Mitochondrial Chronic Progressive External Ophthalmoplegia

Background: Chronic progressive external ophthalmoplegia (CPEO) is a rare disorder that can be at the forefront of several mitochondrial diseases. This review overviews mitochondrial CPEO encephalomyopathies to enhance accurate recognition and diagnosis for proper management. Methods: This study is conducted based on publications and guidelines obtained by selective review in PubMed. Randomized, double-blind, placebo-controlled trials, Cochrane reviews, and literature meta-analyses were particularly sought. Discussion: CPEO is a common presentation of mitochondrial encephalomyopathies, which can result from alterations in mitochondrial or nuclear DNA. Genetic sequencing is the gold standard for diagnosing mitochondrial encephalomyopathies, preceded by non-invasive tests such as fibroblast growth factor-21 and growth differentiation factor-15. More invasive options include a muscle biopsy, which can be carried out after uncertain diagnostic testing. No definitive treatment option is available for mitochondrial diseases, and management is mainly focused on lifestyle risk modification and supplementation to reduce mitochondrial load and symptomatic relief, such as ptosis repair in the case of CPEO. Nevertheless, various clinical trials and endeavors are still at large for achieving beneficial therapeutic outcomes for mitochondrial encephalomyopathies. Key Messages: Understanding the varying presentations and genetic aspects of mitochondrial CPEO is crucial for accurate diagnosis and management.


