Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation

The 1555A→G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A→G mutation. Here, we report that a deafness-susceptibility allele (m.4317A→G) in the tRNAIle gene modulates the phenotype expression of m.1555A→G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A→G and m.1555A→G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A→G mutation. The m.4317A→G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNAIle. We therefore hypothesized that the m.4317A→G mutation alters both structure and function of tRNAIle. Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A→G and m.4317A→G mutations, three harboring only m.1555A→G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A→G mutation. We also found that the m.4317A→G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNAIle. These m.4317A→G mutation-induced alterations in tRNAIle structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A→G mutation. Furthermore, mutant cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A→G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations.

The 1555A3 G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A3 G mutation. Here, we report that a deafness-susceptibility allele (m.4317A3 G) in the tRNA Ile gene modulates the phenotype expression of m.1555A3 G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A3 G and m.1555A3 G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A3 G mutation. The m.4317A3 G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNA Ile . We therefore hypothesized that the m.4317A3 G mutation alters both structure and function of tRNA Ile . Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A3 G and m.4317A3 G mutations, three harboring only m.1555A3 G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A3 G and m.1555A3 G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A3 G mutation. We also found that the m.4317A3 G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNA Ile . These m.4317A3 G mutation-induced alterations in tRNA Ile structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A3 G mutation. Furthermore, mutant cell lines bearing both m.4317A3 G and m.1555A3 G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A3 G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations.
Mutations in mitochondrial DNA (mtDNA) have been associated with both syndromic deafness (hearing loss with other medical problems such as diabetes) and non-syndromic deafness (hearing loss is the only obvious medical problem) (1)(2)(3). Human mtDNA encodes 13 polypeptides (essential components of oxidative phosphorylation complexes), 2 rRNAs, and 22 tRNAs required for mitochondrial translation (4). The mitochondrial tRNA genes are the hot spots for deafness-associated mutations, including the tRNA Leu(UUR) 3243A3G, tRNA Ser(UCN) 7445A3 G, 7511T3 C, tRNA His 12201T3 C, tRNA Asp 7551A3 G, and tRNA Glu 14692A3 G mutations (5)(6)(7)(8)(9)(10). The m.1555A3 G and m.1494C3 T mutations in the 12S rRNA gene have been associated with both aminoglycoside-induced and non-syndromic deafness in many families worldwide (11)(12)(13)(14)(15)(16)(17)(18)(19). The administration of aminoglycosides induced or worsened hearing loss in these subjects carrying the m.1555A3 G or m.1494C3 T mutation. In the absence of aminoglycosides, matrilineal relatives within and among families carrying the m.1555A3 G or m.1494C3 T mutation exhibited a wide range of penetrance, severity, and age-of-onset in hearing impairment (11, 14, 17, 19 -22). Functional characterization of cell lines derived from matrilineal relatives of Arab-Israeli and Chinese families demonstrated that the m.1555A3 G or m.1494C3 T mutation conferred mild mitochondrial dysfunctions and sensitivity to aminoglycosides (23)(24)(25)(26). These find-ings strongly suggest that the m.1555A3 G or m.1494C3 T mutation is the primary causative event, but by itself it is insufficient to produce the deafness phenotype. Modifier factors, including aminoglycosides, nuclear, and mitochondrial genetic modifiers, are required for the phenotype expression of the m.1555A3 G or m.1494C3 T mutation (2,20,25,27). In previous investigations, we showed that MTO1, MSS1/GTPBP3, or MTO2/TRMU genes involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine in the wobble position of several mitochondrial tRNAs were the potential nuclear modifier genes for the phenotypic expression of m.1555A3 G and m.1494C3 T mutations (28 -33). Genetic evidence suggests that several mtDNA variants, including tRNA Glu 14693A3 G, tRNA Thr 15927G3 A, 15908T3 C, tRNA Arg 10454T3 C, tRNA Ser(UCN) 7444G3 A, and tRNA Cys 5821G3 A may act as mitochondrial modifiers to modulate the phenotypic manifestation of the m.1555A3 G or m.1494C3 T mutation (20, 34 -36). However, the role of these mitochondrial modifiers in the deafness expression remains poorly understood.
In this study, we investigated the pathophysiology of a deafness susceptibility allele (m.4317A3 G) in the tRNA Ile gene in the phenotypic expression by taking advantage of a large cohort of 2651 hearing-impaired Han Chinese probands (15,37,38). Among these, 105 pedigrees with non-syndromic and aminoglycoside-induced deafness harbored the homoplasmic m.1555A3 G mutation (20). These families exhibited a wide range of penetrance and expressivity of hearing impairment. The average penetrance of deafness among 105 pedigrees carrying the m.1555A3 G mutation were 29.5 and 17.6%, respectively, when aminoglycoside-induced hearing loss was included or excluded (20). Strikingly, the penetrance of deafness in the pedigree (WZD91) harboring both m.1555A3 G and m.4317A3 G mutations were 73.9 and 60.9%, respectively, when aminoglycoside-induced hearing loss was included or excluded (20,38). It was anticipated that the m.4317A3 G mutation may further deteriorate the mitochondrial dysfunction associated with the m.1555A3 G mutation, thereby increasing the penetrance and risk of deafness expression. As shown in Fig. 1A, the m.4317A3 G mutation was localized at the highly conserved adenine (A59) of the T-loop in tRNA Ile (39,40). The m.4317A3 G mutation may introduce a new G-C (G59 -C54) base pair to the stem of the T-loop and lead to the rearrangement of the T-arm region (41)(42)(43). This T-arm region is important for the 3Ј-end-processing of the tRNA Ile precursor and subsequently the CCA-addition process (42)(43)(44). Therefore, it was hypothesized that the m.4317A3 G mutation altered both the structure and function of tRNA Ile . In particular, the mutation may affect the aminoacylation capacity and stability of this tRNA. A failure in tRNA metabolism may lead to the impairment of mitochondrial translation and respiration (2,3). It was also proposed that mitochondrial dysfunctions caused by the tRNA mutation affect the production of ATP and reactive oxygen species (ROS). 4 To further investigate the effect of the m.4317A3 G mutation on mitochondrial function, lym-phoblastoid cell lines were generated from three members of pedigree WZD91 carrying both m.1555A3G and m.4317A3 G mutations, three individuals of pedigree WZD92 harboring only m.1555A3 G mutation, and three control subjects lacking two mutations belonging to the same mtDNA haplogroup B4. These cell lines were first assessed for the effect of the m.4317A3 G mutation on the conformation, stability, and aminoacylation capacity of tRNA Ile . We then examined whether the m.4317A3 G altered mitochondrial translation, enzymatic activities of electron transport chain complexes, the rate of oxygen consumption, ATP production, mitochondrial membrane potential, and the generation of ROS.

