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1.
Blood Cells Mol Dis ; 86: 102489, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32877852

RESUMEN

INTRODUCTION: Congenital fibrinogen disorders are characterized by heterogeneous clinical manifestations with mutations in the fibrinogen gene cluster. We aimed to describe the molecular genetics and clinical manifestations of fibrinogen abnormalities and perform genotype-phenotype correlations. MATERIALS AND METHODS: Genetic analysis of fibrinogen genes was performed by direct sequencing. The effect of the specific missense variants on fibrinogen structure and function was analyzed using PROVEAN and PolyPhen-2 algorithms and was predicted by protein modeling. RESULTS: Thirteen mutations, including five novel mutations, were identified in the three fibrinogen genes. There was poor correlation between genotypes and phenotypes. All but one of the novel mutations in subjects were predicted to be deleterious. Protein modeling predicted that multiple ienteractions with surrounding residues for novel variants were likely to result in congenital fibrinogen disorders. CONCLUSION: This study in a relatively large cohort of Chinese patients with congenital fibrinogen disorders enabled the identification of five new fibrinogen missense mutations. In silico modeling may represent a valuable tool for understanding amino acid residues from novel variants leading to congenital fibrinogen disorders, but it should be followed by functional studies. Clinical presentation of fibrinogen disorders was variable, possibly due to genetic and environmental modifiers.


Asunto(s)
Afibrinogenemia/genética , Fibrinógeno/genética , Mutación Missense , Adulto , Anciano , Pueblo Asiatico/genética , China , Femenino , Fibrinógeno/química , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación , Mutación Puntual , Adulto Joven
2.
Acta Haematol ; 143(5): 472-477, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31982874

RESUMEN

Both congenital hypodysfibrinogenemia and factor XI deficiency are rare coagulopathies caused by mutations within the fibrinogen and F11 genes, respectively. To investigate the pathogenesis of combined congenital hypodysfibrinogenemia with factor XI (FXI) deficiency in a Chinese family, coagulation assays, FXI activity (the 1-stage method), fibrinogen activity (the Clauss method), and antigen (prothrombin time [PT]-derived method) were performed. The sequences of fibrinogen genes and F11 were amplified by PCR and analyzed by direct sequencing. The proband as well as his grandmother, father, aunt, and sister showed a low plasma concentration of fibrinogen measured by the Clauss method and a slightly decreased result by the PT-derived method; finally, c.1097A>G in exon 8 of FGG was detected in the pedigree, which caused His340Arg mutation. His grandfather had a slightly prolonged activated partial thromboplastin time (APTT) due to low FXI activity. FXI deficiency was a compound heterozygote inherited with missense mutations of c.434A>G in exon 5 as well as c.1253G>T in exon 11 which caused HGV p.His145Arg and Gly400Val mutations, respectively. The grandfather had no qualitative or quantitative defect in fibrinogen. The proband and his father and aunt had c.434A>G at the exon 5 mutation site and no decrease in FXI activity. His mother had no fibrinogen or F11 gene mutations. Plasma fibrin polymerization was delayed. The proband in our study showed typical changes of congenital hypodysfibrinogemia in the clotting analyses with delayed fibrin polymerization, but although he was a heterozygous carrier of the c.434A>G variant in the F11 gene, he had no decrease in FXI activity and no bleeding tendency, thus questioning the pathogenicity of the identified variant in the F11 gene. To our knowledge, this is the first report of a case of combined hypodysfibrinogenemia and FXI deficiency confirmed by molecular genetic tests.


Asunto(s)
Afibrinogenemia/diagnóstico , Pueblo Asiatico/genética , Deficiencia del Factor XI/diagnóstico , Afibrinogenemia/complicaciones , Afibrinogenemia/genética , Niño , Análisis Mutacional de ADN , Exones , Factor XI/genética , Deficiencia del Factor XI/complicaciones , Deficiencia del Factor XI/genética , Fibrinógeno/genética , Heterocigoto , Humanos , Masculino , Mutación Missense , Linaje
3.
Mol Cell Biochem ; 390(1-2): 205-13, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24573885

RESUMEN

Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman's capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.


Asunto(s)
Proteínas de Homeodominio/genética , Riñón/crecimiento & desarrollo , Nefronas/embriología , Neurofibromina 1/genética , Factores de Transcripción/genética , Animales , Apoptosis/genética , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/biosíntesis , Riñón/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Neurofibromina 1/metabolismo , Organogénesis/genética , Factores de Transcripción/biosíntesis
4.
ScientificWorldJournal ; 2014: 682189, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25133251

RESUMEN

BACKGROUND: Ex vivo culture of intact embryonic kidney has become a powerful system for studying renal development. However, few methods have been available for gene manipulation and have impeded the identification and investigation of genes in this developmental process. RESULTS: Here we systemically compared eight different serotypes of pseudotyped self-complementary adenoassociated viruses (scAAVs) transduction in cultured embryonic kidney with a modified culture procedure. We demonstrated that scAAV was highly effective in delivering genes into and expressing in compacted tissues. scAAV serotypes 2 and 8 exhibited higher efficiency of transduction compared to others. Expression kinetics assay revealed that scAAV can be used for gene manipulation at the study of UB branching and nephrogenesis. Repressing WT1 in cultured kidney using shRNA impairs tubule formation. We for the first time employed and validated scAAV as a gene delivery tool in cultured kidney. CONCLUSIONS: These findings are expected to expedite the use of the ex vivo embryonic kidney cultures for kidney development research. For other ex vivo cultured organ models, scAAV could also be a promising tool for organogenesis study.


