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Leber's hereditary optic neuropathy (LHON) is a maternally transmitted eye disease due to the degeneration of retinal ganglion cells (RGCs). Mitochondrial 11778G > A mutation is the most common LHON-associated mitochondrial DNA (mtDNA) mutation. Our recent studies demonstrated some LHON families manifested by synergic interaction between m.11778G > A mutation and YARS2 allele (c.572G > T, p.Gly191Val) encoding mitochondrial tyrosyl-tRNA synthetase. However, the RGC-specific effects of LHON-associated mtDNA mutations remain elusive and there is no highly effective therapy for LHON. Here, we generated patients-derived induced pluripotent stem cells (iPSCs) from fibroblasts derived from a Chinese LHON family (both m.11778G > A and c.572G > T mutations, only m.11778G > A mutation, and control subject). The c.572G > T mutation in iPSC lines from a syndromic individual was corrected by CRISPR/Cas9. Those iPSCs were differentiated into neural progenitor cells and subsequently induced RGC-like cells using a stepwise differentiation procedure. Those RGC-like cells derived from symptomatic individual harboring both m.11778G > A and c.572G > T mutations exhibited greater defects in neuronal differentiation, morphology including reduced area of soma, numbers of neurites and shortened length of axons, electrophysiological properties than those in cells bearing only m.11778G > A mutation. Furthermore, these RGC-like cells revealed more drastic reductions in oxygen consumption rates, levels of mitochondrial ATP and increasing productions of reactive oxygen species than those in other cell models. These mitochondrial dysfunctions promoted the apoptotic process for RGC degenerations. Correction of YARS2 c.572G > T mutation rescued deficiencies of patient-derived RGC-like cells. These findings provide new insights into pathophysiology of LHON arising from RGC-specific mitochondrial dysfunctions and step toward therapeutic intervention for this disease., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In this report, we investigated the molecular mechanism underlying a deafness-associated m.5783C > T mutation that affects the canonical C50-G63 base-pairing of TΨC stem of tRNACys and immediately adjacent to 5' end of light-strand origin of mitochondrial DNA (mtDNA) replication (OriL). Two dimensional agarose gel electrophoresis revealed marked decreases in the replication intermediates including ascending arm of Y-fork arcs spanning OriL in the mutant cybrids bearing m.5783C > T mutation. mtDNA replication alterations were further evidenced by decreased levels of PolγA, Twinkle and SSBP1, newly synthesized mtDNA and mtDNA contents in the mutant cybrids. The m.5783C > T mutation altered tRNACys structure and function, including decreased melting temperature, conformational changes, instability and deficient aminoacylation of mutated tRNACys. The m.5783C > T mutation impaired the 5' end processing efficiency of tRNACys precursors and reduced the levels of tRNACys and downstream tRNATyr. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced effects in the polypeptides harboring higher numbers of cysteine and tyrosine codons. These alterations led to deficient oxidative phosphorylation including instability and reduced activities of the respiratory chain enzyme complexes I, III, IV and intact supercomplexes overall. Our findings highlight the impact of mitochondrial dysfunction on deafness arising from defects in mitochondrial DNA replication and tRNA metabolism., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: To investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation., Methods: Cybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis., Results: Nineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy., Conclusions: Our findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.7516delA mutation affecting the 5' end processing sites of mitochondrial tRNAAsp and tRNASer(UCN). An in vitro processing experiment demonstrated that m.7516delA mutation caused the aberrant 5' end processing of tRNASer(UCN) and tRNAAsp precursors, catalyzed by RNase P. Using cytoplasmic hybrids (cybrids) derived from one hearing-impaired Chinese family bearing the m.7516delA mutation and control, we demonstrated the asymmetrical effects of m.7516delA mutation on the processing of tRNAs in the heavy (H)-strand and light (L)-strand polycistronic transcripts. Specially, the m.7516delA mutation caused the decreased levels of tRNASer(UCN) and downstream five tRNAs, including tRNATyr from the L-strand transcripts and tRNAAsp from the H-strand transcripts. Strikingly, mutant cybrids exhibited the lower level of COX2 mRNA and accumulation of longer and uncleaved precursors of COX2 from the H-strand transcripts. Aberrant RNA metabolisms yielded variable reductions in the mitochondrial proteins, especially marked reductions in the levels of ND4, ND5, CO1, CO2 and CO3. The impairment of mitochondrial translation caused the proteostasis stress and respiratory deficiency, diminished ATP production and membrane potential, increased production of reactive oxygen species and promoted apoptosis. Our findings provide new insights into the pathophysiology of deafness arising from mitochondrial tRNA processing defects., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cyathostomins are one kind of the most important parasites in equids. Cylicostephanus minutus is a member of the subfamily Cyathostominae. In the present study, we determined the complete mitochondrial (mt) genomes from four Cs. minutus isolates and reconstructed the phylogenetic relationship of Strongylidae to test the hypothesis that Cs. minutus represents a species complex. The complete mt genome sequences of Cs. minutus were 13,772-13,822 bp in length, and contained 36 genes (12 protein coding genes, 22 tRNA genes, two rRNA genes), and two non-coding regions (NCRs). The intraspecific identity of nucleotide sequences and amino acid sequences in Cs. minutus (1-4) were 89.3-97.9% and 97.0-98.8%, respectively. Two operational taxonomic units (OTUs) were determined based on the mt genome sequences, OTU 2 (Csm 1 and Csm 2) and OTU 3 (Csm 3 and Csm 4). Sequence analysis showed the divergence between OTU 2 and OTU 3 was 8.9-10.7%. Pairwise comparisons of 12 protein coding genes between OTU 2 and OTU 3 showed a difference of 3.0-13.3% at the nucleotide level and 0-6.7% at the amino acid level. Phylogenetic analysis showed the separation of Cs. minutus isolates from the same host into different distinct clades based on mt genomes. Comparisons of partial mt cox1, nad5, and cytb and ITS2 sequences from 20 Cs. minutus isolates from the same host and the same geographical location with other Cs. minutus sequences available in GenBank revealed significant nucleotide differences. Phylogenetic analysis showed a separation of Cs. minutus into three distinct clades. Thus, the comparative and phylogenetic analyses of mtDNA datasets indicated that Cs. minutus represents a complex of at least three species. Our results have further confirmed the existence of a cryptic Cs. minutus species, and provides a reference for the taxonomical, population genetics, and systematics studies of other cyathostomin species., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ji et al.)

Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNAMet and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5' end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mutations in mitochondrial DNA (mtDNA) have been associated with deafness and their pathophysiology remains poorly understood. In this study, we investigated the pathogenic mechanism of deafness-associated 7505A > G variant in the mitochondrial tRNA Ser(UCN) . The m.7505A > G variant affected the highly conserved adenine at position 11 (A11), disrupted the highly conserved A11-U24 base-pairing of DHU stem of tRNA Ser(UCN) and introduced a tertiary base pairing (G11-C56) with the C56 in the TΨC loop. We therefore hypothesized that the m.7505A > G variant altered both structure and function of tRNA Ser(UCN) . We demonstrated that the m.7505A > G variant perturbed the conformation and stability of tRNA Ser(UCN) , as indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wild type molecule. Using the cybrids constructed by transferring mitochondria from the Chinese family into mitochondrial DNA (mtDNA)-less cells, we demonstrated the m.7505A > G variant led to significantly decreased steady-state levels of tRNA Ser(UCN) in the mutant cybrids, as compared with those of control cybrids. The aberrant tRNA Ser(UCN) metabolism resulted in the variable decreases in mtDNA-encoded polypeptides in the mutant cybrids. Furthermore, we demonstrated that the m.7505A > G variant decreased the activities of mitochondrial respiratory complexes I, III and IV, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the mutant cybrids. These results demonstrated that the m.7505A > G variant affected both structure and function of tRNA Ser(UCN) and consequently altered mitochondrial function. Our findings highlighted critical insights into the pathophysiology of maternally inherited deafness, which is manifested by the aberrant tRNA metabolism., (Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Defective nucleotide modifications of mitochondrial tRNAs have been associated with several human diseases, but their pathophysiology remains poorly understood. In this report, we investigated the pathogenic molecular mechanism underlying a hypertension-associated 4435A→G mutation in mitochondrial tRNA Met The m.4435A→G mutation affected a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon, which is important for the fidelity of codon recognition and stabilization. We hypothesized that the m.4435A→G mutation introduced an m 1 G37 modification of tRNA Met , altering its structure and function. Primer extension and methylation activity assays indeed confirmed that the m.4435A→G mutation created a tRNA methyltransferase 5 (TRMT5)-catalyzed m 1 G37 modification of tRNA Met We found that this mutation altered the tRNA Met structure, indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wildtype molecule. We demonstrated that cybrid cell lines carrying the m.4435A→G mutation exhibited significantly decreased efficiency in aminoacylation and steady-state levels of tRNA Met , as compared with those of control cybrids. The aberrant tRNA Met metabolism resulted in variable decreases in mitochondrial DNA (mtDNA)-encoded polypeptides in the mutant cybrids. Furthermore, we found that the m.4435A→G mutation caused respiratory deficiency, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in mutant cybrids. These results demonstrated that an aberrant m 1 G37 modification of mitochondrial tRNA Met affected the structure and function of its tRNA and consequently altered mitochondrial function. Our findings provide critical insights into the pathophysiology of maternally inherited hypertension, which is manifested by the deficient tRNA nucleotide modification., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Several mitochondrial tRNA mutations have been associated with hypertension, but their pathophysiology remains poorly understood. In this report, we identified a novel homoplasmic 3253T→C mutation in the mitochondrial tRNA Leu(UUR) gene in a Han Chinese family with maternally inherited hypertension. The m.3253T→C mutation affected a highly conserved uridine at position 22 at the D-stem of tRNA Leu(UUR) , introducing a G-C base pairing (G13-C22) at the D-stem and a tertiary base pairing (C22-G46) between the D-stem and the variable loop. We therefore hypothesized that the m.3253T→C mutation altered both the structure and function of tRNA Leu(UUR) Using cytoplasmic hybrid (cybrid) cell lines derived from this Chinese family, we