Abstract
Glioma is the most common malignant primary brain tumor and is associated with poor prognosis. Genetic factors contributing to glioma risk have recently been investigated through genome-wide association studies (GWAS), implicating seven independent glioma risk loci in six chromosomal regions. Here, we performed an in-depth functional analysis of the risk locus proximal to the PHLDB1 gene on 11q23.3. We retrieved all SNPs in linkage disequilibrium (r2 ≥ 0.2) with the glioma-associated SNP (rs498872) and performed a comprehensive bioinformatics and experimental functional analysis for the region. After testing candidate SNPs for allele-specific activity in a luciferase-based enhancer scanning assay, we established a subset of 10 functional SNPs in the promoters of PHLDB1 and DDX6, and in a putative enhancer element. Chromatin conformation capture (3C) identified a physical interaction between the enhancer element containing a functional SNP (rs73001406) and the promoter of the DDX6 gene. Knockdown experiments in cell culture and 3D assays to evaluate the role of PHLDB1 and DDX6 suggest that both genes may contribute to the phenotype. These studies reveal the functional landscape of the 11q23.3 glioma susceptibility locus and identify a network of functional SNPs in regulatory elements and two target genes as a possible mechanism driving glioma risk association.
Highlights
We found that knockdown of DDX6 led to a significant reduction in the migratory phenotype of U87MG cells, while knockdown of pleckstrin homology-like domain family B member 1 (PHLDB1) had no significant effect (Fig. 7B)
We initially identified 41 candidate SNPs, 10 of which lie in regions with enhancer activity in normal human astrocyte (NHA) or U87MG cells
The majority of these SNPs lie within promoter regions, either for PHLDB1 or DDX6, while the remaining SNPs lie in additional enhancer or repressor regions. 3C experiments detected a physical interaction between one enhancer region containing a candidate SNP and the promoter of DDX6
Summary
Functional analysis of cancer susceptibility loci is a field that has developed rapidly over the last few years. We have applied a systematic functional analysis to identify candidate functional SNPs and target genes within the 11q23.3 glioma susceptibility locus, a locus with relatively little annotation related to cancer predisposition. We began with a bioinformatics analysis of all SNPs in linkage disequilibrium (LD) (r2 ≥ 0 .2) with the PHLDB1 tag SNP, rs498872, which led to a total of 41 candidate functional SNPs. We conducted an analysis of genes within the locus in order to identify potential target genes. Experiments using normal human astrocyte (NHA) and human malignant glioma (U87MG) cells were conducted in order to assess the enhancer activity of each SNP and potential influence on protein binding or chromatin interactions. A 3D culture model system (neurospheres) was used to assess two potential target genes within the locus for their functional relevance
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