Abstract

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNATyr were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNATyr metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

Introduction

Leber's hereditary optic neuropathy (LHON) [MIM53500] is the most common mitochondrial disorder leading to severe visual impairment or even blindness by death of retinal ganglion cells, affecting from children to young adults (1–5). In the majority of cases worldwide, LHON is due to one of three point mutations in the mitochondrial DNA (mtDNA) encoding three subunits of respiratory chain complex I (NADH dehydrogenase): ND1 3460G>A, ND4 11778G>A and ND6 14684T>C mutations (5–10). Only ∼50% of male and ∼10% of female matrilineal relatives carrying one of above mutations develop optic neuropathy, reflecting the complex etiology of this disease (5,11–13). Furthermore, only relatively mild mitochondrial dysfunction, especially the reduced activity of complex I, was observed in mutant cells carrying one of these mutations (14–16). These suggest that the LHON-associated mtDNA mutation(s) is the primary causative evident, but the additional genetic factors such as nuclear modifier genes are required for the phenotypic expression of LHON (5,16–19). In particular, a predominance of male patients presenting with visual loss suggests a nuclear encoded X-linked susceptibility allele(s) for the phenotypic manifestation of LHON-associated mtDNA mutations in some pedigrees (20), while the inherited patterns of other families carrying the m.11778G>A or m.14484T>C mutation indicated the involvement of autosomal recessive modifier genes in the phenotypic expression (17,19,21,22).

Huge efforts have been undertaken in the last few years to identify LHON susceptibility alleles in the nuclear genomes. Despite statistical support for the linkages of several putative nuclear modifier loci, no mutations in these modifier genes have been identified (20,21,23). Thus, the involvement of multiple factors and a relatively rare disorder make it very difficult to identify such nuclear modifier genes by use of conventional genetic approaches, such as genomewide linkage analysis. In this study, we used an exome sequencing approach, in combination with a functional cell assay, to identify the LHON susceptibility allele(s) in the nuclear genomes. By taking advantage of a large cohort of 1281 Chinese probands with LHON (24,25), we performed the exome sequencing of DNA from four members (proband III-2, affected mother II-1 and unaffected sibling III-4 and father II-1) of WZ142 pedigree carrying the m.11778G>A mutation (26). As a result, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase (27). The change of highly conserved 191 residue glycine to valine at the catalytic domain may alter tRNA metabolism, thereby causing mitochondrial dysfunction (28). We then performed Sanger sequence analysis of a cohort of 305 symptomatic subjects and 270 asymptomatic individuals of 167 Chinese pedigrees carrying m.11778G>A mutation and 265 controls subjects lacking the m.11778G>A mutation. Functional significance of the p.191G>V mutation in the YARS2 was first assessed for the effects on the stability of YARS2 and tRNATyr metabolism including aminoacylation capacities and stability of tRNA, through use of lymphoblastoid mutant cell lines derived from six matrilineal relatives of Chinese families (individuals carrying only the m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations but lacking a clinical phenotype, and subjects exhibiting visual loss and both m.11778G>A and homozygous p.191Gly>Val mutations) and genetically unrelated control subjects lacking these mutations. This p.191Gly>Val mutation was further evaluated for effect on mitochondrial translation, respiration, production of ATP and reactive oxygen species (ROS) using above cell lines.

Results

Identification of YARS2 mutation by whole exome sequencing

To identify the nuclear modifier gene for the phenotypic manifestation of LHON-associated mtDNA mutation, we performed exome sequencing of DNA from four members (proband III-I, affected mother II-2 and unaffected sibling III-4 and father II-1) of WZ142 family carrying the m.11778G>A mutation (Fig. 1A). The overview of the exome analysis is summarized in Figure 1B and Supplementary Material, Table S1. A total of 77 358 single nucleotide variants (SNVs) were identified as a result of variant calling. Of these, we excluded non-coding variants and then filtered for variants that were absent or present in less than 1% frequency in the 1000 genomes database, resulting in 3451 SNVs. Of these, 152 SNVs were homozygous and 2157 SNVs were heterozygous in proband III-I. Using the SIFT genome tool (http://sift.jcvi.org/www/SIFT_chr_coords_submit.html), seven of the homozygous SNVs were predicted to cause amino acid changes that would be damaging to the protein; and of the heterozygous SNVs, 21 genes were found to contain two or more variants predicted to be damaging. Using the MitoCarta database program for filtering these candidate genes, only one encoded a protein with a known mitochondrial function: YARS2 encoded the mitochondrial tyrosyl-tRNA synthetase (27). The identified SNV c.572G>T is located in the exon I of YARS2 (RefSeq NC_000012.12) and results in the change of highly conserved 191 glycine to valine (p.191Gly>Val) at the catalytic domain (28). Sanger sequence analysis of DNA fragment spanning all exons and their flanking sequences of YARS2 (Fig. 1C) confirmed that symptomatic subjects II-2, III-1 carried the homozygous p.191Gly>Val mutation and asymptomatic individual III-4 and married-in-control II-1 harbored the heterozygous p.191Gly>Val mutation, respectively. Furthermore, we identified additional eight polymorphisms in these subjects (Supplementary Material, Table S2).

Figure 1.

