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1.
Brain ; 146(5): 1831-1843, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-36227727

RESUMO

Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing, an amplification-free method for simultaneous analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR-Cas9, Oxford Nanopore long-read sequencing and a bioinformatics pipeline using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci. Clinical nanopore Cas9-targeted sequencing allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using clinical nanopore Cas9-targeted sequencing we analysed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions on the basis of clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.


Assuntos
Ataxia Cerebelar , Degenerações Espinocerebelares , Adulto , Humanos , Ataxia/genética , Ataxia Cerebelar/genética , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Proteína do X Frágil da Deficiência Intelectual
2.
Neuropediatrics ; 54(3): 206-210, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36693418

RESUMO

Synapsins are neuron-specific phosphoproteins that modulate neurotransmitter release, synaptic plasticity, and molecular processes shaping higher brain functions. Pathogenic synapsin-1 (SYN1) variants are associated with epilepsy, intellectual disabilities, and behavioral problems. We detected a novel SYN1 variant [c.477_479delTGG (p.Gly160del)] in brothers with focal epilepsy with secondary generalization. The deleted amino acid was found to be highly conserved among mammalian species. In electroencephalography, the older brother showed a bioelectrical status epilepticus and was also diagnosed with attention deficit hyperactivity disorder. Behavioral abnormalities were seen before or after the seizures. Both patients responded quickly to treatment with valproate. Our case reports are consistent with the clinical heterogeneity of the pathogenic SYN1 variants described in the literature.


Assuntos
Epilepsias Parciais , Epilepsia , Humanos , Masculino , Epilepsias Parciais/tratamento farmacológico , Epilepsias Parciais/genética , Mamíferos/metabolismo , Irmãos , Sinapsinas/química , Sinapsinas/metabolismo , Ácido Valproico/uso terapêutico
3.
Hum Mol Genet ; 27(7): 1186-1195, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29361167

RESUMO

Mitochondrial dynamics play an important role in cellular homeostasis and a variety of human diseases are linked to its dysregulated function. Here, we describe a 15-year-old boy with a novel disease caused by altered mitochondrial dynamics. The patient was the second child of consanguineous Jewish parents. He developed progressive muscle weakness and exercise intolerance at 6 years of age. His muscle biopsy revealed mitochondrial myopathy with numerous ragged red and cytochrome c oxidase (COX) negative fibers and combined respiratory chain complex I and IV deficiency. MtDNA copy number was elevated and no deletions of the mtDNA were detected in muscle DNA. Whole exome sequencing identified a homozygous nonsense mutation (p.Q92*) in the MIEF2 gene encoding the mitochondrial dynamics protein of 49 kDa (MID49). Immunoblotting revealed increased levels of proteins promoting mitochondrial fusion (MFN2, OPA1) and decreased levels of the fission protein DRP1. Fibroblasts of the patient showed elongated mitochondria, and significantly higher frequency of fusion events, mtDNA abundance and aberrant mitochondrial cristae ultrastructure, compared with controls. Thus, our data suggest that mutations in MIEF2 result in imbalanced mitochondrial dynamics and a combined respiratory chain enzyme defect in skeletal muscle, leading to mitochondrial myopathy.


Assuntos
Fibroblastos/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais , Doenças Musculares , Mutação de Sentido Incorreto , Fatores de Alongamento de Peptídeos , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Cultura Primária de Células
4.
Br J Cancer ; 123(4): 619-623, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32451468

RESUMO

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a cancer predisposition syndrome caused by defects on chromosome 11p15.5. The quantitative cancer risks in BWS patients depend on the underlying (epi)genotype but have not yet been assessed in a population-based manner. METHODS: We identified a group of 321 individuals with a molecularly confirmed diagnosis of BWS and analysed the cancer incidence up to age 15 years and cancer spectrum by matching their data with the German Childhood Cancer Registry. RESULTS: We observed 13 cases of cancer in the entire BWS cohort vs 0.4 expected. This corresponds to a 33-fold increased risk (standardised incidence ratio (SIR) = 32.6; 95% confidence interval = 17.3-55.7). The specific cancers included hepatoblastoma (n = 6); nephroblastoma (n = 4); astrocytoma (n = 1); neuroblastoma (n = 1) and adrenocortical carcinoma (n = 1). The cancer SIR was highest in patients with a paternal uniparental disomy of 11p15.5 (UPDpat). A high cancer risk remained when cases of cancer diagnosed prior to the BWS diagnosis were excluded. CONCLUSIONS: This study confirms an increased cancer risk in children with BWS. Our findings suggest that the highest cancer risk is associated with UPDpat. We were unable to confirm an excessive cancer risk in patients with IC1 gain of methylation (IC1-GOM) and this finding requires further investigation.


