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1.
J Neuromuscul Dis ; 7(1): 41-46, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31744015

RESUMO

The natural history of patients with spinal muscular atrophy (SMA) has changed due to advances in standard care and development of targeted treatments. Nusinersen was the first drug approved for the treatment of all SMA patients. The transfer of clinical trial data into a real-life environment is challenging, especially regarding the advice of patients and families to what extent they can expect a benefit from the novel treatment. We report the results of a modified Delphi consensus process among child neurologists from Germany, Austria and Switzerland about the indication or continuation of nusinersen treatment in children with SMA type 1 based on different clinical case scenarios.

2.
Am J Hum Genet ; 105(2): 302-316, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31256877

RESUMO

Members of a paralogous gene family in which variation in one gene is known to cause disease are eight times more likely to also be associated with human disease. Recent studies have elucidated DHX30 and DDX3X as genes for which pathogenic variant alleles are involved in neurodevelopmental disorders. We hypothesized that variants in paralogous genes encoding members of the DExD/H-box RNA helicase superfamily might also underlie developmental delay and/or intellectual disability (DD and/or ID) disease phenotypes. Here we describe 15 unrelated individuals who have DD and/or ID, central nervous system (CNS) dysfunction, vertebral anomalies, and dysmorphic features and were found to have probably damaging variants in DExD/H-box RNA helicase genes. In addition, these individuals exhibit a variety of other tissue and organ system involvement including ocular, outer ear, hearing, cardiac, and kidney tissues. Five individuals with homozygous (one), compound-heterozygous (two), or de novo (two) missense variants in DHX37 were identified by exome sequencing. We identified ten total individuals with missense variants in three other DDX/DHX paralogs: DHX16 (four individuals), DDX54 (three individuals), and DHX34 (three individuals). Most identified variants are rare, predicted to be damaging, and occur at conserved amino acid residues. Taken together, these 15 individuals implicate the DExD/H-box helicases in both dominantly and recessively inherited neurodevelopmental phenotypes and highlight the potential for more than one disease mechanism underlying these disorders.

3.
Orphanet J Rare Dis ; 14(1): 105, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077250

RESUMO

BACKGROUND: X-linked Duchenne muscular dystrophy (DMD), the most frequent human hereditary skeletal muscle myopathy, inevitably leads to progressive dilated cardiomyopathy. We assessed the effect and safety of a combined treatment with the ACE-inhibitor enalapril and the ß-blocker metoprolol in a German cohort of infantile and juvenile DMD patients with preserved left ventricular function. METHODS TRIAL DESIGN: Sixteen weeks single-arm open run-in therapy with enalapril and metoprolol followed by a two-arm 1:1 randomized double-blind placebo-controlled treatment in a multicenter setting. INCLUSION CRITERIA: DMD boys aged 10-14 years with left ventricular fractional shortening [LV-FS] ≥ 30% in echocardiography. Primary endpoint: time from randomization to first occurrence of LV-FS < 28%. Secondary: changes of a) LV-FS from baseline, b) blood pressure, c), heart rate and autonomic function in ECG and Holter-ECG, e) cardiac biomarkers and neurohumeral serum parameters, f) quality of life, and g) adverse events. RESULTS: From 3/2010 to 12/2013, 38 patients from 10 sites were centrally randomized after run-in, with 21 patients continuing enalapril and metoprolol medication and 17 patients receiving placebo. Until end of study 12/2015, LV-FS < 28% was reached in 6/21 versus 7/17 patients. Cox regression adjusted for LV-FS after run-in showed a statistically non-significant benefit for medication over placebo (hazard ratio: 0.38; 95% confidence interval: 0.12 to 1.22; p = 0.10). Analysis of secondary outcome measures revealed a time-dependent deterioration of LV-FS with no statistically significant differences between the two study arms. Blood pressure, maximal heart rate and mean-NN values were significantly lower at the end of open run-in treatment compared to baseline. Outcome analysis 19 months after randomization displayed significantly lower maximum heart rate and higher noradrenalin and renin values in the intervention group. No difference between treatments was seen for quality of life. As a single, yet important adverse event, the reversible deterioration of walking abilities of one DMD patient during the run-in period was observed. CONCLUSIONS: Our analysis of enalapril and metoprolol treatment in DMD patients with preserved left ventricular function is suggestive to delay the progression of the intrinsic cardiomyopathy to left ventricular failure, but did not reach statistical significance, probably due to insufficient sample size. CLINICAL TRIAL REGISTRATION: DRKS-number 00000115, EudraCT-number 2009-009871-36.


Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Enalapril/uso terapêutico , Metoprolol/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Disfunção Ventricular Esquerda/prevenção & controle , Adolescente , Cardiomiopatias/prevenção & controle , Criança , Método Duplo-Cego , Enalapril/efeitos adversos , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Metoprolol/efeitos adversos , Resultado do Tratamento
4.
Mitochondrion ; 43: 37-42, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30092295

RESUMO

Kearns-Sayre syndrome (KSS) is a multisystemic disorder marked by aerobic cell metabolism dysfunction. Fibroblasts derived from KSS patient skin biopsy exhibit heterogeneous occurrence of mitochondrial genomes as those circular DNA molecules partially carry the common deletion. In our approach, we aim to evaluate the intercellular alterations in respect to mitochondrial DNA integrity by laser capture microdissection and multiplex quantitative real-time PCR in single cells. The obtained results give new insights into the understanding of mitochondrial genetics, e.g. postulated sorting of damaged mitochondria, and heterogeneity of cells. Further, we discuss the relevance of intercellular heterogeneities for human mitochondrial disorders in general.


Assuntos
DNA Mitocondrial/genética , Fibroblastos/patologia , Síndrome de Kearns-Sayre/patologia , Microdissecção e Captura a Laser/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Célula Única/métodos , Biópsia , Pré-Escolar , Feminino , Variação Genética , Humanos , Lactente , Masculino , Deleção de Sequência , Pele/patologia
5.
Trials ; 19(1): 291, 2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29793540

RESUMO

BACKGROUND: Trials in rare diseases have many challenges, among which are the need to set up multiple sites in different countries to achieve recruitment targets and the divergent landscape of clinical trial regulations in those countries. Over the past years, there have been initiatives to facilitate the process of international study set-up, but the fruits of these deliberations require time to be operationally in place. FOR-DMD (Finding the Optimum Steroid Regimen for Duchenne Muscular Dystrophy) is an academic-led clinical trial which aims to find the optimum steroid regimen for Duchenne muscular dystrophy, funded by the National Institutes of Health (NIH) for 5 years (July 2010 to June 2015), anticipating that all sites (40 across the USA, Canada, the UK, Germany and Italy) would be open to recruitment from July 2011. However, study start-up was significantly delayed and recruitment did not start until January 2013. METHOD: The FOR-DMD study is used as an example to identify systematic problems in the set-up of international, multi-centre clinical trials. The full timeline of the FOR-DMD study, from funding approval to site activation, was collated and reviewed. Systematic issues were identified and grouped into (1) study set-up, e.g. drug procurement; (2) country set-up, e.g. competent authority applications; and (3) site set-up, e.g. contracts, to identify the main causes of delay and suggest areas where anticipatory action could overcome these obstacles in future studies. RESULTS: Time from the first contact to site activation across countries ranged from 6 to 24 months. Reasons of delay were universal (sponsor agreement, drug procurement, budgetary constraints), country specific (complexity and diversity of regulatory processes, indemnity requirements) and site specific (contracting and approvals). The main identified obstacles included (1) issues related to drug supply, (2) NIH requirements regarding contracting with non-US sites, (3) differing regulatory requirements in the five participating countries, (4) lack of national harmonisation with contracting and the requirement to negotiate terms and contract individually with each site and (5) diversity of languages needed for study materials. Additionally, as with many academic-led studies, the FOR-DMD study did not have access to the infrastructure and expertise that a contracted research organisation could provide, organisations often employed in pharmaceutical-sponsored studies. This delay impacted recruitment, challenged the clinical relevance of the study outcomes and potentially delayed the delivery of the best treatment to patients. CONCLUSION: Based on the FOR-DMD experience, and as an interim solution, we have devised a checklist of steps to not only anticipate and minimise delays in academic international trial initiation but also identify obstacles that will require a concerted effort on the part of many stakeholders to mitigate.


