# Testing naturally-occurring mutations for impact on brain enhancer function

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2022 · $235,500

## Abstract

SUMMARY
Enhancers are distal non-coding regulatory DNA elements that contribute to the activation of target genes in a
developmental, cell-type, and context-dependent manner. In other words, enhancers act as the genomic
switches that enable the precise control of gene expression required for the development and function of the
brain. There is a consensus that enhancers are a critical for gene expression and that sequence variation
within enhancers contributes to genetic risk. However, there are major barriers towards being able to predict
which non-coding mutations matter in the context of regulatory function and disease risk. To date, the role of
non-coding regions in brain disorders, including autism spectrum disorder, has been largely impenetrable due
to the quantity of non-coding DNA and the difficulty characterizing functional impact of variants outside of
coding sequence in the brain. Whole genome sequencing (WGS) efforts promise comprehensive genome-wide
mutation analysis, and this method has been adopted at scale in ASD. However, computational prediction of
regulatory variant function alone is currently insufficient for functional variant identification, including in ASD.
Massively parallel reporter assays (MPRAs) provide a solution via enabling functional screening of enhancers
and their variants, and quantitative measurement of the regulatory capacity of hundreds to thousands of
individual candidate sequences in a single experiment. For such functional assays to be relevant to the brain
and ASD, it is critical to use models that capture the complexity and organization of the brain. We implemented
a large-scale screen of de novo regulatory variants using in vivo deployment of an MPRA in postnatal mouse
brain. From a pool of ~1000 de novo variants assayed in early postnatal mouse cortex using our innovative
function-based test, we identified strong and weak enhancers, and putative allele-specific activity associated
with these naturally occurring de novo regulatory mutations. Although a significant demonstration of assay
potential, our preliminary results fail to fully take advantage of the ability to interrogating context-dependent
function in the complexity of the brain. Here we proposed work to verify in vivo MPRA performance and extend
this method to generate cell-type specific enhancer readout. In doing this, we will define the regulatory capacity
of candidate enhancers harboring de novo variants from ASD proband and control genomes. In Aim 1, we
propose a screen-centered approach, using our MPRA to define cell-type and allele-specific enhancer activity.
In Aim 2, we propose an enhancer-centered approach, defining in vivo activity of individual enhancers in
mouse postnatal brain via image-based analysis. Our results will generate function-based evaluations of
naturally occurring de novo regulatory mutations, enabling statistical testing of functionally-defined enhancer
activity in tandem with deep single enhancer functional investiga...

## Key facts

- **NIH application ID:** 10357952
- **Project number:** 5R21MH126400-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Alexander Nord
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $235,500
- **Award type:** 5
- **Project period:** 2021-03-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10357952

## Citation

> US National Institutes of Health, RePORTER application 10357952, Testing naturally-occurring mutations for impact on brain enhancer function (5R21MH126400-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10357952. Licensed CC0.

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