Identification of the tRNA Ile 4317A3 G mutation in a large cohort of hearing-impaired subjects
The m.4317A3 G mutation in tRNA Ile gene was identified in only one proband (WZD91-III-3) carrying the m.1555A3 G mutation among 2651 Chinese hearing-impaired probands (37,38) but was absent in 574 Chinese control subjects. As shown in Fig. 1, the m.4317A3 G mutation affected the highly conserved adenine (A59) of the T-loop in tRNA Ile (39 -40). The m.4317A3 G mutation may introduce a new G-C (G59 -C54) base-pairing to the stem of the T-loop and lead to the rearrangement of the T-arm region (41)(42)(43). Therefore, it was anticipated that the m.4317A3 G mutation altered both structure and function of tRNA Ile . In the sequence analysis of the entire mtDNA in the proband (WZD91-III-3), three symptomatic affected matrilineal relatives (III-1, III-9, and III-17) and two asymptomatic matrilineal relatives (IV-5 and IV-10) (Fig. 1B) exhibited the presence of both m.1555A3 G and m.4317A3 G mutations and a set of polymorphisms belonging to the Eastern Asian haplogroups B4 (Table S1) (45). These variants included 16 variants in the D-loop region, four known variants in the 12S rRNA gene, three variants in the 16S rRNA gene, the previously identified COII/tRNA Lys intergenic 9-bp deletion corresponding to mtDNA at positions 8281-8289, 10 known and one novel silent variant in the protein encoding genes, as well as a missense variant m.8573G3 A (p.16G3 D) in the ATP6 gene (46). The phylogenetic analyses showed that there were no other functionally significant variants in their mtDNAs. Further analysis showed that both m.4317A3 G and m.1555A3 G mutations were present in homoplasmy in all matrilineal relatives of pedigree WZD91 but was absent in other members of these families (Fig. 1C).