demonstrated that the m.3253T→C mutation perturbed the conformation and stability of tRNA Leu(UUR) , as suggested by faster electrophoretic mobility of mutated tRNA relative to the wild-type molecule. Northern blot analysis revealed an ∼45% decrease in the steady-state level of tRNA Leu(UUR) in the mutant cell lines carrying the m.3253T→C mutation, as compared with control cell lines. Moreover, an ∼35% reduction in aminoacylation efficiency of tRNA Leu(UUR) was observed in the m.3253T→C mutant cells. These alterations in tRNA Leu(UUR) metabolism impaired mitochondrial translation, especially for those polypeptides with a high proportion of Leu(UUR) codons, such as ND6. Furthermore, we demonstrated that the m.3253T→C mutation decreased the activities of mitochondrial complexes I and V, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the cells. In conclusion, our findings may provide new insights into the pathophysiology of maternally inherited hypertension., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Mitochondria have the profound impact on vascular function in both health and disease. However, mitochondrial genetic determinants for the development of hypertension remain poorly explored., Methods and Results: The Sanger sequence analysis of 22 mitochondrial tRNA genes were performed in a cohort of 2070 Han Chinese hypertensive and 512 control subjects. This analysis identified 165 variants among 22 tRNA genes. These variants were evaluated for the pathogenicity using the following criteria: (1) present in <1% of the controls; (2) evolutional conservation; (3) potential structural and functional alterations. We identified 47 (5 known and 42 novel/putative) hypertension-associated tRNA variants in 80 hypertensive subjects. These variants could have potential structural alterations and functional significance of tRNAs. By using lymphoblastoid cell lines derived from 6 probands carrying one of 6 represented variants (tRNA(Ala) 5655T>C, tRNA(Gly) 10003T>C, tRNA(Leu(UUR)) 3253T>C, tRNA(Asp) 7551A>G, tRNA(Glu) 14692A>G, tRNA(Thr) 15909A>G) and 6 control subjects lacking these variants, we showed marked reductions in the steady-state level of corresponding 5 tRNAs, but not tRNA(Thr), in mutant cell lines, compared with control cells lines. The various decreases in the activities of complexes I, III and IV were observed in mutant cells carrying one of five tRNA variants, except tRNA(Thr) 15909A>G variant. The deficient respirations were responsible for the decrease in the mitochondrial ATP production and increasing production of reactive oxygen species in mutant cell lines carrying one of five tRNA variants., Conclusion: Mitochondrial tRNA variants are the important causes of hypertension, accounting for 3.9% cases of 2070 Han Chinese hypertensive subjects. Our findings may provide new insights into the pathophysiology of hypertension that were manifested by mitochondrial dysfunction., (Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρ o cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial DNA-based diseases which have been studied using Lymphoblastoid cell lines (LCLs) and transmitochondrial cybrids. Individual genetic information is preserved permanently in LCLs while the development of transmitochondrial cybrids provide ex-vivo cellular platform to study molecular mechanism of mitochondrial DNA-based diseases. The cytoplasmic donor cells for previous transmitochondrial cybrids come from patient's tissue or platelet directly. Here, we depicted in details the principle, methods and techniques to establish LCLs from frozen peripheral bloods harboring mitochondrial 4401G > A mutation by infection of Epstein Barr virus, and then to generate cybrids using ρ 0 206 and LCLs. The process of establishing these two cellular models was summarized into four steps as follows: (1) Generation of LCLs; (2) Transformation; (3) Selection; (4) Verification. To faithfully represent the function of mtDNA mutation, we analyzed and identified the sites of mtDNA mutations and copy numbers of each cellular models as well as the karyotype of transmitochondrial cybrids. Those clones with consistent parameters were selected for preservation and future analysis of the function of point mutations of mtDNA. Although these two cellular models play important roles in understanding molecular mechanism of mitochondrial DNA-based diseases on the cellular level, their limitations should be considered when elucidating the character of tissue specificity of mitochondrial DNA-based diseases.

