Mitochondrial disease in children

Review Symposium doi: 10.1111/joim.13054 Mitochondrial disease in children S. Rahman From the Mitochondrial Research Group, UCL Great Ormond Street Institute of Child Health, London, UK Content List – Read more articles from the symposium: “Mitochondria in human disease” Abstract. Rahman S (Paediatric Metabolic Medicine, UCL Great Ormond Street Institute of Child Health, London, UK). Mitochondrial disease in children (Review Symposium). J Intern Med 2020; 287: 609– 633. Mitochondrial disease presenting in childhood is characterized by clinical, biochemical and genetic complexity. Some children are affected by canonical syndromes, but the majority have nonclassical multisystemic disease presentations involving virtually any organ in the body. Each child has a unique constellation of clinical features and disease trajectory, leading to enormous challenges in diagnosis and management of these heterogeneous disorders. This review discusses the classical mitochondrial syndromes presenting most frequently in childhood and then presents an organbased perspective including systems less frequently linked to mitochondrial disease, such as skin and hair abnormalities and immune dysfunction. An approach to diagnosis is then presented, encompassing clinical evaluation and biochemical, neuroimaging and genetic investigations, and emphasizing the problem of phenocopies. The impact of next-generation sequencing is discussed, together with the importance of functional validation of novel genetic variants never previously linked to mitochondrial disease. The review concludes with a brief discussion of currently available and emerging therapies. The field of mitochondrial medicine has made enormous strides in the last 30 years, with approaching 400 different genes across two genomes now linked to primary mitochondrial disease. However, many important questions remain unanswered, including the reasons for tissue specificity and variability of clinical presentation of individuals sharing identical gene defects, and a lack of disease-modifying therapies and biomarkers to monitor disease progression and/or response to treatment. Keywords: diagnostic approach, differential diagnosis, mitochondrial genetics, next-generation sequencing, phenomics, phenocopies. Introduction Architecture of this review Mitochondrial structure, function and dysfunction In this review, I will discuss the clinical complexity of mitochondrial disorders presenting in childhood, starting with a description of some of the classically recognized stereotypic syndromes, arranged according to the age at onset of symptoms. I will then take an organ-based approach to the clinical features of paediatric mitochondrial disease. I will also consider the biochemical and genetic complexity of these disorders and guide the reader to a structured approach to diagnosing these challenging disorders. Finally, I will discuss the problem of phenocopies, and how to distinguish mitochondrial disorders from other complex multisystem disorders presenting in childhood, including the increasing number of disorders in Mitochondria are dynamic subcellular organelles with innumerable functions, including energy generation via oxidative phosphorylation (OXPHOS), calcium homeostasis and regulation of apoptotic cell death, placing them at the centre of cellular metabolism and signalling. There continues to be considerable debate about the precise definition of primary mitochondrial diseases but we have recently attempted to define these as genetic disorders leading either to OXPHOS dysfunction or other disturbances of mitochondrial structure and function [1]. Primary mitochondrial disorders have been estimated to have a minimum birth prevalence of 1 in 5000 [2]. ª 2020 The Association for the Publication of the Journal of Internal Medicine 609 Mitochondrial disease in children / S. Rahman which secondary mitochondrial dysfunction has been reported [3]. Clinical complexity: Canonical syndromic presentations of childhood mitochondrial disease A clinical classification was the first rational classification of mitochondrial disease, since canonical syndromes with particular constellations of symptoms and signs had been recognized for decades before their genetic basis was understood [4-9]. A selection of these syndromes is described below, according to age at presentation (Fig. 1), and an ‘A to Z’ of mitochondrial syndromes is provided in Table 1. However, it is important to remember the enormous clinical heterogeneity of mitochondrial disease (Fig. 2) and that most children affected by mitochondrial disease do not have a classical syndromic presentation, particularly at the earliest stages of their disease when only a single organ may be involved. Congenital lactic acidosis Presentation with lactic acidosis at or shortly after birth may reflect an acquired problem such as hypovolaemia, hypoxia or sepsis, but an underlying inborn error of metabolism should be considered in the differential diagnosis. More common metabolic causes include deficiencies of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), as well as mitochondrial respiratory chain disorders, but lactic acidosis may also be a feature of disorders of gluconeogenesis (e.g. fructose-1,6bisphosphatase deficiency and glycogen storage disease type 1), tricarboxylic acid cycle and long- chain fatty acid oxidation defects, and some organic acidurias including biotinidase deficiency, methylmalonic acidaemia (MMA) and propionic acidaemia (PA). Metabolic clues to the underlying diagnosis include a low lactate/pyruvate ratio in PDH deficiency, hyperammonaemia in PC and carbonic anhydrase VA (CA5A) deficiencies, profound hypoglycaemia in the gluconeogenesis disorders and characteristic organic acid excretion in biotinidase deficiency, MMA and PA. Of the mitochondrial respiratory chain disorders, fatal infantile lactic acidosis (FILA) has most frequently been reported in children with complex I deficiency [10], but is increasingly recognized as a feature of other mitochondrial disorders. Of note, the lactic acidosis may be transient and resolve within a few days of birth in some primary mitochondrial disorders [11], leading to diagnostic difficulties. Although I have started with congenital lactic acidosis as the earliest onset mitochondrial disease, antenatal mitochondrial disease presentations are increasingly recognized. For example, prenatal presentation has been reported in some of the early-onset CoQ10 biosynthesis deficiency syndromes [12,13], defects of mitochondrial ribosomal proteins [14,15] and even in POLG disease [16]. Infantile-onset mitochondrial DNA depletion syndromes The mitochondrial DNA (mtDNA) depletion syndromes (MDDS) are defined by a quantitative reduction of the absolute mtDNA copy number, which was initially arbitrarily set at < 30% of agematched controls [17,18]. However, infants with severe MDDS typically have < 10% residual mtDNA in the most affected tissues, although Fig. 1 Paediatric mitochondrial diseases arranged by age of onset. Canonical syndromic presentations of paediatric mitochondrial disease arranged according to age of onset. 610 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 Mitochondrial disease in children / S. Rahman Table 1 An alphabetical classification of mitochondrial syndromes Clinical features Gene defect(s)a ACAD9 deficiency HCM, exercise intolerance, lactic acidosis ACAD9 Alpers syndrome Intractable epilepsy, psychomotor regression, liver disease POLG Disease A B Barth syndrome DCM, myopathy, neutropaenia TAZ Bjornstad syndrome SNHL, pili torti BCS1L C Cowchock syndrome Axonal neuropathy, SNHL, cognitive impairment AIFM1 D DARS2 deficiency Leukoencephalopathy with brain stem and spinal cord DARS2 E Ethylmalonic Acrocyanosis, petechiae, chronic diarrhoea, developmental delay ETHE1 involvement and lactate elevation (LBSL) encephalopathy F Friedreich ataxia Progressive ataxia, HCM, sensory neuropathy, diabetes mellitus FXN G GRACILE syndrome Growth retardation, aminoaciduria, cholestasis, iron overload, BCS1L lactic acidosis, early death H HUPRA Hyperuricaemia, pulmonary hypertension, renal failure, SARS2 alkalosis I IOSCA Infantile-onset spinocerebellar ataxia TWNK J Juvenile-onset POLG Ataxia neuropathy spectrum (ANS) and Myoclonic epilepsy, POLG K Kearns–Sayre L Leigh syndrome Subacute necrotizing encephalomyelopathy ~100 genes LHON Leber hereditary optic neuropathy MT-ND1, MT-ND4, MT- MEGDEL 3MGA, deafness, encephalopathy, Leigh-like SERAC1 MELAS Mitochondrial encephalomyopathy, lactic acidosis, stroke-like MT-TL1 syndromes syndrome myopathy, sensory ataxia (MEMSA) PEO, pigmentary retinopathy, cardiac conduction defects, ataxia, SLSMD high CSF protein, (onset < 20 years) ND6 M episodes MERRF Myoclonic epilepsy, ragged-red fibres MT-TK MLASA Myopathy, lactic acidosis, sideroblastic anaemia PUS1, YARS2 TYMP MNGIE Mitochondrial neurogastrointestinal encephalopathy N NARP Neurogenic muscle weakness, ataxia, retinitis pigmentosa MT-ATP O OPA1 disease Dominant optic atrophy, variably associated with SNHL and OPA1 multisystemic features P Pearson syndrome Sideroblastic anaemia, lactic acidosis, pancreatic exocrine SLSMD insufficiency PEO Progressive external ophthalmoplegia SLSMD Perrault syndrome Premature ovarian failure, SNHL, variable neurological CLPP, HARS2, LARS2, Q QIL1 deficiency Early-onset fatal mitochondrial encephalopathy, liver disease QIL1 R RARS2 deficiency Pontocerebellar hypoplasia type 6 RARS2 S Sengers syndrome Cataracts, HCM, muscle