Clinical manifestations

Presentation and course

Like other mitochondrial syndromes (ie, Kearns-Sayre syndrome and MERRF), MELAS is a multisystem disorder. To make a clinical diagnosis, the following features must be present: (1) stroke-like episodes typically—but not mandatory—before age 40; (2) encephalopathy, characterized by seizures, dementia, or both; and (3) evidence of a mitochondrial myopathy with lactic acidosis, ragged-red fibers, or both. In addition, to confirm the diagnosis, at least two of the following should be present: normal early development, recurrent headache, or recurrent vomiting (50).

A clinical diagnosis of MELAS can also be made in an individual with at least two category A and two category B criteria (148):

Table 1. Clinical Diagnostic Criteria of MELAS

Category A criteria	Category B criteria
• Headaches with vomiting • Seizures • Hemiplegia • Cortical blindness • Acute focal lesions on neuroimaging (see suggestive findings, brain imaging)	• High plasma or cerebrospinal fluid (CSF) lactate • Mitochondrial abnormalities on muscle biopsy (see suggestive findings, suggestive laboratory findings) • A MELAS-related pathogenic variant (see Table 2)

The diagnosis of MELAS is established in a proband who meets the clinical diagnostic criteria and who is a carrier of a pathogenic variant in one of the genes listed in Table 2 identified by molecular genetic testing.

Over 100 reported cases of MELAS support the validity of this clinical syndrome (49). Besides the obligatory cardinal manifestations, numerous additional symptoms and signs are associated with MELAS. Features reported in the majority of patients include exercise intolerance, limb weakness, short stature, hearing loss, and elevated cerebrospinal fluid protein. The headaches of MELAS have often been described as migrainous and are frequently severe during the acute phase of the strokes. Patients with mtDNA mutation m.3243A > G and with MELAS phenotype showed the highest prevalence of headache (88%, 86%, respectively) among patients affected by mitochondrial disorders in a cohort of a German cross-sectional study (74). The subjects described headaches as short-lasting and often mild or moderate intensity. The high prevalence of migraine in MELAS is independent of sex, phenotype, or genotype (80).

A stroke-like episode may occur not only in individuals with MELAS but also in patients with other forms of mitochondrial syndromes (102) and should be generally suspected if features of an ischemic stroke are accompanied by atypical central nervous system features. The most frequent onset symptoms of a stroke-like episode are migraine or migraine-like headache, seizures, drowsiness, vomiting, and visual field defects. Occasionally, strokes occur during a febrile illness. The strokes are called atypical because the lesions do not conform to the territories supplied by major cerebral arteries. The morphological equivalent of a stroke-like episode is the stroke-like lesion, which are most frequently located in an occipito-temporal distribution. The strokes usually affect the cortex and subjacent white matter and frequently affect posterior cerebral hemispheres. Acute strokes can be detected as increased signal on routine T2-weighted MRI scans. Elevated apparent diffusion coefficient was often reported during these episodes, mainly suggesting vasogenic edema in the acute phase and representing a different pattern than in classic stroke. However, there has been an increase in the number of reports of a decrease in apparent diffusion coefficient, compatibly with cytotoxic edema in acute phase, such as typical stroke. Thus, increased and decreased apparent diffusion coefficient portions are mixed in stroke-like lesions, supporting both cytopathy and vasogenic theories: mild energy failure should result in moderate cellular dysfunction, which is responsible for vasogenic edema (high apparent diffusion coefficient), and severe energy failure should relate to irreversible cellular failure, responsible for cytotoxic edema (low apparent diffusion coefficient) (55).

Features present in fewer than half of the patients are basal ganglia calcifications, myoclonus, ataxia, episodic coma, optic nerve atrophy, cardiomyopathy, pigmentary retinopathy, electrocardiographic evidence of pre-excitation, cardiac conduction block, ophthalmoplegia, diabetes mellitus, hirsutism, gastrointestinal dysmotility, and nephropathy. Cardiomyopathy affects about a third of patients with MELAS: the most common manifestation is hypertrophic cardiomyopathy, which may eventually progress to dilated cardiomyopathy. Individuals affected by MELAS syndrome may more easily run into serious cardiac complications such as malignant arrhythmias, heart failure, and sudden cardiac arrest (51).

Children with MELAS often have normal early psychomotor development until the onset of symptoms between 2 and 10 years of age. Though less common, infantile onset may occur and may present as failure to thrive, growth retardation, and progressive deafness. Onset in older children typically presents as recurrent attacks of a migraine-like headache, anorexia, vomiting, and seizures. Children with MELAS are also frequently found to have short stature (112).

Akinetic rigid parkinsonism/MELAS overlap syndrome was reported in a patient with a unique 4 base-pair deletion in the cytochrome b gene (21). Sandoval and colleagues reported a case of parkinsonism as initial symptom of MELAS syndrome, which is an unusual first manifestation of this disease (120). The association between movement disorders such as parkinsonism and MELAS syndrome is probably due to changes in basal ganglia resulting from stroke-like episodes. Peripheral neuropathy may be an under-recognized manifestation of MELAS; nerve conduction abnormalities were detected in 23 of 30 (77%) patients (62) and in 11 of 12 (92%) patients (82) with the m.3243A>G mtDNA mutation, mostly with a sensory axonal pattern. A 2012 review of the psychiatric manifestations of mitochondrial disorders included 26 cases of MELAS syndrome with one or more of the following symptoms: major depressive disorder (n=5), cognitive impairment (n=11), psychotic disorder (n=15), anxiety disorder (n=6), frontal lobe syndrome (n=3), or personality change (n=2) (04). A study reported how social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission, probably due to abnormalities in GABA signaling transmission (58). Rarely, catatonia may be observed in the acute phase of the disease (96).

