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1.
FEBS Lett ; 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33655490

RESUMO

Mitochondrial disorders are monogenic disorders characterized by a defect in oxidative phosphorylation and caused by pathogenic variants in one of over 340 different genes. The implementation of whole-exome sequencing has led to a revolution in their diagnosis, duplicated the number of associated disease genes, and significantly increased the diagnosed fraction. However, the genetic etiology of a substantial fraction of patients exhibiting mitochondrial disorders remains unknown, highlighting limitations in variant detection and interpretation, which calls for improved computational and DNA sequencing methods, as well as the addition of OMICS tools. More intriguingly, this also suggests that some pathogenic variants lie outside of the protein-coding genes and that the mechanisms beyond the Mendelian inheritance and the mtDNA are of relevance. This review covers the current status of the genetic basis of mitochondrial diseases, discusses current challenges and perspectives, and explores the contribution of factors beyond the protein-coding regions and monogenic inheritance in the expansion of the genetic spectrum of disease.

2.
Nat Protoc ; 16(2): 1276-1296, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462443

RESUMO

RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects in individuals affected by genetically undiagnosed rare disorders. Pioneering studies have shown that RNA-seq could increase the diagnosis rates over DNA sequencing alone by 8-36%, depending on the disease entity and tissue probed. To accelerate adoption of RNA-seq by human genetics centers, detailed analysis protocols are now needed. We present a step-by-step protocol that details how to robustly detect aberrant expression levels, aberrant splicing and mono-allelic expression in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on the detection of RNA outliers pipeline (DROP), a modular computational workflow that integrates all the analysis steps, can leverage parallel computing infrastructures and generates browsable web page reports.


Assuntos
Sequência de Bases/genética , Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Diagnóstico , Técnicas e Procedimentos Diagnósticos , Doença/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/genética , Software , Fluxo de Trabalho
3.
Nat Commun ; 12(1): 529, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483494

RESUMO

Aberrant splicing is a major cause of rare diseases.  However, its prediction from genome sequence alone remains in most cases inconclusive. Recently, RNA sequencing has proven to be an effective complementary avenue to detect aberrant splicing. Here, we develop FRASER, an algorithm to detect aberrant splicing from RNA sequencing data. Unlike existing methods, FRASER captures not only alternative splicing but also intron retention events. This typically doubles the number of detected aberrant events and identified a pathogenic intron retention in MCOLN1 causing mucolipidosis. FRASER automatically controls for latent confounders, which are widespread and affect sensitivity substantially. Moreover, FRASER is based on a count distribution and multiple testing correction, thus reducing the number of calls by two orders of magnitude over commonly applied z score cutoffs, with a minor loss of sensitivity. Applying FRASER to rare disease diagnostics is demonstrated by reprioritizing a pathogenic aberrant exon truncation in TAZ from a published dataset. FRASER is easy to use and freely available.


Assuntos
Algoritmos , Processamento Alternativo , Biologia Computacional/métodos , RNA-Seq/métodos , Análise de Sequência de RNA/métodos , Internet , Íntrons/genética , Software
4.
Front Genet ; 11: 288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32265993

RESUMO

[This corrects the article DOI: 10.3389/fgene.2020.00095.].

5.
Front Genet ; 11: 95, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32180794

RESUMO

The regulation of mitochondrial proteome is unique in that its components have origins in both mitochondria and nucleus. With the development of OMICS technologies, emerging evidence indicates an interaction between mitochondria and nucleus based not only on the proteins but also on the non-coding RNAs (ncRNAs). It is now accepted that large parts of the non-coding genome are transcribed into various ncRNA species. Although their characterization has been a hot topic in recent years, the function of the majority remains unknown. Recently, ncRNA species microRNA (miRNA) and long-non coding RNAs (lncRNA) have been gaining attention as direct or indirect modulators of the mitochondrial proteome homeostasis. These ncRNA can impact mitochondria indirectly by affecting transcripts encoding for mitochondrial proteins in the cytoplasm. Furthermore, reports of mitochondria-localized miRNAs, termed mitomiRs, and lncRNAs directly regulating mitochondrial gene expression suggest the import of RNA to mitochondria, but also transcription from the mitochondrial genome. Interestingly, ncRNAs have been also shown to hide small open reading frames (sORFs) encoding for small functional peptides termed micropeptides, with several examples reported with a role in mitochondria. In this review, we provide a literature overview on ncRNAs and micropeptides found to be associated with mitochondrial biology in the context of both health and disease. Although reported, small study overlap and rare replications by other groups make the presence, transport, and role of ncRNA in mitochondria an attractive, but still challenging subject. Finally, we touch the topic of their potential as prognosis markers and therapeutic targets.