Assuntos
Síndrome de Beckwith-Wiedemann/genética , Cromossomos Humanos Par 11/genética , Neoplasias/epidemiologia , Dissomia Uniparental/genética , Adolescente , Síndrome de Beckwith-Wiedemann/epidemiologia , Criança , Pré-Escolar , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Lactente , Masculino , Neoplasias/classificação , Sistema de Registros , Estudos Retrospectivos
5.
J Med Genet ; 55(7): 497-504, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29574422

RESUMO

BACKGROUND: Genomic imprinting results from the resistance of germline epigenetic marks to reprogramming in the early embryo for a small number of mammalian genes. Genetic, epigenetic or environmental insults that prevent imprints from evading reprogramming may result in imprinting disorders, which impact growth, development, behaviour and metabolism. We aimed to identify genetic defects causing imprinting disorders by whole-exome sequencing in families with one or more members affected by multilocus imprinting disturbance. METHODS: Whole-exome sequencing was performed in 38 pedigrees where probands had multilocus imprinting disturbance, in five of whom maternal variants in NLRP5 have previously been found. RESULTS: We now report 15 further pedigrees in which offspring had disturbance of imprinting, while their mothers had rare, predicted-deleterious variants in maternal effect genes, including NLRP2, NLRP7 and PADI6. As well as clinical features of well-recognised imprinting disorders, some offspring had additional features including developmental delay, behavioural problems and discordant monozygotic twinning, while some mothers had reproductive problems including pregnancy loss. CONCLUSION: The identification of 20 putative maternal effect variants in 38 families affected by multilocus imprinting disorders adds to the evidence that maternal genetic factors affect oocyte fitness and thus offspring development. Testing for maternal-effect genetic variants should be considered in families affected by atypical imprinting disorders.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Síndrome de Beckwith-Wiedemann/genética , Desiminases de Arginina em Proteínas/genética , Síndrome de Silver-Russell/genética , Proteínas Reguladoras de Apoptose , Síndrome de Beckwith-Wiedemann/patologia , Cromossomos Humanos Par 11/genética , Metilação de DNA/genética , Feminino , Impressão Genômica/genética , Mutação em Linhagem Germinativa/genética , Humanos , Recém-Nascido , Doenças do Recém-Nascido/genética , Doenças do Recém-Nascido/fisiopatologia , Herança Materna , Linhagem , Gravidez , Proteína-Arginina Desiminase do Tipo 6 , Síndrome de Silver-Russell/fisiopatologia
6.
Genet Med ; 20(4): 444-451, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29261183

RESUMO

PurposeDiagnosing primary mitochondrial diseases (MDs) is challenging in clinical practice. The mitochondrial disease criteria (MDC) have been developed to quantify the clinical picture and evaluate the probability of an underlying MD and the need for a muscle biopsy. In this new genetic era with next-generation sequencing in routine practice, we aim to validate the diagnostic value of MDC.MethodsWe retrospectively studied MDC in a multicenter cohort of genetically confirmed primary MD patients.ResultsWe studied 136 patients (61 male, 91 nuclear DNA (nDNA) mutations). Forty-five patients (33%) had probable MD and 69 (51%) had definite MD according to the MDC. A muscle biopsy was performed in 63 patients (47%). Patients with nDNA mutations versus mitochondrial DNA mutations were younger (6.4 ± 9.7 versus 19.5 ± 17.3 y) and had higher MDC (7.07 ± 1.12/8 versus 5.69 ± 1.94/8). At a cutoff of 6.5/8, the sensitivity to diagnose patients with nDNA mutations is 72.5% with a positive predictive value of 69.5%. In the nDNA mutation group, whole-exome sequencing could diagnose patients with lower scores (MDC (6.84 ± 1.51/8) compared to Sanger sequencing MDC (7.44 ± 1.13/8, P = 0.025)). Moreover 7/8 patients diagnosed with possible MD by MDC were diagnosed by whole-exome sequencing.ConclusionMDC remain very useful in the clinical diagnosis of MD, in interpreting whole-exome results and deciding on the need for performing muscle biopsy.