Almost all reported cases of MELAS have a mitochondrial DNA point mutation. Maternal relatives, then, are likely to harbor the same mutation. Few families have more than one member with MELAS; more often oligosymptomatic maternally related individuals manifest only some of the features of MELAS (49). In addition, the MELAS point mutation may also be found in asymptomatic family members. Intriguingly, three families have been described where the m.3243A>G mutation has arisen de novo; this finding is of major relevance for both diagnostic investigations and genetic counselling (23).

Moraes and colleagues identified another clinical phenotype associated with the m.3243A>G mutation: progressive external ophthalmoplegia (PEO3243) (97). They identified 16 progressive external ophthalmoplegia patients with this mutation. Maternally inherited diabetes and deafness had been associated with the m.3243A>G mutation (116; 141). Mitochondrial patients may show features of both type 1 and type 2 diabetes. A progression to complete insulin dependency due to beta-cells failure is possible for these subjects, although most patients with mitochondrial diabetes do not require insulin therapy at diagnosis (149). In 770 Chinese patients with diabetes, the m.3243A>G mutation was found in 1.69% of patients (142). Patients with MELAS have also presented with a syndrome mimicking herpes simplex encephalitis, pulmonary artery hypertension, and cardiomyopathy (57; 130; 36).

In 82 m.3243A>G mutation carriers, the most prevalent symptoms were hearing loss (48%), gastrointestinal problems (42%), decreased vision (42%), exercise intolerance (38%), and glucose intolerance (37%), but only 4% had stroke-like episodes (24). Hearing loss may be of both cochlear and retrocochlear origin, and it may be subclinical. Therapeutic implications of early recognition include interventions in the form of hearing aids, cochlear implants, and cautioning the physicians for avoidance of aminoglycosides (140).

More than 50% of m.3243A > G mutation carriers were found to have some form of gait problems. It has been suggested that quantification of gait may be a suitable outcome parameter in the upcoming clinical trials in this patient group (114).

A study on a cohort of 129 English patients harboring the m.3243A>G mutation showed how only 10% of patients exhibited a classical MELAS phenotype (101). In general, the m.3243A>G mutation manifests far more often as mitochondrially inherited diabetes and deafness or as other oligosymptomatic forms than as full-blown MELAS. Patients with mitochondrially inherited diabetes and deafness have insulin-dependent diabetes associated to relatively low BMI; the onset of the diabetes, usually associated with progressive neurosensory deafness, is observed at 30- or 40-years-old (138).

A multicenter UK study identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes, with the possibility of analyzed postmortem cases in 26 cases (103). The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1, followed by recessive pathogenic POLG variants and other rarer pathogenic mtDNA variants. Clinicoradiological, electrophysiological, and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy, and significant radiological and pathological features of neurodegeneration were more evident in patients harboring pathogenic mtDNA variants compared with POLG.

Prognosis and complications

The disease progresses over years with episodic deterioration related to stroke-like events. The course varies from patient to patient. In a cohort of 33 adults with the m.3243A>G MELAS mutation followed for 3 years, deterioration of sensorineural function, cardiac left ventricular hypertrophy, EEG abnormalities, and overall severity were observed (83). In a natural history study in 31 patients with MELAS and 54 carrier relatives over a follow-up period of up to 10.6 years, neurologic examination, neuropsychological testing, and daily living scores significantly declined in all patients with MELAS, whereas no significant deterioration occurred in carrier relatives. The death rate was more than 17-fold higher in fully symptomatic patients than in carrier relatives. The average observed age at death in the MELAS patient group was 34.5+/-19 years (range 10.2 to 81.8 years). Of the deaths, 22% occurred in patients younger than 18 years. The estimated overall median survival time based on fully symptomatic patients was 16.9 years from onset of focal neurologic disease (60). A Japanese prospective cohort study of 96 patients with MELAS confirmed a rapidly progressive course within a 5-year interval, with 20.8% of patients dying within a median time of 7.3 years from diagnosis (148). The studies mentioned above showed how the clinical severity of MELAS is linked to both the age of onset and the duration of the disease. A retrospective Chinese study in which 138 patients were enrolled reported how the main causes of death (observed in 42.2% of patients) were an acute stroke-like episode or status epilepticus. Stroke-like episodes were reported as the most common initial symptoms (152). Thus, a correct management of stroke-like episodes is primary to improve the prognosis.

The severity of the disease probably depends on the percent mutation and the tissue distribution of the mutation. Patients with a younger age of onset tend to have a more severe course. Yearly ECGs and echocardiograms to screen for cardiopathy, particularly in children with MELAS, may be prudent (146).

