E derivative which acts a potent ROS scavenger.  An initial dose–dependent study conducted by Koene et al. showed that the molecule was tolerated at a dose of 800 mg in male participants, causing clinically relevant changes to cardiac electrophysiology tests.  A phase 2 clinical trial is currently being undertaken to investigate this further (Clinical trial identifier: NCT02909400). 

Nutritional Supplementations.  Another small molecule being investigated for treatment of mitochondrial myopathy is KH176, a vitamin E derivative which acts a potent ROS scavenger.  An initial dose–dependent study conducted by Koene et al. showed that the molecule was tolerated at a dose of 800 mg in male participants, causing clinically relevant changes to cardiac electrophysiology tests.  A phase 2 clinical trial is currently being undertaken to investigate this further (Clinical trial identifier: NCT02909400). 

Deoxypyramidine bypass therapy has been trialed in mitochondrial depletion syndromes, such as those caused by a mutation in thymidine kinase 2 (TK2). Garone et al. investigated the effect of dCMP and dTMP supplementation in a TK2 mouse model. This supplementation bypassed the TK2 defect, increasing dTTP levels, and ameliorated biochemical abnormalities in these mice.

Options for future treatment by genetic therapy using mitochondrial genome manipulation in somatic tissues or replacement in the germline are still in the phase of clinical trials or animal models. Restriction endonucleases, transcription activator-like effectors, transcription activator-like effector nucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats all follow the concept of manipulating mtDNA through locating or targeting the mutation and then proceeding to eliminate or cleave the mutation. Replacement therapies that aim to replace mutated mtDNA with wild-type mtDNAinclude pronuclear and oocyte spindle transfer. They have been restricted in many nations due to debates and uncertainties about their outcomes.  

Regular aerobic exercise is recommended and thought to reduce fatigue and improve the quantity of muscle mitochondria and quality of life.

A ketogenic diet, which is high in fat and moderate in protein as well as low in carbohydrates, is an option for epileptics;however,it is contraindicated in patients with mtDNA deletion-related myopathy, so proper consultation with a nutritionist is recommended. The patient should be counseled about avoiding toxic mitochondrial medications such as metformin, propofol, valproic acid, aminoglycosides, linezolid, and nucleoside analog treatments.To counteract the impairment of mitochondrial function in these patients, a common treatment strategy employing a “mitochondrial cocktail” of vitamins, supplements, and antioxidants is used. These include L-carnitine, coenzyme Q10, riboflavin, thiamine,vitamin C, and E. Other pharmaceutical options used are Idebenonefor OPA1,L-arginine and nicotinamide for MELAS,and active dT and dC substrates inTK2d

Abbreviations: CPEO: chronic progressive external ophthalmoplegia, 5MTHF: 5-Methyltetrahydrofolate, CSF: cerebrospinal fluid, KSS: Kearns–Sayre syndrome, mtDNA: mitochondrial Deoxyribonucleic acid, LHON: Leber’s hereditary optic neuropathy, MELAS: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, MRS: magnetic resonance spectroscopy, NAD: nicotinamide adenine dinucleotide, ANT1: adenine nucleotide translocator-1 gene (SLC25A4), TK2d: thymidine kinase 2 deficiency, BCVA: best corrected visual acuity, MIDD: maternally inherited diabetes and deafness, ATP: adenosine triphosphate, ROS: reactive oxygen species, PMD: primary mitochondria disorder, PMM: primary mitochondrial myopathies, NADH: nicotinamide adenine dinucleotide + hydrogen, MNGIE: mitochondrial neurogastrointestinal encephalopathy, HLA: human leukocyte antigens.

https://www.fulgentgenetics.com/mtdna

MITOP - Overview: Mitochondrial Full Genome Analysis, Next-Generation Sequencing (NGS), Varies (mayocliniclabs.com) 

GeneDx: https://providers.genedx.com/tests/detail/combined-mito-genome-plus-mito-focused-nuclear-gene-panel-736

https://mnglabs.labcorp.com/tests/620029/mngenome-trio-sequencing

https://www.mayocliniclabs.com/test-catalog/Overview/617103

https://mnglabs.labcorp.com/tests/NGS301/comprehensive-cellular-energetics-defects-ngs-panel-and-copy-number-analysis-mtdna

https://www.preventiongenetics.com/testInfo?val=PGmito-%252D-Mitochondrial-Genome-Sequencing

https://www.mayocliniclabs.com/test-catalog/Overview/62510#Clinical-and-Interpretive

Why Take a Test - Biostarks 

Assessment of flow-mediated dilation in humans: a methodological and physiological guideline - PMC 

The non-syndromic clinical spectrums of mtDNA 3243A>G mutation - PMC 

Mitochondrial DNA mutation “m.3243A>G”—Heterogeneous clinical picture for cardiologists (“m.3243A>G”: A phenotypic chameleon) - Niedermayr - 2018 - Congenital Heart Disease - Wiley Online Library 

Interpretation of mitochondrial tRNA variants | Genetics in Medicine 

https://www.curemito.org/

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