# Understanding GWAS signals: functional variants or functional haplotypes?

> **NIH NIH R21** · JOHNS HOPKINS UNIVERSITY · 2020 · $450,313

## Abstract

SUMMARY
Genome wide association studies (GWAS) reveal disease associated genetic variation, but more often than
not point to multiple variants with correlated genotypes due to linkage disequilibrium (LD). Many of the variants
in LD are correlated with disease not because they have a functional consequence but because they associate
with others that do. Whether each independent GWAS signal, i.e. each set of disease SNPs in strong LD
includes one or more functional variant driving the association, and whether alleles in LD are related in
function is generally unknown. To accurately model the effects of disease associated variants it is important to
understand the possible dynamic relationship between functionality and LD. From our and others' published
work we have evidence that in some cases LD may be driven by selection for the co-occurrence of functional
variant alleles in cis on the same haplotype. We hypothesize that new regulatory alleles undergo selective
pressure that depends on the existing regulatory alleles on the same haplotype. In this R21 exploratory
research grant application we propose to use the CACNA1C gene, on which we have previously performed
extensive work, as a model to look for evidence of cooperative activity of the SZ-associated SNPs. In that work
we acquired evidence that interaction between multiple SZ variants may be mediated by a transcription factor
(TF) called PKNOX2, a TF highly expressed in the brain and previously associated with substance abuse and
SZ presentation. Here we will use human induced pluripotent stem cells (iPSCs) differentiated into neurons by
NGN2 induction (iNs) and CRISPR/Cas9 editing of each SZ associated variant and the PKNOX2 gene to study
the consequences of modifying each variant and the connecting TF. In particular we propose the following
specific aims: (1) We will employ CRISPR/Cas9 genome editing on iPSC lines from healthy controls carrying
each of the two major haplotypes. We will delete the region surrounding each SZ-SNP (~50 bp) and measure
the effect of each deletion on CACNA1C expression in each background. We expect more than one deletion to
have a consequence on the gene's regulation. (2) We will use Chromatin immunoprecipitation followed by
sequencing (ChIP-Seq) using a PKNOX2 antibody on opposite haplotype homozygotes. We expect to
demonstrate interaction with PKNOX2 with the SNP regions and differential pull down of the two alleles of
multiple CACNA1C SZ SNPs. We will use the same data to identify other genomic sequences interacting with
PKNOX2, allelic differences where the genotypes differ, and possible association of these regions with SZ in
public GWAS data. (3) We will knock out PKNOX2 by genome editing and use the otherwise isogenic iN cells
to: (i) Measure the changes of CACNA1C and the transcriptome by RNAseq on iNs', (ii) Measure the
consequences to open chromatin by ATAC-seq on iNs' (iii) Show the effects of removing PKNOX2 on
chromatin interactions of the CACNA1C prom...

## Key facts

- **NIH application ID:** 9968705
- **Project number:** 1R21MH122936-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Dimitrios Avramopoulos
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $450,313
- **Award type:** 1
- **Project period:** 2020-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9968705

## Citation

> US National Institutes of Health, RePORTER application 9968705, Understanding GWAS signals: functional variants or functional haplotypes? (1R21MH122936-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/9968705. Licensed CC0.

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