Assuntos
Lista de Checagem , Ensaios Clínicos como Assunto/métodos , Estudos Multicêntricos como Assunto/métodos , Distrofia Muscular de Duchenne/tratamento farmacológico , Doenças Raras/tratamento farmacológico , Projetos de Pesquisa , Esteroides/administração & dosagem , Orçamentos , Ensaios Clínicos como Assunto/economia , Ensaios Clínicos como Assunto/legislação & jurisprudência , Contratos , Humanos , Cooperação Internacional , Estudos Multicêntricos como Assunto/economia , Estudos Multicêntricos como Assunto/legislação & jurisprudência , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/economia , Seleção de Pacientes , Doenças Raras/diagnóstico , Doenças Raras/economia , Projetos de Pesquisa/legislação & jurisprudência , Apoio à Pesquisa como Assunto , Esteroides/efeitos adversos , Esteroides/provisão & distribução , Fatores de Tempo , Resultado do Tratamento
7.
Hum Mutat ; 38(11): 1477-1484, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28726266

RESUMO

Biallelic GLDN mutations have recently been identified among infants with lethal congenital contracture syndrome 11 (LCCS11). GLDN encodes gliomedin, a protein required for the formation of the nodes of Ranvier and development of the human peripheral nervous system. We report six infants and children from four unrelated families with biallelic GLDN mutations, four of whom survived beyond the neonatal period into infancy, childhood, and late adolescence with intensive care and chronic respiratory and nutritional support. Our findings expand the genotypic and phenotypic spectrum of LCCS11 and demonstrate that the condition may not necessarily be lethal in the neonatal period.


Assuntos
Artrogripose/diagnóstico , Artrogripose/genética , Genes Letais , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Fenótipo , Artrogripose/mortalidade , Biópsia , Análise Mutacional de DNA , Evolução Fatal , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Raízes Nervosas Espinhais/ultraestrutura , Sequenciamento Completo do Exoma
8.
Contemp Clin Trials ; 58: 34-39, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28450193

RESUMO

Despite corticosteroids being the only treatment documented to improve strength and function in boys with Duchenne muscular dystrophy (DMD) corticosteroid prescription is inconsistent and in some countries, corticosteroids are not prescribed. We are conducting a clinical trial that (1) compares the 3 most frequently prescribed corticosteroid regimes; (2) standardizes treatment of DMD complications; and (3) standardizes prevention of corticosteroid side effects. Investigators at 38 sites in 5 countries plan to recruit 300 boys aged 4-7 who are randomly assigned to one of three regimens: daily prednisone; daily deflazacort; or intermittent prednisone (10days on/10days off). Boys are followed for a minimum of 3years to assess the relative effectiveness and adverse event profiles of the different regimens. The primary outcome is a 3-dimensional variable consisting of log-transformed time to rise from the floor, forced vital capacity, and subject/parent satisfaction with treatment, each averaged over all post-baseline visits. The study protocol includes evidence- and consensus-based treatment of DMD complications and of corticosteroid side effects. This study seeks to establish a standard corticosteroid regimen for DMD. Since all new interventions for DMD are being developed as add-on therapies to corticosteroids, defining the optimum regimen is of importance for all new treatments.


