1,TBX5 Gene And Cardiac Development 2,Analysis Of SNP And Haplotype In HOXC Gene Cluster Within Susceptible Region 12q13 Of Simple Congenital Heart Disease

Congenital Heart Disease ( CHD) belongs to complex disease, whose predisposing genes are still unknown. CHD is a common congenital malformation that does severe harm to newborn's health, occurring in about 1% of live births and 10% of stillbirths. Studies of the developmental genetics in embryogenesis and organogenesis can provide insights into the pathogenesis of many diseases. Cardiac development is a earlier period event, which not only involves the coordinated expression and strict interaction between many genes during embryogenesis, but also requires intricate cell migration, differentiation and proliferation. By studying Drosophila, chicken and mouse embryos, some genes related to cardiac development have been found. Among them, TBX5 is an important transcription factor during heart formation.TBX5 gene is expressed in the developing heart, limb bud, lung and trachea at an early stage. TBX5 expression is precisely controlled in the developing heart and limbs in human and mice. Haploinsufficiency or overexpression of TBX5 gene causes heart abnormity. TBX5 interacts with other two cardiac transcription factors - NKX2.5 and GATA4, playing a central role during early cardiac development. But the regulation mechanisms and pathways are unknown. Studies suggested that inter - regulation mechanisms maybe exist among the 3 transcription factors. The human TBX5 gene was cloned in 1997 and its mutations caused Holt - Oram syndrome ( HOS). Choosing suitable experimental animals to study cardiac development is of great importance. Because it is impossible to make full use of the human embryos, in order to identify TBX5 - related transcription factors and study interaction between them, we choose Wistar rat for further investigation. There was no related reports of rat Tbx5 gene, so we i-solated embryonic heart from E13 rat embryo, cloned Tbx5 cDNA and studied its biological function, such as expression profile, subcellular localization and fusion protein expression.As a marker of cardiac precursor cell differentiation, Nkxl. 5 belongs to Homeobox gene family and plays a key role during cardiac development of fruit, mouse and human. Nkx2. 5 regulates many downstream genes expression during heart formation. There is interaction between NKX2. 5 and TBX5 transcription factors, but it is still not clear whether there exist upstream or downstream regulation between the two transcription factors. Our primary studies suggest that besides of interaction, NKX2. 5 gene maybe locate upstream of TBX5 gene and regulate TBX5 gene expression during the regulative network involved in cardiac development. In this study, we identified two Nkx2. 5 binding sites in 600bp sequence up to transcription start site of TBX5 gene by Electrophoretic Mobility Shift Assay (EMSA) and cell line transfection.Section ICloning of Tbx5 , A Key Gene during Heart Formation and its Expression in Rat Embryonic HeartMaterials and MethodsTotal RNA was extracted from El3 rat embryonic heart and first strand cD-NA was obtained by reverse transcription. Primers was designed according to the predicted sequence in GeneBank. Tbx5 cDNA was cloned by PCR and confirmed by sequencing. RT - PCR and Northern blot was applied to examine the expression pattern of Tbx5 in rat tissues. Subcellular localization of Tbx5 gene was studied by transfection of green fluorescent protein. Finally, we constructed Tbx5 expression vector and induced GST - Tbx5 fusion protein expression in E. coli. BL21.ResultsWe cloned TbxS cDNA sequence and submitted it to GeneBank ( Genbank Ace. No. AY859491). BLAST results showed that sequence homology was 99% compared to the predicted sequence in GeneBank (Ace. No. XM222201) , but lacked base the 705 - 994bp sequence. Analysis of BLAST/P indicated that the rat Tbx5 protein was extremely conserved (94% sequence homology) to that of mouse and human. TbxS was expressed as a single transcript in many rat tissues and had the highest expression level in heart. In subcellular localization of Tbx5 Protein, the fluorescence signal was concentrated in the nuclei. After constructing expression vector of Tbx5 , the GST - Tbx5 fusion protein was obtained by prokaryotic expression.Section IITBX5 Gene Expression is Regulated Through Two Nkx2. 5 Binding ElementsMaterials and methodsUsing P - Match software, we predicted two possible Nkx2. 5 binding elements at - 139-----142 and -312 ~ 315 in 600 bp sequence up to TBXS genetranscription start site. The nucleotide sequences of locus - specific oligonucle-otides and mutation probes were designed;Nuclear extracts were prepared from myocardium of mouse. Two Nkx2.5 binding elements were identified by electro-phoretic mobility shift assay. TBX5 promoter sequence of human and Nkxl. 5 cD-NA of mouse were cloned, respectively. Then we constructed luciferase report vector pGL3 - TBX5 and eukaryotic expression vector pcDNA3. 