RNA-seq profiling and dual-luciferase reporter assay revealed that the novel splicing mutation c.335–1G < A of Nkx2.5 can act upstream of PYK2 to regulate cardiomyocyte apoptosis via miR-19a and miR-19b (miR-19a/b) during the disease progression of atrial septal defects (ASD). This evidence concerns the gene NKX2-5 and atrial septal defect.