Abstract
BackgroundIdentifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells.ResultsWe identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells—followed by differential gene expression studies—revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts.ConclusionsOur efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations.
Highlights
Identifying functional non-coding variation is critical for defining the genetic contributions to human disease
Genome-wide computational predictions of regulatory Single Nucleotide Polymorphism (SNP) To identify and prioritize a set of putative cis-acting regulatory element (CRE) that harbor Regulatory Single Nucleotide Polymorphism (rSNP), we developed a novel computational pipeline (Fig. 1)
Identification of enhancers relevant to the peripheral nerve To validate the efficacy of our computational approach, we selected a set of 160 regions (~ 2.5% of the total dataset; Additional file 3), which includes all genomic segments harboring a SNP that we identified on chromosome X (94 regions), chromosome 22 (29 regions), and chromosome 21 (37 regions)
Summary
Identifying functional non-coding variation is critical for defining the genetic contributions to human disease. The cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. The ability to predict the effect of non-coding variation on gene expression and rapidly validate genomic regions for cis-regulatory activity will aid the identification of modifiers of human disease [4]. The genes implicated in CMT2, which affects motor and sensory axons, include neurofilament light chain (NEFL) [11] and mitofusin 2 (MFN2) [12], which are critical for axon function. Patients with a duplication of PMP22 have variable age of onset (3-73 years of age), variable motor and sensory nerve involvement, and display a broad spectrum of severity, ranging from mild difficulty in walking or running to impairment requiring a wheelchair [14]
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