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1.
Front Mol Biosci ; 7: 590842, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33240932

RESUMO

Mitochondrial diseases (MD) are rare disorders caused by deficiency of the mitochondrial respiratory chain, which provides energy in each cell. They are characterized by a high clinical and genetic heterogeneity and in most patients, the responsible gene is unknown. Diagnosis is based on the identification of the causative gene that allows genetic counseling, prenatal diagnosis, understanding of pathological mechanisms, and personalized therapeutic approaches. Despite the emergence of Next Generation Sequencing (NGS), to date, more than one out of two patients has no diagnosis in the absence of identification of the responsible gene. Technologies currently used for detecting causal variants (genetic alterations) is far from complete, leading many variants of unknown significance (VUS) and mainly based on the use of whole exome sequencing thus neglecting the identification of non-coding variants. The complexity of human genome and its regulation at multiple levels has led biologists to develop several assays to interrogate the different aspects of biological processes. While one-dimension single omics investigation offers a peek of this complex system, the combination of different omics data allows the discovery of coherent signatures. The community of computational biologists and bioinformaticians, in order to integrate data from different omics, has developed several approaches and tools. However, it is difficult to understand which suits the best to predict diverse phenotypic outcome. First attempts to use multi-omics approaches showed an improvement of the diagnostic power. However, we are far from a complete understanding of MD and their diagnosis. After reviewing multi-omics algorithms developed in the latest years, we are proposing here a novel data-driven classification and we will discuss how multi-omics will change and improve the diagnosis of MD. Due to the growing use of multi-omics approaches in MD, we foresee that this work will contribute to set up good practices to perform multi-omics data integration to improve the prediction of phenotypic outcomes and the diagnostic power of MD.

2.
Hum Mutat ; 41(8): 1394-1406, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32419253

RESUMO

Whole mitochondrial DNA (mtDNA) sequencing is now systematically used in clinical laboratories to screen patients with a phenotype suggestive of mitochondrial disease. Next Generation Sequencing (NGS) has significantly increased the number of identified pathogenic mtDNA variants. Simultaneously, the number of variants of unknown significance (VUS) has increased even more, thus challenging their interpretation. Correct classification of the variants' pathogenicity is essential for optimal patient management, including treatment and genetic counseling. Here, we used single muscle fiber studies to characterize eight heteroplasmic mtDNA variants, among which were three novel variants. By applying the pathogenicity scoring system, we classified four variants as "definitely pathogenic" (m.590A>G, m.9166T>C, m.12293G>A, and m.15958A>T). Two variants remain "possibly pathogenic" (m.4327T>C and m.5672T>C) but should these be reported in a different family, they would be reclassified as "definitely pathogenic." We also illustrate the contribution of single-fiber studies to the diagnostic approach in patients harboring pathogenic variants with low level heteroplasmy.


Assuntos
DNA Mitocondrial/genética , Doenças Mitocondriais/genética , Adolescente , Adulto , Idoso , Feminino , Heteroplasmia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Padrões de Herança , Masculino , Pessoa de Meia-Idade , Conformação de Ácido Nucleico , Análise de Sequência de DNA
3.
J Hum Genet ; 64(7): 637-645, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948790

RESUMO

The genetic causes of Leigh syndrome are heterogeneous, with a poor genotype-phenotype correlation. To date, more than 50 nuclear genes cause nuclear gene-encoded Leigh syndrome. NDUFS6 encodes a 13 kiloDaltons subunit, which is part of the peripheral arm of complex I and is localized in the iron-sulfur fraction. Only a few patients were reported with proven NDUFS6 pathogenic variants and all presented with severe neonatal lactic acidemia and complex I deficiency, leading to death in the first days of life. Here, we present a patient harboring two NDUFS6 variants with a phenotype compatible with Leigh syndrome. Although most of previous reports suggested that NDUFS6 pathogenic variants invariably lead to early neonatal death, this report shows that the clinical spectrum could be larger. We found a severe decrease of NDUFS6 protein level in patient's fibroblasts associated with a complex I assembly defect in patient's muscle and fibroblasts. These data confirm the importance of NDUFS6 and the Zn-finger domain for a correct assembly of complex I.


