Fig 1.
NARS2 mutations identified in two unrelated families.
(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).
Table 1.
Clinical findings of affected PKDF406 family members.
Fig 2.
Expression of Nars2 in mouse inner ear and brain.
(A) RT-PCR analysis of Nars2 expression in the C57Bl6/J mouse cochlear (C), vestibular (V) tissues and brain (B) at different developmental stages (P0, P30 and P90). Gapdh expression was used as an internal control. (B-G) Expression of Nars2 in the P2 mouse cochlea is shown. Hybridization signals of Nars2 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.
Fig 3.
NARS2 structure and molecular modeling.
(A) Schematic representation of NARS2 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 Pyrococcus horikoshii AsnRS. The asparagine binding site is represented in beige; Mg2+ ions are shown in red. The human mutations p.Val213Phe and p.Asn381Ser were mapped on the Pyrococcus horikoshii molecule at position p.Lys181 and p.Glu337, respectively. (D) Dimeric representation of Entamoeba histolytica 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 E. histolytica residue corresponding to human NARS2 p.Asn381 is p.Val355 and shown by an orange sphere. The E. histolytica residue corresponding to human p.Val213 is p.Glu197 and is displayed in dark brown.
Fig 4.
NARS2 homodimerization and RNA level: effect of the p.Val213Phe and p.Asn381Ser mutations.
(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-tRNAAsn 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-tRNAAsn is decreased compared to his parents and a control sample. Due to high passage number, we could not measure the mt-tRNAAsn levels in the fibroblast of patient II.3.
Fig 5.
Analysis of the impact of the NARS2 mutations on mitochondrial functions.
(A) Oxygen consumption in intact patient cells with NARS2 overexpression. Oxygen consumption in NARS2 overexpressing cells was analyzed using the Seahorse XF24 analyzer. 2 μM oligomycin, 4 μM FCCP and 2 μM rotenone/antimycin were added at the indicated points. (B) Oxygen consumption rates from fibroblast mitochondria of the indicated genotypes. “Null” corresponds to fibroblasts from patient II.1 of LS06 family. Overexpression of the p.Val213Phe NARS2 construct failed to improve the oxygen consumption rate, but the wild type NARS2 construct significantly rescued it (wild type NARS2: OCR ratio = 0.650 ±0.103, p.Val213Phe NARS2: OCR ratio = 0.459 ±0.027, n = 3, p = 0.003). (C) Enzymatic activity of the individual respiratory chain complexes from fibroblast mitochondria of the indicated genotypes. Complex II activity was unaffected in all genotypes. Overexpression of the wild type NARS2 construct significantly improved the activities of complexes I, III and IV (p = 0.002, p = 0.032 and p = 0.004, respectively), and expression of the p.Val213Phe NARS2 construct had no effect. Students t-test have been performed for statistical analysis. Data are represented as the mean ± SEM.
Table 2.
Summary of the known aminoacyl tRNA-synthetase genes associated with deafness.