Assuntos
Imunossupressores/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Prednisona/administração & dosagem , Pregnenodionas/uso terapêutico , Criança , Pré-Escolar , Avaliação da Deficiência , Método Duplo-Cego , Esquema de Medicação , Testes de Função Cardíaca , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Masculino , Força Muscular , Satisfação do Paciente , Prednisona/efeitos adversos , Prednisona/uso terapêutico , Pregnenodionas/administração & dosagem , Pregnenodionas/efeitos adversos , Amplitude de Movimento Articular , Projetos de Pesquisa , Capacidade Vital
9.
J Inherit Metab Dis ; 38(5): 905-14, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25868664

RESUMO

FBXL4 deficiency is a recently described disorder of mitochondrial maintenance associated with a loss of mitochondrial DNA in cells. To date, the genetic diagnosis of FBXL4 deficiency has been established in 28 individuals. This paper retrospectively reviews proxy-reported clinical and biochemical findings and evaluates brain imaging, morphological and genetic data in 21 of those patients. Neonatal/early-onset severe lactic acidosis, muscular hypotonia, feeding problems and failure to thrive is the characteristic pattern at first presentation. Facial dysmorphic features are present in 67% of cases. Seven children died (mean age 37 months); 11 children were alive (mean age at follow-up 46 months), three children were lost to follow-up. All survivors developed severe psychomotor retardation. Brain imaging was non-specific in neonates but a later-onset, rapidly progressive brain atrophy was noted. Elevated blood lactate and metabolic acidosis were observed in all individuals; creatine kinase was elevated in 45% of measurements. Diagnostic workup in patient tissues and cells revealed a severe combined respiratory chain defect with a general decrease of enzymes associated with mitochondrial energy metabolism and a relative depletion of mitochondrial DNA content. Mutations were detected throughout the FBXL4 gene albeit with no clear delineation of a genotype-phenotype correlation. Treatment with "mitochondrial medications" did not prove effective. In conclusion, a clinical pattern of early-onset encephalopathy, persistent lactic acidosis, profound muscular hypotonia and typical facial dysmorphism should prompt initiation of molecular genetic analysis of FBXL4. Establishment of the diagnosis permits genetic counselling, prevents patients undergoing unhelpful diagnostic procedures and allows for accurate prognosis.


Assuntos
Proteínas F-Box/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Mutação , Ubiquitina-Proteína Ligases/genética , Acidose Láctica/complicações , Acidose Láctica/congênito , Acidose Láctica/genética , Criança , Pré-Escolar , Progressão da Doença , Assimetria Facial/complicações , Assimetria Facial/congênito , Assimetria Facial/genética , Família , Feminino , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/mortalidade , Hipotonia Muscular/complicações , Hipotonia Muscular/congênito , Hipotonia Muscular/genética , Neuroimagem , Prognóstico , Estudos Retrospectivos
10.
Eur J Pediatr ; 173(9): 1253-6, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24973050

RESUMO

UNLABELLED: To date, the genetic basis of Dubowitz syndrome (short stature, microcephaly, facial abnormalities, eczema) is unknown and vascular complications are not known to be associated with this syndrome. In microcephalic osteodysplastic primordial dwarfism type II (MOPD II; disproportionate short statue, microcephaly, facial abnormalities), however, cerebral aneurysms and other vascular abnormalities are frequent complications. MOPD II is a genetic disorder caused by mutations in the pericentrin (PCNT) gene (21q22). We report on a patient who came to our attention as a 22-year-old with subarachnoid bleeding due to a ruptured cranial aneurysm. Until then, the patient was thought and published to have Dubowitz syndrome; previously, he was treated with coronary bypass surgery for extensive coronary angiopathy. Consecutive genetic testing revealed MOPD II. After clinical stabilization, the patient was discharged to a specialized rehabilitation center where he died due to re-rupture of a cranial aneurysm. CONCLUSION: In patients with short stature-especially when clinical features are accompanied by vascular complications-MOPD II should be considered as a differential diagnosis leading to consecutive genetic testing. After detection of mutations in the PCNT gene, a full vascular status including cerebral imaging and cardiac evaluation needs to be determined in order to analyze vascular abnormalities and initiate prophylactic treatment.