Assuntos
Genoma Mitocondrial , Genômica , Mitocôndrias/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Genes Mitocondriais , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mutação , Estudos Retrospectivos , Fluxo de Trabalho , Adulto Jovem
7.
Muscle Nerve ; 54(2): 328-33, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27015314

RESUMO

INTRODUCTION: Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) is caused by m.8993T>G/C mutations in the mitochondrial adenosine triphosphate synthase subunit 6 gene (MT-ATP6). Traditionally, heteroplasmy levels between 70% and 90% lead to NARP, and >90% result in Leigh syndrome. METHODS: In this study we report a 30-year-old man with NARP and m.8993T>G in MT-ATP6. RESULTS: Although the patient carried the mutation in homoplasmic state in blood with similarly high levels in urine (94%) and buccal swab (92%), he presented with NARP and not the expected, more severe Leigh phenotype. The mutation could not be detected in any of the 3 analyzed tissues of the mother, indicating a large genetic shift between mother and offspring. Nerve biopsy revealed peculiar endoneurial Schwann cell nuclear accumulations, clusters of concentrically arranged Schwann cells devoid of myelinated axons, and degenerated mitochondria. CONCLUSIONS: We emphasize the phenotypic variability of the m.8993T>G MT-ATP6 mutation and the need for caution in predictive counseling in such patients. Muscle Nerve 54: 328-333, 2016.


Assuntos
Ataxia/genética , Miopatias Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Debilidade Muscular/genética , Retinose Pigmentar/genética , Adulto , Ataxia/complicações , Humanos , Imageamento por Ressonância Magnética , Masculino , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Miopatias Mitocondriais/complicações , Miopatias Mitocondriais/diagnóstico por imagem , Debilidade Muscular/complicações , Mutação/genética , Retinose Pigmentar/complicações , Retinose Pigmentar/diagnóstico por imagem , Nervo Sural/patologia , Nervo Sural/ultraestrutura
8.
J Inherit Metab Dis ; 39(3): 427-436, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27053192

RESUMO

Liver failure is a heterogeneous condition which may be fatal and the primary cause is frequently unknown. We investigated mitochondrial oxidative phosphorylation in patients undergoing liver transplantation. We studied 45 patients who had liver transplantation due to a variety of clinical presentations. Blue native polyacrylamide gel electrophoresis with immunodetection of respiratory chain complexes I-V, biochemical activity of respiratory chain complexes II and IV and quantification of mitochondrial DNA (mtDNA) copy number were investigated in liver tissue collected from the explanted liver during transplantation. Abnormal mitochondrial function was frequently present in this cohort: ten of 40 patients (25 %) had a defect of one or more respiratory chain enzyme complexes on blue native gels, 20 patients (44 %) had low activity of complex II and/or IV and ten (22 %) had a reduced mtDNA copy number. Combined respiratory chain deficiency and reduced numbers of mitochondria were detected in all three patients with acute liver failure. Low complex IV activity in biliary atresia and complex II defects in cirrhosis were common findings. All six patients diagnosed with liver tumours showed variable alterations in mitochondrial function, probably due to the heterogeneity of the presenting tumour. In conclusion, mitochondrial dysfunction is common in severe liver failure in non-mitochondrial conditions. Therefore, in contrast to the common practice detection of respiratory chain abnormalities in liver should not restrict the inclusion of patients for liver transplantation. Furthermore, improving mitochondrial function may be targeted as part of a complex therapy approach in different forms of liver diseases.


Assuntos
Falência Hepática/patologia , Fígado/patologia , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Adolescente , Adulto , Atresia Biliar/metabolismo , Atresia Biliar/patologia , Criança , Pré-Escolar , DNA Mitocondrial/metabolismo , Transporte de Elétrons/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Lactente , Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Falência Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transplante de Fígado/métodos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Fosforilação Oxidativa , Adulto Jovem
9.
Mol Cell Probes ; 29(5): 319-22, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26327357

RESUMO

Mutations in the DARS2 gene are known to cause leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), a rare autosomal recessive neurological disorder. It was originally described as juvenile-onset slowly progressive ataxia and spasticity, but recent reports suggest a broader clinical spectrum. Most patients were found to carry compound heterozygous DARS2 mutations, and only very few patients with homozygous mutations have been described so far. We present here an 8-month-old boy carrying a homozygous missense mutation in DARS2 who clinically showed severe neurological deterioration after a respiratory tract infection, followed by an almost complete remission of symptoms. This report further extends the knowledge about the clinical and molecular genetic spectrum of LBSL.


