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RJ Rodenburg, FALM EskensErasmus MC Cancer Institute, Department of Medical Oncology, Rotterdam, The NetherlandsAbstract: Tivozanib is an oral selective vascular endothelial growth factors receptor (VEGFR) tyrosine kinase inhibitor that is recently approved by the European Medicines Agency for the treatment of previously untreated patients with metastatic renal cell carcinoma (mRCC) as well as for those patients with disease progression during or after cytokine therapy. Nowadays, in first-line and second-line treatment of mRCC, there is an abundance of options, mainly consisting of VEGFR-directed tyrosinekinase inhibitors. This review focusses on the role of tivozanib with respect to patient selection and future perspectives in this fast-changing landscape.Keywords: tivozanib, metastatic renal cell cancer, tyrosine kinase inhibitor

Leigh syndrome (LS), or subacute necrotizing encephalomyelopathy, is a genetically heterogeneous, relentlessly progressive, devastating neurodegenerative disorder that usually presents in infancy or early childhood. A diagnosis of Leigh-like syndrome may be considered in individuals who do not fulfil the stringent diagnostic criteria but have features resembling Leigh syndrome.We describe a unique presentation of Leigh-like syndrome in a 3-year-old boy with elevated 3-hydroxyisovalerylcarnitine (C5-OH) on newborn screening (NBS). Subsequent persistent plasma elevations of C5-OH and propionylcarnitine (C3) as well as fluctuating urinary markers were suggestive of multiple carboxylase deficiency (MCD). Normal enzymology and mutational analysis of genes encoding holocarboxylase synthetase (HLCS) and biotinidase (BTD) excluded MCD. Biotin uptake studies were normal excluding biotin transporter deficiency. His clinical features at 13 months of age comprised psychomotor delay, central hypotonia, myopathy, failure to thrive, hypocitrullinemia, recurrent episodes of decompensation with metabolic keto-lactic acidosis and an episode of hyperammonemia. Biotin treatment from 13 months of age was associated with increased patient activity, alertness, and attainment of new developmental milestones, despite lack of biochemical improvements. Whole exome sequencing (WES) analysis failed to identify any other variants which could likely contribute to the observed phenotype, apart from the homoplasmic (100%) m.8993T>G variant initially detected by mitochondrial DNA (mtDNA) sequencing.Hypocitrullinemia has been reported in patients with the m.8993T>G variant and other mitochondrial disorders. However, persistent plasma elevations of C3 and C5-OH have previously only been reported in one other patient with this homoplasmic mutation. We suggest considering the m.8993T>G variant early in the diagnostic evaluation of MCD-like biochemical disturbances, particularly when associated with hypocitrullinemia on NBS and subsequent confirmatory tests. An oral biotin trial is also warranted.

Leigh syndrome (LS), or subacute necrotizing encephalomyelopathy, is a genetically heterogeneous, relentlessly progressive, devastating neurodegenerative disorder that usually presents in infancy or early childhood. A diagnosis of Leigh-like syndrome may be considered in individuals who do not fulfil the stringent diagnostic criteria but have features resembling Leigh syndrome.We describe a unique presentation of Leigh-like syndrome in a 3-year-old boy with elevated 3-hydroxyisovalerylcarnitine (C5-OH) on newborn screening (NBS). Subsequent persistent plasma elevations of C5-OH and propionylcarnitine (C3) as well as fluctuating urinary markers were suggestive of multiple carboxylase deficiency (MCD). Normal enzymology and mutational analysis of genes encoding holocarboxylase synthetase (HLCS) and biotinidase (BTD) excluded MCD. Biotin uptake studies were normal excluding biotin transporter deficiency. His clinical features at 13 months of age comprised psychomotor delay, central hypotonia, myopathy, failure to thrive, hypocitrullinemia, recurrent episodes of decompensation with metabolic keto-lactic acidosis and an episode of hyperammonemia. Biotin treatment from 13 months of age was associated with increased patient activity, alertness, and attainment of new developmental milestones, despite lack of biochemical improvements. Whole exome sequencing (WES) analysis failed to identify any other variants which could likely contribute to the observed phenotype, apart from the homoplasmic (100%) m.8993T>G variant initially detected by mitochondrial DNA (mtDNA) sequencing.Hypocitrullinemia has been reported in patients with the m.8993T>G variant and other mitochondrial disorders. However, persistent plasma elevations of C3 and C5-OH have previously only been reported in one other patient with this homoplasmic mutation. We suggest considering the m.8993T>G variant early in the diagnostic evaluation of MCD-like biochemical disturbances, particularly when associated with hypocitrullinemia on NBS and subsequent confirmatory tests. An oral biotin trial is also warranted.

