# Functional tests of non-coding DNA variants associated with risk for orofacial clefting

> **NIH NIH R01** · UNIVERSITY OF IOWA · 2021 · $452,376

## Abstract

Orofacial clefting (primarily cleft lip and/or cleft palate) is a relatively common structural birth
defect with environmental and genetic contributions to etiology. Genome wide association
studies (GWAS) and linkage studies have identified many gene variants that are associated with
elevated risk for isolated oral facial clefting (OFC). However, our understanding of the
pathogenic mechanisms underlying this disease remains poor because, one, we have yet to
distinguish DNA variants that directly influence risk for OFC (i.e., causal variants) from those
that are merely in linkage disequilibrium with them, and two, the functions of the regulatory
molecules encoded y OFC-associated genes in craniofacial development are largely unknown.
At each locus, there are multiple identified multiple SNPs that are statistically associated with
OFC – and all of these reside in non-coding DNA. In Aim 1, we will prioritize the SNPs for
functional tests by performing fine mapping of GWAS data, and identifying de novo mutations in
new whole genome sequence data from 800 case-parent trios. In Aim 2 we propose to identify
the OFC-associated SNPs that are functional (causal). We hypothesize that pathogenic SNPs
reside in enhancers that drive expression in oral tissues, and that risk alleles of such SNPs
quantitatively affect activity of the enhancers. To test this hypothesis, we will amplify genomic
DNA containing risk-associated SNPs and test them for allele-dependent enhancer activity in
vitro (cell-based reporter assays). We will also test the tissue specificity of the enhancers
(zebrafish and mouse-based reporter assays). In Aim 3, we will determine the effect that
altering the allele of pathogenic SNPs has on expression of the relevant OFC-risk gene
(genome engineering with CRISPR/Cas9 in vitro). Finally, we apply chromatin immuno
precipitation and chromatin configuration capture in the oral epithelium cell line to deduce the
mechanism by which functional SNPs change expression of the OFC-risk genes. The expected
outcome of the proposed experiments is identification of the mechanisms by which genetic risk
variants cause a common birth defect.

## Key facts

- **NIH application ID:** 10145645
- **Project number:** 5R01DE027362-04
- **Recipient organization:** UNIVERSITY OF IOWA
- **Principal Investigator:** Robert Aaron Cornell
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $452,376
- **Award type:** 5
- **Project period:** 2018-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145645

## Citation

> US National Institutes of Health, RePORTER application 10145645, Functional tests of non-coding DNA variants associated with risk for orofacial clefting (5R01DE027362-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10145645. Licensed CC0.

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