Assuntos
Aspartato-tRNA Ligase/genética , Leucoencefalopatias/genética , Mutação de Sentido Incorreto , Idade de Início , Predisposição Genética para Doença , Homozigoto , Humanos , Lactente , Leucoencefalopatias/diagnóstico , Masculino , Linhagem , Análise de Sequência de DNA
10.
JAMA ; 312(1): 68-77, 2014 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-25058219

RESUMO

IMPORTANCE: Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE: To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN, SETTING, AND PARTICIPANTS: We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. RESULTS: Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). CONCLUSIONS AND RELEVANCE: Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.


Assuntos
Análise Mutacional de DNA , Exoma , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Análise de Sequência de DNA/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Biologia Computacional , Feminino , Haplótipos , Humanos , Lactente , Masculino
11.
Med Genet ; 35(2): 91-104, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38840862

RESUMO

The rapid and dynamic implementation of Next-Generation Sequencing (NGS)-based assays has revolutionized genetic testing, and in the near future, nearly all molecular alterations of the human genome will be diagnosable via massive parallel sequencing. While this progress will further corroborate the central role of human genetics in the multidisciplinary management of patients with genetic disorders, it must be accompanied by quality assurance measures in order to allow the safe and optimal use of knowledge ascertained from genome diagnostics. To achieve this, several valuable tools and guidelines have been developed to support the quality of genome diagnostics. In this paper, authors with experience in diverse aspects of genomic analysis summarize the current status of quality assurance in genome diagnostics, with the aim of facilitating further standardization and quality improvement in one of the core competencies of the field.

12.
J Neuromuscul Dis ; 10(5): 835-846, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37424474

RESUMO

BACKGROUND: The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1 deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods. OBJECTIVE: The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy. METHODS: A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV). RESULTS: 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients). CONCLUSIONS: Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.


Assuntos
Atrofia Muscular Espinal , Doenças Neuromusculares , Humanos , Homozigoto , Deleção de Sequência , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Doenças Neuromusculares/genética , Sequenciamento de Nucleotídeos em Larga Escala
13.
BMC Med Genomics ; 14(1): 94, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33789662

RESUMO

BACKGROUND: Molecular autopsy represents an efficient tool to save the diagnosis in up to one-third of sudden unexplained death (SUD). A defined gene panel is usually used for the examination. Alternatively, it is possible to carry out a comprehensive genetic assessment (whole exome sequencing, WES), which also identifies rare, previously unknown variants. The disadvantage is that a dramatic number of variants must be assessed to identify the causal variant. To improve the evaluation of WES, the human phenotype ontology (HPO) annotation is used internationally for deep phenotyping in the field of rare disease. However, a HPO-based evaluation of WES in SUD has not been described before. METHODS: We performed WES in tissue samples from 16 people after SUD. Instead of a fixed gene panel, we defined a set of HPO terms and thus created a flexible "virtual gene panel", with the advantage, that recently identified genes are automatically associated by HPO terms in the HPO database. RESULTS: We obtained a mean value of 68,947 variants per sample. Stringent filtering ended up in a mean value of 276 variants per sample. Using the HPO-driven virtual gene panel we developed an algorithm that prioritized 1.4% of the variants. Variant interpretation resulted in eleven potentially causative variants in 16 individuals. CONCLUSION: Our data introduce an effective diagnostic procedure in molecular autopsy of SUD with a non-specific clinical phenotype.


Assuntos
Sequenciamento do Exoma , Autopsia , Biologia Computacional , Exoma , Humanos , Masculino , Fenótipo
14.
Neurol Genet ; 6(1): e393, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32042921

RESUMO

OBJECTIVE: To delineate the phenotypic and genotypic spectrum in carriers of mitochondrial MT-ATP6 mutations in a large international cohort. METHODS: We analyzed in detail the clinical, genetical, and neuroimaging data from 132 mutation carriers from national registries and local databases from Europe, USA, Japan, and China. RESULTS: We identified 113 clinically affected and 19 asymptomatic individuals with a known pathogenic MT-ATP6 mutation. The most frequent mutations were m.8993 T > G (53/132, 40%), m.8993 T > C (30/132, 23%), m.9176 T > C (30/132, 23%), and m.9185 T > C (12/132, 9%). The degree of heteroplasmy was high both in affected (mean 95%, range 20%-100%) and unaffected individuals (mean 73%, range 20%-100%). Age at onset ranged from prenatal to the age of 75 years, but almost half of the patients (49/103, 48%) became symptomatic before their first birthday. In 28 deceased patients, the median age of death was 14 months. The most frequent symptoms were ataxia (81%), cognitive dysfunction (49%), neuropathy (48%), seizures (37%), and retinopathy (14%). A diagnosis of Leigh syndrome was made in 55% of patients, whereas the classic syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP) was rare (8%). CONCLUSIONS: In this currently largest series of patients with mitochondrial MT-ATP6 mutations, the phenotypic spectrum ranged from asymptomatic to early onset multisystemic neurodegeneration. The degree of mutation heteroplasmy did not reliably predict disease severity. Leigh syndrome was found in more than half of the patients, whereas classic NARP syndrome was rare. Oligosymptomatic presentations were rather frequent in adult-onset patients, indicating the need to include MT-ATP6 mutations in the differential diagnosis of both ataxias and neuropathies.

