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.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its different subtypes are linked to mutations in dozens of different genes. Mutations in gangliosideinduced differentiation-associated protein 1 (GDAP1) cause two types of CMT, demyelinating CMT4A and axonal CMT2K. The GDAP1-linked CMT genotypes are mainly missense point mutations. Despite clinical profiling and in vivo studies on the mutations, the etiology of GDAP1-linked CMT is poorly understood. Here, we describe the biochemical and structural properties of the Finnish founding CMT2K mutation H123R and CMT2K-linked R120W, both of which are autosomal dominant mutations. The disease variant proteins retain close to normal structure and solution behavior, but both present a significant decrease in thermal stability. Using GDAP1 variant crystal structures, we identify a side-chain interaction network between helices.3,.6, and.7, which is affected by CMT mutations, as well as a hinge in the long helix.6, which is linked to structural flexibility. Structural analysis of GDAP1 indicates that CMT may arise from disruption of specific intra- and intermolecular interaction networks, leading to alterations in GDAP1 structure and stability, and, eventually, insufficient motor and sensory neuron function.