6.
Am J Hum Genet ; 106(1): 102-111, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31883641

RESUMO

Isolated complex III (CIII) deficiencies are among the least frequently diagnosed mitochondrial disorders. Clinical symptoms range from isolated myopathy to severe multi-systemic disorders with early death and disability. To date, we know of pathogenic variants in genes encoding five out of 10 subunits and five out of 13 assembly factors of CIII. Here we describe rare bi-allelic variants in the gene of a catalytic subunit of CIII, UQCRFS1, which encodes the Rieske iron-sulfur protein, in two unrelated individuals. Affected children presented with low CIII activity in fibroblasts, lactic acidosis, fetal bradycardia, hypertrophic cardiomyopathy, and alopecia totalis. Studies in proband-derived fibroblasts showed a deleterious effect of the variants on UQCRFS1 protein abundance, mitochondrial import, CIII assembly, and cellular respiration. Complementation studies via lentiviral transduction and overexpression of wild-type UQCRFS1 restored mitochondrial function and rescued the cellular phenotype, confirming UQCRFS1 variants as causative for CIII deficiency. We demonstrate that mutations in UQCRFS1 can cause mitochondrial disease, and our results thereby expand the clinical and mutational spectrum of CIII deficiencies.


Assuntos
Alopecia/patologia , Cardiomiopatias/patologia , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Proteínas com Ferro-Enxofre/genética , Doenças Mitocondriais/patologia , Mutação , Alelos , Alopecia/genética , Cardiomiopatias/genética , Criança , Complexo III da Cadeia de Transporte de Elétrons/genética , Humanos , Lactente , Masculino , Doenças Mitocondriais/genética , Linhagem
7.
J Clin Invest ; 130(1): 108-125, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31550240

RESUMO

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.


Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Proteínas Mitocondriais/genética , Mutação , Atrofias Ópticas Hereditárias/genética , Animais , Polimerase do DNA Mitocondrial/fisiologia , Replicação do DNA , Proteínas de Ligação a DNA/química , Exoma , Feminino , Humanos , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Atrofias Ópticas Hereditárias/etiologia , Peixe-Zebra
8.
Hum Mutat ; 40(11): 1985-1992, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31209944

RESUMO

We report four unrelated children with homozygous loss-of-function variants in TASP1 and an overlapping phenotype comprising developmental delay with hypotonia and microcephaly, feeding difficulties with failure-to-thrive, recurrent respiratory infections, cardiovascular malformations, cryptorchidism, happy demeanor, and distinctive facial features. Two children had a homozygous founder deletion encompassing exons 5-11 of TASP1, the third had a homozygous missense variant, c.701 C>T (p.Thr234Met), affecting the active site of the encoded enzyme, and the fourth had a homozygous nonsense variant, c.199 C>T (p.Arg67*). TASP1 encodes taspase 1 (TASP1), which is responsible for cleaving, thus activating, the lysine methyltransferases KMT2A and KMT2D, which are essential for histone methylation and transcription regulation. The consistency of the phenotype, the critical biological function of TASP1, the deleterious nature of the TASP1 variants, and the overlapping features with Wiedemann-Steiner and Kabuki syndromes respectively caused by pathogenic variants in KMT2A and KMT2D all support that TASP1 is a disease-related gene.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/genética , Homozigoto , Mutação com Perda de Função , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Fenótipo , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Éxons , Facies , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Linhagem , Síndrome , Sequenciamento Completo do Exoma
9.
Mol Syst Biol ; 15(2): e8513, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777893

RESUMO

Despite their importance in determining protein abundance, a comprehensive catalogue of sequence features controlling protein-to-mRNA (PTR) ratios and a quantification of their effects are still lacking. Here, we quantified PTR ratios for 11,575 proteins across 29 human tissues using matched transcriptomes and proteomes. We estimated by regression the contribution of known sequence determinants of protein synthesis and degradation in addition to 45 mRNA and 3 protein sequence motifs that we found by association testing. While PTR ratios span more than 2 orders of magnitude, our integrative model predicts PTR ratios at a median precision of 3.2-fold. A reporter assay provided functional support for two novel UTR motifs, and an immobilized mRNA affinity competition-binding assay identified motif-specific bound proteins for one motif. Moreover, our integrative model led to a new metric of codon optimality that captures the effects of codon frequency on protein synthesis and degradation. Altogether, this study shows that a large fraction of PTR ratio variation in human tissues can be predicted from sequence, and it identifies many new candidate post-transcriptional regulatory elements.


Assuntos
Proteínas/genética , Proteoma/genética , Distribuição Tecidual/genética , Transcriptoma/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Espectrometria de Massas/métodos , Proteômica/métodos , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos
10.
Am J Hum Genet ; 103(5): 817-825, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30401461

RESUMO

ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD+ to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals' fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.


