# Deciphering Mechanisms of Limb Malformations Caused by Noncoding Variants In Vivo

> **NIH NIH F30** · UNIVERSITY OF CALIFORNIA-IRVINE · 2024 · $45,134

## Abstract

PROJECT SUMMARY/ABSTRACT
 Limb malformations are the second most common congenital abnormality, occurring in 1 in every 500
live births. Mounting evidence implicates rare noncoding mutations to underlie non-syndromic (isolated) limb
malformations. Many of these variants map to transcriptional enhancers, regions of regulatory DNA that tune
gene expression. However, a fundamental gap remains in our understanding of the mechanisms by which these
variants alter enhancer activity and their role in causing limb defects. The most frequently affected noncoding
loci is the limb-specific enhancer of Sonic hedgehog (Shh). With over 30 independent rare variants linked to limb
malformations, the Shh limb enhancer is particularly susceptible to so-called Gain-Of-Function (GOF) variants.
GOF variants cause enhancer overactivity that leads to ectopic expression of their target genes. However, why
GOF variants only cause ectopic gene expression in specific cell types and why only a small subset of
enhancers are susceptible to GOF variants are both unknown.
 GOF variants are among the least understood enhancer mutations that cause human disease. Much of
our lack of understanding of how GOF variants contribute to disease is owed to a lack of suitable model systems.
In vitro cell culture and organoid-based systems fail to recapitulate ectopic expression from GOF variants nor
model their phenotypic consequences. Thus, it is essential to use in vivo systems to determine the functional
and clinical significance of GOF variants. To address this major need, our group recently developed a novel
mouse enhancer reporter assay that enables highly-reproducible detection of ectopic gene expression in the
cells of the anterior limb domain where Shh is normally not expressed. The overall goal of this proposal is to
determine the genetic factors mediating the unique susceptibility of anterior limb bud cells and the Shh limb
enhancer to GOF variants.
I will test the hypothesis that susceptibility to GOF variants is dictated by the regulatory landscape of
anterior limb bud cells and a unique, stable higher-order chromatin structure of the Shh locus. To identify the
genetic factors that mediate ectopic Shh expression, I will characterize the regulatory landscapes and local
chromatin architecture of anterior limb bud cells in which Shh is ectopically active at single-cell resolution. To
determine genetic factors that predispose specific enhancers to pathogenesis, I will test the requirement of
higher-order chromatin structure for limb malformations resulting from GOF variants. By identifying targetable
genetic factors mediating ectopic gene expression, these studies will provide mechanistic insights into how GOF
variants in the limb-specific Shh enhancer contribute to limb malformations. Findings resulting from this proposal
can also be applied to predict the clinical significance of noncoding variants from patient sequencing data and
will have implications for other developmental di...

## Key facts

- **NIH application ID:** 10675480
- **Project number:** 5F30HD110233-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** Ethan W Hollingsworth
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $45,134
- **Award type:** 5
- **Project period:** 2023-01-01 → 2026-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10675480

## Citation

> US National Institutes of Health, RePORTER application 10675480, Deciphering Mechanisms of Limb Malformations Caused by Noncoding Variants In Vivo (5F30HD110233-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10675480. Licensed CC0.

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