(A) Six Chinese pedigrees with LHON. Vision-impaired individuals are indicated by blackened symbols. Individuals who harbored homozygous (−/−), heterozygous (+/−) or wild-type (+/+) YARS2 mutations are indicated. (B) Summary of exome sequencing of patient (WZ142 III-1). The identified SNV c.572G>T (p.191Gly>Val) is located in YARS2, a gene encoding a mitochondrial tyrosyl-tRNA synthetase. (C) Partial sequence chromatograms of YARS2 gene. Sanger sequencing of affected individuals II-2, III-1, unaffected matrilineal relatives IV-2, III-4 and a married-in-control (II-1) of the WZ142 family. The arrow indicates the location of the nucleotide changes at position 572. (D) RFLP analysis for the c.572G>T mutation in some members of WZ142 and WZ562 families. Genotyping for the c.572G>T mutation in other subjects was PCR amplified for exon 1 of YARS2 and followed by digestion of the 623-bp segment with the restriction enzyme Tsp45I.

Figure 1.

(A) Six Chinese pedigrees with LHON. Vision-impaired individuals are indicated by blackened symbols. Individuals who harbored homozygous (−/−), heterozygous (+/−) or wild-type (+/+) YARS2 mutations are indicated. (B) Summary of exome sequencing of patient (WZ142 III-1). The identified SNV c.572G>T (p.191Gly>Val) is located in YARS2, a gene encoding a mitochondrial tyrosyl-tRNA synthetase. (C) Partial sequence chromatograms of YARS2 gene. Sanger sequencing of affected individuals II-2, III-1, unaffected matrilineal relatives IV-2, III-4 and a married-in-control (II-1) of the WZ142 family. The arrow indicates the location of the nucleotide changes at position 572. (D) RFLP analysis for the c.572G>T mutation in some members of WZ142 and WZ562 families. Genotyping for the c.572G>T mutation in other subjects was PCR amplified for exon 1 of YARS2 and followed by digestion of the 623-bp segment with the restriction enzyme Tsp45I.

We further analyzed the presence of the c.572G>T mutation in a cohort of 305 (212 male and 93 female) symptomatic and 270 asymptomatic subjects from 167 Chinese pedigrees carrying m.11778G>A mutation and 285 Chinese control subjects, by restriction fragment length polymorphism (RFLP) analysis, since the c.572G>T mutation disrupted a Tsp45I site, as shown in Figure 1D. Of 305 symptomatic subjects, 5 (3 male and 2 female) subjects who belonged to 4 pedigrees, as shown in Figure 1A, were homozygous for the c.572G>T mutation, 74 (48 male and 26 female) individuals were heterozygous for the c.572G>T mutation and the others lacked this mutation. In particular, six (2 male and 4 female) symptomatic individuals in the pedigree WZ1011 carried two subjects with homozygous mutation (two subjects), heterozygous mutation (three subjects) and lacked the mutation (one subject). However, homozygous c.572G>T mutation was not detected in all asymptomatic individuals and control individuals, while 49 asymptomatic individuals and 32 control subjects carried the heterozygous c.572G>T mutation. This translates to ∼28.7 and 18.5% frequency of this variant in the symptomatic subjects and asymptomatic subjects in this cohorts, respectively. Furthermore, 11.5% in the Chinese control subjects carrying this c.572G>T mutation was comparable with that of 12.66% in 1000 genome database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=11539445). The co-segregation of LHON phenotype with the presence of homozygous c.572G>T and m.11778G>A mutations and higher incidence of this c.572G>T variant in these symptomatic subjects indicates that this variant is a LHON susceptibility allele.

To further investigate the role of mitochondrial variants in the phenotypic manifestation of m.11778G>A mutations in five families carrying homozygous c.572G>T mutation, we examined sequence analysis of entire mtDNA. As shown in Supplementary Material, Table S3, there were no functional significant variants in these mtDNA genomes. These suggested that other mtDNA variants may not play an important role in the phenotypic manifestation of m.11778G>A mutation.

The p.191Gly>Val mutation causes the instability of YARS2

Human YARS2 comprises 477-amino acid protein containing a 16-amino acid mitochondrial targeting signal and catalytic site motifs characteristic of aminoacyl-tRNA synthetases (27). The mutated residue G191 locates within the catalytic domain and is highly conserved among vertebrates (Fig. 2A). The protein prediction program SIFT (http://sift.jcvi.org) predicts the change of Gly191 to Asp or Val to be deleterious effect. As shown in Figure 2B, the Gly191 together with Gly190 form a Gly-Gly motif, located at the c-terminal end of a short α-helix of this protein (28). The Gly190-Gly191 motif, same as other Gly-Gly motifs exhibiting a strong propensity to terminate an α-helical structure, may play an important role in maintaining the structural scaffold of YARS2. Thus, the p.191Gly>Val mutation could affect the structure and stability of the molecule. Furthermore, the Gly191 localizes at the interface between two YARS2 molecules in its dimeric form necessary for the function. The change of small Gly191 residue with the much bulkier Val could alter the association between two molecules of YARS2 in the dimer form.

Figure 2.

The p.191Gly>Val mutation affects the levels of YARS2. (A) Scheme for the structure of YARS2 and multiple sequence alignment of the YARS2 homologs. Position of p.191Gly>Val mutation is marked with an arrow. (B) Modeled structure of partial domain around G190-G191 residues of human YARS2 (28). (C) Western blot analysis of six mutant and two control cell lines. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with YARS2 and with VDAC as a loading control. Quantification of YARS2 levels were determined as described elsewhere (30). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. Cell lines harboring homozygous (−/−), heterozygous (+/−) or wild-type (+/+) YARS2 mutations are indicated. Cell lines carrying MT-ND4 Arg340His (+) or wild-type (−) are indicated.

Figure 2.

