Advances in Primary Mitochondrial Myopathies (PMM)

Abstract

Purpose of review

Although mitochondrial diseases impose a significant functional limitation in the lives of patients, treatment of these conditions has been limited to dietary supplements, exercise, and physical therapy. In the past few years, however, translational medicine has identified potential therapies for these patients.

Recent findings

For patients with primary mitochondrial myopathies, preliminary phase I and II multicenter clinical trials of elamipretide indicate safety and suggest improvement in 6-minute walk test (6MWT) performance and fatigue scales. In addition, for thymidine kinase 2 Deficient (TK2d) myopathy, compassionate-use oral administration of pyrimidine deoxynucleosides have shown preliminary evidence of safety and efficacy in survival of early onset patients and motor functions relative to historical TK2d controls.

Summary

The prospects of effective therapies that improve the quality of live for patients with mitochondrial myopathy underscores the necessity for definitive diagnoses natural history studies for better understanding of the diseases.

Introduction:

Mitochondria are organelles responsible for generating most of the ATP in cells, via oxidative phosphorylation (OxPhos). Mitochondria require dual genomic expression of proteins which originate both in the nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The nDNA diseases manifest Mendelian inheritance (autosomal dominant or recessive) or X-linked transmission while mtDNA diseases are maternally inherited (mutations of the mtDNA) or sporadic (mitochondrial DNA single deletions syndromes). Furthermore, the multicopy nature of mitochondrial DNA gives rise to the concept of heteroplasmy (when both mutated and wild type mtDNA molecules coexist in the same cell) and homoplasmy (only mutant mtDNA is present in the mitochondria of the cell). For a disease to manifest symptoms, the mutated mtDNA molecules in tissue must increase to a critical threshold above which OxPhos function is impaired. Due to dual genome control of mitochondria, mutations in the nuclear genes can impair the mtDNA homeostasis causing mtDNA multiple deletions, depletion, or both. Deletions in mtDNA are more readily detected in muscle and urine than in blood, oral swabs, and fibroblasts. The predilection for muscle involvement in mitochondrial disease is due to the relatively high mutation load as well as the high energy requirements of this tissue [1, 2].

Primary Mitochondrial Myopathies (PMM) are genetic disorders that impair the oxidative phosphorylation with muscles being one of the most affected tissue. The prevalence of mitochondrial disorders has been estimated to be 1:4300 affected individuals [3] with most displaying prominent muscle involvement. Myopathy is not only one of the most prevalent manifestations, but is also a very debilitating feature of this diseases because of weakness and exercise intolerance that impair mobility as well as the capability of the patient to perform daily activities). This review does not cover secondary muscle mitochondrial dysfunction observed in other neuromuscular diseases (like Duchenne, Ullrich myopathy, Bethlem myopathy, and inclusion body myositis) [4].

PMM has become more important in the past five years because of the emergence of new potential treatments like elamipretide for symptomatic treatment and deoxycytidine (dC) and deoxythymidine (dT) for modification of TK2d [5, 6].

Diagnosis:

The gold standard for confirming PMM is based upon molecular genetic tests, which may reveal pathogenic variants in either nDNA or mtDNA in the buccal swab, urine, fibroblasts, and muscle fragments. Pathological examinations of muscle following a muscle biopsy can complement abnormal molecular findings [7, 8]. The important histological muscle findings are: 1) ragged-red fibers (RRF), 2) proliferation of mitochondria observed with histochemical stain for succinate dehydrogenase (SDH or complex II) often described as ragged-blue fibers, 3) cytochrome c oxidase (COX or complex IV) deficient or negative , and 4) ultrastructurally abnormal mitochondria often with paracrystalline inclusions. The activities of respiratory chain enzymes (complexes I-IV) can be measured spectrophotometrically or in native gels while western blot, native gel, or two-dimensional gel assays can assess levels of OxPhos proteins.

Additional complementary tests can help to confirm a mitochondrial disease and include: elevated lactate in blood, cerebropinal fluid, or brain MR spectroscopy, increased blood lactate/pyruvate ratio (>20:1), elevated creatine kinase (CK), increased growth differentiation factor 15 (GDF-15), fibroblast growth factor 21 (FGF-21), decreased glutathione (antioxidant), respiratory chain enzyme analyses in blood or skin fibroblasts, brain and spine MRI, exercise physiology study, and electromyography [2, 9].