weakness, lactic acidosis AGK T TMEM70 deficiency 3MGA, HCM, hyperammonaemia, lactic acidosis TMEM70 U Unclassified The majority of children with mitochondrial disease do not have Nearly 400 genes manifestations TWNK, ERAL1 classical presentations ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 611 Mitochondrial disease in children / S. Rahman Table 1 (Continued ) Disease Clinical features Gene defect(s)a V VARS2 deficiency Severe encephalomyopathy, HCM VARS2 W Wolfram syndrome Diabetes insipidus, diabetes mellitus, optic atrophy, deafness WFS1 (DIDMOAD) X X-linked deafness– dystonia syndrome Y YARS2 deficiency Mohr–Tranebjaerg syndrome (deafness, dystonia, optic TIMM8A neuronopathy) Myopathy, lactic acidosis, sideroblastic anaemia YARS2 3MGA, 3-methylglutaconic aciduria; DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; SLSMD, single large-scale mitochondrial DNA deletion; SNHL, sensorineural hearing loss. a The most frequently associated gene defects are listed for each syndrome. some causes of MDDS are associated with less severe mtDNA depletion, for example 30-40% residual mtDNA in SUCLA2 deficiency [19]. The MDDS may be grouped according to clinical presentation (myopathic, encephalomyopathic, hepatocerebral or multisystem disorder) or underlying genetic mechanism (defect of the mtDNA replication apparatus, mtDNA repair, nucleoside metabolism or mitochondrial dynamics) [20,21]. Infantile-onset MDDS include RRM2B deficiency, which presents shortly after birth with progressive myopathy variably associated with sensorineural hearing loss (SNHL), renal tubulopathy and seizures [22]; Alpers–Huttenlocher syndrome (see below) and deficiencies of DGUOK and MPV17, which cause infantile liver failure with/without encephalopathic features; and TK2 deficiency, which presents as a severe progressive myopathy with elevated lactate and creatine kinase [20]. – – - Fig. 2 Clinical complexity of paediatric mitochondrial disease. Mitochondrial disease presenting in childhood may affect any tissue or organ system in any combination. The right-hand panel highlights the symptoms and signs of mitochondrial disease associated with each organ system. 612 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 Mitochondrial disease in children / S. Rahman Benign reversible mitochondrial myopathy Infants with so-called benign reversible mitochondrial myopathy present after a symptom-free interval at about 6 weeks to 3 months’ age with lactic acidosis and a profound myopathy affecting the limb and respiratory musculature, frequently leading to a requirement for gastrostomy feeding and artificial ventilation. The syndrome has been linked to two homoplasmic mtDNA variants at the same nucleotide in the MT-TE gene encoding the mitochondrial tRNA for glutamic acid [23,24]. Not all individuals with these variants have myopathy, so it seems that other factors are needed for expression of the disease. It has been proposed that the phenotype may be induced by a relative cysteine deficiency in early infancy [25], but possible efficacy of L-cysteine supplementation in this disorder remains to be determined. There is usually a remarkable recovery with supportive care, which may include invasive ventilation for up to 18 months, with only a mild residual myopathy persisting into adult life. Pearson syndrome Patients with Pearson marrow–pancreas syndrome typically present in early infancy, after a brief symptom-free interval, with a transfusion-dependent anaemia and lactic acidosis, variably associated with feeding difficulties and developmental delay. Age of onset ranged from birth to 36 months (mean 7 months) and birth to 16 months (mean 2.5 months) in two large series [26,27]. Ringed sideroblasts and vacuolated myeloid precursors are seen in the bone marrow aspirate, and diagnosis is made by the identification of a single largescale mtDNA deletion (SLSMD) in peripheral blood, bone marrow aspirate, urinary epithelial cells or muscle biopsy. Transfusion requirement is usually 2-3 weeks initially and gradually becomes less frequent over time, with complete resolution of anaemia typically occurring around 2 years. Other bone marrow lineages are frequently affected, leading to neutropaenia, thrombocytopaenia or pancytopaenia. Some patients with Pearson syndrome are extremely unwell with severe faltering growth, metabolic acidosis and hepatic impairment; there is an extremely high mortality in this group. Twelve of 21 cases (57%) died before 4 years in one series [26], and mean age at death was 2.5 years in the second series, with only 22% of cases alive at 18 years [27]. In surviving patients, resolution of the anaemia is associated with a ‘honeymoon’ period of relatively good health before the onset of multisystem problems in the Kearns– Sayre syndrome (KSS) spectrum (see below). These multisystem features may include ptosis, progressive external ophthalmoplegia (PEO), renal tubulopathy, pancreatic exocrine and/or endocrine insufficiency, and gastrointestinal (GI) disturbance (dysmotility or partial villous atrophy leading to malabsorption). Barth and Sengers syndromes Two syndromic cardiomyopathies presenting in infancy are Barth syndrome and Sengers syndrome. Barth syndrome is an X-linked disorder characterized by 3-methylglutaconic aciduria associated with dilated cardiomyopathy (DCM), skeletal myopathy (mainly proximal), impaired growth and (cyclical) neutropaenia [28]. Other cardiac features include endocardial fibroelastosis, left ventricular noncompaction and hypertrophic cardiomyopathy (HCM). TAZ mutations lead to impaired cardiolipin production in the inner mitochondrial membrane. Sengers syndrome is a rare autosomal recessive disorder caused by mutations in AGK encoding acylglycerol kinase, which is also a component of the mitochondrial import machinery [29,30]. Affected infants present with congenital cataract and HCM. Other clinical features include skeletal myopathy, exercise intolerance and lactic acidosis. Progressive cardiac failure may lead to death in infancy, but long-term survival has been reported in some patients [31]. Leigh syndrome This syndrome, also known as subacute necrotizing encephalomyelopathy, was first described as a neuropathological entity by Denis Leigh in 1951 [5]. Leigh reported the post-mortem brain findings of a 7-month-old baby who had died after a 6-week illness involving somnolence and visual and hearing impairment. Leigh described bilateral symmetrical spongiform lesions, especially in the brainstem, representing vacuolation of the neuropil with relative neuronal preservation and associated with demyelination, gliosis and capillary proliferation. During the last six decades, Leigh syndrome, as it has come to be known, has been recognized as the most frequent presentation of mitochondrial disease in childhood. Criteria for Leigh syndrome without the need for neuropathology have been developed, namely a characteristic clinical course (including neurodevelopmental ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 613 Mitochondrial disease in children / S. Rahman regression and symptoms and signs related to basal ganglia and/or brainstem dysfunction), elevated lactate levels in blood or cerebrospinal fluid (CSF) or other evidence of mitochondrial dysfunction (e.g. PDH or OXPHOS deficiency) and characteristic neuroimaging features of bilateral symmetrical T2 signal hyperintensity variably involving the basal ganglia, midbrain and brainstem structures [32]. Leigh syndrome appears to be a common end-point of deficient cerebral energy generation and has been linked to ~ 100 different mitochondrial and nuclear gene defects, frequently with multisystemic disease involvement (www. vmh.life/#leighmap). Alpers–Huttenlocher syndrome Intractable epilepsy and developmental delay associated with characteristic neuropathology were first reported by Bernard Alpers in 1931, and the association with hepatic cirrhosis was noted by Peter Huttenlocher in 1976 [4,33]. Onset is typically in infancy or early childhood with initially focal motor seizures that evolve to bilateral convulsive seizures and often to epilepsia partialis continua and status epilepticus. The prognosis is extremely poor; most cases have a rapidly progressive course leading to death from status epilepticus or hepatic failure within a few months of presentation [34]. Hepatic involvement is not universal and may be triggered by sodium valproate therapy. Most cases are caused by biallelic pathogenic variants in POLG encoding the catalytic subunit of DNA polymerase gamma leading to severe mtDNA depletion, but rarer causes include defects of TWNK (encoding the mitochondrial DNA helicase) or of FARS2, NARS2 and PARS2 (encoding three mitochondrial aminoacyl-tRNA synthetases) [35]. Kearns–Sayre syndrome KSS was initially defined clinically as a triad of PEO, pigmentary retinopathy and heart block presenting before the age of 20 years, with minor criteria including cerebellar ataxia and elevated CSF protein levels [36]. In the intervening four decades, the disorder has been recognized to be a multisystem condition with heterogeneous clinical manifestations including SNHL, renal tubulopathy, GI dysmotility, endocrine disturbance, cardiomyopathy, basal ganglia calcification and leukoencephalopathy, often associated with cerebral folate deficiency [37] for reasons that remain 614 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 obscure. Some patients have a prior history of Pearson syndrome, but many present with KSS without a history of documented anaemia [27]. KSS is usually caused by SLSMDs but has also been reported with multiple mtDNA deletions caused by RRM2B mutations [38]. Progressive external ophthalmoplegia PEO as an isolated finding is a rare presentation of mitochondrial disease in childhood but does occur, usually in association with SLSMDs. More often, paediatric-onset PEO is part of a complex presentation, for example KSS or another multisystem mitochondrial disorder. There has been a long debate about distinction between ‘PEO-plus’ and KSS [36], and the current consensus is that there is a continuous spectrum of clinical phenotype associated with SLSMDs, ranging from Pearson syndrome at the most severe end through Kearns– Sayre syndrome to isolated PEO as the mildest manifestation of SLSMDs [27]. Exercise intolerance Exercise intolerance is a frequent symptom of mitochondrial myopathies in childhood, again usually in the context of a multisystem disorder. It may be an isolated finding in some children with a sporadic mtDNA mutation, particularly affecting a subunit of complex III or IV [39-42]. Characteristic features include muscle cramps and fatigue on exertion but patients may also present with vomiting after exercise related to lactic acidosis. Frank rhabdomyolysis with severe muscle pain and myoglobinuria is a rare presentation of mitochondrial myopathies, being more frequently seen in fatty acid oxidation disorders and glycolytic defects, but does occasionally occur [40,42]. Mitochondrial rhabdomyolysis was most recently reported in association with autosomal dominant multiple mtDNA deletions caused by heterozygous variants in DNA2, encoding a mitochondrial helicase/nuclease [43]. MELAS The syndrome of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) was defined clinically in 1984 [9]. Strokelike episodes may be heralded by migraine headache, homonymous hemianopia or quadrantanopia and seizures, which are often focal but may subsequently generalize as the stroke-like episode progresses. The first seizure or stroke-like Mitochondrial disease in children / S. Rahman episode occurred at a mean of 13.7 years in those with childhood onset in a prospective natural history study, with another peak at a mean of 35.7 years for adult-onset stroke-like episodes [44]. Additional symptoms include short stature, cognitive decline, exercise intolerance, SNHL, ptosis, optic atrophy, GI dysmotility and diabetes mellitus [44]. Eighty per cent of cases have the same pathogenic mtDNA variant, m.3243A>G in the MT-TL1 gene [45]. This variant is very common in the population, with a point prevalence of 1 in 400 [46], and is associated with a wide range of clinical phenotypes other than MELAS, both in childhood and in adult life [47]. life, with a median age of onset of ~20 years [55]. Painless subacute central visual loss typically affects both eyes sequentially. The disorder is most commonly caused by one of three homoplasmic mutations (m.3460G>A, m.11778G>A and m.14484T>C) in mtDNA genes encoding complex I subunits [55]. There is an increased risk of visual loss in males with these mtDNA variants, which is thought to reflect a protective effect of oestrogen in female carriers [56]. Most affected individuals have isolated ophthalmological symptoms, but there may be other clinical features such as dystonia [57] or cardiac conduction problems (Wolff–Parkinson–White) [58], and occasionally, LHON may overlap with Leigh syndrome and/or MELAS [59]. MERRF Myoclonus epilepsy with ragged-red fibres (MERRF) is another canonical mitochondrial syndrome associated with a single so-called common mtDNA mutation (m.8344A>G in the MT-TK gene) in ~ 80% of cases [48]. MERRF frequently presents in childhood with an insidious onset that may include ataxia, SNHL and endocrine disturbance. Onset was before 16 years in one-third of a large Italian cohort [49]. Myoclonus may not be apparent initially and may present later, with additional seizure types. Other clinical features of MERRF include cognitive impairment, multiple lipomatosis, ptosis/PEO, myopathy, peripheral neuropathy and cardiomyopathy [49]. Overlap with MELAS is well recognized, including stroke-like episodes [50,51]. Juvenile-onset POLG syndromes POLG disease presenting in adolescence may resemble Alpers–Huttenlocher syndrome, with severe intractable epilepsy and liver disease [52]. However, juvenile POLG disease may also mimic MERRF, with prominent symptoms of myoclonus and ataxia. Two acronyms have been used to describe the main phenotypes of POLG disease presenting in adolescence: myoclonic epilepsy, myopathy, sensory ataxia (MEMSA) and ataxia neuropathy syndrome (ANS) (reviewed in Ref. [21]). Rare presentations of POLG disease in childhood/adolescence are with a distal myopathy [53] or a disorder resembling mitochondrial neurogastrointestinal encephalopathy (MNGIE) [54]. Leber hereditary optic neuropathy Leber hereditary optic neuropathy (LHON) most frequently presents in the second or third decade of Other phenotypes: A systems approach to mitochondrial disease in childhood An alternative approach to clinical classification of paediatric mitochondrial disorders is to take an organ-based approach, as follows. Mitochondrial encephalomyopathies The three main, overlapping groups of mitochondrial encephalomyopathy are Leigh syndrome, mitochondrial epilepsies and a rapidly growing group of mitochondrial leukoencephalopathies. Leigh syndrome is the most frequently recognized mitochondrial encephalomyopathy, whilst the most prevalent mitochondrial epilepsy is Alpers– Huttenlocher syndrome. Epilepsy is also a prominent feature of the canonical syndromes MELAS and MERRF and may be seen in many cases of Leigh syndrome and in mitochondrial leukoencephalopathies. Epilepsy is estimated to have a prevalence of ~40-60% in paediatric mitochondrial disease and has been reported in association with defects in >140 mitochondrial disease genes, frequently as an adverse prognostic factor [60]. Causes of mitochondrial leukoencephalopathy include deficiencies of complex I and II assembly, defects of iron–sulphur cluster and lipoic acid biosynthesis and MNGIE. Mitochondrial myopathies Muscle is one of the most affected tissues in primary mitochondrial disease, reflecting the high energy demands of muscle contraction [61]. Isolated muscle involvement is a more frequent feature of adult-onset disease but may be observed in paediatric mitochondrial disease, as either the ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 615 Mitochondrial disease in children / S. Rahman initial or sole manifestation, although more usually myopathy is part of a complex multisystem disorder in childhood. There is a predilection for the external eye muscles, and the myopathy is usually proximal rather than distal, but exceptions include a distal myopathy in some cases of POLG disease [53]. The most frequent symptoms of mitochondrial muscle disease are fatigability, weakness, exercise intolerance and pain. Mitochondrial neuropathies Mitochondrial neuropathy may be axonal motor, axonal sensorimotor, sensory ataxia or demyelinating [62]. The classical mitochondrial neuropathy syndrome neurogenic muscle weakness, ataxia, retinitis pigmentosa (NARP) has been linked to pathogenic variants in the MT-ATP6 gene encoding a subunit of complex V [63]. Peripheral neuropathy is a frequent feature of multisystem mitochondrial disease in childhood, including deficiencies of SURF1, POLG, PDH and MPV17 [62,64]. Defects of two proteins involved in mitochondrial dynamics MFN2 and GDAP1 cause Charcot–Marie– Tooth (CMT) disease types 2A and 4A, respectively [65]. In addition, variants in genes previously recognized to cause mitochondrial encephalomyopathies have more recently been reported to cause isolated neuropathies resembling CMT, including MT-ATP6, SURF1, SCO2 and C12orf65 [65-69]. Mitochondrial eye disease In addition to eye muscle involvement (ptosis/ PEO), primary mitochondrial disease can affect any layer of the eye, ranging from corneal opacification (Pearson/KSS) to cataracts (e.g. Sengers syndrome, CLPB deficiency), optic neuropathy (e.g. LHON) and pigmentary retinopathy (e.g. NARP). Optic neuropathy is frequently seen in complex I deficiencies and may be an isolated finding, as in LHON, or be a feature of an encephalomyopathy such as the Leigh syndrome spectrum [70]. The most frequent mitochondrial optic neuropathy is dominant optic atrophy caused by OPA1 mutations [70]. Recently, variants in SSBP1 encoding the single-stranded binding protein needed for mtDNA replication have been shown to cause dominant optic neuropathy and retinopathy [71]. Mitochondrial hearing loss Mitochondrial hearing loss is sensorineural and may be syndromic or nonsyndromic (i.e. an isolated 616 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 finding) and is usually bilateral, symmetrical and progressive. It may be of cochlear origin or be caused by an auditory neuropathy. One study suggested that 40% of individuals with mitochondrial disease have hearing loss [72]. The most frequent genetic variant predisposing to mitochondrial nonsyndromic SNHL is m.1555A>G in the MT-RNR gene encoding a mitochondrial rRNA. This variant is prevalent in ~1 in 500 of the population and confers exquisite sensitivity to aminoglycoside-induced SNHL [73]. Without aminoglycoside exposure, individuals with this variant may have normal hearing until well into adult life, implying a role for newborn screening for the variant followed by lifelong avoidance of aminoglycosides in at-risk individuals [74]. Childhood-onset mitochondrial syndromes in which hearing loss is a prominent feature include MERRF, MELAS, KSS and deficiencies of SUCLA2, BCS1L, RRM2B, SERAC1, RMND1 and TRNT1 [19,22,27,38,47,49,75-78]. Mitochondrial heart disease The most frequent childhood presentation of mitochondrial heart disease is with severe HCM in infancy, although rarely this may be end-stage DCM at presentation. Pinpointing a mitochondrial aetiology to an infantile cardiomyopathy can be extremely challenging, since lactic acidosis may be secondary to hypoxia or hypovolaemia rather than an indicator of an underlying primary mitochondrial disorder. However, specific echocardiographic appearances are increasingly recognized, namely symmetrical hypertrophy with deep trabeculation of the myocardium, sometimes fulfilling criteria for noncompaction of the left ventricle. Barth and Sengers syndromes are described above but other causes of mitochondrial cardiomyopathy presenting in infancy are increasingly recognized, either as an isolated finding or as part of a complex multisystem disorder. These include disorders of mitochondrial translation and defects of coenzyme Q10 biosynthesis [79]. Cardiomyopathy may be seen in some children and adults with the m.3243A>G mutation and may be a cause of sudden death in these individuals [47,80]. Cardiomyopathy has been linked to a handful of mutations in the mitochondrial genome, but in many cases, these mutation reports are unconfirmed (www.mitomap. org). Nuclear gene defects appear to be a more frequent cause of mitochondrial cardiomyopathy, particularly in infancy, as recently reviewed in Ref. [79]. Cardiac conduction defects have been reported frequently in children and adults with Mitochondrial disease in children / S. Rahman KSS, and complete heart block may be a lifethreatening event in these individuals, indicating a need for regular screening ECGs [81]. Sudden death is also a feature of PPA2 mutations, and implantation of a cardiac defibrillator may be lifepreserving in affected individuals [82]. Wolff– Parkinson–White has been reported in LHON, MELAS and Leigh syndrome caused by the m.13513G>A mutation [44,58,83]. Valvular heart disease is a rare feature of mitochondrial disease but has been documented in individuals with mutations in PDSS1 encoding a coenzyme Q10 biosynthetic enzyme [84]. Recently, we have shown that children with Leigh syndrome caused by ADAR mutations are at risk of developing fatal cardiac valve calcification [85]. Mitochondrial endocrine disorders Any endocrine organ can be involved by primary mitochondrial disease, as a result of decreased intracellular production or extracellular secretion of hormones [86]. Diabetes mellitus is the most frequent endocrine manifestation of adult mitochondrial disease in the maternally inherited diabetes and deafness (MIDD) syndrome [47], but in childhood is more often seen in Pearson syndrome and KSS, together with growth hormone deficiency, adrenal insufficiency, hypothyroidism and hypoparathyroidism [27]. Premature ovarian failure is associated with SNHL in Perrault syndrome, a genetically heterogeneous disorder linked to defects of several mitochondrial genes including HARS2, LARS2, TWNK and CLPP, and is also observed in POLG disease and in association with leukoencephalopathy in AARS2 deficiency [86-88]. Mitochondrial endocrine dysfunction most frequently occurs in the context of multisystem disease, but isolated endocrine involvement may be the initial presentation of a mitochondrial disorder [86]. Mitochondrial renal disease Mitochondrial disease frequently affects the kidneys in children, most commonly in the form of a renal tubulopathy or as steroid-resistant nephrotic syndrome (SRNS) associated with focal segmental glomerulosclerosis (FSGS) on renal histology. Mitochondrial renal tubulopathy is most often seen in Pearson syndrome/KSS, and replacement of electrolyte losses can be a severe management problem in affected individuals [27]. The tubulopathy may be Fanconi-type or be more reminiscent of Gitelman syndrome with severe hypomagnesaemia. Tubulopathy is also observed in many other mitochondrial diseases, including defects of assembly of OXPHOS complexes III and IV (deficiencies of BCS1L, COX10 and SURF1), MDDS (notably RRM2B deficiency but also seen in some cases with DGUOK, MPV17, SUCLA2 and TK2 deficiencies) and disorders of mitochondrial translation (e.g. TFSM) [89]. SRNS is a major feature of several disorders of coenzyme Q10 biosynthesis (PDSS2, COQ2, COQ6 and COQ8B deficiencies), variably associated with SNHL, seizures and encephalopathy [89,90]. Recognition of this group and prompt initiation of high-dose coenzyme Q10 supplementation may prevent progression of the renal disease [91]. FSGS is relatively frequently observed in adults with m.3243A>G disease but rarely seen in children [92]. Some children with RMND1 mutations have a pseudohypoaldosteronism-like picture with hyperkalaemia, hyponatraemia and progressive renal impairment [77]. Mitochondrial gastrointestinal disease Nonspecific GI symptoms are a frequent feature of mitochondrial disease in childhood and may be amongst the first clinical features, presenting in early infancy with vomiting, feeding difficulties and faltering growth [93,94]. In ethylmalonic encephalopathy, caused by hydrogen sulphide toxicity resulting from ETHE1 mutations, persistent diarrhoea is associated with acrocyanosis, petechiae and developmental delay [95]. The archetypal mitochondrial syndrome with GI disturbance is MNGIE, caused by deficiency of thymidine phosphorylase leading to toxic accumulation of thymidine and deoxyuridine, which in turn stalls the mtDNA replication apparatus. Affected individuals have severe dysmotility and episodes of pseudo-obstruction associated with demyelinating peripheral neuropathy and a relatively asymptomatic leukoencephalopathy [96]. Although symptoms frequently start in childhood, the diagnosis is not usually made until adult life. Dysmotility is also a major feature of mitochondrial disease related to the m.3243A>G mutation, and a MNGIE-like phenotype has been reported in some patients with POLG mutations and RRM2B mutations [54,97,98]. Exocrine pancreatic insufficiency is a major feature of Pearson syndrome but has also been reported in other primary mitochondrial diseases, including deficiencies of COX4I2, TRNT1 and PTRH2 [78,99,100]. ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 617 Mitochondrial disease in children / S. Rahman Mitochondrial hepatopathies Hepatic involvement is a frequent feature of earlyonset mitochondrial disease and has been estimated to affect ~20% of cases with respiratory chain dysfunction [11]. Genetically confirmed mitochondrial disease was observed in 17% of a large cohort of children presenting with acute liver failure before the age of 2 years [12]. Presentation may be with acute or chronic liver failure and is typically progressive and fatal. However, severe neonatal hepatic dysfunction that usually resolves with time has been reported in approaching 50% of children with SERAC1 mutations [76]. Hepatic involvement may be part of a multisystem disorder, but in neonatal or early infantile mitochondrial liver disease demise from liver failure may occur before neurological symptoms become apparent. Characteristic histological features of mitochondrial liver disease include micro- and macrovesicular steatohepatitis, which may progress to micro- or macronodular cirrhosis [13]. The most frequent causes of mitochondrial liver disease are the hepatocerebral MDDS associated with pathogenic variants in DGUOK, MPV17, POLG, TWNK or SUCLG1, defects of complex III and IV assembly and disorders of mitochondrial translation (reviewed in Ref. [14]). Of these, defects of the mitochondrial translation factor TRMU have been reported to be potentially reversible in some cases [15]. Haematological involvement in mitochondrial disease Pearson syndrome is the most common cause of mitochondrial sideroblastic anaemia (see syndromic presentations above). Other marrow lineages are frequently also involved in Pearson syndrome; affected children may have neutropaenia and/or thrombocytopaenia in addition to the anaemia [27]. Less frequent causes of mitochondrial sideroblastic anaemia include TRNT1 deficiency [78,106] and the syndrome of myopathy, lactic acidosis and sideroblastic anaemia (MLASA), which has been linked to deficiencies of PUS1 and YARS2 [17,108]. Anaemia is a frequent finding in children with mitochondrial disease and is emerging as an adverse prognostic indicator [19]. Interestingly, a laboratory-based study identified iron as the most important stimulator of mitochondrial biogenesis [110]. Immune dysfunction and primary mitochondrial disease B-cell immune deficiency appears to be a consistent feature of TRNT1 deficiency, which is 618 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 sometimes known by the acronym SIFD, for sideroblastic anaemia, immune deficiency, fever and developmental delay [16]. This condition is characterized by recurrent debilitating episodes of severe fever. Other features of TRNT1 deficiency include cerebellitis, SNHL and pigmentary retinopathy [78]. TRNT1 is also required to modify cytosolic tRNAs, and it is not clear whether the Bcell immune deficiency observed in this disorder is because of mitochondrial dysfunction or whether it is a manifestation of disturbed cytosolic translation. Abnormal innate immunity occurs in deficiency of the JAK-STAT cytokine STAT2, which is part of a phosphorylation