The p.191Gly>Val mutation affects the levels of YARS2. (A) Scheme for the structure of YARS2 and multiple sequence alignment of the YARS2 homologs. Position of p.191Gly>Val mutation is marked with an arrow. (B) Modeled structure of partial domain around G190-G191 residues of human YARS2 (28). (C) Western blot analysis of six mutant and two control cell lines. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with YARS2 and with VDAC as a loading control. Quantification of YARS2 levels were determined as described elsewhere (30). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. Cell lines harboring homozygous (−/−), heterozygous (+/−) or wild-type (+/+) YARS2 mutations are indicated. Cell lines carrying MT-ND4 Arg340His (+) or wild-type (−) are indicated.

To experimentally test the predicted effect of p.191Gly>Val mutation for YARS2, we analyzed the levels of YARS2 protein by western blotting in these mutant cell lines carrying only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous YARS2 p.191Gly>Val mutations and two control cell lines (Fig. 2C). In general, the levels of YARS2 in cell lines carrying only m.11778G>A, both m.11778G>A and heterozygous or homozygous YARS2 p.191Gly>Val mutations were 107.8, 76.4 and 50.8%, relative to the average control values. These results strongly support a deleterious effect of p.191Gly>Val mutation on YARS2 structure. However, this mutation did not affect the subcellular location of YARS2 (Supplementary Material, Fig. S1).

Altered aminoacylation capacity of mitochondrial tRNATyr

We investigated whether the YARS2 c.572G>T mutation altered aminoacylation of mitochondrial tRNA. The aminoacylation capacities of tRNATyr, tRNAThr, tRNALys, tRNALeu(CUN) and tRNASer(AGY) in control and mutant cell lines were examined by the use of electrophoresis in an acid polyacrylamide/urea gel system to separate uncharged tRNA species from the corresponding charged tRNA, electroblotting and hybridizing with above tRNA probes (29,30). As shown in Figure 3, the upper band represented the charged tRNA, and the lower band was uncharged tRNA. Electrophoretic patterns showed no obvious differences in electrophoretic mobility of these tRNAs between control and mutant cell lines. Notably, the efficiencies of aminoacylated tRNATyr in these mutant cell lines carrying only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous c.572G>T mutations were 87.12% (P = 0.07), 75.4% (P = 0.01) and 56.7% (P = 0.01), relative to the average control values. Furthermore, the levels of aminoacylated tRNASer(AGY) in these mutant cell lines carrying the m.11778G>A and heterozygous or homozygous c.572G>T mutations were 82%, and 74% of control values. However, the levels of aminoacylation in tRNAThr, tRNALys, tRNALeu(CUN) and tRNASer(AGY) in mutant cell lines were comparable with those in control cell lines (Supplementary Material, Fig. S2).

Figure 3.

In vivo aminoacylation assays. (A) Two micrograms of total mitochondrial RNA purified from six mutant and two control cell lines under acid conditions were electrophoresed at 4°C through an acid (pH 5.2) 10% polyacrylamide–7 M urea gel, electroblotted and hybridized with a DIG-labeled oligonucleotide probe specific for the tRNATyr. The blots were then stripped and rehybridized with tRNAThr, tRNALys, tRNALeu(CUN) and tRNASer(AGY), respectively. (B) In vivo aminoacylated proportions of tRNATyr in mutant and control cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the Student t-test, of the differences between the mean of mutant and mean of control cell lines.

Figure 3.

In vivo aminoacylation assays. (A) Two micrograms of total mitochondrial RNA purified from six mutant and two control cell lines under acid conditions were electrophoresed at 4°C through an acid (pH 5.2) 10% polyacrylamide–7 M urea gel, electroblotted and hybridized with a DIG-labeled oligonucleotide probe specific for the tRNATyr. The blots were then stripped and rehybridized with tRNAThr, tRNALys, tRNALeu(CUN) and tRNASer(AGY), respectively. (B) In vivo aminoacylated proportions of tRNATyr in mutant and control cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the Student t-test, of the differences between the mean of mutant and mean of control cell lines.

Decreased level of mitochondrial tRNATyr

To examine whether the deficient aminoacylation of tRNATyr affects the steady-state level of tRNAs, we subjected total mitochondrial RNA from cell lines to Northern blots and hybridized them with the probes specific for tRNATyr, tRNAThr, tRNALys, tRNALeu(CUN), tRNASer(AGY) and tRNAGly. As shown in Figure 4A, the levels of tRNATyr in mutant cells carrying both m.11778G>A and heterozygous or homozygous YARS2 c.572G>T mutations were markedly decreased, as compared with those in control cells. For comparison, the average level of each tRNA in control or mutant cell lines was normalized to the average levels in the same cell line for reference 5S RNA (31). As shown in Figure 4B, the average steady-state levels of tRNATyr in mutant cell lines carrying only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous c.572G>T mutations were 92.5% (P = 0.14), 68.5% (P =0.004) and 44.3% (P = 0.01) of control cell lines after normalization to 5S RNA, respectively. However, the average steady-state levels of tRNAThr, tRNALys, tRNALeu(CUN), tRNASer(AGY) and tRNAGly in mutant cell lines were comparable with those of two control cell lines lacking the mutations (Supplementary Material, Fig. S3).

Figure 4.

Northern-blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mtRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with the DIG-labeled oligonucleotide probes specific for tRNATyr, tRNAThr, tRNALys, tRNALeu(CUN), tRNASer(AGY) and 5S RNA, respectively. (B) Quantification of the tRNATyr level. Average relative tRNATyr content per cell was normalized to the average content per cell of 5S RNA in two control cell lines and six mutant cell lines, respectively. The values for the latter are expressed as percentages of the average values for the control cell line. The calculations were based on three independent determinations in each cell line. Graph details and symbols are explained in the legend to Figure 3.

