Introduction. Overview

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1 Leigh syndrome Redi Rahmani BA ( Dr. Rahmani of Dartmouth-Hitchcock Medical Center/Geisel School of Medicine at Dartmouth has no relevant financial relationships to disclose. ) Brandon Root MD ( Dr. Root of Dartmouth-Hitchcock Medical Center/Geisel School of Medicine at Dartmouth has no relevant financial relationships to disclose. ) Robert J Singer MD ( Dr. Singer of Dartmouth-Hitchcock Medical Center/Geisel School of Medicine at Dartmouth has no relevant financial relationships to disclose. ) Originally released July 30, 1998; last updated September 12, 2016; expires September 12, 2019 Introduction This article includes discussion of Leigh syndrome, Leigh disease, Leigh encephalomyelopathy, and subacute necrotizing encephalomyelopathy. The foregoing terms may include synonyms, similar disorders, variations in usage, and abbreviations. Overview Leigh disease is an expression form of mitochondrial disease, not specific for age, although most patients are infants or children. An ever increasing number of genes are associated with Leigh disease. Leigh disease is characterized by its dramatic and often lethal neurologic regression in a previously healthy baby or child. The author highlights the typical diagnostic findings, including MRI. The underlying cause of Leigh disease is a mutated or deleted gene that affects the pyruvate dehydrogenase complex or the mitochondrial respiratory chain. Genetic counseling of family members and a rational approach to treatment require a molecular diagnosis as a follow-up to clinical diagnosis. This article presents developments in mitochondrial genetics, new emphasis on genetic techniques for diagnosis, and includes an update on available treatments, especially the administration of thiamine and biotin to all patients with suspected Leigh syndrome. Key points Leigh syndrome is a clinical expression of cerebral pathology due to genetic mitochondrial dysfunction that occurs mostly in infancy or childhood. It is named after the first author who described its course and pathology in young infants, which was morphologically similar to adult Wernicke disease. Its etiology is a mitochondrial dysfunction due to 1 of several mutations in nuclear mitochondrial DNA. Although its presentation and course are generally variable, symptoms may include failure to thrive, weakness/hypotonia, ataxia, oculomotor palsy, seizures, and episodes of lactic acidosis. Its typical course is relapsing, often in response to stress, mostly due to incidental infections. Outcome is often fatal. The characteristic finding on MRI is T1 and T2 prolongation, with symmetrical involvement in the putamen, globus pallidus, caudate, thalami, substantia nigra, inferior olivary nuclei, periaqueductal gray matter, superior cerebellar peduncles, tegmentum of the brainstem, and less commonly, periventricular white matter and corpus callosum. Treatment generally consists of high dose thiamine (> mg/kg/day), biotin (>5-10 mg/kg/day), and supportive measures. In some cases, other agents may be useful depending on the specific genetic mutation. Historical note and terminology Subacute necrotizing encephalomyelopathy was first described in 1951 by Dr. Denis Leigh, a British neuropathologist who reported the case of an infant with developmental regression at 6.5 months that progressed rapidly and resulted in death over a 6-week period (Leigh 1951). Dr. Leigh performed an extensive autopsy and described multiple bilateral foci of spongy degeneration and microscopic vascular proliferation in the brainstem tegmentum, basal ganglia, thalamus, cerebellum, optic nerves, and spinal cord. He speculated on the pathophysiology, noting the similarity between subacute necrotizing encephalomyelopathy (SNE) and Wernicke syndrome. Wernicke syndrome may arise as a result of thiamine deficiency due to nutritional defects such as in alcoholism. Thiamine is a cofactor of the pyruvate dehydrogenase complex. Inherited pyruvate dehydrogenase deficiency causes brain lesions similar to Wernicke syndrome. Though not appreciated at the time, the similarity between the acquired (Wernicke) and the genetic SNE is due to a shared biochemical mechanism. In 1954 a genetic basis (autosomal recessive) was proposed for the disease

2 with a report of 2 affected siblings with consanguineous parents (Feigin and Wolf 1954). Montpetit and colleagues presented an overview of 50 cases in infants and children studied neuropathologically (Montpetit et al 1971). The lesions were bilateral, sharply delimited, and mainly affected nuclear regions such as basal ganglia, mostly the putamina, and brainstem nuclei. The cerebral cortex was involved only in small minority (10%). The white matter was affected less often, with the internal capsule affected in about one third of cases. The spinal cord was affected in one third of the population, with involvement of both grey and white matter. Subsequent research proved associations with lactic acidosis (Worsley et al 1965) and the first detected mitochondrial enzyme deficiencies: pyruvate dehydrogenase complex (PDHC) deficiency (Farmer et al 1973), and cytochrome C oxidase (COX) deficiency (Willems et al 1977). The last named authors in fact proved that Leigh syndrome was heterogenous in origin. Further developments followed the general progress in mitochondrial research, by which it was shown that Leigh syndrome could be caused by mutations in respiratory chain complex subunits in mitochondrial DNA as well as in nuclear DNA. Diagnosis was facilitated enormously by the introduction of MRI, making diagnosis possible without autopsy confirmation and paving the way for prenatal diagnosis. Clinical manifestations Presentation and course The microscopic aspect of the lesions in Leigh disease has the following characteristics: (1) they are sharply demarcated; (2) there is spongiosis of the neuropil, ie, the tissue constituents between neuronal cell bodies mainly consisting of dendrites and axons; (3) capillary thickening and proliferation; and (4) reactive changes involving macrophages (early) and astrocytes (late) (Davis and Robertson 1985; Cavanagh and Harding 1994). Peripheral nerves may show involvement (Goebel et al 1986; Jacobs et al 1990; Grunnet et al 1991; Chabrol et al 1994; Menezes and Ouvrier 2012). According to Menezes and Ouvrier, the cause of the mitochondrial disease may decide on the character of the neuropathy (Menezes and Ouvrier 2012): SURF1 mutations causing a demyelinating neuropathy, MTATP6 mutations causing axonal neuropathy, and late onset disease due