Mitochondrial Disorders Affecting the Nervous System

R.H. Haas, MB, BChir; Z. Zolkipli, MB, ChB

Disclosures

Semin Neurol. 2014;34(3):321-340. 

In This Article

Classical Phenotypes

Leigh Syndrome

Leigh syndrome (LS; Online Mendelian Inheritance in Man, OMIM #256000) or subacute necrotizing encephalopathy is a genetically heterogeneous neurodegenerative disorder associated with defects involving mitochondrial oxidative phosphorylation (OXPHOS). Baertling et al have recently proposed a definition based on three of the most commonly described and characteristic aspects: (1) neurodegenerative disease with variable symptoms due to (2) mitochondrial dysfunction caused by a hereditary genetic defect accompanied by (3) bilateral CNS lesions that can be associated with further abnormalities in diagnostic imaging.[79] Disease onset is mainly in infancy or early childhood.[19,80–95] Prenatal[19,96] and late onset in adolescence and adulthood have been described.[19,80,97,98] The initial presentation is often triggered by intercurrent illness. The subsequent disease course is stepwise deterioration and psychomotor regression, precipitated by episodic metabolic decompensation commonly triggered by infection, prolonged fasting and any other factors that increase metabolic demands; there may be elevated blood and/or CSF lactate.[99] Neurologic features include hypotonia, spasticity, dystonia, ataxia, seizures, developmental delay, developmental regression, ptosis, nystagmus, and poor feeding. In most cases, clinical deterioration leads to death by 3 years of age commonly from central respiratory failure,[95,99] although survival into adulthood has been described.[82] The clinical presentation can be highly variable, and some patients with Leigh syndrome, can have an atypical course without lactic acidosis or characteristic brain lesions.[100]

Sofou et al recently reported the largest natural history study of LS to date. This was a retrospective study of 150 patients in Europe followed for a median period of 9.6 years from disease onset. The median age of onset was 7 months and 80% had presented by the age of 2 years.[19] The most frequent clinical features at presentation in descending order of frequency were motor and ocular abnormalities, feeding difficulty, seizures and failure to thrive.[19] The commonest motor abnormalities were hypotonia, abnormal deep tendon reflexes, and dystonia. The ocular abnormalities most frequently seen were nystagmus, strabismus, visual impairment, optic atrophy, ptosis, and ophthalmoplegia.[19] Seizures occurred in 39.2%, respiratory dysfunction (hyperventilation, abnormal breathing pattern, apnea, obstructive or restrictive respiratory disease and central hypoventilation) in 37.7% and cardiac dysfunction (hypertrophic or dilated cardiomyopathy and conduction defects) in 17.7% of patients. Hypertrichosis and peripheral neuropathy, more commonly demyelinating, were found in 41% and 81%, respectively, in a SURF-1 deficient LS patient series, in addition to the most prevalent symptoms of hypotonia and failure to thrive.[95] Mild dysmorphic features have also been described.[79,95]

Sofou et al found that 56.9% of patients experienced at least one acute exacerbation requiring hospitalization in their disease course; intensive care was required in 39.2% of hospitalized patients.[19] Acute exacerbations were related to infection (60.8%), respiratory complications (13.5%), stroke-like episodes (4.0%), and poor nutrition or hydration (4.0%). Onset of disease before 6 months, failure to thrive, brainstem lesions on MRI scan, and intensive care requirement were significant predictors of poor survival. The median age at death was 2.4 years from respiratory complications (51%), disease progression (18%), or infection (17.6%).

The initial description of LS was based on a postmortem diagnosis. The diagnosis has since evolved into a clinical entity characterized by neurodegenerative symptoms and typical radiologic findings, along with biochemical and molecular genetic testing. Lactic acidosis is not always present in LS patients. Of the LS patients in the European cohort with measured lactate levels, 25% had normal (< 2.4 mmol/L) blood and/or CSF lactate levels.[19] With the availability of radiologic, biochemical, and molecular analysis techniques, the diagnosis no longer weighs upon postmortem findings.

Leigh Syndrome Mimics

Awareness of the conditions that could mimic LS is crucial. Metabolic conditions such as the organic acidurias, propionic academia, methylmalonic academia, and Wilson disease can be excluded by plasma and urine determination. Postinfectious acute necrotizing encephalopathy and bilateral striatal necrosis are other examples. The most significant disorders to exclude would be those amenable to treatment. Biotin-responsive basal ganglia disease (BBGD) is an autosomal recessive disorder caused by mutations in the SLC19A3 gene.[101–103] Biotin-responsive basal ganglia disease typically causes subacute episodes with encephalopathy and subsequent neurologic deterioration that responds to thiamin and biotin treatment. Without treatment, patients have been reported with mild to moderate neurologic deficit, and some deteriorate and die.[103] Neuroradiological findings include bilateral signal hyperintensity in the basal ganglia. Biotinidase deficiency has been reported with a Leigh phenotype.[104,105] The creatine deficiency syndromes can also present with bilateral basal ganglia hyperintensities and mimic LS,[106–108] and in the case of AGAT and GAMT deficiencies, symptoms can be amenable to therapy.[109]

If there is clinical suspicion for LS, laboratory investigations should include plasma and/or CSF lactate searching for lactic acidosis; plasma amino acids searching for hyperalaninemia and homocitrullinemia, the latter could suggest the presence of mtDNA mutation T8993G; and urine organic acids searching for generalized organic aciduria that may signify renal proximal tubulopathy, 3-methylglutaconic aciduria as a secondary feature in LS, and methylmalonic aciduria (MMA), which is found in a Leigh-like encephalopathy caused by SUCLA2 and SUCLG1 mutations.[79]