0 - Nkxl. 5 . By transfecting different plasmids into C0S7 cell line, the roles of Nkx2.5 proe-in to TBX5 gene was observed.ResultsEMS A confirmed the protein - DNA interaction at NKX2. 5 A and NKX2. 5B sites. These two complexes were competed away by the unlabeled probes, but not by the oligonucleotide carrying mutations at the TAAT sequences. The bands were identical with NKX2. 5 consensus. Enzyme digesting and sequencing confirmed that we had constructed luciferase reporter vector pGL3 - TBX5 and eukaryotic expression vector pcDNA - Nkx2. 5 . The cell transfection results clearly demonstrate that 650bp up to transcription start site of TBX5 gene are a-ble to drive luciferase reporter expression. The luciferase activity of COS7 cells increase significantly when co - transfection of pcDNA - Nkx2. 5 constructs.Conclusion1. Rat Tbx5 cDNA sequence was firstly cloned ( GenBank Ace. No. AY859491).2. Tbx5 was expressed as a single transcript in many rat tissues and had the highest expression level in heart. Tbx5 protein was expressed in the nuclei.3. Expression vector of Tbx5 was constructed, and GST — Tbx5 fusion protein was obtained by prokaryotic expression.4. Luciferase reporter vector pGL3 - TBX5 and eukaryotic expression vector pcDNA - Nkx2. 5 were constructed, respectively.5. Two Nkx2. 5 binding elements were identified which located in 600bp sequence up to transcription start site of TBX5 . It was confirmed that Nkx2. 5 could regulate TBX5 gene expression as enhancer.Part II Analysis of SNP and haplotype in HOXC genecluster within candidate region 12ql3 of simple congenital heart diseaseAbstractCongenital heart disease (CHD) is the most common congenital malformation that does severe harm to the newborn s health, occurring in about 1 % of live births and 10% of stillbirths. About 80% of CHD only manifest malformations in cardiovascular system without any abnormality of other systems, which is named simple CHD. CHD is a complex genetic disorder with heritability estimated from 55% to 65% , and it is the clinical manifestation of anomalies in heart development. But the model of inheritance and penetrance of CHD is unclear, the predisposing genes of which is still unknown.We have located a 3.56cM candidate region of CHD within 12ql3.1 — 13. 3 by TDT for 61 nuclear family trios consisting of simple CHD patients and their parents . There is an important gene cluster in the region - HOXC gene cluster. In this study, we chose 4 single nucleotide polymorphisms in the region oiHOXC 4,5,6 to assess whether these HOXC gene were associated with simple CHD. We performed an independent case - control association study and haplotype a-nalysis in 108 CHD patients and 200 normal controls by restriction fragment length polymorphism ( RFLP) and denatured high - performance liquid chroma-tography ( DHPLC ) combined with sequencing.Materials and MethodsFourSNPs (rs894737, rs2071448, rs2071450, rs2277371) were chosenfrom the region of H0XC4 gene, which overlap another two HOXC genes- H0XC5 and H0XC6 , the related location of which in H0XC4 gene areG7471T, C16476T, A17860G, A36130G. PCR products were amplified afterthe genomic DNA was extracted from peripheral leukocytes. The genotypes ofG7471T and A36130G were identified by Mspl restriction enzyme. The genotype of C16476T and A17860G were identified using WAVE 2100 DNA fragment a-nalysis system ( DHPLC ). The genotypic frequencies, allele frequencies and haplotype frequencies in different groups were analyzed by statistical software.ResultsThe length of PCR products was consistent with prediction. G7471T, A36130G and A17860G polymorphisms were confirmed and C16476T polymorphism was not existed. The association analysis at single locus showed significant differences in the genotype and allele frequency of SNP G17860A between patients and controls. The analytical results of haplotype frequencies showed that G7471/G17860/G36130 and G7471/G17860/A36130 were common haplo-types. Four haplotype frequencies of these three polymorphisms showed significant differences between patients and controls. Haplotype frequencies of G7471/ G17860/G36130 and T7471/A17860/A36130 were higher in patients than those in control subjects.Conclusion1. A17860G located in H0XC5 gene 3' - flanking sequence is associated with simple CHD, and the risk to acquire CHD would increase in the individuals with G allele.2. Haplotype frequencies of G7471/G17860/G36130 and T7471/A17860/ A36130 are higher in CHD patients than those in normal controls. Suscepitibile predisposing genes of simple CHD maybe locate within this haplotype region.