Congenital heart defect causing mutation in Nkx2.5 displays in vivo functional deficit

The Nkx2.5 gene encodes a transcription factor that plays a critical role in heart development. In humans, hetero- zygous mutations in NKX2.5 result in congenital heart defects (CHDs). However, the molecularmechanisms by which these mutations cause the disease remain unknown. NKX2.5-R142C is a mutation that was reported to be associated with atrial septal defect (ASD) and atrioventricular (AV) block in 13-patients from one family. The R142C mutation is located within both the DNA-binding domain and the nuclear localization sequence of NKX2.5 protein. The pathogenesis of CHDs in humanswith R142C pointmutation is notwell understood. To ex- amine the functional deficit associated with this mutation in vivo, we generated and characterized a knock-in mouse that harbours the humanmutation R142C. Systematic structural and functional examination of the em- bryonic, newborn, and adult mice revealed that the homozygous embryos Nkx2.5R141C/R141C are developmentally arrested around E10.5with delayed heartmorphogenesis and downregulation of Nkx2.5 target genes, Anf, Mlc2v, Actc1 and Cx40. Histological examination of Nkx2.5R141C/+ newborn hearts showed that 36% displayed ASD,with at least 80% 0f adult heterozygotes displaying a septal defect. Moreover, heterozygous Nkx2.5R141C/+ newborn mice have downregulation of ion channel genes with 11/12 adult mice manifesting a prolonged PR interval that is indicative of 1st degree AV block. Collectively, the present study demonstrates thatmicewith the R141C pointmutation in the Nkx2.5 allele phenocopies humanswith the NKX2.5 R142C point mutation.