Assuntos
Antígenos/sangue , Erros de Diagnóstico , Nanismo/diagnóstico , Nanismo/genética , Retardo do Crescimento Fetal/diagnóstico , Retardo do Crescimento Fetal/genética , Aneurisma Intracraniano/diagnóstico , Aneurisma Intracraniano/genética , Microcefalia/diagnóstico , Microcefalia/genética , Mutação , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/genética , Adulto , Biomarcadores/sangue , Diagnóstico Diferencial , Eczema/diagnóstico , Facies , Evolução Fatal , Transtornos do Crescimento/diagnóstico , Humanos , Deficiência Intelectual/diagnóstico , Aneurisma Intracraniano/terapia , Masculino , Transferência de Pacientes , Recidiva
11.
Hum Mutat ; 35(7): 779-90, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24692096

RESUMO

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.


Assuntos
Estudos de Associação Genética , Doenças Musculares/congênito , Doenças Musculares/genética , Mutação , Tropomiosina/genética , Actinas/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Bases de Dados Genéticas , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/diagnóstico , Fenótipo , Fosforilação , Ligação Proteica , Alinhamento de Sequência , Tropomiosina/química , Tropomiosina/metabolismo , Adulto Jovem
12.
Mol Genet Metab ; 111(3): 342-352, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24461907

RESUMO

Defects of mitochondrial oxidative phosphorylation (OXPHOS) are associated with a wide range of clinical phenotypes and time courses. Combined OXPHOS deficiencies are mainly caused by mutations of nuclear genes that are involved in mitochondrial protein translation. Due to their genetic heterogeneity it is almost impossible to diagnose OXPHOS patients on clinical grounds alone. Hence next generation sequencing (NGS) provides a distinct advantage over candidate gene sequencing to discover the underlying genetic defect in a timely manner. One recent example is the identification of mutations in MTFMT that impair mitochondrial protein translation through decreased formylation of Met-tRNA(Met). Here we report the results of a combined exome sequencing and candidate gene screening study. We identified nine additional MTFMT patients from eight families who were affected with Leigh encephalopathy or white matter disease, microcephaly, mental retardation, ataxia, and muscular hypotonia. In four patients, the causal mutations were identified by exome sequencing followed by stringent bioinformatic filtering. In one index case, exome sequencing identified a single heterozygous mutation leading to Sanger sequencing which identified a second mutation in the non-covered first exon. High-resolution melting curve-based MTFMT screening in 350 OXPHPOS patients identified pathogenic mutations in another three index cases. Mutations in one of them were not covered by previous exome sequencing. All novel mutations predict a loss-of-function or result in a severe decrease in MTFMT protein in patients' fibroblasts accompanied by reduced steady-state levels of complex I and IV subunits. Being present in 11 out of 13 index cases the c.626C>T mutation is one of the most frequent disease alleles underlying OXPHOS disorders. We provide detailed clinical descriptions on eleven MTFMT patients and review five previously reported cases.


Assuntos
Hidroximetil e Formil Transferases/genética , Doença de Leigh/genética , Fosforilação Oxidativa , Biossíntese de Proteínas , Adolescente , Adulto , Criança , Pré-Escolar , Exoma , Feminino , Estudos de Associação Genética , Humanos , Hidroximetil e Formil Transferases/metabolismo , Lactente , Recém-Nascido , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , RNA de Transferência de Metionina/genética , Análise de Sequência de DNA
13.
J Med Genet ; 49(4): 277-83, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22499348

