[The mechanism of TBX5 abnormal expression in simple congenital heart disease].

To explore the mechanism of TBX5 abnormal expression in simple congenital heart disease (CHD), 100 CHD venous blood, 50 CHD heart tissues, and 5 non-CHD heart tissues were involved in this study. The mutation and methylation in the 1 200 bp region upstream of TBX5 gene were detected by high-performance liquid chromatography (DHPLC) and methylation-sensitive restriction endonuclease (MS-RE), respectively. The binding site of NKX2-5 to Tbx5 predicted by P-MATCH software was validated by EMSA (Electrophoretic mobility shift assay). Tbx5 gene expression in mouse cardiac muscle cell H9C2(2-1) transfected with NKX2-5 expression vector was evaluated. No mutation was found in all patients. Both non-CHD and CHD heart tissues had the same methylation in the two CpG islands. Exogenous Nkx2-5 efficiently activated the transcription of the endogenous Tbx5 gene in H9C2 (2-1) cells. EMSA showed that the special binding band appeared when Nkx2-5 existed. These results indicates that the down expression of TBX5 might not be caused by mutation and methylation in the 1 200 bp region upstream of gene, and might be regulated by abnormal expression of NKX2-5 gene in heart muscle of CHD.

Association of the GLI gene with ventricular septal defect after the susceptibility gene being narrowed to 3.56 cM in 12q13.

BACKGROUND: Our previous research has suggested that genes around D12S1056 in 12q13 may confer susceptibility to ventricular septal defect (VSD) in humans. The present study was to define the chromosome region assignment by transmission disequilibrium test (TDT), and to identify the important candidate gene by family-based association study and haplotype analysis. METHODS: Surrounding D12S1056, ten microsatellite markers including D12S329, D12S305, D12S1662, D12S1056, D12S1293, D12S334, D12S102, D12S83, D12S1655 and D12S1691 were chosen, and TDT was performed in 62 nuclear family trios each consisting of an affected child and two healty parents. Subsequently, the GLI gene, a positional candidate gene that maps to the target region, was selected for further analysis. Three single nucleotide polymorphisms (SNPs), G11888C, G11388A, and G11625T, were selected for family-based association study and haplotype analysis. RESULTS: VSD was significantly associated with all selected markers except D12S1691 [72.2 centi morgen (cM)] and D12S1700 (75.76 cM). VSD was also significantly associated with G11888C (chi(2) = 5.918, P = 0.015), G11388A (chi(2) = 8.067, P = 0.005), and G11625T (chi(2) = 11.842, P = 0.001). Haplotype analysis showed a strong linkage disequilibrium between G11888C and G11388A (D' = 0.999), but in significant (chi(2) = 1.035, df = 2, P > 0.05). CONCLUSIONS: The susceptibility gene of VSD was mapped to 3.56 cM in 12q13 by TDT, and the GLI gene, an important candidate in the target region, was associated with VSD.

Cloning of TBX5, a key gene during heart formation and its expression in rat embryonic heart.

To study the expression of TBX5, a key gene during heart formation, full-length TBX5 cDNA was cloned from Wistar rat embryonic heart (GenBank Accession No. AY859491). The cDNA and predicted amino acid sequences were different from those previously reported in GenBank. The expression profile of TBX5 in rat tissues was studied by RT-PCR and Northern blot. TBX5 was expressed in many rat tissues as a single transcript, and the highest level of expression was found in the heart. For subcellular localization, TBX5 was expressed as an EGFP fusion protein in rat hepatocarcinoma cells. Results showed that TBX5 was nuclear. In addition, TBX5-GST fusion protein was obtained by prokaryotic expression. These findings provide a good basis for further identification of TBX5-related transcription factors and protein-protein interaction studies among each other.

Analysis of single nucleotide polymorphisms and haplotypes in HOXC gene cluster within susceptible region 12q13 of simple congenital heart disease.

OBJECTIVE: In the candidate region 12q13 of simple congenital heart disease(CHD), four single nucleotide polymorphisms(SNPs) in HOXC4 gene were chosen in order to investigate the distribution of SNP and haplotypes in simple CHD patients and normal people. METHODS: The genotype of 4 SNPs in 108 simple CHD patients and 200 normal people were analyzed by restriction fragment length polymorphism(RFLP) and denaturing high-performance liquid chromatography(DHPLC). The statistical contingency table method was used to analyze SNP genotype frequency and gene frequency in patients and control group; then, the haplotypes were established and their frequencies in the two groups were assessed by PHASE software. RESULTS: C16476T polymorphism was not detected; A17860G located in 3' flanking sequence of HOXC5 gene displayed significant difference between the two groups. The G allele frequency in simple CHD patients was higher than that in healthy controls(P < 0.05); the distribution of frequencies of 4 haplotypes showed significant difference(P < 0.01). CONCLUSION: The A17860G located in 3'flanking sequence of HOXC5 gene is associated with simple CHD; the risk of CHD in the persons with G17860 is higher than that in those with A17860. the haplotype of 3 SNPs may be linked with the susceptible gene of simple CHD.

