RESUMO
Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Bases de Dados de Proteínas , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Proteínas Mitocondriais/genética , Interferência de RNA , Transdução de Sinais , Transfecção , Ubiquinona/metabolismoRESUMO
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.
Assuntos
Complexo I de Transporte de Elétrons/deficiência , Fibroblastos/patologia , Doença de Leigh/etiologia , Doenças Mitocondriais/etiologia , Proteínas Mitocondriais/genética , Mutação , NADH Desidrogenase/genética , Alelos , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Doença de Leigh/patologia , Masculino , Doenças Mitocondriais/patologia , Linhagem , FenótipoRESUMO
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% â 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% â 57%), exclusive enteral feeding (22% â 46%), and one-to-one assistance for self-care (25% â 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.
Assuntos
Doença de Leigh , Criança , Pré-Escolar , Estudos de Coortes , Efeitos Psicossociais da Doença , Progressão da Doença , Feminino , Humanos , Lactente , Estudos Longitudinais , MasculinoRESUMO
Mitochondria play essential roles in numerous metabolic pathways including the synthesis of adenosine triphosphate through oxidative phosphorylation. Clinically, mitochondrial diseases occur when there is mitochondrial dysfunction - manifesting at any age and affecting any organ system; tissues with high energy requirements, such as muscle and the brain, are often affected. The clinical heterogeneity is parallel to the degree of genetic heterogeneity associated with mitochondrial dysfunction. Around 10% of human genes are predicted to have a mitochondrial function, and defects in over 300 genes are reported to cause mitochondrial disease. Some involve the mitochondrial genome (mtDNA), but the vast majority occur within the nuclear genome. Except for a few specific genetic defects, there remains no cure for mitochondrial diseases, which means that a genetic diagnosis is imperative for genetic counselling and the provision of reproductive options for at-risk families. Next-generation sequencing strategies, particularly exome and whole-genome sequencing, have revolutionised mitochondrial diagnostics such that the traditional muscle biopsy has largely been replaced with a minimally-invasive blood sample for an unbiased approach to genetic diagnosis. Where these genomic approaches have not identified a causative defect, or where there is insufficient support for pathogenicity, additional functional investigations are required. The application of supplementary 'omics' technologies, including transcriptomics, proteomics, and metabolomics, has the potential to greatly improve diagnostic strategies. This review aims to demonstrate that whilst a molecular diagnosis can be achieved for many cases through next-generation sequencing of blood DNA, the use of patient tissues and an integrated, multidisciplinary multi-omics approach is pivotal for the diagnosis of more challenging cases. Moreover, the analysis of clinically relevant tissues from affected individuals remains crucial for understanding the molecular mechanisms underlying mitochondrial pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Mitocôndrias/patologia , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Animais , Genômica/métodos , Humanos , Metabolômica/métodos , Proteômica/métodosRESUMO
BCS1L encodes a homolog of the Saccharomyces cerevisiae bcs1 protein, which has a known role in the assembly of Complex III of the mitochondrial respiratory chain. Phenotypes reported in association with pathogenic BCS1L variants include growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death (GRACILE syndrome), and Björnstad syndrome, characterized by abnormal flattening and twisting of hair shafts (pili torti) and hearing problems. Here we describe two patients harbouring biallelic variants in BCS1L; the first with a heterozygous variant c.166C>T, p.(Arg56*) together with a novel heterozygous variant c.205C>T, p.(Arg69Cys) and a second patient with a novel homozygous c.325C>T, p.(Arg109Trp) variant. The two patients presented with different phenotypes; the first patient presented as an adult with aminoaciduria, seizures, bilateral sensorineural deafness and learning difficulties. The second patient was an infant who presented with a classical GRACILE syndrome leading to death at 4 months of age. A decrease in BCS1L protein levels was seen in both patients, and biochemical analysis of Complex III revealed normal respiratory chain enzyme activities in the muscle of both patients. A decrease in Complex III assembly was detected in the adult patient's muscle, whilst the paediatric patient displayed a combined mitochondrial respiratory chain defect in cultured fibroblasts. Yeast complementation studies indicate that the two missense variants, c.205C>T, p.(Arg69Cys) and c.325C>T, p.(Arg109Trp), impair the respiratory capacity of the cell. Together, these data support the pathogenicity of the novel BCS1L variants identified in our patients.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Doenças Mitocondriais/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acidose Láctica/genética , Adulto , Sequência de Aminoácidos , Colestase/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Fibroblastos/metabolismo , Hemossiderose/genética , Humanos , Lactente , Masculino , Erros Inatos do Metabolismo/genética , Doenças Mitocondriais/congênito , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mutação , Fenótipo , Aminoacidúrias Renais/genéticaRESUMO
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 â¼65 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 â¼830 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.