Clinical presentation of two Chinese families and derived cell lines
All available members of two hearing-impaired Han Chinese families (pedigree WZD91 carrying both m.1555A3 G and m.4317A3 G mutations and pedigree WZD92 harboring only m.1555A3 G mutation), as shown in Fig. 1D, underwent comprehensive evaluations of their medical histories and physical examination with the aim to identify any clinical abnormalities and genetic factors related to the deafness. The audiological tRNA modifier for deafness expression of 12S rRNA mutation examination was performed as detailed elsewhere (14). As shown in Fig. 1D, 17 of 23 matrilineal relatives of pedigree WZD91 suffered from hearing impairment as the sole clinical symptom. Three matrilineal relatives with profound hearing loss had a history of exposure to aminoglycosides, and the other 14 affected matrilineal relatives exhibited the variable degrees of hearing impairment (two members with severe hearing loss, eight subjects with moderate hearing loss, and four individuals with mild hearing loss). In pedigree WZD92, four matrilineal relatives exhibited severe hearing loss due to administration with aminoglycosides, whereas other members of this family had normal hearing. There is no evidence that any member of these families had any other known cause to account for hearing impairment. Comprehensive family medical histories of these individuals showed no other clinical abnormalities, including diabetes, visual impairment, and neuromuscular disorders.

Altered conformation of tRNA Ile
To test whether the m.4317A3 G mutation affected the conformation of tRNA Ile , total mitochondrial RNAs were electrophoresed through 10% polyacrylamide gel (native condition) in Tris-glycine buffer and then electroblotted onto a positively charged nylon membrane for hybridization analysis with digoxigenin (DIG)-labeled oligodeoxynucleotide probes for tRNA Ile , tRNA Ser(AGY) , and tRNA Met , respectively. As shown in Fig. 2A, electrophoretic patterns under native condition showed that the tRNA Ile in three mutant cell lines carrying both m1555A3 G and m.4317A3 G mutations migrated faster than those of three control cell lines. However, there were no obvious electrophoretic mobility differences between the mutant cell lines harboring only m1555A3 G mutation and control cell lines. These data indicated that the m.4317A3 G mutation changed the conformation of tRNA Ile .

Marked reduction in the steady-state levels of tRNA Ile
To examine whether the m.4317A3 G mutation perturbed the metabolism of tRNA Ile , we subjected mitochondrial RNAs from mutant and control cell lines to Northern blottings and hybridized them with DIG-labeled oligodeoxynucleotide probes for tRNA Ile , tRNA Leu(UUR) , tRNA Val , tRNA Gln , tRNA Ala ,

tRNA modifier for deafness expression of 12S rRNA mutation
and tRNA Ser(UCN) , respectively. As shown in Fig. 2B, the average steady-state levels of tRNA Ile in the mutant cells were significantly decreased, as compared with the control cell lines. As shown in Fig. 2C, the average levels of tRNA Ile in these mutant cell lines carrying both m.1555A3 G and m.4317A3 G mutations were 42% (p ϭ 0.007), 48% (p ϭ 0.002), 55% (p ϭ 0.008), 58% (p ϭ 0.011), and 51% (p ϭ 0.004) of average values of three control cell lines after normalization to tRNA Leu(UUR) , tRNA Val , tRNA Gln , tRNA Ala , and tRNA Ser(UCN) , respectively. Furthermore, the average levels of tRNA Ile in mutant cell lines carrying only m.1555A3 G mutation were comparable with those of three control cell lines.

Deficient aminoacylation of tRNA Ile
To evaluate whether the m.4317A3 G mutation aberrated the aminoacylation of tRNA Ile , we examined the aminoacylation capacities of tRNA Ile , tRNA Leu(UUR) , tRNA His , tRNA Met , and tRNA Ser(AGY) in control and mutant cell lines by the use of electrophoresis in an acidic polyacrylamide/urea gel system. As shown in Fig. 3A, there was less acylated tRNA Ile in the double mutant cell lines than those in control cell lines. The upper band represented the charged tRNA, and the lower band represented uncharged tRNA. To further distinguish non-aminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated by heating for 10 min at 60°C (pH 8.3) and then run in parallel. As shown in Fig. 3B, only one band (uncharged tRNA) was present in both mutant and control cell lines after deacylation. As shown in Fig. 3C, the efficiencies of aminoacy-lated tRNA Ile in mutant cell lines carrying only m.1555A3 G mutation and both m.1555A3 G and m.4317A3 G mutations were 94% (p ϭ 0.316) and 54% (p ϭ 0.001) of those in control cell lines, respectively. However, the levels of aminoacylation in tRNA Leu(UUR) , tRNA His , tRNA Met , and tRNA Ser(AGY) in mutant cell lines were comparable with those in the control cell lines.

