Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.

BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

De novo variants in RNF213 are associated with a clinical spectrum ranging from Leigh syndrome to early-onset stroke.

PURPOSE: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations. METHODS: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts. RESULTS: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect. CONCLUSION: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.

Pathogenic Bi-allelic Mutations in NDUFAF8 Cause Leigh Syndrome with an Isolated Complex I Deficiency.

Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.

Genotypic and phenotypic spectrum of infantile liver failure due to pathogenic TRMU variants.

PURPOSE: This study aimed to define the genotypic and phenotypic spectrum of reversible acute liver failure (ALF) of infancy resulting from biallelic pathogenic TRMU variants and determine the role of cysteine supplementation in its treatment. METHODS: Individuals with biallelic (likely) pathogenic variants in TRMU were studied within an international retrospective collection of de-identified patient data. RESULTS: In 62 individuals, including 30 previously unreported cases, we described 47 (likely) pathogenic TRMU variants, of which 17 were novel, and 1 intragenic deletion. Of these 62 individuals, 42 were alive at a median age of 6.8 (0.6-22) years after a median follow-up of 3.6 (0.1-22) years. The most frequent finding, occurring in all but 2 individuals, was liver involvement. ALF occurred only in the first year of life and was reported in 43 of 62 individuals; 11 of whom received liver transplantation. Loss-of-function TRMU variants were associated with poor survival. Supplementation with at least 1 cysteine source, typically N-acetylcysteine, improved survival significantly. Neurodevelopmental delay was observed in 11 individuals and persisted in 4 of the survivors, but we were unable to determine whether this was a primary or a secondary consequence of TRMU deficiency. CONCLUSION: In most patients, TRMU-associated ALF was a transient, reversible disease and cysteine supplementation improved survival.

Natural History of Leigh Syndrome: A Study of Disease Burden and Progression.

OBJECTIVE: This observational cohort study aims to quantify disease burden over time, establish disease progression rates, and identify factors that may determine the disease course of Leigh syndrome. METHODS: Seventy-two Leigh syndrome children who completed the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS) at baseline at 3.7 years (interquartile range [IQR] = 2.0-7.6) and follow-up assessments at 7.5 years (IQR = 3.7-11.0) in clinics were enrolled. Eighty-two percent of this cohort had a confirmed genetic diagnosis, with pathogenic variants in the MT-ATP6 and SURF1 genes being the most common cause. The total NPMDS scores denoted mild (0-14), moderate (15-25), and severe (>25) disease burden. Detailed clinical, neuroradiological, and molecular genetic findings were also analyzed. RESULTS: The median total NPMDS scores rose significantly (Z = -6.9, p < 0.001), and the percentage of children with severe disease burden doubled (22% → 42%) over 2.6 years of follow-up. Poor function (especially mobility, self-care, communication, feeding, and education) and extrapyramidal features contributed significantly to the disease burden (τb  ≈ 0.45-0.68, p < 0.001). These children also deteriorated to wheelchair dependence (31% → 57%), exclusive enteral feeding (22% → 46%), and one-to-one assistance for self-care (25% → 43%) during the study period. Twelve children (17%) died after their last NPMDS scores were recorded. These children had higher follow-up NPMDS scores (disease burden; p < 0.001) and steeper increase in NPMDS score per annum (disease progression; p < 0.001). Other predictors of poor outcomes include SURF1 gene variants (p < 0.001) and bilateral caudate changes on neuroimaging (p < 0.01). INTERPRETATION: This study has objectively defined the disease burden and progression of Leigh syndrome. Our analysis has also uncovered potential influences on the trajectory of this neurodegenerative condition. ANN NEUROL 2022;91:117-130.

Prion-like α-synuclein pathology in the brain of infants with Krabbe disease.

Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene that causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (n = 4) compared to infant controls (n = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease.

Forecasting stroke-like episodes and outcomes in mitochondrial disease.

In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors. Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk. The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82-0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005]. Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.

Delineating selective vulnerability of inhibitory interneurons in Alpers' syndrome.

