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Case Reports
. 2014 Jan;1842(1):56-64.
doi: 10.1016/j.bbadis.2013.10.008. Epub 2013 Oct 24.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency

Abdulraheem Almalki  1 , Charlotte L Alston, Alasdair Parker, Ingrid Simonic, Sarju G Mehta, Langping He, Mojgan Reza, Jorge M A Oliveira, Robert N Lightowlers, Robert McFarland, Robert W Taylor, Zofia M A Chrzanowska-Lightowlers
Affiliations
Case Reports

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency

Abdulraheem Almalki et al. Biochim Biophys Acta. 2014 Jan.

Abstract

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe).

Keywords: Aminoacyl-tRNA synthetase; Aminoacylation; LBSL; MLASA; MRI; Mitochondria; Mitochondrial disease; Mitochondrial translation; OXPHOS; PCH6; Protein synthesis; aaRS; aminoacyl-tRNA synthetase; leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation; magnetic resonance imaging; mitochondrial; mitochondrial DNA; mt-; mtDNA; myopathy, lactic acidosis and sideroblastic anaemia; oxidative phosphorylation; pontocerebellar hypoplasia type 6.

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Figures

Fig. 1
Fig. 1
Cranial MRI performed at age 2.5 years. A. Transverse T1 FLAIR image illustrating symmetrical, anterior predominant, white matter signal changes. B. Sagittal T1 weighted image demonstrating thinning of the anterior and mid portions of the corpus callosum.
Fig. 2
Fig. 2
Molecular characterisation of novel FARS2 mutations. A. Heterozygous 88 kb deletion identified on Array CGH; B. Sequencing chromatograms, arrow indicates the maternally transmitted novel c.973G > T, p.Asp325Tyr FARS2 variant. C. Alignment of the FARS2 protein sequence flanking the position of the amino acid change from eukaryotic organisms (yeast, worms, flies, fish and mammals) indicating the conservation of the p.Asp325 amino acid.
Fig. 3
Fig. 3
Effect of FARS2 mutation on mitochondrial homeostasis. A. Cytochrome c oxidase (COX) histochemistry of the patient muscle showed a generalised loss of enzyme activity compared to age-matched control tissue. B. Respiratory chain enzyme activity in muscle biopsy, fibroblast and myoblast: activities of complex I, complex II, and complex IV were determined in control (blue) and patient (red) and normalised to citrate synthase. Results are based on three independent measurements and are shown as percent of the mean control value ± standard deviation C. Steady state levels of RC proteins in fibroblasts (left panel) and myoblasts (right panel) were determined by Western blotting. 10% SDS-PAGE was performed with cell lysates (30 μg) from control (C1, C2) and patient (P), except for FARS2 and SDHA in the bottom 2 panels where 80 μg mitochondrial protein was loaded per lane. Western blots were decorated with antibodies to the proteins indicated. Secondary α-antibodies were HRP conjugated and detection was by ECL + and ImageQuant software. D. High resolution northern blot analysis was performed on total RNA (2 μg) from control (C1, C2) and patient (P). Membranes were hybridised with radiolabelled probes for mt-tRNAPhe, mt-tRNAVal and mt-tRNALeu(UUR). Densitometric analyses were performed on all blots. A representative example is presented with the values relative to controls for the signals derived for each tRNA below the sample. E. De novo mitochondrial protein synthesis in control myoblast (lane C) and the patient myoblast cells (lane P). Designation of proteins is as described by . A section of Coomassie blue (CBB) stained gel is shown indicate equal loading.
Fig. 4
Fig. 4
Mitochondrial morphology, mtDNA and nucleoid distribution. A. Nucleoid and mitochondrial morphology imaging was performed with TMRM and PicoGreen in control and patient myoblasts. TMRM accumulated in the mitochondria allowing visualisation of the mitochondrial network while PicoGreen staining localised to nucleoids indicating the position/distribution of non-supercoiled DNA, scale bar = 5 μm B. Relative quantification of mtDNA copy number in controls and patient myoblasts: quantitative real-time PCR was performed using 10 ng of total DNA for three controls (blue, red and green) and the patient (violet). MT-ND4 and 18S probes were used for the quantification of the mitochondrial and the nuclear copy number. Results shown are the mean of three measurements from three independent DNA preparations.
Fig. 5
Fig. 5
Aminoacylation activity and ATP binding ability of wild-type and mutant FARS2. A. Wild-type (blue) and p.Asp325Tyr mutant (violet) FARS2 proteins were assessed for activity in the presence or absence of ATP using either E. coli or human mt-tRNA a substrate. Incorporation of radioactive phenylalanine was measured by liquid scintillation counting n = 4. B. Wild-type (blue) and mutant (violet) FARS2 protein were assessed for ability to bind ATP. Isocitric dehydrogenase (IDH, black) was used as a control. Incorporation of radioactive ATP was measured by Cerenkov counter, n = 3.
Fig. 6
Fig. 6
Lentiviral transduction of myoblasts with wildtype FARS2. Both control (C1) and patient (P) myoblasts were transduced with lentivirus designed to express wildtype FARS2. Images are representative of a minimum of n = 3. A. Following selection cell lysates (40 μg) were subjected to Western blot analysis to determine the levels of FARS2 and mt-encoded complex IV proteins. B. High resolution northerns were performed on RNA from control (C1) and patient (P) prior to (− LV; lanes 1 and 2) or post (+ LV; lanes 3 and 4) lentiviral transduction with wild type FARS2.
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