RESUMO

BACKGROUND: Next generation sequencing has become the core technology for gene discovery in rare inherited disorders. However, the interpretation of the numerous sequence variants identified remains challenging. We assessed the application of exome sequencing for diagnostics in complex I deficiency, a disease with vast genetic heterogeneity. METHODS: Ten unrelated individuals with complex I deficiency were selected for exome sequencing and sequential bioinformatic filtering. Cellular rescue experiments were performed to verify pathogenicity of novel disease alleles. RESULTS: The first filter criterion was 'Presence of known pathogenic complex I deficiency variants'. This revealed homozygous mutations in NDUFS3 and ACAD9 in two individuals. A second criterion was 'Presence of two novel potentially pathogenic variants in a structural gene of complex I', which discovered rare variants in NDUFS8 in two unrelated individuals and in NDUFB3 in a third. Expression of wild-type cDNA in mutant cell lines rescued complex I activity and assembly, thus providing a functional validation of their pathogenicity. Using the third criterion 'Presence of two potentially pathogenic variants in a gene encoding a mitochondrial protein', loss-of-function mutations in MTFMT were discovered in two patients. In three patients the molecular genetic correlate remained unclear and follow-up analysis is ongoing. CONCLUSION: Appropriate in silico filtering of exome sequencing data, coupled with functional validation of new disease alleles, is effective in rapidly identifying disease-causative variants in known and new complex I associated disease genes.


Assuntos
Exoma , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Análise de Sequência de DNA , Substituição de Aminoácidos , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Expressão Gênica , Humanos , Mutação , NADH Desidrogenase/genética
14.
Mitochondrion ; 11(3): 413-20, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21167962

RESUMO

Surf1 gene mutations were detected as a main cause for Leigh syndrome (LS), also known as infantile subacute necrotizing encephalomyelopathy. This syndrome which is commonly associated with systemic cytochrome c oxidase (COX) deficiency manifests in early childhood and has an invariable poor prognosis. Progressive disturbances of the respiratory function, for which both the metabolic condition and necrotizing brainstem lesions contribute, belong to the major symptoms of LS. A constitutive knockout (KO) mouse for Surf1 enables invasive investigations of distinct aspects of LS. In the present study the respiratory function was analyzed applying an arterially perfused brainstem preparation. Compared to wild type (WT) preparations Surf1 KO preparations had a higher baseline respiratory frequency and abnormal responses to hypoxia and hypercapnia that involved both respiratory frequency and motor nerve discharge pattern. These data suggest that COX deficiency impairs peripheral and/or central chemoreceptor function.


Assuntos
Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Doença de Leigh/diagnóstico , Doença de Leigh/fisiopatologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismo , Animais , Células Quimiorreceptoras/fisiologia , Humanos , Doença de Leigh/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Modelos Animais , Taxa Respiratória
15.
PLoS One ; 5(10): e13513, 2010 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-20976001

RESUMO

Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.


Assuntos
DNA Mitocondrial/genética , Síndrome MERRF/genética , Adolescente , Sequência de Bases , Western Blotting , Primers do DNA , Feminino , Humanos , Síndrome MERRF/patologia , Masculino , Linhagem , Reação em Cadeia da Polimerase
16.
Lancet Neurol ; 9(11): 1053-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20801085