Reactive oxygen species (ROS) are known to be involved in the pathophysiology of complex regional pain syndrome type I (CRPS I). Since the mitochondrial respiratory chain is a major source of ROS, we hypothesized that mitochondria play a role in the pathophysiology of CRPS I. The hypothesis was tested by studying mitochondrial energy metabolism in muscle tissue from amputated limbs of CRPS I patients. We observed that mitochondria obtained from CRPS I muscle tissue displayed reduced mitochondrial ATP production and substrate oxidation rates in comparison to control muscle tissue. Moreover, we observed reactive oxygen species evoked damage to mitochondrial proteins and reduced MnSOD levels. It remains to be established if the mitochondrial dysfunction that is apparent at the end-stage of CRPS I is also present in earlier stages of the disease, or are secondary to CRPS I. The observation of a reduced mitochondrial energy production combined with reactive oxygen species induced damage in muscle tissue from CRPS I patients warrants further studies into the involvement of mitochondrial dysfunctioning in the pathophysiology of CRPS I. [ABSTRACT FROM AUTHOR]

Mitochondrial oxidative phosphorylation (OXPHOS) represents the final step in the conversion of nutrients into cellular energy. Genetic defects in the OXPHOS system have an incidence between 1:5,000 and 1:10,000 live births. Inherited isolated deficiency of the first complex (CI) of this system, a multisubunit assembly of 45 different proteins, occurs most frequently and originates from mutations in either the nuclear DNA, encoding 38 structural subunits and several assembly factors, or the mitochondrial DNA, encoding 7 structural subunits. The deficiency is associated with devastating multisystemic disorders, often affecting the brain, with onset in early childhood. There are currently no rational treatment strategies. Here, we present an overview of the genetic origins and cellular consequences of this deficiency and discuss how these insights might aid future development of treatment strategies. [ABSTRACT FROM AUTHOR]

Objective: Mitochondrial complex I deficiency is the commonest diagnosed respiratory chain defect, being genetically heterogeneous. The male preponderance of previous patient cohorts suggested an X-linked underlying genetic defect. We investigated mutations in the X-chromosomal complex I structural genes, NDUFA1 and NDUFB11, as a novel cause of mitochondrial encephalomyopathy.Methods: We sequenced 12 nuclear genes and the mitochondrial DNA-encoded complex I genes in 26 patients with respiratory chain complex I defect. Novel mutations were confirmed by polymerase chain reaction restriction length polymorphism. Assembly/stability studies in fibroblasts were performed using two-dimensional blue native gel electrophoresis.Results: Two novel p.Gly8Arg and p.Arg37Ser hemizygous mutations in NDUFA1 were identified in two unrelated male patients presenting with Leigh's syndrome and with myoclonic epilepsy and developmental delay, respectively. Two-dimensional blue native gel electrophoresis showed decreased levels of intact complex I with no accumulation of lower molecular weight subcomplexes, indicating that assembly, stability, or both are compromised.Interpretation: Mutations in the X-linked NDUFA1 gene result in complex I defect and encephalomyopathy. Assembly/stability analysis might give an explanation for the different clinical phenotypes and become useful for future diagnostic purposes. [ABSTRACT FROM AUTHOR]

Background: The MRPS36 gene encodes a recently identified component of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the Krebs cycle catalyzing the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. Defective OGDHC activity causes a clinically variable metabolic disorder characterized by global developmental delay, severe neurological impairment, liver failure, and early-onset lactic acidosis., Methods: We investigated the molecular cause underlying Leigh syndrome with bilateral striatal necrosis in two siblings through exome sequencing. Functional studies included measurement of the OGDHC enzymatic activity and MRPS36 mRNA levels in fibroblasts, assessment of protein stability in transfected cells, and structural analysis. A literature review was performed to define the etiological and phenotypic spectrum of OGDHC deficiency., Results: In the two affected brothers, exome sequencing identified a homozygous nonsense variant (c.283G>T, p.Glu95*) of MRPS36. The variant did not affect transcript processing and stability, nor protein levels, but resulted in a shorter protein lacking nine residues that contribute to the structural and functional organization of the OGDHC complex. OGDHC enzymatic activity was significantly reduced. The review of previously reported cases of OGDHC deficiency supports the association of this enzymatic defect with Leigh phenotypic spectrum and early-onset movement disorder. Slightly elevated plasma levels of glutamate and glutamine were observed in our and literature patients with OGDHC defect., Conclusions: Our findings point to MRPS36 as a new disease gene implicated in Leigh syndrome. The slight elevation of plasma levels of glutamate and glutamine observed in patients with OGDHC deficiency represents a candidate metabolic signature of this neurometabolic disorder. © 2024 International Parkinson and Movement Disorder Society., (© 2024 International Parkinson and Movement Disorder Society.)