Typical MRI changes in LS are focal, bilateral, and symmetrical lesions on T2-weighted imaging of the basal ganglia, diencephalon, and/or brainstem (Fig. 1B). Magnetic resonance spectroscopy may reveal a lactate peak in the brain parenchyma or CSF. Skeletal muscle biopsy may reveal ragged red fibers (RRFs) on a modified Gomori trichrome stain, myofibers that fail to stain with the histochemical reaction for cytochrome c oxidase (COX) or paracrystalline inclusions on EM. Biochemical analysis of skeletal muscle biopsies from the quadriceps femoris may reveal OXPHOS deficiencies. Isolated deficiency of complex I is the most common cause of OXPHOS disease, resulting in variable phenotypes affecting infants and adults and LS is one such phenotype. Isolated complex I deficiency is the most frequently observed biochemical abnormality in LS, accounting for 34% of cases.[100] Sofou et al identified abnormal electron transport chain (ETC) activity in 70% of LS patients, the most prevalent being complex I deficiency in 25 of 77 patients (32.5%).[19] A muscle biopsy may be avoided if the clinical and biochemical phenotype is suggestive of a particular gene mutation, or if pyruvate dehydrogenase (PDH) deficiency is suspected, for which enzymatic analysis can be performed on fibroblasts from a skin biopsy.

A reflexive testing approach to molecular analysis is helpful. Peripheral blood or if available, skeletal muscle should first be screened for mtDNA deletions and common point mutations. If negative, the next step would be mtDNA sequencing, followed by nuclear DNA sequencing or whole exome sequencing. Leigh syndrome can be maternally inherited due to mutations in genes encoded by mitochondrial DNA (mtDNA).[99] More often, LS is inherited in a Mendelian manner that includes X-linked forms due to PDH deficiency or autosomal recessive inheritance due to mutations in nuclear genes encoding mitochondrial respiratory chain complex subunits or complex assembly proteins. Mutations causing disturbances in CoQ10 metabolism or dysregulation in mitochondrial RNA/DNA maintenance may also cause LS.[110]

Genetic etiology was confirmed in 77 of the 150 European LS patients (59.2%): Nuclear DNA mutations were more common than mitochondrial DNA mutations (37.7% and 21.5%, respectively).[19] Among the nuclear genes in which pathogenic mutations have been identified are NDUFS1, NDUFS4, NDUFS7, NDUFS8, NDUFV1, SDHA1, SURF1, LRPPRC, GFM1, TACO1, PDSS2, FOXRED1, and BCS1L.[110,111]

Neuropathy Ataxia and Retinitis Pigmentosa

Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) (OMIM 551500) was first described in a family heteroplasmic for the m.8993T > G mutation in the MT-ATP6 gene.[112] Although several mutations throughout MT-ATP6, which encodes subunit 6 of ATPase, have been associated with LS, only mt.8993T > G and mt.8993T > C are established causes of the classical NARP phenotype.

Neuropathy ataxia and retinitis pigmentosa is characterized by retinal, central, and peripheral nervous system neurodegeneration.[113] The clinical features are a length-dependent sensorimotor axonal polyneuropathy, ataxia, retinitis pigmentosa that manifests clinically as nyctalopia, sensorineural hearing loss, seizures, and cognitive deficit. Other described clinical features include short stature, progressive external ophthalmoplegia, cardiac conduction defects and a mild anxiety disorder. Visual symptoms may be the only clinical feature.[99] The clinical course of NARP may be stable and slowly progressive over decades.

The m.8993T > C and mt.8993T > G mutations are associated with variable disease phenotype within a clinical spectrum ranging from NARP to childhood maternally inherited Leigh syndrome (MILS). Some affected individuals with NARP are often diagnosed after their children have been found to have LS.[62] Mitochondrial heteroplasmy may explain some of this phenotypic variability. Greater degrees of mutant heteroplasmy tend to lead to more severe clinical deficits.[9,114] In NARP/MILS, the risk of developing severe functional disability as is the case in MILS increases greatly past a threshold of 60% to 70% mutant blood heteroplasmy for the m.8993T > G ATPase 6 mutation and 80% to 90% blood heteroplasmy for the m.8993T> C mutation.[115,116] However, there is both intra- and interfamilial phenotypic variability, even in families with the same mutation and similar levels of heteroplasmy. The disease phenotype is reported to be milder with the m.8993T > C point mutation.

The MRI scan in NARP may show pontocerebellar atrophy. The skeletal muscle biopsy may show myopathic changes on histopathology, and any of the abnormalities on histochemistry or EM as outlined above for LS. Analysis of mtDNA targeted to the NARP common point mutations can be performed in peripheral blood and if absent, in skeletal muscle. The absence of deletions in peripheral blood does not exclude the diagnosis, as the pathogenic mutation may be undetectable in mtDNA from leukocytes.

Kearns-sayre Syndrome and Chronic Progressive External Ophthalmoplegia

Kearns–Sayre syndrome (KSS; OMIM #530000) is at the severe end of the spectrum of mitochondrial deletion disease. Kearns–Sayre syndrome is a multisystem disorder defined by the triad of onset before age 20 years, pigmentary retinopathy, and progressive external ophthalmoplegia (PEO). In addition, affected individuals have at least one of the following: cardiac conduction block, CSF protein concentration >100 mg/dL, or cerebellar ataxia. Other clinical features include nystagmus, ataxia, corticospinal dysfunction, kyphoscoliosis, bulbar, and limb girdle muscle weakness. Kearns–Sayre syndrome is also associated with a variety of endocrinopathies such as short stature, gonadal failure, diabetes mellitus, thyroid disease, hyperaldosteronism, hypomagnesaemia, and hypoparathyroidism.[117,118] Kearns–Sayre syndrome is usually heralded by ptosis and muscle weakness followed in a few years by PEO. Neurodegeneration with ataxia and dementia is common with spongiform white matter changes typically found.[20,31] Multisystemic involvement at presentation is less frequent and is associated with rapid disease progression.[119,120] The disease usually progresses to death in young adulthood.[121] There are no natural history studies to date.