signalling cascade. We demonstrated abnormal mitochondrial fission in muscle and cultured skin fibroblasts of a series of children with STAT2 deficiency and went on to show that STAT2 is needed for activation of mitochondrial fission by phosphorylation of a critical serine at position 616 of the DRP1 protein [111]. Immune function has not been well studied in children with mitochondrial diseases, and it is possible that immune dysfunction may be a feature of other paediatric mitochondrial diseases. Dysmorphology Pattern recognition is an essential tool for the diagnostician. Characteristic facial appearances have been reported for a few mitochondrial disorders, including deficiencies of RARS2, FBXL4, TAZ and TMEM70 [28,112-114]. It is possible that other mitochondrial disorders may also be associated with specific facial appearances, but the ultrararity of most individual mitochondrial disorders means that no one clinician will see sufficient cases in their career to become adept at recognizing mitochondrial disease by facial appearance alone. However, it is possible that artificial intelligence tools may enable this sort of facial recognition in the future. For example, facial recognition software has been used to highlight characteristic facial appearances in congenital disorders of glycosylation [115]. Abnormalities of skin and hair Hypertrichosis is frequently a striking feature in children with SURF1 deficiency and was documented in 41% of cases in a large multicentre cohort [93]. The mechanism driving the hypertrichosis remains unknown, but we are increasingly recognizing this as a feature of other causes of Leigh syndrome and indeed of other mitochondrial Mitochondrial disease in children / S. Rahman disorders. Excess hair is typically observed on the forearms, thighs, shins and upper back. The presence of a low hairline, synophrys and pedal oedema in children with deficiency of the mitoribosomal protein MRPS22 led to the suggestion that this disorder mimics Cornelia de Lange syndrome [116]. Hair abnormalities (pili torti) have been reported in Bjornstad syndrome, one of the clinical manifestations of deficiency of the complex III assembly factor BCS1L [75]. Interestingly, not all patients with BCS1L mutations have pili torti, and it is not clear why this phenomenon is present in some patients but not others. Even more intriguingly, alopecia totalis was recently reported in children with deficiency of the complex III subunit UQCRFS1 [117], implying that complex III may be critical for hair growth. Cutis laxa syndromes have been associated with pathogenic variants in PYCR1 and ALDH18A1, encoding enzymes involved in de novo mitochondrial proline synthesis [118]. Genetic complexity of mitochondrial disease The genetics of mitochondrial disease is complex, with contributions from two genomes. Mitochondrial disease may be inherited by a number of different genetic mechanisms including maternal (mtDNA mutations) and autosomal recessive, autosomal dominant and X-linked for nuclear gene mutations. Sporadic mtDNA mutations have been recognized for more than three decades, but recently, an increasing number of de novo nuclear gene variants have been linked to mitochondrial disease, initially for DNM1L variants [119]. More recently, de novo variants have been reported in ATAD3A, CTBP1, ISCU, SLC25A4 and SLC25A24 [120]. DiMauro and colleagues were the first to classify the nuclear-encoded disorders rationally according to the underlying molecular defect [121]. Genetic causes of primary mitochondrial disease may be subdivided into defects of OXPHOS subunits and complex assembly, disorders of mitochondrial DNA maintenance, defects of mitochondrial gene expression, deficiencies of cofactor biosynthesis and transport, defects of mitochondrial solute and protein import, disorders of mitochondrial lipid membranes and organellar dynamics (fission/ fusion) and disorders of mitochondrial quality control (Table 2), but new disease mechanisms continue to be discovered. Since the advent of next-generation sequencing methods, there has been a rapid pace of gene discovery for mitochondrial disease and currently approaching 400 different gene defects have been linked to primary mitochondrial disease (Table 2). Approach to diagnosis When to suspect mitochondrial disease in childhood? There are probably no absolutely pathognomonic clinical features of mitochondrial disease in childhood. Clinical presentations that should arouse suspicion of an underlying mitochondrial disorder include stroke-like episodes, acquired ptosis and/ or ophthalmoplegia, sideroblastic anaemia and epilepsia partialis continua. However, it is frequently the combination of disease pathologies affecting multiple seemingly unrelated organs that triggers the clinical recognition of a mitochondrial disorder [122]. In other cases, the initial diagnostic clue may be a biochemical abnormality such as elevation of blood or CSF lactate, plasma alanine, urinary 3-methylglutaconic acid or other mitochondrial disease biomarkers [123]. Increasingly, a mitochondrial disorder is not suspected until potentially pathogenic genetic variants are identified in a known mitochondrial disease gene during nextgeneration sequencing of an exome or genome. Importance of history taking With so much focus on the enormous complexity of mitochondrial disease and the ‘differentness’ of it, the similarities to the rest of medicine may easily be overlooked. For example, very often the diagnosis rests in the history; careful attention to detail can allow the observant physician to win the diagnostic lottery. The patient, even a young nonverbal child, provides many clues about their personal story. The order of events is extremely important. What happened first? Was he or she born with the cataracts or squint or ptosis or did these features develop later? How does the mother, especially an experienced mother, feel about her child? Are things as expected? Or subtly different? So much of mitochondrial disease starts subtly, insidiously, in the first days and weeks of life. Did the baby not feed quite as well as they should, was there more than the usual amount of gastro-oesophageal reflux? Were they thrown off course by the mildest of infections? How do they compare to peers and siblings at the same age? Are developmental milestones being met? Has there been a loss of skills with intercurrent illnesses? Are they slower to recover from intercurrent illnesses than siblings, peers, other family members? How many systems are affected? It is important to enquire specifically about vision, hearing, language acquisition, cardiorespiratory and gastrointestinal symptoms, as well as about ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 619 Mitochondrial disease in children / S. Rahman Table 2 Classification of mitochondrial disease genes Mode of Disease mechanism Gene defects inheritance Disorders of Oxidative Phosphorylation (OXPHOS) Complexes and their Assembly Complex I Subunits and Assembly Factors MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6 mtDNA NDUFA1 XL NDUFA2, NDUFA6, NDUFA9, NDUFA10, NDUFA11, NDUFA12, AR NDUFA13 NDUFB3, NDUFB8, NDUFB9, NDUFB10, NDUFB11, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, NDUFAF7, NDUFAF8, ACAD9, ECSIT, FOXRED1, NUBPL, TIMMDC1, TMEM126B Complex II Subunits and Assembly Factors Complex III Subunits and Assembly Factors SDHA, SDHAF1 AR SDHB AR/AD SDHC, SDHD, SDHAF2 AD MT-CYB mtDNA UQCRB, UQCRC2, UQCRFS1, UQCRQ, UQCC2, UQCC3, CYC1, AR BCS1L, HCCS, TTC19, LYRM7 Complex IV Subunits and Assembly Factors MT-CO1, MT-CO2, MT-CO3 mtDNA COX7B XL COX4I1, COX4I2, COX5A, COX6A1, COX6A2, COX6B1, COX8A, AR NDUFA4, SURF1, SCO1, SCO2, COX10, COX14, COX15, COX20, COA3, COA5, COA6, COA7, FASTKD2, PET100, PET117, CEP89 Complex V Subunits and Assembly Factors Assembly of Multiple OXPHOS MT-ATP6, MT-ATP8 mtDNA ATP5F1A, ATP5F1D, ATP5F1E, ATP5MD, ATPAF2, TMEM70 AR OXA1L AR Complexes Disorders of Mitochondrial DNA Maintenance Nucleotide Pool Maintenance Replication RRM2B AR/AD DGUOK, TK2, MPV17, TYMP, ABAT, SAMHD1 AR POLG, POLG2, TWNK, DNA2 AR/AD SSBP1 AD MGME1, RNASEH1, TOP3A, FBXL4 AR Disorders of Mitochondrial Gene Expression Mitochondrial transfer RNAs MT-TA, MT-TC, MT-TD, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, mtDNA MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MTTS, MT-TT, MT-TV, MT-TW, MT-TY, MT-DEL* Mitochondrial Aminoacyl-tRNA Synthetases AARS2, CARS2, DARS2, EARS2, FARS2, GARS, HARS2, IARS2, AR KARS, LARS2, MARS2, NARS2, PARS2, RARS2, SARS2, TARS2, VARS2, WARS2, YARS2, QRSL1, GATB, GATC Mitochondrial Transcript Processing and Modification HSD17B10 XL TFAM, POLRMT, MTFMT, TRIT1, TRMT5, TRMT10C, TRNT1, PNPT1, AR MTO1, TRMU, GTPBP3, PUS1, THG1L, ELAC2, MTPAP, NSUN3, PDE12 620 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 Mitochondrial disease in children / S. Rahman Table 2 (Continued ) Mode of Disease mechanism Mitoribosome (Ribosomal RNA and Protein Subunits; Assembly and Gene defects inheritance MT-RNR1, MT-RNR2 mtDNA MRPL3, MRPL12, MRPL44, MRPS2, MRPS7, MRPS14, MRPS16, AR Recycling Factors; Translation MRPS22, MRPS23, MRPS25, MRPS28, MRPS34, PTCD3 (MRPS39), Initiation, Elongation and MRM2, ERAL1, RMND1, C12orf65, GFM1, GFM2, GUF1, LRPPRC, Termination Factors) TACO1, TSFM, TUFM Disorders of Mitochondrial Membrane Lipids, Import, Dynamics and Quality Control Mitochondrial Membrane Phospholipid Metabolism and Protein Import Machinery Mitochondrial Solute Carriers TAZ, TIMM8A XL AGK, CHKB, DNAJC19, GFER, PAM16, SERAC1, PLA2G6, TIMM22, AR