Figure 4.

Northern-blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mtRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with the DIG-labeled oligonucleotide probes specific for tRNATyr, tRNAThr, tRNALys, tRNALeu(CUN), tRNASer(AGY) and 5S RNA, respectively. (B) Quantification of the tRNATyr level. Average relative tRNATyr content per cell was normalized to the average content per cell of 5S RNA in two control cell lines and six mutant cell lines, respectively. The values for the latter are expressed as percentages of the average values for the control cell line. The calculations were based on three independent determinations in each cell line. Graph details and symbols are explained in the legend to Figure 3.

Reduction in the level of mitochondrial proteins

To determine whether a failure in tRNA metabolism impaired mitochondrial translation, a western blot analysis was carried out to examine the steady-state levels of seven respiratory complex subunits in mutant and control cells with Tom20 (a nuclear gene encoding mitochondrial protein) as a loading control. As shown in Figure 5, the levels of MT-CO2, subunit 2 of cytochrome c oxidase; MT-ND1, MT-ND4, MT-ND5 and MT-ND6, subunits 1, 4, 5 and 6 of NADH dehydrogenase; MT-CYTB, apocytochrome b; MT-ATP6 and subunit 6 of the H+-ATPase were decreased in mutant cell lines, as compared with those of control cell lines. As shown in Figure 5B, the overall levels of seven mitochondrial translation products in the mutant cell carrying only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous c.572G>T mutations were 115.8% (P = 0.14), 56.4% (P = 0.004) and 35.6% (P = 0.01) of two control cell lines, respectively. Notably, the average levels of MT-ND1, MT-ND4, MT-ND5, MT-ND6, MT-ATP6, MT-CO2 and MT-CYTB in the mutant cells carrying both m.11778G>A and homozygous c.572G>T mutations were 16.9, 27, 40.4, 28.6, 105, 39.6 and 76.4% of the average values of control cells, respectively, while the average levels of MT-ND1, MT-ND4, MT-ND5, MT-ND6, MT-ATP6, MT-CO2 and MT-CYTB in the mutant cells carrying both m.11778G>A and heterozygous c.572G>T mutations were 39.6, 65.5, 36.4, 28.3, 123, 52.9, and 76% of the average values of control cells, respectively. However, the average levels of these polypeptides in the mutant cells carrying only m.11778G>A mutations were comparable to those of control cells.

Figure 5.

Western blot analysis of mitochondrial proteins. (A) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with seven respiratory complex subunits in mutant and control cells with Tom20 as a loading control. MT-CO2 indicates subunit II of cytochrome c oxidase; MT-ND1, MT-ND4, MT-ND5 and MT-ND6, subunits 1, 4, 5 and 6 of the reduced nicotinamide–adenine dinucleotide dehydrogenase; MT-ATP6, subunit 6 of the H+-ATPase and MT-CYTB, apocytochrome b. (B) Quantification of mitochondrial protein levels. The levels of mitochondrial proteins in six mutant cell lines and two control cell lines were determined as described elsewhere (30). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 3. (C) Quantification of seven respiratory complex subunits. The levels of MT-ND1, MT-ND4, MT-ND5, MT-ND6, MT-CO2 and MT-CYTB in six mutant cell lines and two control cell lines were determined as described elsewhere (30).

Figure 5.

Western blot analysis of mitochondrial proteins. (A) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with seven respiratory complex subunits in mutant and control cells with Tom20 as a loading control. MT-CO2 indicates subunit II of cytochrome c oxidase; MT-ND1, MT-ND4, MT-ND5 and MT-ND6, subunits 1, 4, 5 and 6 of the reduced nicotinamide–adenine dinucleotide dehydrogenase; MT-ATP6, subunit 6 of the H+-ATPase and MT-CYTB, apocytochrome b. (B) Quantification of mitochondrial protein levels. The levels of mitochondrial proteins in six mutant cell lines and two control cell lines were determined as described elsewhere (30). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 3. (C) Quantification of seven respiratory complex subunits. The levels of MT-ND1, MT-ND4, MT-ND5, MT-ND6, MT-CO2 and MT-CYTB in six mutant cell lines and two control cell lines were determined as described elsewhere (30).

Reduced activity of complexes I and IV

To investigate the effect of the YARS2 c.572G>T mutation on the oxidative phosphorylation, we measured the activities of respiratory complexes by isolating mitochondria from mutant and control cell lines. Complex I (NADH ubiquinone oxidoreductase) activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor (32,33). Complex III (ubiquinone cytochrome c oxidoreductase) activity was measured as the reduction of cytochrome c (III) using d-ubiquinol-2 as the electron donor. The activity of complex IV (cytochrome c oxidase) was monitored by following the oxidation of cytochrome c (II). As shown in Figure 6, the activity of complex I in the mutant cell carrying only 11778G>A mutation, both m.11778G>A and heterozygous or homozygous c.572G>T mutations were 92.4, 75 and 51.4% of two control cell lines, respectively. Notably, the activity of complex IV in the mutant cell carrying only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous c.572G>T mutations were 108.8, 75 and 38.2% of two control cell lines, respectively. However, the activities of complexes II and III in all mutant cell lines were comparable with those of control cell lines.

Figure 6.

Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were investigated by enzymatic assay on complexes I, II, III and IV in mitochondria isolated from various cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 3.

Figure 6.

Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were investigated by enzymatic assay on complexes I, II, III and IV in mitochondria isolated from various cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 3.

Respiration defects

To evaluate if the YARS2 c.572G>T mutation alters cellular bioenergetics, we examined the oxygen consumption rates (OCR) of mutant and control cells lines (34). As shown in Figure 7, the basal OCR in the mutant cell lines carrying only m.11778G>A or both m.11778G>A with homozygous or heterozygous c.572G>T mutations was 106.6, 95.6 and 63.1% relative to the mean value measured in the control cell lines, respectively. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cell lines, OCR were measured after the sequential addition of oligomycin (inhibit the ATP synthase), FCCP (to uncouple the mitochondrial inner membrane and allow for maximum electron flux through the ETC), rotenone (to inhibit complex I) and antimycin (to inhibit complex III) (complex V). The difference between the basal OCR and the drug-insensitive OCR yields the amount of ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR. As shown in Figure 7, the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR in mutant cell lines carrying the m.11778G>A with homozygous c.572G>T mutations were 51.9, 118.1, 33.7, 25.3 and 95.1%, those cell lines carrying both m.11778G>A with heterozygous c.572G>T mutations were 93, 117.8, 56.6, 41.4 and 119%, cell lines carrying only m.11778G>A mutations were 98, 143.7, 63.1, 48.3 and 160%, relative to the mean value measured in the control cell lines, respectively.

Figure 7.

Respiration assays. (A) An analysis of O2 consumption in the various cell lines using different inhibitors. The rates of O2 (OCR) were first measured on 2 × 104 cells of each cell line under basal condition and then sequentially added to oligomycin (1.5 μM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (0.5 μM), rotenone (1 μM) and antimycin A (1 μM) at indicated times to determine different parameters of mitochondrial functions. (B) Graphs presented the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR in mutant and control cell lines. Non-mitochondrial OCR was determined as the OCR after rotenone/antimycinA treatment. Basal OCR was determined as OCR before oligomycin minus OCR after rotenone/antimycin A. ATP-lined OCR was determined as OCR before oligomycin minus OCR after oligomycin. Proton leak was determined as Basal OCR minus ATP-linked OCR. Maximal was determined as the OCR after FCCP minus non-mitochondrial OCR. Reserve capacity was defined as the difference between Maximal OCR after FCCP minus Basal OCR. The average values of four determinations for each cell line were shown, and the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to Figure 3.

Figure 7.

Respiration assays. (A) An analysis of O2 consumption in the various cell lines using different inhibitors. The rates of O2 (OCR) were first measured on 2 × 104 cells of each cell line under basal condition and then sequentially added to oligomycin (1.5 μM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (0.5 μM), rotenone (1 μM) and antimycin A (1 μM) at indicated times to determine different parameters of mitochondrial functions. (B) Graphs presented the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR in mutant and control cell lines. Non-mitochondrial OCR was determined as the OCR after rotenone/antimycinA treatment. Basal OCR was determined as OCR before oligomycin minus OCR after rotenone/antimycin A. ATP-lined OCR was determined as OCR before oligomycin minus OCR after oligomycin. Proton leak was determined as Basal OCR minus ATP-linked OCR. Maximal was determined as the OCR after FCCP minus non-mitochondrial OCR. Reserve capacity was defined as the difference between Maximal OCR after FCCP minus Basal OCR. The average values of four determinations for each cell line were shown, and the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to Figure 3.

Reduced level in mitochondrial ATP production

The capacity of oxidative phosphorylation in mutant and wild-type cells was examined by measuring the levels of cellular and mitochondrial ATP production using a luciferin/luciferase assay. Populations of cells were incubated in the media in the presence of glucose (reflects total cellular ATP production), or 2-deoxy-d-glucose with pyruvate (reflects mitochondrial ATP production) (35–36). In the presence of glucose, the levels of total cellular ATP production in mutant cells were comparable with those measured in the control cell lines (data now shown). In the presence of pyruvate and 2-deoxy-d-glucose to inhibit the glycolysis, the levels of mitochondrial ATP production in the mutant cell lines harboring only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous YARS2 c.572G>T mutations were 80.2% (P = 0.10), 71.6% (P = 0.04) and 49% (P = 0.018) of average value of two control cell lines (Fig. 8A).

Figure 8.

Measurement of ATP and ROS levels. (A) ATP level in mitochondria. Cells were incubated with 5 mM 2-deoxy-d-glucose plus 5 mM pyruvate to determine ATP generation under mitochondrial ATP synthesis. Average rates of ATP level per cell line were determined as elsewhere (34). Three determinations were made for each cell line. (B) Relative levels of ROS production. The rates of production in ROS from six mutant cell lines and two control cell lines were analyzed by BD-LSR II flow cytometer system with or without H2O2 stimulation. The relative ratio of intensity (stimulated versus unstimulated with H2O2) was calculated. The average of three determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 3.

Figure 8.

Measurement of ATP and ROS levels. (A) ATP level in mitochondria. Cells were incubated with 5 mM 2-deoxy-d-glucose plus 5 mM pyruvate to determine ATP generation under mitochondrial ATP synthesis. Average rates of ATP level per cell line were determined as elsewhere (34). Three determinations were made for each cell line. (B) Relative levels of ROS production. The rates of production in ROS from six mutant cell lines and two control cell lines were analyzed by BD-LSR II flow cytometer system with or without H2O2 stimulation. The relative ratio of intensity (stimulated versus unstimulated with H2O2) was calculated. The average of three determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 3.

