Ventricular Septal Defect In Patients With Myocardial Tissue Related Gene Selection And Validation

Background Congenital heart defects (CHDs) account for the largestnumber of birth defects in human, with an incidence of 0.6%～0.9% inlive births and 5%～10% in spontaneously aborted pregnancies. As asingle cardiac malformation, the ventricular septal defect (VSD) is themost frequent form of CHDs and is present in the 33% of all affectedinfants.It is well established that environmental and genetic causes are bothcontributing to the development of VSD. Although the defect of VSD canbe repaired through surgery operation or trans-catheter intervention, thebasic mechanism of VSD is still unknown.Using SSH in the present study to identify differentially expressedgenes related to VSD could partly elucidate the mechanism of thedevelopment of VSD and lead to new aspect of diagnosis and primaryprevention of VSD, even the mechanism in cardiac development, aging,and disease.Objective Ventricular septal defect (VSD) accounts for the largestnumber of birth congenital heart defects in human, but the geneticprograms that control ventricular septation are poorly understood. UsingSSH in the present study to identify differentially expressed genes related to VSD could elucidate the mechanism of the development of VSD.Methods To identify differentially expressed genes between ventricularseptal defect and normal ventricular septum myocardium, we haveundertaken suppression subtractive hybridization (SSH) and generatedreciprocal cDNA collections of representative mRNAs specific to humanheart with ventricular septal defect versus normal control. The products ofSSH were inserted to vectors and transferred to competent Cells. Then,the clones were obtained after amplifying and sequenced, bioinfonnaticsanalysis later. Some randomly selected genes were further validated byRT-PCR.Results (1) Following SSH, forward and backward subtract librariesincluding 648 and 730 recons were built, and the length of insert partswas between 200bp and 600bp; (2) 1378 clones (expressed sequence tag,EST) were sequenced and found to derive from 551 different genes. Ofthese 551 unique genes, 299 genes were up-regulated and 252 genes weredown-regulated in VSD patient's heart. These predominately expressedgenes included genes involved in energy metabolism, cell cycle andgrowth, cytoskeleton and cell adhesion, LIM protein, zinc finger proteinand development; (3) The validation of differential expression of genesby RT-PCR demonstrated the low false positive rate associated with SSHin this experiment.Conclusions The trends of validation of differential expression of genes by RT-PCR were consistent to the results of SSH, which suggestedthat the library was highly reliability and credibility. It is anticipated thatfurther study of genes identified will provide insights into their specificroles in the etiology of VSD, even in cardiac development, aging, anddisease.