11 research outputs found

    Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome.

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    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome

    Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome.

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    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome

    <i>NARS2</i> mutations identified in two unrelated families.

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    <p>(A) Pedigree of the LS06 family. Filled symbols represent affected individuals and small circles represent carrier individual. The pedigree shows autosomal recessive inheritance of compound heterozygous NARS2 variants [c.969T>A; p.Tyr323*] and [c.1142A>G; p.Asn381Ser]. (B) SDS PAGE and Western blot of control and patient II.1 muscle homogenates (10μg and 20μg of protein), samples were probed for mitochondrial respiratory chain complexes via MitoProfile total OXPHOS human WB antibody cocktail. The result showed significantly decreased amounts of mitochondrial respiratory complex I and IV. (C) SDS PAGE and Western blot of fibroblast lysates from both affected probands (II.1, II.3), their parents (I.1, I.2) and controls using anti-NARS2 antibody and anti-GAPDH antibody as loading control. The expected position of a truncated NARS2 protein product (Δ154aa) stemming from the p.Tyr323* allele is indicated with a black arrow. (D) Pedigree of the PKDF406 family. Filled symbols represent affected individuals, and a double horizontal line represents a consanguineous marriage. Alleles forming the risk haplotypes are boxed. The short tandem repeat (STR) markers, their relative map positions (Mb) according to UCSC Genome Bioinformatics build GRCh37 (hg19), and their genetic positions (cM) based on the Marshfield genetic map are shown next to the pedigree. A haplotype analysis revealed a linkage region delimited by a proximal meiotic recombination at marker D11S911 in individual IV:4 (arrowhead) and distal recombination at marker D11S4082 in individuals IV:8 and IV:9 (arrowhead).</p

    NARS2 homodimerization and RNA level: effect of the p.Val213Phe and p.Asn381Ser mutations.

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    <p>(A) Immunoprecipitates (IP) with anti-GFP antibodies from HEK293T cells transiently transfected with GFP-tagged (arrowhead) and HA-tagged NARS2 (arrow) constructs. Precipitates were immunoblotted with antibodies to the GFP and HA tags. NARS2 homodimerizes, and the p.Val213Phe mutation does not affect the dimerization process. No dimerization was detected with p.Asn381Ser NARS2 construct. (B) Steady state level for mt-tRNA<sup>Asn</sup> was assessed by Northern blot and the results were validated by two independent laboratories. 5S-rRNA probe was used as a loading control on the same membrane. In fibroblasts of patient II.1, from LS06 family, the level of mt-tRNA<sup>Asn</sup> is decreased compared to his parents and a control sample. Due to high passage number, we could not measure the mt-tRNA<sup>Asn</sup> levels in the fibroblast of patient II.3.</p

    Expression of <i>Nars2</i> in mouse inner ear and brain.

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    <p>(A) RT-PCR analysis of <i>Nars2</i> expression in the C57Bl6/J mouse cochlear (C), vestibular (V) tissues and brain (B) at different developmental stages (P0, P30 and P90). <i>Gapdh</i> expression was used as an internal control. (B-G) Expression of <i>Nars2</i> in the P2 mouse cochlea is shown. Hybridization signals of <i>Nars2</i> antisense (B, D, F) and sense (control) probes (C) in mid-modiolar sections of P2 cochlea are shown. Positive signals were detected in the cochlear epithelium, including the region of the organ of Corti, (D) as indicated by the Myo15a-positive hair cells (E, bracket). Positive signals were also detected in the cells surrounding the cochlear duct (D, asterisks) and neurofilament-positive spiral ganglion (G, SG). B, C, E and G are 12 mm adjacent sections. Abbreviations: cd, cochlear duct, SG, spiral ganglion. The scale bar in C is 100 μm and applies to B and C. The scale bar in G is 100 μm and applies to D-G.</p

    <i>NARS2</i> structure and molecular modeling.

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    <p>(A) Schematic representation of <i>NARS2</i> gene and predicted encoded protein product. Exons are represented with boxes. The anti-codon binding domain is shown in pink, and the catalytic domain is shown in beige. The c.637G>T, c.969T>A, c.1142A>G mutations are located in exons 6, 10 and 11, respectively, which are all coding for a part of the catalytic domain of the protein. (B) Protein sequence alignments show the evolutionary conservation of the mutated residues (arrows). (C) Mapping of homologs of the p.Val213Phe and p.Asn381Ser mutations in a 3D structure of NARS2. This 3D model of the NARS2 protein is based on the structure of <i>Pyrococcus horikoshii</i> AsnRS. The asparagine binding site is represented in beige; Mg<sup>2+</sup> ions are shown in red. The human mutations p.Val213Phe and p.Asn381Ser were mapped on the <i>Pyrococcus horikoshii</i> molecule at position p.Lys181 and p.Glu337, respectively. (D) Dimeric representation of <i>Entamoeba histolytica</i> AsnRS (PDB: 3M4Q) using PyMol molecular graphics system. Anticodon binding domain is represented in green, hinge region in pink, catalytic core in blue and second monomer is shown in grey. The <i>E</i>. <i>histolytica</i> residue corresponding to human NARS2 p.Asn381 is p.Val355 and shown by an orange sphere. The <i>E</i>. <i>histolytica</i> residue corresponding to human p.Val213 is p.Glu197 and is displayed in dark brown.</p
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