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1.
Cell Stem Cell ; 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31588047

RESUMO

To investigate the contribution of parental genomes to early embryogenesis, we profiled the single-cell transcriptomes of human biparental and uniparental embryos systematically from the 1-cell to the morula stage. We observed that uniparental embryos exhibited variable and decreased embryonic genome activation (EGA). Comparative transcriptome analysis identified 807 maternally biased expressed genes (MBGs) and 581 paternally biased expressed genes (PBGs) in the preimplantation stages. MBGs became apparent at the 4-cell stage and contributed to the initiation of EGA, whereas PBGs preferentially appeared at the 8-cell stage and might affect embryo compaction and trophectoderm specification. Regulatory network analysis revealed that DUX4, EGR2, and DUXA are key transcription factors in MBGs' expression; ZNF263 and KLF3 are important for PBGs' expression. We demonstrated that parent-specific DNA methylation might account for the expression of most PBGs. Our results provide a valuable resource to understand parental genome activation and might help to elucidate parent-of-origin effects in early human development.

2.
Cell Res ; 28(10): 996-1012, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30127373

RESUMO

Dysregulated intracellular pH is emerging as a hallmark of cancer. In spite of their acidic environment and increased acid production, cancer cells maintain alkaline intracellular pH that promotes cancer progression by inhibiting apoptosis and increasing glycolysis, cell growth, migration, and invasion. Here we identify signal transducer and activator of transcription-3 (STAT3) as a key factor in the preservation of alkaline cytosol. STAT3 associates with the vacuolar H+-ATPase in a coiled-coil domain-dependent manner and increases its activity in living cells and in vitro. Accordingly, STAT3 depletion disrupts intracellular proton equilibrium by decreasing cytosolic pH and increasing lysosomal pH, respectively. This dysregulation can be reverted by reconstitution with wild-type STAT3 or STAT3 mutants unable to activate target genes (Tyr705Phe and DNA-binding mutant) or to regulate mitochondrial respiration (Ser727Ala). Upon cytosolic acidification, STAT3 is transcriptionally inactivated and further recruited to lysosomal membranes to reestablish intracellular proton equilibrium. These data reveal STAT3 as a regulator of intracellular pH and, vice versa, intracellular pH as a regulator of STAT3 localization and activity.

3.
Gigascience ; 7(3): 1-19, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635374

RESUMO

Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.


Assuntos
Metilação de DNA/genética , Genoma/genética , RNA Guia/genética , Sistemas CRISPR-Cas/genética , Ilhas de CpG/genética , Células HEK293 , Humanos , Metiltransferases/genética
4.
Front Cell Dev Biol ; 6: 5, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29468158

RESUMO

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca2+ regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca2+ changes, which can ultimately correspond to changes in specific stem cell transcription states.

5.
Gigascience ; 7(1): 1-7, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29267855

RESUMO

Background: DNA methylation plays a key role in the regulation of gene expression and carcinogenesis. Bisulfite sequencing studies mainly focus on calling single nucleotide polymorphism, different methylation region, and find allele-specific DNA methylation. Until now, only a few software tools have focused on virus integration using bisulfite sequencing data. Findings: We have developed a new and easy-to-use software tool, named BS-virus-finder (BSVF, RRID:SCR_015727), to detect viral integration breakpoints in whole human genomes. The tool is hosted at https://github.com/BGI-SZ/BSVF. Conclusions: BS-virus-finder demonstrates high sensitivity and specificity. It is useful in epigenetic studies and to reveal the relationship between viral integration and DNA methylation. BS-virus-finder is the first software tool to detect virus integration loci by using bisulfite sequencing data.


