# Derepression of Hedgehog Signaling in Congenital Heart Disease Patients

> **NIH NIH F31** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2020 · $45,520

## Abstract

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 Sh...

## Key facts

- **NIH application ID:** 9992623
- **Project number:** 1F31HL152659-01
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Jonathan Klonowski
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $45,520
- **Award type:** 1
- **Project period:** 2020-05-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9992623

## Citation

> US National Institutes of Health, RePORTER application 9992623, Derepression of Hedgehog Signaling in Congenital Heart Disease Patients (1F31HL152659-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9992623. Licensed CC0.

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