The goal of the proposed study is to discern the functional and biological relevance of gliomas risk variants
identified through genome wide association studies (GWAS). GWAS have led to the discovery of 8
susceptibility loci in glioma: 8q24.11, 11q23.3, 5p15.33, 9p21.3, 20q13.33, 7p11.2 (2 independent loci) and
3q26.2. None of the GWAS SNPs map to exons and we hypothesize that the associated SNPs are non-
functional, are in linkage disequilibrium with casual/functional SNPs, and map to risk enhancers that in turn
regulate target gene expression in an allele specific manner. To date no functional/causal variants for glioma
GWAS have been identified. However, our team has identified some candidate target genes for 6 of the 8
GWAS loci using expression quantitative trait loci (eQTL) mapping in multiple brain regions and allelic specific
gene expression (ASE) analyses. Based on these promising findings, we propose a comprehensive post
GWAS analysis of glioma risk loci using a series of complementary approaches. In Aim 1 we will identify
candidate target genes of glioma risk loci using data from two sources: publicly available RNA-Seq and
genotyping data from multiple brain regions of 400 post-mortem brains of the Genotype-Tissue Expression
Project's (GTEx); we will also generate RNA-Seq and genotyping data from an additional 300 pathologically
verified, fresh-frozen autopsied normal brain tissues (multiple brain regions) from the University of Miami Brain
Bank (UMBB). eQTL mapping, eQTL meta-analyses and eQTL-ASE will be performed, using the UMBB
samples as the discovery set and GTEx dataset as a validation dataset. In Aim 2 we will use publicly available
ChIP-Seq and DNAse1 hypersensitivity chromatin data to identify candidate regulatory elements within GWAS
loci. We will: (a) validate candidate regulatory elements using enhancer/promoter luciferase vector activity
assays in multiple glioma cell lines. (b) Assess allele specific effects on enhancer/promoter activity using either
site-directed mutagenesis or naturally occurring haplotypes (c). Identify and validate novel candidate target
genes of risk enhancers identified in Aim 1 by knocking out risk enhancers using CRISPR-Cas9 gene editing
technology in glioma cell lines and assessing changes in target gene expression using RNA-Seq. In Aim 3 we
will use existing data and novel data generated through Aims 1 and 2, to identify and validate the physical
interaction between risk enhancers and candidate target genes. Interacting loci of candidate risk enhancers will
be identified using 4C-Seq using five glioma cell lines, and will be compared to candidate target genes
identified through Aims 1 and Aim 2. Any significant interactions, especially those with candidate local and
distal target genes identified through Aims 1 and 2 will be further validated by 3C and fluorescent in situ
hybridization (FISH). Through these efforts we will develop a mechanistic and biological understanding of
glioma risk that will be an essential first step in our translational efforts to develop preventive therapeutics
approaches for glioma.
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