Charcot-Marie-Tooth disease type 1J

Charcot-Marie-Tooth disease (CMT) is an incurable neurodegenerative disease of the peripheral nervous system, characterized by progressive distal motor and sensory impairment. CMT is the most common inherited neurological disease, affecting 1:2500 people. It is a heterogenous group of disorders, caused by variants in over 100 identified disease genes. This thesis describes the identification of ITPR3 variants as a cause for CMT. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular signaling hubs that regulate the release of Ca2+ from the endoplasmic reticulum (ER) to the cytosol. Humans have three subtypes of IP3Rs (IP3R1-3), which form homo- or heterotetrametric calcium channels at the ER. Study I identified a new genetic cause and disease type of CMT. The disease is caused by variants in ITPR3. A previously unknown ITPR3 variant was found in Finnish family with CMT, and an international collaboration led to the identification of an additional CMT patient with another previously unknown ITPR3 variant. Evidence for disease causality of these variants included their absence in population databases of genetic variation, effects on conserved amino acids in functionally important protein domains, and functional analyses of patient fibroblasts. This newly characterized form of CMT was subsequently classified as CMT1J. Study II identified ITPR3 as a canine disease gene in a cohort of Lancashire Heeler dogs. The dogs displayed a severe developmental enamel defect and reduced nerve conduction velocity, indicating the role of IP3Rs in oral health and neuropathy. The affected dogs had a homozygous nonsense variant in ITPR3. Immunoblotting of the affected dogs’ skin fibroblasts revealed a complete loss of IP3R3 protein. In addition, the protein levels of IP3R1 and IP3R2 were clearly decreased, which suggests codependency in the regulation of different IP3R subtypes. As expected, Ca2+ imaging assays revealed decreased IP3R-mediated Ca2+ flux upon stimulation of G-protein-coupled-receptors. These results highlight the importance of IP3Rs in enamel formation and peripheral nerves, and expand the phenotypic spectrum related to ITPR3. This was the first study to report a spontaneous large animal model of CMT1J, suggesting that the variants with the most severe consequences on channel function may be associated with broader disease phenotypes. Study III describes the first knockout (KO) of the ITPR1-3 genes in human induced pluripotent stem cells (iPSC), and the consequences of lost IP3Rs on iPSC function. ITPR1-3 were successfully knocked out with CRISPR/Cas9 genome editing. Previous studies have suggested that Ca2+ signaling is a major regulator of stem cell proliferation and differentiation. In KO-iPSC the differentiation potential and stemness were retained, indicating the adaptability of the iPSC in the changes in their Ca2+ signaling machinery. However, metabolomics analyses showed that triple-KO iPSC lacking all the subtypes displayed abnormal mitochondrial function. Specifically, the pyruvate utilization was affected to favor pyruvate carboxylase over the pyruvate dehydrogenase pathway. These data show that KO iPSC can be used as models for exploring the consequences of IP3R loss in various somatic cell types through differentiation in the future. Metabolic changes might manifest upon differentiation into specialized cell types, which may lead to the identification of cell type specific dysfunction that are relevant for understanding CMT1J disease mechanisms. Understanding the molecular mechanisms behind pathogenic variants, as well as generating the appropriate cell models are requisite for the development of effective treatment for rare diseases. Peripheral nervous system cells are inherently challenging to study due the unavailability of the cell types. Identification of the ITPR3 variants as a cause of CMT1J and generation of iPSC models for studying the IP3R function pave the way for the development of new treatments for these debilitating conditions.