Defining the Genetic and Molecular Mechanisms of Disease-Associated Mutations in Aminoacyl-tRNA Synthetase Genes

Abstract

Protein synthesis is an essential process that allows the nucleic acid code to be translated in the cell. For protein translation to occur, the ribosome must be supplied with tRNA molecules charged with amino acids. The enzymes responsible for ligating tRNA and amino acids are the aminoacyl-tRNA synthetases (ARS), encoded by a family of 37 genes. Variants in all 37 ARS genes can cause dominant and/or recessive human genetic diseases. The recessive diseases are severe, multi-system disorders that primarily affect the central nervous system, as well as the muscles, lung, and liver. The dominant diseases are axonal peripheral neuropathies commonly classified as Charcot-Marie-Tooth disease. The full clinical and genetic spectrum for both dominant and recessive ARS-related disease has yet to be determined. Additionally, the mechanism of these diseases is poorly understood. This dissertation seeks to expand the locus, allelic, and phenotypic heterogeneity of ARS-mediated diseases, and to define the mechanism of ARS-mediated disease, through 1) characterizing newly identified patient alleles; 2) developing a pipeline of model organisms to predict novel ARS disease candidates and define their associated dominant and recessive phenotypes, and 3) testing pathogenic alanyl-tRNA synthetase (AARS1) alleles for a dominant-negative effect in a yeast model.

Here, we characterized 15 variants across glycyl-(GARS1), histidyl-(HARS1), methionyl-(MARS1), asparginyl-(NARS1), and threonyl-tRNA synthetase (TARS1) identified in patients with dominant peripheral neuropathy or multisystem recessive diseases. Through synthesizing genetic and functional evidence, we expanded the allelic spectrum of GARS1- and HARS1- mediated dominant neuropathy, and the allelic and phenotypic spectrum of MARS1- and TARS1-mediated recessive disease. We also identified NARS1 as a candidate gene for dominant peripheral neuropathy. To complement these efforts, we developed a predictive pipeline using the defined phenotypes of pathogenic ARS alleles in yeast, C. elegans, and mouse. We used this pipeline to design deleterious mutations in TARS1 and assess them for a dominant peripheral neuropathy or multi-system recessive phenotypes. Through studies in yeast and worm, we identified a hypomorphic TARS1 allele, R433H. When tested in mouse, in trans with a null TARS1 allele, R433H causes a recessive phenotype of lung failure, growth restriction, and hair defects. This model will be an asset to determine how reduced TARS1 function differentially impacts mammalian tissues, and can inform clinical efforts to identify and treat patients with bi-allelic TARS1 mutations.

Finally, we directly tested the hypothesis that dominant ARS variants are dominant-negative alleles. We focused on two variants in alanyl-tRNA synthetase (AARS1) with strong genetic evidence for pathogenicity, R329H and G102R AARS1. These variants reduce gene function in a yeast complementation assay, indicating that they are loss-of-function alleles. However, they repress yeast growth when co-expressed with wild-type AARS1, indicating that they are also dominantly toxic. To determine if this dominant toxicity was due to dimerization with wild-type AARS1, we designed a dimer domain mutation that impaired dimerization, and placed it in cis with R329H and G102R. This double-mutant rescued yeast growth, showing that dimerization is required for toxicity and that R329H and G102R are dominant-negative alleles. We also assessed three additional AARS1 variants, and found that they also are dominant-negative alleles in this assay.

In sum, this work significantly contributes to defining the known genetic and phenotypic spectrum of ARS-mediated diseases, to expanding the role of model organisms in identifying candidate pathogenic ARS variants, and to defining the mechanism of dominant ARS-mediated peripheral neuropathy.