Clinical history

Mitochondrial myopathies present with exercise intolerance, generalized or localized progressive muscle weakness including extraocular muscles (ptosis, ophthalmoparesis, or both), cramps, myalgia, shortness of breath, dyspnea on exertion, elevated creatine kinase, and myoglobinuria. Patient frequently complain of specific symptoms including: fatigue, tiredness, lack of energy, exercise intolerance, and exhaustion. PMM is often accompanied by multiorgan dysfunction with variable failure to thrive, developmental delay, regression, dementia, encephalomyopathy, stroke-like episodes, seizures, ataxia, optic atrophy, sensorineural hearing loss, cardiomyopathy, diabetes, hepatopathy, nephropathy, and peripheral neuropathy. In the pediatric population, PMM can present as floppy infant syndrome, motor developmental delay or retardation, hypotonia, respiratory insufficiency, and hyporeflexia/areflexia. Patients with multiorgan involvement typically present during infancy with more severe phenotypes while organ-specific symptoms tend to occur in adulthood onset forms. It is important to note that mitochondrial diseases show a large intra and inter- familiar clinical heterogeneity, which sometimes makes it difficult to establish an accurate genotype/phenotype association [2, 10–12].

Detailed family history is required and can help uncover the pattern of inheritance. Mitochondrial red flags should be directly asked (e.g., early onset diabetes, hearing loss, retinopathy, cardiac disease, ptosis, ophthalmoparesis) [10].

Clinical Syndromes

Although there are some well-described nosological mitochondrial syndromes in which myopathy is typically a symptom, there is a heterogeneous cohort of patients with PMM that does not fit any specific syndrome [11]. Classic mitochondrial syndromes with muscle involvement are summarized in table 1.

Table 1.

Primary mitochondrial classical syndromes with muscle involvement

DISEASE	INHERITANCE	GENES	MOLECULAR DEFECTS	CLINICAL FEATURES	LABORATORY TESTS	REFERENCES
CPEO	Autosomal dominant, recessive, sporadic (common 4,977 base-pair deletion), or maternally inherited (mtDNA)	POLG1 POLG2 ANT1 C10ORF2 OPA1 TK2	1/3 of all single large-scale deletions of mtDNA (4.9-kb)	• Bilateral ophthalmoparesis • Ptosis • Oropharyngeal weakness • Myopathy	• Detection of mtDNA single deletion or multiple mtDNA deletions with primary nDNA pathogenic variant	[13]
MELAS	Maternal inheritance (mtDNA)	MT-TL1 MT-ND5	80% of all cases have the m.3243A>G mutation in the MT-TL1	• Stroke-like episodes, • Myopathy • Encephalopathy with seizures, cognitive impairment, or both	• Lactic acidemia in blood and CSF • Muscle biopsies with ragged red fibers • MRI Stroke-like lesion(s)	[16]
Kearns-Sayre syndrome	Maternal inheritance (mtDNA), sporadic	Variable deletion ranging from 1.1 to 10 kb	mtDNA single deletions	• Onset before 20 years • Pigmentary retinopathy • CPEO • Myopathy • Cardiac conduction block	• CSF protein higher than 100 mg/dL • Lactic acidemia in blood and CSF • Muscle biopsy RRF • Cardiac conduction block	[13]
MERRF	Maternal inheritance, sporadic	MT-TK	m.8344A>G	• Myoclonus • Generalized epilepsy • Ataxia • Myopathy	• CSF protein higher than 100 mg/dL • Lactic acid elevation in blood, CSF, or both	[20]
CoQ10 deficiency	Autosomal recessive	COQ2, COQ4, COQ6, COQ7, COQ8A, COQ8B, COQ9, PDSS1, PDSS2	CoQ10 biosynthesis defect	• Early infantile onset encephalomyopathy • Steroid-resistant nephrotic syndrome • Multiple-system atrophy-like neurodegenerative phenotype	• Lactic acidemia in blood and CSF • Muscle biopsies with reduced CoQ10 levels	[21]
TK2 deficiency	Autosomal recessive	TK2	Cause mitochondrial DNA (mtDNA) maintenance defect	• Infantile-onset myopathy • Juvenile/childhood proximal weakness  • Late-onset myopathy with facial and limb weakness	• Multiple respiratory chain complex deficiency • Reduced mitochondrial DNA content • Elevated CK	[23, 24]

