Genetic and Functional Evaluation of Aminoacyl-tRNA Synthetase Mutations in Patients with Peripheral Neuropathy.

Abstract

Charcot-Marie-Tooth (CMT) disease comprises a group of clinically and genetically heterogeneous peripheral neuropathies mainly characterized by distal muscle weakness and wasting, and impaired sensation in the extremities. CMT disease is the most common inherited peripheral neuropathy, with a prevalence of ~1 in 2,500 individuals. While CMT disease is relatively common, our understanding of the genes mutated in patients with CMT disease and the mechanism by which these mutations lead to peripheral neuropathy remains incomplete. Genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease with an axonal pathogenesis. ARSs are ubiquitously expressed, essential enzymes, responsible for covalently attaching amino acids to their cognate tRNA molecules, thus completing an essential step of protein translation. To improve our understanding of the genetic and functional mechanisms by which ARS mutations lead to disease, we sought to: (1) identify and characterize ARS gene mutations in a cohort of patients with CMT disease and no known disease-causing mutation; (2) evaluate the functional consequences of disease-associated ARS mutations; and (3) develop a multi-cellular model system for assessing the toxicity of ARS alleles in neurons. Our genetic analyses led to the discovery of missense and frameshift lysyl-tRNA synthetase (KARS) gene mutations in a compound heterozygous patient with CMT disease and additional non-neurological sequelae, and the discovery of missense alanyl-tRNA synthetase (AARS) gene mutations in two families with CMT2N. Using a combination of biochemical analyses and yeast viability assays, we determined that disease-associated AARS and KARS mutations lead to impaired enzyme activity. Finally, we developed a C. elegans model system for evaluating the effect of ARS mutations on peripheral nerve axons in vivo, and discovered that overexpression of a mutant AARS enzyme leads to morphological defects. Together, these studies underscore the critical role of ARS enzymes in peripheral nerve function and suggest that impaired aminoacylation may be a central component to disease pathology. In summary, my dissertation research has advanced our understanding of the genetic and functional role of ARS enzymes in peripheral neuropathies and will provide a platform for further investigating the molecular pathology of ARS-related CMT disease.