In a large prospective cohort study comprising 135 clinically heterogeneous A3243G mutation carriers and 30 healthy volunteers, proton magnetic resonance spectroscopic imaging ((1)H MRSI) showed promise in identifying disease biomarkers as well as individuals at risk of developing the MELAS phenotype (144). Mutation carriers included 45 patients with MELAS, 11 participants who would develop the MELAS syndrome during follow-up (converters), and 79 participants who would not develop the MELAS syndrome during follow-up (nonconverters). Higher lactate and total choline levels predicted the risk of individual mutation carriers to develop the MELAS phenotype.

Clinical vignette

The girl was born after a normal pregnancy and delivery. Early development was normal, but weakness of the legs was evident at age 2 when she was noted to have difficulty climbing stairs. At age 5 years, examination revealed short stature (92 cm) and low weight (15 kg), both below the third percentile. There was mild proximal muscle weakness with a Gowers sign. Her parents were nonconsanguineous and healthy. Her 4-year-old brother had mental retardation, short stature, and hearing loss. Her two sisters, 1 year and 11 years old, were clinically normal. At age 5.5 years, after strenuous exercise, she lost consciousness for 30 minutes. She subsequently had multiple episodes of dimmed vision, each lasting a few minutes. At age 6 years she had 30-minute to 60-minute periods of right body paresthesias followed by blindness after physical exertion. Following one episode, she had a generalized tonic-clonic seizure, and phenobarbital therapy was initiated. Postictally, she had a right hemiparesis that resolved after 3 days. A second right focal seizure was followed by hemiparesis for 5 days. Phenobarbital dosage was increased. Lactic acidosis was noted. After another generalized seizure she became blind. The loss of vision was thought to be cortical in origin because pupillary reactions to light and funduscopy were normal. A brain MRI revealed bilaterally increased T2-signal in the occipital cortex. Leukocyte DNA analysis revealed an A-to-G mutation at nucleotide 3243 of the mitochondrial DNA. L-carnitine (100 mg/kg per day) was initiated without clinical improvement; the disorder continued to progress. At age 9 years she was severely demented and deaf. Audiometry revealed neurosensory hearing loss. At age 10 years she died due to congestive heart failure.

Biological basis

Etiology and pathogenesis

Thirty-three different mitochondrial DNA point mutations have been associated with the MELAS clinical phenotype (Table 2), and this list is still growing (MITOMAP). The most prevalent of these mutations is an A-to-G transition mutation at nucleotide 3243 (m.3243A> G) of the tRNALeu(UUR) (39). This mutation has been found in about 80% of cases of MELAS (39; 17). Five additional mutations in the tRNALeu(UUR), a hot-spot for mtDNA mutations, have been reported in patients with MELAS (Table 1). Eight other tRNA point mutations associated with MELAS have been described. In addition, mutations in polypeptide-encoding genes have been identified. The gene encoding subunit 5 of complex I (ND5) is a second hot spot for MELAS mutations as 6-point mutations in this gene have been associated with this phenotype, including the m.13513G>A, which may be the second most common cause of MELAS (121; 125). One patient with a MELAS phenotype had compound heterozygous mutations in the nuclear DNA encoded polymerase gamma (POLG1) gene (25). At least two mitochondrial DNA mutations have been associated with overlap MERRF-MELAS syndromes; one is at nucleotide 8356 in tRNA-Lys (151) and the other is at nucleotide 7512 in tRNA-Ser(UCN) (100). Several patients with features of both MELAS and Leigh syndrome have been reported with the m.13513G>A and a ND3 gene mutation (19; 75; 143). A case of stroke-like episodes/Leigh syndrome associated with a novel m.3482A>G mutation in MT-ND1 was reported (45). A sporadic patient with a multisystem disorder with features of MELAS (stroke-like episodes), MERRF, Leber hereditary optic neuropathy, and Kearns-Sayre syndrome had a missense mutation in subunit 3 of complex I (137). In very rare cases, MERRF-like phenotypes have been described with the MELAS m.3243A> G mutation (11).

Table 2. MtDNA Mutations causing MELAS

mtDNA nucleotide change	Gene symbol	Protein Change	Reference
m.3243A> G	MT-TL1	No protein translated	(39)
m.3271T>C			(40)
m.3252A>G			(98)
m.3291T>C			(41)
m.3256C>T			(123)
m.3260A>G			(104)
m.583G>A	MT-TF		(44)
m.1642G>A	MT-TV		(136)
m.1644G>A			(134)
m.4332G>A	MT-TQ		(06)
m.5521G>A	MT-TW		(48)
m.5814A>G	MT-TC		(87)
m.7512T>C	MT-TS1		(76)
m.8316T>C	MT-TK		(14)
m.8296A>G			(119)
m.12146 A>G	MT-TH		(13)
m.12299A>C	MT-TL2		(02)
m.3481G>A	MT-ND1	p.Gln59Lys	(84)
m.3697G>A		p.Gly131Ser	(66)
m.3946G>A		p.Gln214Lys	(66)
m.3949T>C		p.Tyr215His	(66)
m.7023G>A	MT-CO2	p.Val374Met	(133)
m.9957T>C	MT-CO3	p.Phe251Leu	(88)
m.12770A>G	MT-ND5	p.Glu145Gly	(77)
m.13042G>A		p.Ala236Thr	(99)
m.13084A>T		p.Ser250Cys	(19)
m.13513G>A		p.Asp393Asn	(121)
m.13514A>G		p.Asp393Gly	(18)
m.13528A>G		p.Thr398Ala	(92)
m.14453G>A	MT-ND6	p.Ala74Val	(115)
m.14787_14790del		p.Ile14Thr+fs	(21)
m.14864T>C	MT-CYB	p.Cys40Arg	(31)
m.15092G>A	MT-CYB	p.G116S	(86)
m.3482A>G	MT-ND1	p.E59G	(45)