Introduction
Chronic progressive external ophthalmoplegia (CPEO) is the most common manifestation of mitochondrial diseases and is characterized by bilateral symmetrical progressive ptosis and reduced ocular motility.CPEO can be isolated or accompanied by a clinical feature of systemic involvement of mitochondrial dysfunction (CPEO plus syndrome).The worldwide prevalence of CPEO is unknown; however, the incidence of CPEO is 1-2 per 100,000.In the United Kingdom's cohort database, the estimated prevalence of CPEO recorded was 1 in 30,000 [1].
Von Graefe, in 1868, originally described CPEO, and later, in 1958, Kearns and Sayre first described their triad of CPEO, retinal degeneration, and heart block (Kearns-Sayre syndrome) [2].Microscopical findings of pathological mitochondria in 1967 and ragged red fibers in 1972 [3,4], as well as increased venous pyruvate and lactate in 1976 [5], were significant in understanding the disease's path.More recently, singular and multiple mitochondrial DNA (mtDNA) deletions were detected in 1988 and 1989 [6,7].Finally, in 2000, the first nuclear DNA (nDNA) mutation was detected alongside multiple mtDNA deletions [8].
Brain Sci.2024, 14, 135 2 of 26 amount of ATP due to defective oxidative phosphorylation.The extraocular muscles are particularly susceptible due to their high mitochondrial volume and lower mutational threshold [10].Their susceptibility is expressed in multiple mitochondrial disorders, highlighting the significance of examining other manifestations in patients with PEO.
skeletal muscles, and the eye [9] (Figure 1).In CPEO, the ocular findings of ptosis and ophthalmoplegia occur due to the inability of the abnormal mitochondria to supply an adequate amount of ATP due to defective oxidative phosphorylation.The extraocular muscles are particularly susceptible due to their high mitochondrial volume and lower mutational threshold [10].Their susceptibility is expressed in multiple mitochondrial disorders, highlighting the significance of examining other manifestations in patients with PEO.Both mtDNA and nDNA can be affected in mitochondrial diseases.mtDNA has a unique trait of heteroplasmy since numerous copies are distributed in varying numbers to each oocyte before fertilization.This ensures a fluctuation of mutant mtDNA load in the characteristic maternal inheritance of mtDNA.Point mutations occurring in an asymptomatic mother have a chance of either being diluted to her child or transmitted with a higher load [11,12].When mutated mtDNA loads exceed a certain threshold, symptoms occur, and, in this case, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) can be inherited.Large-scale deletions can occur during embryogenesis, giving rise to mtDNA deletion syndromes such as CPEO, Kearns-Sayre syndrome (KSS), Both mtDNA and nDNA can be affected in mitochondrial diseases.mtDNA has a unique trait of heteroplasmy since numerous copies are distributed in varying numbers to each oocyte before fertilization.This ensures a fluctuation of mutant mtDNA load in the characteristic maternal inheritance of mtDNA.Point mutations occurring in an asymptomatic mother have a chance of either being diluted to her child or transmitted with a higher load [11,12].When mutated mtDNA loads exceed a certain threshold, symptoms occur, and, in this case, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) can be inherited.Large-scale deletions can occur during embryogenesis, giving rise to mtDNA deletion syndromes such as CPEO, Kearns-Sayre syndrome (KSS), and Pearson syndrome (PS).In the case of Leigh syndrome (LS), both mtDNA and nDNA can be affected, giving rise to a similar clinical phenotype with different genetic variants.
Brain Sci.2024, 14, 135 3 of 26 Mutations of nDNA can interfere with mitochondrial maintenance by affecting mtDNA synthesis, the mitochondrial nucleotide pool, and mitochondrial fusion.Nuclear genes responsible for synthesis include POLG, TWNK, RNASEH1, MGME1, and DNA2.The nucleotide pool is maintained through nucleotide metabolism with TYMP and RRM2B, the salvage pathway that includes TK2 and DGUOK, and nucleotide import such as ANT1 and MPV17.Finally, the OPA1 gene aids in preventing the loss of mitochondrial components, contributing to mitochondrial fusion [13] (Figure 2).
Brain Sci.2024, 14, 135 3 of 27 and Pearson syndrome (PS).In the case of Leigh syndrome (LS), both mtDNA and nDNA can be affected, giving rise to a similar clinical phenotype with different genetic variants.Mutations of nDNA can interfere with mitochondrial maintenance by affecting mtDNA synthesis, the mitochondrial nucleotide pool, and mitochondrial fusion.Nuclear genes responsible for synthesis include POLG, TWNK, RNASEH1, MGME1, and DNA2.The nucleotide pool is maintained through nucleotide metabolism with TYMP and RRM2B, the salvage pathway that includes TK2 and DGUOK, and nucleotide import such as ANT1 and MPV17.Finally, the OPA1 gene aids in preventing the loss of mitochondrial components, contributing to mitochondrial fusion [13] (Figure 2).  1) is shown and is responsible for salvaging deoxyribonucleosides (dNs) and converting them into deoxyribonucleotide triphosphates (dNTPs) used in mtDNA replication.Along this pathway, Thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGK) convert dNs into deoxyribonucleotide monophosphates (dNMPs) that later convert into deoxyribonucleoside diphosphates(dNDPs) by nucleotide monophosphate kinase (NMPK), then into dNTPs by nucleotide diphosphate kinase (NDPK).The cytosolic nucleotide metabolism pathway (2) includes thymidine phosphorylase (TP), which converts thymidine into thymine, and ribonucleotide reductase (RNR), which converts NDPs into dNDPs, supplying the nucleotide salvage pathway.RNR consists of two catalytic and two R2 or p53-induced small subunits.The nucleotide transport proteins (3) supply the nucleotide salvage pathway from the cytosol.MPV17 protein supplies dNTPs, while adenine nucleotide transporter (ANT1) supplies ADPs with the assistance of acylglycerol kinase (AGK), which are later converted to deoxyadenosine diphosphate (dADPs) feeding into the dNDPs.Mitochondrial DNA synthesis (4) requires the enzymes TWINKLE, a helicase, and the synthesis initiator, DNA polymerase gamma (POLG), which needs an RNA primer that is supplied by mitochondrial transcription factor A (TFAM).POLG consists of one catalytic subunit and two subunits encoded from POLG2.(5) The removal of RNA primers and flap intermediates is then achieved via ribonuclease H1 (RNase H1), DNA helicase/ nuclease 2 (DNA2), and mitochondrial genome maintenance exonuclease 1 (MGME1).( 6) Mitochondrial fusion is mediated by the proteins optic atrophy 1 (OPA1), F-box and leucine-rich repeat 4 (FBXL4), mitofusin 1 and 2 (MFN 1 and 2).  1) is shown and is responsible for salvaging deoxyribonucleosides (dNs) and converting them into deoxyribonucleotide triphosphates (dNTPs) used in mtDNA replication.Along this pathway, Thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGK) convert dNs into deoxyribonucleotide monophosphates (dNMPs) that later convert into deoxyribonucleoside diphosphates(dNDPs) by nucleotide monophosphate kinase (NMPK), then into dNTPs by nucleotide diphosphate kinase (NDPK).The cytosolic nucleotide metabolism pathway (2) includes thymidine phosphorylase (TP), which converts thymidine into thymine, and ribonucleotide reductase (RNR), which converts NDPs into dNDPs, supplying the nucleotide salvage pathway.RNR consists of two catalytic and two R2 or p53-induced small subunits.The nucleotide transport proteins (3) supply the nucleotide salvage pathway from the cytosol.MPV17 protein supplies dNTPs, while adenine nucleotide transporter (ANT1) supplies ADPs with the assistance of acylglycerol kinase (AGK), which are later converted to deoxyadenosine diphosphate (dADPs) feeding into the dNDPs.Mitochondrial DNA synthesis (4) requires the enzymes TWINKLE, a helicase, and the synthesis initiator, DNA polymerase gamma (POLG), which needs an RNA primer that is supplied by mitochondrial transcription factor A (TFAM).POLG consists of one catalytic subunit and two subunits encoded from POLG2.(5) The removal of RNA primers and flap intermediates is then achieved via ribonuclease H1 (RNase H1), DNA helicase/nuclease 2 (DNA2), and mitochondrial genome maintenance exonuclease 1 (MGME1).( 6) Mitochondrial fusion is mediated by the proteins optic atrophy 1 (OPA1), F-box and leucine-rich repeat 4 (FBXL4), mitofusin 1 and 2 (MFN 1 and 2).
In this review, we will review clinical entities of mitochondrial encephalomypoathies based on the corresponding genetics of each syndrome rather than symptomology because of the wide overlap of presentations and varying modes of inheritance accompanying each syndrome (Table A1).

