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1.
J Biol Chem ; 295(14): 4383-4397, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32094224

RESUMO

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the "mitochondrial gas pedal." Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.


Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Animais , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Coração/fisiologia , Malatos/química , Malatos/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Fosforilação Oxidativa , Ratos , Especificidade por Substrato , Sinaptossomos/metabolismo
2.
Am J Hum Genet ; 99(1): 188-94, 2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-27292112

RESUMO

In the PI(3,5)P2 biosynthetic complex, the lipid kinase PIKFYVE and the phosphatase FIG4 are bound to the dimeric scaffold protein VAC14, which is composed of multiple heat-repeat domains. Mutations of FIG4 result in the inherited disorders Charcot-Marie-Tooth disease type 4J, Yunis-Varón syndrome, and polymicrogyria with seizures. We here describe inherited variants of VAC14 in two unrelated children with sudden onset of a progressive neurological disorder and regression of developmental milestones. Both children developed impaired movement with dystonia, became nonambulatory and nonverbal, and exhibited striatal abnormalities on MRI. A diagnosis of Leigh syndrome was rejected due to normal lactate profiles. Exome sequencing identified biallelic variants of VAC14 that were inherited from unaffected heterozygous parents in both families. Proband 1 inherited a splice-site variant that results in skipping of exon 13, p.Ile459Profs(∗)4 (not reported in public databases), and the missense variant p.Trp424Leu (reported in the ExAC database in a single heterozygote). Proband 2 inherited two missense variants in the dimerization domain of VAC14, p.Ala582Ser and p.Ser583Leu, that have not been previously reported. Cultured skin fibroblasts exhibited the accumulation of vacuoles that is characteristic of PI(3,5)P2 deficiency. Vacuolization of fibroblasts was rescued by transfection of wild-type VAC14 cDNA. The similar age of onset and neurological decline in the two unrelated children define a recessive disorder resulting from compound heterozygosity for deleterious variants of VAC14.


Assuntos
Alelos , Proteínas de Membrana/genética , Mutação , Doenças do Sistema Nervoso/genética , Idade de Início , Sequência de Aminoácidos , Criança , Pré-Escolar , Exoma/genética , Éxons/genética , Feminino , Genes Recessivos , Heterozigoto , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/química , Mutação de Sentido Incorreto/genética , Linhagem
3.
Brain ; 139(Pt 2): 338-45, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26685157

RESUMO

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Epilepsia/diagnóstico , Epilepsia/genética , Doença de Leigh/diagnóstico , Doença de Leigh/genética , Subunidades Proteicas/genética , Criança , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Epilepsia/complicações , Evolução Fatal , Feminino , Humanos , Doença de Leigh/complicações , Mutação/genética
4.
Brain ; 136(Pt 10): 3140-50, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24014518

RESUMO

Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10(-9), odds ratio (A) = 1.42, 95% confidence interval: 1.26-1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures.


Assuntos
Epilepsia do Lobo Temporal/genética , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Esclerose/genética , Convulsões Febris/genética , Epilepsia do Lobo Temporal/etiologia , Estudo de Associação Genômica Ampla/métodos , Hipocampo/patologia , Humanos , Estudos Prospectivos , Convulsões Febris/diagnóstico , Lobo Temporal/patologia
5.
Nat Genet ; 33(4): 527-32, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12612585

RESUMO

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.


Assuntos
Canais de Cloreto/genética , Epilepsia Generalizada/genética , Mutação , Adolescente , Adulto , Sequência de Bases , Membrana Celular/metabolismo , Códon de Terminação , Análise Mutacional de DNA , DNA Complementar/metabolismo , Eletrofisiologia , Saúde da Família , Feminino , Heterozigoto , Humanos , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Linhagem , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
6.
Cells ; 12(2)2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36672163

RESUMO

Epilepsy and mental retardation are known to be associated with pathogenic mutations in a broad range of genes that are expressed in the brain and have a role in neurodevelopment. Here, we report on a family with three affected individuals whose clinical symptoms closely resemble a neurodevelopmental disorder. Whole-exome sequencing identified a homozygous stop-gain mutation, p.Gln19*, in the BATF2 gene in the patients. The BATF2 transcription factor is predominantly expressed in macrophages and monocytes and has been reported to modulate AP-1 transcription factor-mediated pro-inflammatory responses. Transcriptome analysis showed altered base-level expression of interferon-stimulated genes in the patients' blood, typical for type I interferonopathies. Peripheral blood mononuclear cells from all three patients demonstrated elevated responses to innate immune stimuli, which could be reproduced in CRISPR-Cas9-generated BATF2-/- human monocytic cell lines. BATF2 is, therefore, a novel disease-associated gene candidate for severe epilepsy and mental retardation related to dysregulation of immune responses, which underscores the relevance of neuroinflammation for epilepsy.