Purpose: The purpose of this study was to investigate the mutational incidence and spectrum of mitochondrial ND1 gene in subjects with Leber's hereditary optic neuropathy (LHON)., Methods: A cohort of 1281 Han Chinese probands and 478 control subjects underwent sequence analysis of mitochondrial (mt)DNA. Resultant variants were evaluated for evolutionary conservation, allelic frequencies, and structural and functional consequences. Respiratory complex activities were measured using lymphoblastoid cell lines derived from 25 probands carrying the mtDNA mutation and 3 controls., Results: Mutational analysis identified 178 (70 missense and 108 silent) variants in the MT-ND1 gene. The incidences of known m.3460G>A, m.3635G>A, m.3733G>A, m.3866T>C, and m.3394T>C mutations were 1.33%, 0.86%, 0.08%, 0.55%, and 2.97%, respectively. Fifteen novel putative mutations were identified in 27 probands, translated into 2.1% cases of this cohort. The activity of complex I in mutant cell lines carrying one of putative mutations ranged from 66% to 76% of the average values in control cell lines, whereas activities of complexes II, III, and IV in mutant cells were comparable with those in controls. The low penetrances of optic neuropathy were observed in pedigrees carrying novel putative mutation(s). Moreover, mtDNAs in 101 probands carrying the MT-ND1 mutation(s) were widely dispersed among 15 Eastern Asian haplogroups. In particular, the occurrences of haplogroups M, M9, and M10 in patients carrying the ND1 mutations were higher than those in controls., Conclusions: These data demonstrated that the MT-ND1 gene is a hot spot for mutations associated with LHON. Our findings may provide valuable information for pathophysiology, management, and genetic counseling of LHON.

