Function of TBX20 gene mutation T262M in children with congenital heart disease
Objective To explore the function of TBX20 missense mutation C.785C＞T(T262M),newly correlated with congenial heart disease(CHD),and further examine the correlation between T262M and the pathogenesis of CHD.Methods The pathogenicity of gene mutation T262M was predicted by PolyPhen2 and Mutation Taster.The protein structure models of wild-type TBX20 and mutant TBX20-T262M were constructed by homology modeling technology.And the three-dimensional structures of mutant and wild-type proteins were compared for predicting the changes and effects of protein spatial conformation after mutation.Molecular docking simulation was utilized for predicting the functional changes of mutant T262M as compared with wild-type TBX20.Real-time fluorescent quantitative polymerase chain reaction(PCR)was employed for detecting the expression of wild-type and mutant TBX20 mRNA.Dual luciferase reporter system was utilized for detecting the effect of T262M on the promoter activity of downstream target gene ANF.Results As predicted by PolyPhen2 and Mutation Taster software,T262M was pathogenic and protein function might be affected.Homology modeling indicated that T262M caused a loss of hydrogen bonds of side chains,resulting in altered protein conformation.Molecular docking simulation revealed that the binding force of mutant T262M with NKX2.5,GATA4 was higher than that of wild-type TBX20.The results of real-time fluorescent quantitative PCR showed that the mRNA expression of mutant T262M was significantly higher than that of wild-type TBX20,regardless of whether or not TBX20 expression plasmid was transfected alone or co-transfected with GATA4 and NKX2.5(P＜0.05).The results of dual luciferase reporter gene detection in vitro cell experiments showed that the effect of mutant TBX20-T262M on the activity of downstream target gene ANF promoter was not significantly different from wild-type TBX20(P＞0.05).Conclusion The newly discovered TBX20 gene missense mutation T262M is pathogenic,resulting in altered protein spatial conformation and improving binding force with NKX2.5 and GATA4.It may be involved in the occurrence of CHD through up-regulating mRNA expression.
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