to POLG1 mutations causing a predominant sensory neuropathy with ataxia. PDHc deficiency may cause a mixed or axonal sensorimotor neuropathy. These reported series emphasize the need to test peripheral nerve function in Leigh syndrome patients, especially because the clinical severity of brain disease may overshadow the symptoms of peripheral nerve involvement. Muscle biopsies generally show nonspecific myopathic changes with little or no abnormal appearance to the mitochondria, in contrast to many other mitochondrial disorders. The median onset of disease presentation is 7 months (Sofou et al 2014), with onset in 80% of children before 2 years of age, 55% in the first year and the additional 25% by 14 months. An additional 17% have onset of the disease between 3 and 15 years of age, and about 2% after 15 years of age (Lyons et al 1996). Disease onset prior to 6 months of age, failure to thrive, brainstem lesions on imaging, and need for intensive care treatment are all associated with poorer survival (Sofou et al 2014). Once started, the disease may progress slowly, plateau, or (rarely) rapidly progress to death. A relapsing and remitting course is frequent in Leigh disease. The onset is often seemingly acute, in the setting of an illness or after a seizure from which recovery is incomplete, prolonged, or associated with unexplained coma. Neurologic dysfunction typically results from variable involvement of basal ganglia, brainstem, and spinal cord. The most common clinical features are motor symptoms followed by ocular findings (Sofou et al 2014). Early findings may be poor sucking ability, the loss of head control, loss of motor skills, vomiting, and irritability. This may progress to hypotonia and failure to thrive. Ataxia, cerebellar tremor, and incoordination may be evident if the child is walking, and movement disorders reflective of basal ganglia involvement including dystonia or choreoathetosis are not uncommon (Macaya et al 1993). Abnormalities of respiration due to bilateral brainstem involvement are often present at some stage of Leigh disease and consist of periodic hyperventilation, apnea, gasping, sighing, and irregular breathing. Respiratory failure in Leigh disease may become fatal unless treated. Oculomotor palsies also typically result from the brainstem involvement in the disease. There are multiple oculomotor palsies, and supranuclear palsy of vertical or lateral gaze often occurs.

3 Optic atrophy may be encountered as part of a mixture of 2 syndromes, each due to primary mitochondrial DNA mutation: Leigh syndrome and Leber hereditary optic neuropathy (LHON) (Fruhman et al 2011; Leshinsky-Silver et al 2011). In a study on the ophthalmologic manifestation in 44 patients with Leigh syndrome, Han and colleagues found strabismus to be the most common manifestation (40.9%), then pigmentary retinopathy (22.5%), followed by optic atrophy (22.5%), ptosis (15.9%), and finally, nystagmus (13.6%) (Han et al 2015). Variably, there may be additional cranial nerve involvement, dysphagia due to paralysis of swallowing, or deafness. Sudden onset of Leigh disease with involvement of peripheral nervous system and spinal cord may closely mimic Guillain-Barré syndrome. Epileptic seizures affect 40% of patients with Leigh disease and are more often seen in early onset disease when they may present as infantile spasms. Patients who have a history of seizures from birth have a higher occurrence of disease relapse (Kamoshita et al 1970; Tsao et al 1997; Sofou et al 2014). The most common forms of seizures in Leigh syndrome are generalized tonic-clonic and complex partial, found in 11% of patients in 1 study of 60 patients (Chevallier et al 2014). Further studies documented variable multisystem involvement, such as hypertrophic cardiomyopathy, renal tubulopathy, and fatty liver infiltration (Agapitos et al 1997). Later studies have assigned such visceral involvement to specific genetic etiologies. Childhood moyamoya disease has also been reported in patients with Leigh syndrome (Cullu et al 2013), as has Fukuyama congenital muscular dystrophy (Kondo et al 2014). Episodes of lactic acidosis are also common, and may lead to further respiratory and/or renal impairment. MRI has aided in the diagnosis of Leigh disease in the living patient, to the extent that postmortem examination of the brain is no longer required to confirm the diagnosis. The findings on MRI are characteristic, corresponding to the pathologic findings. MRI shows T1 and T2 prolongation that is remarkable for its symmetrical involvement, most commonly in the putamen, globus pallidus, caudate, thalami, substantia nigra, inferior olivary nuclei, periaqueductal gray matter, superior cerebellar peduncles, tegmentum of the brainstem, and less commonly periventricular white matter and corpus callosum. CT scans have been less helpful than MRI but nevertheless will detect the characteristic basal ganglia involvement. Magnetic resonance spectroscopy has demonstrated decreased N-acetylaspartate and increased lactate levels, which are most apparent in the areas showing the most involvement on MRI (Medina et al 1990; Barkovich et al 1993). An increase of lactic acid and pyruvate in blood and cerebrospinal fluid is an important indicator for involvement of the pyruvate dehydrogenase complex or mitochondrial respiratory chain. The degree of these elevations is, however, variable, and normal levels do not exclude Leigh syndrome in the presence of a clear history and typical MRI findings. Prognosis and complications The prognosis in Leigh disease is generally poor, but dependent to a great degree on the etiology and the presence or absence of seizures. For infants whose disease has its onset before 24 months of age, death generally occurs within 2 years of onset. For childhood and adolescent Leigh disease, the prognosis is more variable, with death occurring 3 to 10 years after onset. Children of all ages may exceptionally have a fulminant course lasting only days to weeks. For those patients with thiamine-responsive forms, the prognosis may be better. Complications include the sequelae of seizures and their treatment, failure to thrive, aspiration pneumonia, joint contractures, and skin breakdown from decreased movement. Clinical vignette An 18-month-old girl was referred for evaluation of global developmental delay, growth retardation, acquired microcephaly, hypotonia, and episodic hyperventilation and ataxia. She had been born at 42 weeks' gestation and weighed 2550 gm. The pregnancy had been complicated only by hyperemesis requiring intravenous hydration. Labor, delivery, and the perinatal period had no complications. The infant did well until 3 months of age when she developed significant vomiting, which was treated with thickened feeds and antacids with some improvement. However, by 9 months there was an obvious decline in her growth parameters, and she was intolerant of solid foods. At 18 months she had hypotonia, tremulousness, and ataxia. On examination at that time, her weight was at the 50th percentile for a 12 month old, her height at the 50th percentile for a 16 month old, and her head circumference at the 50th percentile for a 10 month old. She had global developmental delay, generally functioning at an 11- to 13-month level,