15.
Mol Genet Genomic Med ; 6(6): 1188-1198, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30406974

RESUMO

BACKGROUND: The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a robust high-throughput platform for nuclear and mitochondrial DNA (mtDNA) analyses. METHOD: We developed a custom Agilent SureSelect Mitochondrial and Nuclear Disease Panel (Mito-aND-Panel) capture kit that allows parallel enrichment for subsequent NGS-based sequence analysis of nuclear mitochondrial disease-related genes and the complete mtDNA genome. Sequencing of enriched mtDNA simultaneously with nuclear genes was compared with the separated sequencing of the mitochondrial genome and whole exome sequencing (WES). RESULTS: The Mito-aND-Panel permits accurate detection of low-level mtDNA heteroplasmy due to a very high sequencing depth compared to standard diagnostic procedures using Sanger sequencing/SNaPshot and WES which is crucial to identify maternally inherited mitochondrial disorders. CONCLUSION: We established a NGS-based method with combined sequencing of the complete mtDNA and nuclear genes which enables a more sensitive heteroplasmy detection of mtDNA mutations compared to traditional methods. Because the method promotes the analysis of mtDNA variants in large cohorts, it is cost-effective and simple to setup, we anticipate this is a highly relevant method for sequence-based genetic diagnosis in clinical diagnostic applications.


Assuntos
Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças Mitocondriais/genética , Análise de Sequência de DNA/métodos , Custos e Análise de Custo , DNA Mitocondrial/química , DNA Mitocondrial/genética , Testes Genéticos/economia , Testes Genéticos/normas , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Doenças Mitocondriais/diagnóstico , Sensibilidade e Especificidade , Análise de Sequência de DNA/economia , Análise de Sequência de DNA/normas
16.
Sci Rep ; 8(1): 11682, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-30076399

RESUMO

Deletions in mitochondrial DNA (mtDNA) are an important cause of human disease and their accumulation has been implicated in the ageing process. As mtDNA is a high copy number genome, the coexistence of deleted and wild-type mtDNA molecules within a single cell defines heteroplasmy. When deleted mtDNA molecules, driven by intracellular clonal expansion, reach a sufficiently high level, a biochemical defect emerges, contributing to the appearance and progression of clinical pathology. Consequently, it is relevant to determine the heteroplasmy levels within individual cells to understand the mechanism of clonal expansion. Heteroplasmy is reflected in a mosaic distribution of cytochrome c oxidase (COX)-deficient muscle fibers. We applied droplet digital PCR (ddPCR) to single muscle fibers collected by laser-capture microdissection (LCM) from muscle biopsies of patients with different paradigms of mitochondrial disease, characterized by the accumulation of single or multiple mtDNA deletions. By combining these two sensitive approaches, ddPCR and LCM, we document different models of clonal expansion in patients with single and multiple mtDNA deletions, implicating different mechanisms and time points for the development of COX deficiency in these molecularly distinct mitochondrial cytopathies.


Assuntos
DNA Mitocondrial/genética , Células Musculares/metabolismo , Reação em Cadeia da Polimerase/métodos , Deleção de Sequência/genética , Adolescente , Adulto , Idoso , Biópsia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , Dosagem de Genes , Genes Recessivos , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Fosforilação Oxidativa , Reprodutibilidade dos Testes , Succinato Desidrogenase/metabolismo , Adulto Jovem
18.
Neurology ; 88(13): 1226-1234, 2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28251916

