Connexin 43 ; genetics ; Connexins ; genetics ; Heart Defects, Congenital ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Infant, Newborn ; Mutation ; genetics ; Myocardium ; metabolism ; Transcription Factors ; genetics

OBJECTIVEThe purpose of this investigation was to identify the novel genetic mutations in patients with a congenital atrial septal defect (ASD).

METHODSClinical data and blood specimens from a total of 12 unrelated ASD pedigrees and a cohort of 168 unrelated subjects with sporadic ASD were collected and evaluated in contrast to 200 healthy individuals. The whole exons and partial flanking introns of NKX2-5 gene were amplified by polymerase chain reaction and sequenced using the di-deoxynucleotide chain termination approach. The acquired sequences were aligned with those publicized in GenBank by the aid of programme BLAST to identify the sequence variations. The software ClustalW was applied for analysis of the conservative of the altered amino acids.

RESULTSA novel heterozygous mutation of NKX2-5 gene, i.e., a substitution of thymine for cytosine at nucleotide 536, predicting the conversion of serine into phenylalanine at amino acid residue 179, was identified initially in a proband. The same missense mutation was thereafter detected in other 3 affected members of the identical family but neither in the healthy members of the kindred nor in the 200 normal controls. A cross-species alignment of the protein sequences encoded by NKX2-5 gene displayed that the mutated amino acid was highly conserved evolutionarily. No mutation of NKX2-5 gene was observed in the other 11 ASD pedigrees or in 168 patients with sporadic ASD. Additionally, a common synonymous single nucleotide polymorphism, a transition of adenine (A) into guanine (G) at nucleotide 63, was found in NKX2-5 gene. However, there were no significant differences in the prevalence of alleles A and G between ASD patients and healthy controls (chi(2) = 2.8641, P = 0.0906).

CONCLUSIONA novel mutation of NKX2-5 gene identified in an ASD family suggests that mutated NKX2-5 gene is responsible for familial ASD.

Adolescent ; Amino Acid Sequence ; Child ; Child, Preschool ; Exons ; Female ; Heart Septal Defects, Atrial ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Male ; Mutation ; Transcription Factors ; genetics

OBJECTIVETo explore the differences of NKX2.5 and TBX5 gene mutations between in vitro fertilization (IVF) children with congenital heart disease (CHD) and naturally conceived children with CHD.

METHODSBlood samples from 68 IVF children with CHD and 98 naturally conceived children with CHD were collected. The mutations in coding regions 1 and 2 of the NKX2.5 gene, and coding regions 4, 5, and 8 of the TBX5 gene were examined by polymerase chain reaction (PCR) and DNA sequencing.

RESULTSAn A-to-G mutation at nucleotide 63 (c.63A>G) in coding region 1 of the NKX2.5 gene was found in both IVF and naturally conceived children with CHD. There were no significant differences in genotype and allele frequencies at c.63A>G locus of the NKX2.5 gene between the two groups. No mutations were detected in coding region 2 of the NKX2.5 gene and coding regions 4, 5 and 8 of the TBX5 gene.

CONCLUSIONSThere is no difference in NKX2.5 and TBX5 gene mutations between IVF and naturally conceived children with CHD. Therefore, it is presumed that assisted reproductive technology may not lead to mutations in the NKX2.5 and TBX5 genes.

Child, Preschool ; Female ; Fertilization in Vitro ; Heart Defects, Congenital ; genetics ; Homeobox Protein Nkx-2.5 ; genetics ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; T-Box Domain Proteins ; genetics