4 microcephaly, normal deep tendon reflexes, mild hypotonia, intention tremor, and a wide-based gait. She exhibited some self-stimulating and self-injurious behaviors, poking at her eyes and banging her head on hard surfaces. A peculiar breathing pattern was noted, with episodes of hyperventilation. There was no history of injury or major illness, and she was an otherwise healthy child. There was no family history of similarly affected members. Previous tests included a barium swallow, CT and MRI of the brain, EEG, chromosomes, serum quantitative amino acids, urine organic and amino acids, serum ammonia, chemistries including glucose, biotinidase, and arterial blood gas measurement, all of which were normal. A serum lactate was elevated at 28.8 mmol/l (normal < 23); pyruvate and ophthalmological examination were normal. CSF was normal except for an elevated lactate of 3.5 mmol/l (normal < 2.0). A repeat brain MRI was performed and showed characteristic, symmetrical increased T2-weighted signal in the putamina and tegmentum of the brainstem, supportive of the tentative diagnosis of Leigh disease. She was enrolled in an early intervention program where she received physical therapy, occupational therapy, and speech services. Over the next 2 years she had periods of worsening, and she developed seizures. She was treated empirically with B vitamins and coenzyme Q and given anticonvulsants for seizures. Despite a gastrostomy tube, she failed to gain weight adequately. She continued to deteriorate, becoming less alert and interactive and losing all language. She eventually died at 4 years of age of respiratory failure. No autopsy was performed. Biological basis Etiology and pathogenesis Leigh syndrome represents the clinical and radiological expression of a group of inherited disorders of energy metabolism. Two major subgroups exist: (1) disorders of oxidative phosphorylation caused by dysfunction of components of the mitochondrial respiratory chain; (2) disorders of pyruvate oxidation through an impairment of the pyruvate dehydrogenase complex (PDHc). The ultimate cause of Leigh syndrome is a failure of ATP production through a mutation specifically affecting an oxidative phosphorylation or pyruvate dehydrogenase complex related gene. Debray and colleagues present a useful overview of its diverse etiologies (Debray et al 2008). Table 1 summarizes the mitochondrial and nuclear genes that have been associated with Leigh syndrome. Table 1. Genetic Etiology of Leigh Syndrome (A) Mitochondrial DNA Gene mutations Remarks Large-scale deletions of mitochondrial DNA Gene mutations affecting subunits Complex I: MT-ND1, ND6, MT-TK, TLI, and assembly factors: complexes I, TV, TW III, IV, V Complex IV: COX III Complex V: MTATP6 (ATPase 6 subunit) trna mutations Most common: trnalys (B) Nuclear DNA (1) Mutations affecting subunits, assembly factors and coenzyme Q Complex I: NDUFS1,2,3,4,7,8, NDUFV1, NDUFA1,2,10,11,12, NDUFAF2, C8ORF38, C20ORF7, FOXRED1, C12ORF65 Complex II: SDHA (Fp subunit), SDHAF1 Complex III: BCS1L, TTC19, UQCRQ Complex IV: SURF1, NDUFA4, COX10, COX15, SCO2, PET100 Coenzyme Q: PDSS2 ATP synthase: LRPPRC? Rare Variable intrafamilial disease expression due to heteroplasmy Nuclear encoded subunits summarized in (Koene et al 2012; Ruhoy and Saneto 2014). PET100 is found in Lebanese with Leigh syndrome

5 (2) Mutations affecting synthesis and translation of mitochondrial DNA. Resultant defects may affect 1 or more complexes mtdna depletion: SUCLA2, POLG; translation of mtdna transcript: TSFM, EFG1 (mitochondrial elongation factor), LRPPRC (posttranscription defect specifically affecting COX subunits), TACO1 (translation activator gene of COX1), formation of τm5(u) in anticodon wobble position: GTBPB3; methionyl-trna formyltransferase defect: MTF LRPPRC mutation is found in French- Canadian Leigh syndrome (3) Pyruvate dehydrogenase complex (PDHc) (4) Thiamine pyrophosphokinase deficiency PDHA1, PDHB, DLAT, DLD, PDHX TPK1 PDHA1 encoding E1 alpha located on the X-chromosome (5) Thiamine transport deficiency SLC19A3 Biotin-thiamine-responsive basal ganglia disease mimics Leigh syndrome (6) β-oxidation of fatty acids ECHS1 (short chain enoyl coenzyme A hydratase, that catalyzes the second step of β-oxidation) (7) Glycogen synthesis GYG2: initiation of glycogen synthesis X-linked recessive Our knowledge of Leigh syndrome and its background started with the astute observation of common morphological features linking Wernicke disease, the cerebral disease caused by an acquired thiamine deficiency, to Leigh syndrome, a genetic disorder (Leigh 1951; Feigin and Wolf 1954). Deficiency of thiamine (vitamin B1), a cofactor of the pyruvate dehydrogenase complex, causes deficiency of acetyl-coa, necessary to sustain the citric acid cycle, and leads to accumulation of pyruvate and lactate. Genetic deficiency of pyruvate dehydrogenase has the same biochemical consequences and constitutes 1 of the defined causes of Leigh syndrome. Today most of the known biochemical defects that cause Leigh syndrome are due to defects in oxidative phosphorylation. There are different ways to catalogue mitochondrial disorders: by inheritance, Mendelian inheritance versus mitochondrial inheritance; by single respiratory chain complex versus multiple complexes; or by the type of operation of the defective gene product. A useful handle for the subdivision of etiologies is to differentiate on the basis of involvement of single complexes, versus multiple complexes. Dysfunction of single respiratory chain complexes. Mitochondrial energy generation is based on the interaction of 5 complexes (I to V) together forming the respiratory chain. The complexes are attached to the inner mitochondrial membrane. Energy delivered as electron pairs is fed to the respiratory chain by the citric acid cycle. The transport of electron pairs along the respiratory chain from 1 complex to the next leads to stepwise loss of energy. The