Bi-allelic TARS Mutations Are Associated with Brittle Hair Phenotype

Abstract

Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEβ), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638^∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.

Keywords: TTD; aminoacyl tRNA synthetase; brittle hair; non-photosensitive trichothiodystrophy; protein translation; tiger tail; transcription.

Figure 1

Identification of TARS Variants (A) Sanger sequencing profiles of TARS cDNA. Subject TTD5VI shows homozygosity for the c.680T>C missense variant (right panel), and subject TTD18PV shows compound heterozygosity for missense variant c.826A>G (left panel) and nonsense variant c.1912C>T (middle panel). (B) Amino acid conservation across different species orthologs. (C) Schematic representation (created with InterPro) of the domain structure of the TARS protein with the location of the subjects’ variants.

Figure 2

Functional Consequence of TARS Variants (A) Immunoblot analysis used for determining TARS protein amounts in lysates of primary fibroblasts TTD5VI and TTD18PV and two wild-type controls; anti-tubulin was used as a loading control. (B) Quantification of the immunoblot. The band intensities of TARS were normalized to tubulin and expressed as percentage of the intensities for control cells (C4RO). The error bars indicate the SEM of three independent experiments. (C) Representative picture of a yeast complementation assay. Wild-type and mutant strains were grown on solid medium without leucine and uracil (−LU; left panels) or in the presence of 5-FOA (right panels). Cells were spotted with serial dilutions and pictures were taken after 96 h at 30°C. (D) The cytosolic fraction from TTD5VI, TTD18PV, and two control fibroblasts was used for determining TARS (blue) and KARS (green; internal control) amino acid charging on tRNA. Threonine and lysine charging in control fibroblast C4RO was set at 100%, and error bars indicate SD of three independent experiments.