Assuntos
Deficiência Intelectual , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Leucócitos Mononucleares/metabolismo , Imunidade , Fenótipo
7.
Biochim Biophys Acta ; 1812(3): 321-5, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21138766

RESUMO

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.


Assuntos
Replicação do DNA , DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/genética , Fibroblastos/enzimologia , Mitocôndrias/fisiologia , Mutação/genética , Adulto , Substituição de Aminoácidos , Estudos de Casos e Controles , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/metabolismo , Esclerose Cerebral Difusa de Schilder/genética , Esclerose Cerebral Difusa de Schilder/patologia , Inibidores Enzimáticos/farmacologia , Epilepsia/genética , Epilepsia/patologia , Etídio/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Heterozigoto , Homozigoto , Humanos , Lactente , Masculino , Mitocôndrias/efeitos dos fármacos , Doenças Musculares/genética , Doenças Musculares/patologia , Inibidores da Síntese de Ácido Nucleico , Timidina Quinase/genética
8.
Genes (Basel) ; 13(3)2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35327983

RESUMO

Here, we report a consanguineous family harboring a novel homozygous frame-shift mutation in ASPM leading to a truncation of the ASPM protein after amino acid position 1830. The phenotype of the patients was associated with microcephaly, epilepsy, and behavioral and cognitive deficits. Despite the obvious genetic similarity, the affected patients show a considerable phenotypic heterogeneity regarding the degree of mental retardation, presence of epilepsy and MRI findings. Interestingly, the degree of mental retardation and the presence of epilepsy correlates well with the severity of abnormalities detected in brain MRI. On the other hand, we detected no evidence for substantial nonsense-mediated ASPM transcript decay in blood samples. This indicates that other factors than ASPM expression levels are relevant for the variability of structural changes in brain morphology seen in patients with primary hereditary microcephaly caused by ASPM mutations.


Assuntos
Epilepsia , Deficiência Intelectual , Microcefalia , Variação Biológica da População , Cognição , Transtornos Cognitivos/genética , Epilepsia/genética , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/genética , Microcefalia/genética , Mutação , Proteínas do Tecido Nervoso/genética
9.
Brain ; 133(Pt 1): 23-32, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19843651

RESUMO

Idiopathic generalized epilepsies account for 30% of all epilepsies. Despite a predominant genetic aetiology, the genetic factors predisposing to idiopathic generalized epilepsies remain elusive. Studies of structural genomic variations have revealed a significant excess of recurrent microdeletions at 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 in various neuropsychiatric disorders including autism, intellectual disability and schizophrenia. Microdeletions at 15q13.3 have recently been shown to constitute a strong genetic risk factor for common idiopathic generalized epilepsy syndromes, implicating that other recurrent microdeletions may also be involved in epileptogenesis. This study aimed to investigate the impact of five microdeletions at the genomic hotspot regions 1q21.1, 15q11.2, 16p11.2, 16p13.11 and 22q11.2 on the genetic risk to common idiopathic generalized epilepsy syndromes. The candidate microdeletions were assessed by high-density single nucleotide polymorphism arrays in 1234 patients with idiopathic generalized epilepsy from North-western Europe and 3022 controls from the German population. Microdeletions were validated by quantitative polymerase chain reaction and their breakpoints refined by array comparative genomic hybridization. In total, 22 patients with idiopathic generalized epilepsy (1.8%) carried one of the five novel microdeletions compared with nine controls (0.3%) (odds ratio = 6.1; 95% confidence interval 2.8-13.2; chi(2) = 26.7; 1 degree of freedom; P = 2.4 x 10(-7)). Microdeletions were observed at 1q21.1 [Idiopathic generalized epilepsy (IGE)/control: 1/1], 15q11.2 (IGE/control: 12/6), 16p11.2 IGE/control: 1/0, 16p13.11 (IGE/control: 6/2) and 22q11.2 (IGE/control: 2/0). Significant associations with IGEs were found for the microdeletions at 15q11.2 (odds ratio = 4.9; 95% confidence interval 1.8-13.2; P = 4.2 x 10(-4)) and 16p13.11 (odds ratio = 7.4; 95% confidence interval 1.3-74.7; P = 0.009). Including nine patients with idiopathic generalized epilepsy in this cohort with known 15q13.3 microdeletions (IGE/control: 9/0), parental transmission could be examined in 14 families. While 10 microdeletions were inherited (seven maternal and three paternal transmissions), four microdeletions occurred de novo at 15q13.3 (n = 1), 16p13.11 (n = 2) and 22q11.2 (n = 1). Eight of the transmitting parents were clinically unaffected, suggesting that the microdeletion itself is not sufficient to cause the epilepsy phenotype. Although the microdeletions investigated are individually rare (<1%) in patients with idiopathic generalized epilepsy, they collectively seem to account for a significant fraction of the genetic variance in common idiopathic generalized epilepsy syndromes. The present results indicate an involvement of microdeletions at 15q11.2 and 16p13.11 in epileptogenesis and strengthen the evidence that recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 confer a pleiotropic susceptibility effect to a broad range of neuropsychiatric disorders.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 16/genética , Epilepsia Generalizada/genética , Predisposição Genética para Doença/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia Generalizada/etiologia , Feminino , Humanos , Masculino , Linhagem , Adulto Jovem
10.
BMC Evol Biol ; 10: 270, 2010 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-20813043

RESUMO

BACKGROUND: We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee) individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens. RESULTS: We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. CONCLUSIONS: Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans.


Assuntos
Genoma Mitocondrial/genética , Pan paniscus/classificação , Pan paniscus/genética , Animais , DNA Mitocondrial/genética , Humanos , Filogenia , ATPases Translocadoras de Prótons/genética
11.
Epilepsia Open ; 2(3): 334-342, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-29588962

RESUMO

Objective: Genetic generalized epilepsy (GGE) encompasses seizure disorders characterized by spike-and-wave discharges (SWD) originating within thalamo-cortical circuits. Hyperpolarization-activated (HCN) and T-type Ca2+ channels are key modulators of rhythmic activity in these brain regions. Here, we screened HCN4 and CACNA1H genes for potentially contributory variants and provide their functional analysis. Methods: Targeted gene sequencing was performed in 20 unrelated familial cases with different subtypes of GGE, and the results confirmed in 230 ethnically matching controls. Selected variants in CACNA1H and HCN4 were functionally assessed in tsA201 cells and Xenopus laevis oocytes, respectively. Results: We discovered a novel CACNA1H (p.G1158S) variant in two affected members of a single family. One of them also carried an HCN4 (p.P1117L) variant inherited from the unaffected mother. In a separate family, an HCN4 variant (p.E153G) was identified in one of several affected members. Voltage-clamp analysis of CACNA1H (p.G1158S) revealed a small but significant gain-of-function, including increased current density and a depolarizing shift of steady-state inactivation. HCN4 p.P1117L and p.G153E both caused a hyperpolarizing shift in activation and reduced current amplitudes, resulting in a loss-of-function. Significance: Our results are consistent with a model suggesting cumulative contributions of subtle functional variations in ion channels to seizure susceptibility and GGE.

12.
Epilepsy Res ; 114: 47-51, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26088884

RESUMO

In the recent years, several neurological syndromes related to defects of the glucose transporter type 1 (Glut1) have been descried. They include the glucose transporter deficiency syndrome (Glut1-DS) as the most severe form, the paroxysmal exertion-induced dyskinesia (PED), a form of spastic paraparesis (CSE) as well as the childhood (CAE) and the early-onset absence epilepsy (EOAE). Glut1, encoded by the gene SLC2A1, is the most relevant glucose transporter in the brain. All Glut1 syndromes respond well to a ketogenic diet (KD) and most of the patients show a rapid seizure control. Ketogenic Diet developed to an established treatment for other forms of pharmaco-resistant epilepsies. Since we were interested in the question if those patients might have an underlying Glut1 defect, we sequenced SLC2A1 in a cohort of 28 patients with different forms of pharmaco-resistant epilepsies responding well to a KD. Unfortunately, we could not detect any mutations in SLC2A1. The exact action mechanisms of KD in pharmaco-resistant epilepsy are not well understood, but bypassing the Glut1 transporter seems not to play an important role.