The increase of ROS production

The levels of the ROS generation in the vital cells derived from control and mutant cell lines were measured with flow cytometry under normal and H2O2 stimulation (36,37). Geometric mean intensity was recorded to measure the rate of ROS of each sample. The ratio of geometric mean intensity between unstimulated and stimulated with H2O2 in each cell line was calculated to delineate the reaction upon increasing level of ROS under oxidative stress. As shown in Figure 8B, the levels of ROS generation in the mutant cell lines carrying only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous YARS2 c.572G>T mutations were 111.5% (P = 0.30), 114.8% (P = 0.07) and 147.2% (P = 0.015) of two control cell lines.

Discussion

Nuclear modifier genes were proposed to increase the susceptibility to LHON-associated mtDNA mutations (38,39). Using an exome sequencing approach, in combination with functional assays, we identified the first LHON susceptibility allele (c.572G>T, p.191Gly>Val) in the YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase for the phenotypic expression of the m.11778G>A mutation. Strikingly, all matrilineal relatives carrying both homozygous YARS2 p.191Gly>Val and m.11778G>A mutations exhibited visual failure, while 56 matrilineal relatives harboring both heterozygous YARS2 p.191Gly>Val and m.11778G>A mutations and 32 genetically unrelated subjects carrying only heterozygous YARS2 p.191Gly>Val mutation have normal vision. These suggested that the coexistence of homozygous YARS2 p.191Gly>Val mutation with the m.11778G>A mutation led to the expression of optic neuropathy in these subjects. Notably, the observations that an equal numbers of symptomatic male and female subjects carrying the YARS2 p.191Gly>Val mutation strongly support the hypothesis that an autosomal recessive modifier gene such as YARS2 is involved in the phenotypic expression the m.11778G>A or m.14484T>C mutation (5,21,22). However, the majority of other symptomatic individuals, who harbored the heterozygous YARS2 p.191Gly>Val or lacked this mutation, may result from the interaction between m.11778G> mutation and other putative modifier genes, particularly X-linked modifiers for a predominance of male patients (20,23).

The highly conserved Gly191 residue locates within the catalytic domain of YARS2, which interacts with the base-pairing (1G-72C) of tRNATyr acceptor stem (28,40,41). It was anticipated that the p.191Gly>Val mutation affected structure and function of this enzyme, as in the case of p.191Gly>Asp mutation (42). The instability of mutated YARS2 was supported by the reduced levels of YARS2 observed in cell lines carrying both m.11778G>A and heterozygous or homozygous p.191Gly>Val mutations. The primary defect in the p.191Gal>Val mutation was the reduced synthetic rate of aminoacylated tRNATyr. In this study, 46 and 25% decreases in aminoacylated tRNATyr were observed in mutant cell lines carrying both m.11778G>A and homozygous or heterozygous p.191Gly>Val mutations, independent of mitochondrial genotype. This result was consistent with a marked loss in catalytic efficiency of YARS2 in a recombinant YARS2 carrying the p.191Gly>Asp mutation by the in vitro aminacylation assay (42). In this investigation, it is interesting that the lowered efficiency of aminoacylated tRNASer(AGY) was also observed in the cell lines carrying the YARS2 p.191Gly>Val mutation. It is likely that the mutated YARS2 may mediate mitochondrial tRNA metabolism, thereby reducing the aminoacylated efficiencies of other mitochondrial tRNAs (31). An inefficiently aminoacylated tRNA caused by the YARS2 mutations may then make the tRNATyr to be metabolically less stable and more subject to degradation, thereby lowering the level of the tRNA. Indeed, the decreases of 56% and ∼31% in the steady-state levels of tRNATyr in those mutant cells carrying both m.11778G>A and homozygous or heterozygous p.191Gly>Val mutations were significantly correlated with the degree of decreased aminoacylation of tRNATyr. However, these reduced levels of tRNATyr in mutant cells were indeed above the proposed threshold level (>70% reduction) to produce a clinical phenotype associated with tRNA mutations (43,44). This suggested that the YARS2 p.191Gly>Val mutation is by itself insufficient to produce a clinical phenotype, as in the case of the nuclear modifier TRMU p.10Ala>Ser mutation for deafness-associated 12S rRNA 1555A>G mutation (31).