Decreases in levels of mitochondrial proteins
To determine whether the m.4317A3 G mutation alters mitochondrial translation, the Western blot analysis was conducted to examine the levels of seven mtDNA encoding polypeptides in mutant and control cell lines with ⌻⌷⌴20 as a loading control. As shown in Fig. 4A, the levels of ND1, ND4, and ND5, ND6 (subunits 1, 4, 5, and 6 of NADH:ubiquinone oxidoreductase), CO2 (subunit II of cytochrome c oxidase), and CYTB (apocytochrome b) exhibited the variable reductions in the mutant cell lines, whereas the levels of ATP8 (subunit 8 of the H ϩ -ATPase) in the mutant cell lines were comparable with those of control cell lines. As shown in Fig. 4B, the overall levels of seven mitochondrial translation products in mutant cell lines carrying both m.1555A3 G and m.4317A3 G mutations were 65% (p ϭ 0.001), relative to the mean value measured in the control cell lines. As shown in Fig. 4C, the average levels of ND1, ND4, ND5, ND6, CO2, ATP8, and CYTB in these mutant cells carrying both m.1555A3 G and m.4317A3 G mutations were 58, 74, 62, 68, 59, 102, and 29% of the average values of control cells, respectively. Furthermore, the overall levels of seven mitochondrial translation products in the mutant cell  (Table S2).
We then examined the levels of five subunits of phosphorylation system (OXPHOS) in control and mutant cell lines by Western blot analysis. As shown in Fig. 5, the average level of mtDNA-encoded subunit CO2 of cytochrome c oxidase in the mutant cell carrying only the m.1555A3 G mutation, both m.1555A3 G and m.4317A3 G mutations were 86% (p ϭ 0.019) and 63% (p ϭ 0.007) of control cell lines, respectively. However, the levels of other four polypeptides (NDUFB8 of NADH:ubiquinone oxidoreductase; SDHB of succinate ubiquinone oxidoreductase; UQCRC2 of ubiquinol cytochrome c reductase and ATP5A of H ϩ -ATPase), encoded by nuclear genes, in mutant cell lines were comparable with those in control cell lines.

Reduced activities of complex I, III, and IV
To investigate the effect of the m.4317A3 G 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 (47)(48)(49). The activity of complex II (succinate ubiquinone oxidoreductase) exclusively encoded by the nuclear DNA was examined by the artificial electron acceptor DCPIP (50,51). 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 Fig. 6, the activity of complex I in the mutant cells carrying only m.1555A3 G mutation, both m.1555A3 G and m.4317A3 G mutations were 79% (p ϭ 0.016) and 55% (p ϭ 0.001) of the average values of three control cell lines, respectively. The activity of complex III in the mutant cell carrying only m.1555A3 G mutation, both m.1555A3 G and m.4317A3 G mutations were 80% (p ϭ 0.017) and 42% (p Ͻ 0.001) of control cell lines, respectively. Moreover, the activity of complex IV in the mutant cell carrying only the m.1555A3 G mutation, both m.1555A3 G and m.4317A3 G mutations were 85% (p Ͻ 0.001) and 59% (p ϭ 0.022) of controls, respectively. However, the activities of complexes II in all mutant cell lines were comparable with those of control cell lines.

Respiration deficiency
To evaluate whether the m.4317A3 G mutation affects cellular bioenergetics, we examined the oxygen consumption rates (OCRs) of mutant and control cell lines. OCR values were expressed as the relative value normalized to protein content. As shown in Fig. 7, the average basal OCRs in mutant cell lines carrying both m.1555A3 G and m.4317A3 G, and only m.1555A3 G mutation were 52% (p ϭ 0.002) and 80% (p ϭ 0.042), relative to the mean value measured in the control cell . Western blot analysis of mitochondrial proteins. A, 5 g of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with antibodies specific for ND1, ND4, ND5, ND6, CO2, CYTB, and ATP8 and with ⌻⌷⌴20 as a loading control, respectively. B, quantification of total mitochondrial protein levels. The levels of mitochondrial proteins in mutant and control cell lines were determined as described elsewhere (8). C, quantification of seven polypeptides. The levels of ND1, ND4, ND5, ND6, CO2, CYTB, and ATP8 in mutant and control cell lines were determined as described elsewhere (8 -10). Graph details and symbols are explained in the legend to Fig. 2.

Figure 5. Western blot analysis of OXPHOS subunits.
A, 5 g of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with antibody mixture specific for subunits of each OXPHOS complex and with TOM20 as a loading control. B, quantification of the levels of ATP5A, UQCRC2, SDHB, CO2, and NDUFB8 in mutant and control cell lines was determined as described elsewhere (8 -10). Graph details and symbols are explained in the legend to Fig. 2. tRNA modifier for deafness expression of 12S rRNA mutation lines, respectively. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cell lines, OCRs 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 A (to inhibit complex III). 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 Fig.  7B, the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and non-mitochondrial OCR were 50% (p ϭ 0.001), 62% (p ϭ 0.027), 51% (p ϭ 0.020), 52% (p ϭ 0.010), and 69% (p ϭ 0.123) in the mutant cell lines carrying both m.1555A3 G and m.4317A3 G mutations, and 79% (p ϭ 0.031), 94% (p ϭ 0.106), 81% (p ϭ 0.045), 82% (p ϭ 0.301), and 82% (p ϭ 0.740) in the mutant cell lines carrying only m.1555A3 G mutation.