Assuntos
Dieta Cetogênica , Epilepsia/dietoterapia , Epilepsia/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia Resistente a Medicamentos/dietoterapia , Epilepsia Resistente a Medicamentos/genética , Feminino , Transportador de Glucose Tipo 1/deficiência , Humanos , Masculino , Mutação/genética , Adulto Jovem
13.
Arch Neurol ; 59(7): 1137-41, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12117362

RESUMO

CONTEXT: Missense mutations in the GABRG2 gene, which encodes the gamma 2 subunit of central nervous gamma-aminobutyric acid (GABA)(A) receptors, have recently been described in 2 families with idiopathic epilepsy. In one of these families, the affected individuals predominantly exhibited childhood absence epilepsy and febrile convulsions. OBJECTIVE: To assess the role of GABRG2 in the genetic predisposition to idiopathic absence epilepsies. DESIGN: The GABRG2 gene was screened by single-strand conformation analysis for mutations. Furthermore, a population-based association study assessing a common exon 5 polymorphism (C588T) was carried out. PATIENTS: The sample was composed of 135 patients with idiopathic absence epilepsy and 154 unrelated and ethnically matched controls. RESULTS: A point mutation (IVS6 + 2T-->G) leading to a splice-donor site mutation in intron 6 was found. The mutation, which is predicted to lead to a nonfunctional protein, cosegregates with the disease status in a family with childhood absence epilepsy and febrile convulsions. The association study did not find any significant differences in the allele and genotype frequencies of the common exon 5 polymorphism (C588T) between patients with idiopathic absence epilepsy and controls (P>.35). CONCLUSIONS: Our study identified a splice-donor-site mutation that was probably causing a nonfunctional GABRG2 subunit. This mutation occurred in heterozygosity in the affected members of a single nuclear family, exhibiting a phenotypic spectrum of childhood absence epilepsy and febrile convulsions. The GABRG2 gene seems to confer a rare rather than a frequent major susceptibility effect to common idiopathic absence epilepsy syndromes.


Assuntos
Epilepsia Tipo Ausência/genética , Mutação de Sentido Incorreto , Receptores de GABA-A/genética , Convulsões Febris/genética , Adolescente , Alelos , Epilepsia Tipo Ausência/metabolismo , Éxons , Feminino , Genótipo , Humanos , Masculino , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões Febris/metabolismo
14.
Neurology ; 83(23): 2183-7, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25361775

RESUMO

OBJECTIVE: We report a consanguineous family with 2 affected individuals whose clinical symptoms closely resembled MERRF (myoclonus epilepsy with ragged red fibers) syndrome including severe myoclonic epilepsy, progressive spastic tetraparesis, progressive impairment of vision and hearing, as well as progressive cognitive decline. METHODS: After excluding the presence of pathogenic mitochondrial DNA mutations, whole-exome sequencing of blood DNA from the index patient was performed. Detected homozygous mutations and their cosegregation were confirmed by Sanger sequencing. CARS2 (cysteinyl-tRNA synthetase 2, mitochondrial) messenger RNA analysis was performed by reverse transcription PCR and sequencing. RESULTS: We identified a homozygous c.655G>A mutation in the CARS2 gene cosegregating in the family. The mutation is localized at the last nucleotide of exon 6 and thus is predicted to cause aberrant splicing. Analysis of the CARS2 messenger RNA showed that the presence of the mutation resulted in removal of exon 6. This leads to an in-frame deletion of 28 amino acids in a conserved sequence motif of the protein involved in stabilization of the acceptor end hairpin of tRNA(Cys). CONCLUSION: CARS2 is a novel disease gene associated with a severe progressive myoclonic epilepsy most resembling MERRF syndrome.


Assuntos
Aminoacil-tRNA Sintetases/genética , Epilepsias Mioclônicas/genética , Síndrome MERRF/genética , Mutação/genética , Adulto , DNA Mitocondrial/genética , Epilepsias Mioclônicas/etiologia , Feminino , Homozigoto , Humanos , Síndrome MERRF/complicações , Síndrome MERRF/diagnóstico , Masculino , Mitocôndrias/genética , Linhagem , Splicing de RNA/genética , Adulto Jovem
15.
Nat Genet ; 45(2): 214-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23313956

RESUMO

Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG, POLG2 and C10orf2) or the biosynthesis pathways of deoxyribonucleoside 5'-triphosphates for mtDNA synthesis. However, in many of these disorders, the underlying genetic defect has yet to be discovered. Here, we identify homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We show that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Fibroblasts from affected individuals do not repopulate after chemically induced mtDNA depletion. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Thus, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance.


Assuntos
Replicação do DNA/genética , DNA Mitocondrial/genética , Exodesoxirribonucleases/genética , Doenças Mitocondriais/genética , Modelos Moleculares , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon sem Sentido/genética , Primers do DNA/genética , Componentes do Gene , Células HeLa , Humanos , Doenças Mitocondriais/enzimologia , Dados de Sequência Molecular , Análise de Sequência de DNA
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