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. 2023 Nov;25(11):100938.
doi: 10.1016/j.gim.2023.100938. Epub 2023 Jul 13.

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder

Andrea Accogli  1 , Sheng-Jia Lin  2 , Mariasavina Severino  3 , Sung-Hoon Kim  4 , Kevin Huang  2 , Clarissa Rocca  5 , Megan Landsverk  6 , Maha S Zaki  7 , Almundher Al-Maawali  8 , Varunvenkat M Srinivasan  9 , Khalid Al-Thihli  8 , G Bradly Schaefer  10 , Monica Davis  10 , Davide Tonduti  11 , Chiara Doneda  12 , Lara M Marten  13 , Chris Mühlhausen  13 , Maria Gomez  14 , Eleonora Lamantea  15 , Rafael Mena  16 , Mathilde Nizon  17 , Vincent Procaccio  18 , Amber Begtrup  19 , Aida Telegrafi  19 , Hong Cui  19 , Heidi L Schulz  20 , Julia Mohr  20 , Saskia Biskup  21 , Mariana Amina Loos  22 , Hilda Verónica Aráoz  23 , Vincenzo Salpietro  24 , Laura Davis Keppen  6 , Manali Chitre  25 , Cassidy Petree  2 , Lucy Raymond  25 , Julie Vogt  26 , Lindsey B Sawyer  27 , Alice A Basinger  27 , Signe Vandal Pedersen  28 , Toni S Pearson  29 , Dorothy K Grange  30 , Lokesh Lingappa  31 , Paige McDunnah  32 , Rita Horvath  33 , Benjamin Cognè  17 , Bertrand Isidor  34 , Andreas Hahn  35 , Karen W Gripp  32 , Seyed Mehdi Jafarnejad  36 , Elsebet Østergaard  37 , Carlos E Prada  38 , Daniele Ghezzi  39 , Vykuntaraju K Gowda  9 , Robert W Taylor  40 , Nahum Sonenberg  4 , Henry Houlden  5 , Marie Sissler  41 , Gaurav K Varshney  42 , Reza Maroofian  43
Affiliations

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder

Andrea Accogli et al. Genet Med. 2023 Nov.

Abstract

Purpose: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype but with limited neuroradiological data and insufficient evidence for causality of the variants.

Methods: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays and a zebrafish model.

Results: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs, and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model.

Conclusion: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity.

Keywords: Cerebellar atrophy; Mitochondrial dysfunction; Mitochondrial threonyl-tRNA-synthetase; TARS2; mTORC1 signaling.

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Conflict of interest statement