CPEO
In its isolated form, CPEO is typically a sporadic disorder characterized by progressive bilateral ptosis and ophthalmoparesis [9] (Figure 3).Ptosis examination yields poor levator palpebrae superioris (LPS) muscle function, where eyelid excursion is often less than 8-10 mm rather than the normal ≥12 mm.Slowed, incomplete, and omnidirectional saccades can be a subtle early clinical sign that is frequently missed.Later on, as the disease progresses, ophthalmoplegia becomes more evident.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 [14,15].Retinal examination could reveal pigmentary retinopathy that is typical in Kearns-Sayre syndrome, characterized as salt and pepper retinopathy, where clumps of retinal pigment epithelium (RPE) alternate with areas devoid of RPE [16].However, these retinal changes rarely harbor field defects or a change in visual acuity.In this review, we will review clinical entities of mitochondrial encephalomypoathies based on the corresponding genetics of each syndrome rather than symptomology because of the wide overlap of presentations and varying modes of inheritance accompanying each syndrome (Table A1).

CPEO
In its isolated form, CPEO is typically a sporadic disorder characterized by progressive bilateral ptosis and ophthalmoparesis [9] (Figure 3).Ptosis examination yields poor levator palpebrae superioris (LPS) muscle function, where eyelid excursion is often less than 8-10 mm rather than the normal ≥12 mm.Slowed, incomplete, and omnidirectional saccades can be a subtle early clinical sign that is frequently missed.Later on, as the disease progresses, ophthalmoplegia becomes more evident.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 [14,15].Retinal examination could reveal pigmentary retinopathy that is typical in Kearns-Sayre syndrome, characterized as salt and pepper retinopathy, where clumps of retinal pigment epithelium (RPE) alternate with areas devoid of RPE [16].However, these retinal changes rarely harbor field defects or a change in visual acuity.Treatment of CPEO is focused on the correction of ptosis.It can start with eyelid crutches as a non-surgical solution, which usually is not preferred due to discomfort or intolerable aesthetics [17].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 [18,19].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 [19].

