Researchers at University of Michigan developed a new method that can help identify potential targets relevant for normal peripheral nerve activity and Charcot-Marie-Tooth (CMT) disease therapy development.
Based on genetic analysis of non-coding regions in genes coupled with experimental cell line testing, the new method revealed the Tubb2b gene as an important regulator of Schwann cells and a potential therapeutic target. Schwann cells, affected in CMT type 1, are responsible for the production of the myelin layer that protects neurons and ensures correct communication.
The method was described in the study “A genome-wide assessment of conserved SNP alleles reveals a panel of regulatory SNPs relevant to the peripheral nerve,” which was published in the journal BMC Genomics.
The change of a single DNA base, also known as SNPs, in the genetic code can lead to serious alterations of the coded information. This also holds true if it happens in genomic regions that do not encode any protein, but instead regulate how the information is read (regulatory SNPs).
It has been known that regulatory SNPs can have important roles in disease development and progression. However, there is still lacking information validating the biological function and activity of most of these SNPs.
To fill this gap, researchers at UM developed a new analysis pipeline that may facilitate the discovery of relevant regulatory SNPs and disease-related genes.
They performed an initial genetic analysis of the entire genetic information of humans, mice, and chicken and looked for conserved regions among them, which are usually indicative of important regulatory activity. They found a total of 6,197 SNPs located within 6,164 conserved, non-coding sequences.
To validate that this approach allowed selecting SPNs with regulatory function, they tested 160 identified regions in three experimental cell lines relevant for peripheral nerves and CMT disease: Schwann cells, motor neurons, and muscle cells.
The team identified 13, 11, and 21 sequences, respectively, that showed a significant response in Schwann cells, motor neurons, and muscle cells. Additional experiments allowed to restrict the list to only three, one and five genetic regions that showed an unique active response in each cell type.
To further validate the potential of this analytical strategy to find relevant regulatory SNPs, the team repeated the pipeline, but restricted the analysis to regions that could interact with SOX10 — an important regulator of myelin production in Schwann cells.
This approach revealed 22 regions with regulatory potential linked to SOX10 activity, two of which were validated in the cell line experiments. “We then selected one of these elements for further characterization to assess the biological relevance of our approach,” the researchers wrote.
They found that deletion of the identified region changed the ability of the cell to read the Tubb2b gene in Schwann cells, suggesting that this gene could be a potential target. Interestingly, Tubb2b is known to be mutated in patients with asymmetric polymicrogyria, a neurological disorder.
“This strategy, combined with functional analyses, can yield candidate target genes,” the researchers concluded.
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