Assuntos
Doença de Leigh/genética , NADH Desidrogenase/genética , Acidose Láctica/genética , Núcleo Celular/genética , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Fibroblastos/enzimologia , Estudos de Associação Genética , Humanos , Lactente , Doença de Leigh/diagnóstico por imagem , Doença de Leigh/enzimologia , Masculino , Mitocôndrias/genética , Músculos/enzimologia , NADH Desidrogenase/metabolismo , Domínios Proteicos/genética , Análise de Sequência de DNA
5.
BMC Med Genet ; 19(1): 57, 2018 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-29625556

RESUMO

BACKGROUND: Since the advent of next generation sequencing (NGS), several studies have tried to evaluate the relevance of targeted gene panel sequencing and whole exome sequencing for molecular diagnosis of mitochondrial diseases. The comparison between these different strategies is extremely difficult. A recent study analysed a cohort of patients affected by a mitochondrial disease using a NGS approach based on a targeted gene panel including 132 genes. This strategy led to identify the causative mutations in 15.2% of cases. The number of novel genes responsible for respiratory chain deficiency increases very rapidly. METHODS: In order to determine the impact of larger panels used as a first screening strategy on molecular diagnosis success, we analysed a cohort of 80 patients affected by a mitochondrial disease with a first mitochondrial DNA (mtDNA) NGS screening and secondarily a targeted mitochondrial panel of 281 nuclear genes. RESULTS: Pathogenic mtDNA abnormalities were identified in 4.1% (1/24) of children and 25% (14/56) of adult patients. The remaining 65 patients were analysed with our targeted mitochondrial panel and this approach enabled us to achieve an identification rate of 21.7% (5/23) in children versus 7.1% (3/42) in adults. CONCLUSIONS: Our results confirm that larger gene panels do not improve diagnostic yield of mitochondrial diseases due to (i) their very high genetic heterogeneity, (ii) the ongoing discovery of novel genes and (iii) mutations in genes apparently not related to mitochondrial function that lead to secondary respiratory chain deficiency.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Análise de Sequência de DNA/métodos , Idoso , Pré-Escolar , Feminino , Heterogeneidade Genética , Variação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade
7.
Hum Mol Genet ; 26(9): 1599-1611, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28335035

RESUMO

Wolfram syndrome (WS) is a progressive neurodegenerative disease characterized by early-onset optic atrophy and diabetes mellitus, which can be associated with more extensive central nervous system and endocrine complications. The majority of patients harbour pathogenic WFS1 mutations, but recessive mutations in a second gene, CISD2, have been described in a small number of families with Wolfram syndrome type 2 (WFS2). The defining diagnostic criteria for WFS2 also consist of optic atrophy and diabetes mellitus, but unlike WFS1, this phenotypic subgroup has been associated with peptic ulcer disease and an increased bleeding tendency. Here, we report on a novel homozygous CISD2 mutation (c.215A > G; p.Asn72Ser) in a Moroccan patient with an overlapping phenotype suggesting that Wolfram syndrome type 1 and type 2 form a continuous clinical spectrum with genetic heterogeneity. The present study provides strong evidence that this particular CISD2 mutation disturbs cellular Ca2+ homeostasis with enhanced Ca2+ flux from the ER to mitochondria and cytosolic Ca2+ abnormalities in patient-derived fibroblasts. This Ca2+ dysregulation was associated with increased ER-mitochondria contact, a swollen ER lumen and a hyperfused mitochondrial network in the absence of overt ER stress. Although there was no marked alteration in mitochondrial bioenergetics under basal conditions, culture of patient-derived fibroblasts in glucose-free galactose medium revealed a respiratory chain defect in complexes I and II, and a trend towards decreased ATP levels. Our results provide important novel insight into the potential disease mechanisms underlying the neurodegenerative consequences of CISD2 mutations and the subsequent development of multisystemic disease.