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours arising from chromaffin cells. Pathogenic variants in the gene succinate dehydrogenase subunit B (SDHB) are associated with malignancy and poor prognosis. When metastases arise, limited treatment options are available. The pathomechanism of SDHB -associated PPGL remains largely unknown, and the lack of suitable models hinders therapy development. Germline heterozygous SDHB pathogenic variants predispose to developing PPGLs with a life-long penetrance of around 50%. To mimic the human disease phenotype, we characterised adult heterozygous sdhb mutant zebrafish as a potential model to study SDHB -related PPGLs. Adult sdhb mutant zebrafish did not develop an obvious tumour phenotype and were anatomically and histologically like their wild-type siblings. However, sdhb mutants showed significantly increased succinate levels, a major hallmark of SDHB -related PPGLs. While basal activity was increased during day periods in mutants, mitochondrial complex activity and catecholamine metabolite levels were not significantly different. In conclusion, we characterised an adult in vivo zebrafish model, genetically resembling human carriers. Adult heterozygous sdhb mutants mimicked their human counterparts, showing systemic elevation of succinate levels despite the absence of a tumour phenotype. This model forms a promising basis for developing a full tumour phenotype and gaining knowledge of the pathomechanism behind SDHB -related PPGLs.

Brody disease is a rare autosomal recessive myopathy, caused by pathogenic variants in the ATP2A1 gene. It is characterized by an exercise-induced delay in muscle relaxation, often reported as muscle stiffness. Children may manifest with an abnormal gait and difficulty running. Delayed relaxation is commonly undetected, resulting in a long diagnostic delay. Almost all published cases so far were adults with childhood onset and adult diagnosis. With diagnostic next-generation sequencing, an increasing number of patients are diagnosed in childhood. We describe the clinical and genetic features of 9 children from 6 families with Brody disease. All presented with exercise-induced delayed relaxation, reported as difficulty running and performing sports. Muscle strength and mass was normal, and several children even had an athletic appearance. However, the walking and running patterns were abnormal. The diagnostic delay ranged between 2 and 7 years. Uniformly, a wide range of other disorders were considered before genetic testing was performed, revealing pathogenic genetic variants in ATP2A1 . To conclude, this case series is expected to improve clinical recognition and timely diagnosis of Brody disease in children. We propose that ATP2A1 should be added to gene panels for congenital myopathies, developmental and movement disorders, and muscle channelopathies.

Background: Congenital disorders of the mitochondrial respiratory chain are a heterogeneous group of inborn errors of metabolism. Among them, NADH:ubiquinone oxidoreductase (complex I, CI) deficiency is the most common. Biallelic pathogenic variants in NDUFAF2, encoding the nuclear assembly CI factor NDUFAF2, were initially reported to cause progressive encephalopathy beginning in infancy. Since the initial report in 2005, less than a dozen patients with NDUFAF2-related disease have been reported., Methods: Clinical, biochemical, and neuroradiological features of four new patients residing in Northern Israel were collected during 2016-2022 at Emek Medical Center. Enzymatic activities of the five respiratory-chain complexes were determined in isolated fibroblast mitochondria by spectrophotometric methods. Western blot analyses were conducted with anti-human NDUFAF2 antibody; antibody against the mitochondrial marker VDAC1 was used as a loading control. Genetic studies were performed by chromosome microarray analysis using Affymetrix CytoScan 750 K arrays., Results: All four patients presented with infantile-onset growth retardation, ophthalmological impairments with nystagmus, strabismus (starting between 5 and 9 months), and further progressed to life-threatening episodes of apnea usually triggered by trivial febrile illnesses (between 10 and 18 months) with gradual loss of acquired developmental milestones (3 of 4 patients). Serial magnetic-resonance imaging studies in two of the four patients showed a progressive pattern of abnormal T2-weighted hyperintense signals involving primarily the brainstem, the upper cervical cord, and later, the basal ganglia and thalami. Magnetic-resonance spectroscopy in one patient showed an increased lactate peak. Disease progression was marked by ventilatory dependency and early lethality. 3 of the 4 patients tested, harbored a homozygous 142-kb partial interstitial deletion that omits exons 2-4 of NDUFAF2. Mitochondrial CI activity was significantly decreased in the only patient tested. Western blot analysis disclosed the absence of NDUFAF2 protein compared to normal controls. In addition, we reviewed all 10 previously reported NDUFAF2-deficient cases to better characterize the disease., Conclusions: Biallelic loss-of-function mutations in NDUFAF2 result in a distinctive phenotype in the spectrum of Leigh syndrome with clinical and neuroradiological features that are primarily attributed to progressive brainstem damage., (© 2024. The Author(s).)

