Derepression of Hedgehog Signaling in Congenital Heart Disease Patients

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Congenital heart disease (CHD) is one of the most prevalent birth defects, affecting up to 1% of live births. A previous mouse forward genetic screen for mutations causing CHD conducted in our laboratory recovered 91 genes causing CHD, with more than 50 being cilia and ciliary signaling related. Interestingly of the 15 genes recovered causing CHD in cilia transduced cell signaling pathways, at least 5 are associated with Sonic hedgehog (Shh) signaling. Shh signaling in fact is known to play an important role in development of the outflow tract and semilunar valve via the regulation of the cardiac neural crest and second heart field cell lineages. While the role of Shh in CHD pathogenesis is now well described in mouse models, its role in human CHD remains poorly understood. Hence, to examine the potential importance of Shh signalng in human CHD pathogenesis, we conducted a screen for defect in Shh signaling using CHD patient fibroblasts. Surprisingly, our analysis showed 17 of 41 CHD patients exhibited altered hedgehog signaling without overt ciliogenesis defects, suggesting hedgehog signaling but not ciliary structure is affected. Real time PCR analysis showed 9 of the 17 patients had abnormal basally activated transcription of GLI, a hegehog responsive gene, in the absence of pathway stimulation. This suggests disruption in the negative regulation of Shh signaling may play an important role in CHD pathogenesis. Interestingly, 6 of the 9 patients with basal GLI activation exhibited CHD of a similar spectrum comprising left ventricular outflow obstructions. As Shh signaling also plays an essential role in patterning development of the central nervous system, it is worth noting that 3 of the CHD patients with basally activated GLI transcription also had brain dysplasia. Based on these findings, we hypothesize mutations in negative regulators of Shh signaling may play a significant role in CHD pathogenesis and CHD associated brain dysplasia. To test this hypothesis, we developed studies in 3 aims. In Aim 1, we will identify determinants of negative Shh regulation contributing to CHD by intersecting patient fibroblast RNAseq and whole exome sequencing data with Shh negative regulators identified in previous Shh CRISPR screens. In Aim 2, we will conduct siRNA and CRISPR gene KO to assess the potential role of negative regulators on Shh signaling. In Aim 3, we will use mouse CRISPR gene editing and F0 founder embryo analysis to assess the role of negative Shh regulators on CHD pathogenesis and abnormal neurodevelopment. Phenotyping for cardiac and brain abnormalities will include hemodynamic assessments using Doppler echocardiography, followed by anatomical analyses using microCT, MRI, and serial histopathological 3D reconstruction. These studies will provide new insights into how negative regulation of Shh signaling may contribute to heart development and the pathogenesis of CHD. These studies also may suggest a paradigm shift linking dysregulated Shh signaling with structural heart defects and abnormal neurodevelopment, findings that may have therapeutic implications long term.