Kearns–Sayre syndrome typically causes cardiac conduction defects, progressing to complete heart block manifesting as congestive heart failure, syncope, or sudden death in up to 57% of patients.[118] Abnormalities in cardiac conduction might begin with left fascicular block with or without right bundle branch block. Patients with KSS might have an unpredictable progression to complete heart block with an associated mortality of 20%.[118] Annual surveillance including an electrocardiogram and echocardiogram is required. If heart block is detected, early intervention with a pacemaker is warranted.

The phenotype varies from mild myopathy to KSS syndrome in deletion disease. Progression and symptomatology is correlated with the degree of heteroplasmy and size of the deletion.[122] Kearns–Sayre syndrome is due to single, large-scale deletions of mtDNA and is almost always sporadic.[123] More than 150 mtDNA deletions have been associated with KSS.[121] Approximately 90% of individuals with KSS have a large-scale (i.e., 1.1- to 10-kb) mtDNA deletion that is usually present in all tissues; however, mutant mtDNA is often undetectable in blood cells, necessitating examination of muscle.[121] A 5 kb "common" deletion is present in approximately one-third of patients.[124] Pearson's marrow-pancreas syndrome (PS), characterized by pancreatic, hepatic, and renal insufficiency with refractory sideroblastic anemia, is diagnosed early in life and usually fatal in infancy. Some of the patients that survive this disease develop KSS.[125,126] It is well established that single large mtDNA deletions are generally the cause of KSS, PS, and chronic progressive external ophthalmoplegia (CPEO) syndromes.

The diagnosis of KSS is based on clinical features and laboratory investigations. Lactic acidosis may be present. There may be a myopathic appearance on EMG. A nerve conduction study may reveal a neuropathy. The brain MRI may show bilateral basal ganglia lesions, and cerebral or cerebellar atrophy.[121] Skeletal muscle biopsy typically shows RRFs with the modified Gomori trichrome stain and hyperactive fibers with the SDH stain. Both RRFs and some non-RRFs fail to stain with the histochemical reaction for COX (Fig. 4).[121] MtDNA deletion may be found in blood by long-range-polymerase chain reaction analysis or Southern blot testing, but muscle is usually required. There may be OXPHOS deficiencies on biochemical analysis. A duplication/deletion mtDNA analysis should be performed on peripheral blood, and if absent, on skeletal muscle to identify mtDNA deletions. The absence of deletions in peripheral blood does not exclude the diagnosis.

Chronic progressive external ophthalmoplegia is characterized by slowly progressive, often asymmetric ptosis and limitation of ocular motility in all directions of gaze.[127] This condition typically starts in childhood or early adulthood with a bilateral ptosis in up to 90% of patients. The ptosis can be asymmetric at onset.[128] Ptosis typically precedes ophthalmoparesis by months to years and may progress to become complete. As the disease progresses, the ptotic eyelids occlude the pupils and interfere with vision; patients may adopt a backward head tilt, accompanied by elevation of the frontalis muscles, to compensate.[129] Orbicularis oculi muscles may also become weak in CPEO, resulting in lagophthalmos and ectropion and predisposing patients to exposure keratopathy.[129] The ophthalmoparetic process is painless and affects all extraocular muscles symmetrically, and patients do not commonly complain of diplopia until a convergence insufficiency on fixation develops. Visual acuity and papillary function is usually not affected in CPEO. Skeletal muscle weakness is present in most patients with CPEO and may involve the neck, limb, or bulbar musculature, with bifacial weakness the rule.[129]

Chronic progressive external ophthalmoplegia is the most common manifestation of mitochondrial syndromes associated with the presence of single or multiple deletions in mitochondrial DNA. Patients with CPEO have variable presentations ranging from pure CPEO to a CPEO "plus" syndrome with other accompanying multisystem features of mitochondrial disease,[128] commonly KSS. Differential diagnoses include myasthenia gravis, oculopharyngeal muscular dystrophy, and myotonic dystrophy.

A skeletal muscle biopsy may show abnormalities on histopathology, histochemistry, and/or EM suggestive of mitochondrial disease. As in the case of KSS, the molecular diagnosis of PEO requires analysis of a muscle biopsy. Chronic progressive external ophthalmoplegia is either due to a point mutation or a single, large-scale mtDNA deletion or multiple mtDNA deletions secondary to a nuclear mutation disrupting mtDNA replication or repair. The nuclear genes that have been implicated in CPEO include TP, which encodes for thymidine phosphorylase, resulting in the autosomal recessive disorder, MNGIE. Mutations in ANT1, Twinkle, POLG1, POLG2, and OPA1 have been implicated in autosomal dominant PEO (adPEO).[129] RRM2B mutations have been reported in adult-onset adCPEO[130] and arCPEO[131] recently, SPG7 mutations have been identified in sporadic adult-onset CPEO with spastic ataxia.[132]

Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS; OMIM #540000) is an often devastating multisystem syndrome characterized by progressive encephalopathy and stroke-like episodes, leading to disability and early death.[133] It causes significant morbidity, with the life-time prevalence of stroke-like episodes approaching 99%.[134] The pathophysiology underlying the stroke-like episodes remains questionable.