Assuntos
DNA Viral/genética , Genoma Humano , Vírus da Hepatite B/genética , Hepatócitos/virologia , Software , Integração Viral , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sensibilidade e Especificidade , Sulfitos/química , Sequenciamento Completo do Genoma
6.
Genet Med ; 20(7): 697-707, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29095815

RESUMO

PURPOSE: Recent studies demonstrate that whole-genome sequencing enables detection of cryptic rearrangements in apparently balanced chromosomal rearrangements (also known as balanced chromosomal abnormalities, BCAs) previously identified by conventional cytogenetic methods. We aimed to assess our analytical tool for detecting BCAs in the 1000 Genomes Project without knowing which bands were affected. METHODS: The 1000 Genomes Project provides an unprecedented integrated map of structural variants in phenotypically normal subjects, but there is no information on potential inclusion of subjects with apparent BCAs akin to those traditionally detected in diagnostic cytogenetics laboratories. We applied our analytical tool to 1,166 genomes from the 1000 Genomes Project with sufficient physical coverage (8.25-fold). RESULTS: With this approach, we detected four reciprocal balanced translocations and four inversions, ranging in size from 57.9 kb to 13.3 Mb, all of which were confirmed by cytogenetic methods and polymerase chain reaction studies. One of these DNAs has a subtle translocation that is not readily identified by chromosome analysis because of the similarity of the banding patterns and size of exchanged segments, and another results in disruption of all transcripts of an OMIM gene. CONCLUSION: Our study demonstrates the extension of utilizing low-pass whole-genome sequencing for unbiased detection of BCAs including translocations and inversions previously unknown in the 1000 Genomes Project.

7.
FEBS Lett ; 591(13): 1892-1901, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28580607

RESUMO

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR-Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9-mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design.


Assuntos
Sistemas CRISPR-Cas/genética , Cromatina/genética , Edição de Genes/métodos , Clivagem do DNA , Loci Gênicos/genética , Genômica , Células HEK293 , Células HeLa , Humanos , Mutação Puntual
8.
Stem Cell Reports ; 8(3): 648-658, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28216144

RESUMO

The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B) is causative for frontotemporal dementia linked to chromosome 3 (FTD3). CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT) and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Demência Frontotemporal/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Neurônios/metabolismo , Diferenciação Celular , Reprogramação Celular , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Ferro/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/citologia , Estresse Oxidativo/genética , Transcriptoma
9.
Virus Genes ; 52(5): 620-4, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27314269

RESUMO

Adenovirus is a leading cause of respiratory infection in children. Salivirus/klassevirus was first identified as an etiologic agent of gastroenteritis and was never reported in respiratory infection cases. The case being discussed here caught our attention because, although it is a common respiratory infection, it was fatal, while similar cases were mild. In order to find potential causes in the fatal case, we describe the clinical diagnosis and treatment, the sequencing analysis of the salivirus/klassevirus, and the co-infectious adenovirus. Metagenomics sequencing was conducted on the samples from a nasopharyngeal swab of the children with adenovirus infection. Sequences were assembled using IDBA-ud (1.1.1); phylogenetic analysis was performed using MEGA 5.2. RT-PCR and quantitative PCR were performed to verify the existence of the virus in the samples. A nearly full genome of this new virus strain was obtained with 7633 nt encoding a polyprotein of 2331 aa. Meanwhile, it was detected specifically in the nasopharyngeal swab by RT-PCR. Further, homology analysis indicated that the virus has a closer relationship with Salivirus A strain in Shanghai (GU245894). Our study reports the first case of Human salivirus/klassevirus in respiratory specimens of a child with fatal adenovirus infection in Shenzhen, China. The finding and investigation of the virus will provide more useful information for the clinical diagnosis of unexplained lethal infection and expand our knowledge of the new family, salivirus/klassevirus in picornavirus.


Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/classificação , Adenoviridae/genética , Fezes/virologia , Infecções Respiratórias/virologia , China , Coinfecção/virologia , Gastroenterite/virologia , Genoma Viral/genética , Humanos , Lactente , Masculino , Filogenia , Análise de Sequência de DNA/métodos
10.
Nat Genet ; 48(7): 740-6, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27213287