release of energy by electron transport is coupled to the energetically uphill transport of protons from the mitochondrial matrix to the intermembrane space. The transmembrane proton gradient thus created is reversed in a final step through backflow in a channel formed by complex V. This backflow of protons drives the reaction that phosphorylates ADP to ATP. The creation and availability of this ubiquitous energy carrier is known as energy conservation. DiMauro and Schon provide an excellent review of the functioning of the respiratory chain (DiMauro and Schon 2003). Biogenesis of the 5 complexes involves the assembly of multiple peptide subunits. Mitochondrial complex subunits are encoded on either mitochondrial or nuclear genes. Because of this dual origin of respiratory chain complexes, there are essentially 2 modes of inheritance of Leigh syndrome: mitochondrial and Mendelian. In the former case, (also known as MILS, or maternally inherited Leigh syndrome), the disease is transmitted through the mother, whose oocyte contributes her child's mitochondria at the moment of fertilization, but such pathogenic mitochondrial mutations may also arise de novo. Typical for Leigh syndrome with maternal inheritance is the variability of the ratio between normal and affected mtdna in each individual, causing wide variability within affected families. In the case of Mendelian inheritance, the mode of inheritance is autosomal recessive, with both parents heterozygous for the mutated gene or, X-linked. As in all autosomal recessive disorders consanguinity enhances the risk of disease. After the clinical diagnosis of Leigh disease is reached, the next step is to define its biochemical background through the search for an enzymatic defect, which resides either in the pyruvate dehydrogenase complex or the respiratory

6 chain. The biochemical findings lead the direction of the final step, which is the identification of a coding defect in 1 of the mitochondrial or nuclear genes as the ultimate cause of the disorder. Leigh disease has become associated with defects in all of the respiratory chain complexes as well as pyruvate dehydrogenase complex. Complex I (NADH:ubiquinone oxidoreductase). Complex I, the largest complex, accounts for up to 30% of mitochondrial disorders in childhood (Fassone and Rahman 2012). It comprises 7 subunits encoded by mitochondrial DNA and 38 encoded by nuclear genes. Complex I deficiency may cause diverse mitochondrial syndromes, including progressive leukoencephalopathy, cardiomyopathy, and severe lactic acidosis. The most common association, however, is Leigh disease. Because complex I is assembled from mitochondrial- and nuclear-encoded subunits, both Mendelian inheritance and mitochondrial inheritance are involved. This renders the mode of inheritance, as derived from the family history, important for the choice of genes to investigate. In complex I dysfunction, the frequency ratio of mitochondrial and nuclear gene defects diagnosed is about equal. Among the mitochondrial genes encoding complex I, especially ND5, mutations are related to Leigh disease (Chol et al 2003). Heteroplasmy, ie, the presence of a normal and a mutated set of mitochondria in an individual due to maternal (mitochondrial) inheritance was detected in MILS (Maternal Inherited Leigh Syndrome), contrasting a common notion that associates heteroplasmy in mitochondrial disorders with adult onset. An extreme example is the case of a 1-month-old infant with acute Leigh disease due to an ND3 mutation, which was also found in the asymptomatic mother (Leshinsky-Silver et al 2005). In another study, heteroplasmy was encountered but not found in the parents, suggesting de novo mutation (Lebon et al 2003). The importance of heteroplasmy as a cause of variable expression within a single pedigree is illustrated by the report of van Karnebeek and colleagues of a family with ND5 mutation with MELAS (mitochondrial inherited lactic acidosis and stroke like episodes) in a 10-year-old female and a fatal neonatal course in a younger sister (van Karnebeek et al 2011). Some insight into how these mutations lead to the pathophysiology of Leigh syndrome is described in Burman and colleagues' work with Drosophilia (Burman et al 2014). They show in their research that the ND2 gene subunit of C1 is directly involved in the proton-pumping mechanism of C1. A defect in this subunit then causes a respiratory pathway deficiency. The first nuclear gene encoding a respiratory chain subunit, NDUFS8, a subunit of complex I, was identified in 1998 (Loeffen et al 1998). By 2012, mutations in 16 nuclear genes mostly encoding subunits and assembly factors of complex I are associated with Leigh syndrome (Koene et al 2012). According to Koene and colleagues, almost all children with nuclear-encoded complex I mutations ultimately develop Leigh syndrome or leukoencephalopathy. Mortality is high, with 75% dying before the age of 10 years. It should be noted that 1 complex I gene, NDUFA1, is encoded on the X-chromosome and its associated Leigh syndrome X-linked (Fernandez-Moreira et al 2007). Complex II, succinate-ubiquinone oxidoreductase. Complex II is composed of 4 subunits, all encoded on nuclear DNA. Reports on complex II deficiency and Leigh syndrome are rare. Bourgeron and colleagues reported siblings with Leigh disease, complex II deficiency, and a mutation in the flavoprotein (Fp) subunit (Bourgeron et al 1995). Brockmann and colleagues reported elevated succinate by MR spectroscopy of affected white matter (Brockmann et al 2002). This finding may offer an interesting approach to the detection of complex II deficiency in brain. In a further study, mutation of the complex II assembly factor SDHAF1 was identified as causative gene defect (Ohlenbusch et al 2012). Interestingly, SDHA mutations have also been found in paragangliomas and pheochromocytomas, and in the rare instance a patient is diagnosed with Leigh syndrome due to CII defect, specifically SDHA, tumor screening should be considered (Renkema et al 2015). Coenzyme Q (CoQ) and complex III (ubiquinol cytochrome c reductase). Complex III is made up of 11 subunits of which 10 are encoded by nuclear genes. CoQ transfers electrons from complexes I and II to complex III. Indirect enzymatic methods measuring I+III and II+III do not distinguish between deficiencies of CoQ and complex III. Leshinsky-Silver and colleagues reported a neonate with severe lactic acidosis, hyperammonemia, and a brain MRI compatible with Leigh disease (Leshinsky-Silver et al 2003). In liver severely reduced activities of II+III and I+III were restored in vitro by CoQ. In another study Q10 (coenzyme Q) administration ameliorated clinical findings in an adult with Leigh disease, deficient succinate:cytochrome c oxidoreductase (complex II-III) and decreased CoQ levels in tissues and CSF (Van Maldergem et al 2002). A gene defect affecting the synthesis of CoQ leading to Leigh syndrome and nephropathy was first identified in 2006 by Lopez and colleagues (Lopez et al 2006). Mutations were found in the PDSS2 subunit gene of CoQ. Complex III has rarely been associated with Leigh disease. De Lonlay and colleagues reported mutations in a complex III nuclear encoded subunit (BCS1L) in patients with a multisystem disorder affecting liver, kidney, and brain (De

