RESUMO
Isolated complex I (CI) deficiencies are a major cause of primary mitochondrial disease. A substantial proportion of CI deficiencies are believed to arise from defects in CI assembly factors (CIAFs) that are not part of the CI holoenzyme. The biochemistry of these CIAFs is poorly defined, making their role in CI assembly unclear, and confounding interpretation of potential disease-causing genetic variants. To address these challenges, we devised a deep mutational scanning approach to systematically assess the function of thousands of NDUFAF6 genetic variants. Guided by these data, biochemical analyses and cross-linking mass spectrometry, we discovered that the CIAF NDUFAF6 facilitates incorporation of NDUFS8 into CI and reveal that NDUFS8 overexpression rectifies NDUFAF6 deficiency. Our data further provide experimental support of pathogenicity for seven novel NDUFAF6 variants associated with human pathology and introduce functional evidence for over 5,000 additional variants. Overall, our work defines the molecular function of NDUFAF6 and provides a clinical resource for aiding diagnosis of NDUFAF6-related diseases.
Assuntos
Complexo I de Transporte de Elétrons , Doenças Mitocondriais , Proteínas Mitocondriais , Humanos , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Mitocôndrias/metabolismo , Mitocôndrias/genéticaRESUMO
Pathogenic variants in both mitochondrial and nuclear genes contribute to the clinical and genetic heterogeneity of mitochondrial diseases. There are now pathogenic variants in over 300 nuclear genes linked to human mitochondrial diseases. Nonetheless, diagnosing mitochondrial disease with a genetic outcome remains challenging. However, there are now many strategies that help us to pinpoint causative variants in patients with mitochondrial disease. This chapter describes some of the approaches and recent advancements in gene/variant prioritization using whole-exome sequencing (WES).
Assuntos
Exoma , Doenças Mitocondriais , Humanos , Genômica , Doenças Mitocondriais/genética , Sequenciamento do Exoma , Núcleo CelularRESUMO
Primary mitochondrial disease describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation. Diagnosis is challenging; >350 genes, both nuclear and mitochondrial DNA (mtDNA) encoded, are known to cause mitochondrial disease, leading to all possible inheritance patterns and further complicated by heteroplasmy of the multicopy mitochondrial genome. Technological advances, particularly next-generation sequencing, have driven a shift in diagnostic practice from 'biopsy first' to genome-wide analyses of blood and/or urine DNA. This has led to the need for a reference framework for laboratories involved in mitochondrial genetic testing to facilitate a consistent high-quality service. In the United Kingdom, consensus guidelines have been prepared by a working group of Clinical Scientists from the NHS Highly Specialised Service followed by national laboratory consultation. These guidelines summarise current recommended technologies and methodologies for the analysis of mtDNA and nuclear-encoded genes in patients with suspected mitochondrial disease. Genetic testing strategies for diagnosis, family testing and reproductive options including prenatal diagnosis are outlined. Importantly, recommendations for the minimum levels of mtDNA testing for the most common referral reasons are included, as well as guidance on appropriate referrals and information on the minimal appropriate gene content of panels when analysing nuclear mitochondrial genes. Finally, variant interpretation and recommendations for reporting of results are discussed, focussing particularly on the challenges of interpreting and reporting mtDNA variants.
Assuntos
Genoma Mitocondrial , Doenças Mitocondriais , Gravidez , Feminino , Humanos , Estudo de Associação Genômica Ampla , Doenças Mitocondriais/genética , DNA Mitocondrial/genética , Testes Genéticos/métodos , Mitocôndrias/genéticaRESUMO
Imbalances in mitochondrial and peroxisomal dynamics are associated with a spectrum of human neurological disorders. Mitochondrial and peroxisomal fission both involve dynamin-related protein 1 (DRP1) oligomerisation and membrane constriction, although the precise biophysical mechanisms by which distinct DRP1 variants affect the assembly and activity of different DRP1 domains remains largely unexplored. We analysed four unreported de novo heterozygous variants in the dynamin-1-like gene <i>DNM1L</i>, affecting different highly conserved DRP1 domains, leading to developmental delay, seizures, hypotonia, and/or rare cardiac complications in infancy. Single-nucleotide DRP1 stalk domain variants were found to correlate with more severe clinical phenotypes, with in vitro recombinant human DRP1 mutants demonstrating greater impairments in protein oligomerisation, DRP1-peroxisomal recruitment, and both mitochondrial and peroxisomal hyperfusion compared to GTPase or GTPase-effector domain variants. Importantly, we identified a novel mechanism of pathogenesis, where a p.Arg710Gly variant uncouples DRP1 assembly from assembly-stimulated GTP hydrolysis, providing mechanistic insight into how assembly-state information is transmitted to the GTPase domain. Together, these data reveal that discrete, pathological <i>DNM1L</i> variants impair mitochondrial network maintenance by divergent mechanisms.
Assuntos
Dinâmica Mitocondrial , Proteínas Mitocondriais , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
BACKGROUND: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. METHODS: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. RESULTS: We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. CONCLUSION: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.
Assuntos
RNA , Transcriptoma , Alelos , Humanos , Análise de Sequência de RNA/métodos , Sequenciamento do ExomaRESUMO
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
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
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
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
BACKGROUND: Acquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease. RESULTS: To decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns. CONCLUSIONS: These patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.
Assuntos
DNA Polimerase gama/genética , DNA Mitocondrial/análise , Técnicas Genéticas , Doenças Mitocondriais/genética , Deleção de Sequência , Software , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Replicação do DNA , DNA Mitocondrial/metabolismo , Células HEK293 , Humanos , Pessoa de Meia-Idade , Músculo Quadríceps/química , Músculo Quadríceps/patologia , Adulto JovemRESUMO
Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects' fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects' fibroblasts with wild-type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module.
Assuntos
Doença de Leigh , Doenças Mitocondriais , Alelos , Criança , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Doença de Leigh/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , MutaçãoRESUMO
Coenzyme Q10 (CoQ10) deficiency is a clinically and genetically heterogeneous subtype of mitochondrial disease. We report two girls with ataxia and mitochondrial respiratory chain deficiency who were shown to have primary CoQ10 deficiency. Muscle histochemistry displayed signs of mitochondrial dysfunction-ragged red fibers, mitochondrial paracrystalline inclusions, and lipid deposits while biochemical analyses revealed complex II+III respiratory chain deficiencies. MRI brain demonstrated cerebral and cerebellar atrophy. Targeted molecular analysis identified a homozygous c.1015G>A, p.(Ala339Thr) COQ8A variant in subject 1, while subject 2 was found to harbor a single heterozygous c.1029_1030delinsCA variant predicting a p.Gln343_Val344delinsHisMet amino acid substitution. Subsequent investigations identified a large-scale COQ8A deletion in trans to the c.1029_1030delinsCA allele. A skin biopsy facilitated cDNA studies that confirmed exon skipping in the fibroblast derived COQ8A mRNA transcript. This report expands the molecular genetic spectrum associated with COQ8A-related mitochondrial disease and highlights the importance of thorough investigation of candidate pathogenic variants to establish phase. Rapid diagnosis is of the utmost importance as patients may benefit from therapeutic CoQ10 supplementation.
RESUMO
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
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
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
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
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
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.