A failure in the tRNATyr metabolism caused by the YARS2 p.191Gly>Val mutation then affects mitochondrial protein synthesis. In fact, the mtDNA encoded 13 polypeptides in the complexes of the oxidative phosphorylation system (MT-ND1-6, ND4L of complex I, MT-CYTB of complex III, MT-CO1, CO2, CO3 of complex IV, and MT-ATP6 and MT-ATP8 of complex V) (45,46). In this study, 39 and 61% reductions in the levels of mitochondrial proteins were observed in mutant cell lines carrying both m.11778G>A and heterozygous or homozygous YARS2 p.191Gly>Val mutations, respectively. These results were comparable with those in cell lines carrying YARS2 p.52Phe>Leu or p.49Gly>Asp mutation (45,46). Notably, variable decreases in the levels of seven mtDNA-encoded polypeptides were observed in mutant cell lines. As shown in Supplementary Material, Table S4, cell lines carrying both m.11778G>A and homozygous YARS2 p.191Gly>Val mutations exhibited marked reductions (59 to 83%) in the levels of five polypeptides (MT-ND1, MT-ND4, MT-ND5, MT-ND6 and MT-CO2) harboring 9–16 tyrosine codons, a relative mild reduction (23%) in the level of MT-CYTB carrying 17 tyrosine codons, but a mild increased level (5%) in the level of MT-ATP6 with only three tyrosine codons. In the other studies, the in vivo Pulse-labeling mitochondrial protein synthesis assay also showed much less reductions in the levels of MT-ND3, MT-ND4L, MT-ATP6 and MT-ATP8 than those of other polypeptides with high tyrosine codons in mutant cell lines carrying the YARS2 p.52Phe>Leu and p.49Gly>Asp mutations (47,48). However, these data were not fully comparable with the case of MERRF-associated m.8344A>G mutation in tRNALys gene (49). Thus, the impaired synthesis of MT-ND1, MT-ND4, MT-ND5 and MT-ND6, subunits of complex I and MT-CO1, MT-CO2 and MT-CO3, subunits of complex IV may alter the activities of complexes I and IV and then worsen the respiratory phenotypes associated with m.11778G>A mutation. In this study, 25 and 49.4% decreases of complex I activity, 25 and 62% reductions in complex IV activities were observed in cell lines harboring both m.11778G>A and heterozygous or homozygous YARS2 p.191Gly>Val mutations, while ∼10% decrease in the activity of complex I but not those of complexes III and IV in these mutant cell lines carrying only m.11778G>A mutation (14,15). Furthermore, alteration in mitochondrial protein synthesis was apparently responsible for the reduced rates in the basal OCR, or ATP-linked OCR, reserve capacity and maximal OCR among the control and mutant cell lines. In particular, the 37, 43 and 66% decreases in maximal OCR reduction were observed in cell lines harboring only m.11778G>A mutation, both m.11778G>A and heterozygous or homozygous YARS2 p.191Gly>Val mutations, respectively. This correlation is clearly consistent with the importance that a failure in tRNA metabolism plays a critical role in producing their respiration defects, as in the cases of cells carrying both TRMU 10Ala>Ser and deafness-associated 12S rRNA 1555A>G mutations (31).

The respiratory deficiency then affects the efficiency of mitochondrial ATP synthesis. In this investigation, 20% reduction of mitochondrial ATP production in lymphoblastoid cell lines carrying only m.111778G>A mutation may be caused by the defective activity of complex I, as in the cases of cybrid cell lines bearing the m.3460G>A, m.14484T>C or m.11778G>A mutations (50,51). In contrast, ∼51% drop in mitochondrial ATP production in lymphoblastoid cell lines bearing both m.11778G>A and homozygous YARS2 p.191Gly>Val mutations may result from the defective activities of Complexes I and IV. Indeed, our previous investigation implicated that marked decreases in mitochondrial ATP production in lymphoblastoid cell lines derived from a large Chinese pedigree with high penetrance of LHON resulted from altered activity of Complexes I and III (35). The impairment of oxidative phosphorylation can lead to more electron leakage from electron transport chain, and in turn, elevate the production of ROS in mutant cells (52). Here, the mildly reduced production of mitochondrial ATP in the lymphoblastoid cell lines carrying only m.11778G>A mutation was in a good agreement with the previous observation in cybrid cell lines harboring the same mutation (51,53). However, 47% increase of ROS production in cells carrying both m.11778G>A and homozygous YARS2 p.191Gly>Val mutations was the consequence of the altered activities of Complexes I and IV. The overproduction of ROS can establish a vicious cycle of oxidative stress in the mitochondria, thereby damaging mitochondrial and cellular proteins, lipids and nuclear acids (54). The retinal ganglion cells may be preferentially involved because they are somehow exquisitely sensitive to subtle imbalance in cellular redox state or increased level of free radicals (54,55). This would lead to dysfunction or apoptosis of retinal ganglion cells carrying both m.11778G>A and p.191Gly>Val mutations, thereby producing a phenotype of visual loss (5,56).

In summary, our study has identified the first nuclear modifier gene YARS2 for the phenotypic expression of LHON-associated mtDNA mutations. The mutated YARS2 causes the inefficient aminoacylation and lower level of tRNATyr, subsequently impairing mitochondrial translation, specially for polypeptides with high content of tyrosine codon. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with m.11778G>A mutation, below the threshold for normal cell function, thereby expressing the LHON phenotype. Therefore, the mutated YARS2, acting as a nuclear modifier, triggers the optic neuropathy in individuals harboring the m.11778G>A mutation. Thus, our findings provide new insights into the understanding of pathophysiology of not only mitochondrial disease such as LHON but also neurodegenerative diseases such as Parkinson's.

Materials and Methods

Families and subjects

DNA samples used for this investigation were from members of families who carried the m.11778G>A mutation: 307 symptomatic and 271 asymptomatic members from 167 Han Chinese pedigrees with LHON (19,22,25,26). A total of 285 control DNA samples lacking the m.11778G>A mutation were obtained from adult Han Chinese from same area. The ophthalmic examinations and other clinical evaluations of probands, other members of these families and 285 control subjects were conducted as detailed elsewhere (22,25,26). Informed consent, blood samples and clinical evaluations were obtained from all participants and families, under protocols approved by the Institutional Review Board of Cincinnati Children's Hospital, Ethic Committees of Zhejiang University and the Wenzhou Medical University.