Decreased levels of mitochondrial ATP production
The capacities of oxidative phosphorylation in mutant and control cell lines were measured by examining the levels of cellular and mitochondrial ATP using a luciferin/luciferase assay. Populations of cells were incubated in the media in the presence of glucose, and 2-deoxy-D-glucose with pyruvate (8). As shown in Fig. 8A, in the presence of glucose (total cellular levels of ATP), the average levels of ATP production in mutant cells carrying both m.1555A3 G and m.4317A3 G mutations and only m.1555A3 G mutation were 92% (p ϭ 0.729) and 98% (p ϭ 0.243), relative to the mean value measured in the control cell lines, respectively. In the presence of pyruvate and 2-deoxy-D-glucose to inhibit the glycolysis (mitochondrial levels of ATP), as shown in Fig. 8B, the levels of mitochondrial ATP production in mutant cell lines carrying both m.1555A3 G and m.4317A3 G mutations and only m.1555A3 G mutation were 56% (p Ͻ 0.001) and 81% (p ϭ 0.001) of the mean value measured in the control cell lines, respectively.

Reductions in mitochondrial membrane potential
To examine whether the m.4317A3 G mutation affects mitochondrial membrane potential (⌬⌿m), a fluorescence probe JC-10 assay system was used to measure the ⌬⌿m in mutant and control cell lines. The ratios of fluorescence intensities of excitation/emission ϭ 490/590 and 490/530 nm (FL590/FL530) were recorded to delineate the ⌬⌿m of each sample. The relative ratios of the FL590/FL530 geometric mean between mutant and control cell lines were calculated to represent the level of ⌬⌿m, as described elsewhere (8). As shown in Fig. 9, the levels of ⌬⌿m in mutant cell lines carrying both m.1555A3 G and m.4317A3 G mutations and only m.1555A3 G mutation were 59% (p Ͻ 0.001) and 76% (p Ͻ 0.001) of the mean value measured in the control cell lines, respectively. By contrast, the levels of ⌬⌿m in mutant cell lines in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, to dissipate the mitochondrial membrane potential) were comparable with those of control cell lines.

Increase in mitochondrial ROS production
The levels of mitochondrial ROS among the lymphoblastoid cells were determined using a MitoSOX assay via flow cytometry (52). Geometric mean intensity was recorded to measure and delineate the rate of ROS of each sample. As shown in Fig.  10, the levels of ROS generation in the mutant cell lines cells carrying both m.1555A3 G and m.4317A3 G mutations ranged from 165 to 181%, with an average 171% (p Ͻ 0.001) of the mean value measured in the control cell lines. Moreover, levels of ROS generation in the mutant cell lines carrying only m.1555A3 G mutation was 133% (p ϭ 0.001) of the mean value measured in control cell lines.

