Defining the Role of Cysteinyl-tRNA Synthetase (CARS1) in Human Recessive Disease

Abstract

Proteins are essential to nearly all aspects of biology, and the synthesis of proteins from genetic information (i.e., translation) is a vital cellular process. The first step of translation is performed by aminoacyl-tRNA synthetases (ARSs), a group of essential enzymes that ligate tRNA molecules to cognate amino acids. Mutations in all 37 human ARS-encoding loci have been associated with two distinct clinical presentations: (i) dominant axonal peripheral neuropathy; and (ii) severe, early-onset, multi-system recessive disease. However, our understanding of the allelic, locus, and clinical heterogeneity of ARS-related phenotypes is incomplete, and the molecular, cellular, and organismal consequences of ARS variants are poorly defined. In this dissertation, we: (1) expand the allelic and phenotypic heterogeneity of ARS-mediated dominant and recessive disease; (2) provide the first report of pathogenic cysteinyl-tRNA synthetase 1 (CARS1) variants in a multi-system recessive human disease; and (3) investigate the downstream consequences of CARS1 variants on translation.

In Chapter 2, we evaluate the evidence for pathogenicity of 13 previously unreported ARS variants. For each variant, we assess segregation with disease, frequency in the general population, conservation of the affected amino-acid residues, and effects on gene and protein function. We expand the allelic heterogeneity of alanyl-tRNA synthetase 1 (AARS1)- and glycyl-tRNA synthetase 1 (GARS1)-associated dominant disease. Additionally, we expand the phenotypic spectrum of alanyl-tRNA synthetase 2 (AARS2)- and histidyl-tRNA synthetase 1 (HARS1)-associated recessive disease to include ataxia. Functional studies reveal that the majority of ARS variants result in reduced function and suggest that impaired tRNA charging may contribute to disease pathogenesis in both dominant and recessive ARS-associated diseases.

In Chapter 3, we present clinical, genetic, and functional data that implicate cysteinyl-tRNA synthetase 1 (CARS1) variants in a complex, multi-system, recessive disease that includes microcephaly, developmental delay, and brittle hair and nails. Functional studies reveal that each variant results in complete or partial loss-of-function effects, suggesting that tRNACys charging is impaired, which may lead to defects in translation. In Chapter 4, we utilize cell, yeast, and mouse models to investigate effects of variants on global translation and specifically on the translation of cysteine-rich proteins. Assessments of patient fibroblast cells reveal no significant differences in global translation between patient and control cells; however, preliminary data from dual luciferase assays in yeast expressing disease-associated CARS1 variants suggest that translation of cysteine-rich sequences may be impaired. Furthermore, to evaluate the effects of CARS1 variants in a tractable mammalian model with disease-relevant tissues, a mouse model homozygous for a disease-associated CARS1 variant is generated. Homozygous mice recapitulate the growth restriction phenotype observed in patients but display no other phenotypes. Future investigation of a mouse that is compound heterozygous for the disease-associated CARS1 variant and a null allele will test if this more severe genotype causes effects on translation in patient-relevant tissues.

Overall, this thesis research expands the clinical, locus, and allelic heterogeneity of ARS-associated disease, provides insight into the pathogenic mechanisms and downstream consequences of ARS variants, and may lead to potential avenues for therapeutic development.