RESUMO

BACKGROUND: Duchenne muscular dystrophy is a rare X-linked progressive disease characterised by loss of ambulation at about age 10 years, with death in early adulthood due to respiratory and cardiac insufficiency. Steroids are effective at slowing the progression of muscle weakness; however, their use is limited by side-effects, prompting the search for alternatives. We assessed the effect of ciclosporin A as monotherapy and in combination with intermittent prednisone for the treatment of ambulant patients with this disorder. METHODS: Our study was a parallel-group, placebo-controlled, double-blind, multicentre trial at trial sites of the German muscular dystrophy network, MD-NET, over 36 months. Ambulant patients with Duchenne muscular dystrophy who were aged 5 years or older were randomly assigned to receive either ciclosporin A (3·5-4·0 mg/kg per day) or matching placebo. Allocation was done centrally with computer-generated random numbers. Patients and investigators were masked to the allocated treatment. After 3 months of treatment, both groups were also given intermittent prednisone for a further 12 months (0·75 mg/kg, alternating 10 days on with 10 days off). All patients who received at least one dose of study drug or placebo were included in the primary analysis. The primary outcome measure was manual muscle strength measured on the Medical Research Council (MRC) scale. This trial is registered with the German clinical trial register DRKS, number DRKS00000445. FINDINGS: 77 patients were randomly assigned to the ciclosporin A group and 76 to the placebo group; 73 patients on ciclosporin A and 73 on placebo received at least one dose and were available for efficacy analyses. 3 months of treatment with ciclosporin A alone did not show any significant improvement in primary outcome measures (mean change in the proportion of a possible total MRC score [%MRC] was -2·6 [SD 6·0] for patients on ciclosporin A and -0·8 [4·9] for patients on placebo; adjusted group difference estimate -0·88, 97·5% CI -2·6 to 0·9; p=0·26). The combination of ciclosporin A with intermittent steroids was not better than intermittent steroids alone over 12 months (mean change in %MRC was 0·7 [7·1] for patients on ciclosporin A and -0·3 [7·9] for patients on placebo; adjusted group difference estimate -0·85, -3·6 to 1·9; p=0·48). Numbers of adverse events (75 in patients on ciclosporin A and 74 on placebo) and serious adverse events (four with ciclosporin A and four with placebo) did not differ significantly between groups. INTERPRETATION: Ciclosporin A alone or in combination with intermittent prednisone does not improve muscle strength or functional abilities in ambulant boys with Duchenne muscular dystrophy, but is safe and well tolerated. FUNDING: German Federal Ministry of Education and Research, Action Benni and co eV, Novartis Pharma AG, and Deutsche Gesellschaft für Muskelkranke eV.


Assuntos
Ciclosporina/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Criança , Método Duplo-Cego , Humanos , Masculino , Distrofia Muscular de Duchenne/fisiopatologia , Literatura de Revisão como Assunto , Resultado do Tratamento
17.
Hum Mol Genet ; 19(8): 1413-24, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20080937

RESUMO

A new type of congenital disorders of glycosylation, designated CDG-Ip, is caused by the deficiency of GDP-Man:Man3GlcNAc2-PP-dolichol-alpha1,2-mannosyltransferase, encoded by the human ortholog of ALG11 from yeast. The patient presented with a multisystemic disorder characterized by muscular hypotonia, seizures, developmental retardation and death at the age of 2 years. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains, which is a characteristic sign for CDG-I. Analysis of dolichol-linked oligosaccharides in patient-derived fibroblasts revealed an accumulation of Man3GlcNAc2-PP-dolichol and Man4GlcNAc2-PP-dolichol. Determination of mannosyltransferase activities of early steps of lipid-linked oligosaccharide biosynthesis in fibroblasts indicated that the patient was deficient in elongating Man3GlcNAc2-PP-dolichol. These findings gave rise to genetic analysis of the hALG11 cDNA, in which homozygosity for mutation c.T257C (p.L86S) was identified. Verification of the mutation as a primary cause for the genetic defect was proved by retroviral expression of human wild-type and mutated ALG11 cDNA in patient-derived fibroblasts as well as using a yeast alg11 deletion strain as a heterologous expression system for hALG11 variants. Immunofluorescence examinations combined with western blotting showed no differences of intracellular localization or expression of ALG11 between control and patient fibroblasts, respectively, indicating no mislocalization or degradation of the mutated transferase.