Discussion
Mitochondrial genetic modifiers were proposed to increase the susceptibility to deafness-associated m.1555A3 G or m.1494T3 C mutation (2,53). Using genetic and molecular approaches, in combination with functional assays, we demonstrated that a deafness susceptibility allele (m.4317A3 G mutation) in the tRNA Ile gene modulated the phenotypic expression of deafness-associated 12S rRNA 1555A3 G mutation. The m.4317A3 G mutation (conventional position 59 of tRNA) affected the highly conserved adenine of the T-loop in tRNA Ile (39 -40). We hypothesized that the anticipated alteration of the tertiary structure of tRNA Ile by the m.4317A3 G mutation led to a failure in tRNA metabolism. In vitro assays showed that the m.4317A3 G mutation impaired the 3Ј-end-processing of the tRNA Ile precursor and led to the decreased CCA-addition of tRNA Ile (41)(42)(43). Alternatively, the aberrant structure of the T-arm makes mutant tRNA Ile more unstable, inefficiently aminoacylated, and finally subject to degradation (39,40). The instability of mutant tRNA Ile was evidenced by the altered conformation observed in mutant tRNA Ile derived from cell lines carrying the m.4317A3 G mutation and in mutant tRNA Ile transcript (G59). Moreover, the altered tertiary structure caused by the m.4317A3 G mutation may affect the aminoacylated efficiency of tRNA Ile (42). In this study, a 46% decrease in aminoacylated tRNA Ile was observed in mutant cell lines carrying both m.4317A3 G and m.1555A3 G mutations. These results were consistent with a slightly but significantly decreased aminoacylated efficiency of tRNA Ile in vitro transcripts (42). In particular, mutant cell lines bearing both tRNA modifier for deafness expression of 12S rRNA mutation m.4317A3 G and m.1555A3 G mutations exhibited ϳ50% decreases in the steady-state level of tRNA Ile , although there was no significant reduction in the level of tRNA Ile in mutant cell lines carrying only the m.1555A3 G mutation. This level of total tRNA Ile in mutant cell lines is, however, above a proposed threshold level, which is 30% of the control level of tRNA, to support the normal rate of mitochondrial translation (5,6,8,54). These data strongly indicate that the m.4317A3 G mutation alone is insufficient to produce a deafness phenotype, as in the case of deafness-associated tRNA Glu 14692A3 G, tRNA Asp 7551A3 G, 12S rRNA 1555A3 G, and 1494C3 T mutations (9, 10, 23, 26).
The altered tRNA Ile metabolism led to a defect in mitochondrial translation. Alternatively, the mutant tRNA Ile may interact faultily with translation machinery, thereby affecting the mitochondrial translation (55,56). In fact, the mtDNA encoded 13 polypeptides in the complexes of the oxidative phosphorylation system (ND1-6; ND4L of complex I; CYTB of complex III; CO1, CO2, CO3 of complex IV; and ATP6 and ATP8 of complex V) (4,46). In this investigation, 35% reductions in the average levels of seven mtDNA-encoded proteins were observed in mutant cell lines carrying both m.4317A3 G and m.1555A3 G mutations, respectively. In contrast, the cell lines carrying only m.1555A3 G mutation exhibited 18% reduction 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/antimycin A treatment. Basal OCR was determined as OCR before oligomycin minus OCR after rotenone/antimycin A. ATP-linked 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. OCR values were expressed in picomoles of oxygen/min/microgram of protein The average values of three determinations for each cell line were shown. Graph details and symbols are explained in the legend to Fig. 2. tRNA modifier for deafness expression of 12S rRNA mutation in the average levels of seven mtDNA-encoded proteins. However, both m.4317A3 G and m.1555A3 G mutations did not reduce the levels of four subunits (NDUFB8 of NADH:ubiquinone oxidoreductase; SDHB of succinate ubiquinone oxidoreductase; UQCRC2 of ubiquinol cytochrome c reductase; and ATP5A of H ϩ -ATPase), encoded by nuclear genes. These data were consistent with our previous data that 27 and 58% decreases in the levels of seven mtDNA-encoding proteins were observed in the lymphoblastoid cell lines carrying only the m.1555A3 G mutation or both m.1555A3 G and TRMU A10S mutations, derived from matrilineal relatives of an Arab-Israeli family, respectively (33). Notably, there were variable decreases in the levels of seven mtDNA-encoded polypeptides in mutant cell lines carrying the both m.1555A3 G and m.4317A3 G mutations, ranging from 29 to 102% of the average values of control cell lines. As shown in Table S2, cell lines harboring both m.1555A3 G and m.4317A3 G mutations exhibited marked reductions (71%) in the levels of CYTB, mild reductions (26% to 42%) in the level of ND1, ND4, ND5, ND6, and CO2, but a comparable level in the level of ATP8, as compared with those in control cell lines. In contrast to what was previously shown in cells carrying the tRNA Lys 8344A3 G mutation (56) and tRNA Ser(UCN) 7445A3 G mutation (5), polypeptides levels in mutant cell lines, relative to those in control cell lines, did not significantly correlate with the number or proportion of isoleucine codons. Hence, the impaired synthesis of ND1, ND4, and ND5, ND6 subunits of complex I, CYTB, subunit of complex III and CO2, subunits of complex IV may perturb the activities of complexes I, III, and IV and then worsen the defects in mitochondrial translation and respiratory phenotypes associated with the m.1555A3 G mutation. In this study, there were more severe decreases of complexes I, III, and IV activities observed in cell lines carrying both m.1555A3 G and m.4317A3 G mutations than those bearing only m.1555A3 G mutation. In addition, impairment of mitochondrial translation resulted in the decreased rates in the basal OCR, or ATP-linked OCR, reserved capacity, and maximal OCR among the control and mutant cell lines. These data highlighted that aberrant tRNA metabolism played a critical role in producing their respiration defects, as in the cases of cells carrying deafness-associated m.7511T3 C, m.7551A3 G, and m.14692A3 G mutations (6,9,10).
The respiratory deficiency may lead to the uncoupling of the oxidative pathway for ATP synthesis, oxidative stress, and subsequent failure of cellular energetic process (57). In this investigation, ϳ19 and 44% decreases in mitochondrial ATP production were observed in these mutant cell lines carrying only m.1555A3 G or both m.1555A3 G and m.4317A3 G mutations, respectively. However, 44% reduction of mitochondrial ATP production in mutant cell lines harboring both m.1555A3 G and m.4317A3 G mutations were comparable with those in lymphoblastoid cell lines bearing both m.1555A3 G and homozygous TRMU A10S mutations (33). As a result, cochlear hair cells bearing both m.1555A3 G and m.4317A3 G mutations may be particularly sensitive to increased ATP demand (1,2,58). Furthermore, the deficient activities of respiratory chain complexes often impaired mitochondrial membrane potentials, which is a key indicator of cellular viability (8,10). Indeed, mitochondrial membrane potentials reflect the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation (49,59). In this study, 24 and 41% reductions in mitochondrial membrane potential in lymphoblastoid cell lines carrying the only m.1555A3 G mutation and both m.1555A3 G and m.4317A3 G mutations was much lower than those in cell lines carrying the m.12201T3 C mutation (8). The abnormal oxidative phosphorylation and mitochondrial membrane potential led to the increased production of reactive oxygen species and the subsequent failure of cellular energetic processes in mutant cells carrying m.1555A3 G and m.4317A3 G mutations. In particular, 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 and increasing apoptotic signaling (60,61). Hair cells and neurons in the cochlea may be preferentially affected, because they are somehow exquisitely sensitive to subtle imbalances in the cellular redox state or tRNA modifier for deafness expression of 12S rRNA mutation increased level of free radicals (58,62). This would lead to the dysfunction or death of hair cells in the cochlea carrying both m.1555A3 G and m.4317A3 G mutations.
In summary, our study demonstrated the role of a deafness susceptibility allele (m.4317A3 G mutation) in the tRNA Ile gene in the phenotypic manifestation of the deafness-associated m.1555A3 G mutation. The m.4317A3 G mutation altered both structure and function of tRNA Ile . The aberrant tRNA metabolism further deteriorated the defective mitochondrial translation associated with the 12S rRNA 1555A3 G mutation. These alterations resulted in the respiratory deficiency, decreasing ATP production and increasing ROS pro-duction. These biochemical defects led to the high penetrance and occurrence of deafness in these Chinese families carrying both m.1555A3 G and m.4317A3 G mutations. Therefore, the tRNA Ile 4317A3 G mutation acts in synergy with 12S rRNA 1555A3 G mutation, modulating phenotypic manifestation.