Ionic calcium (Ca 2+ ) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca 2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca 2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca 2+ uptake., Competing Interests: Declaration of competing interest WJHK is a scientific advisor of Khondrion B.V. (Nijmegen, The Netherlands). This company was not involved in the data analysis and interpretation, writing of the manuscript, and in the decision to submit the manuscript for publication., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analyses. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 van Strien et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Statin use may exacerbate exercise-induced skeletal muscle injury caused by reduced coenzyme Q10 (CoQ10) levels, which are postulated to produce mitochondrial dysfunction., Objectives: We determined the effect of prolonged moderate-intensity exercise on markers of muscle injury in statin users with and without statin-associated muscle symptoms. We also examined the association between leukocyte CoQ10 levels and muscle markers, muscle performance, and reported muscle symptoms., Methods: Symptomatic (n = 35; age 62 ± 7 years) and asymptomatic statin users (n = 34; age 66 ± 7 years) and control subjects (n = 31; age 66 ± 5 years) walked 30, 40, or 50 km/d for 4 consecutive days. Muscle injury markers (lactate dehydrogenase, creatine kinase, myoglobin, cardiac troponin I, and N-terminal pro-brain natriuretic peptide), muscle performance, and reported muscle symptoms were assessed at baseline and after exercise. Leukocyte CoQ10 was measured at baseline., Results: All muscle injury markers were comparable at baseline (P > 0.05) and increased following exercise (P < 0.001), with no differences in the magnitude of exercise-induced elevations among groups (P > 0.05). Muscle pain scores were higher at baseline in symptomatic statin users (P < 0.001) and increased similarly in all groups following exercise (P < 0.001). Muscle relaxation time increased more in symptomatic statin users than in control subjects following exercise (P = 0.035). CoQ10 levels did not differ among symptomatic (2.3 nmol/U; IQR: 1.8-2.9 nmol/U), asymptomatic statin users (2.1 nmol/U; IQR: 1.8-2.5 nmol/U), and control subjects (2.1 nmol/U; IQR: 1.8-2.3 nmol/U; P = 0.20), and did not relate to muscle injury markers, fatigue resistance, or reported muscle symptoms., Conclusions: Statin use and the presence of statin-associated muscle symptoms does not exacerbate exercise-induced muscle injury after moderate exercise. Muscle injury markers were not related to leukocyte CoQ10 levels. (Exercise-induced Muscle Damage in Statin Users; NCT05011643)., Competing Interests: Funding Support and Author Disclosures Dr Allard is financially supported by a grant from the Radboud Institute for Health Sciences. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, including Mcat , is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants in MCAT . Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Re-expression of wild-type MCAT rescued the phenotype in patient fibroblasts. This is the first report of a patient with MCAT pathogenic variants and combined oxidative phosphorylation deficiency., Competing Interests: BW, SN, AS, JG, RR, JL, AE, HV, TN, BG, RD, JR, SH No competing interests declared, (© 2023, Webb, Nowinski et al.)

Purpose: For patients with inherited metabolic disorders (IMDs), any diagnostic delay should be avoided because early initiation of personalized treatment could prevent irreversible health damage. To improve diagnostic interpretation of genetic data, gene function tests can be valuable assets. For IMDs, variant-transcending functional tests are readily available through (un)targeted metabolomics assays. To support the application of metabolomics for this purpose, we developed a gene-based guide to select functional tests to either confirm or exclude an IMD diagnosis., Methods: Using information from a diagnostic IMD exome panel, Kyoto Encyclopedia of Genes and Genomes, and Inborn Errors of Metabolism Knowledgebase, we compiled a guide for metabolomics-based gene function tests. From our practical experience with this guide, we retrospectively selected illustrative cases for whom combined metabolomic/genomic testing improved diagnostic success and evaluated the effect hereof on clinical management., Results: The guide contains 2047 metabolism-associated genes for which a validated or putative variant-transcending gene function test is available. We present 16 patients for whom metabolomic testing either confirmed or ruled out the presence of a second pathogenic variant, validated or ruled out pathogenicity of variants of uncertain significance, or identified a diagnosis initially missed by genetic analysis., Conclusion: Metabolomics-based gene function tests provide additional value in the diagnostic trajectory of patients with suspected IMD by enhancing and accelerating diagnostic success., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP + ) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C 10 -TPP + ). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP&#95;001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