TIMM50, TIMMDC1 SLC25A11, SLC25A24 AD SLC25A4 AD/AR SLC25A1, SLC25A3, SLC25A10, SLC25A12, SLC25A13, SLC25A15, AR SLC25A19, SLC25A20, SLC25A21, SLC25A22, SLC25A26, SLC25A32, SLC25A38, SLC25A42, SLC25A46, GOT2, MICU1, MICU2 Mitochondrial Dynamics Intermembrane Space and MICOS DNM1L, MFN2, OPA1, GDAP1, MSTO1 AD/AR MFF, STAT2, TRAK1, MIEF2 AR CHCHD10, CHCHD2 AD QIL1 AR ER–Mitochondrial Tethering EMC1 AR Mitochondrial Protein Quality CLPX, HSPE1 AD Complex Control AFG3L2, ATAD3A, SPG7, HSPA9, HSPD1, HTRA2 AD/AR PMPCA, PMPCB, MIPEP, XPNPEP3, CLPB, CLPP, LONP1, PITRM1, AR SACS, TRAP1, PRKN, PINK1, YME1L Toxicity ETHE1, HIBCH, ECHS1, SQOR AR Antioxidant Defense TXN2, TXNIP AR IDH2, DLST AD FH AD/AR ACO2, IDH3A, IDH3B, MDH2, OGDH, SUCLA2, SUCLG1 AR PDHA1 XL DLAT, DLD, MPC1, PC, PDHB, PDHX, PDK3, PDP1, PDPR AR CRAT, ETFA, ETFB, ETFDH, FA2H, PYCR1 AR Other Disorders of Energy Metabolism Tricarboxylic Acid Cycle Enzymes Pyruvate Metabolism Fatty Acid Metabolism Disorders of Vitamin and Cofactor Metabolism Coenzyme Q10 Biosynthesis PDSS1, PDSS2, COQ2, COQ4, COQ5, COQ6, COQ7, COQ8A, COQ8B, AR COQ9 Iron–Sulphur Cluster Protein Biosynthesis ABCB7 XL ISCA1, ISCA2, ISCU, FDXR, FDX2, FXN, LYRM4, NFS1, NFU1 AR Lipoic Acid Biosynthesis BOLA3, GLRX5, IBA57, LIAS, LIPT1, LIPT2, MECR AR Cytochrome c Synthesis CYCS AD Biotin Metabolism BTD, HLCS AR ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 621 Mitochondrial disease in children / S. Rahman Table 2 (Continued ) Mode of Disease mechanism Gene defects inheritance TPK1, SLC19A2, SLC19A3 AR SLC52A1 AD FLAD1, SLC52A2, SLC52A3 AR Nicotinamide Metabolism NMNAT1, NADK2, NAXD, NAXE, NNT AR Coenzyme A Metabolism COASY, PANK2, PPCS AR Heavy Metal Metabolism (copper, SLC33A1 AD/AR CCS, SLC39A8 AR SECISBP2, SEPSECS AR Thiamine Metabolism and Transport Riboflavin Metabolism and Transport manganese) Selenocysteine Metabolism Other Cellular Defects Associated with Mitochondrial Dysfunction Calcium Homeostasis Haem Biosynthesis Apoptosis Defects WFS1 AD/AR ANO10, C19ORF70, CISD2, CYP24A1 AR ALAS2 XL ABCB6 AD SFXN4 AR AIFM1 XL DIABLO AD APOPT1, PTRH2 AR DNA Repair APTX, XRCC4 AR Miscellaneous or Unknown PNPLA4 XL CTBP1, FGF12, KIF5A, STXBP1 AD Function ALDH18A1, C19ORF12, DCC, DIAPH1, OPA3 AD/AR CA5A, C1QBP, PNPLA8, POP1, PPA2, ROBO3, RTN4IP1, SPART, AR SPATA5, TANGO2, TMEM65, TMEM126A AD, autosomal dominant; AR, autosomal recessive; ER, endoplasmic reticulum; mtDNA, mitochondrial DNA; MICOS, mitochondrial contact site and cristae organizing system; 3MGA, 3-methylglutaconic aciduria; XL, X-linked. exercise tolerance, fatigue and the presence of migraines or seizures. Many children with mitochondrial disease do not have symptoms and signs that align closely with canonical mitochondrial syndromes (Table 1) but the pattern of organ involvement can be a powerful clue to the underlying diagnosis (Table 3). The family history may also provide useful diagnostic clues, for example if there is consanguinity, previous stillbirths or early neonatal deaths, or a strong history of problems running through the maternal lineage. on fundoscopy, hypotonia, muscle weakness, dystonia, spasticity, extrapyramidal and cerebellar signs, and evidence of peripheral neuropathy. Muscle strength may be normal on static testing, and repeated testing may be needed to elicit the muscle fatigability typical of mitochondrial myopathy. Tachypnoea may be a clue to lactic acidosis, and there may be a hyperdynamic precordium if there is significant cardiomyopathy. A search for multisystem features should include assessment by a cardiologist (including ECG and echocardiography), ophthalmologist and audiological physician. Physical examination A thorough physical examination should be performed, including auxology and full neurological assessment, searching carefully for ptosis, ophthalmoplegia, optic atrophy or pigmentary retinopathy 622 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 Diagnostic investigations Diagnosis of a mitochondrial disorder requires a multidisciplinary approach, which may include metabolic investigations, neuroimaging, muscle Mitochondrial disease in children / S. Rahman Table 3 Examples of symptom constellations that aid in differential diagnosis of mitochondrial disease Cardinal clinical feature Variable additional clinical features Syndrome Gene defect Epilepsia partialis Movement disorder (choreoathetosis); Alpers POLG Leigh ~100 different nuclear continua Stepwise neurodevelopmental hepatic impairment Bilateral symmetrical basal ganglia and/or brainstem lesions, elevated lactate and mtDNA gene regression defects Stroke-like episodes SNHL, lactic acidosis MELAS MT-TL1 (m.3243A>G) Ptosis/ Ataxia, cardiac conduction defect, Kearns–Sayre SLSMD ophthalmoplegia pigmentary retinopathy Progressive myopathy Elevated lactate and CK Myopathic MDDS TK2 Progressive myopathy SNHL, respiratory muscle weakness, renal Encephalomyopathic RRM2B tubulopathy Dilated MDDS Neutropaenia (cyclical) and 3MGA Barth TAZ Cataracts Sengers AGK Sideroblastic anaemia Lactic acidosis, pancreatic insufficiency Pearson SLSMD Sideroblastic anaemia Myopathy, lactic acidosis MLASA PUS1, YARS2 Sideroblastic anaemia B-cell immune deficiency, SNHL, recurrent SIFD TRNT1 cardiomyopathy Hypertrophic cardiomyopathy fevers, RP Renal impairment Profound SNHL, myopathy RMND1 deficiency RMND1 Steroid-resistant Seizures, SNHL CoQ10 biosynthesis COQ6, COQ8B, PDSS2, disorder nephrotic syndrome GI pseudo-obstruction Foot drop (peripheral neuropathy), COQ2, MNGIE TYMP leukoencephalopathy Liver failure Status epilepticus Alpers POLG Liver failure Peripheral neuropathy Hepatocerebral MPV17 Liver failure Recovery TRMU deficiency TRMU Acrocyanosis Diarrhoea, petechiae, neurodevelopmental Ethylmalonic ETHE1 MDDS delay encephalopathy 3MGA, 3-methylglutaconic aciduria; CK, creatine kinase; GI, gastrointestinal; MDDS, mitochondrial DNA depletion syndrome; MELAS, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; MLASA, myopathy, lactic acidosis and sideroblastic anaemia; MNGIE, mitochondrial neurogastrointestinal encephalopathy; RP, retinitis pigmentosa; SIFD, sideroblastic anaemia, immune deficiency, fever and developmental delay; SLSMD, single large-scale mitochondrial DNA deletion; SNHL, sensorineural hearing loss. biopsy and genetic testing. Blood, urine and CSF metabolites that can help in the delineation of a specific mitochondrial disorder or in the identification of a nonmitochondrial multisystem disorder (‘phenocopy’) are detailed in Table 4, together with a list of histological investigations and enzyme assays. Neuroimaging phenotypes Magnetic resonance imaging of the brain can be extremely helpful in the diagnosis of mitochondrial disease. For example, in MELAS there are typically parieto-occipital stroke-like lesions not corresponding to vascular territories, whilst Leigh ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 623 Mitochondrial disease in children / S. Rahman syndrome is characterized by bilateral symmetric T2 hyperintense lesions in the basal ganglia, midbrain and/or brainstem, with variable lesions elsewhere in the brain and spinal cord. MRS may reveal a lactate peak in many mitochondrial disorders or a succinate peak in complex II deficiency. A retrospective review of a large French series of children and adults with biochemical evidence of mitochondrial dysfunction and a genetic diagnosis (152 mitochondrial, 37 nonmitochondrial) suggested that the presence of a lactate peak and hyperintensity in the basal ganglia/pallidum/ brainstem had a high positive predictive value for mitochondrial disease, whereas lesions elsewhere in the brain were not helpful in either diagnosing or excluding a mitochondrial disorder [124]. A normal MRI brain was found to decrease the likelihood of an underlying mitochondrial disorder but did not completely exclude it [124]. Cerebral white matter disturbance is increasingly being recognized as a manifestation of mitochondrial disease. There are frequently cystic changes, and another diagnostic clue pointing to an underlying disorder may be the involvement of grey matter structures (e.g. basal ganglia) as well as white matter lesions. Leukoencephalopathy was initially reported in deficiencies of complexes I, II and IV but is now recognized to be a feature of many of the mitochondrial translation disorders, particularly the aminoacyl-tRNA synthetase deficiencies [125]. Some mitochondrial disease genes appear to be associated with very specific neuroimaging appearances (Table 5), which can be used to direct genetic investigations and expedite genetic diagnoses of these cases. However, it could be argued that there may be an ascertainment bias in identifying patients with certain gene defects associated with particular radiological phenotypes, and it will therefore be interesting to see whether the observed correlations are upheld in multiple large series over time. The role of muscle biopsy in the 21st century With the increasingly widespread use of nextgeneration sequencing as a first-line diagnostic test, muscle biopsies are being performed less frequently in the diagnostic work-up of suspected mitochondrial disease in children [126]. In my own practice, we now tend to reserve first-line muscle biopsies for infants and children presenting critically unwell to the intensive care unit, where the risk of demise is great and there is insufficient time to wait for a genetic diagnosis. Other reasons to perform a muscle biopsy include searching for 624 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 