Kearns-Sayre Syndrome
Kearns-Sayre syndrome is a syndrome of CPEO and pigmentary retinopathy, with onset before the age of 20 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" [20].
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 Treatment of CPEO is focused on the correction of ptosis.It can start with eyelid crutches as a non-surgical solution, which usually is not preferred due to discomfort or intolerable aesthetics [17].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 [18,19].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 [19].

Kearns-Sayre Syndrome
Kearns-Sayre syndrome is a syndrome of CPEO and pigmentary retinopathy, with onset before the age of 20 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" [20].
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 [21].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 [22,23].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 of a case report by Shaker et al. [24].
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.

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 [25].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 [26].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 [27].
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 [28].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 [29].
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 [26,30].

Leigh Syndrome
Leigh syndrome is a fatal, progressive neurodegenerative disease that typically manifests in infants and young children of 3 months to 2 years of age [31].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 [32,33].
The clinical features of LS vary, with the most common characteristics, according to a meta-analysis by Chang et al., being developmental delay, hypotonia, respiratory dysfunction, epilepsy, reduced feeding, and weakness [34].The ocular features of LS include nystagmus, ptosis, ophthalmoplegia, strabismus, pigmentary retinopathy, and optic atrophy [34,35].Common cardiac abnormalities are hypertrophic or dilated cardiomyopathy and conduction defects such as Wolff-Parkinson-White syndrome [36,37].
Consensus on the clinical diagnosis is yet to be determined; however, LS is suspected through the hallmarks of the disease along with findings suggestive of brainstem dysfunc-tion in addition to T2 weighted brain MRI lesions and accessory laboratory findings [34].Brain MRI findings typically show bilateral symmetrical supra-tentorial (basal ganglia, thalamus, and sub-thalamus) and/or infra-tentorial (brainstem and dentate nuclei) lesions.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 [38].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 [39].
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 [40].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 [41].
The clinical presentations vary widely, usually in childhood, with neurological symptoms that include SLEs, sensorineural hearing loss, and cognitive impairment associated with diffuse white matter injury.Less commonly, it can present with gastrointestinal manifestations that include gastric perforation, ischemic colitis, segmental ileal paralysis, pseudo-obstruction, or megacolon.Endocrine manifestations, such as diabetes mellitus, have also been reported in MELAS [44].
The transient SLEs of the disease are characterized by nausea, vomiting, a migrainelike headache, encephalopathy, and focal seizures with or without neurological deficits.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) [46].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 [47].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 [48].MRI findings of SLE 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 [49].Another distinctive finding in neuroimaging was reported in some cases of MELAS as cerebellar lesions SLLs [49,50].
Since MELAS is associated with reduced levels of citrulline and arginine, which are NO precursors, and decreased NO that contributes to SLEs, supplement replacement with arginine was proposed.A systematic review by Argudo et al. concluded that the studies conducted showed promising results in managing SLEs [51].Acute phase management consists of giving an intravenous dose of 500 mg/kg/day or 10 g/m 2 in 24 h for 3-5 days.Whereas chronically, 150-300 mg/kg/day (maximum of 500 mg) is used instead [52].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 [42].For treating epilepsy, levetiracetam is considered to be the first-line anticonvulsant in mitochondrial encephalomyopathy due to the mitochondrial toxicity of other anticonvulsant agents [53].

4.
Nuclear DNA Gene Mutations/Protein Dysfunction Causing CPEO 4.1.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 [54].
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 [55].
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 [55].