Assuntos
Senilidade Prematura/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Atrofia Óptica/genética , Cálcio/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Linhagem , Síndrome de Wolfram/genética
8.
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
9.
Neuromuscul Disord ; 26(12): 885-889, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27816331

RESUMO

An 11-year-old boy with psychomotor delay, exercise intolerance, ptosis and growth delay had a muscle biopsy showing typical mitochondrial alterations (60% of ragged-red fibers and 90% of cytochrome-c oxidase-deficient fibers). Next-generation sequencing revealed a novel heteroplasmic mutation (m.15958A>T) in the MTTP gene that encodes tRNAPro. The mutation was not present in the accessible non-muscle tissues of the patient's asymptomatic mother. Mutations in the rarely affected MTTP gene are responsible for different clinical presentations. We report the third early-onset case associated with a mutation in this gene. The severity of myopathy is likely related to the high mutation rate (96%) found in the patient's muscle. The clinical heterogeneity associated with MTTP mutations illustrates the value of the next-generation sequencing in routine diagnosis of mitochondrial diseases.


Assuntos
Genes Mitocondriais , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologia , Mutação , RNA de Transferência de Prolina/genética , Criança , DNA Mitocondrial , Humanos , Masculino , Miopatias Mitocondriais/fisiopatologia , Músculo Esquelético/patologia , Fenótipo
10.
Biol Res ; 49: 4, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26742794

RESUMO

BACKGROUND: Coenzyme Q10 (CoQ10 or ubiquinone) deficiency can be due either to mutations in genes involved in CoQ10 biosynthesis pathway, or to mutations in genes unrelated to CoQ10 biosynthesis. CoQ10 defect is the only oxidative phosphorylation disorder that can be clinically improved after oral CoQ10 supplementation. Thus, early diagnosis, first evoked by mitochondrial respiratory chain (MRC) spectrophotometric analysis, then confirmed by direct measurement of CoQ10 levels, is of critical importance to prevent irreversible damage in organs such as the kidney and the central nervous system. It is widely reported that CoQ10 deficient patients present decreased quinone-dependent activities (segments I + III or G3P + III and II + III) while MRC activities of complexes I, II, III, IV and V are normal. We previously suggested that CoQ10 defect may be associated with a deficiency of CoQ10-independent MRC complexes. The aim of this study was to verify this hypothesis in order to improve the diagnosis of this disease. RESULTS: To determine whether CoQ10 defect could be associated with MRC deficiency, we quantified CoQ10 by LC-MSMS in a cohort of 18 patients presenting CoQ10-dependent deficiency associated with MRC defect. We found decreased levels of CoQ10 in eight patients out of 18 (45 %), thus confirming CoQ10 disease. CONCLUSIONS: Our study shows that CoQ10 defect can be associated with MRC deficiency. This could be of major importance in clinical practice for the diagnosis of a disease that can be improved by CoQ10 supplementation.


Assuntos
Ataxia/genética , Transporte de Elétrons/genética , Doenças Mitocondriais/genética , Debilidade Muscular/genética , Mutação , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Adolescente , Adulto , Idoso , Ataxia/diagnóstico , Ataxia/metabolismo , Biópsia , Células Cultivadas , Criança , Pré-Escolar , Cromatografia Líquida , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismo , Debilidade Muscular/diagnóstico , Debilidade Muscular/metabolismo , Músculos/patologia , Espectrofotometria/métodos , Espectrometria de Massas em Tandem/métodos , Ubiquinona/biossíntese , Ubiquinona/genética , Ubiquinona/metabolismo , Adulto Jovem
12.
Biol. Res ; 49: 1-9, 2016. tab
Artigo em Inglês | LILACS | ID: lil-774431