functional evidence to support pathogenicity of variants of uncertain significance observed on next-generation sequencing, or where genetic investigations are ‘negative’ yet clinical suspicion of an underlying mitochondrial disorder remains high. Biochemical phenotypes in muscle Until recently, assessment of respiratory chain enzyme activities in biopsies of skeletal muscle (or another affected tissue such as heart or liver) was considered a first-line investigation of mitochondrial disease. The ‘diagnostic rate’ of respiratory chain abnormalities in suspected mitochondrial disease varied widely in historical cohorts, from 28 to 71 % [127,128]. Observed abnormalities included isolated deficiencies of single respiratory chain enzymes (most frequently complex I or complex IV) or deficiencies of multiple respiratory chain enzymes. Initial dogma was that an isolated enzyme deficiency was suggestive of a defect in a subunit or assembly factor of that enzyme, whereas multiple respiratory chain enzyme deficiencies indicated a disorder of mtDNA maintenance or gene expression. However, there appear to be an increasing number of exceptions to these rules. For example, we have observed isolated complex I deficiency with defects of ELAC2 and isolated complex IV deficiency associated with AARS2 mutations. Both genes encode factors required for mitochondrial translation so theoretically should be associated with multiple respiratory chain deficiencies. Thus, the dogmatic rules of thumb are no longer considered so useful. Another issue is the increasing population of patients with genetically confirmed mitochondrial disease who have normal respiratory chain enzyme activities in muscle biopsies. The explanations may be that the defects are not expressed in skeletal muscle (the tissue that is most frequently biopsied in suspected mitochondrial disease) or possibly that the underlying molecular defect does not impair the activities of the respiratory chain enzymes in the way that they are assayed. Genetic testing Ultimately, identification of pathogenic genetic variants is required to confirm the diagnosis of a primary mitochondrial disorder. Recognition of particular constellations of clinical features (Table 3) or knowledge of a geographically prevalent disorder (e.g. LRPPRC mutations causing Leigh Mitochondrial disease in children / S. Rahman Table 4 Investigation of suspected mitochondrial disease in childhood Test Tissue Indication Lactate Blood, CSF Useful if elevateda but may be normal Lactate/pyruvate ratio Blood, CSF Decreased in PDH deficiency Blood gas, bicarbonate Blood To determine whether there is acid–base disturbance Glucose Blood, CSF Hypoglycaemia is a feature of some PMDs, diabetes of others Amino acids Blood, CSF Alanine, proline and glycine may be increased and arginine and citrulline decreased in PMD Acylcarnitines Blood Characteristic elevations of acylcarnitine species in SUCLA2, SUCLG1, HIBCH and ECHS1 defects; differential diagnosis of complex multisystem disease; may be secondary carnitine deficiency in PMD FGF21, GDF15 Blood Considered biomarkers of PMD, useful if elevated but may be normal Organic acids Urine Presence of 3MGA and Krebs cycle intermediates may be helpful in Thymidine, deoxyuridine Blood, Urine If clinical suspicion of MNGIE differential diagnosis Thymidine phosphorylase assay Platelets If clinical suspicion of MNGIE Transferrin electrophoresis Blood Differential diagnosis of complex multisystem disease Very long-chain fatty acids Blood Differential diagnosis of complex multisystem disease Creatine and related metabolites Blood, Urine Differential diagnosis of complex multisystem disease; may be Lysosomal enzymes WBC Differential diagnosis of complex multisystem disease Neurotransmitters CSF If movement disorder 5-methyltetrahydrofolate CSF May be low, especially in KSS PDH assay Fibroblasts If clinical suspicion of PDH deficiency Histology Muscleb Ragged-red, ragged-blue and COX-negative fibres? Electron microscopy Muscleb Ultrastructural mitochondrial abnormalities? Respiratory chain enzyme assays Muscleb To determine which complex(es) is/are deficient Coenzyme Q10 WBC, Muscle Disorder of coenzyme Q10 biosynthesis? secondary creatine deficiency in PMD 3MGA, 3-methylglutaconic aciduria; COX, cytochrome c oxidase; FGF21, fibroblast growth factor 21; GDF15, growth/ differentiation factor 15; KSS, Kearns–Sayre syndrome; MNGIE, mitochondrial neurogastrointestinal encephalopathy; PDH, pyruvate dehydrogenase; PMD, primary mitochondrial disease; WBC, white blood cells. a Providing secondary and artefactual causes of hyperlactataemia have been excluded. b or other affected tissue. syndrome in French Canada) may lead to directed testing of a specific pathogenic variant. However, the rapid rate of gene discovery for mitochondrial disease and rarity of some of the newly identified gene defects makes it difficult to accumulate clinical experience of these ultra-rare disorders and consider them in the differential diagnosis. Therefore, genome-wide next-generation sequencing is increasingly the genetic testing method of choice. Often, this is exome or genome sequencing but sometimes large gene panels are tested such as the MitoExome [129] or the Genomics England PanelApp (https://panelapp.genomicsengland.co. uk/). Trio (parent/child) sequencing is invaluable in the identification of de novo variants. MtDNA can be sequenced separately or captured within the exome or genome, and mtDNA can be quantified by qPCR or droplet digital PCR of DNA extracted from an affected tissue. The importance of functional validation of genetic variants identified by next-generation sequencing methods cannot be overemphasized. Methods to confirm the pathogenicity of novel variants include Western blotting, biochemical assays (e.g. of mitochondrial ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 625 Mitochondrial disease in children / S. Rahman Table 5 Examples of mitochondrial gene defects associated with specific neuroimaging phenotypes Gene defect Neuroimaging features AARS2 Leukoencephalopathy with particular involvement of left–right connections, descending References [88] tracts and cerebellar atrophy DARS2 Leukoencephalopathy with brainstem and spinal cord involvement and lactate [157] elevation (LBSL) EARS2 Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) [158] IBA57 Diffuse cavitating leukoencephalopathy, most prominent posteriorly and in fronto-parietal [159] ISCA2 Diffusely abnormal white matter signal in cerebrum, cerebellum, brainstem and spinal cord [160] LYRM7 Multifocal cavitating leukoencephalopathy with multiple small cavitations in [161] regions periventricular and deep cerebral white matter MT-TL1 Parieto-occipital stroke-like lesions, not conforming to vascular territories [9] m.3243A>G NDUFV1 Progressive macrocystic leukoencephalopathy (may be confused with vanishing white [162] matter disease) NUBPL Predominant signal abnormalities of cerebellar cortex, deep cerebral white matter, basal [163] ganglia, thalami and corpus callosum RARS2 Severe progressive pontocerebellar atrophy (rarely pons may be spared) SDHA SDHAF1 Leukoencephalopathy with signal abnormalities in central corticospinal tracts and spinal [11] [164] cord, cerebral white matter (sparing of U fibres), corpus callosum, pons, middle cerebellar peduncles and cerebellar white matter; succinate peak on MRS SERAC1 Bilateral basal ganglia T2 hyperintensities, initially sparing dorsal putamen TYMP Asymptomatic leukoencephalopathy (demyelination) [165] [96] MRS, 1H-magnetic resonance spectroscopy. translation), mass spectrometric profiling of OXPHOS complexes, immunocytochemical assays (e.g. in disorders of mitochondrial dynamics) and functional complementation studies (e.g. lentiviral transduction with the wild-type gene of interest to rescue the biochemical defect). sequencing methods. We initially used Leigh syndrome as a prototype paediatric mitochondrial disorder and showed that use of the ‘Leigh Map’ computational resource placed the correct disease gene amongst the top-ranked genes in 80% of test cases [134]. Harnessing phenomics Differential diagnosis and phenocopies Phenomics is the systematic study of all the measurable physical and biochemical attributes of an individual [130,131] and is a particularly attractive tool for application in the field of inborn errors of metabolism [132]. The development of the Human Phenotype Ontology has allowed for the quantitation of phenomic traits [133]. We harnessed this information to determine whether phenomics could be used to create a computational diagnostic resource for mitochondrial disease, to be used in concert with next-generation Mitochondrial diseases are notorious mimics, long recognized to present with any combination of symptoms affecting one or multiple organs at any age, from intrauterine life to late adulthood (Fig. 2). This phenotypic heterogeneity, coupled with the biochemical and genetic complexity of mitochondria, leads to enormous diagnostic challenges. Next-generation sequencing has introduced unparalleled opportunities for enhanced diagnosis of mitochondrial disease and led to the discovery of new disease genes at an astonishing rate, but has 626 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 Mitochondrial disease in children / S. Rahman also revealed that mitochondrial dysfunction may in many cases be secondary to nonprimary mitochondrial genetic defects, leading to even more complexity [3]. Thus, mitochondrial disease could be thought of as a modern-day