Twinkle mtDNA Helicase (TWNK)
The TWNK gene, also known as C10orf2 or PEO1, is responsible for encoding TWIN-KLE, an mtDNA helicase, an enzyme that unwinds DNA temporarily for replication [56].The dysfunction in TWINKLE is thought to pause or stall mtDNA replication and accumulate many mtDNA deletions over time [57].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 [58].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 [59,60].It is characterized by CPEO, cachexia, severe gastrointestinal dysmotility, sensorineural hearing loss, peripheral neuropathy, and leukoencephalopathy [59].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 [61].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 [62,63].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 [62].

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 [64].
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 [64].On the other hand, arPEO is a childhood-onset disease that is more severe with associated retinopathy, myopathy, and mood disorders [64,66].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 [67].Finally, MNGIE-like RRM2B is a rare phenotype that can occur with cachexia, gastrointestinal dysmotility, and peripheral neuropathy [68].

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 [69].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 [70][71][72].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 [73].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].
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 [76].
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 [77].
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 [78].

Deoxyguanosine Kinase
Deoxyguanosine kinase (DGK) phosphorylates purine deoxyribonucleosides and contributes to the deoxyribonucleoside salvage pathway in the mitochondrial matrix [79].Two forms of DGUOK gene deficiency have been described in the literature, with neonatal multisystem disorder being the most common [80].It is characterized by hepatic and neurological manifestations, including developmental delay, hypotonia, nystagmus, jaundice, cholestasis, and hepatomegaly [81].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 [82][83][84].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 [81,85].

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 [86,87].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 [88].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 [87].

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 singlestranded DNA (ssDNA) flaps and enabling the ligation of new DNA strands [89].MGME1 affects the turnover of 7S DNA and causes its accumulation when depleted or causes 7S DNA attrition when overexpressed [90].7S DNA is a ssDNA arising from the noncoding region and is postulated to contribute to the mtDNA displacement loop (D-loop) as an intermediate of premature termination of mtDNA replication [91].Patients with this mutation have shown an increase in 7S DNA and a multisystemic phenotype of PEO, muscle wasting, emaciation, and respiratory failure [92].A similar phenotype was reported recently with skeletal malformations, atrioventricular block, and cerebellar atrophy in magnetic resonance imaging [93] (Figure 4).loop) as an intermediate of premature termination of mtDNA replication [91].Patients with this mutation have shown an increase in 7S DNA and a multisystemic phenotype of PEO, muscle wasting, emaciation, and respiratory failure [92].A similar phenotype was reported recently with skeletal malformations, atrioventricular block, and cerebellar atrophy in magnetic resonance imaging [93] (Figure 4).

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 [94].The complete loss of this gene causes the characteristic clinical phenotype of cardiomyopathy and myopathy [95].On the other hand, the overexpression of this gene results in cardioprotective features [96].Other clinical features of this disorder include exercise intolerance, muscle weakness, ptosis, and lactic acidosis [97].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 [98].Recent studies suggest that reducing protein leak can be an effective treatment option for aged cardiomyocytes, which may assist in ANT1-related symptoms [99].Another showed improvement in exercise intolerance using nicotinamide riboside in ANT1-deficient mice [100].

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 [101,102].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 [103].Brain MRI findings may harbor abnormalities in lower brainstem reticular formation, reticulospinal tracts at the cervicocranial junction, and cerebral leukoencephalopathy [102,104].

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 [94].The complete loss of this gene causes the characteristic clinical phenotype of cardiomyopathy and myopathy [95].On the other hand, the overexpression of this gene results in cardioprotective features [96].Other clinical features of this disorder include exercise intolerance, muscle weakness, ptosis, and lactic acidosis [97].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 [98].Recent studies suggest that reducing protein leak can be an effective treatment option for aged cardiomyocytes, which may assist in ANT1-related symptoms [99].Another showed improvement in exercise intolerance using nicotinamide riboside in ANT1-deficient mice [100].

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 [101,102].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 [103].Brain MRI findings may harbor abnormalities in lower brainstem reticular formation, reticulospinal tracts at the cervicocranial junction, and cerebral leukoencephalopathy [102,104].