Whole exome sequencing

Whole exome sequencings of four subjects (III-1, III-4, II-2 and II-1) of WZ142 pedigree carrying the m.11778G>A mutation were performed by BGI (Shenzhen, China). High-quality genomic DNA (3 μg) was captured by hybridization using the SureSelect XT Human All Exon 50 Mb kit (Agilent Technologies). Samples were prepared according to the manufacturer's instructions. Each captured library was run on a HiSeq 2000 instrument and sequences generated as 90 bp pair-end reads. An average of 82 million paired reads was generated per sample, the mean duplication rate was 6.37, and 98% of the targeted region was covered by at least 50 × mean depth. All sequencing reads were mapped to the human reference genome (GRCh37) at UCSC. Software SOAPsnp was used to assemble the consensus sequence and call genotypes in target regions. GATK (Indel Genotyper V1.0) was used to indel detection. The threshold for filtering single-nucleotide polymorphisms (SNPs) included the following criterion: SNP quality score should be ≥20; sequencing depth should be between 4 and 200; estimated copy number should be no more than 2 and the distance between two SNPs should be larger than 5.

Mutation analysis of YARS2 gene

Five pairs of primers for PCR-amplifying exons and their flanking sequences, including splicing-donor and acceptor-consensus sequences of YARS2, were used for this analysis. The forward and reverse primers for PCR amplification and sequence analysis are shown in Supplementary Material, Table S5. Fragments spanning five exons and flanking sequences from three matrilineal relatives (II-2, III-1 and III-4) and one spouse (II-1) of WZ142 pedigree, and two genetically unrelated Chinese controls were PCR amplified, purified and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer with use of the Big Dye Terminator Cycle sequencing reaction kit (Applied Biosystems). These sequence results were compared with the YARS genomic sequence (RefSeq NC_000012.12). Genotyping for the c.572G>T mutation in other subjects was PCR amplified for exon 1 and followed by digestion of the 623-bp segment with the restriction enzyme Tsp45I. The forward and reverse primers for exon 1 are 5′- GACTCGCTTCATGTGGGTCAT-3′ and 5′- CGAAGGGCAGCAACTACAATC-3′, respectively. The Tsp45I-digested products were analyzed on 1.5% polyacrymide gel.

Sequence analysis of mitochondrial genomes

The entire mitochondrial genomes of five Chinese families carrying homozygous YARS2 c.572G>T and m.11778G>A mutations were analyzed as described elsewhere (57). The resulting sequence data were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920) (45).

Cell lines and culture conditions

Lymphoblastoid cell lines were immortalized by transformation with the Epstein–Barr virus, as described previously (58). Cell lines derived from two matrilineal members of WZ142 pedigree (III-1, III-4), four matrilineal members of WZ1011 and two genetically unrelated control individuals C1 and C2 were grown in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum.

Mitochondrial tRNA analysis

Mitochondria were isolated from various cell lines (∼2.0 × 108 cells), as described previously (59). For the aminoacylation assays, total mitochondrial RNAs were isolated under acid conditions, and 2 μg of total mitochondrial RNAs was electrophoresed at 4°C through an acid (pH 5.2) 10% polyacrylamide–7 M urea gel to separate the charged and uncharged tRNA as detailed elsewhere (29,60). For the tRNA Northern analysis, total mitochondrial RNAs were isolated using TOTALLY RNA™ kit (Ambion), as described previously, 2 μg of total mitochondrial RNA were electrophoresed through a 10% polyacrylamide/7 M urea gel in Tris–borate–EDTA buffer (TBE) (after heating the sample at 65°C for 10 min). The gels were then electroblotted onto a positively charged nylon membrane (Roche) for the hybridization analysis with non-radioactive digoxin (DIG)-labeled oligodeoxynucleotide probes for tRNATyr, tRNAThr, tRNALys, tRNALeu(CUN), tRNASer(AGY) or 5S RNA, as detailed previously (30,60,61,62). DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide Tailing kit (Roche). The hybridization and quantification of density in each band were performed as detailed previously (30).

Western blot analysis

Western blotting analysis was performed as detailed previously (30). The antibodies used for this investigation were from Abcam [anti YARS2 (ab68725), TOM20 (ab56783), VDAC (ab14734), MT-ND1 (ab74257), MT-ND5 (ab92624) and MT-ATP6 (ab101908), MT-CO2 (ab110258)], Santa Cruz Biotechnology [MT-ND4 (sc-20499-R) and MT-ND6 (sc-20667)] and Proteintech [MT-CYTB (55090-1-AP)]. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously (30).

Measurements of oxygen consumption

The rates of oxygen consumption in cybrid cell lines were measured with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience), as detailed previously (30,34).

Enzymatic assays

The enzymatic activities of complexes I, II, III and IV were assayed as detailed elsewhere (32,33,63).

ATP measurements

The Cell Titer-Glo® Luminescent Cell Viability Assay kit (Promega) was used for the measurement of cellular and mitochondrial ATP levels, according to the modified manufacturer's instructions (30,35,36).

ROS measurements

ROS measurements were performed following the procedures detailed previously (35–37).

Computer analysis

Statistical analysis was carried out using the unpaired, two-tailed Student's t-test contained in the Microsoft-Excel program or Macintosh (version 2007). Differences were considered significant at a P < 0.05.

Supplementary Material

Supplementary Material is available at HMG online.

Funding

This work was supported by the National Key Technologies R&D Program grant 2012BAI09B03 from the Ministry of Science and Technology of China to M.-X.G. and P.J., and grants 31471191, 81200724 and 81400434 from the Natural Science Foundation of China to M.-X.G., J.Z. and Y.J., respectively.

Acknowledgements

We are grateful to patients and their family members for their participation.

Conflict of Interest statement. All authors have no proprietary or commercial interest in any of materials discussed in this article.

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