SUPV3L1 encodes a helicase that is mainly localized in the mitochondria. It has been shown in vitro to possess both double-stranded RNA and DNA unwinding activity that is ATP-dependent. Here we report the first two patients for this gene who presented with a homozygous preliminary stop codon resulting in a C-terminal truncation of the SUPV3L1 protein. They presented with a characteristic phenotype of neurodegenerative nature with progressive spastic paraparesis, growth restriction, hypopigmentation, and predisposition to autoimmune disease. Ophthalmological examination showed severe photophobia with corneal erosions, optic atrophy, and pigmentary retinopathy, while neuroimaging showed atrophy of the optic chiasm and the pons with calcification of putamina, with intermittent and mild elevation of lactate. We show that the amino acids that are eliminated by the preliminary stop codon are highly conserved and are predicted to form an amphipathic helix. To investigate if the mutation causes mitochondrial dysfunction, we examined fibroblasts of the proband. We observed very low expression of the truncated protein, a reduction in the mature ND6 mRNA species as well as the accumulation of double-stranded RNA. Lentiviral complementation with the full-length SUPV3L1 cDNA partly restored the observed RNA phenotypes, supporting that the SUPV3L1 mutation in these patients is pathogenic and the cause of the disease., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na + -Cl - cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB , HNF1B , FXYD2 , or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown., Methods: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF , which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22 Na + transport., Results: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T ( n =7), m.616T>C ( n =1), m.643A>G ( n =1) (all in MT-TF ), and m.4291T>C ( n =4, in MT-TI ). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake., Conclusion: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies., (Copyright © 2022 by the American Society of Nephrology.)

Background: The combination of statin therapy and physical activity reduces cardiovascular disease risk in patients with hyperlipidemia more than either treatment alone. However, mitochondrial dysfunction associated with statin treatment could attenuate training adaptations., Objectives: This study determined whether moderate intensity exercise training improved muscle and exercise performance, muscle mitochondrial function, and fiber capillarization in symptomatic and asymptomatic statin users., Methods: Symptomatic (n = 16; age 64 ± 4 years) and asymptomatic statin users (n = 16; age 64 ± 4 years) and nonstatin using control subjects (n = 20; age 63 ± 5 years) completed a 12-week endurance and resistance exercise training program. Maximal exercise performance (peak oxygen consumption), muscle performance and muscle symptoms were determined before and after training. Muscle biopsies were collected to assess citrate synthase activity, adenosine triphosphate (ATP) production capacity, muscle fiber type distribution, fiber size, and capillarization., Results: Type I muscle fibers were less prevalent in symptomatic statin users than control subjects at baseline (P = 0.06). Exercise training improved muscle strength (P < 0.001), resistance to fatigue (P = 0.01), and muscle fiber capillarization (P < 0.01), with no differences between groups. Exercise training improved citrate synthase activity in the total group (P < 0.01), with asymptomatic statin users showing less improvement than control subjects (P = 0.02). Peak oxygen consumption, ATP production capacity, fiber size, and muscle symptoms remained unchanged in all groups following training. Quality-of-life scores improved only in symptomatic statin users following exercise training (P < 0.01)., Conclusions: A moderate intensity endurance and resistance exercise training program improves muscle performance, capillarization, and mitochondrial content in both asymptomatic and symptomatic statin users without exacerbating muscle complaints. Exercise training may even increase quality of life in symptomatic statin users. (The Effects of Cholesterol-Lowering Medication on Exercise Performance [STATEX]; NL5972/NTR6346)., Competing Interests: Funding Support and Author Disclosures Dr Allard was financially supported by a grant from the Radboud Institute for Health Sciences. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC&#95;012920.1:m.3291T > C; NC&#95;012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line., (© 2021. The Author(s).)

Individuals with mitochondrial disease often present with psychopathological comorbidity, and mitochondrial dysfunction has been proposed as the underlying pathobiology in various psychiatric disorders. Several studies have suggested that medications used to treat neuropsychiatric disorders could directly influence mitochondrial function. This review provides a comprehensive overview of the effect of these medications on mitochondrial function. We collected preclinical information on six major groups of antidepressants and other neuropsychiatric medications and found that the majority of these medications either positively influenced mitochondrial function or showed mixed effects. Only amitriptyline, escitalopram, and haloperidol were identified as having exclusively adverse effects on mitochondrial function. In the absence of formal clinical trials, and until such trials are completed, the data from preclinical studies reported and discussed here could inform medication prescribing practices for individuals with psychopathology and impaired mitochondrial function in the underlying pathology., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Antidepressants have been shown to influence mitochondrial function directly, and suboptimal mitochondrial function (SMF) has been implicated in complex psychiatric disorders. In the current study, we used a mouse model for trait SMF to test the hypothesis that chronic fluoxetine treatment in mice subjected to chronic stress would negatively impact brain bioenergetics, a response that would be more pronounced in mice with trait SMF. In contrast, we hypothesized that chronic ketamine treatment would positively impact mitochondrial function in both WT and mice with SMF. We used an animal model for trait SMF, the Ndufs4 GT/GT mice, which exhibit 25% lower mitochondrial complex I activity. In addition to antidepressant treatment, mice were subjected to chronic unpredictable stress (CUS). This paradigm is widely used to model complex behaviours expressed in various psychiatric disorders. We assayed several physiological indices as proxies for the impact of chronic stress and antidepressant treatment. Furthermore, we measured brain mitochondrial complex activities using clinically validated assays as well as established metabolic signatures using targeted metabolomics. As hypothesized, we found evidence that chronic fluoxetine treatment negatively impacted brain bioenergetics. This phenotype was, however, not further exacerbated in mice with trait SMF. Ketamine did not have a significant influence on brain mitochondrial function in either genotype. Here we report that trait SMF could be a moderator for an individual's response to antidepressant treatment. Based on these results, we propose that in individuals with SMF and comorbid psychopathology, fluoxetine should be avoided, whereas ketamine could be a safer choice of treatment., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca 2+ , sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD + ] ratio, which was corroborated by using the NADH/NAD + redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD + redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD + redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