Assuntos
Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Retículo Endoplasmático/enzimologia , Manosiltransferases/deficiência , Sequência de Aminoácidos , Erros Inatos do Metabolismo dos Carboidratos/genética , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Linhagem Celular , Células Cultivadas , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Fibroblastos/metabolismo , Glucanos/metabolismo , Glicosilação , Humanos , Lactente , Manosiltransferases/química , Manosiltransferases/genética , Dados de Sequência Molecular , Transporte Proteico , Alinhamento de Sequência
18.
Am J Pathol ; 175(3): 1019-29, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19661442

RESUMO

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor beta-aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl-alpha-phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both beta-aminopropionitrile and N-tert-butyl-alpha-phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin.


Assuntos
DNA Mitocondrial/genética , Fibroblastos/fisiologia , Síndrome de Kearns-Sayre/genética , Proteína-Lisina 6-Oxidase/metabolismo , Aminopropionitrilo/farmacologia , Animais , Células Cultivadas , Colágeno , Óxidos N-Cíclicos/farmacologia , Dano ao DNA , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Síndrome de Kearns-Sayre/metabolismo , Camundongos , Mitocôndrias/genética , Oxirredução , Estresse Oxidativo , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Deleção de Sequência , Pele/metabolismo , Pele/patologia , Envelhecimento da Pele
19.
Mol Cytogenet ; 2: 10, 2009 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-19284615

RESUMO

BACKGROUND: Submicroscopic imbalances in the subtelomeric regions of the chromosomes are considered to play an important role in the aetiology of mental retardation (MR). The aim of the study was to evaluate a quantitative PCR (qPCR) protocol established by Boehm et al. (2004) in the clinical routine of subtelomeric testing. RESULTS: 296 patients with MR and a normal karyotype (500-550 bands) were screened for subtelomeric imbalances by using qPCR combined with SYBR green detection. In total, 17 patients (5.8%) with 20 subtelomeric imbalances were identified. Six of the aberrations (2%) were classified as causative for the symptoms, because they occurred either de novo in the patients (5 cases) or the aberration were be detected in the patient and an equally affected parent (1 case). The extent of the deletions ranged from 1.8 to approximately 10 Mb, duplications were 1.8 to approximately 5 Mb in size. In 6 patients, the copy number variations (CNVs) were rated as benign polymorphisms, and the clinical relevance of these CNVs remains unclear in 5 patients (1.7%). Therefore, the overall frequency of clinically relevant imbalances ranges between 2% and 3.7% in our cohort. CONCLUSION: This study illustrates that the qPCR/SYBR green technique represents a rapid and versatile method for the detection of subtelomeric imbalances and the option to map the breakpoint. Thus, this technique is highly suitable for genotype/phenotype studies in patients with MR/developmental delay and/or congenital defects.

20.
Pediatr Neurol ; 34(1): 35-40, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16376276

RESUMO

Megalencephaly with dilated Virchow-Robin spaces has been suggested to represent a new clinical entity. This report describes two males and a female who have been monitored from pregnancy. The patients manifest a relatively normal psychomotor development with some minor neurologic symptoms such as mild muscle hypotonia and clumsy motor performance. Biochemical and electrophysiologic tests were normal. In the white matter of the brain, a prominent dilatation of the Virchow-Robin spaces with some adjacent signal alterations could be demonstrated by magnetic resonance imaging. Magnetic resonance spectroscopy revealed normal metabolite concentrations in the cortical and deep gray matter and normal-appearing white matter. Affected white matter was characterized by mildly reduced to normal levels of myo-inositol and a decrease of all other metabolites including total N-acetyl moieties, choline-containing compounds, and total creatine. These data indicate that the dilatation of Virchow-Robin spaces reflects an underlying brain pathology causing neuroaxonal damage. Possible differential diagnoses are discussed.


Assuntos
Encefalopatias/metabolismo , Encefalopatias/patologia , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Criança , Pré-Escolar , Colina/metabolismo , Creatina/metabolismo , Dilatação Patológica/metabolismo , Dilatação Patológica/patologia , Dipeptídeos/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Inositol/metabolismo , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Tamanho do Órgão , Fosfocreatina/metabolismo
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