CSF: cerebrospinal fluid

RRF: ragged-red fibers

EKG: electrocardiogram

CK: creatine phosphokinase

CPEO: chronic progressive external ophthalmoplegia

MELAS: mitochondrial encephalomyopathy lactic acidosis stroke-like episodes

MERRF: myoclonus epilepsy ragged-red fibers

POLG1 or POLG2: encoding mtDNA-specific polymerase gamma

ANT1: adenine nucleotide translocator1

C10ORF2: encoding a mtDNA helicase called Twinkle

OPA1: Optic atrophy type 1

TK2: Thymidine kinase 2

Chronic progressive external ophthalmoplegia (CPEO) is responsible for 2/3 of PMM and is characterized by symmetric, slowly progressive, bilateral paresis of extrinsic ophthalmic muscle, with diplopia when asymmetric, and ptosis. Patients tend to have a compensatory contraction of frontalis muscle with backward head tilting and tendency to turn the head to see. This condition, in general, is followed by oropharyngeal weakness (dysphagia and dysarthria) and proximal weakness (neck flexors, shoulders, and hips). Myasthenia gravis is the main differential diagnosis of this disease [13, 14].

Kearns-Sayre syndrome (KSS) is historically defined by the triad of pigmentary retinopathy, CPEO with ptosis, and cardiac conduction alterations, typically starting before 20 years of age. Proximal muscle weakness is almost invariably present. Other manifestations include failure to thrive, dysphagia or achalasia, levels of proteins in the cerebrospinal fluid higher than 0.1 g/L, cognitive decline, cerebellar ataxia, sensorineural hearing loss, and endocrine dysfunction (diabetes mellitus, hypoparathyroidism, adrenal insufficiency). These patients require a close cardiac follow-up, because of the risk of a cardiac block and the possible need of a prophylactic pacemaker. CPEO, KSS, and Pearson syndrome (which manifests typically during early infancy with sideroblastic anemia and exocrine pancreas insufficiency) represent the spectrum of mitochondrial deletion syndrome. [13, 15].

Mitochondrial Encephalomyopathy Lactic Acidosis, and Stroke-like episodes (MELAS) is a complex multisystemic syndrome characterized by encephalopathy, seizures, headaches, stroke-like episodes (with onset usually before the age of 40 years), dementia, hearing impairment, gastrointestinal symptoms, muscle weakness, exercise intolerance, peripheral neuropathy, and diabetes. When the symptoms start during infancy, there is typically a period of normal early psychomotor development, followed by failure to thrive, and learning disability [16, 17]. For this condition, experimental data suggest that arginine and citrulline could be effective in reducing stroke-like episodes and treat acute stroke [18, 19].

Myoclonic epilepsy with ragged red fibers (MERRF) often starts during infancy with myoclonus followed by generalized epilepsy, progressive ataxia, weakness, and dementia. Other possible findings include, short stature, optic atrophy, pigmentary retinopathy, hearing loss, lipomatosis, and cardiac involvement (specifically Wolff-Parkinson-White syndrome) [20].

Primary coenzyme Q10 (CoQ10) deficiencies are clinically and genetic heterogeneous syndromes. Clinical manifestations encompass: central nervous system involvement, with encephalopathy, hypotonia, seizures, dystonia, cerebellar ataxia, epilepsy, stroke-like episodes, spasticity or intellectual disability, steroid-resistant nephrotic syndrome, isolated or in association with central nervous system involvement; peripheral neuropathy and sensory neural hearing loss; hypertrophic cardiomyopathy; retinopathy and optic atrophy. Muscle involvement is not uncommon, especially in patients with a multisystemic phenotype. Typical findings include weakness and exercise intolerance. Some muscle biopsies have shown non-specific signs of lipid accumulation and mitochondrial proliferation. Patients with CoQ10 deficiency might respond well to high dose of oral CoQ10 (5-50 mg/Kg/day) [2, 21].

