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. 2019 Feb 15;28(4):525-538.
doi: 10.1093/hmg/ddy344.

Homozygosity for a Mutation Affecting the Catalytic Domain of tyrosyl-tRNA Synthetase (YARS) Causes Multisystem Disease

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Free PMC article

Homozygosity for a Mutation Affecting the Catalytic Domain of tyrosyl-tRNA Synthetase (YARS) Causes Multisystem Disease

Katie B Williams et al. Hum Mol Genet. .
Free PMC article

Abstract

Aminoacyl-tRNA synthetases (ARSs) are critical for protein translation. Pathogenic variants of ARSs have been previously associated with peripheral neuropathy and multisystem disease in heterozygotes and homozygotes, respectively. We report seven related children homozygous for a novel mutation in tyrosyl-tRNA synthetase (YARS, c.499C > A, p.Pro167Thr) identified by whole exome sequencing. This variant lies within a highly conserved interface required for protein homodimerization, an essential step in YARS catalytic function. Affected children expressed a more severe phenotype than previously reported, including poor growth, developmental delay, brain dysmyelination, sensorineural hearing loss, nystagmus, progressive cholestatic liver disease, pancreatic insufficiency, hypoglycemia, anemia, intermittent proteinuria, recurrent bloodstream infections and chronic pulmonary disease. Related adults heterozygous for YARS p.Pro167Thr showed no evidence of peripheral neuropathy on electromyography, in contrast to previous reports for other YARS variants. Analysis of YARS p.Pro167Thr in yeast complementation assays revealed a loss-of-function, hypomorphic allele that significantly impaired growth. Recombinant YARS p.Pro167Thr demonstrated normal subcellular localization, but greatly diminished ability to homodimerize in human embryonic kidney cells. This work adds to a rapidly growing body of research emphasizing the importance of ARSs in multisystem disease and significantly expands the allelic and clinical heterogeneity of YARS-associated human disease. A deeper understanding of the role of YARS in human disease may inspire innovative therapies and improve care of affected patients.

