Genetics and mechanisms of rare inherited neuromuscular disorders : a focus on neurofilament light

Hereditary neuromuscular disorders are a heterogenous group of diseases affecting motor nerves and musculature. The clinical distinction between nerve or muscle originating disorders can be difficult since both neuronal loss and muscle disruption lead to muscle weakness and atrophy. The underlying genetic cause of a neuromuscular disease and its pathological molecular mechanism need to be unraveled to develop disease modifying treatments. Fortunately, next-generation sequencing (NGS) technologies have revolutionized the molecular diagnostics of hereditary diseases. High throughput technologies can be used to efficiently sequence all possible pathogenic variants in an individual’s genome to discover the affected gene. However, the gene variant behind the disorder does not directly indicate the mechanism of disease. To discover treatable processes, the cellular and molecular alterations caused by the mutant gene must be modelled in appropriate systems. In this dissertation I aimed to discover genetic causes behind rare hereditary neuromuscular disease in adult patients, evaluate the efficacy of NGS in this patient group, and model NEFL (neurofilament light) nonsense variants that cause Charcot-Marie-Tooth (CMT) disease with induced pluripotent stem cell (iPSC) derived motor neurons (iPSC-MN) to discover treatable mechanisms of disease. In a cohort of 100 adult patients, we discovered pathogenic variants in 27% of patients with varied hereditary disorders such as myopathy, neuropathy, spasticity, ataxia, and parkinsonism using clinical whole-exome sequencing (WES). Twelve of the variants in 13 patients were novel changes in known disease-associated genes, thus we expanded the genetic causes and phenotypes of these disorders. I showed WES to be efficient and potentially cost-effective in the molecular diagnosis of adult patients with suspected hereditary origin of disease. In addition, we discovered Finnish patients with previously uncharacterized phenotypes linked to PYROXD1 variants. The Finnish patients had a late-onset limb-girdle muscular dystrophy (LGMD)-like phenotype in contrast to the previously described congenital myopathy with myofibrillar changes. Nonsense variants in the NEFL gene cause a severe, early-onset form of axonal CMT through an uncharacterized mechanism. Studies on dominant NEFL variants indicate the axon degeneration to be caused by mutant protein aggregation, however, the disease mechanism behind nonsense cases is still elusive. I characterized a novel pathogenic recessive nonsense variant p.R367X in NEFL causing CMT. I showed the novel variant to lead to nonsense-mediated decay of mRNA and to neurofilament light polypeptide (NFL) protein loss in iPSC-MN of the patient. We generated isogenic NEFL knockout cell lines from control iPSC to more accurately assay the effects of NFL loss. I characterized the patient and isogenic gene-edited iPSC-MN devoid of NFL and described specific molecular alterations in the neurons. I showed the absence of the integral neurofilament (NF) protein NFL to be causative in recessive NEFL CMT but not to affect iPSC-MN differentiation or the growth of elaborate axonal connections in patient or isogenic gene-edited models. Furthermore, the neurons were able to produce NFs without compensation from other intermediate filament proteins. However, there were fewer NFs and their composition was altered by NFL absence and reduction of neurofilament heavy polypeptide (NFH). The loss of NFL reduced axon diameter, decreased amplitude of miniature post-synaptic currents (EPSCs) and increased movement of axonal mitochondria in iPSC-MNs. Clinical studies on patients and Nefl knockout mouse models have shown similar mechanisms of disease as our iPSC-MN model. The caliber of large axons is also reduced in patients with NEFL nonsense mutations. In the patients the reduction of axon size and loss of large myelinated axons leads to decreased nerve conduction velocity. The synaptic function of NFL has not been previously described in human models, but NFL has been shown to bind glutamatergic receptors in mice. The reduced amplitude in EPSCs suggests a synaptic regulatory role of NFL also in humans warranting further research. The loss of NFL in the synapse could reduce the excitability of spinal motor neurons leading to reduced activity. Increase of mitochondrial movement in axons without NFL adds to previous research on mice indicating NFs and NFL working as scaffolds for organelles in axons. The increase in mitochondrial movement could reduce the amount of correctly localized active mitochondria and lead to local metabolic deficiencies predisposing axons to degeneration. My iPSC-MN modelling studies show the usability of stem cell-based models in studying the mechanisms of axonal neuropathy and its pathological processes. Further research is needed to pinpoint the translatable pathological alterations in the human axons that could be targeted for treatments in patients.