Decoding the Mechanism of Pathogenic Enhancer Mutations In Congenital Limb Disorders

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.