7 Lonlay et al 2001). MRI characteristics were compatible with Leigh syndrome. TTC19 gene mutation in a patient with Leigh syndrome was reported and believed to cause either a loss of protein function or mrna decay. TTC19 encodes tetratricopeptide 19, a CIII assembly factor on the inner mitochondrial membrane, thus affecting CIII function (Atwal 2014). Complex IV (cytochrome c oxidase, COX). Human COX comprises 13 subunits: 3 are mitochondrial DNA-encoded, whereas the remaining are encoded by nuclear genes. A major cause of Leigh disease and COX deficiency are mutations in SURF1 gene, which encodes an assembly factor for complex IV (Zhu et al 1998; Tiranti et al 1999). In a large retrospective study of 180 patients diagnosed with complex IV deficiency from Eastern Europe, 47 had a mutation in SURF1 (Böhm et al 2006). All patients with a mutation in SURF1 suffered from Leigh syndrome. Mutations in other assembly factors usually do not cause Leigh disease but other mitochondrial syndromes (Shoubridge 2001). Two published reports associate COX assembly factor COX10 with Leigh disease (Antonicka et al 2003; Coenen et al 2004), whereas mutations in another assembly factor, COX15, have been associated with early onset (Oquendo et al 2004) and adult Leigh disease (Bugiani et al 2005). Interestingly, the most common nuclear gene mutation associated with Leigh disease, SURF1, may show distinctive MRI abnormalities with predominant abnormalities in MR involvement of the subthalamic nuclei, medulla, inferior cerebellar peduncles, and substantia nigra. It is claimed that this pattern may predict SURF1 mutations (Farina et al 2002; Rossi et al 2003). SURF1 has even been associated with hypertrichosis in Leigh syndrome, and according to the group who studied this relationship, should prompt a work-up for SURF1 mutation (Baertling et al 2013). Mitochondrial mutations as a cause of biochemical COX deficiency are relatively rare. Tiranti and colleagues reported a mutation in the mitochondrial COX III gene in a patient with Leigh disease (Tiranti et al 2000). Darin and colleagues in a series of COX deficient patients detected 1 with a mitochondrial trna (trp) mutation (Darin et al 2003), and others are on record (DiMauro and Schon 2003). LRPPRC is attributed to complex IV deficiencies due to defects in mitochondrial translation process affecting specific subunits (Sasarman et al 2010) as it is for TACO1 (Weraarpachai et al 2009). LRPPRC mutations are associated with the French-Canadian variant of Leigh syndrome, a severe early-onset type with involvement of the liver, also known as Saguenay-Lac-Saint-Jean cytochrome c oxidase deficiency (SLSJ-COX) (Debray et al 2011). Interestingly, work by Mourier and associates has shown that in LRPPRC conditional knockout mice hearts, there is instead a defect that results in ATP synthase deficiency (Mourier et al in 2014). The role of LRPPRC protein in mrna stabilization has been well documented at this point (Sasarman et al 2010; Sasarman et al 2015), and these findings may need further exploration. SCO2 (synthesis of cytochrome c oxidase) gene mutations have also been reported to cause Leigh syndrome, and the mechanism has been further elucidated. Chadha and colleagues propose that the documented variants of SCO2, which encodes for an assembly factor for COX that acts to transport copper ions into the complex, alter the structure of the protein, thus leading to functional COX deficiency (Chadha et al 2014). NDUFA4 mutations, originally believed to cause complex I defects, have now been shown to be associated with complex IV instead. Using polyacrylamide gel analysis, Pitceathly and colleagues showed that NDUFA4 is a subunit of COX and defect can lead to disease (Pitceathly et al 2013). PET100 was shown to encode a complex IV biogenesis factor that is located on the inner mitochondrial membrane and forms a subcomplex with complex IV subunits. Mutation in this gene in 8 Lebanese individuals was causative for Leigh syndrome and has been unrecognized until this point because of the small size of the gene (Lim et al 2014). Complex V (ATP synthase). Complex V comprises approximately 16 subunits of which 2 are encoded by mitochondrial DNA; the remaining are encoded on nuclear genes. A point mutation, affecting nucleotide 8993 within the ATPase 6 subunit, is associated with Leigh disease (Santorelli et al 1993; Carrozzo et al 2001; Akagi et al 2002; Jacobs et al 2005; Moslemi et al 2005). Heteroplasmy determines the phenotype, with high (greater than 90%) mutation loads associated with Leigh disease and lesser mutation loads present in asymptomatic relatives. Two other mitochondrial disorders associated with the ATPase 6 gene are NARP (neuropathy-ataxia-retinitis pigmentosa) and familial bilateral striatal necrosis. In the first patient with NARP, Holt and colleagues discovered the heteroplasmic 8993 point mutation that was subsequently found also to cause Leigh disease (Holt et al 1990; Santorelli et al 1993). It is remarkable that the same m.8993tà mutation causes different disorders, Leigh syndrome and NARP, dependent on the degree of heteroplasmy (ie, mutation load). Familial striatal necrosis is mainly expressed as late infantile or juvenile onset dystonia with putaminal lesions and a slowly progressive course. The MRI lesions are stable and do not proceed to involvement of the brainstem as seen in Leigh disease. In this disorder different point mutations in the ATPase 6 gene have been found (De Meirleir et al 1995). As mentioned above, work now potentially places LRPPRC mutations as a causative deficiency in ATP synthase as well. Interestingly, Arun and colleagues reported on the association of neurofibromin (NF1) and LRPPRC. They showed that the tubulin binding domain of NF1 is a binding partner of the LRPPRC protein and may be a link that can help to further explain each disorder (Arun et al 2013).