syphilis and Sir William Osler’s aphorism ‘Know syphilis in all its manifestations and relations, and all other things clinical will be added unto you’ [135] could be updated to ‘S/he who knows mitochondrial disease knows all of medicine’ to reflect the protean manifestations and complexity of mitochondrial disease. Disease mechanisms The mechanisms underlying mitochondrial disease remain incompletely understood. Whilst energy insufficiency is widely cited as the cause of mitochondrial disease presentations, it is increasingly considered that disturbance of other key mitochondrial functions, including intracellular signalling, maintaining redox balance, calcium homeostasis and regulation of apoptosis, is likely to contribute to disease pathogenesis [1]. In addition, seminal work in cell and animal models of mitochondrial disease has implicated one-carbon metabolism and an integrated stress response in the pathogenesis of primary mitochondrial disease [136-138], and recent studies have suggested that oxygen may exert direct neuronal toxicity in Leigh syndrome [139]. What influences the phenotype? Mitochondrial disease is different in every individual that I have encountered in my clinical practice of nearly 30 years. Even siblings sharing the same homozygous mutation rarely (I would go so far as to say never) manifest with an identical set of problems and rate of progression. Each has his/her own unique problems and responses to environmental stressors. Whether these are the result of their genetic background or environmental factors (e.g. timing of exposure to first viral infection [140]) is unknown in the majority of cases. But sometimes we are given small clues. I saw two brothers with the same homozygous mutation in a complex III assembly gene but one had worse development, more challenging behaviour, higher blood lactate values and a more severe movement disorder. This brother’s karyotype was 47XXY, whereas his more mildly affected brother had the usual male chromosomal complement of 46XY. However, it would be extremely challenging to prove that the extra X chromosome is indeed the cause of the differing phenotypes in these two siblings. Work on differentiated cells and organoids derived from induced pluripotent stem cells may eventually help to unravel the complex relationship between genotype and phenotype in mitochondrial diseases [141,142]. Monitoring Annual investigations should aim to screen for known treatable complications of mitochondrial disease. In my clinic, we routinely screen for anaemia, renal impairment and renal tubulopathy, hepatic impairment, endocrine dysfunction (diabetes, hypothyroidism, cortisol insufficiency and growth hormone deficiency using IGF1 and IGFBP3 as proxy markers) and measure levels of plasma amino acids and acylcarnitines. Children are reviewed annually or every two years (depending on the disorder) in a specialist cardiology clinic, where echocardiography and ECG are performed. More frequent cardiac screening may be indicated in specific disorders. For example, a systematic review of electrical heart disease in KSS led to 6 monthly ECGs being advocated for this disorder [81]. Regular ophthalmological and audiological review is also recommended. Difficulties with swallowing or episodes of choking should be investigated by videofluoroscopy with input from a specialist speech and language therapist. In addition, prominent GI symptoms should prompt investigation for pancreatic exocrine insufficiency (intractable diarrhoea), pancreatitis (severe abdominal pain) or GI dysmotility (abdominal pain, vomiting, constipation, diarrhoea). Because of a lack of evidence-based guidelines for mitochondrial monitoring, a Delphi panel was convened recently to obtain consensus recommendations [143]. Management The multi-layered complexity of mitochondrial disease, particularly in childhood, has meant that therapeutic advances have lagged a long way behind the genetic discoveries. A few gene defects have suggested clear treatment strategies, such as coenzyme Q10 supplementation for disorders of coenzyme Q10 biosynthesis [90], and riboflavin for defects of riboflavin transport and metabolism as well as various flavoprotein disorders, for example deficiencies of ACAD9, AIFM1 and some complex I subunits [144]. However, precision medicine is not yet available for the vast majority of mitochondrial ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 627 Mitochondrial disease in children / S. Rahman disorders presenting in childhood, and for these children, supportive measures remain of paramount importance. Adjunctive therapies may include hearing aids, cochlear implants, brow suspension for ptosis, gastrostomy feeds, pancreatic enzyme supplementation, pacemaker insertion, medical treatment of cardiomyopathy, electrolyte supplementation to replace renal tubular losses, blood transfusion for sideroblastic anaemia, antiepileptic drugs and (in selected situations, after careful multidisciplinary assessment) renal, cardiac or hepatic transplantation [21,145,146]. Emerging therapies Many research groups across the globe are exploring novel experimental strategies, which fall broadly into two categories: pharmacological and genetic approaches. These emerging therapies have been the subject of a number of recent reviews, to which the reader is directed [147-150]. Disease-agnostic pharmacological approaches can be grouped into antioxidants, interventions aimed to increase mitochondrial biogenesis, molecules to stabilize the mitochondrial membrane lipids and targeting mitophagy via the mTOR pathway. Some of these approaches are currently in clinical trial [150,151]. Targeted pharmacological approaches include the use of nucleosides to restore mtDNA synthesis in myopathic MDDS caused by TK2 deficiency [152]. Genetic therapies targeting the mtDNA include selective elimination of mutant mtDNA using restriction enzymes delivered as mitoTALENs [153] or zinc finger nucleases [154], or replacing a mitochondrial protein by expressing it from the nucleus, an approach that is currently in clinical trial for LHON [155]. AAV-mediated gene therapy directed at nuclear-encoded mitochondrial defects has been reported in a number of mouse models [150,156], but has not yet been translated to human mitochondrial disease. Prognosis What about prognosis? Nothing, not the magnitude of lactate elevation in blood or CSF, the activities of respiratory chain enzymes in biopsied skeletal muscle, not even the definitive genetic diagnosis, can speak to a child’s prognosis as much as his/ her own trajectory to this point in time. Has he or she slowly been making progress? Or progressively deteriorating and acquiring multisystem problems? Has there been developmental regression 628 ª 2020 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2020, 287; 609–633 with intercurrent illnesses? Have they had a previous episode of respiratory failure necessitating intensive care admission? Whichever of these is true for an individual child, this is the likely trajectory of their disease going forwards. With the publication of a number of (largely retrospective) natural history studies in recent years, we can finally begin to add some more precise detail to these general observations, at least for some gene defects. It is sobering to note the continuing high mortality of paediatric mitochondrial disease. A systematic review of retrospective natural history studies reporting mortality data for 23 mitochondrial disorders revealed a mean/median age of death < 1 year in 13% of disorders, <5 years in 57% and < 10 years in 74% of the diseases studied [94]. Final thoughts What has been the predominant weather for the mitochondrial disease field in the last 30 years? I would say mainly overcast with a lot of thundershowers and occasional bursts of sunshine when breakthroughs are made. No rainbows yet, and certainly no pots of gold at the end of the rainbow. Is the weather finally changing? I think it may be too early to tell for sure. What have been the three most important landmark moments in this period? The discovery of mtDNA mutations, the demonstration of the power of next-generation sequencing and the possibility of effective therapy for at least one disorder (COQ2 deficiency) have been the highlights for me. The discovery of the first mtDNA mutations heralded the birth of a new field of medicine, one defined by complexity and uncertainty at every level (clinical, biochemical, genetic, diagnostic, therapeutic). The identification of nuclear gene mutations as a cause of mitochondrial disease was the opening of a window, letting in light and fresh air, whilst the subsequent introduction of next-generation sequencing brought in unprecedented diagnostic power. Where previously the journey had been undertaken in the back of a beaten-up truck with rusty suspension and inadequate fuel, this was a switching of gears, in fact a switching to an entirely different vehicle, more like a brand new Ferrari. Are we collectively responsible for the lack of progress in finding effective therapies? Had we been better scientists and physicians would we be further forward? Or do the difficulties lie in the complex nature of mitochondrial diseases? I favour the latter view and believe that the passion and determination of the Mitochondrial disease in children / S. Rahman mitochondrial scientific and medical community will find effective therapies for these devastating diseases in the not too distant future. 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