RESUMO

OBJECTIVE: To study the prevalence, molecular cause, and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the North of England. METHODS: Detailed neurologic and electrophysiologic assessments and next-generation panel testing or whole exome sequencing were performed in 105 patients with clinical symptoms of distal hereditary motor neuropathy (dHMN, 64 patients), axonal motor neuropathy (motor Charcot-Marie-Tooth disease [CMT2], 16 patients), or complex neurologic disease predominantly affecting the motor nerves (hereditary motor neuropathy plus, 25 patients). RESULTS: The prevalence of dHMN is 2.14 affected individuals per 100,000 inhabitants (95% confidence interval 1.62-2.66) in the North of England. Causative mutations were identified in 26 out of 73 index patients (35.6%). The diagnostic rate in the dHMN subgroup was 32.5%, which is higher than previously reported (20%). We detected a significant defect of neuromuscular transmission in 7 cases and identified potentially causative mutations in 4 patients with multifocal demyelinating motor neuropathy. CONCLUSIONS: Many of the genes were shared between dHMN and motor CMT2, indicating identical disease mechanisms; therefore, we suggest changing the classification and including dHMN also as a subcategory of Charcot-Marie-Tooth disease. Abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies.


Assuntos
Doença de Charcot-Marie-Tooth/epidemiologia , Heterogeneidade Genética , Neuropatia Hereditária Motora e Sensorial/epidemiologia , Neuropatia Hereditária Motora e Sensorial/genética , Mutação/genética , Adolescente , Adulto , Idoso , Análise de Variância , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Estudos de Coortes , Conexinas/genética , Análise Mutacional de DNA , Eletromiografia , Inglaterra/epidemiologia , Saúde da Família , Feminino , GTP Fosfo-Hidrolases/genética , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Proteínas da Mielina/genética , Condução Nervosa/genética , Adulto Jovem , Proteína beta-1 de Junções Comunicantes
19.
J Neurol ; 262(7): 1673-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25957632

RESUMO

Whole exome sequencing (WES) is a recently developed technique in genetics research that attempts to identify causative mutations in complex, undiagnosed genetic conditions. Causative mutations are usually identified after filtering the hundreds of variants on WES from an individual's DNA selected by the phenotype. We investigated a patient with a slowly progressive chronic axonal distal motor neuropathy and extrapyramidal syndrome using WES, in whom common genetic mutations had been excluded. Variant filtering identified potentially deleterious mutations in three known disease genes: DCTN1, KIF5A and NEFH, which have been all associated with similar clinical presentations of amyotrophic lateral sclerosis, Parkinsonism and/or hereditary spastic paraplegia. Predicting the functional effect of the mutations were analysed in parallel with detailed clinical investigations. This case highlights the difficulties and pitfalls of applying WES in patients with complex neurological diseases and serves as an instructive tale.


Assuntos
Doenças dos Gânglios da Base/complicações , Doenças dos Gânglios da Base/genética , Transtornos dos Movimentos/complicações , Transtornos dos Movimentos/genética , Análise de Sequência de DNA , Idoso , Dor nas Costas/complicações , Progressão da Doença , Complexo Dinactina , Humanos , Cinesinas/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas de Neurofilamentos/genética , Ciática/complicações , Ciática/genética
20.
Biomed Res Int ; 2015: 462592, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26550569

RESUMO

TMEM70 is involved in the biogenesis of mitochondrial ATP synthase and mutations in the TMEM70 gene impair oxidative phosphorylation. Herein, we report on pathology and treatment of ATP synthase deficiency in four siblings. A consanguineous family of Roma (Gipsy) ethnic origin gave birth to 6 children of which 4 were affected presenting with dysmorphic features, failure to thrive, cardiomyopathy, metabolic crises, and 3-methylglutaconic aciduria as clinical symptoms. Genetic testing revealed a homozygous mutation (c.317-2A>G) in the TMEM70 gene. While light microscopy was unremarkable, ultrastructural investigation of muscle tissue revealed accumulation of swollen degenerated mitochondria with lipid crystalloid inclusions, cristae aggregation, and exocytosis of mitochondrial material. Biochemical analysis of mitochondrial complexes showed an almost complete ATP synthase deficiency. Despite harbouring the same mutation, the clinical outcome in the four siblings was different. Two children died within 60 h after birth; the other two had recurrent life-threatening metabolic crises but were successfully managed with supplementation of anaplerotic amino acids, lipids, and symptomatic treatment during metabolic crisis. In summary, TMEM70 mutations can cause distinct ultrastructural mitochondrial degeneration and almost complete deficiency of ATP synthase but are still amenable to treatment.


Assuntos
Proteínas de Membrana/genética , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/deficiência , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Criança , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Doenças Mitocondriais/terapia , ATPases Mitocondriais Próton-Translocadoras/genética , Resultado do Tratamento
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