The origin of the mtDNA mutations is uncertain, but nucleotide substitutions occur about 10 times more frequently in mtDNA than in nDNA (12), and the repair mechanisms for mtDNA may not be as efficient as for nDNA (78). In support of this concept is the high frequency (one in 200 live births) of de novo m.3243A> G and other common mtDNA mutations detected in a survey of umbilical cord blood samples in the Northeast of England (29). The m.3243A> G mutation is clearly transmitted in a maternal inheritance pattern (17).

Even in the first stages of embryogenesis, there is a compensatory cellular response to the respiratory chain defect. MELAS embryos have been shown to harbor approximately 3-fold higher amounts of mtDNA, suggesting that mtDNA can replicate in early embryos and emphasizing the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans (95).

Clinical expression of the mutation depends on three factors: (1) mtDNA heteroplasmy, (2) mtDNA tissue distribution, and (3) tissue threshold. Each mitochondrion contains multiple copies of mtDNA, and each cell contains multiple mitochondria. Therefore, each cell can contain a variable proportion of normal and mutant mtDNA molecules (heteroplasmy). The tissue distribution of mutant mtDNA is also heterogeneous. In addition, the most metabolically active tissues are most susceptible to the deleterious effect of the mtDNA mutations (tissue threshold). The tissue threshold probably does not vary significantly among individuals, but mtDNA heteroplasmy and variable tissue distribution may account for the clinical diversity of patients with MELAS. As yet undefined nuclear DNA factors may also modify the phenotypic expression of the mtDNA mutations (97).

Analyses of respiratory chain enzymes have demonstrated complex I deficiency or combined defects of multiple complexes (50).

Skeletal muscle biopsies demonstrate mitochondrial abnormalities. Modified Gomori trichrome stain shows ragged-red fibers; the red stain in the subsarcolemmal regions of muscle fibers is due to accumulations of mitochondria. Similarly, the succinate dehydrogenase histochemical stain reveals darker than normal fibers, which are equivalent to ragged-red fibers. Ultrastructural studies have confirmed the increased number of mitochondria, as well as morphologically abnormal mitochondria that sometimes contain paracrystalline inclusions. MELAS may be distinguished from other mitochondrial diseases thanks to the identification of a large proportion of ragged-red fibers, with variable activity of COX and the presence of strongly succinate dehydrogenase-stained vessels (79). Ragged-red fibers are commonly seen in patients with MELAS, myoclonus epilepsy with ragged-red fibers (MERRF), Kearn-Sayre syndrome, and chronic progressive external ophthalmoplegia, and are absent in patients with Leber hereditary optic neuropathy, neuropathy, ataxia, and retinitis pigmentosa, and some nuclear defects associated mitochondrial diseases. Exceptionally, in a Chinese study, patients negative for ragged-red fibers were identified with similar disease course, clinical symptoms, and neuroimaging results to ragged-red fibers-positive patients with MELAS, suggesting that in cases of highly suspected MELAS, yet without positive myopathological findings, combined immunofluorescence and genetic studies should be used to achieve final diagnosis (81).

Pathological studies have demonstrated a spongiform encephalopathy predominantly in the cerebral cortex (128). These cortical lesions are characterized by necrosis, loss of neurons, gliosis, and microcystic formations (status spongiosus). Subcortical white matter may be involved, but deep white matter is usually spared. Other regions of the brain that can be affected include the cerebellum, pontine nuclei, and inferior olives. Mineral deposits of calcium and iron have been noted in and around blood vessel walls in the basal ganglia and thalamus. Immunohistochemical staining for mtDNA-encoded subunits of the respiratory chain enzymes revealed decreased staining in multiple focal areas of the brain, predominantly in the cerebral cortex (129). Abnormal and excessive mitochondria were observed in smooth muscle and endothelial cells of small arterioles (less than 250 microns in diameter) and capillaries of MELAS brains (105); this vasculopathy may contribute to the pathogenesis of stroke-like episodes. In addition to endothelial dysfunction, diseased endothelial cells were found to be proatherogenic and proinflammation due to high levels of reactive oxygen species and Ox-LDLs, and high basal expressions of VCAM-1, in particular isoform b, respectively. Consistently, more monocytes were found to adhere to MELAS endothelial cells as compared to the isogenic control, suggesting the presence of an atherosclerosis-like pathology in MELAS (111).

Autopsy studies of brains from two patients with MELAS provided further evidence of vasculopathy as it revealed cytochrome c oxidase-deficiency with high levels (95% or higher) of the m.3243A> G mtDNA mutation in leptomeningeal and cortical blood vessels (07). An in vitro study suggests that the m.3243A> G mutation impairs integrity of the blood-brain barrier (20). Large extracranial blood vessels may also be affected in patients with MELAS as demonstrated in a patient with the common m.3243A> G mutation who had a fatal aortic dissection and whose mother also died of large vessel rupture (135).

Postmortem brain tissues from four patients with MELAS showed reduced hippocampal expression of calbindin D, a high affinity calcium-binding protein. As similar reductions have been associated with aging and neurodegenerative disorders, including Alzheimer disease, this may play a role in the cognitive abnormalities associated with MELAS (30).

The pathogenesis of MELAS is not fully understood and believed to result from several interacting mechanisms including impaired mitochondrial energy production, microvasculature angiopathy, nitric oxide (NO) deficiency, and neuronal hyperexcitability. Nitric oxide deficiency in MELAS syndrome is likely to be multifactorial in origin, with the decreased availability of the nitric oxide precursors, arginine and citrulline, playing a major role. When compared to control subjects, children with MELAS syndrome were found to have lower nitric oxide production, arginine flux, plasma arginine, and citrulline flux. Arginine supplementation and more so citrulline supplementation resulted in increased nitric oxide production, arginine flux, and arginine concentration. The greater effect of citrulline in increasing nitric oxide production is due to its greater ability to increase arginine availability, particularly in the intracellular compartment in which nitric oxide synthesis happens. Controlled clinical trials to assess the therapeutic effects of arginine and citrulline on clinical complications of MELAS syndrome are needed (27). In addition, neuronal hyperexcitability may play a crucial role in the acute stroke-like lesions (53). According to this, Sakai and colleagues reported a case of a patient in which treatment with the anticonvulsant levetiracetam was effective against the stroke-like episodes (118). The neuronal hyperexcitability theory primarily supposes the astrocytic dysfunction due to ATP deficiency as the underlying mechanism: astrocytic dysfunction causes defective uptake of glutamate through glutamate transporters and of potassium through Na+/ K+ ATPase into astrocytes and the elevation of glutamate and potassium concentration in the synaptic clefts that can induce not only vasodilatation in small vessels but also neuronal death by excitotoxicity.

In addition, neuronal hyperexcitability may play a role in the acute stroke-like lesions (53), even if the exact pathophysiology of acute cortical lesions in MELAS remains unclear. According to the cytopathic theory, defects in oxidative phosphorylation resulting from the mitochondrial mutation cause neuronal and glial cellular dysfunction, which may result in cell death during periods of higher metabolic activity. This theory is also supported by the higher baseline metabolic activity in the occipital cortex, the area in which the presence of lesions is more frequent. Otherwise, the angiopathic theory proposes that abnormal mitochondrial function in the arteriolar endothelium results in impaired autoregulation and subsequent ischemia (08). A detailed histopathological study of the temporal bone of a MELAS patient indicated that degeneration of the stria vascularis and spiral ganglion cells cause sensorineural hearing loss (132).

The pathomechanism whereby defects in mitochondrial DNA cause the histological and clinical features of MELAS is not known. Muscle cells with the m.3243A> G mutation grown in tissue culture demonstrate respiratory deficiency (68). King and Attardi developed a cell line called rho-0 that grows in the absence of mtDNA (64). The rho-0 cells can be fused with cytoplasts harboring mutant mtDNA forming cybrids. Cybrids with greater than 95% m.3243A> G mtDNA showed decreased rates of protein synthesis, lower levels of steady-state mitochondrial translational products, reduced oxygen consumption, and increased amounts of an unprocessed RNA fragment containing the mutant gene (designated RNA-19) (65). Kaufmann and colleagues confirmed the presence of elevated levels of RNA-19 in vivo by studying tissues from patients with MELAS (61). Other investigators have demonstrated that high levels of the mutant tRNA decreased aminoacylation (covalent attachment of leucine to the tRNA) and were associated with hypomodification of the D-stem; these alterations may contribute to the decreased protein synthesis (47; 10; 16; 108). An alternative theory, also based on cybrid work, is that the mutant tRNALeu (UUR) is less efficiently modified at the base corresponding to the wobble base of the codon, thereby leading to misreading of leucine codons as phenylalanine codons (147) due to lack of a post-transcriptional methyl-taurine modification of the anticodon wobble base (67). Cultured myoblasts from a patient with the m.3243A> G mutation revealed evidence of both amino acid misincorporation and moderate reduction of protein synthesis (122).

Obviously, the mitochondrial dysfunction in MELAS leads to complex mitochondria-to-nucleus signaling pathways whose components are, however, mostly unknown. It was demonstrated that oxidative stress mediates an NFkB-dependent induction of microRNA-9/9*, which acts as a post-transcriptional negative regulator of the mt-tRNA-modification enzymes GTPBP3, MTO1, and TRMU (94). Down-regulation of these enzymes affects the modification status of non-mutant tRNAs and contributes to the MELAS phenotype. Importantly, anti-microRNA-9 treatments of MELAS cybrids reverse the phenotype. These data represent the first evidence that an mt-DNA disease can directly affect microRNA expression and that cells respond to stress by modulating the expression of mt-tRNA-modifying enzymes. Hence, microRNA-9/9* is a crucial player in mitochondria-to-nucleus signaling as it regulates expression of nuclear genes in response to changes in the functional state of mitochondria (93).

Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation can be reproduced in human induced pluripotent stem cells (43). Of note, complex I was specifically sequestered in perinuclear PTEN-induced putative kinase 1 (PINK1) and Parkin-positive autophagosomes on neuronal differentiation, suggesting active degradation through mitophagy. These data show that cellular context actively modifies respiratory chain deficiency in MELAS and that autophagy is a significant component of neuronal MELAS pathogenesis.