Figures

Figure 1
Figure 1
Pedigree, physical features and growth for YARS c.499C > A, p.Pro167Thr homozygotes. (A) Family pedigree for seven related children homozygous for the novel YARS variant. Solid shapes represent YARS p.Pro167Thr homozygotes (M/M) described in the current report. Red asterisks denote related YARS p.Pro167Thr heterozygotes (M/+) who underwent detailed neurologic examination and electrophysiological studies. (B) All children were microcephalic with poor weight gain. Five children had linear growth below the third percentile for age. For growth curves, shaded areas represent reference ranges for age. Colors represent individual children. (CE) An 18-month-old girl homozygous for YARS p.Pro167Thr with a long forehead, strabismus, deeply set eyes (C) and small hands (E). (FH) A five-year-old girl homozygous for YARS p.Pro167Thr with a long forehead (F), thin hair, low-set ears (G) and digital clubbing (H).
Figure 2
Figure 2
Brain imaging and histology for YARS c.499C > A, p.Pro167Thr homozygotes. (A) Brain MRI at 6 months of age with multiple areas of dysmyelination. Axial diffusion weighted imaging (bottom) and apparent diffusion coefficient (ADC) mapping (top) with restricted diffusion along the white matter of the temporal, occipital and parietal lobes (green arrows), medial temporal lobe (blue arrows) and central tegmental tracts (purple arrows). (B) Abnormal T2 hyperintensity of the optic nerves (white arrow), tegmental tracts (orange arrow) and superior cerebellar peduncles (red arrow) and absent myelination of the corpus callosum (yellow arrow). (C) Hematoxylin and eosin staining of brain biopsy with diffuse vacuolar changes in the gray matter neurons (black arrow) and neurofilament (pink arrow).
Figure 3
Figure 3
Chronic liver disease in YARS c.499C > A, p.Pro167Thr homozygotes. (A and B) Affected children had chronic direct hyperbilirubinemia, coagulopathy and hypoalbuminemia accompanied by modest elevations in ALT, GGT and ammonia. (C) Trichrome staining of liver biopsy for child (age 3 months) initially revealed diffuse micro- and macrovesicular steatosis but no evidence of fibrosis. (D) Repeat biopsy (age 18 months) showed disrupted liver architecture, bridging fibrosis and nodule formation. (E) Electron microscopy showed poorly defined cristae within the mitochondria (white arrows). Blue arrows identify normal-appearing mitochondria. For laboratory values, colors represent individual children. Shaded areas reflect laboratory reference ranges.
Figure 4
Figure 4
Chronic anemia in YARS c.499C > A, p.Pro167Thr homozygotes. (A) Anemia was noted on presentation (age 5 weeks) for one child and required multiple blood transfusions (red triangles) throughout life. Reticulocyte count was initially low in the setting of anemia, then increased robustly without the use of erythropoietin. (B) MCV was initially low, then increased to macrocytic range around 8 months of age. RDW remained chronically elevated. (C) Blood smear at age 9 months shows abnormal erythrocyte morphology, including central pallor (white arrow), target cells (red arrow) and tear drop cells (black arrow). (D) Bone marrow biopsy at age 3 months with normocellular bone marrow and trilineage hematopoeiesis, but increased myeloid to erythroid ratio with a paucity of erythroid precursors. For laboratory studies, shaded regions represent reference ranges.
Figure 5
Figure 5
YARS p.Pro167Thr results in a loss-of-function in yeast complementation assays. Yeast lacking endogenous TYS1 (the yeast ortholog of YARS) and harboring a maintenance vector containing wild-type (Wt) TYS1 and URA3 were transformed with an experimental vector containing LEU2 and Wt TYS1, TYS1 p.Gly41Arg (a known null allele), TYS1 p.Glu196Gln (a known hypomorphic allele), TYS1 p.Pro167Thr (in triplicate; A, B, and C) or a vector with no TYS1 insert (`Empty’). (A) All yeast lines grew on solid media deficient in leucine and uracil (lower panel), confirming proper uptake of both vectors. Adding 5-FOA to select against the URA3-containing maintenance vector allowed an assessment of growth supported by the experimental vector alone (upper panel). Wt TYS1 (‘Wt’) supported robust cellular growth while the vector with no TYS1 insert (‘Empty’) did not support any cellular growth. TYS1 p.Gly41Arg did not support any cellular growth and TYS1 p.Glu196Gln supported severely reduced cellular growth, consistent with previous assessments of these alleles. In a manner similar to TYS1 p.Glu196Gln, TYS1 p.Pro167Thr supported severely reduced cellular growth. (B) Yeast carrying the TYS1 p.Gly41Arg allele or an empty vector was unable to grow in liquid 5-FOA media. Note the 15-h lag between mid-log phase of wild-type and TYS1 p.Pro167Thr expressing yeast. Error bars represent standard deviations of the average OD600 of three biological replicates for each strain. TSY1 p.Glu196Gln was excluded from growth curve analysis to simplify the figure.
Figure 6
Figure 6
Substitution of threonine for proline impairs YARS homodimerization. (A) Schematic of the functional domains of human TyrRS (35,52). YARS c.499C > A causes a substitution of Thr for Pro at amino acid position 167, within the Rossmann fold necessary for YARS homodimerization. Neighboring amino acids 159–161 of connective polypeptide 1 (CP1) (Pro, Leu, Leu, − PLL; yellow highlight) are critical for dimer formation (35), and the amino acids highlighted in green are highly conserved members of the dimer interface. Recognition of tRNA-Tyr occurs at the anticodon binding domain, and the C-terminus is homologous to the proinflammatory cytokine endothelial-monocyte-activating polypeptide II (EMAP II) (41,42,53). (B and C) Recombinant YARS-FLAG p.Pro167Thr overexpressed in HEK-293 T cells (C) localizes to the cytosol in a manner indistinguishable from that of TyrRS-FLAG wild-type (wt) (B) (n = 3). Scale bar in (C) = 10 μm. Green fluorescence: anti-FLAG immunoreactivity; blue fluorescence: DAPI. (D and E) Human YARS-FLAG wt and p.Pro167Thr constructs were co-overexpressed with human YARS-V5 wt and p.Pro167Thr constructs in HEK-293 T cells (as indicated). YARS-FLAG proteins were immunoprecipitated (IP) from whole cell lysates (input) (n = 5). YARS-FLAG wt co-precipitated YARS-V5 wt abundantly [normalized to 1 in the immunoblotted (IB) protein band optical densities displayed in E]. Co-precipitation of YARS-V5 p.Pro167Thr by YARS-FLAG wt and YARS-V5 wt by YARS-FLAG p.Pro167Thr were reduced roughly 5-fold. Co-precipitation of YARS-V5 p.Pro167Thr by YARS-FLAG p.Pro167Thr was reduced 10-fold. wt: wild type; m: YARS c.499C > A, p.Pro167Thr mutant; empty: ‘empty vector’ encoding the FLAG epitope tag peptide sequence followed by a stop codon. IB protein band optical density (E) was measured from three replicates of the co-immunoprecipitation data shown in D. Error bars represent one standard deviation.
Figure 7
Figure 7
Protein structural modeling of the YARS p.Pro167Thr substitution. (A) Structural models for YARS wt (Uniprot P54577) (left) and YARS p.Pro167Thr (right) generated using SWISS-MODEL/ProMod3 v1.1.0 (54–58) based on template PDB 5thl.1.A (best fit from 72 templates; GMQE = 0.7; identity = 99.73). In both cases, YARS is displayed as a homodimer. The models assume homozygosity for YARS wt (left) and YARS p.Pro167Thr (right). Homodimerization is preserved in the YARS Pro167Thr model, but with a 3.1% reduction of the QSQE oligomerization score (from 0.65 for wt to 0.63 for Pro167Thr). (BE) Models in B–E are displayed as monomers for greater resolution. ‘Overlay’ (center) highlights differences between the models. Proline 167 (Pro167) is situated within the Rossmann fold necessary for YARS homodimerization. Neighboring amino acids of CP1 (Pro159, Leu160, Leu161; blue labels) are important for dimer formation, although C-terminal amino acids also stabilize the dimer (59). Amino acids labeled in green are highly conserved members of the dimer interface (34). The YARS Pro167Thr substitution induces reorganization of amino acids 119–123 (Lys-Gly-Thr-Asp-Tyr) (purple box in B and asterisk (*) near Thr121 in C–E) which normally folds as a small beta-sheet/alpha-helix/turn motif in close proximity to the dimer interface and tyrosine (Tyr) binding pocket. Tyrosine166 (Tyr166), adjacent to Pro167 and the helix formed by amino acids 120–122, is one of five amino acids in this region that coordinate Tyr binding (60). (C–E) Rotations of the model from the perspectives of amino acids Tyr166 (C), Leu160 (D) and Leu 169 (E) demonstrating the impact of the p.Pro167Leu substitution on local structure within the dimer interface and tyrosine (Tyr) binding pocket.

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