RESUMO

The human major histocompatibility complex (MHC) region has been shown to be associated with numerous diseases. However, it remains a challenge to pinpoint the causal variants for these associations because of the extreme complexity of the region. We thus sequenced the entire 5-Mb MHC region in 20,635 individuals of Han Chinese ancestry (10,689 controls and 9,946 patients with psoriasis) and constructed a Han-MHC database that includes both variants and HLA gene typing results of high accuracy. We further identified multiple independent new susceptibility loci in HLA-C, HLA-B, HLA-DPB1 and BTNL2 and an intergenic variant, rs118179173, associated with psoriasis and confirmed the well-established risk allele HLA-C*06:02. We anticipate that our Han-MHC reference panel built by deep sequencing of a large number of samples will serve as a useful tool for investigating the role of the MHC region in a variety of diseases and thus advance understanding of the pathogenesis of these disorders.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Complexo Principal de Histocompatibilidade/genética , Polimorfismo de Nucleotídeo Único/genética , Psoríase/genética , Butirofilinas/genética , Estudos de Casos e Controles , China/epidemiologia , Predisposição Genética para Doença , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Cadeias beta de HLA-DP/genética , Humanos , Psoríase/epidemiologia
11.
Biol Trace Elem Res ; 168(1): 252-60, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25937150

RESUMO

Cadmium (Cd) is an environmental toxicant and an inflammation-related xenobiotic. Selenium (Se) is a well-known nutritional trace element and a potent chemopreventive agent. The present study aimed to investigate the effect of Se on the cytotoxicity of Cd in bird immunocytes in vitro. Chicken splenic lymphocytes exposed to CdCl2 (10(-6) mol/L), Na2SeO3 (10(-7) mol/L), or a mixture of the two (10(-7) mol/L Na2SeO3 and 10(-6) mol/L CdCI2) were incubated for 12, 24, 36, 48, or 60 h. Cd significantly increased (P < 0.05 or P < 0.01) the messenger RNA (mRNA) expression levels of nuclear factor kappaB (NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2), and similar results were observed in the protein expression levels of NF-κB and COX-2. In addition, the nitric oxide (NO) content and the inducible iNOS activity were increased in the Cd-treated group compared to the control group. Furthermore, the protective effects of Se against Cd toxicity in chicken splenic lymphocytes were illustrated by the increase in select cytokines (NF-κB, iNOS, COX-2, TNF-α, and PGE2), NO content and iNOS activity. The biochemical parameters exhibited sensitivity to Se and Cd, suggesting that they may act as potential biomarkers for assessing the effects of Se and Cd risk on chicken splenic lymphocytes.


Assuntos
Cádmio/toxicidade , Citocinas/biossíntese , Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Linfócitos/metabolismo , Óxido Nítrico/farmacologia , Compostos de Selênio/farmacologia , Baço/citologia , Animais , Galinhas , Citocinas/genética , Inflamação/genética , Linfócitos/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase Tipo II/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Baço/efeitos dos fármacos
12.
Biol Trace Elem Res ; 165(2): 214-21, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25653004

RESUMO

Cadmium (Cd) is an environmental pollutant that is considered to be a potent toxin to organisms. Selenium (Se) has been known for its concomitant biological effects and characteristics with Cd. Due to the lack of the research regarding how the duality of Cd/Se affects immune cytokines in poultry, this paper aims to partly tackle this question. Chicken splenic lymphocytes with Cd (10(-6) mol/L CdCl2), Se (10(-7) mol/L Na2SeO3), Cd + Se (10(-7) mol/L Na2SeO3 and 10(-6) mol/L CdCl2), and a control group were incubated for 12, 24, 36, 48, and 60 h, respectively. At each time point, the cells were collected and the messenger RNA (mRNA) expression levels of interleukin (IL)-1ß, IL-2, IL-4, IL-10, IL-17, and interferon-γ (IFN-γ) were also examined. Compared with the control group and the Se-alone-treated group, the mRNA expression levels of IL-2, IL-4, IL-10, IL-17, and IFN-γ decreased significantly in the Cd-alone-treated group. By contrast, the mRNA expression level of IL-1ß markedly increased. Levels of IL-2, IL-4, IL-10, IL-17, and IFN-γ in Cd + Se-treated groups were significantly higher than those in Cd-alone-treated groups; however, the levels were not as high as the Se-alone-treated groups and the control group. The mRNA expression level of IL-1ß in the Cd + Se-treated group was lower than in the Cd-alone-treated group. The relationships with IL-2, IL-4, and IL-10 were found to be closer in the PC 1 matrix and 3D plot of the principal component analysis (PCA) loadings. IL-17 and IFN-γ were closer in the matrix of PC 2. However, IL-1ß gene expression appeared to be isolated in the matrix of PC 3. In addition, the results of cytokine cluster analysis showed that IL-2, IL-4, IL-10, IL-17, and IFN-γ were in the first group and that IL-1ß was in the second group. Therefore, Se partly attenuate immune toxicity induced by Cd in chicken splenic lymphocytes.