Epidemiology

Patients with MELAS have been found worldwide. This disorder has no known ethnic predilection. An Italian work observed that male gender could represent a risk factor for the development of stroke-like episodes in m.3243A>G patients (85).

An epidemiological study of a northern province of Finland estimated the prevalence of the m.3243A>G MELAS mutation is at least 18.4:100,000 (95% confidence interval, 10.9-29.1:100,000) (139). In a large Caucasian population in Australia, the population prevalence was 236/100,000 (ie, approximately 1 in 400 people) (89). Apart from the classical MELAS phenotype, there are protean manifestations of the m.3243A>G mutation. In the largest cohort study to date comprising 129 mutation carriers, 10% exhibited classical MELAS, 30% had mitochondrially inherited diabetes and deafness (MIDD), 6% MELAS/MIDD, 2% MELAS/chronic PEO (CPEO), and 5% MIDD/CPEO overlap syndromes (101). Isolated sensorineural hearing loss occurred in 3%, and 6% had PEO and other mixed features. Twenty-eight percent of patients demonstrated a panoply of clinical features, which were not consistent with any of the classical syndromes. Nine percent of individuals harboring the mutation were clinically asymptomatic.

Prevention

To investigate the applicability of preimplantation genetic diagnosis (PGD), trophectoderm biopsy was used to determine the mutation load in a 35-year-old female with MELAS (46). Transfer of a mutation-free blastocyst gave birth to a healthy boy with undetectable mutation in any of the analyzed tissues. This case supports the applicability of blastocyst PGD for patients with mtDNA disorders to establish healthy offspring.

Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA (mitochondrial replacement) may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease (52).

Differential diagnosis

When a patient with MELAS presents with an acute stroke-like episode, the differential diagnosis includes other causes of stroke in a young person: heart disease, carotid or vertebral artery disorders, sickle cell anemia, vasculopathies, lipoprotein disorders, cancer, venous thrombosis, moyamoya disease, complicated migraine, and homocystinuria. Because there is often an antecedent history of migraine headache and of headache with the acute stroke, patients with MELAS might be diagnosed as having migraine with prolonged aura, basilar migraine, or hemiplegic migraine. In one patient with MELAS, the angiographic findings were compatible with the diagnosis of moyamoya disease (73).

A MELAS-like phenotype with defects in nuclear genes including MRM2, FASTKD2, and POLG has been reported, suggesting the genetic screening of these genes in patients with a m.3243A>G negative MELAS-like presentation (15; 35; 150).

When patient has a family history concerning for maternal mitochondrial inheritance, physicians should consider other mitochondrial diseases as possible diagnosis, especially when clinic presentations of disorders may be similar. For example, Kearns-Sayre syndrome, myoclonus epilepsy associated with ragged-red fibers (MERRF) or Leigh syndrome are mitochondrial disorders that may present, depending on the disease, with visual disturbance, short stature, hearing loss, ataxia, cardiac involvement, seizures, mental deterioration, and myopathy with ragged-red fibers on biopsy. Thus, to dispel any doubt, genetic testing represents a fundamental diagnostic item (112).

Diagnostic workup

Screening patients for MELAS should begin with routine blood tests including complete blood count, serum electrolytes, liver function tests, blood urea nitrogen, creatinine, lactate, and pyruvate. These tests may reveal kidney or liver dysfunction. Lactate and pyruvate at rest are commonly elevated in patients with MELAS; these values may increase dramatically after moderate exercise. The blood leukocyte DNA may be screened for an mtDNA point mutation. The m.3243A>G mutation is detectable in about 80% of patients with MELAS. Age-adjusted blood heteroplasmy may be the most convenient heteroplasmy measure to use routinely within the clinical setting (42). Urine heteroplasmy levels should be adjusted for sex and are highly variable, and thus, must be interpreted with caution, even if urinary epithelial cells show m.3243A>G mutation levels that are comparable to levels in skeletal muscle and higher than in leukocytes (126). Therefore, urinary epithelial cells may be the tissue of choice to confirm the diagnosis of MELAS without an invasive procedure (24).

MELAS has a higher prevalence of ECG abnormalities compared with other mitochondrial clinical syndromes, and the m.3243A>G mutation is associated with a higher prevalence of electrocardiogram abnormalities than other genetic defects (113). Electrocardiogram may reveal pre-excitation or incomplete heart block. The latter is much less common than in Kearns-Sayre syndrome; there are no reports of complete heart block in MELAS. Lumbar puncture may show elevated cerebrospinal fluid protein, but only one case was reported to have a protein greater than 100 mg/dL. Electromyography and nerve conduction studies are typically consistent with a myogenic process, although a neuropathy may also be present.

Brain imaging with CT or MRI scans often reveals lesions compatible with strokes, frequently affecting the posterior cerebrum, and not conforming to the distribution of major arteries; in addition, basal ganglia calcification and atrophy are commonly seen (03; 01; 90). A brain magnetic resonance spectroscopy study of MELAS patients and their relatives demonstrated a direct correlation between the neurologic impairment and cerebral lactic acidosis (63). Moreover, abnormal cortical vein T2/FLAIR signal has been observed in patients with MELAS, correlating with cumulative brain lesion severity but not acuity. As venous hyperintensity was present prior to, during, and after lesion onset, it may even be predictive of lesion development. Possible mechanisms of brain injury include cortical venous stenosis, congestion, and venous ischemia (145).