HIV infection and antiretroviral therapy have been linked to mitochondrial dysfunction. The role of platelet mitochondrial dysfunction in thrombosis, immunoregulation and age-related diseases is increasingly appreciated. Here, we studied platelet mitochondrial DNA content (mtDNA pl ) and mitochondrial function in people living with HIV (PLHIV) and related this to platelet function. In a cohort of 208 treated PLHIV and 56 uninfected controls, mtDNA pl was quantified, as well as platelet activation, platelet agonist-induced reactivity and inflammation by circulating factors and flow cytometry. In a subgroup of participants, the metabolic activity of platelets was further studied by mitochondrial function tests and the Seahorse Flux Analyzer. PLHIV had significantly lower mtDNA pl compared to controls (8.5 copies/platelet (IQR: 7.0-10.7) vs. 12.2 copies/platelet (IQR: 9.5-16.6); p < 0.001), also after correction for age, sex and BMI. Prior zidovudine-use (n = 46) was associated with a trend for lower mtDNA pl . PLHIV also had reduced ex vivo platelet reactivity and mean platelet volume compared to controls. MtDNA pl correlated positively with both platelet parameters and correlated negatively with inflammatory marker sCD163. Mitochondrial function tests in a subgroup of participants confirmed the presence of platelet mitochondrial respiration defects. Platelet mitochondrial function is disturbed in PLHIV, which may contribute to platelet dysfunction and subsequent complications. Interventions targeting the preservation of normal platelet mitochondrial function may ultimately prove beneficial for PLHIV.

Introduction: The m.3243A > G mitochondrial DNA mutation is one of the most common mitochondrial disease-causing mutations, with a carrier rate as high as 1:400. This point mutation affects the MT-TL1 gene, ultimately affecting the oxidative phosphorylation system and the cell's energy production. Strikingly, the m.3243A > G mutation is associated with different phenotypes, including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD) and myopathy., Objectives: We investigated urine metabolomes of MELAS, MIDD and myopathy patients in order to identify affected metabolic pathways and possible treatment options., Methods: A multiplatform metabolomics approach was used to comprehensively analyze the metabolome and compare metabolic profiles of different phenotypes caused by the m.3243A > G mutation. Our analytical array consisted of NMR spectroscopy, LC-MS/MS and GC-TOF-MS., Results: The investigation revealed phenotypic specific metabolic perturbations, as well as metabolic similarities between the different phenotypes. We show that glucose metabolism is highly disturbed in the MIDD phenotype, but not in MELAS or myopathy, remodeled fatty acid oxidation is characteristic of the MELAS patients, while one-carbon metabolism is strongly modified in both MELAS and MIDD, but not in the myopathy group. Lastly we identified increased creatine in the urine of the myopathy patients, but not in MELAS or MIDD., Conclusion: We conclude by giving novel insight on the phenotypes of the m.3243A > G mutation from a metabolomics point of view. Directives are also given for future investigations that could lead to better treatment options for patients suffering from this debilitating disease.