Isolated mitochondrial complex III deficiency is a relatively rare cause of respiratory chain dysfunction. Complex III consists of 11 subunits, only one of which, cytochrome b, is encoded by mtDNA (MTCYB). Mutations in MTCYB are most frequently associated with sporadic myopathy characterized by exercise intolerance with or without myoglobinuria [22]. Multisystemic involvement has also been described.

Thymidine kinase 2, encoded by the nuclear gene TK2, is the first enzyme in the deoxypyrimidine salvage pathway in mitochondria. These deoxynucleosides are required for mtDNA replication and maintenance. Autosomal recessive mutations in TK2 gene cause TK2d, with consequent nucleotide imbalance that in turn produces mtDNA depletion, multiple deletions, or both.

TK2 deficiency, also known as Mitochondrial DNA Depletion Syndrome 2, has three main phenotypes depending on the age of presentation of symptoms. The infantile-onset form is characterized by myopathy with early death usually due to respiratory failure and severe mtDNA depletion. In general, cognitive function is spared, but some patient have encephalopathy, seizures, or both. Patients may also manifest dysarthria, dysphagia, and hearing loss. The childhood-onset presentation, beginning between 1 and 12 years-old, manifests generalized proximal limb weakness with prominent respiratory insufficiency and median survival at least 13 years. In the late-onset form, beginning at age 12 years-old or older, the presentation is typically bulbar and limb myopathy with variable respiratory muscle weakness and mtDNA deletions. The bulbar manifestations include chronic progressive external ophthalmoplegia, dysphagia, and dysarthria [23, 24].

Clinical Care

In general, clinical care for mitochondrial diseases includes a complete initial screen with complete blood count, basic metabolic panel, lactate at rest, creatine kinase (CK), hepatic function panel, hemoglobin A1c (HgbA1c), thyroid-stimulating hormone (TSH), free thyroxine level (FT4), parathyroid function (parathyroid hormone, serum calcium, magnesium, and phosphate), vitamin D, urea, creatinine, and urinary albumin/creatinine ratios [10].

Initial follow-up every 6 to 12 months should include neurologic, cardiologic (with electrocardiogram and echocardiogram tests), and endocrinologic assessments (screen for diabetes and thyroid-stimulating hormone). Depending on the particular mitochondrial disease and mutation, adjustments in the follow up are required. For example, patients with single mtDNA deletions, close follow is necessary to screen for cardiac conduction block with a low threshold for placement of an implantable cardioverter-defibrillator because increased risk for sudden death. In contrast, patients with either m.3243A>G or m.8344A>G variants, should be screened for ventricular pre-excitation (m.3243A> G and m.8344A>G) and cardiomyopathy. In patients with new-onset fatigue, screening for anemia and adrenal insufficiency should be considered. Depending on the clinical demand of the patient, other specialist evaluations are needed, such as: ophthalmology, audiology, gastroenterology, pulmonology, immunology, and nephrology [3, 10]. Genetic counseling is important to convey critical information about the possibility of the relatives being affected (and alternatives for future pregnancy) and screening of relatives at risk [9].

Treatment

Regular aerobic exercise has been showed to improve the number of mitochondria in muscle, fatigue, and quality of life scales. A multi-disciplinary approach, including physical therapy, occupation therapy, speech therapy and nutritionist, is advised. Avoidance of medications toxic to mitochondria such as valproic acid (risk of hepatotoxicity particularly in Alpers disease), aminoglycosides, linezolid, metformin, and nucleoside reverse transcriptase inhibitors is generally advised. Because of the high risk of propofol infusion syndrome in patients with mitochondrial diseases, this medication should be used with caution [10, 11, 25–30].

Dietary supplements including antioxidants and mitochondrial cofactors are frequently used although long-term efficacy has not been definitively proven. Often described as mito-cocktails, the supplements often include a variety of different compounds such as complex B vitamins (mainly thiamine [vitamin B1], and riboflvin [vitamin B2]), creatine, ubiquinol (the reduced form of coenzyme Q₁₀ [CoQ₁₀] with higher bioavailability than coenzyme Q₁₀), alpha lipoic acid, N-acetylcysteine, folinic acid, vitamin C and, vitamin E. One small randomized placebo-controlled trial indicated evidence of the combination of creatine, CoQ10, and lipoic acid [31–33].

