Aminoacyl-tRNA synthetases (ARSs) are a ubiquitously expressed, essential class of enzymes responsible for ligating amino acids to cognate tRNA molecules. A subset of ARS enzymes are mutated in recessive and dominant human genetic diseases and additional ARS alleles of unknown significance are being identified at a rapid pace. It is now important to proactively develop disease-relevant functional data for ARS variants to rapidly inform patient diagnosis and prognosis, and to distinguish ARS variants relevant for recessive diseases from those relevant for dominant diseases. More than 20 missense mutations in glycyl-tRNA synthetase (GARS) and alanyl-tRNA synthetase (AARS) have been implicated in recessive, early-onset neurodevelopmental diseases or in dominant, late-onset peripheral neuropathy. We have shown that all disease-associated mutations cause a loss-of-function effect, which can be detected in yeast complementation assays. Importantly, these assays can distinguish between pathogenic and non-pathogenic alleles. We have also shown that GARS and AARS missense mutations associated with dominant neuropathy are neurotoxic when over-expressed in a worm model system; the combined loss-of-function and neurotoxic effects of these missense mutations support a dominant-negative mechanism. To fully assess these two loci for pathogenic mutations, improve our ability to predict the subset of mutations that will cause dominant neuropathy, and advance our understanding of the molecular pathology, we will:
(Aim 1) perform massively parallel mutagenesis of each gene and identify all loss-of-function mutations in allelically heterogeneous yeast cultures;
and (Aim 2) prioritize the identified mutations and test them for an effect on enzyme dimerization, for a dominant-negative effect in yeast, and for dominant neurotoxicity in C. elegans. If impaired tRNA charging is an important component of ARS-related dominant neuropathy then loss-of-function missense mutations in any ARS should cause this disease. To address this, we will identify loss-of-function missense mutations in an ARS enzyme that has not been implicated in neuropathy, test these mutations for dominant toxicity in worm, and assess the most promising mutations for a role in dominant neuropathy in mouse (Aim 3). These efforts will provide a complete catalog of pathogenic GARS and AARS mutations for research and diagnostic purposes, and will more broadly evaluate the role of ARS enzymes in peripheral nerve health.
The employment of rapid, affordable `next-generation' sequencing technologies has provided a wealth of human genetic variation. A major challenge is to rapidly determine the subset of this variation that causes genetic disease. Here, we initiate efforts to comprehensively catalog all possible pathogenic mutations in a class of 37 human genes (aminoacyl-tRNA synthetases) associated with various disease phenotypes. These efforts will improve our understanding of the pathogenicity of the associated human diseases and provide datasets for rapid patient diagnosis.