Assuntos
Complexo I de Transporte de Elétrons/deficiência , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Alelos , Sequência de Aminoácidos , Complexo I de Transporte de Elétrons/genética , Feminino , Fibroblastos/patologia , Heterogeneidade Genética , Humanos , Lactente , Masculino , Mitocôndrias/genética , Fenótipo , Alinhamento de SequênciaRESUMO
The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.
Assuntos
Aminoacil-tRNA Sintetases/genética , Pneumopatias/genética , Mutação/genética , Adolescente , Alelos , Doença de Charcot-Marie-Tooth/genética , Pré-Escolar , Feminino , Genes Recessivos/genética , Heterozigoto , Humanos , Lactente , Masculino , Biossíntese de Proteínas/genéticaRESUMO
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.
Assuntos
Cardiomiopatias , Morte Súbita Cardíaca , Adolescente , Alelos , Cardiomiopatias/genética , Pré-Escolar , Morte Súbita Cardíaca/etiologia , Humanos , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Proteínas Mitocondriais/genética , MutaçãoRESUMO
Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.
Assuntos
DNA Mitocondrial/genética , Genes Mitocondriais/genética , Mitocôndrias Musculares/genética , Doenças Mitocondriais/genética , Fibras Musculares Esqueléticas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Deleção de Sequência , Análise de Célula ÚnicaRESUMO
Mitochondrial disease is a challenging area of genetics because two distinct genomes can contribute to disease pathogenesis. It is also challenging clinically because of the myriad of different symptoms and, until recently, a lack of a genetic diagnosis in many patients. The last five years has brought remarkable progress in this area. We provide a brief overview of mitochondrial origin, function, and biology, which are key to understanding the genetic basis of mitochondrial disease. However, the primary purpose of this review is to describe the recent advances related to the diagnosis, genetic basis, and prevention of mitochondrial disease, highlighting the newly described disease genes and the evolving methodologies aimed at preventing mitochondrial DNA disease transmission.
Assuntos
Doenças Mitocondriais/diagnóstico , DNA Mitocondrial , Feminino , Humanos , Masculino , Doenças Mitocondriais/genética , Doenças Mitocondriais/prevenção & controle , MutaçãoRESUMO
Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp-/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp-/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.
Assuntos
Cardiomiopatias/genética , Proteínas de Transporte/genética , Transporte de Elétrons/fisiologia , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação , Adulto , Idade de Início , Idoso , Alelos , Sequência de Aminoácidos , Animais , Cardiomiopatias/complicações , Cardiomiopatias/patologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Células Cultivadas , Pré-Escolar , Estudos de Coortes , DNA Mitocondrial , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Recém-Nascido , Masculino , Camundongos , Pessoa de Meia-Idade , Doenças Mitocondriais/complicações , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Linhagem , Conformação Proteica , Homologia de Sequência , Índice de Gravidade de Doença , Adulto JovemRESUMO
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.