Assuntos
Cádmio/química , Regulação da Expressão Gênica , Linfócitos/efeitos dos fármacos , Selênio/química , Baço/efeitos dos fármacos , Animais , Galinhas , Citocinas/metabolismo , Perfilação da Expressão Gênica , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Linfócitos/metabolismo , Masculino , Aves Domésticas , Análise de Componente Principal , RNA Mensageiro/metabolismo , Baço/metabolismo
13.
Hemoglobin ; 39(1): 18-23, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25548039

RESUMO

We have developed a new method for non-invasive prenatal testing (NIPT) of paternally inherited fetal mutants for ß-thalassemia (ß-thal). Specially designed primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) were used to detect four major mutations [IVS-II-654, HBB: c.316-197C > T; codon 17 (A > T), HBB: c.52A > T; -28 (A > G), HBB: c.-78A > G and codons 41/42 (-TTCT), HBB: c.126_129delCTTT] causing ß-thal in China. The PIRA-PCR assay was first tested in a series of mixed DNA with different concentrations and mixed proportions. Subsequently, this assay was further tested in 10 plasma DNA samples collected from pregnant women. In the DNA mixture simulation test, the PIRA-PCR assay was able to detect 3.0% target genomic DNA (gDNA) mixed in 97.0% wild-type gDNA isolated from whole blood. For plasma DNA testing, the results detected by PIRA-PCR assay achieved 100.0% consistency with those obtained from the amniocentesis analysis. This new method could potentially be used for NIPT of paternally inherited fetal mutants for ß-thal.


Assuntos
Mutação , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Natal/métodos , Globinas beta/genética , Talassemia beta/genética , Sequência de Bases , Análise Mutacional de DNA/métodos , Primers do DNA/genética , Feminino , Humanos , Masculino , Gravidez , Talassemia beta/diagnóstico
14.
Mol Psychiatry ; 20(5): 563-572, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25113377

RESUMO

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of post-mortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q⩽0.01, 53 genes; q⩽0.05, 213 genes; q⩽0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (P<10(-7)) and BPD (P=0.029). To our knowledge, this is the first time that a substantially large number of genes show concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor-mediated phagocytosis, regulation of actin cytoskeleton pathways, along with several cancer pathways. Functional analyses of these genes revealed an interconnected pathway network centered on lysosomal function and the regulation of actin cytoskeleton. These pathways and their interacting network were principally confirmed by an independent transcriptome sequencing data set of the hippocampus. Dysregulation of lysosomal function and cytoskeleton remodeling has direct impacts on endocytosis, phagocytosis, exocytosis, vesicle trafficking, neuronal maturation and migration, neurite outgrowth and synaptic density and plasticity, and different aspects of these processes have been implicated in SCZ and BPD.


Assuntos
Citoesqueleto de Actina/genética , Transtorno Bipolar/genética , Predisposição Genética para Doença/genética , Lisossomos/fisiologia , Dobramento de Proteína , Esquizofrenia/genética , Citoesqueleto de Actina/metabolismo , Transtorno Bipolar/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Modelos Biológicos , Análise Multivariada , Esquizofrenia/patologia , Transdução de Sinais/genética , Estatística como Assunto
15.
Biol Chem ; 396(1): 27-33, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25060345

RESUMO

Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management.


Assuntos
Exoma/genética , Raquitismo Hipofosfatêmico Familiar/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Adolescente , Adulto , Criança , China , Feminino , Aconselhamento Genético , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Análise de Sequência
16.
Ecotoxicol Environ Saf ; 110: 95-102, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25202854