By 7T magnetic resonance spectroscopy (MRS), it was possible to noninvasively quantify intramyocellular lipid accumulation (38). This may serve as a novel biomarker in the future.

In case of suspicion of stroke-like episode, physicians should obtain a detailed history from the patient or caregiver. Focal neurologic deficit that shows which hyper-acute onset, especially pure motor weakness (facial weakness or hemiparesis) that evolves rapidly within minutes, is unusual for a mitochondrial stroke-like episode and more suggestive of a vascular stroke. Otherwise, complex visual symptoms, perceptive problems, and hearing disturbances persisting for hours or days are features more suggestive of mitochondrial stroke-like episode. It is also important to try to identify potential triggers such as infection, gut dysmotility, dehydration, prolonged fasting, and nonadherence to the antiepileptic drug(s). For any suspected stroke-like episodes in patients who are known to harbor primary mtDNA or recessive POLG pathogenic variants, physicians should perform MRI head scan (proposed protocol: T1, T2, FLAIR, DWI, and ADC; if MRI head is contraindicated, CT head can be performed), electroencephalogram (EEG), blood and laboratory tests, chest radiograph (if aspiration pneumonia suspected), abdominal radiography (if intestinal pseudo-obstruction is suspected), and electrocardiogram (102).

Finally, skeletal muscle biopsy can be performed to confirm the diagnosis. Ragged-red fibers on modified Gomori trichrome stain are the hallmark histological feature. The ragged-red fibers often show positive histochemical staining for cytochrome c oxidase activity. In addition, other features characteristic of abnormal mitochondria may be found. Mitochondrial enzyme activities can be measured in whole muscle homogenate, or in isolated mitochondria. The muscle mtDNA should be screened for the mutations associated with MELAS.

Management

No treatment for the genetic defect is currently available. Coenzyme Q10 (50 to 100 mg 3 times a day) and L-carnitine (330 mg 3 times a day) have been used to improve mitochondrial function. Idebenone, another quinone compound similar to coenzyme Q10, has the theoretical advantage of crossing the blood-brain barrier and has been reported to be beneficial in isolated patients with MELAS; a study suggested that the benefit use of idebenone is due to one of its longer-lasting metabolites, QS-10, QS-6, and QS-4 (37). β-Lapachone, a natural quinone compound, was found to attenuate mitochondrial dysfunction in MELAS cybrid cells more efficiently than the quinones idebenone and CoQ10 that are already in clinical use (56). β-Lapachone restored energy production and mitochondrial membrane potential, normalized the elevated ROS level, reduced lactic acidosis, and activated Sirt1 by increasing the intracellular NAD(+)/NADH ratio, which was accompanied by increased mtDNA content. Further studies are needed to establish the efficacy of this compound in vivo.

Dichloroacetate inhibits the pyruvate dehydrogenase-specific kinase, thereby activating pyruvate dehydrogenase complex and reducing lactate levels. Unfortunately, a double-blind randomized placebo-controlled trial of DCA showed no treatment benefit and the trial was terminated due to frequent peripheral nerve toxicity (59).

Supplementation with L-cysteine (which is required for the 2-thiomodification of mitochondrial tRNAs) partially rescued the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations (05). Clinical studies on L-cysteine have not yet been performed.

In cybrid cells with almost homoplasmic m.3243A>G mutation, the addition of ketone bodies for 4 weeks significantly restored complex I activity and ATP synthesis, and it increased mtDNA copy number. Hence, ketogenic diet may be a promising therapy for MELAS (32).

L-arginine, a nitric oxide precursor, has been used to stimulate vasodilation in MELAS (72; 71). In open-label studies, intravenous L-arginine administered shortly after the onset of stroke-like episode seemed to improve outcomes whereas oral administration during interictal phases appeared to decrease the frequency and severity of stroke-like events (72; 71). Maintaining plasma arginine concentration at least 168 mmol/l has been reported to prevent the ictuses through the putative pathophysiologic mechanism and optimal normalization of endothelial dysfunction (54). There is even more improvement in nitric oxide production with arginine and citrulline supplementation (27; 26). Interestingly, NO production seems to become measurable now by use of a stable isotope tracer infusion technique (28). Controlled studies assessing the potential beneficial effects of arginine or citrulline supplementation, their optimum dosing, and safety ranges in stroke-like episodes of MELAS syndrome are urgently required.

In the meantime, recommendations for the treatment of stroke-like episodes have been published by the Mitochondrial Medicine Society (107; 69):

	• Patients with known MELAS who present with any symptoms suggestive of a metabolic stroke should receive a loading dose of intravenous arginine hydrochloride. Although the optimal dose has not been defined, a bolus of 0.5 g/kg given within 3 hours of symptom onset is recommended.
	• After the initial arginine bolus, an additional 0.5 g/kg should be administered as a continuous infusion for 24 hours for the next 3 to 5 days.
	• Patients may be transitioned to oral L-arginine at a 1-to-1 dose once swallowing safety is ensured and they are able to tolerate oral intake.
	• Prophylaxis: Once a patient with MELAS has experienced a first stroke, arginine should be administered prophylactically to reduce the risk of recurrent stroke-like episodes. A daily dose of 0.15 to 0.30 g/kg administered orally in three divided doses is recommended.
	• Moreover, in the acute episode, the patient should be given dextrose-containing fluids as soon as possible to reverse ongoing or impending catabolism (even in the setting of euglycemia). In the acute stroke-like episode, patients should immediately undergo electroencephalography to assess for subclinical status epilepticus.
	• Seizures respond to conventional anticonvulsant therapy; there is no known drug regimen of choice.