Assuntos
Complexo II de Transporte de Elétrons/deficiência , Erros Inatos do Metabolismo/genética , Doenças Mitocondriais/genética , Succinato Desidrogenase/genética , Adolescente , Adulto , Criança , Pré-Escolar , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Feminino , Fumaratos/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Redes e Vias Metabólicas/genética , Erros Inatos do Metabolismo/epidemiologia , Erros Inatos do Metabolismo/metabolismo , Pessoa de Meia-Idade , Doenças Mitocondriais/epidemiologia , Doenças Mitocondriais/metabolismo , Fosforilação Oxidativa , Proteínas/genética , Ácido Succínico/metabolismoRESUMO
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.
Assuntos
Variação Genética/genética , Doenças Mitocondriais/epidemiologia , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos de Coortes , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/diagnóstico , Reino Unido/epidemiologia , Adulto JovemRESUMO
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.
Assuntos
Sequenciamento do Exoma/métodos , Genoma Mitocondrial , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças Mitocondriais/diagnóstico , Técnicas de Diagnóstico Molecular , Humanos , Doenças Mitocondriais/genética , Análise de Sequência de RNA , TranscriptomaRESUMO
Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11-13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged-red/cytochrome c oxidase-negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10 ) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum-Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions.
Assuntos
Encefalopatias Metabólicas/genética , Doenças Mitocondriais/genética , Debilidade Muscular/genética , Mutação , Proteômica/métodos , Rabdomiólise/genética , Encefalopatias Metabólicas/diagnóstico , Ácidos Graxos/metabolismo , Feminino , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Homozigoto , Humanos , Lactente , Masculino , Doenças Mitocondriais/diagnóstico , Fosforilação Oxidativa , Fenótipo , Rabdomiólise/diagnóstico , Sequenciamento Completo do GenomaRESUMO
Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.
Assuntos
Cardiomiopatia Hipertrófica/genética , Genes Mitocondriais , Predisposição Genética para Doença , Mutação , Proteínas de Neoplasias/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , Alelos , Substituição de Aminoácidos , Biomarcadores , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/terapia , Estudos de Coortes , Ativação Enzimática , Feminino , Expressão Gênica , Estudos de Associação Genética , Genótipo , Humanos , Lactente , Cinética , Masculino , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fenótipo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5' ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m(1)R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants-compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2-was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m(1)R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function.
Assuntos
Genes Recessivos/genética , Metiltransferases/genética , Doenças Mitocondriais/etiologia , Mutação/genética , Processamento Pós-Transcricional do RNA/genética , RNA/genética , Ribonuclease P/genética , Sequência de Aminoácidos , Transporte de Elétrons/genética , Feminino , Humanos , Recém-Nascido , Masculino , Mitocôndrias/metabolismo , Doenças Mitocondriais/patologia , Linhagem , Biossíntese de Proteínas/fisiologia , RNA/metabolismo , RNA Mitocondrial , RNA de Transferência/genética , Homologia de Sequência de AminoácidosRESUMO
Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs(∗)2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.
Assuntos
Alelos , Complexo I de Transporte de Elétrons/deficiência , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Mutação/genética , Fenótipo , Adolescente , Adulto , Idade de Início , Sequência de Aminoácidos , Criança , Complexo I de Transporte de Elétrons/genética , Feminino , Humanos , Lactente , Masculino , Proteínas de Membrana/química , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.
Assuntos
Morte Súbita Cardíaca/etiologia , Pirofosfatase Inorgânica/deficiência , Pirofosfatase Inorgânica/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Mutação de Sentido Incorreto/genética , Acidose Láctica/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Arritmias Cardíacas/genética , Cardiomiopatias/enzimologia , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Criança , Pré-Escolar , Morte Súbita Cardíaca/patologia , Etanol/efeitos adversos , Exoma/genética , Feminino , Fibroblastos/citologia , Fibroblastos/patologia , Fibrose/enzimologia , Fibrose/genética , Fibrose/patologia , Humanos , Lactente , Recém-Nascido , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/metabolismo , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Linhagem , Fenótipo , Convulsões , Adulto JovemRESUMO
Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.