# Decoding the Mechanism of Pathogenic Enhancer Mutations In Congenital Limb Disorders

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA-IRVINE · 2024 · $400,369

## Abstract

PROJECT SUMMARY
Regulatory regions of the genome play crucial roles in ensuring appropriate spatiotemporal expression of genes.
Like other regions of the genome, regulatory DNA sequences exhibit variations that could influence their
functions. Yet, there is a significant gap in our understanding of how variants in non-coding gene regulatory
regions alter gene expression and what impact these variants have on an individual’s development or fitness. In
particular, some rare variants confer gain-of-function enhancer activity, causing ectopic gene expression that
contributes to limb malformations, intellectual disabilities, and autism among other congenital conditions.
Determining how these gain-of-function enhancer variants cause ectopic gene expression and adverse
outcomes is critical for understanding the etiology of underlying diseases. The main objective of this project is to
use limb development as a model to uncover transcriptional mechanisms of pathogenic Sonic Hedgehog (SHH)
activation in anterior limb bud cells that occurs in patients with rare variants in the ZRS limb enhancer of SHH.
More than 30 independent rare variants in ZRS are linked to human polydactyly. However, these gain-of-function
enhancer variants are challenging to study because cell culture- or organoid-based models typically do not
recapitulate ectopic gene expression observed in vivo. In preliminary studies, we used our newly developed,
reproducible transgenic mouse assay to show that most of these rare variants cause ectopic enhancer activity
in the anterior margin of developing mouse limb buds, suggesting a common mechanism for pathogenic
enhancer de-repression. The proposed project will dissect the mechanism(s) by which gain-of-function enhancer
variants result in pathogenic developmental phenotypes. We will utilize a modified version of our novel transgenic
assay, which enables visualisation of pathogenic enhancer activity in live embryos, together with our enhancer
variant knockin mouse model that faithfully recapitulates limb malformations observed in patients, to delineate
transcriptional mechanisms that cause ectopic Shh expression and pathogenic phenotypes. In Aim 1 we will
characterize chromatin and spatial mechanism of ectopic gene activation resulting from gain-of-function
mutations in the ZRS enhancer. In Aim 2 we will identify transcription factors that cause ectopic Shh activation.
The high-resolution and rigorous quantitative characterization of novel genetic factors that contribute to enhancer
pathogenicity will significantly advance our knowledge of enhancer malfunction in disease. Ultimately, this
knowledge can be used in combination with information about epigenome state from single-cell studies to predict
the clinical significance of novel non-coding variants emerging from rapidly expanding whole-genome
sequencing studies.

## Key facts

- **NIH application ID:** 10890388
- **Project number:** 1R01HD115268-01A1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** Evgeny Kvon
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $400,369
- **Award type:** 1
- **Project period:** 2024-07-01 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10890388

## Citation

> US National Institutes of Health, RePORTER application 10890388, Decoding the Mechanism of Pathogenic Enhancer Mutations In Congenital Limb Disorders (1R01HD115268-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10890388. Licensed CC0.

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