8 Dysfunction of multiple respiratory chain complexes. Disease caused by defective trna genes. Twenty-two trna genes form part of the mitochondrial genome. Aminoacids, each specified by a particular trna, are incorporated during the production of many mitochondrial proteins. Commonly found in MELAS and MERRF, trna mutations are only rare causes of Leigh disease (Sue et al 1999; Darin et al 2003; Tulinius et al 2003), the most common affecting trnalys (Schapira 2006). Different phenotypes are caused by mitochondrial DNA mutations. The outcome of such mutations depends on the percentage of affected versus normal mtdna (degree of heteroplasmy) and the yet unexplained tissue proclivity. Several excellent reviews reflect on these aspects (DiMauro and Schon 2003; Schapira 2006; Zeviani and Carelli 2007). trna mutations affect overall functioning rather than specific complexes, and the result of mitochondrial studies in muscle may show more than 1 complex being involved, eg, a combined decrease of activities of complexes I and IV together with a decrease in overall ATP production. Tissue heteroplasmy also results in non-specific decline of ATP production. Techniques that make use of immunochemistry against complexes may reveal heteroplasmy in biopsy material, eg, cultured fibroblasts by showing a mosaic pattern of staining for each complex (De Paepe et al 2006). Occasionally, homoplasmic single-base mtdna mutations may occur that affect both a mother and all children, but with different degrees of illness, despite the fact that all carry the same mutation load. An impressive example reported by McFarland and colleagues describes a mother and her 6 children, who all carried the same mt-trnaval mutation, which resulted in mild symptoms in the mother and lethal disease in all 6 children, including a case of Leigh syndrome (McFarland et al 2002). In rare instances, large-scale deletions of mitochondrial DNA may cause Leigh disease. This contrasts with adult-onset progressive external ophthalmoplegia in which large-scale mutations of mtdna are commonly found (Yamashita et al 2008). MtDNA depletion as a cause of Leigh syndrome. A growing group of disorders involves mutations to nuclear genes involved in mitochondrial nucleotide synthesis. This causes depletion of mitochondrial DNA. Multicomplex involvement is usual in mtdna depletion. mtdna depletion has various clinical effects that may include Leigh syndrome. An example of the latter is SUCLA2 mutation (Elpeleg et al 2005; Ostergaard et al 2007). SUCLA2 encodes 1 subunit of the citric acid cycle enzyme succinyl-coa ligase that reversibly catalyses the formation of succinate and ATP from succinyl- CoA and ADP. Its role in mitochondrial nucleotide synthesis is not solved completely. Deficiency causes both depletion of mtdna with a reduction of respiratory chain complexes I, III and IV and increased excretion of methylmalonic acid, formed from succinyl-coa. Another nuclear gene, POLG, encodes polymerase-gamma, which forms part of the mitochondrial DNA replication machinery. Its deficiency also causes mtdna depletion. Its most common consequence in infants and children is Alpers syndrome, a progressive disease affecting liver and cerebral cortex. Rarely, a mutation of POLG may result in Leigh syndrome (Taanman et al 2009). Large-scale mtdna mutations as a cause of Leigh syndrome. Chae and colleagues report a heteroplasmic large-scale deletion of mtdna resulting in Leigh syndrome, renal tubulopathy, and hypoparathyroidism (Chae et al 2010). Defects in mitochondrial DNA translation. Another emerging group of disorders with impairment of multiple respiratory chain complexes is due to mutations in nuclear genes that encode proteins involved in the translation of mitochondrial RNA transcripts. This subgroup of nuclear genes that affect mitochondrial protein translation processes includes mitochondrial elongation factor (EFG1) associated with infantile onset encephalopathy with characteristics of Leigh syndrome (Valente et al 2007). Ahola and associates found that defects in mitochondrial translation elongation factor Ts (EFTs) were responsible for infantile mitochondrial cardiomyopathy that progressed to juvenile Leigh syndrome in 2 siblings (Ahola et al 2014). The mutation was found in the TSFM gene that encodes for mitochondrial EFTs, leading to protein instability and mitochondrial translation defect. Interestingly, a high carrier frequency of 1:80 for this mutation was found in a Finnish population of 35,000 with no homozygotes, likely because it is homozygous lethal. Another nuclear gene encoding a protein required to stabilize mrna transcripts for mitochondrial DNA translation, LRPPRC, is mutated in the French-Canadian Leigh syndrome, a severe early-onset type with typical involvement of the liver, and acute crises, either due to lactic acidosis or stroke-like episodes against a backdrop of moderately impaired development, a pattern unusual in other types of Leigh syndrome. The mitochondrial dysfunction typically affects COX (complex IV) (Debray et al 2011). The function of LRPPRC has become elucidated (Ruzzenente et al 2012). However, there now exists an alternative explanation of the mechanism of LRPPRC. Work by Mourier and colleagues, also done in mice cardiac tissue as done by Ruzzenente and associates, showed instead inactivate subassembled ATP synthase

9 complexes causing mitochondrial membrane hyperpolarization and increased mitochondrial ROS species production (Mourier et al 2014). DiMauro and Garone thoroughly review the impact of genetic mitochondrial disorders on the fetus and newborn (Dimauro and Garone 2011). Further support of the need for proper translational of mitochondrial DNA came from the work by Kopajtich and associates (Kopajtich et al 2014). They showed that individuals carrying compound heterozygous or homozygous mutations in GTPBP3 had combined respiratory chain complex deficiencies in skeletal muscle, lactic acidosis, hypertrophic cardiomyopathy, neurologic symptoms, and MRI involvement of the thalamus, putamen, and brainstem. These mutations resulted from mitochondrial translational defects due to the role of GTPBP3 in forming τm5(u) in the anticodon wobble position of 5 mitochondrial trnas. In the initiation of translation, mitochondria rely on the N-formylation of initiator methionyl-trna (Met-tRNA) by mitochondrial methionyl-trna formyltransferase (mt-mtf). Sinha and colleagues showed that compound heterozygous mutations within the nuclear genome for mt-mtf resulted in reduced mitochondrial translation efficiency and combined oxidative phosphorylation deficiency leading to Leigh syndrome (Sinha et