The present study aimed to observe the morphological distribution of bone marrow (BM)-derived Nkx2-5(+) cardiac progenitor cells (CPCs) in bone marrow niche and evaluate the effect of acute myocardial ischemia (AMI) on the mobilizion of BM-derived Nkx2-5(+) CPCs. Animal models of BALB/c mouse AMI, cerebral and hind-limb ischemia were established. Nanogold labeling method, immunofluorescence and Western blot were used to identify the distribution of BM-derived Nkx2-5(+) CPCs and the expressions of Nkx2-5 protein in peripheral blood and BM after AMI. Meanwhile, in different ischemia organ models and after AMD3100 (SDF-1/CXCR4 antagonist) pretreatment in AMI model, Nkx2-5 protein expressions in peripheral blood were also assayed. Nkx2-5(+) CPCs were found to locate in cavitas medullaris. The percentage of Nkx2-5(+) CPCs in blood increased immediately after AMI. Nkx2-5 protein expression in peripheral blood was also upregulated at the timepoint of 24 h post-AMI (P<0.01) and kept stable without further enhancement from day 1 to day 7 post-AMI. In BM, Nkx2-5 protein expression was upregulated immediately after AMI and downregulated afterwards (P<0.01). After AMD3100 pretreatment in AMI group, Nkx2-5 protein expression was significantly inhibited in peripheral blood (P<0.05). In cerebral and hind-limb ischemia models, Nkx2-5 protein expressions were significantly lower than that in AMI group (P<0.01), but with no significant difference to control group. These results suggest that Nkx2-5(+) CPCs are physiologically resident in BM and AMI initiates mobilization of BM-derived Nkx2-5(+) CPCs in a predominant organ-specific manner. In the procedure of mobilization, SDF-1 may play a critical role in a chemoattracted manner.
Animals ; Bone Marrow ; metabolism ; Hematopoietic Stem Cell Mobilization ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Myocardial Infarction ; metabolism ; Myocardium ; cytology ; Stem Cells ; cytology ; Transcription Factors ; metabolism

OBJECTIVETo study the association of single nucleotide polymorphisms (SNPs) of transcription factors (NKX2.5, GATA4, TBX5, and FOG2) with congenital heart disease (CHD) in the Chinese population.

METHODSPubMed, Google Scholar, CNKI, Wanfang Data, and Weipu Data were searched for articles on the association of SNPs of target genes with CHD in the Chinese population. If one locus was mentioned in at least two articles, the random or fixed effect model was used to perform a pooled analysis of study results and to calculate the pooled OR and its 95%CI. If a locus was mentioned in only one article, related data were extracted from this article to analyze the association between the SNPs of this locus and CHD.

RESULTSTwenty-three articles were included. The Meta analysis showed that there were significant differences between the CHD and control groups in the genotype and allele frequencies of GATA4 rs1139244 and rs867858 and the genotype frequency of GATA4 rs904018, while there were no significant differences in the SNPs of the other genetic loci between the two groups. The single-article analysis showed that there were significant differences between the two groups in the allele frequencies of NKX2.5 rs118026695/rs703752, GATA4 rs884662/rs12825/rs12458/rs3203358/rs4841588, and TBX5 rs6489956. There were no significant differences in the SNPs of FOG2 locus between the two groups.

CONCLUSIONSThe SNPs of some loci in NKX2.5, GATA4, and TBX5 are associated with CHD in the Chinese population, but the association between the SNPs of FOG2 locus and the development of CHD has not been found yet.

Asian Continental Ancestry Group ; genetics ; DNA-Binding Proteins ; genetics ; GATA4 Transcription Factor ; genetics ; Genetic Predisposition to Disease ; Heart Defects, Congenital ; genetics ; Homeobox Protein Nkx-2.5 ; genetics ; Humans ; Polymorphism, Single Nucleotide ; T-Box Domain Proteins ; genetics ; Transcription Factors ; genetics

To study the function and potential application of nkx2.5, a critical gene for heart development, we constructed a recombinant adenovirus overexpressing nkx2.5 gene (Ad-Nkx2.5) with the AdEasy system. To evaluate the effect and mechanism of Ad-Nkx2.5 against oxidative injury, the H9c2 myocardial cells were infected with the recombinant adenoviruses Ad-Nkx2.5 or Ad-EGFP, and subsequently exposed to H2O2 to induce apoptosis. The anti-apoptotic potential of Ad-Nkx2.5 was validated by MTT assay for cell viability, Hoechst33342 staining for cellular morphology, and immunoblotting for caspase-3 activity. Ad-Nkx2.5 infection led to an increased survival rate of H9c2 cells and decreased the amount of caspase-3 in an active form. Additionally, overexpression of Nkx2.5 inhibited the release of cytochrome C from the mitochondria into the cytosol. Mechanismic studies showed that Nkx2.5 upregulated bcl-2 gene expression and significantly repressed H2O2-induced expression of bax detected by Real-time PCR. Additionally, H2O2 treatment did not affect the nuclear localization of Nkx2.5. These findings indicate that adenovirus-mediated nkx2.5 gene transfer exerted a protective effect on H9c2 cells against H2O2-induced apoptosis via mitochondrial pathway, and the Nkx2.5-mediated expression modulation of apoptosis-associated genes could be involved in this event.
Adenoviridae ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Genetic Vectors ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; biosynthesis ; genetics ; Hydrogen Peroxide ; pharmacology ; Myocytes, Cardiac ; cytology ; Oxidative Stress ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the role of homeobox gene Nkx2-5 in cardiac myogenesis.