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 tRNA(Tyr) 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 tRNA(Tyr) 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., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mitochondrial genome is responsible for multiple human diseases in a maternal inherited pattern, yet phenotypes of patients in a same pedigree frequently vary largely. Genes involving in epigenetic modification, RNA processing, and other biological pathways, rather than "threshold effect" and environmental factors, provide more specific explanation to the aberrant phenotype. Thus, the double hit theory, mutations both in mitochondrial DNA and modifying genes aggravating the symptom, throws new light on mitochondrial dysfunction processes. In addition, mitochondrial retrograde signaling pathway that leads to reconfiguration of cell metabolism to adapt defects in mitochondria may as well play an active role. Here we review selected examples of modifier genes and mitochondrial retrograde signaling in mitochondrial disorders, which refine our understanding and will guide the rational design of clinical therapies.

Purpose: To investigate the prevalence and spectrum of mitochondrial ND4 mutations in subjects with Leber's hereditary optic neuropathy (LHON)., Methods: A cohort of 1281 Chinese Han probands and 478 control subjects underwent clinical and genetic evaluation, and sequence analysis of mitochondrial (mt) DNA, as well as enzymatic assay of NADH:ubiquinone oxidoreductase., Results: In this cohort, 503 probands had a family history of optic neuropathy and 778 subjects were sporadic cases. Mutational analysis of ND4 gene identified 149 (102 known and 47 novel) variants. The prevalence of known m.11778G>A mutation was 35.36%. Furthermore, we identified the known m.11696G>A and m.11253T>C mutations and five novel putative LHON-associated mutations. These mutations accounted for 2.74% of cases of LHON subjects. By enzymatic assay, we showed a mild decrease in the activity of NADH:ubiquinone oxidoreductase in mutant cell lines carrying only one putative mtDNA mutation. The low penetrance of optic neuropathy and mild biochemical defects in these pedigrees carrying only m.11696G>A mutation and one putative LHON-associated mutation suggested that the mutation(s) is(are) necessary but is(are) itself(themselves) insufficient to produce a visual failure. Moreover, mtDNAs in 169 probands carrying the LHON-associated mutation(s) were widely dispersed among 13 Eastern Asian haplogroups. In particular, the frequencies of haplogroups D, M8, M10, M11, and H in probands carrying the LHON-associated mtDNA mutation(s) were higher than those in Chinese controls., Conclusions: These results suggested that the ND4 gene is the hot spot for mutations associated with LHON. Thus, these findings may provide valuable information for the further understanding of pathogenic mechanism of LHON.

Purpose: To investigate the molecular pathogenesis of Leber's hereditary optic neuropathy (LHON) in Chinese families., Methods: A cohort of 1218 Han Chinese subjects with LHON and 316 control subjects underwent the clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA., Results: The age at onset of optic neuropathy in these subjects ranged from 5 to 55 years, with the average of 18 years. Mutational analysis of ND6 gene identified 92 (73 known and 19 novel) variants in these subjects. These variants included 29 (9 novel and 20 known) missense mutations and 63 silence variants. A total of 94 subjects carrying one of the known T14484C, T14502C, and G14459A mutations accounted for 7.7% cases of this cohort, particularly 4.4% for T14484C mutation. Furthermore, eight putative LHON-associated ND6 mutations accounted for 1.1% case of this cohort. Thus, 106 subjects carrying one of ND6 mutations accounted for 8.7% cases of this cohort. Low penetrance of optic neuropathy in pedigrees carrying one of eight putative mutations indicated that the mutation(s) is necessary, but itself insufficient to produce a clinical phenotype. Mitochondrial DNAs in 98 probands carrying the ND6 mutation(s) were widely dispersed among 12 Eastern Asian subhaplogroups. In particular, the occurrences of haplogroups M9, M10, M11, and H2 in patients carrying the ND6 mutations were higher than those in controls., Conclusions: These data further support that the ND6 gene is the hot spot for mutations associated with LHON. Thus, our findings may provide valuable information for the further understanding of pathophysiology and management of LHON.

Background: Coronary heart disease is the leading cause of death worldwide. Mitochondrial genetic determinants for the development of this disorder remain less explored., Methods and Results: We performed a clinical and genetic evaluation and mutational screening of 22 mitochondrial tRNA genes in a cohort of 80 genetically unrelated Han Chinese subjects and 125 members of 4 families with coronary heart disease and 512 Chinese control subjects. This analysis identified 16 nucleotide changes among 9 tRNA genes. Of these, the T5592C mutation creates a highly conservative base pairing (5G-68C) on the acceptor stem of tRNA(Gln), whereas the G15927A mutation destabilizes a highly conserved base pairing (28C-42G) in the anticodon stem of tRNA(Thr). However, the other tRNA variants were polymorphisms. The pedigrees of BJH24 carrying the T5592C mutation, BJH15, and BJH45 harboring the G15927A mutation exhibited maternal transmission of coronary heart disease. Sequence analysis of their mitochondrial genomes revealed the presence of T5592C or G15927A mutation but the absence of other functionally significant mutations in all matrilineal relatives of these families., Conclusions: Our previous observations showed that altered structures of tRNAs by these mtDNA mutations caused mitochondrial dysfunction. These may be the first evidence that mtDNA mutations increase the risk of coronary heart disease. Our findings may provide new insights into the pathophysiology of this disorder.