RESUMO

BACKGROUND: Coenzyme Q10 (CoQ10 or ubiquinone) deficiency can be due either to mutations in genes involved in CoQ10 biosynthesis pathway, or to mutations in genes unrelated to CoQ10 biosynthesis. CoQ10 defect is the only oxidative phosphorylation disorder that can be clinically improved after oral CoQ10 supplementation. Thus, early diagnosis, first evoked by mitochondrial respiratory chain (MRC) spectrophotometric analysis, then confirmed by direct measurement of CoQ10 levels, is of critical importance to prevent irreversible damage in organs such as the kidney and the central nervous system. It is widely reported that CoQ10 deficient patients present decreased quinone-dependent activities (segments I + III or G3P + III and II + III) while MRC activities of complexes I, II, III, IV and V are normal. We previously suggested that CoQ10 defect may be associated with a deficiency of CoQ10-independent MRC complexes. The aim of this study was to verify this hypothesis in order to improve the diagnosis of this disease. RESULTS: To determine whether CoQ10 defect could be associated with MRC deficiency, we quantified CoQ10 by LC-MSMS in a cohort of 18 patients presenting CoQ10-dependent deficiency associated with MRC defect. We found decreased levels of CoQ10 in eight patients out of 18 (45 %), thus confirming CoQ10 disease. CONCLUSIONS: Our study shows that CoQ10 defect can be associated with MRC deficiency. This could be of major importance in clinical practice for the diagnosis of a disease that can be improved by CoQ10 supplementation.


Assuntos
Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Ataxia/genética , Transporte de Elétrons/genética , Mutação , Doenças Mitocondriais/genética , Debilidade Muscular/genética , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Ataxia/diagnóstico , Ataxia/metabolismo , Biópsia , Células Cultivadas , Cromatografia Líquida , Fibroblastos/enzimologia , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismo , Debilidade Muscular/diagnóstico , Debilidade Muscular/metabolismo , Músculos/patologia , Espectrofotometria/métodos , Espectrometria de Massas em Tandem/métodos , Ubiquinona/biossíntese , Ubiquinona/genética , Ubiquinona/metabolismo
13.
Endokrynol Pol ; 66(3): 198-206, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26136127

RESUMO

INTRODUCTION: Insulin gene VNTR was associated with polycystic ovary syndrome (PCOS) in some studies but not in others. This couldb be due to the heterogeneity of the definition of PCOS and/or the use of inappropriate gene mapping strategies. MATERIAL AND METHODS: In this investigation, the association of VNTR with PCOS was explored in a population of women from Central Europe (377 cases and 105 controls) in whom PCOS was diagnosed according to Rotterdam criteria. Seven SNPs: rs3842756 (G/A), rs3842755 (G/T), rs3842754 (C/T), rs3842753 (A/C), rs3842752 (C/T), rs3842748 (G/C), and rs689 (T/A) were genotyped in a portion of the population (160 cases and 95 controls) by sequencing or by SSO-PCR. Analysis of linkage disequilibrium (LD) pattern allowed selecting three tagSNPs (rs3842754, rs3842748, and rs689), which were genotyped in the rest of the population by KASPar. RESULTS: Six haplotypes were reconstructed, among which three (h1, h2 and h6) were more frequent. Statistical analysis allowed observation of the association of the SNP rs3842748, through its GC genotype, with obesity in PCOS (P = 0.049; OR CI95% 1,59 [1.00-2.51]) and in classical PCOS (YPCOS) (P = 0.010), as well as the correlation of the SNP rs689 and the pair of haplotypes h1/h1 with higher levels of testosteronaemia in the PCOS group, although this was at the limit of significance (P = 0.054) CONCLUSION: These results are in accordance with some studies in literature and highlight the role of insulin gene VNTR in complex metabolic disorders.


Assuntos
Insulina/genética , Repetições Minissatélites , Síndrome do Ovário Policístico/metabolismo , Polimorfismo de Nucleotídeo Único , Adulto , Feminino , Haplótipos , Humanos , Obesidade/metabolismo , Síndrome do Ovário Policístico/genética , Romênia , Análise de Sequência de DNA , População Branca/genética , Adulto Jovem
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