RESUMO

The expression of heat shock proteins (Hsps) commonly increases to provide neuroprotection when brain tissues are under stress conditions. Residues of avermectins (AVMs) have neurotoxic effects on a number of non-target organisms. The aim of this study was to investigate the effects of AVM exposure on the expression levels of Hsp 60, Hsp 70 and Hsp 90 for pigeon (Columba livia) neurons both in vivo and in vitro. The results showed that in general, the mRNA and protein levels of Hsps were increased in treated groups relative to control groups after AVM exposure for 30d, 60d and 90d in the cerebrum, cerebellum and optic lobe in vivo. However, AVM exposure had no significant effects on the transcription expression of Hsps for 90d in the optic lobe and decreased the translation expression of Hsps significantly for 90d in the optic lobe. In vitro, the LC50 of avermectin for King pigeon neurons is between 15µgL(-1) and 20µgL(-1). Following AVM (2.5-20µgL(-1)) exposure, the mRNA expression of the 3 Hsps was up-regulated to different degrees. Compared with the control groups, a significant decrease, a remarkable increase and a non-significant change was found in the protein expression of Hsp 60, Hsp 70 and Hsp 90 separately following AVM (2.5-20µgL(-1)) exposure. Based on these results, we conclude that AVM exposure can induce a protective stress response in pigeons by means of promoting the mRNA and protein expression of Hsps under in vivo and in vitro conditions, thus easing the neurotoxic effects of AVM to some extent.


Assuntos
Chaperonina 60/genética , Columbidae/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Ivermectina/análogos & derivados , Animais , Encéfalo/metabolismo , Chaperonina 60/metabolismo , Columbidae/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Ivermectina/toxicidade , Dose Letal Mediana , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética
17.
Biomed Res Int ; 2014: 186048, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25110662

RESUMO

Alport syndrome (AS) is a monogenic disease of the basement membrane (BM), resulting in progressive renal failure due to glomerulonephropathy, variable sensorineural hearing loss, and ocular anomalies. It is caused by mutations in the collagen type IV alpha-3 gene (COL4A3), the collagen type IV alpha-4 gene (COL4A4), and the collagen type IV alpha-5 gene (COL4A5), which encodes type IV collagen α3, α4, and α5 chains, respectively. To explore the disease-related gene in a four-generation Chinese Han pedigree of AS, exome sequencing was conducted on the proband, and a novel deletion mutation c.499delC (p.Pro167Glnfs*36) in the COL4A5 gene was identified. This mutation, absent in 1,000 genomes project, HapMap, dbSNP132, YH1 databases, and 100 normal controls, cosegregated with patients in the family. Neither sensorineural hearing loss nor typical COL4A5-related ocular abnormalities (dot-and-fleck retinopathy, anterior lenticonus, and the rare posterior polymorphous corneal dystrophy) were present in patients of this family. The phenotypes of patients in this AS family were characterized by early onset-age and rapidly developing into end-stage renal disease (ESRD). Our discovery broadens the mutation spectrum in the COL4A5 gene associated with AS, which may also shed new light on genetic counseling for AS.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Colágeno Tipo IV/genética , Grupos Étnicos/genética , Exoma/genética , Mutação/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , China , Colágeno Tipo IV/química , Análise Mutacional de DNA , Família , Feminino , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , Nefrite Hereditária/genética , Linhagem
18.
Biol Trace Elem Res ; 160(3): 340-51, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25035189

RESUMO

Cadmium (Cd) is an important environmental pollutant present in soil, water, air, and food. Selenium (Se) can antagonize some metal element toxicity including Cd. To investigate the cytotoxicity of Cd and the protective effects of Se on bird immunocytes in vitro, chicken splenic lymphocytes with CdCl2 (10(-6) mol/L), Na2SeO3 (10(-7) mol/L), and the mixture (10(-7) mol/L Na2SeO3 and 10(-6) mol/L CdCI2) were incubated for 12, 24, 36, and 48 h, respectively. A high level of malondialdehyde (MDA) and reactive oxygen species (ROS) productions were observed in Cd treatment group; the activities of catalase (CAT), glutathione peroxidise (GSH-Px), superoxide dismutase (SOD), and the mitochondrial inner transmembrane potential (ΔΨm) were significantly lower in Cd treatment group than those in controls (P < 0.05 or P < 0.01). In contrast, Se significantly improved the activities of antioxidant enzymes and reduced MDA and ROS levels compared to Cd treatment alone group, although not restored to the levels of control group. The population of apoptosis cells demonstrated that Cd induces the apoptosis of chicken splenic lymphocytes; in addition, increased mRNA level of Bak, p53, caspase-3, caspase-9, and cytochrome c (Cyt c) and decreased Bcl-2, Bcl-xl, and CaM were observed in Cd treatment group. Se ameliorated ΔΨm and [Ca(2+)]i for mitochondria function restoring, and Se was able to modulate the expression of relative genes. In conclusion, concurrent treatment with Se reduced the Cd-induced morphological changes and oxidative stress, ion disorder, and apoptosis, suggesting that the toxic effects of Cd on the chicken splenic lymphocytes were partly meliorated by Se.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Linfócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Selênico/farmacologia , Baço/metabolismo , Animais , Cádmio/toxicidade , Galinhas , Linfócitos/patologia , Malondialdeído/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Oxirredutases/metabolismo , Selênio/farmacologia , Baço/patologia
19.
J Neurol Neurosurg Psychiatry ; 85(10): 1149-52, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24828896