Assuntos
Ataxia Cerebelar/genética , Deficiências do Desenvolvimento/genética , Glicosídeo Hidrolases/genética , Mutação/genética , Doenças Neurodegenerativas/genética , ADP-Ribosilação/genética , Adenosina Difosfato Ribose/genética , Adolescente , Alelos , Criança , Pré-Escolar , Exoma/genética , Feminino , Humanos , Lactente , Masculino , Malformações do Sistema Nervoso/genética , Processamento de Proteína Pós-Traducional/genética
11.
PLoS One ; 13(7): e0199938, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29995917

RESUMO

The accurate quantification of cellular and mitochondrial bioenergetic activity is of great interest in medicine and biology. Mitochondrial stress tests performed with Seahorse Bioscience XF Analyzers allow the estimation of different bioenergetic measures by monitoring the oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of the statistical best practices for determining aggregated OCR measurements and comparisons have been lacking. Therefore, to understand how OCR behaves across different biological samples, wells, and plates, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines. We show that the noise of OCR is multiplicative, that outlier data points can concern individual measurements or all measurements of a well, and that the inter-plate variation is greater than the intra-plate variation. Based on these insights, we developed a novel statistical method, OCR-Stats, that: i) robustly estimates OCR levels modeling multiplicative noise and automatically identifying outlier data points and outlier wells; and ii) performs statistical testing between samples, taking into account the different magnitudes of the between- and within-plate variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 45% and of maximal respiration by 29%. Moreover, using positive and negative controls, we show that our statistical test outperforms the existing methods, which suffer from an excess of either false positives (within-plate methods), or false negatives (between-plate methods). Altogether, this study provides statistical good practices to support experimentalists in designing, analyzing, testing, and reporting the results of mitochondrial stress tests using this high throughput platform.


Assuntos
Mitocôndrias/metabolismo , Análise Serial de Tecidos/métodos , Linhagem Celular , Respiração Celular , Metabolismo Energético , Fibroblastos/citologia , Modelos Estatísticos , Consumo de Oxigênio
12.
Am J Hum Genet ; 102(3): 460-467, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29429571

RESUMO

Respiratory chain complex I deficiency is the most frequently identified biochemical defect in childhood mitochondrial diseases. Clinical symptoms range from fatal infantile lactic acidosis to Leigh syndrome and other encephalomyopathies or cardiomyopathies. To date, disease-causing variants in genes coding for 27 complex I subunits, including 7 mitochondrial DNA genes, and in 11 genes encoding complex I assembly factors have been reported. Here, we describe rare biallelic variants in NDUFB8 encoding a complex I accessory subunit revealed by whole-exome sequencing in two individuals from two families. Both presented with a progressive course of disease with encephalo(cardio)myopathic features including muscular hypotonia, cardiac hypertrophy, respiratory failure, failure to thrive, and developmental delay. Blood lactate was elevated. Neuroimaging disclosed progressive changes in the basal ganglia and either brain stem or internal capsule. Biochemical analyses showed an isolated decrease in complex I enzymatic activity in muscle and fibroblasts. Complementation studies by expression of wild-type NDUFB8 in cells from affected individuals restored mitochondrial function, confirming NDUFB8 variants as the cause of complex I deficiency. Hereby we establish NDUFB8 as a relevant gene in childhood-onset mitochondrial disease.


Assuntos
Encefalopatias/genética , Complexo I de Transporte de Elétrons/deficiência , Doença de Leigh/genética , Doenças Mitocondriais/genética , Mutação/genética , Sequência de Aminoácidos , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Imagem por Ressonância Magnética , Masculino , Fosforilação Oxidativa , Linhagem , Porinas/metabolismo
13.
Neuropediatrics ; 49(1): 59-62, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28915517

RESUMO

Recently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2-related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism.


Assuntos
Epilepsia/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Mutação/genética , Pré-Escolar , Transportador 2 de Aminoácido Excitatório , Saúde da Família , Humanos , Masculino , Fenótipo
14.
Mitochondrion ; 36: 15-20, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-27721048

RESUMO

Leber's hereditary optic neuropathy (LHON) is an inherited mitochondrial disease that usually leads to acute or subacute bilateral central vision loss. In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene. Here we characterize clinically, genetically, and biochemically a LHON family with multiple patients harboring two of these primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic. The unusually low male-to-female ratio of affected family members is also seen among the other patients previously reported with two primary LHON mutations m.11778G>A and m.14484T>C. While the index patient had very late onset of symptoms at 75years and severe visual loss, her two daughters had both onset in childhood (6 and 9years), with moderate to mild visual loss. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family. Ours is the first LHON family harboring two primary LHON mutations where functional studies were performed in several affected family members. A more pronounced bioenergetic defect was found to correlate with an earlier age at onset. The patient with the earliest age at onset had a more significant complex I dysfunction than all controls, including the LHON patient with only the m.11778G>A mutation, suggesting a synergistic effect of the two primary LHON mutations in this patient.


Assuntos
DNA Mitocondrial/genética , Saúde da Família , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/patologia , Mutação Puntual , Adulto , Fatores Etários , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Masculinidade
15.
Am J Hum Genet ; 100(1): 151-159, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27989324

RESUMO

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children.


Assuntos
Encefalopatias/genética , Ciclo do Ácido Cítrico , Malato Desidrogenase/genética , Mutação , Idade de Início , Alelos , Sequência de Aminoácidos , Criança , Pré-Escolar , Ciclo do Ácido Cítrico/genética , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fumaratos/metabolismo , Teste de Complementação Genética , Humanos , Lactente , Recém-Nascido , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Masculino , Metabolômica , Modelos Moleculares
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