Onset of symptoms is frequently between the ages of 2 and 10 years, some are delayed between 10 and 40 years,[135] and 90% have disease onset before 30 years of age.[136] In those with disease onset before age 6 years, the most common presenting symptom is developmental delay, in those with onset between 6 and 10 years, it is muscle cramping or pain, and in those with onset within the second and third decades, hearing loss is the commonest presentation.[136]

The diagnosis is based on diagnostic criteria proposed by Hirano et al in 1992: (1) Stroke-like episodes, typically before age 40 years; (2) encephalopathy with seizures and/or dementia; (3) mitochondrial myopathy, evidenced by lactic acidosis and/or RRF on muscle biopsy; and (4) two of the following are required: (a) normal early psychomotor development, (b) recurrent headache, (c) recurrent vomiting.[137] Other features of MELAS are exercise intolerance, GI problems, muscle weakness, dementia, short stature, cardiomyopathy, and sensorineural hearing loss.[137] Psychiatric manifestations such as hallucinations, depression, and behavior difficulties are also prevalent in MELAS. The cumulative residual effects of the stroke-like episodes gradually impair motor abilities, vision, and cognition, often by adolescence or young adulthood.[135]

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes is usually associated with the m.3243A > G mutation in the mtDNA gene MTTL1 encoding mt tRNALeu(UUR).[138] This mutation is predominant in approximately 80% of the patients with MELAS. At least 39 distinct mtDNA mutations have been associated with MELAS, among these are m.3271T > C and m.3252A > G in the MTTL1 gene, and identified with increasing frequency are mutations in ND5 of complex I, the commonest of which is m.13513G > A.[135] Population-based studies suggest the m.3243A > G mutation is the most common disease-causing mtDNA mutation, with a carrier rate of 1 in 400 people.[139] It is important to note that m.3243A > G is responsible for several other syndromes aside from MELAS, which include maternally inherited deafness and diabetes (MIDD), progressive external ophthalmoplegia (PEO), and LS. It is also important to note that maternal relatives without a classical MELAS phenotype may suffer from a variety of clinical symptoms involving the central nervous system, muscle, heart, and the endocrine system. In vitro studies in cultured skin fibroblasts have shown that the m.3243A > G mutation impairs mitochondrial respiratory chain and protein synthesis when a certain threshold (~85%) of mutant mtDNA is exceeded.[140] Because the level of mutant mtDNA varies both among individuals and in different organs and tissues of single individuals, it is thought that the load of mutant mtDNA is in part responsible for the varied clinical expression of mtDNA defects in general and of the M.3243A > G MELAS mutation in particular.[140]

Of 129 symptomatic patients and asymptomatic carriers (age range 11 months–74 years; 39% male) identified to have m.3243A > G from the MRC Centre for Neuromuscular Diseases Mitochondrial Disease Patient Cohort Study UK, 13 patients (10%) exhibited a classical MELAS phenotype, a lower prevalence compared with previous cohorts; 39 (30%) had MIDD, 8 (6%) MELAS/MIDD, 2 (2%) MELAS/CPEO, and 6 (5%) MIDD/CPEO overlap syndromes.[139] Sensorineural hearing loss was evident in 66 patients (51%), and this was an isolated symptom in 4 patients (3%). Diabetes (type I or II) was present in 54 patients (42%). Overall, 36 patients (28%) did not conform to any of the classical syndromes and presented with a diverse phenotypic spectrum that included proximal myopathy (27%), ataxia (24%), migraine (23%), and seizures (18%). Twelve individuals (9%) carrying the mutation were clinically asymptomatic.[139] These results highlight the phenotypic variability of the m.3243A > G mutation and the need to identify individuals with this mutation in anticipation of future disease expression such as diabetes. Kaufmann et al conducted a cross-sectional study of symptomatic and asymptomatic carriers with controls; carrier relatives carrying the m.3243A > G without the full MELAS phenotype displayed a high prevalence of disease manifestations.[141]

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes progresses over years with episodic deterioration related to stroke-like events. In a prospective natural history study of 31 individuals with MELAS (age mean ± standard deviation [SD], 30 ± 15 years at baseline, range 4–61 years, 48% male) and 54 symptomatic and asymptomatic carrier relatives (age 38 ± 17 years, range 4–76 years, 28% male) harboring the m.3243A > G over a follow-up period of up to 10.6 years, neurologic examination, neuropsychological testing, and daily living scores significantly declined in all affected individuals with MELAS, whereas no significant deterioration occurred in carrier relatives.[136] In patients with MELAS, there was a progressive decline in neuropsychological and neurologic findings, worsening MRI abnormalities and progressively increased CNS lactate levels as measured by MRS[136] (Fig. 5). The death rate was more than 17-fold higher in fully symptomatic individuals compared with carrier relatives. The average observed age at death in the affected MELAS group was 34.5 ± 19 years (range 10.2–81.8 years). Of the deaths, 22% occurred in those younger than 18 years and were attributed to MELAS-related medical complications in all patients except for three patients. Seizures, stroke-like episodes, sepsis, and GI pseudo-obstruction were noted during the end-of-life period. In the three patients in whom death was considered sudden and unexpected, two were found to have hypertrophic cardiomyopathy on postmortem, and one had status epilepticus and was on medication for Wolff-Parkinson-White syndrome. The estimated overall median survival time based on fully symptomatic individuals was 16.9 years from onset of focal neurologic disease, for example, seizures or stroke.[136]

Figure 5.

Magnetic resonance imaging and magnetic resonance spectroscopy of 12-year-old boy with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) due to m.3243A > G mutation at 90% in blood. (A) T2-weighted magnetic resonance image shows atrophy with ventriculomegaly, caudate nucleus and putaminal bright signal, and bilateral occipital subacute metabolic strokes in a nonvascular distribution, and parietal and left frontal stroke lesions. (B) Magnetic resonance spectroscopy (voxel in posterior paramedian parietal lobe) shows high lactate doublet (1.3 ppm) and a significant decreased N-acetylaspartate (2.0 ppm)/creatine (3.2 ppm) ratio.