RESUMO

BACKGROUND: Autosomal dominant (AD) central core disease (CCD) is a congenital myopathy characterised by the presence of cores in the muscle fibres which correspond to broad areas of myofibrils disorganisation, Z-line streaming and lack of mitochondria. Heterozygous mutations in the RYR1 gene were observed in the large majority of AD-CCD families; however, this gene was excluded in some of AD-CCD families. OBJECTIVE: To enlarge the genetic spectrum of AD-CCD demonstrating mutations in an additional gene. PATIENTS AND METHODS: Four affected AD family members over three generations, three of whom were alive and participate in the study: the mother and two of three siblings. The symptoms began during the early childhood with mild delayed motor development. Later they developed mainly tibialis anterior weakness, hypertrophy of calves and significant weakness (amyotrophic) of quadriceps. No cardiac or ocular involvement was noted. RESULTS: The muscle biopsies sections showed a particular pattern: eccentric cores in type 1 fibres, associated with type 1 predominance. Most cores have abrupt borders. Electron microscopy confirmed the presence of both unstructured and structured cores. Exome sequencing analysis identified a novel heterozygous missense mutation p.Leu1723Pro in MYH7 segregating with the disease and affecting a conserved residue in the myosin tail domain. CONCLUSIONS: We describe MYH7 as an additional causative gene for AD-CCD. These findings have important implications for diagnosis and future investigations of AD-congenital myopathies with cores, without cardiomyopathy, but presenting a particular involvement of distal and quadriceps muscles.


Assuntos
Miosinas Cardíacas/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto/genética , Miopatia da Parte Central/genética , Cadeias Pesadas de Miosina/genética , Adulto , Idoso , Feminino , Heterozigoto , Humanos , Masculino , Fibras Musculares de Contração Lenta/diagnóstico por imagem , Fibras Musculares de Contração Lenta/patologia , Fibras Musculares de Contração Lenta/ultraestrutura , Miopatia da Parte Central/diagnóstico por imagem , Miopatia da Parte Central/patologia , Linhagem , Radiografia
20.
Hum Mutat ; 35(5): 625-36, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24610732

RESUMO

Balanced chromosomal rearrangement (or balanced chromosome abnormality, BCA) is a common chromosomal structural variation. Next-generation sequencing has been reported to detect BCA-associated breakpoints with the aid of karyotyping. However, the complications associated with this approach and the requirement for cytogenetics information has limited its application. Here, we provide a whole-genome low-coverage sequencing approach to detect BCA events independent of knowing the affected regions and with low false positives. First, six samples containing BCAs were used to establish a detection protocol and assess the efficacy of different library construction approaches. By clustering anomalous read pairs and filtering out the false-positive results with a control cohort and the concomitant mapping information, we could directly detect BCA events for each sample. Through optimizing the read depth, BCAs in all samples could be blindly detected with only 120 million read pairs per sample for data from a small-insert library and 30 million per sample for data from nonsize-selected mate-pair library. This approach was further validated using another 13 samples that contained BCAs. Our approach advances the application of high-throughput whole-genome low-coverage analysis for robust BCA detection-especially for clinical samples-without the need for karyotyping.


Assuntos
Aberrações Cromossômicas , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Translocação Genética , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Mapeamento Cromossômico , Humanos , Cariotipagem
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