Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
1.
Am J Hum Genet ; 110(2): 251-272, 2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36669495

RESUMO

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.


Assuntos
Perfilação da Expressão Gênica , Transtornos do Neurodesenvolvimento , Humanos , RNA-Seq , Cicloeximida , Análise de Sequência de RNA/métodos , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética
2.
Eur J Hum Genet ; 29(12): 1789-1795, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34426662

RESUMO

In a Dutch non-consanguineous patient having mitochondrial encephalomyopathy with complex I and complex IV deficiency, whole exome sequencing revealed two compound heterozygous variants in SLIRP. SLIRP gene encodes a stem-loop RNA-binding protein that regulates mitochondrial RNA expression and oxidative phosphorylation (OXPHOS). A frameshift and a deep-intronic splicing variant reduced the amount of functional wild-type SLIRP RNA to 5%. Consequently, in patient fibroblasts, MT-ND1, MT-ND6, and MT-CO1 expression was reduced. Lentiviral transduction of wild-type SLIRP cDNA in patient fibroblasts increased MT-ND1, MT-ND6, and MT-CO1 expression (2.5-7.2-fold), whereas mutant cDNAs did not. A fourfold decrease of citrate synthase versus total protein ratio in patient fibroblasts indicated that the resulting reduced mitochondrial mass caused the OXPHOS deficiency. Transduction with wild-type SLIRP cDNA led to a 2.4-fold increase of this ratio and partly restored OXPHOS activity. This confirmed causality of the SLIRP variants. In conclusion, we report SLIRP variants as a novel cause of mitochondrial encephalomyopathy with OXPHOS deficiency.


Assuntos
Encefalomiopatias Mitocondriais/genética , Proteínas de Ligação a RNA/genética , Células Cultivadas , Criança , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Genes Recessivos , Humanos , Masculino , Encefalomiopatias Mitocondriais/patologia , Mutação , Proteínas de Ligação a RNA/metabolismo
3.
Biochim Biophys Acta ; 1761(9): 1070-7, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16935026

RESUMO

Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.


Assuntos
Aterosclerose/metabolismo , Lipoproteínas HDL/sangue , Proteínas de Transferência de Fosfolipídeos/metabolismo , Triglicerídeos/biossíntese , Animais , Valva Aórtica/patologia , Aterosclerose/patologia , Proteínas de Transferência de Ésteres de Colesterol/biossíntese , Proteínas de Transferência de Ésteres de Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol/genética , Dieta Aterogênica , Feminino , Humanos , Lipoproteínas VLDL/biossíntese , Masculino , Camundongos , Camundongos Transgênicos , Proteínas de Transferência de Fosfolipídeos/sangue , Proteínas de Transferência de Fosfolipídeos/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Fatores Sexuais , Triglicerídeos/sangue
4.
J Lipid Res ; 45(5): 805-11, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-14993244

RESUMO

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins and mediates HDL conversion. PLTP-overexpressing mice have increased atherosclerosis. However, mice do not express cholesteryl ester transfer protein (CETP), which is involved in the same metabolic pathways as PLTP. Therefore, we studied atherosclerosis in heterozygous LDL receptor-deficient (LDLR(+/-)) mice expressing both human CETP and human PLTP. We used two transgenic lines with moderately and highly elevated plasma PLTP activity. In LDLR(+/-)/huCETPtg mice, cholesterol is present in both LDL and HDL. Both are decreased in LDLR(+/-)/huCETPtg/huPLTPtg mice (>50%). An atherogenic diet resulted in high levels of VLDL+LDL cholesterol. PLTP expression caused a strong PLTP dose-dependent decrease in VLDL and LDL cholesterol (-26% and -69%) and a decrease in HDL cholesterol (-70%). Surprisingly, atherosclerosis was increased in the two transgenic lines with moderately and highly elevated plasma PLTP activity (1.9-fold and 4.4-fold, respectively), indicating that the adverse effect of the reduction in plasma HDL outweighs the beneficial effect of the reduction in apolipoprotein B (apoB)-containing lipoproteins. The activities of the antiatherogenic enzymes paraoxonase and platelet-activating factor acetyl hydrolase were both PLTP dose-dependently reduced ( approximately -33% and -65%, respectively). We conclude that expression of PLTP in this animal model results in increased atherosclerosis in spite of reduced apoB-containing lipoproteins, by reduction of HDL and of HDL-associated antioxidant enzyme activities.


Assuntos
Apolipoproteínas B/metabolismo , Arteriosclerose/sangue , Suscetibilidade a Doenças , Proteínas de Membrana/sangue , Proteínas de Transferência de Fosfolipídeos/sangue , Regulação para Cima , Animais , Proteínas de Transporte/metabolismo , Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol , Dieta , Glicoproteínas/metabolismo , Humanos , Hipercolesterolemia/sangue , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA