In this study, functional analyses demonstrated that the mutation identified in patients with ECD and ARS abolished the transcriptional activation of ANF- or PLOD1-driven luciferase reporter by PITX2 and eliminated the transcriptionally synergistic activation between PITX2 and NKX2.5, indicating that functionally impaired PITX2 is potentially an alternative molecular mechanism underpinning CHD and ARS. This evidence concerns the gene NKX2-5 and Axenfeld-Rieger syndrome.