A Diagnostic Approach to Mitochondrial Encephalomyopathies
There is no consensus on a specific algorithm for diagnosing mitochondrial encaphalomyopathies, and a general approach of clinical suspicion was followed by clinical and biochemical findings, which were then forwarded to targeted or exploratory sequencing.Clinical suspicion requires knowledge of inheritance, varying phenotypical findings, and syndromes along the myopathic, encephalomyopathic, hepatocerebral, and neurogastrointestinal forms, among others.When clinical suspicion arises, an array of clinical and biochemical tests can be carried out to narrow down differential diagnoses and focus on targeted sequencing (Table 1).LGMD with ophthalmoplegia Abbreviations: OPMD: Oculopharyngeal muscular dystrophy, OPDM: Oculopharyngodistal myopathy, LGMD: Limb-girdle muscular dystrophy, WEBINO: Wall-eyed bilateral internuclear ophthalmoplegia, 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, HSP: Hereditary spastic paraplegia, SCA: Spinocerebellar ataxia, CFEOM: congenital fibrosis of the extraocular muscles.
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 [105].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 [106,107].Other biomarkers are plasma lactate for metabolic crisis, creatine kinase for myopathy, an endocrinological panel for diabetes, thyroid and parathyroid screening, and a urine dipstick with a renal function test.Neuroimaging, electroencephalography, and nerve conduction studies are used to screen and assess neuropathies.Cardiomyopathies are less frequent but require screening nonetheless with echocardiography, in addition to an electrocardiogram for arrhythmias.Muscle biopsies are less commonly performed now due to genetic testing being the reliable gold standard of diagnosis.They 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 [108].
Next-generation sequencing (NGS) has been a significant development in diagnosing mitochondrial diseases, being faster, more accurate, and cost-effective.Watson et al. proposed a genetics-first approach towards confirming diagnoses using it along with ancillary non-invasive testing before moving to more invasive tests in case of unclear diagnoses.Their approach depends on identifying and running existing phenotype-genotype correla-tions through a targeted sequencing panel, followed by exploratory whole-exome (WES) or whole-genome sequencing (WGS) if no correlation was noted.This method also identifies novel mutations and confirms their pathogenicity; if not, a review or more invasive tests such as a muscle biopsy can be performed [109] (Figure 5).
Next-generation sequencing (NGS) has been a significant development in diagnosing mitochondrial diseases, being faster, more accurate, and cost-effective.Watson et al. proposed a genetics-first approach towards confirming diagnoses using it along with ancillary non-invasive testing before moving to more invasive tests in case of unclear diagnoses.Their approach depends on identifying and running existing phenotype-genotype correlations through a targeted sequencing panel, followed by exploratory whole-exome (WES) or whole-genome sequencing (WGS) if no correlation was noted.This method also identifies novel mutations and confirms their pathogenicity; if not, a review or more invasive tests such as a muscle biopsy can be performed [109] (Figure 5).

Treatment of Mitochondrial Diseases
Patients with mitochondrial diseases are generally treated with supportive and symptomatic multi-disciplinary therapy.Regular aerobic exercise is recommended and thought to reduce fatigue and improve the quantity of muscle mitochondria and quality of life [110,111].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 [112].The patient should be counseled about avoiding toxic mitochondrial medications such as metformin, propofol, valproic acid, aminoglycosides, linezolid, and nucleoside analog treatments [113,114].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,

Treatment of Mitochondrial Diseases
Patients with mitochondrial diseases are generally treated with supportive and symptomatic multi-disciplinary therapy.Regular aerobic exercise is recommended and thought to reduce fatigue and improve the quantity of muscle mitochondria and quality of life [110,111].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 [112].The patient should be counseled about avoiding toxic mitochondrial medications such as metformin, propofol, valproic acid, aminoglycosides, linezolid, and nucleoside analog treatments [113,114].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. [115].Other pharmaceutical options used are Idebenone for OPA1, L-arginine and nicotinamide for MELAS, and active dT and dC substrates in TK2d (Table A2).
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 mtDNA include pronuclear and oocyte spindle transfer.They have been restricted in many nations due to debates and uncertainties about their outcomes [116].