A clinical trial demonstrated that oral supplementation with high-dose taurine was effective in preventing stroke-like episodes and was able to ameliorate taurine modification defect in mitochondrial tRNALeu(UUR) in peripheral blood leucocytes in vivo (106). However, as for arginine, no adequately controlled clinical studies determining the efficacy of these compounds in treating or preventing stroke-like episodes have been published.

In 2018, in Newcastle, a European group of experts met to develop a consensus-based guidance for the management of mitochondrial stroke-like episodes (102). The consensus recommends managing patients with stroke-like episodes at the intensive care unit in the following circumstances:

	• Generalized, convulsive status epilepticus
	• Focal motor seizures with breakthrough generalized seizures, which fail to respond to intravenous AEDs
	• Severe encephalopathy with a high risk of aspiration
	• Focal motor status epilepticus with retained consciousness failing to respond to benzodiazepine and other intravenous AEDs

Possible manifestations that may be observed during a stroke-like episode are focal seizures (including nonmotor seizures such as occipital seizures) with or without evolution to bilateral convulsive seizures. In case of first episode, an early administration of benzodiazepine if the patient is not in hospital should be considered. Once in hospital patients should be treated urgently with an intravenous antiepileptic drug such as intravenous levetiracetam (20-40 mg/kg, max 4500 mg), phenytoin (15-20 mg/kg with cardiac monitoring), phenobarbitone (10-15 mg/kg with respiratory monitoring), or lacosamide (200-400 mg).

Midazolam is the first choice of general anesthetics agent for treating refractory status epilepticus associated with stroke-like episodes.

Sodium valproate is contraindicated for patients with POLG mutations and should also be avoided in patients with MELAS epilepsy caused by other genotypes if an alternative drug is available and efficient.

Maintenance intravenous fluid should be administered for patients who are at risk of dehydration, especially in those with encephalopathy or those presenting with vomiting due to intestinal pseudo-obstruction. A better response to rehydration may be observed in subjects with elevated plasma lactate.

Sodium bicarbonate can be used with care in severe lactic acidosis (pH < 7.1). However, the management of severe metabolic acidosis needs a multidisciplinary approach (ie, with intensivist or nephrologist). As mentioned above, the use of dichloroacetate on lowering lactic acid is not recommended because it may cause toxic neuropathy in patients with MELAS.

Hearing loss caused by isolated cochlear dysfunction has been successfully treated with cochlear implantation (131; 124). Aminoglycosides and platinum-based anticancer drugs, due to the increase of reactive oxygen species levels following their administration, may precipitate the development of hearing loss and should be used with caution. Consequently, mitochondria-targeted antioxidants could be useful for the prevention and treatment of diseases associated with mitochondrial dysfunction, although clinical trials are necessary to confirm the otoprotective role of these supplements in patients (33).

Special considerations

Pregnancy

Infertility may preclude this issue in some patients. Women with mtDNA point mutations are at risk of passing the mutation to their children. Prenatal testing of the fetus is of uncertain utility. In a small study of nine pregnancies in five women from families with the m.3243A>G mtDNA mutation, mutation loads in amniocytes and chorionic villi were stable at two to three stages of pregnancy (09). Eight pregnancies with fetal mutation levels 35% or lower mutation produced healthy children who have been followed for 3 months to 6 years, whereas the pregnancy with 63% mutation in the fetus was terminated. Additional studies are necessary to establish the value of prenatal testing.

In a retrospective cohort study on obstetric complications in carriers of the m.3243A>G mutation, 98 pregnancies in 46 women were reported. Of those, 25.3% had premature delivery (including 5.5% with gestation ≤ 32 weeks), 12% suffered from preeclampsia, and 11% were complicated by gestational diabetes. Hence, proper guidance during pregnancies and early detection of possible obstetric complications are needed in patients with MELAS and carriers. As techniques to prevent transmission of mitochondrial mutations are studied, it is important to know the possible complications patients may experience from the ensuing pregnancy (22).

Anesthesia

In theory, drugs that lower seizure threshold should be avoided. Because propofol can inhibit the mitochondrial respiratory chain, some authors have advocated avoidance of this anesthetic agent in patients with mitochondrial diseases (91); however, patients with MELAS have received propofol without complications (109). We recommend midazolam as the first choice of general anesthetics agent for treating refractory status epilepticus associated with stroke-like episodes.

The greater susceptibility of patients affected by mitochondrial diseases to developing lactic acidosis should be considered by physicians during surgical procedures and perioperative fasting, which may be possible metabolic stress factors and exacerbate this condition. Routine, perioperative use of lactate-free intravenous fluids (such as 5% dextrose-0.9% saline) is recommended in all patients with mitochondrial diseases undergoing general anesthesia, including those affected by MELAS.