Stroke-like lesions occur in approximately 90% of MELAS patients and correlate with focal neurologic symptoms.[138] During stroke-like episodes, the MRI brain scan reveals areas of T2 prolongation, with predominant involvement of the cerebral cortex, predominantly in the occipital and parietal lobes and not confined to vascular territories and sparing of the deep white matter[142] (Fig. 5). An EMG may show myopathy and a nerve conduction study frequently shows neuropathy, and is predominantly axonal and sensory.[135] Muscle biopsy often shows RRFs on the modified Gomori trichrome stain, "ragged blue fibers" due to the hyperintense reaction with SDH,[135] COX-positive RRFs, and succinate dehydrogenase reactive vessels.[143] Electron transport chain activity may be normal, or a deficiency may be detected. Molecular analysis targeted to the common point mutations could first be performed on peripheral blood. If negative, other tissues such as saliva, skin fibroblasts, urinary sediment, or preferably skeletal muscle can be tested. If the common point mutations are absent, the next step would be to proceed with full mtDNA sequencing.

Myoclonic Epilepsy With Ragged Red Fibers

Myoclonic epilepsy with ragged red fibers (MERRF; OMIM #545000) is a multisystem disorder that was first described in 1980.[144] Myoclonic epilepsy with ragged red fibers is characterized by myoclonus, which is often the first symptom, followed by generalized epilepsy, ataxia, weakness, and dementia. Onset is usually in childhood, occurring after normal early development.[145] Common findings are hearing loss, short stature, optic atrophy, and cardiomyopathy with Wolff-Parkinson-White (WPW) syndrome. Occasionally pigmentary retinopathy and lipomatosis are observed.

Myoclonic epilepsy with ragged red fibers is associated with various mtDNA point mutations, the most frequent being the 8344A > G change in the transfer RNA lysine,[146] present in 80% of patients with MERRF. This mutation can also be associated with other phenotypes, such as LS and myopathy with truncal lipomas.[147] Three additional mutations, m.8356T > C, m.8363G > A, and m.8361G > A, are present in 10% of affected individuals.[145]

Mancuso et al conducted a retrospective review of a cohort of patients harboring the 8344A.G mutation who were registered in the database of the Nation-wide Italian Collaborative Network of Mitochondrial Diseases.[148] Four of 39 subjects (10.3%) were asymptomatic mutation carriers. The mean age at onset of disease in the 35 symptomatic patients was 30.1 ± 18.4 years (range 0–66; 11/35 with clinical onset before age 16). At the last evaluation, mean age and disease duration were 45.6 ± 15.3 years (range 5–72), and 21.6 6 15.3 years (range 2–59), respectively.[148]

The CNS features observed in decreasing order of frequency were generalized seizures in 35.3%, cerebellar ataxia (23.5%), myoclonus (23.5%), cognitive involvement (5.9%), and stroke-like episodes (2.9%). In this cohort, myoclonus was a less frequent feature compared with a previous report of 61%.[149] In terms of neuromuscular symptoms, muscle weakness was evident in 58.8%, as well as exercise intolerance (44.1%), increased creatine kinase (CK) levels (44.1%), ptosis (29.4%), and ophthalmoparesis (5.9%). Other features included cardiomyopathy with arrhythmia (11.8%), arrhythmia (5.9%), peripheral neuropathy (14.7%), swallow impairment (2.9%), respiratory impairment (2.9%), hearing loss (35.3%), and diabetes mellitus (11.8%).[148]

The authors then conducted a systematic review of the literature and identified 282 previously reported 8344A > G-positive individuals (male/female ratio 0.85; mean age at the last evaluation 34.2 ± 19.1 years).[148] In 321 patients with a mean age of 35 years, the clinical picture was characterized by the following signs/symptoms in descending order: myoclonus, muscle weakness, ataxia (35%–45% of patients); generalized seizures, hearing loss (25%–34.9%); cognitive impairment, multiple lipomatosis, neuropathy, exercise intolerance (15%–24.9%); and increased CK levels, ptosis/ophthalmoparesis, optic atrophy, cardiomyopathy, muscle wasting, respiratory impairment, diabetes, muscle pain, tremor, migraine (5%–14.9%).[148]

Lactic acidosis is frequent in blood and CSF. An MRI scan of the brain often shows atrophy and basal ganglia calcification. A skeletal muscle biopsy may show RRF, hyperactive staining on SDH and COX-negative fibers.[145] Electron transport chain activity may be normal.

Leber's Hereditary Optic Neuropathy

Leber's hereditary optic neuropathy (LHON; OMIM #535000) is characterized by acute, painless loss of central vision, which is bilateral in approximately 25% of cases.[150] If unilateral, the contralateral eye is usually affected within 6 to 8 weeks. The majority of carriers become symptomatic in the second and third decades of life, typically young adult males, and over 90% of carriers who will experience visual failure will have onset before the age of 50 years.[150] However, visual deterioration can occur at any time during the first to the seventh decade of life; LHON should be part of the differential diagnosis for all cases of bilateral, simultaneous, or sequential optic neuropathy, irrespective of age, and particularly in male patients.[150]

The visual loss usually begins with defective color vision and a central scotoma. The loss of visual acuity is profound and levels off below 20/400 within a few months; the visual field defects show large centrocecal absolute scotomas.[151] Funduscopy reveals the characteristic signs of circumpapillary telangiectatic microangiopathy, swelling of the retinal nerve fiber layer (RNFL) around the disc and lack of leakage on fluorescein angiography, consistent with pseudoedema.[151] The optic disc appears hyperemic initially, though the axonal loss in the papillomacular bundle leads to severe temporal pallor of the optic disc, and in time, optic disc atrophy develops (Fig. 3). The endpoint of LHON is usually optic atrophy associated with permanent loss of central vision, although there is relative sparing of pupillary light responses.[152,153] Loss of visual acuity stabilizes within the first year after onset, leaving the patient, in most cases, legally blind. Microangiopathy and fundus changes such as RNFL swelling may be present in asymptomatic matrilineal relatives.[154]