Conclusions
In conclusion, understanding the pathophysiology of mitochondrial disorders has significantly advanced over the years, with the identification of mitochondrial and nuclear DNA mutations and their impact on different tissues and organs.This knowledge has led to improved classification and diagnosis of these disorders and to the knowledge that CPEO is one of their most common manifestations.Unfortunately, definitive phenotype-genotype correlations are still far out of reach, and physicians should familiarize themselves with these, maintain high clinical suspicion to diagnose them, and stop focusing on a solitary finding.Since no definitive treatment option is available, medical therapy focuses on alleviating symptoms and relieving defective mitochondrial expression.Genetic therapy is the future for treating these conditions, and this is an area where ongoing research is directed.Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflicts of interest.[154]

Figure 1 .
Figure 1.The diverse clinical outcomes of mitochondrial encephalomyopathies necessitate thorough screening of patients under suspicion.

Figure 1 .
Figure 1.The diverse clinical outcomes of mitochondrial encephalomyopathies necessitate thorough screening of patients under suspicion.

Figure 2 .
Figure 2.This diagram shows the proteins involved in the maintenance of mitochondrial DNA and the pathways involved.The mitochondrial nucleotide salvage pathway (1) is shown and is responsible for salvaging deoxyribonucleosides (dNs) and converting them into deoxyribonucleotide triphosphates (dNTPs) used in mtDNA replication.Along this pathway, Thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGK) convert dNs into deoxyribonucleotide monophosphates (dNMPs) that later convert into deoxyribonucleoside diphosphates(dNDPs) by nucleotide monophosphate kinase (NMPK), then into dNTPs by nucleotide diphosphate kinase (NDPK).The cytosolic nucleotide metabolism pathway (2) includes thymidine phosphorylase (TP), which converts thymidine into thymine, and ribonucleotide reductase (RNR), which converts NDPs into dNDPs, supplying the nucleotide salvage pathway.RNR consists of two catalytic and two R2 or p53-induced small subunits.The nucleotide transport proteins (3) supply the nucleotide salvage pathway from the cytosol.MPV17 protein supplies dNTPs, while adenine nucleotide transporter (ANT1) supplies ADPs with the assistance of acylglycerol kinase (AGK), which are later converted to deoxyadenosine diphosphate (dADPs) feeding into the dNDPs.Mitochondrial DNA synthesis (4) requires the enzymes TWINKLE, a helicase, and the synthesis initiator, DNA polymerase gamma (POLG), which needs an RNA primer that is supplied by mitochondrial transcription factor A (TFAM).POLG consists of one catalytic subunit and two subunits encoded from POLG2.(5)The removal of RNA primers and flap intermediates is then achieved via ribonuclease H1 (RNase H1), DNA helicase/ nuclease 2 (DNA2), and mitochondrial genome maintenance exonuclease 1 (MGME1).(6) Mitochondrial fusion is mediated by the proteins optic atrophy 1 (OPA1), F-box and leucine-rich repeat 4 (FBXL4), mitofusin 1 and 2 (MFN 1 and 2).