Subjects and audiological examinations
A total of 2651 genetically unrelated Han Chinese subjects with hearing impairment and 574 normal hearing Han Chinese control subjects for this study were described elsewhere (37).

tRNA modifier for deafness expression of 12S rRNA mutation
Two hearing-impaired Chinese Han pedigrees for this study were ascertained at the Otology Clinic of the First Affiliated Hospital, Wenzhou Medical University. This study was in compliance with the Declaration of Helsinki. Informed consent, blood samples, and clinical evaluations were obtained from all participants and families, under protocols approved by the Ethics Committees of Zhejiang University and Wenzhou Medical University. Audiological and neurological examinations of hearing impairment were performed as detailed previously (14). All available members of two pedigrees and three control subjects were evaluated at length to identify both of personal and family medical history of hearing loss, the history of the use of aminoglycosides, and other clinical abnormalities.

Mutational analysis of mitochondrial DNA
Genomic DNA was isolated from whole blood of participants using QIAamp DNA Blood Mini Kit (Qiagen, No. 51104). The subject's DNA fragments spanning the mitochondrial 12S rRNA and tRNA Ile genes were PCR-amplified by the use of oligodeoxynucleotides corresponding to mtDNA at positions 618 -2007 and 3796 -4693, respectively (4). Each fragment was purified and then analyzed by direct sequencing. These sequence results were compared with the updated consensus Cambridge sequence (GenBank TM accession number NC_ 012920) (4). The entire mtDNAs of two probands WZD91 III-3 and WZD92 II-8 and three Chinese control subjects (A23, A22, and A21) were PCR-amplified in 24 overlapping fragments using sets of the light and heavy strand oligonucleotide primers, as described previously (63). These sequence results were compared with the updated consensus Cambridge sequence, as described above. To quantify the m.4317A3 G mutation, PCR segment (898 bp) spanning the tRNA Ile gene was amplified and subsequently digested with restriction enzyme Af1II, as the m.4317A3 G mutation created the site for this enzyme. Equal amounts of various digested samples were then analyzed by electrophoresis through 3% agarose gel. The proportions of digested and undigested PCR products were determined by the ImageQuant program after ethidium bromide staining to determine whether the m.4317A3G mutation is in homoplasmy in these subjects.