Spontaneous recovery of visual acuity may infrequently occur even years after onset, with contraction of the scotoma or reappearance of small islands of vision within it (fenestration).[151] The most favorable prognostic factors are young age of onset and the 14484/ND6 mutation.[155] Leber's hereditary optic neuropathy causes significant visual impairment; in the majority of cases, visual recovery is minimal.[156]

The majority of patients with LHON (90%–95%) harbor one of three primary mtDNA point mutations: m.3460G > A, m.11778G > A, and m.14484T > C, which affect key complex I subunits of the mitochondrial respiratory chain.[156] Leber's hereditary optic neuropathy can be also due to rare pathogenic mtDNA point mutations affecting different subunits of complex I, more commonly the MT-ND6 and MT-ND1 subunit genes.[157,158] The mtDNA pathogenic mutations, which are in most cases homoplasmic (100% of the mtDNA molecules are mutated), do not explain at least two features of LHON: the male prevalence and the incomplete penetrance. Not all LHON mutation carriers will experience visual loss during their lifetime. The conversion rate for male carriers is 50% compared with 10% for female carriers. The primary LHON mutation is therefore a prerequisite, but secondary factors are clearly modulating the risk of visual loss.[151,159] Their identification has proven challenging, and the evidence favors a complex disease model, with both genetic and environmental factors interacting to precipitate optic nerve dysfunction.[159–161] The only strong evidence of a genetic modifier is currently the association of LHON with a European mtDNA background, known as haplogroup J, which increases the penetrance of the m.11778G > A and m.14484T > C LHON mutations.[159,162]

The differential diagnoses of LHON include LS, MELAS, Friedrich ataxia, and Wolfram syndrome.

mtDNA Depletion Syndromes

MtDNA depletion syndromes (MDS) are heterogenous autosomal recessive disorders characterized by a severe reduction in mtDNA content in affected tissues and organs.[163] Onset is mainly in infancy leading to an early death. MtDNA depletion syndromes are associated with defects in mtDNA maintenance caused by mutations in nuclear genes that function in either mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis or mtDNA replication. Thymidine kinase 2 (TK2), adenosine diphosphate- (ADP-) forming succinyl CoA ligase β subunit (SUCLA2), guanosine diphosphate (GDP)-forming succinyl CoA ligase α subunit (SUCLG1), ribonucleotide reductase M2 B subunit (RRM2B), deoxyguanosine kinase (DGUOK), and thymidine phosphorylase (TYMP) encode proteins that maintain the mitochondrial dNTP pool.[163] Mutations in any of these genes result in mtDNA depletion.

MtDNA is replicated by the concerted action of DNA polymerase γ (pol γ) encoded by POLG, or its accessory subunit, p55 encoded by POLG2. Pol γ is the only known DNA polymerase to be found in mammalian mitochondria and thus bears the burden of DNA replication and DNA repair functions.[6] The mitochondrial helicase, C10orf2 or Twinkle, is a replication factor. Mutations in POLG, POLG2, and C10orf2 (Twinkle) result in insufficient mtDNA synthesis and a reduction of mtDNA content.[163] Clinically, MDS are usually classified as one of four forms: a myopathic form associated with mutations in TK2; an encephalomyopathic form associated with mutations in SUCLA2, SUCLG1, or RRM2B; a hepatocerebral form associated with mutations in DGUOK, MPV17, POLG, or C10orf2; and a neurogastrointestinal form associated with mutations in TYMP.[164,165]

TK2-related Myopathic MDS

Typically, initial development is normal and the majority of affected children present before the age of 2 years with gradual onset of hypotonia, generalized fatigue, proximal muscle weakness, and feeding difficulty, with normal cognitive function.[163] Muscle weakness rapidly progresses to respiratory failure and death within a few years of onset. The adult form of TK-2 related myopathy is slowly progressive.[166] Rarely, there are encephalomyopathic, hepatomyopathic, or spinal muscular atrophy-like forms of presentation.[6] Serum CPK is usually elevated. Electromyography usually shows nonspecific myopathic changes. Histopathological hallmarks for mitochondrial disease such RRF, COX-negative fibers, and SDH hyperstaining may be seen. Electron transport chain activity assays are decreased in multiple complexes, commonly complexes I, I + III, and IV.[163] mtDNA content is severely reduced in muscle.

SUCLA2 and SUCLG1-related Encephalomyopathic MDS

Mutations in the succinyl-CoA synthase gene SUCLA2 cause infants to present with hypotonia typically before the age of 6 months. All affected children develop hypotonia, muscle atrophy, and psychomotor delay. Other features are feeding difficulty, gastroesophageal reflux, sensorineural hearing loss, and epilepsy. Most patients die in childhood.[163] SUCLG1 deficiency has a similar phenotype characterized by intrauterine growth retardation, hepatomegaly, hypotonia, respiratory insufficiency due to acidosis, and severe hypothermia.[166] In the neonatal form, death ensues within a few days. No adult forms have been reported so far. Lactic acidosis is severe. Hypoglycemia and mild to moderate elevation of MMA and methylcitrate may be present. Plasma and urine amino acid determination reveals highly elevated taurine and glycine and moderately elevated lysine and alanine.[166] Electromyography may reveal findings suggestive of motor neuron involvement, whereas a brain MRI scan may show cortical atrophy, bilateral basal ganglia involvement, and delayed myelination.[163] Electron transport chain assays commonly show combined deficiency of complexes I, III, and IV. Quantitation of mtDNA shows a decreased mtDNA content in muscle.