Objective: We reported here the clinical and genetic evaluations as well as mutational analysis of mitochondrial DNA(mtDNA) in a Chinese family with maternally transmitted non-syndromic hearing loss and investigated the influence of the mitochondrial tRNA(Asp) A7551G mutation to the phenotypic manifestation of the deafness., Methods: One Chinese Han pedigrees of maternally transmitted nonsyndromic hearing loss were collected. The proband and family members underwent clinical, genetic, and molecular evaluations, such as audiological examinations, mutational analysis of mitochondrial genome and mutational analysis of GJB2 gene., Results: Six people of this pedigree suffered from hearing loss, including four matrilineal members, and others did not have significant clinical abnormalities. Sequence analysis of the complete mitochondrial genome in the proband showed that there were 28 mtDNA polymorphisms belonging to East -Asian haplogroup A4.In addition to the A7551G homogeneity mutation, there were no other functionally significant variants found in this family. The A7551G mutation located immediately at the three prime end to the anticodon, corresponding with the conventional position 37 of tRNA(Asp), and its' CI value was 100% compared with other 15 primate species. The A7551G mutation was absent in other Chinese controls. The mutations on GJB2 were detected by direct sequence analysis,GJB2 235delC and 299delAT which was associated with hearing loss were found in the genomic DNA of the proband and some matrilineal members. Clinical evaluation showed a variable phenotype of severity, age-at-onset and audiometric configuration of hearing loss in the matrilineal relatives in these families., Conclusions: The A7551G mutation may modify the secondary structure of the tRNA, and affect the stabilization of tRNA(Asp), produce non-normal functional tRNA(Asp) ultimately. And it may cause the phenotypic manifestation of the deafness that associated with A7551G mutation. Therefore, the mitochondrial tRNA(Asp) A7551G mutation may be a new mitochondrial mutation for hearing loss.

Objective: To evaluate the effect of mitochondrial DNA(mtDNA) secondary mutations, haplotypes, GJB2 gene mutations on phenotype of 1494C>T mutation, and to study the molecular pathogenic mechanism of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss., Methods: Two Chinese Han pedigrees of maternally transmitted aminoglycoside induced and nonsyndromic hearing loss were collected. The two probands and their family members underwent clinical, genetic and molecular evaluations including audiological examinations and mutational analysis of mitochondrial genome and GJB2 gene., Results: Clinical evaluation revealed wide range of severity, age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in both families, for which the penetrance of hearing loss was respectively 42.9% and 28.6% when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss were 14.3% and 14.3%. Sequence analysis of mitochondrial genomes identified a known 12S rRNA 1494C>T mutation, in addition with distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroups C4a1a and B4b1c, respectively., Conclusion: Mitochondrial 12S rRNA 1494C>T mutation probably underlie the deafness in both families. Lack of significant mutation in the GJB2 gene ruled out involvement of GJB2 in the phenotypic expression. However, aminoglycosides and other nuclear modifier genes may still modify the phenotype of the 1494C>T mutation in these families. The B4b1c is a newly identified haplogroup in aminoglycoside-induced and nonsyndromic hearing loss family carrying the 1494C>T mutation. The 1494C>T mutation seems to have occurred sporadically through evolution.

We reported here the clinical, genetic, and molecular characterization of Leber's hereditary optic neuropathy (LHON) with C5601T mutation in seven Chinese families. The ophthalmologic examinations of seven Chinese families who were clinically diagnosed LHON were conducted. Strikingly, these families exhibited very low penetrance of visual impairment, and the penetrance was 9.5%, 14.3%, 4.5%, 8.3%, 10.0%, 22.2% and 25.0%. Meanwhile, entire mitochondrial genome of seven probands was amplified by PCR using 24 pairs of oligonucleotide primers with overlapping fragments. Molecular analysis of mitochondrial DNA (mtDNA) in these pedigrees revealed the absence of three common LHON associated G11778A, G3460A and T14484C mutations but the presence of homoplastic LHON associated tRNAAla C5601T mutation in probands and other matrilineal relatives. These mtDNA polymorphism sites belongs to the Asian haplogroups G2, G2a1, G2a1, G2, G2b, G2a1 and G2. By analyzing mitochondrial genome, seven LHON families all carry the C5601T mutation. The C5601T mutation occurs at the highly conserved nucleotide (conventional position 59) of tRNAAla, thereby contributing to the structural formation and stabilization of functional tRNAs and leading to mitochondrial dysfunction involved in visual impairment. The incomplete penetrance of visual loss in these seven Chinese pedigrees strongly indicates that the tRNAAla C5601T mutation was itself insufficient to produce a clinical phenotype. The lack of functional mtDNA variants in these pedigrees ruled out the role of mitochondrial background in the phenotypic expression of visual loss. Therefore, nuclear backgrounds and environmental factors seem to be modifying factors for the phenotypic manifestation of the tRNAAla C5601T mutation in the seven Chinese families.