Expression of MTLC gene in gastric carcinoma.

AIM: To investigate the expression of c-myc target from laryngeal cancer cells (MTLC) gene in gastric carcinoma (GC) tissues and the effect of MTLC over-expression on gastric carcinoma cell line BGC823. METHODS: RT-PCR was performed to determine the expression of MTLC mRNA in GC and matched control tissues. BGC823 cells were transfected with an expression vector pcDNA3.1-MTLC by liposome and screened by G418. Growth of cells expressing MTLC was observed daily by manual counting. Apoptotic cells were determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: The expression of MTLC mRNAs was down-regulated in 9(60%) of 15 cases of GC tissues. The growth rates of the BGC823 cells expressing MTLC were indistinguishable from that of control cells. A marked acceleration of apoptosis was observed in MTLC-expressing cells. CONCLUSION: MTLC was down-regulated in the majority of GC tissues and could promote apoptosis of GC cell lines, which suggests that MTLC may play an important role in the carcinogenesis of gastric carcinoma.

[Analysis on genetic polymorphism of 14 short tandem repeat loci on chromosome 7p14-15 and 12q13 in Chinese north Hans].

OBJECTIVE: To analyze the genetic polymorphism of 6 short tandem repeat (STR) loci on chromosome 7p14-15 and 8 STR loci on chromosome 12q13 in Chinese north Hans. METHODS: Fluorescence-labeling polymerase chain reaction and capillary electrophoresis were used to analyze the genetic polymorphism of 100 randomly selected individuals from Chinese north Han nationality at 6 STR loci (D7S1808, D7S2250, D7S2251, D7S683, D7S656 and D7S528) on chromosome 7p14-15 and 8 STR loci(D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137 and D12S102) on chromosome 12q13. RESULTS: In the Chinese north Han population, 7 alleles and 24 genotypes, 8 alleles and 27 genotypes, 7 alleles and 22 genotypes, 4 alleles and 10 genotypes, 6 alleles and 17 genotypes, 5 alleles and 13 genotypes were observed at D7S1808, D7S2250, D7S2251, D7S683, D7S656 and D7S528. The heterozygosities at the above 6 STR loci were 86%, 88%, 83%, 79%, 85% and 80%, respectively. Five alleles and 15 genotypes, 5 alleles and 15 genotypes, 8 alleles and 29 genotypes, 6 alleles and 17 genotypes, 6 alleles and 17 genotypes, 6 alleles and 19 genotypes, 5 alleles and 13 genotypes, 7 alleles and 24 genotypes were observed at D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137 and D12S102. The heterozygosities at the above 8 STR loci were 86%, 84%, 87%, 82%, 84%, 85%, 81% and 89%, respectively. CONCLUSION: The distributions of allele frequencies of 6 STR loci on chromosome 7p14-15 and of 8 STR loci on chromosome 12q13 were consistent with the Hardy-Weinberg equilibrium. The highly genetic polymorphism was observed in Chinese north Han population.

Cloning of hTERT cDNA fragment and application of anti-hTERT monoclonal antibody in mechanism of laryngeal carcinogenesis.

The mechanism of telomerase activation is still not clear till now. In order to understand the expressional mode of telomerase and the mechanism of telomerase activation in laryngeal carcinogenesis, we cloned a fragment of hTERT cDNA and prepared a monoclonal antibody against hTERT. We performed immuno-histochemical staining in laryngeal cancer tissues using this antibody. We found that the frequencies of hTERT positive cells were positively correlated with undifferentiation of cancer tissues and that the expression of hTERT was positively correlated with levels of c-Myc, which suggested that c-Myc might play an important role in activation of telomerase. These results revealed that overexpression of c-Myc upregulated telomerase, which in turn resulted in immortalization of laryngeal squamous cells, and this mechanism existed not only in the initiation of laryngeal carcinogenesis but also in the whole process of cancer development.

[Studies on the mutation and expression of TBX5 gene in human simple congenital heart disease].

This work is to investigate the mutation and expression of TBX5 gene in human simple congenital heart disease. The mutations of eight exons of TBX5 gene in 61 CHD family members (a total of 216 individuals including 65 patients and 151 normal relatives) were examined by PCR-DGGE. Using beta-actin as internal control, the differential expression between 34 myocardium samples from simple congenital heart disease patients and three normal controls was conducted by RT-PCR. There is no mutation detected in all samples; The mRNA expression levels of TBX5 gene show descent tendency in samples of simple congenital heart disease compared with normal controls. The mutations in coding region of TBX5 gene do not cause human simple congenital heart disease, but the abnormality in transcription level of TBX5 gene maybe a kind of mechanism causing human simple congenital heart disease.