RRM2B-related Encephalomyopathic MDS

Mutations in the nuclear-encoded mitochondrial maintenance gene RRM2B cause mtDNA depletion, and multiple mtDNA deletions. RRM2B-related mtDNA depletion usually manifests as severe multisystem disease (encephalomyopathy with proximal renal tubulopathy) and is often fatal in early life. Inheritance is autosomal recessive. Affected individuals typically present during the first months of life with hypotonia, lactic acidosis, failure to thrive, tubulopathy, microcephaly, psychomotor delay, sensorineural hearing loss, and profound mtDNA depletion in muscle.[163] The disease progresses rapidly, leading to death within a few months. Multiple mtDNA deletions cause tissue-specific COX deficiency. Inheritance can be either autosomal recessive, with PEO and multisystem involvement manifesting during early childhood or adulthood,[167] or autosomal dominant with tissue specific manifestations such as PEO developing in late adulthood.[130,131] RRM2B mutations have been reported to cause a mitochondrial neurogastrointestinal encephalopathy- (MNGIE-) like phenotype with mtDNA depletion,[168] and a KSS phenotype.[167] Lactic acidosis may be present. Muscle biopsy may show RRF and COX-negative fibers. Electron transport chain assays commonly show combined deficiency of complexes I, III, and IV[163] or combined I, III, IV, and V.[166] In addition to mtDNA depletion, RRM2B mutations also cause multiple mtDNA deletions in adults.[169] This mutation is the third most common cause of multiple mtDNA deletions following POLG and C10orf2 (Twinkle).[169]

DGUOK-related Hepatocerebral MDS

There are two phenotypes of deoxyguanosine kinase (DGUOK) deficiency: multiorgan disease in neonates with isolated hepatic disease later in infancy or childhood, and progressive liver disease is the most common cause of death in both forms.[170] The majority of affected individuals have a neonatal-onset multiorgan disease that presents with lactic acidosis and hypoglycemia in the first week of life.[163] Within weeks of birth, all infants develop hepatic disease and neurologic dysfunction. Severe myopathy, developmental regression, and typical rotary nystagmus developing into opsoclonus are also seen. Liver involvement may cause neonatal- or infantile-onset liver failure that is generally progressive with ascites, edema, and coagulopathy.[163] The majority of affected newborns with the multiorgan form of the disease show elevated serum tyrosine or phenylalanine on newborn screening.[163] A brain MRI scan is usually normal in DGUOK deficiency, but some patients show moderate hyperintensity of the globus pallidus bilaterally and subtentorial abnormal myelination.[171] mtDNA content is reduced in liver and muscle, and electron transport chain activity assays are commonly decreased in complexes I, I + III, and IV.[163] Hepatic dysfunction is progressive in the majority of individuals with both forms of DGUOK-related MDS and is the most common cause of death. For children with the multiorgan form, liver transplantation provides no survival benefit.[163]

MPV17-related Hepatocerebral MDS

The gene MPV17 encodes an inner mitochondrial membrane protein. Mutations cause an infantile-onset disorder, which can present with a spectrum of combined hepatic, neurologic, and metabolic manifestations.[163] The phenotype is characterized by recurrent episodes of severe hypoglycemia, hepatopathy evolving toward cirrhosis and liver failure, and growth retardation. Patients present with poor feeding, failure to thrive, diarrhea, and recurrent vomiting.[166] Within the first few months of life they develop generalized muscle wasting and hypotonia. Common neurologic phenotypic features include microcephaly, ataxia, developmental delay, muscle weakness, seizures, ischemic stroke, or dystonia. Patients who survive develop polyneuropathy and lesions of the cerebellum and the cerebral cortex.[166] In the vast majority of affected individuals, liver disease progresses to liver failure in infancy or childhood, and liver transplant is the only treatment option. Neurologic manifestations include developmental delay, hypotonia, muscle weakness, and motor and sensory peripheral neuropathy. Lactic acidosis and hypoglycemia is present in most cases, which typically presents during the first 6 months of life. A brain MRI may show cortical and subcortical hyperintensities involving the cerebellar white matter.[166] Navajo neurohepatopathy is another recognized phenotype.[59]

C10orf2-related Hepatocerebral MDS

Mutations in C10orf2 (Twinkle mtDNA helicase) have been associated with variable phenotypes, including infantile-onset spinocerebellar ataxia (IOSCA), adPEO, and hepatocerebral MDS.[163] Onset is usually in infancy, although adult-onset adPEO has been reported. The neurologic involvement progresses to include hyporeflexia, muscular atrophy, ophthalmoplegia, nystagmus, athetosis, ataxia, epilepsy, sensory neuropathy, sensorineural hearing loss, and psychomotor regression. Prognosis is poor. Mutations in the Twinkle gene most frequently cause multiple mtDNA deletions, but also cause mtDNA depletion, which is more evident in the liver than in skeletal muscle.[166]