Cell lines and culture conditions
Lymphoblastoid cell lines derived from members of two Chinese families (three subjects harboring only m.1555A3 G mutation (WZD92 III-13, WZD92 III-17, and WZD92 III-18) and three individuals (WZD91 III-1, WZD91 II-3, and WZD91 III-7) carrying both m.1555A3 G and m.4317A3 G mutations) and three control individuals (A21, A22, and A23) lacking both mutations belonging to the same mtDNA haplogroup were immortalized by transformation with the Epstein-Barr virus, as described elsewhere (64). Lymphoblastoid cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Mitochondrial tRNA analysis
Total mitochondrial RNAs were obtained from mitochondria isolated from various cell lines (ϳ2.0 ϫ 10 8 cells) using the Totally RNA TM kit (Ambion), as described previously (65). For the tRNA Northern blot analysis, 2 g of total mitochondrial RNA were electrophoresed through a urea denaturing 10% PAGE with 8 M urea in Tris borate/EDTA buffer. The gels were electroblotted onto a positively charged nylon membrane (Roche Applied Science) for the hybridization analysis with DIG-labeled oligodeoxynucleotide probes for tRNA Ile , tRNA Leu(UUR) , tRNA Val , tRNA Gln , tRNA Ala , and tRNA Ser(UCN) , as detailed previously (8 -10, 66, 67). DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide tailing kit (Roche Applied Science). The hybridization and quantification of density in each band were performed as detailed previously (8 -10).
For the aminoacylation assays, total mitochondrial RNAs were isolated under acid conditions, and 2 g of RNAs were electrophoresed at 4°C through an acid (pH 5.0) 10% polyacrylamide-8 mM urea gel to separate the charged and uncharged tRNA as detailed elsewhere (56,68). To further distinguish non-aminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated by being heated for 10 min at 60°C (pH 8.3) and then run in parallel (9, 68). The gels were then electroblotted onto a positively charged nylon membrane (Roche Applied Science) for the hybridization analysis with oligodeoxynucleotide probes as described above. Quantification of density in each band was performed as detailed previously (68).
For the tRNA mobility shift assay, two g of RNAs were electrophoresed through a 10% polyacrylamide native gel at 4°C with 50 mM Tris-glycine buffer. After electrophoresis, the gels were treated according to the Northern blot analysis as described above (66).

Enzymatic assays
The enzymatic activities of complexes I-IV were measured as detailed elsewhere (47)(48)(49)(50)(51). In brief, citrate synthase activity was analyzed by the reduction of 5,5Ј-dithiobis-2-nitrobenzoic acid at 412 nm in the assay buffer containing 0.1 mM 5,5Ј-dithiobis-2-nitrobenzoic acid, 50 M acetyl coenzyme A, and 250 M oxaloacetate. Complex I activity was determined with 10 g/ml antimycin A and 2 mM KCN by following the decrease in the absorbance due to the NADH oxidation at 340 nm in assay buffer. The activity of complex II was analyzed by tracking the tRNA modifier for deafness expression of 12S rRNA mutation secondary reduction of DCPIP by decylubiquinone at 600 nm in the assay buffer. Complex III activity was determined in the presence of 2 g/ml antimycin A and 2 mM KCN by measuring the reduction of cytochrome c at 550 nm with reduced decylubiquinone in the assay buffer. Complex IV activity was measured by monitoring the oxidation of reduced cytochrome c as a decrease of absorbance at 550 nm in the assay buffer. All assays were performed by using Synergy H1 (Biotek, Winooski, VT). Complex I-IV activities were normalized by citrate synthase activity.

Measurements of oxygen consumption
The rates of oxygen consumption in lymphoblastoid cell lines were assayed with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience), as detailed previously (8,69). The protein content of each well was then measured to normalize OCR values.

ATP measurements
The Cell Titer-Glo luminescent cell viability assay kit (Promega) was used for the measurement of cellular and mitochondrial ATP levels, following the modified manufacturer's instructions (8,66).

Assessment of mitochondrial membrane potential
Mitochondrial membrane potential was assessed with JC-10 assay kit microplate (Abcam) according to general manufacturer's recommendations with some modifications, as detailed elsewhere (8,66).

Computer analysis
Statistical analysis was carried out using the unpaired, twotailed Student's t test contained in the Microsoft Excel program or Macintosh (version 2007). Differences were considered significant at a p Ͻ 0.05.