AIMS: Alpers' syndrome is a severe neurodegenerative disease typically caused by bi-allelic variants in the mitochondrial DNA (mtDNA) polymerase gene, POLG, leading to mtDNA depletion. Intractable epilepsy, often with an occipital focus, and extensive neurodegeneration are prominent features of Alpers' syndrome. Mitochondrial oxidative phosphorylation (OXPHOS) is severely impaired with mtDNA depletion and is likely to be a major contributor to the epilepsy and neurodegeneration in Alpers' syndrome. We hypothesised that parvalbumin-positive(+) interneurons, a neuronal class critical for inhibitory regulation of physiological cortical rhythms, would be particularly vulnerable in Alpers' syndrome due to the excessive energy demands necessary to sustain their fast-spiking activity. METHODS: We performed a quantitative neuropathological investigation of inhibitory interneuron subtypes (parvalbumin+, calretinin+, calbindin+, somatostatin interneurons+) in postmortem neocortex from 14 Alpers' syndrome patients, five sudden unexpected death in epilepsy (SUDEP) patients (to control for effects of epilepsy) and nine controls. RESULTS: We identified a severe loss of parvalbumin+ interneurons and clear evidence of OXPHOS impairment in those that remained. Comparison of regional abundance of interneuron subtypes in control tissues demonstrated enrichment of parvalbumin+ interneurons in the occipital cortex, while other subtypes did not exhibit such topographic specificity. CONCLUSIONS: These findings suggest that the vulnerability of parvalbumin+ interneurons to OXPHOS deficits coupled with the high abundance of parvalbumin+ interneurons in the occipital cortex is a key factor in the aetiology of the occipital-predominant epilepsy that characterises Alpers' syndrome. These findings provide novel insights into Alpers' syndrome neuropathology, with important implications for the development of preclinical models and disease-modifying therapeutics.

Identification and characterization of novel MPC1 gene variants causing mitochondrial pyruvate carrier deficiency.

Pyruvate, the end product of glycolysis, is a key metabolic molecule enabling mitochondrial adenosine triphosphate synthesis and takes part in multiple biosynthetic pathways within mitochondria. The mitochondrial pyruvate carrier (MPC) plays a vital role in transporting pyruvate from the cytosol into the organelle. In humans, MPC is a hetero-oligomeric complex formed by the MPC1 and MPC2 paralogs that are both necessary to stabilize each other and form a functional MPC. MPC deficiency (OMIM#614741) due to pathogenic MPC1 variants is a rare autosomal recessive disease involving developmental delay, microcephaly, growth failure, and increased serum lactate and pyruvate. To date, two MPC1 variants in four cases have been reported, though only one with a detailed clinical description. Herein, we report three novel pathogenic MPC1 variants in six patients from three unrelated families, identified within European, Kuwaiti, and Chinese mitochondrial disease patient cohorts, one of whom presented as a Leigh-like syndrome. Functional analysis in primary fibroblasts from the patients revealed decreased expression of MPC1 and MPC2. We rescued pyruvate-driven oxygen consumption rate in patient's fibroblasts by reconstituting with wild-type MPC1. Complementing homozygous MPC1 mutant cDNA with CRISPR-deleted MPC1 C2C12 cells verified the mechanism of variants: unstable MPC complex or ablated pyruvate uptake activity. Furthermore, we showed that glutamine and beta-hydroxybutyrate were alternative substrates to maintain mitochondrial respiration when cells lack pyruvate. In conclusion, we expand the clinical phenotypes and genotypes associated with MPC deficiency, with our studies revealing glutamine as a potential therapy for MPC deficiency.

Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy.

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency.

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

PPA2-associated sudden cardiac death: extending the clinical and allelic spectrum in 20 new families.

PURPOSE: Biallelic hypomorphic variants in PPA2, encoding the mitochondrial inorganic pyrophosphatase 2 protein, have been recently identified in individuals presenting with sudden cardiac death, occasionally triggered by alcohol intake or a viral infection. Here we report 20 new families harboring PPA2 variants. METHODS: Synthesis of clinical and molecular data concerning 34 individuals harboring five previously reported PPA2 variants and 12 novel variants, 11 of which were functionally characterized. RESULTS: Among the 34 individuals, only 6 remain alive. Twenty-three died before the age of 2 years while five died between 14 and 16 years. Within these 28 cases, 15 died of sudden cardiac arrest and 13 of acute heart failure. One case was diagnosed prenatally with cardiomyopathy. Four teenagers drank alcohol before sudden cardiac arrest. Progressive neurological signs were observed in 2/6 surviving individuals. For 11 variants, recombinant PPA2 enzyme activities were significantly decreased and sensitive to temperature, compared to wild-type PPA2 enzyme activity. CONCLUSION: We expand the clinical and mutational spectrum associated with PPA2 dysfunction. Heart failure and sudden cardiac arrest occur at various ages with inter- and intrafamilial phenotypic variability, and presentation can include progressive neurological disease. Alcohol intake can trigger cardiac arrest and should be strictly avoided.

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Mutations of the mitochondrial carrier translocase channel subunit TIM22 cause early-onset mitochondrial myopathy.