POLG-related Hepatocerebral MDS

Also referred to as Alpers syndrome, Alpers–Huttenlocher syndrome (AHS) presents most frequently during infancy (similar to MCHS) and is fatal.[7] Mitochondrial polymerase γ (POLG) deficiency with mtDNA depletion was reported as the cause[15] leading to the identification of the first mutations in POLG in two unrelated families.[172] Alpers–Huttenlocher syndrome is one of the most severe phenotypic manifestations in the spectrum of POLG-related disorders. The hallmark features of AHS include the clinical triad of refractory seizures, developmental regression, hepatopathy, and 2 of 11 other clinical or laboratory findings.[173] The typical age of onset is between 2 and 4 years of age, but can range from 3 months to 36 years of age. Children with AHS appear healthy at birth and may develop normally until the onset of their illness.[173] Seizures are the first sign of AHS in approximately 50% of affected children,[173] with an early predilection of epileptiform discharges over the occipital region of the brain, and generalized discharges as the disease progresses.[7] Over time, the seizures become increasingly resistant to anticonvulsant therapy. Valproic acid can precipitate fatal liver failure[174] in AHS and should be avoided.[174] Migraine headaches are also an early symptom. Early in the disease course areflexia and hypotonia are present and are later followed by spastic paraparesis that evolves over months to years, leading to psychomotor regression.[163] Ataxia develops in all persons with AHS, unless the disease process is so rapid that it results in early death or the corticospinal tract involvement precludes the evaluation for ataxia.[173] Motor and sensory neuropathy may be present. A rapid and stepwise cognitive deterioration often occurs in the setting of an infectious illness.[173] The clinical manifestations may include somnolence, loss of concentration, loss of expressive and receptive language, irritability with loss of normal emotional responses, and memory deficits. Cortical visual loss leading to blindness is common in fully developed AHS, but may not appear for months to years after the onset of other neurologic manifestations.[173] Other neurologic features may include stroke and stroke-like episodes, myoclonus, choreoathetosis, parkinsonism, and nystagmus.[163] Cerebrospinal fluid protein is generally elevated. A brain MRI scan may show gliosis and generalized brain atrophy.

Liver involvement can progress rapidly to end-stage liver failure within a few months.[173]

Disease progression is variable, with life expectancy from onset of symptoms ranging from 3 months to 12 years.[173] Muscle or liver mtDNA depletion (defined as < 35% of age-matched normal mtDNA content) can lag behind the onset of disease symptoms.[173] With disease progression, all patients will eventually have mtDNA depletion. Therefore, the absence of mtDNA depletion early in the course of disease cannot be used to exclude POLG disease.[173]

Mitochondrial Neurogastrointestinal Encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder first described in 1976.[175] Mitochondrial neurogastrointestinal encephalomyopathy is caused by mutations in the nuclear gene TYMP, encoding thymidine phosphorylase (TP).[176] Hirano et al defined the diagnostic criteria in 1994: severe gastrointestinal dysmotility; cachexia; ptosis, ophthalmoparesis, or both due to extraocular muscle weakness; peripheral neuropathy; and leukoencephalopathy with TP activity < 10% of the normal control mean.[134] Severe biochemical defects of thymine phosphorylase activity lead to marked increases in thymidine and deoxyuridine nucleoside levels in blood, urine, and tissues.[176–179] These elevated nucleoside levels cause mtDNA instability and ETC dysfunction.[180] The condition is relentlessly progressive, and patients usually die at an average age of 37 years.

Garone et al reported a cohort of 102 patients with MNGIE (50% female, mean age 32.4 years, age range 11–59 years).[180] Ninety-two patients were confirmed to have TYMP mutations. The average age of disease onset was 17.9 years (range 5 months–35 years), but the majority of patients reported the first symptoms in childhood. Surprisingly, among patients with severe thymine phosphorylase deficiency in the buffy coat (< 10% of normal control mean), early onset did not correlate with a short life expectancy.[180] Gastrointestinal symptoms were confirmed as the most frequent first feature of the disease as found in 36 of 63 patients (57.1%). These symptoms included diarrhea, abdominal pain, nausea/vomiting, abdominal cramps, weight loss, borborygmi, cachexia, intestinal pseudo-obstruction, bloating, and intestinal invagination. Ocular symptoms such as ptosis and external ophthalmoplegia preceded GI disease in 14 of 63 patients (22.2%). It is not uncommon that the clinical suspicion for a diagnosis of MNGIE is triggered by abdominal imaging that reveals a large and dilated stomach, consistent with pseudo-obstruction, in the context of weight loss and cachexia. This occurred in 9 of 63 patients (14%). Patients presented with peripheral neuropathy manifesting as numbness or foot drop, limb weakness, and paraesthesia or cramps. The neuropathy was predominantly a demyelinating neuropathy.

Kaplan–Meier analysis revealed significant mortality between the ages of 20 and 40 years. The average age of death was 35 years (range 15–54 years) and identified causes included pneumonia due to aspiration (eight patients), peritonitis due to intestinal rupture (two patients), suicide (two patients), electrolyte imbalance (two patients), sepsis (one patient), malignant melanoma (one patient), cardiac arrhythmia (one patient), metabolic acidosis (one patient), cardiopulmonary arrest (one patient), and oesophageal varicocele bleeding (one patient).[180]

The leukoencephalopathy in MNGIE involves the cerebral hemispheres, but basal ganglia, cerebellum, and brainstem are often affected. Typically, patients do not display cognitive manifestations despite the diffuse white matter involvement of the brain, although 20% of patients in this cohort did have mild neurologic symptoms such as cognitive impairment, dementia, seizure, headache, or psychiatric symptoms such as depression.

Mitochondrial neurogastrointestinal encephalomyopathy can be diagnosed by demonstrating severely reduced thymine phosphorylase activity in the buffy coat; marked elevations of thymidine, deoxyuridine, or both in plasma or urine; or by detecting TYMP gene mutations in peripheral blood.[180] A demyelinating neuropathy may be evident on a nerve conduction study. Muscle biopsy typically reveals mitochondrial alterations such as RRFs, COX-deficient fibers, or decreased activities of mitochondrial OXPHOS activity; however, the skeletal muscle may not show any mitochondrial abnormalities.[180]

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers a permanent cure, but it carries a significantly high mortality risk and is limited by matched donor availability.[181]

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