DNA Sequence Analysis And Expression Of TGFβ2Gene In Children With Congenital Conotruncal Defects

Congenital heart disease (CHD) is the most common birth defect, which affects9.1in1000live births worldwide and has became the congenital malformations with the highest incidence in our country. Congenital conotruncal defects (CTD) accounts for30%of the CHD and is caused by the abnormal development of the outflow tract (OFT), which including tetralogy of Fallot (TOF), double outlet right ventricle (DORV), transposition of the great arteries (TGA), persistent truncus arteriosus (PTA) and pulmonary atresia (PA) and is associated with hypoxemia and serious acidosis. CTD could lead to high mortality and bring many social problems. Approximately80%of the CHD is multifactorial, which results from abnormal interactions between genetic and environmental factors. In the past few decades, pathogenic mutations in several genes including transforming growth factor β2(TGFβ2) gene have been proposed to play an important role in the pathogenesis of CHD. TGFβ2is a member of the highly conserved TGFP superfamily with various effects on cell growth and differentiation. Targeted disruption of the TGFP2gene in the TGFβ2-knockout mice leads to a wide spectrum of cardiovascular abnormalities including double-outlet right ventricle, atrioventricular canal defects, and aortic arch interruption. Downregulation of the TGFP2expression in the retinoic acid-treated mice has also been shown to induce defect of the OFT septum. Moreover, Kubalak et al. have shown that elevated TGFP2expression enhances apoptosis and contributes to the abnormal development of outflow tract and aortic sac in embryos of retinoic X receptor alpha (RXRA) knockout mice. Although much evidence indicates that TGFβ2is a critical mediator of the OFT formation and differentiation during cardiac development in animals, limited data are available regarding whether TGFP2gene is mutated and/or abnormally expressed during the pathogenesis of congenital conotruncal defects (CTD) in human. To address this question, we analyzed the sequence and expression of TGFβ2gene in400children with CTD. The results will help to illustrate the pathogenesis of conotruncal defects. Part ⅠSequence analysis in the coding region of TGFβ2gene in conotruncal defects patientsObjectives:To detect the sequence variation in the coding region of TGFβ2gene with CTD patients and clarify whether the variations are susceptible for CTD.Methods:A cohort of400pediatric patients with CTD was recruited in the study. PCR and DNA sequencing were performed for the detections of DNA variations of TGFβ2gene in CTD patients.Results:The diagnoses of these400patients include TOF, DOLV (double outlet left ventricle), DORV, TGA and PA. We did not find any pathogenic mutations of the TGFβ2gene except for one silent variation (c.597T>C) in the exon4in one patient. The encoded protein remained to be isoleucine (p.I1991) in this case.Conclusions:Detected the coding region of TGFβ2gene in400CTD patients, we did not find any pathogenic mutations or single nucleotide polymorphisms (SNPs) except for one silent variation in one case. The result indicates that germline mutation of the TGFβ2gene is not a common cause of CTD in human.Part ⅡExpression of TGFP2mRNA in the outflow tract myocardium tissues of conotruncal defects patientsObjectives:To understand whether TGFβ2gene was abnormally expressed in the myocardial tissue of OFT in children with CTD, we investigate the mRNA expression of TGFP2in the outflow tract myocardium tissues between healthy people and children with CTD. Moreover, we also detect TGFβ2mRNA expression with normal fetuses.Methods:The mRNA expression of TGFβ2in the myocardium tissues obtained from57children with CTD,18healthy people (13adults and5age-matched children), and20aborted fetuses were analyzed with real-time PCR.Results: 1. No difference was observed for the TGFβ2mRNA expression between children with CTD and the controls.2. The expression levels of TGFβ2mRNA in the myocardium of healthy people and children with CTD were both significantly lower than the levels in the fetal myocardium.3. Expression of mRNA was gradually reduced as the gestational age increased. That is, expression of TGFβ2in myocardium was negatively correlated with gestation.Conclusions:1. There was no significant difference between the children with CTD and the controls, which suggested that downstream molecular rather than TGFβ2itself work directly on heart development. Members of the TGFβ gene superfamily are similar in structure, sometimes overlapping expression patterns, and qualitatively similar activities, which suggest that TGFβ1-3have redundant functions and lack of one TGFβ isoform may be compensated by other isoforms. Our results indicate that TGFβ2gene may be less critical in humans in the pathogenesis of heart abnormalities.2. We found that human expression pattern of TGFβ2was time-specific during embryogenesis, and the mRNA expression was maintained throughout postnatal life, which may partly reflect the expression level in embryonic.Part ⅢExpression of TGFβ2protein in the outflow tract myocardium tissues of CTD patientsObjectives:To examine the protein expression level of TGFβ2in the outflow tract myocardium tissues of children with CTD, and to illustrate its association with the formation of CTD.Methods:The protein expression of TGFβ2gene was detected by immunohistochemistry with12children of CTD and3age-matched healthy children.Results:The protein expression was not significant different between CTD group and control group. Conclusions:The protein expression of TGFβbetween patients with CTD and healthy children showed no significant difference, which provided further evidence for the concept that TGF02gene may be less critical in humans in the pathogenesis of heart abnormalities.