# Functional analysis of ESRP1/2 and CTNND1 gene variants in orofacial cleft

> **NIH NIH R01** · CHILDREN'S HOSP OF PHILADELPHIA · 2024 · $659,562

## Abstract

Technical abstract
Genetic study of orofacial clefts (OFC) is foundational to genetics of congenital structural
birth defects. Most OFC cases are non-syndromic and involve complex genetic
mechanisms that are yet to be fully elucidated. Currently, genetic diagnosis for cleft
anomalies is hampered by two critical knowledge gaps. First, genes essential for
palate formation are incompletely identified. Second, even when the cleft risk genes are
associated, algorithms used to impute deleterious from benign gene variants via
computational and statistical methods remain unreliable. There is a critical need to
translate genome sequencing to clinically actionable data, where functional studies
provide the highest-level evidence to impute pathogenicity. We showed that Esrp1 and
its paralog Esrp2 (hereafter Esrp1/2) operate in the periderm of mouse and zebrafish to
regulate craniofacial development. Esrp1/2 mediates alternative splicing of RNA
transcripts, creating epithelial isoforms that function in oral epithelium and periderm. This
proposal tests the central hypothesis that Esrp1/2 is required to generate epithelial
isoform of Ctnnd1, which maintains periderm integrity necessary for craniofacial
morphogenesis.
 We will impute pathogenicity of ESRP1/2 and CTNND1 human gene variants
associated with OFC. Using completed genome sequencing projects and projects in
progress, we curate large numbers of ESRP1, ESRP2 and CTNND1 gene variants to
ascertain their function. We employ complementary in vivo zebrafish esrp1/2 mutant
assay and in vitro Esrp1/2 murine Py2T cell lines to optimize rigor of approach. We
will also discover and functionally validate Esrp-regulated genes, using zebrafish
epithelial transgenic reporter lines. We discovered that Esrp1/2 regulates alternative
splicing of CTNND1 and will functionally interrogate human CTNND1 gene variants in
the zebrafish ctnnd1 mutant in a rescue assay.
 The expected outcome of this project is to gain mechanistic insights by
leveraging genome sequencing data associated with orofacial cleft cohorts, to
functionally analyze ESRP1/2 and CTNND1 gene variants in craniofacial development.
We will also identify and functionally validate Esrp-regulated genes acting in the
periderm. This work will have broader impact by elucidating how regulation of RNA
alternative splicing and cell signaling mechanisms are important in periderm and
craniofacial morphogenesis.

## Key facts

- **NIH application ID:** 10782469
- **Project number:** 5R01DE032332-02
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Eric Chien-Wei Liao
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $659,562
- **Award type:** 5
- **Project period:** 2023-02-15 → 2028-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10782469

## Citation

> US National Institutes of Health, RePORTER application 10782469, Functional analysis of ESRP1/2 and CTNND1 gene variants in orofacial cleft (5R01DE032332-02). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10782469. Licensed CC0.

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