al 2014). It is not known, though, if this mutation causes reduced mitochondrial translation because of the reduced activity of the mt-mtf and/or because of reduced levels of the mutated mt-mtf. Impact of radical oxygen species due to respiratory chain impairment. Quantitative studies on the impact of complex I deficiencies in patient fibroblasts demonstrated that decrease of complex I activity is inversely correlated with a rise in the production of radical oxygen species (Distelmaier et al 2009). This finding supports the use of antioxidants in therapy. Furthermore, the oxidative damage reserve has been found to be significantly reduced in all patients with mitochondrial disorders, including Leigh syndrome. It was shown that such patients have significant redox imbalance, increased levels of oxidation, lower blood reduced glutathione, and higher oxidized glutathione levels (Enns et al 2014). In fact, Enns and colleagues and Pastore and associates suggest that measuring peripheral whole blood GSH and GSSH levels may be indicated in monitoring mitochondrial dysfunction (Pastore et al 2013; Enns et al 2014). Pyruvate dehydrogenase complex deficiency. Pyruvate dehydrogenase complex (PDHc) deficiency is an important cause of progressive and relapsing inherited brain disorders, including Leigh disease, but also typically may cause brain malformations including callosal dysgenesis, gyral abnormalities, and periventricular cysts. PDHc is a mitochondrial enzyme that converts pyruvate into acetyl CoA. It links the glycolytic pathway to the citric acid cycle. It is at an important control point determining the balance of energy generation from carbohydrates, fatty acids, and amino acids as all produce acetyl-coa as final product (Brown et al 1994). Acetyl-CoA is the starting point of the citric acid cycle, which delivers energy equivalents to the mitochondrial respiratory chain. The brain in its early developmental stage is mainly dependent on glycolysis. Therefore, PDHc deficiency affects the developing brain at the embryo-fetal stage as opposed to respiratory chain defects where the time of onset is usually around birth or later. This difference in timing of energy processes affecting brain development has important consequences. It explains malformative effects of PDHc deficiency as opposed to deficiencies of the respiratory chain. Leigh syndrome and brain malformations are not mutually exclusive; when seen together, PDHc deficiency is very likely to be the cause. The site of the PDHc enzyme complex is in the mitochondrial matrix. The complex is made up of up 3 enzyme activities: E1, E2, and E3, (pyruvate dehydrogenase E1, dihydrolipoamide acetyl transferase E2, and lipoamide dehydrogenase E3) to which are attached a kinase and a phosphatase that act as on-off switches for the complex; the X-protein or E3 binding protein, which binds E2 to E3. E1, pyruvate dehydrogenase, is a tetramer constructed from 2 units E1alpha and 2 units E1beta. Thiamine pyrophosphate acts as its coenzyme. The E1alpha subunit is encoded on the X-chromosome by the PDHA1 gene, the E1beta subunit by PDHB, E2 by DLAT, E3 by DLD, E3-binding protein by PDHX and PDH-phosphatase by PDP1. With the exception of PDH-kinase, all parts have become associated with clinical metabolic defects. The most severe mutations cause fatal neonatal lactic acidosis. Female neonates with microcephaly, cerebral malformations including callosal agenesis, and neuronal migration disorder are another distinctive group with E1alpha mutations (Brown 1992). Although E1alpha is encoded on the X-chromosome, affected females are symptomatic because of non-random X-inactivation favoring the affected X-chromosome. E3 participates in 2 other reactions beside PDH: 2-oxoglutarate dehydrogenase and in breakdown of branched-chain amino acid. Deficiency of E3, therefore, causes an increase in the excretion of 2-oxoglutarate and an increase of branched-chain

10 amino acids in plasma (Grafakou et al 2003). Four neurologic subtypes were identified in a group of 22 patients diagnosed with PDHc dysfunction (Barnerias et al 2010): (1) neonatal encephalopathy with lactic acidosis, (2) nonprogressive infantile encephalopathy, (3) Leigh syndrome, and (4) recurrent ataxia. Leigh syndrome was diagnosed in 8 males. Cerebral malformations diagnosed by MRI were found in both PDHA and PDHX mutations, consisting of periventricular pseudocysts, gyral abnormalities, polymicrogyria, and total or partial callosal agenesis. In a retrospective analysis of 371 published PDHc cases, patients with a defect in the X-linked E1alpha subunit (PDHA1) were the largest subgroup with the ratio between females and males close to equal (Patel et al 2012). Neuroimaging of PDHc deficient patients showed lesions compatible with Leigh syndrome in 27% of available studies (n=186). The most common manifestation was ventriculomegaly. Hypogenesis or agenesis of the corpus callosum was found in 31%, the latter finding typically found in E1alpha deficiency. Lethality was high, with most of the deaths in early childhood. In another large serial study of 59 patients affected by various mutations in PDHA1, PDHB, and DLAT, no correlation was found with genotype, but males affected by PDHA1 mutations were more severely affected than females with more prolonged survival in the latter (Debrosse et al 2012). Thiamine transporter defect. There is current debate in this area on the pathophysiology and treatment for these defects. In a reply to Haack and colleagues' work in 2014, Gerards summarizes the multiple manifestations resulting from thiamine transporter defects including Warnicke-like encephalopathy, leukoencephalopathy, and Leigh syndrome (Gerards et al 2014). In 2013, Gerards and colleagues described the SCL19A3 mutation resulting in Leigh syndrome. SCL19A3 is a low affinity, high capacity transporter and 1 of 2 known thiamine transporters (the other being SCL19A2) that bring thiamine into the cell (Gerards et al 2013). Though defects in this gene have been reported previously, Gerards and colleagues showed that Leigh syndrome developed in Moroccan patients because of a nonsense c.20c>a mutation leading to complete absence of the protein. These patients showed response to thiamine administration. Because thiamine transport defects may result in biotin-thiamine responsive basal ganglia disease, Leigh syndrome, or 1 of the other reported phenotypes, it is justified that all patients suspected of Leigh syndrome should be given high doses of thiamine and biotin. Thiamine pyrophosphokinase (TPK) deficiency. In a report, a defect in thiamine pyrophosphokinase was found to be present in a