Purpose: To investigate the pathophysiology of Leber's hereditary optic neuropathy (LHON)., Methods: Seventy-one subjects from three Chinese families with LHON underwent clinical, genetic, molecular, and biochemical evaluations. Biochemical characterizations included the measurements of the rates of endogenous, substrate-dependent respirations, the adenosine triphosphate (ATP) production and generation of reactive oxygen species using lymphoblastoid cell lines derived from five affected matrilineal relatives of these families and three control subjects., Results: Ten of 41 matrilineal relatives exhibited variable severity and age at onset of optic neuropathy. The average age at onset of optic neuropathy in matrilineal relatives of the three families was 5, 11, and 24 years, respectively. Molecular analysis identified the ND1 T3866C (I187T) mutation and distinct sets of polymorphisms belonging to the Eastern Asian haplogroups D4a, M10a, and R, respectively. The I187T mutation is localized at the highly conserved isoleucine at a transmembrane domain of the ND1 polypeptide. The marked reductions in the rate of endogenous, malate/glutamate-promoted and succinate/glycerol-3-phosphate-promoted respiration were observed in mutant cell lines carrying the T3866C mutation. The deficient respiration is responsible for the reduced ATP synthesis and increased generation of reactive oxygen species., Conclusions: Our data convincingly show that the ND1 T3866C mutation leads to LHON. This mutation may be insufficient to produce a clinical phenotype. Other modifier factors may contribute to the phenotypic manifestation of the T3866C mutation. The T3866C mutation should be added to the list of inherited factors for molecular diagnosis of LHON. Thus, our findings may provide new insights into the understanding of pathophysiology and valuable information on the management of LHON.

Mitochondrial DNA (mtDNA) mutations are one of the important causes of deafness. In particular, the 12S rRNA gene is the hot spots for mutations associated with both aminoglycoside ototoxicity and nonsyndromic deafness. In this report, a total of 318 Chinese pediatric hearing-impaired subjects were recruited from otology clinics in the Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and molecular analysis of 12S rRNA gene. Mutational analysis identified 34 variants in the 12S rRNA gene in this cohort. The incidences of the known deafness-associated 1555A>G, 1494C>T and 1095T>C mutations were 9.1%, 0.6% and 1.25% in this cohort, respectively. Other mtDNA variants were evaluated by structural and phylogenetic analysis. Of these, the 839A>G and 1452T>C variants could confer increased sensitivity to aminoglycosides or nonsyndromic deafness as they were not present in 449 Chinese controls and localized at highly conserved nucleotides of the 12S rRNA. However, other variants appeared to be polymorphisms. These data further support the idea that mitochondrial 12S rRNA is one of major targets for aminoglycoside ototoxicity. These data have been providing valuable information to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

Mitochondrial DNA (mtDNA) exhibits matrilineal inherence. Familial mitochondrial diseases caused by mtDNA mutations are generally involved in organs featuring high energy consumption, which include heart, brain and skeletal muscle. Recently, it has been found that some essential hypertension patients featured classical maternal inheritance, which has confirmed and enriched mtDNA mutations as one of the molecular mechanisms underlying maternally inherited hypertension. Nevertheless, more general as well as radical questions are still to be answered. This article reviews recent advance in mitochondrial genome evolution, mtDNA genetics and the role of mtDNA mutations in maternally inherited hypertension.