Pheochromocytomas and paragangliomas (PPGLs) caused by mutations in the B-subunit of the succinate dehydrogenase (SDHB) have the highest metastatic rate among PPGLs, and effective systemic therapy is lacking. To unravel underlying pathogenic mechanisms, and to evaluate therapeutic strategies, suitable in vivo models are needed. The available systemic Sdhb knock-out mice cannot model the human PPGL phenotype: heterozygous Sdhb mice lack a disease phenotype, and homozygous Sdhb mice are embryonically lethal. Using CRISPR/cas9 technology, we introduced a protein-truncating germline lesion into the zebrafish sdhb gene. Heterozygous sdhb mutants were viable and displayed no obvious morphological or developmental defects. Homozygous sdhb larvae were viable, but exhibited a decreased lifespan. Morphological analysis revealed incompletely or non-inflated swim bladders in homozygous sdhb mutants at day 6. Although no differences in number and ultrastructure of the mitochondria were observed. Clear defects in energy metabolism and swimming behavior were observed in homozygous sdhb mutant larvae. Functional and metabolomic analyses revealed decreased mitochondrial complex 2 activity and significant succinate accumulation in the homozygous sdhb mutant larvae, mimicking the metabolic effects observed in SDHB-associated PPGLs. This is the first study to present a vertebrate animal model that mimics metabolic effects of SDHB-associated PPGLs. This model will be useful in unraveling pathomechanisms behind SDHB-associated PPGLs. We can now study the metabolic effects of sdhb disruption during different developmental stages and develop screening assays to identify novel therapeutic targets in vivo. Besides oncological syndromes, our model might also be useful for pediatric mitochondrial disease caused by loss of the SDHB gene.

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4 -/- mouse tissues. Ndufs4 -/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4 -/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4 -/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4 -/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4 GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi-platform targeted mass spectrometry-based metabolomics. Ndufs4 GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4 GT/GT from WT mice. Specifically, we observed a reversed TCA cycle flux and rewiring of amino acid metabolism in the prefrontal cortex. Next, exposing mice to chronic variable stress (a model for depression-like behavior), we found that Ndufs4 GT/GT mice showed altered stress response and coping strategies with a robust stress-associated reprogramming of amino acid metabolism. Our data suggest that impaired mitochondrial CI function is a candidate driver for altered stress reactivity and stress-induced brain metabolic reprogramming. These changes result in unique phenomic and metabolomic signatures distinguishing groups based on their mitochondrial genotype.