patient with Leigh syndrome-like features including progressive, early-onset developmental delay. TPK produces thiamine pyrophosphate once thiamine is transported into the cell, and is a cofactor for pyruvate dehydrogenase, transketolase, 2-ketoglutarate dehydrogenase, and branched chain α-keto acid dehydrogenase. In this case, Banka and colleagues reported that lactic acidosis may not be present and 2-ketoglutaric aciduria may be the only marker present (Banka et al 2014). However, unlike patients with typical TPK deficiency or thiamine transporter defects, their Leigh syndrome-like patient was not responsive to early thiamine supplementation. Fatty acid oxidation and valine catabolism defects. Short chain enoyl coenzyme A hydratase is a key enzyme in multiple pathways including β-oxidation of short- and medium-chain fatty acids and catabolism of isoleucine and valine from methacrylyl-coa. In regards to fatty acid metabolism, compound heterozygous mutations in the ECHS1 gene were found in a 21-month-old boy with hypotonia, metabolic acidosis, and developmental delay. A combined respiratory chain deficiency was also observed in this patient. Function was restored when the patient-derived myoblasts were made to express exogenous, wild-type ECHS1 (Sakai et al 2015). In regards to the valine metabolism, build-up of methacrylyl-coa and acryloyl-coa was attributed to 2 siblings with fatal Leigh syndrome. Interestingly enough, build-up of fatty acid pathway metabolites were not found in these patients, speaking to both the diversity and metabolic specificity within 1 enzyme (Peters et al 2014). Glycogen synthesis. Two male siblings were investigated with findings of Leigh syndrome and ketonemia without elevation of lactate and pyruvate. X-linked recessive mutations were found in GYG2. GYG2 encodes glycogenin-2 protein, which is involved in glycogen synthesis initiation. The mutation was found to alert the protein structure, resulting in destabilization and malfunction (Imagawa et al 2014). Epidemiology" The described incidence of the disorder is approximately 1 in 40,000 births, though this is highly population specific, as an isolated group in Quebec, Canada has been found to have an incidence of 1 in 2000 births and a Faroese population an incidence of 1 in 2500 births. In another study of the northern European population, the incidence was found to be

11 1 in 32,000 (Ruhoy and Saneto 2014). Prevention The only prevention of Leigh disease lies in genetic counseling or prenatal diagnosis. For prenatal diagnosis a pathogenic mutation in a nuclear or mitochondrial gene should be identified. In the case of an inherited mitochondrial mutation all offspring may become clinically affected depending on the ultimate degree of the mutation load. Differential diagnosis The age of presentation and the pattern of neurologic involvement in Leigh disease are highly variable. The usual presentation is a subacute encephalopathy, often with brainstem involvement. This kind of presentation usually is sufficiently alarming to prompt MRI investigation. The most encountered finding on MRI is bilateral involvement of basal ganglia, the brainstem, or both. The spinal cord may be involved initially as well. Bilateral basal ganglia involvement may be seen in several inborn errors beside respiratory chain anomalies and involvement of the pyruvate dehydrogenase complex. Lactic acid elevation in blood, CSF, or both, is usual, but may be minimal or even absent. Each of the typical findings in Leigh disease may be seen in other disorders as well. The clinical presentation with stepwise regression is also seen in vanishing white matter disease, an autosomal recessive disorder. In this disorder no involvement is seen of the basal ganglia and the white matter proceeds to cavitation. Lactic acidosis may be seen in other disorders, eg, biotinidase deficiency, disorders of gluconeogenesis, and occasionally in congenital disorders of glycosylation. CDG1A may present with lactic acidosis (Briones et al 2001). Diagnostic exclusion of this group is by sialotransferrin electrophoresis in plasma. Pure involvement of the caudate nucleus and putamen combined with progressive dystonia during childhood is seen in infantile striatal necrosis, an autosomal recessive disorder. Associated gene defects have been found in mitochondrial ATPase 6 gene (De Meirleir et al 1995) and in the nuclear pore gene nup62 (Basel-Vanagaite et al 2006). It is worth addressing biotin responsive and biotin-thiamine responsive basal ganglia disease, as these fully imitate in clinical and imaging findings. As such, it is important to treat these as Leigh syndrome for all intents and purposes. Thus, it is suggested that all patients with suspected Leigh syndrome should receive thiamine and biotin (Baertling et al 2014). The diagnosis should be considered in nearly all cases of progressive encephalopathy in infants and especially in cases with associated failure to thrive, unexplained vomiting, or diarrhea, particularly when there is a suggestive MRI profile. Diagnostic workup Presentation and course of Leigh disease are highly variable (Rahman et al 1996). The following profile will aid in selecting patients for specific diagnostic work-up: (1) Stepwise, subacute neurologic regression, often following an infectious period, often involving the brainstem. (2) MRI findings of bilateral involvement of basal ganglia, mostly the putamina, eventually also involving the brainstem nuclei and white matter. Some patterns of MRI findings may be associated with certain deficiencies. Patients with complex I deficiencies have been found to have bilateral brainstem lesions as well as anomalies of the putamen. Interestingly, supratentorial stroke-like lesions were found only in patients with mitochondrial DNA complex I deficiencies. Meanwhile, necrotizing leukoencephalopathy was found in patients with nuclear DNA mutations. Conversely, patients with MT-TLI and PDH deficiencies demonstrated stroke-like images or corpus callosum malformations, respectively, that were not found in complex I deficiencies (Lebre et al 2011). Proton MR-spectroscopy may show elevated lactate in the most affected areas (eg, basal ganglia). (3) A positive family history including evidence of individuals with mild phenotypic expression. There may be a history of parental consanguinity, similar cases, or recurrent miscarriages (Baertling et al 2014). The family history may also help to establish the mode of inheritance. A pedigree supporting mitochondrial (maternal) inheritance may greatly aid in the molecular genetic approach (mitochondrial vs. nuclear genes). Laboratory investigations.