Figure 2 .
Figure 2.This diagram shows the proteins involved in the maintenance of mitochondrial DNA and the pathways involved.The mitochondrial nucleotide salvage pathway (1) is shown and is responsible for salvaging deoxyribonucleosides (dNs) and converting them into deoxyribonucleotide triphosphates (dNTPs) used in mtDNA replication.Along this pathway, Thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGK) convert dNs into deoxyribonucleotide monophosphates (dNMPs) that later convert into deoxyribonucleoside diphosphates(dNDPs) by nucleotide monophosphate kinase (NMPK), then into dNTPs by nucleotide diphosphate kinase (NDPK).The cytosolic nucleotide metabolism pathway (2) includes thymidine phosphorylase (TP), which converts thymidine into thymine, and ribonucleotide reductase (RNR), which converts NDPs into dNDPs, supplying the nucleotide salvage pathway.RNR consists of two catalytic and two R2 or p53-induced small subunits.The nucleotide transport proteins (3) supply the nucleotide salvage pathway from the cytosol.MPV17 protein supplies dNTPs, while adenine nucleotide transporter (ANT1) supplies ADPs with the assistance of acylglycerol kinase (AGK), which are later converted to deoxyadenosine diphosphate (dADPs) feeding into the dNDPs.Mitochondrial DNA synthesis (4) requires the enzymes TWINKLE, a helicase, and the synthesis initiator, DNA polymerase gamma (POLG), which needs an RNA primer that is supplied by mitochondrial transcription factor A (TFAM).POLG consists of one catalytic subunit and two subunits encoded from POLG2.(5)The removal of RNA primers and flap intermediates is then achieved via ribonuclease H1 (RNase H1), DNA helicase/nuclease 2 (DNA2), and mitochondrial genome maintenance exonuclease 1 (MGME1).(6) Mitochondrial fusion is mediated by the proteins optic atrophy 1 (OPA1), F-box and leucine-rich repeat 4 (FBXL4), mitofusin 1 and 2 (MFN 1 and 2).

Figure 3 .
Figure 3. Patient A, diagnosed with mitochondrial encephalomyopathy, presents with chronic progressive external ophthalmoplegia with limited eye movements in all gazes and cerebellar signs (intention tremor in finger-to-nose test and tandem walking), in addition to areas of pigment hyperplasia on fundoscopy.The yellow discoloration shown in the image is from fluorescein eye staining.

Figure 3 .
Figure 3. Patient A, diagnosed with mitochondrial encephalomyopathy, presents with chronic progressive external ophthalmoplegia with limited eye movements in all gazes and cerebellar signs (intention tremor in finger-to-nose test and tandem walking), in addition to areas of pigment hyperplasia on fundoscopy.The yellow discoloration shown in the image is from fluorescein eye staining.

Figure 4 .
Figure 4. Patient B is a 27-year-old male with a recent diagnosis of a homozygous pathogenic variant of MGME1, presenting with chronic progressive external ophthalmoplegia (limitation with horizontal and vertical gazes), refractory errors, pigmentary retinopathy, exercise intolerance, myopathy, fatigue, attention-deficit/ hyperactivity disorder, and right bundle branch block.He underwent ptosis repair at the ages of 15 and 17, but ptosis recurred over time.He has a long family history of consanguinity and a similar clinical phenotype presented in his cousin and past uncles.

Figure 4 .
Figure 4. Patient B is a 27-year-old male with a recent diagnosis of a homozygous pathogenic variant of MGME1, presenting with chronic progressive external ophthalmoplegia (limitation with horizontal and vertical gazes), refractory errors, pigmentary retinopathy, exercise intolerance, myopathy, fatigue, attention-deficit/hyperactivity disorder, and right bundle branch block.He underwent ptosis repair at the ages of 15 and 17, but ptosis recurred over time.He has a long family history of consanguinity and a similar clinical phenotype presented in his cousin and past uncles.

Figure 5 .
Figure 5.A summary of the proposed diagnostic pathway by Watson et al., where invasive testing is preceded by genetic sequencing, the gold standard of diagnosis.The green line indicates a yes, while the red line indicates no.

Figure 5 .
Figure 5.A summary of the proposed diagnostic pathway by Watson et al., where invasive testing is preceded by genetic sequencing, the gold standard of diagnosis.The green line indicates a yes, while the red line indicates no.

Author Contributions:
Conceptualization, A.A. and A.E.; resources, A.A., A.E. and R.B.; writingoriginal draft preparation, A.A. and A.E.; writing-review and editing, A.A., A.E. and R.B.; supervision, R.B.All authors have read and agreed to the published version of the manuscript.

Table 1 .
This table shows the differential diagnosis of chronic progressive external ophthalmoplegia, which should be excluded to avoid misdiagnosis.

Table A1 .
The clinical features of mitochondrial encephalomyopathies.

Table A2 .
Proposed therapeutic options for mitochondrial encephalomyopathies.