Characterization of a novel gene variant in Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is a collective name for inherited neuropathies affecting the peripheral nerves. CMT affects 1:2500 children and adults worldwide. The disease is genetically highly heterogeneous, and the pathogenic mechanisms are largely unknown. Thus far, there is no cure known for the Charcot-Marie-Tooth disease. Therefore, the study of the genetic factors involved in the disease and the understanding of the underlying molecular mechanisms will benefit the development of strategies to prevent or treat these diseases. In this thesis, a new candidate gene for CMT was investigated in patient fibroblasts. The novel gene variant was originally found at University of Helsinki in a pair of Finnish brothers with CMT; and in later examinations, in their affected family members. The gene encodes an ER calcium channel receptor that is responsible for Ca2+ release from the endoplasmic reticulum (ER) and plays an important role in the regulation of various cellular processes. In this thesis, I studied the effect of the variant in patient fibroblasts by Western blotting, quantitative reverse transcriptase PCR (RT-qPCR) and calcium imaging. I also knocked down the gene using siRNA in healthy fibroblasts to investigate if the loss of the receptor has a similar effect on calcium signaling as the patient variant. My results showed that siRNA treatment significantly decreased the targeted protein levels and delayed the ATP-evoked Ca2+ release from ER without profound effect on the amplitude of the release. Similar effects of the studied mutation were observed in one patient cell line, but not in the other. Patient cell line, which did not have alterations in the levels of the protein and Ca2+ release, had elevated levels of mRNA of the affected gene. The results suggest that the gene variant does not impair the total volume of the ATP-evoked Ca2+ release from ER. The possible effect of the studied mutation may be related to the decreased levels of the mutated protein, which at the functional level may affect the timing of total Ca2+ release from ER. However, the functional effect of the variant could not be confirmed with the fibroblast cells; further experiments are needed to clearly confirm the variant’s effect on calcium signaling.