A few mitochondrial diseases may be responsive to vitamin/cofactor treatments. For example, in primary CoQ₁₀ deficiencies due to CoQ₁₀ biosynthetic defects, CoQ₁₀ supplementation in high doses can ameliorate symptoms. ACAD9 deficiency myopathy may respond to riboflavin while some cases of ETFDH myopathy have responsive to riboflavin, CoQ₁₀, or both [25, 34]. New supplement approaches are under study, but not available clinically yet [26]. Relevant supplements are summarized in table 2.

Table 2:

General doses of vitamins/co-factors in mitochondrial diseases.

SUBSTANCE	DOSE	REFERENCE
Arginine	Acute: 500 mg/kg IV per day (for 1–3 days) Maintenance dose: 150–300 mg/kg per day oral divided 2–3 times a day	[19, 32]
Alpha lipoic acid	50–200 mg/d	[32]
B50 or B100 (B vitamin complexes)	1 tablet oral given 1–2 times a day	[32]
Creatine	Paediatric: 0.1 g/kg per day oral. Adult: 10 g per day oral. Divided 1–2 times per day	[32]
CoQ10 (Ubiquinol)	Pediatric: 2–8 mg/kg per day oral divided in two doses Adult: 50–600 mg per day oral	[32]
Riboflavin (B2)	50–400 mg per day oral	[32]

IV: intra venous

Immunization is encouraged to prevent neurodegeneration and decompensations associated with infection in the case of mitochondrial patients [32].

Elamipretide, an aromatic-cationic tetrapeptide, has emerged as a possible symptomatic treatment for mitochondrial myopathy. This molecule penetrates cell membranes and target the inner mitochondrial membrane, where it binds with cardiolipin and may stabilize mitochondrial OxPhos enzyme supercomplexes. A Phase I dose escalation study indicated safety at 3 doses and, at the highest dose, increased exercise performance (6MWT increased 64.5 meters compared to 20.4 meters in the placebo group, p = 0.053) after 5 days of daily intravenous infusions of elamipretide in 36 adult participants with genetically confirmed PMM [5]. A phase III randomized placebo-controlled trial is currently being conducted [5].

In contrast to the broad symptomatic approach of elamipretide, a focused disease modifying approach to TK2d has emerged as a promising therapy. Treatment dC and dT provides substrates for cytosolic TK1 and deoxycytidine kinase, which can compensate for TK2d as demonstrated in a mouse model that demonstrated improvements in mtDNA depletion, OxPhos defects, disease onset and survival. In 2019, a compassionate use (expanded access) study of 16 patients demonstrated safety and improved survival in early onset TK2d patient (onset <2 years-old) as well as well as motor functions in all forms of TK2d relative to the natural history studies [6, 35, 36].

Conclusion

Mitochondrial myopathies are caused by impaired OxPhox due to a variety of mutations in the nuclear or mitochondrial genome. Skeletal muscle involvement can be present in isolation or as part of a complex multisystemic syndrome and can manifest as muscle weakness (most commonly proximal), PEO with or without ptosis, exercise intolerance, fatigue, or rhabdomyolysis. A focused clinical history can help directing the diagnostic tools toward a specific diagnosis (e.g. mtDNA vs nDNA defects). The introduction of next generation sequencing techniques has revolutionized the field of mitochondrial disease widely expanding the number of genetic etiologies. However, there are currently no effective treatments available for the majority of patients with mitochondrial myopathies and no FDA approved treatments. Existing options focus on the management of symptoms and on improving the quality of life of patients. The establishment of patients’ registries has facilitated the possibility of investigating therapeutic options in large cohort of patients. The increasing number of clinical trials being conducted on mitochondrial disease offer hope for the future.

Key points:

1. Myopathic symptom is one of the most prevalent and disabling symptoms of Mitochondrial diseases.

2. Multisystemic involvement should be investigated in patients with mitochondrial disease due to the potentiality of multi-organ life-threatening involvement.

3. Although therapies did not prove to be curative, it is important to identify asymptomatic patients to provide adequate surveillance.

4. In the past few years, medications emerged to help patients with mitochondrial diseases, Elemipretide for myopathy in general and deoxycytidine (dC) + deoxythymidine (dT) for TK2 deficiency. Gene therapy is a promise for future treatments.

5. Physical conditioning was proved to improve mitochondrial myopathy symptoms.