Preliminary Functional Study Of The C.T182A Mutation In NODAL Gene That Associated With Congenital Heart Disease

Background: Congenital heart disease is a disease that involves both environmental and genetic factors.More and more genes and signaling pathways have been found to play a role in the pathogenesis of congenital heart disease.NODAL is the major ligand of Nodal signaling pathway which can precisely induce the formation of mesoderm and endoderm,as well as indirectly regulate the directional differentiation of cardiac progenitor cells during the development of cardiac embryos.Previous studies have reported that a rare mutation(c.T182 A,p.L61Q)in the coding sequence of the NODAL gene was screened in patients with congenital heart disease.The present study was aimed to investigate the functional changes of NODAL protein caused by this mutation.Methods: The NODAL gene was analyzed by bioinformatics programs.The wild-type and mutant NODAL expression plasmids were constructed respectively.The transcriptional activity of the NODAL mutation on downstream genes was analyzed by the dual-luciferase reporter assay.Subcellular localization and Western blot were used to analyze the effect of the NODAL mutation on its localization and expression.Results: The NODAL mutation,c.T182 A,was predicted to be disease-causing by bioinformatics programs.The NODAL mutant protein doesn't change its normal subcellular distribution.However,p.L61 Q mutation reduced the transcriptional activity of NODAL to activate its downstream target genes,and inhibits the expression of NODAL protein.Conclusions: Our study identified that the gene mutation of NODAL transcription factor(c.T182A)was damaging which altered its regulation on the downstream gene and also inhibited its expression of NODAL protein without changing its normal subcellular distribution.It is suggested that the c.T182 A mutation in NODAL gene is probably a pathogenic gene mutation.And expanded the mutation information spectrum of NODAL gene,and provided a new clue for the genetic mechanism of congenital heart disease.