NGLY1 encodes the enzyme N-glycanase that is involved in the degradation of glycoproteins as part of the endoplasmatic reticulum-associated degradation pathway. Variants in this gene have been described to cause a multisystem disease characterized by neuromotor impairment, neuropathy, intellectual disability, and dysmorphic features. Here, we describe four patients with pathogenic variants in NGLY1. As the clinical features and laboratory results of the patients suggested a multisystem mitochondrial disease, a muscle biopsy had been performed. Biochemical analysis in muscle showed a strongly reduced ATP production rate in all patients, while individual OXPHOS enzyme activities varied from normal to reduced. No causative variants in any mitochondrial disease genes were found using mtDNA analysis and whole exome sequencing. In all four patients, variants in NGLY1 were identified, including two unreported variants (c.849T>G (p.(Cys283Trp)) and c.1067A>G (p.(Glu356Gly)). Western blot analysis of N-glycanase in muscle and fibroblasts showed a complete absence of N-glycanase. One patient showed a decreased basal and maximal oxygen consumption rates in fibroblasts. Mitochondrial morphofunction fibroblast analysis showed patient specific differences when compared to control cell lines. In conclusion, variants in NGLY1 affect mitochondrial energy metabolism which in turn might contribute to the clinical disease course., (© 2020 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Brody disease is an autosomal recessive myopathy characterized by exercise-induced muscle stiffness due to mutations in the ATP2A1 gene. Almost 50 years after the initial case presentation, only 18 patients have been reported and many questions regarding the clinical phenotype and results of ancillary investigations remain unanswered, likely leading to incomplete recognition and consequently under-diagnosis. Additionally, little is known about the natural history of the disorder, genotype-phenotype correlations, and the effects of symptomatic treatment. We studied the largest cohort of Brody disease patients to date (n = 40), consisting of 22 new patients (19 novel mutations) and all 18 previously published patients. This observational study shows that the main feature of Brody disease is an exercise-induced muscle stiffness of the limbs, and often of the eyelids. Onset begins in childhood and there was no or only mild progression of symptoms over time. Four patients had episodes resembling malignant hyperthermia. The key finding at physical examination was delayed relaxation after repetitive contractions. Additionally, no atrophy was seen, muscle strength was generally preserved, and some patients had a remarkable athletic build. Symptomatic treatment was mostly ineffective or produced unacceptable side effects. EMG showed silent contractures in approximately half of the patients and no myotonia. Creatine kinase was normal or mildly elevated, and muscle biopsy showed mild myopathic changes with selective type II atrophy. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity was reduced and western blot analysis showed decreased or absent SERCA1 protein. Based on this cohort, we conclude that Brody disease should be considered in cases of exercise-induced muscle stiffness. When physical examination shows delayed relaxation, and there are no myotonic discharges at electromyography, we recommend direct sequencing of the ATP2A1 gene or next generation sequencing with a myopathy panel. Aside from clinical features, SERCA activity measurement and SERCA1 western blot can assist in proving the pathogenicity of novel ATP2A1 mutations. Finally, patients with Brody disease may be at risk for malignant hyperthermia-like episodes, and therefore appropriate perioperative measures are recommended. This study will help improve understanding and recognition of Brody disease as a distinct myopathy in the broader field of calcium-related myopathies., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: Idiopathic dilated cardiomyopathy (DCM) is recognised to be a heritable disorder, yet clinical genetic testing does not produce a diagnosis in >50% of paediatric patients. Identifying a genetic cause is crucial because this knowledge can affect management options, cardiac surveillance in relatives and reproductive decision-making. In this study, we sought to identify the underlying genetic defect in a patient born to consanguineous parents with rapidly progressive DCM that led to death in early infancy., Methods and Results: Exome sequencing revealed a potentially pathogenic, homozygous missense variant, c.542G>T, p.(Gly181Val), in SOD2 . This gene encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase, a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O 2 -· ) into H 2 O 2 . Measurement of hydroethidine oxidation showed a significant increase in O 2 -· levels in the patient's skin fibroblasts, as compared with controls, and this was paralleled by reduced catalytic activity of SOD2 in patient fibroblasts and muscle. Lentiviral complementation experiments demonstrated that mitochondrial SOD2 activity could be completely restored on transduction with wild type SOD2., Conclusion: Our results provide evidence that defective SOD2 may lead to toxic increases in the levels of damaging oxygen radicals in the neonatal heart, which can result in rapidly developing heart failure and death. We propose SOD2 as a novel nuclear-encoded mitochondrial protein involved in severe human neonatal cardiomyopathy, thus expanding the wide range of genetic factors involved in paediatric cardiomyopathies., Competing Interests: Competing interests: PHGMW is scientific advisor of Khondrion, Nijmegen, the Netherlands. This SME had no involvement in the data collection, analysis and interpretation, writing of the manuscript and in the decision to submit the manuscript for publication., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Mitochondrial complex I deficiency is the most frequent mitochondrial disorder presenting in childhood and the mutational spectrum is highly heterogeneous. The NDUFB11 gene is one of the recently identified genes, which is located in the short arm of the X-chromosome. Here we report clinical, biochemical, functional and genetic findings of two male patients with lactic acidosis, hypertrophic cardiomyopathy and isolated complex I deficiency due to de novo hemizygous mutations (c.286C > T and c.328C > T) in the NDUFB11 gene. Neither of them had any skin manifestations. The NDUFB11 gene encodes a relatively small integral membrane protein NDUFB11, which is essential for the assembly of an active complex I. The expression levels of this protein was decreased in both patient cells and a lentiviral complementation experiment also supported the notion that the complex I deficiency in those two patients is caused by NDUFB11 genetic defects. Our findings together with a review of the thirteen previously described patients demonstrate a wide spectrum of clinical features associated with NDUFB11-related complex I deficiency. However, histiocytoid cardiomyopathy and/or congenital sideroblastic anemia could be indicative for mutation in the NDUFB11 gene, while the clinical manifestation of the same mutation can be highly variable., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS., Competing Interests: The authors declare no competing interests.

Mitochondrial diseases are a group of rare life-threatening diseases often caused by defects in the oxidative phosphorylation system. No effective treatment is available for these disorders. Therapeutic development is hampered by the high heterogeneity in genetic, biochemical, and clinical spectra of mitochondrial diseases and by limited preclinical resources to screen and identify effective treatment candidates. Alternative models of the pathology are essential to better understand mitochondrial diseases and to accelerate the development of new therapeutics. The fruit fly Drosophila melanogaster is a cost- and time-efficient model that can recapitulate a wide range of phenotypes observed in patients suffering from mitochondrial disorders. We targeted three important subunits of complex I of the mitochondrial oxidative phosphorylation system with the flexible UAS-Gal4 system and RNA interference (RNAi): NDUFS4 (ND-18), NDUFS7 (ND-20), and NDUFV1 (ND-51). Using two ubiquitous driver lines at two temperatures, we established a collection of phenotypes relevant to complex I deficiencies. Our data offer models and phenotypes with different levels of severity that can be used for future therapeutic screenings. These include qualitative phenotypes that are amenable to high-throughput drug screening and quantitative phenotypes that require more resources but are likely to have increased potential and sensitivity to show modulation by drug treatment.

Objective: To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS , encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively., Methods: We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS . Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts., Results: Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity., Conclusion: This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies., (© 2019 American Academy of Neurology.)