Conflict of Interest Amber Begtrup, Hong Cui, and Aida Telegrafi are employees of GeneDx, LLC. All other authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Clinical summary of individuals with biallelic TARS2 variants. Pedigree of families 1-15 (A) and previously reported individuals (B). Genotypes of tested individuals are indicated under the symbols. Variants observed in more than 1 family have been shown in different colors. In the pedigree, squares represent males, circles represent females, black shaded symbols denote affected individuals harboring biallelic TARS2 variants, and the gray shaded symbol refer to individuals presumably affected by the same disorder yet not tested. C. Schematic depiction of TARS2 transcript (ENSG00000143374) and the modular human mitochondrial threonyl-tRNA synthetase (TARS2) protein (ENSP00000358060) with localization of the disease associated variants. The different functional domains are shown to scale, named, and colored. MTS and AB stand for mitochondrial targeting sequence and anticodon-binding, respectively. m1, m2, and m3 are catalytic motifs 1, 2, and 3, respectively. Missense variants are indicated in red for those reported in the present study and in black for those reported elsewhere. Allelic compositions, as identified in individuals, are linked through black lines. Variants that do not lead to missense variants are indicated in brackets (because they correspond to genomic modifications). Although amino acid conversion of a given mutation is generally preceded by the letter “p.,” this is omitted for sake of simplicity. D. Clinical features of individuals with biallelic TARS2 variants. Subtle and non-specific dysmorphic features are indicated, such as thin upper lip vermilion in individual II:1 of family 5; mild coarse facial features in individuals II:1 and II:3 of family 6; short nose, long philtrum, and thin upper lip vermilion in individual II:2 of family 7; left eye strabismus and right uplifted earlobe in individual II:1 of family 9; broad and prominent forehead, sparse eyebrows, infraorbital creases, thin upper lip vermilion, and dimple chin in patient II:2 of family 10; thick eyebrows in individual 3; and deep set eyes in individual 5 previously reported by Zheng et al., E. Phenotypic bar graph showing the most relevant clinical and radiological features among all individuals so far identified with biallelic TARS2 variants. Red: number of individuals out of 28 showing each feature. Blue: number of individuals without each specific feature. Gray: number of individuals for whom a specific feature or the brain MRI was not available.
Figure 2
Figure 2
Neuroradiological features of individuals with biallelic TARS2 variants and of a control subject for comparison. Most of the individuals present cerebellar atrophy (open arrows) variably associated with cerebellar cortex signal alterations and dentate nuclei T2/FLAIR hyperintensity (arrowheads). There is mild-to moderate white matter volume loss and/or enlargement of the cerebral CSF spaces in most individuals. The corpus callosum is thin in 8 individuals (thick arrows). Note the faint T2 signal abnormalities in the globi pallidi (dashed arrows) and putamen/caudate nuclei (thin arrows). CSF, cerebrospinal fluid.
Figure 3
Figure 3
Protein modeling and interaction of mutant TARS2 with Rag GTPase. A. Structure of the dimeric ThrRS from Staphylococcus aureus (PDB: 1NYR;14). Functional domains are named and colored in 1 monomer. B. Structure of one monomer of the Escherichia coli ThrRS, in complex with cognate tRNA with the CCA extremity of the tRNA pointing into the catalytic core (PDB: 1QF6;15). C-G. Structural environment of the wild-type residues and theoretical impact of their variants. Structural templates are indicated in light gray. The 3D model of human TARS2 is from misynpat.org., In all models, motifs 1, 2, and 3 that build the catalytic core are colored in white. Residues found mutated are shown in stick representation and colored in red for those reported in the present study and in black for those reported elsewhere (see Figure 1 and Supplemental Table 2 for references), with the corresponding amino acid from either S. aureus or E. coli indicated in brackets. C. Zoom into the catalytic core where catalytic Zn2+ and small substrates (aminoacyl-adenylate aa∼AMP and PPi) are shown in cyan. D. Zoom into the editing domain. Catalytic residues for editing, as identified in, are shown in orange. E. Zoom into the anticodon-binding domain. Key nucleotides from the tRNA anticodon loop are numbered (34-37). F. Zoom into the interface between the catalytic domain and the anticodon-binding domain. G. Example of possible local distortion engendered by the L544F variant. All molecular representations were prepared with PyMOL (Schrödinger, Inc.). H. Correspondence of residues of human (Uniprot: Q9BW92), E. coli (Uniprot: P0A8M3), S. aureus (Uniprot: Q2YTA7), and D. rerio (NCBI: XP_021322466.1) in the 3 structural templates as established using the multiple sequence alignment. I. Co-immunoprecipitation (co-IP) interaction of mutant TARS2 variants with Rag GTPases. HEK293T cells were transfected with the indicated plasmids. Co-IP was performed using an anti-hemagglutinin (HA) antibody. Triangles on the right indicate the full-length RagA (blue) and RagC (red). α-tubulin is shown as a loading control. EV, empty vector; WT, wildtype. J. Mean ± SEM of relative binding of TARS2 with RagA (left) or RagC (right) from 3 independent co-IP experiments is shown. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (one-way ANOVA followed by Dunnett’s post hoc test).
Figure 4
Figure 4
Functional testing of TARS2 variants in zebrafish. A. Representative images of an embryo injected with control MO (ctrl MO), tars2 MO, or tars2 MO rescue at 3 dpf. Lateral view, anterior to the left. The blue line indicates brain size, and the black arrow indicates heart edema. B. Quantification of head size from uninjected (n = 30 animals), ctrl MO (n = 31 animals), tars2 MO (n = 52 animals), tars2 MO + zebrafish tars2 RNA (+ tars2) (n = 41 animals), and tars2 MO + human TARS2 RNA (+ TARS2) (n = 52 animals) embryos at 3 dpf. Each symbol represents 1 animal. Values were calculated as percentage of the mean value of uninjected embryos. Error bars = mean ± SD. C. Quantification of heart edema. Animals were collected according to phenotype as shown in the image and calculated as the percentage of total animals. D. Representative cerebellum image of Tg(olig2:DsRed) of ctrl MO, tars2 MO, and tars2 MO rescue at 3 dpf. The red fluorescence color was replaced with pseudo-magenta color. E. Quantification of cerebellum area as indicated in (D). n = 10 animals. Error bars = mean ± SD. F. Acridine orange was used to stain brain in ctrl MO, tars2 MO, and tars2 MO rescue at 3 dpf. Scale bar = 100 μm. G. Confocal projections of phalloidin stained muscle fibers in the trunk region of ctrl MO, tars2 MO, and tars2 MO rescue. The anterior is shown to the left and dorsal at the top. The right panels show orthogonal views, dorsal to the top. H. Experimental approach for functionally characterizing human TARS2 variants. In vitro synthetic human TARS2 variant mRNA was mixed with zebrafish tars2 splice-blocking MO and microinjected into 1-cell stage embryos, followed by phenotypic evaluation at 3 dpf. The blue line indicates head size and the black arrow indicates heart edema. I. Summary of rescue results with the mean value for each group (from Figure S6). No rescue means the mean value of the group is close to the mean of tars2 MO group, and the statistic shows no significance compared with tars2 MO group. Partially rescued means the mean value of the group is higher than the mean of tars2 MO group but lower than the mean of uninjected control group, and the statistical difference shows significance compared with tars2 MO but also shows significance compared with the uninjected control group. Rescue means the mean value of the group is close to the uninjected control and the statistical difference shows no significance compared with uninjected control group. J. Quantification of heart edema. Animal’s phenotypes were quantified as shown in the image and the percentage of total animals was calculated. In (B and E), one-way ANOVA with Tukey’s multiple comparison correction is shown: ns, not significant P ≥ .05, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 and ∗∗∗∗P < .0001 compared with tars2 MO group.
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