METHODSTwo P19 cell lines, namely cells transfected with exogenous expression of Nkx2-5 gene and non-transfected cells, were cultured in suspension for 4 days to induce cell aggregation, and the cell aggregates were transferred to the Petri dish for further adherent culture. On days 4, 8, 12 and 16 of adherent culture, the expressions of α-sarcomeric actin (α-SA) and cardiac troponin T (cTnT) protein were detected by immunocytochemistry, and the mRNA expressions of GATA-4, α-myosin heavy chain (α-MHC) and atrial natriuretic factor (ANF) genes by RT-PCR.

RESULTSIn the transfected cells, α-SA and cTnT protein expressions were detected on days 8, 12 and 16 of adhere culture, and their expressions increased gradually with time. α-SA and cTnT expression was significantly higher on day 16 than on day 8 of culture (P<0.01). RT-PCR analysis of the transfected cell showed the presence of GATA-4 expression on day 4 of adherent culture, and the expression increased on days 8 and 12 but decreased on day 16. ANF and α-MHC expressions were found on days 8, 12, and 16, increasing gradually over time and showing significant differences from those on day 4 (P<0.05 or P<0.01). The expression of α-MHC was significantly higher on days 12 and 16 than on day 8 (P<0.05 or P<0.01), and ANF expression was significantly higher on day 16 than on days 8 and 12 (P<0.01). The non-transfected cells were negative for the expressions of all these genes.

CONCLUSIONExogenous expression of Nkx2-5 gene can induce P19 cells to express cardiac markers in vitro.

Actins ; metabolism ; Animals ; Atrial Natriuretic Factor ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cell Line ; GATA4 Transcription Factor ; metabolism ; Gene Expression ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; metabolism ; Myosin Heavy Chains ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection ; Troponin T ; metabolism

BACKGROUNDCongenital heart disease (CHD) is the most common developmental anomaly in newborns. The germline mutations in GATA4 and NKX2.5 genes have been identified as responsible for CHD. The frequency of GATA4 and NKX2.5 mutations in Chinese Uygur patients with CHD and the correlation between their genotype and CHD phenotype are unknown.

METHODSWe examined the coding region of GATA4 and NKX2.5 genes in 62 Chinese Uygur patients with CHD and 117 Chinese Uygur individuals as the controls by denaturing high performance liquid chromatography (DHPLC) and sequencing.

RESULTSTwo heterozygous missense mutations of c.1220C > A and c.1273G > A in GATA4 gene, which cause the amino acid residue changes of P407Q and D425N in GATA4, were found in a patient with tetralogy of Fallot and a patient with ventricular septal defect, respectively. The two patients did not have atrioventricular conduct defects or non-cardiac abnormalities. The two mutations are expected to affect the protein function. There were no reported NKX2.5 mutations in the patients.

CONCLUSIONOur results provided the primary data on CHD phenotype associated with GATA4 mutation in the Chinese Uygur population.

Asian Continental Ancestry Group ; genetics ; DNA Mutational Analysis ; Female ; GATA4 Transcription Factor ; genetics ; Genetic Predisposition to Disease ; Heart Defects, Congenital ; genetics ; Heart Septal Defects, Ventricular ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; Humans ; Male ; Mutation, Missense ; genetics ; Polymerase Chain Reaction ; Tetralogy of Fallot ; genetics ; Transcription Factors ; genetics

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.
Animals ; Antioxidants ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclic GMP ; metabolism ; Embryoid Bodies ; drug effects ; metabolism ; physiology ; Enzyme Activators ; pharmacology ; Gene Expression ; drug effects ; Guanylate Cyclase ; genetics ; metabolism ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Mice ; Myosin Heavy Chains ; genetics ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Donors ; pharmacology ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Nitroso Compounds ; pharmacology ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Second Messenger Systems ; Transcription Factors ; genetics ; metabolism ; Troponin ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; metabolism