Protein import into mitochondria is facilitated by translocases within the outer and the inner mitochondrial membranes that are dedicated to a highly specific subset of client proteins. The mitochondrial carrier translocase (TIM22 complex) inserts multispanning proteins, such as mitochondrial metabolite carriers and translocase subunits (TIM23, TIM17A/B and TIM22), into the inner mitochondrial membrane. Both types of substrates are essential for mitochondrial metabolic function and biogenesis. Here, we report on a subject, diagnosed at 1.5 years, with a neuromuscular presentation, comprising hypotonia, gastroesophageal reflux disease and persistently elevated serum and Cerebrospinal fluid lactate (CSF). Patient fibroblasts displayed reduced oxidative capacity and altered mitochondrial morphology. Using trans-mitochondrial cybrid cell lines, we excluded a candidate variant in mitochondrial DNA as causative of these effects. Whole-exome sequencing identified compound heterozygous variants in the TIM22 gene (NM_013337), resulting in premature truncation in one allele (p.Tyr25Ter) and a point mutation in a conserved residue (p.Val33Leu), within the intermembrane space region, of the TIM22 protein in the second allele. Although mRNA transcripts of TIM22 were elevated, biochemical analyses revealed lower levels of TIM22 protein and an even greater deficiency of TIM22 complex formation. In agreement with a defect in carrier translocase function, carrier protein amounts in the inner membrane were found to be reduced. This is the first report of pathogenic variants in the TIM22 pore-forming subunit of the carrier translocase affecting the biogenesis of inner mitochondrial membrane proteins critical for metabolite exchange.

The genetic basis of isolated mitochondrial complex II deficiency.

Mitochondrial complex II (succinate:ubiquinone oxidoreductase) is the smallest complex of the oxidative phosphorylation system, a tetramer of just 140 kDa. Despite its diminutive size, it is a key complex in two coupled metabolic pathways - it oxidises succinate to fumarate in the tricarboxylic acid cycle and the electrons are used to reduce FAD to FADH2, ultimately reducing ubiquinone to ubiquinol in the respiratory chain. The biogenesis and assembly of complex II is facilitated by four ancillary proteins, all of which are autosomally-encoded. Numerous pathogenic defects have been reported which describe two broad clinical manifestations, either susceptibility to cancer in the case of single, heterozygous germline variants, or a mitochondrial disease presentation, almost exclusively due to bi-allelic recessive variants and associated with an isolated complex II deficiency. Here we present a compendium of pathogenic gene variants that have been documented in the literature in patients with an isolated mitochondrial complex II deficiency. To date, 61 patients are described, harbouring 32 different pathogenic variants in four distinct complex II genes: three structural subunit genes (SDHA, SDHB and SDHD) and one assembly factor gene (SDHAF1). Many pathogenic variants result in a null allele due to nonsense, frameshift or splicing defects however, the missense variants that do occur tend to induce substitutions at highly conserved residues in regions of the proteins that are critical for binding to other subunits or substrates. There is phenotypic heterogeneity associated with defects in each complex II gene, similar to other mitochondrial diseases.

Pathogenic variants in MT-ATP6: A United Kingdom-based mitochondrial disease cohort study.

Distinct clinical syndromes have been associated with pathogenic MT-ATP6 variants. In this cohort study, we identified 125 individuals (60 families) including 88 clinically affected individuals and 37 asymptomatic carriers. Thirty-one individuals presented with Leigh syndrome and 7 with neuropathy ataxia retinitis pigmentosa. The remaining 50 patients presented with variable nonsyndromic features including ataxia, neuropathy, and learning disability. We confirmed maternal inheritance in 39 families and demonstrated that tissue segregation patterns and phenotypic threshold are variant dependent. Our findings suggest that MT-ATP6-related mitochondrial DNA disease is best conceptualized as a mitochondrial disease spectrum disorder and should be routinely included in genetic ataxia and neuropathy gene panels. ANN NEUROL 2019;86:310-315.

Recent advances in understanding the molecular genetic basis of mitochondrial disease.

Mitochondrial disease is hugely diverse with respect to associated clinical presentations and underlying genetic causes, with pathogenic variants in over 300 disease genes currently described. Approximately half of these have been discovered in the last decade due to the increasingly widespread application of next generation sequencing technologies, in particular unbiased, whole exome-and latterly, whole genome sequencing. These technologies allow more genetic data to be collected from patients with mitochondrial disorders, continually improving the diagnostic success rate in a clinical setting. Despite these significant advances, some patients still remain without a definitive genetic diagnosis. Large datasets containing many variants of unknown significance have become a major challenge with next generation sequencing strategies and these require significant functional validation to confirm pathogenicity. This interface between diagnostics and research is critical in continuing to expand the list of known pathogenic variants and concomitantly enhance our knowledge of mitochondrial biology. The increasing use of whole exome sequencing, whole genome sequencing and other "omics" techniques such as transcriptomics and proteomics will generate even more data and allow further interrogation and validation of genetic causes, including those outside of coding regions. This will improve diagnostic yields still further and emphasizes the integral role that functional assessment of variant causality plays in this process-the overarching focus of this review.