Asunto(s)
Dependovirus/genética , Riñón/metabolismo , Transducción Genética/métodos , Animales , Células HEK293 , Humanos , Riñón/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos/métodos
6.
Mol Med Rep ; 11(1): 503-8, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25334019

RESUMEN

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 and G1 in human THP-1 macrophage-derived foam cells. Furthermore, the present study aimed to investigate the role of the liver X receptor (LXR) α in this process. A model of oxidized low-density lipoprotein-induced foam cells from human THP-1 cells was established. The effect of FGF21 on cholesterol efflux was analyzed using a liquid scintillation counter. The expression of ABCA1 and ABCG1 was determined using quantitative polymerase chain reaction and western blot analyses. FGF21 was found to enhance apolipoprotein A1- and high-density lipoprotein-mediated cholesterol efflux. FGF21 was also observed to increase the mRNA and protein expression of ABCA1 and ABCG1. Furthermore, LXRα-short interfering RNA attenuated the stimulatory effects induced by FGF21. These findings suggest that FGF21 may have a protective effect against atherosclerosis by enhancing cholesterol efflux through the induction of LXRα-dependent ABCA1 and ABCG1 expression.


Asunto(s)
Colesterol/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Línea Celular , Femenino , Humanos , Receptores X del Hígado , Ratones , Receptores Nucleares Huérfanos/metabolismo , Regulación hacia Arriba
7.
PLoS One ; 9(1): e84893, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24416307

RESUMEN

The transcription activator-like effector nucleases (TALENs) strategy has been widely used to delete and mutate genes in vitro. This strategy has begun to be used for in vivo systemic gene manipulation, but not in an organ-specific manner. In this study, we developed a modified, highly efficient TALEN strategy using a dual-fluorescence reporter. We used this modified strategy and, within 5 weeks, we successfully generated kidney proximal tubule-specific gene Ttc36 homozygous knockout mice. Unilateral nephrectomy was performed on the 6-week-old founders (F0) to identify the knockout genotype prior to the birth of the offspring. This strategy was found to have little effect on reproduction in the knockout mice and inheritability of the knockout genotypes. The modified TALEN knockout strategy in combination with unilateral nephrectomy can be readily used for studies of gene function in kidney development and diseases.


Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Técnicas de Inactivación de Genes/métodos , Riñón/crecimiento & desarrollo , Animales , Secuencia de Bases , Núcleo Celular/genética , Mutación del Sistema de Lectura , Genes Reporteros/genética , Células HEK293 , Recombinación Homóloga/genética , Homocigoto , Humanos , Riñón/citología , Riñón/metabolismo , Riñón/cirugía , Ratones , Nefrectomía , Fenotipo , Factores de Tiempo
8.
Proteomics Clin Appl ; 7(11-12): 829-38, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23687078

RESUMEN

PURPOSE: Apolipoprotein E knockout (apoE-/- ) mouse is one of the most popular models for cardiovascular research, especially in the study of atherosclerosis. Naturally, large amount of studies try to uncover the role of apoE in atherosclerosis, and indeed apoE plays an important role in this pathogenesis. Kidney is an organ that contains lots of capillaries and also largely expresses apoE. Moreover, a protective role of apoE in kidney as an autocrine regulator has been demonstrated previously, however, the underlying mechanism is largely unknown. EXPERIMENTAL DESIGN: In this study, comparative proteomics is for the first time used to identify the differential proteins in kidneys of apoE-/- and wild type (WT) mice, respectively, and we try to reveal the signaling network of apoE in mice kidney using bioinformatics analysis. RESULTS: Our findings show that approximately 80 proteins are significantly differentially expressed in kidneys of apoE-/- and WT mice, and the signaling network correlated to apoE is successfully established by employing bioinformatics assay. CONCLUSIONS AND CLINICAL RELEVANCE: Taken together, we originally identify the proteins with differential expression and propose an apoE correlated molecular network in mice kidney. These findings further provide evidence of the role of apoE in mice kidney and a brand new perspective in the protection and treatment of kidney disease.


Asunto(s)
Apolipoproteínas E/deficiencia , Riñón/metabolismo , Proteómica/métodos , Transducción de Señal , Animales , Apolipoproteínas E/metabolismo , Análisis por Conglomerados , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica , Genotipo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Ionización de Electrospray
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