Mutations in mitochondrial DNA (mtDNA) have been associated with hypertension in several pedigrees with maternal inheritance. However, the pathophysiology of maternally inherited hypertension remains poorly understood. We reported here clinical, genetic evaluations and molecular analysis of mtDNA in a three-generation Han Chinese family with essential hypertension. Eight of 17 matrilineal relatives exhibited a wide range of severity in essential hypertension, whereas none of the offsprings of the affected father had hypertension. The age-at-onset of hypertension in the maternal kindred varied from 31 to 65 years, with an average of 52 years. Sequence analysis of mtDNA in this pedigree identified the known homoplasmic 4435A>G mutation, which is located at immediately 3' end to the anticodon, corresponding to the conventional position 37 of tRNA(Met), and 41 variants belonging to the Asian haplogroup G2a1. In contrast, the 4435A>G mutation occurred among mtDNA haplogroups B5a, D, M7a2 and J. The adenine (A37) at this position of tRNA(Met) is extraordinarily conserved from bacteria to human mitochondria. This modified A37 was shown to contribute to the high fidelity of codon recognition, structural formation and stabilization of functional tRNAs. However, 41 other mtDNA variants in this pedigree were the known polymorphisms. The occurrence of the 4435A>G mutation in two genetically unrelated families affected by hypertension indicates that this mutation is involved in hypertension. Our present investigations further supported our previous findings that the 4435A>G mutation acted as an inherited risk factor for the development of hypertension. Our findings will be helpful for counseling families of maternally inherited hypertension.

The authors report here the clinical, genetic, molecular and biochemical characterisation of a large five-generation Han Chinese pedigree with maternally transmitted non-syndromic hearing loss. 17 of 35 matrilineal relatives exhibited variable severity and age at onset of sensorineural hearing loss. The average age at onset of hearing loss in matrilineal relatives of this family is 29 years, while matrilineal relatives among families carrying other mitochondrial DNA mutations developed hearing loss with congenital conditions or early age at onset. Molecular analysis of their mitochondrial genome identified the novel heteroplasmic T12201C mutation in the transfer RNA (tRNA)(His) gene. The levels of T12201C mutation in matrilineal relatives of this family correlated with the severity and age at onset of non-syndromic hearing loss. By contrast, other heteroplasmic mitochondrial DNA mutations often cause syndromic hearing loss. The T12201C mutation destabilises a highly conservative base-pairing (5A-68U) on the acceptor stem of tRNA(His). tRNA northern analysis revealed that the T12201C mutation caused an ∼75% reduction in the steady-state level of tRNA(His). An in vivo protein labeling analysis showed an ∼47% reduction in the rate of mitochondrial translation in cells carrying the T12201C mutation. Impaired mitochondrial translation is apparently a primary contributor to the marked reduction in the rate of overall respiratory capacity, malate/glutamate-promoted respiration, succinate/glycerol-3-phosphate-promoted respiration or N,N,Ń,Ń-tetramethyl-p-phenylenediamine/ascorbate-promoted respiration. These data provide the first direct evidence that mitochondrial dysfunctions caused by the heteroplasmic tRNA(His) mutation lead to late-onset non-syndromic deafness. Thus, the authors' findings provide new insights into the understanding of pathophysiology and valuable information on the management and treatment of maternally inherited hearing loss.

Mutations in mitochondrial DNA (mtDNA) are one of the molecular bases of hypertension. Among these, the tRNAMet A4435G, tRNAMet/tRNAGln A4401G, tRNAIle A4263G, T4291C and A4295G mutations have been reported to be associated with essential hypertension. These mutations alter the structure of the corresponding mitochondrial tRNAs and cause failures in tRNA metabolism. These shortages of these tRNAs lead to an impairment of mitochondrial protein synthesis and a failure in the oxidative phosphorylation function. These result in a deficit in ATP synthesis and an increase of generation of reactive oxygen species. As a result, these mitochondrial dysfunctions may contribute to the development of hypertension. Furthermore, the tissue specificity of these pathogenic mtDNA mutations might be associated with tRNA metabolism and nuclear modifier genes. These mtDNA mutations should be considered as inherited risk factors for future molecular diagnosis. Thus, these findings provide new insights into the molecular mechanism, management and treatment of maternally inherited hypertension. This review summarized the association between mtDNA mutations and hypertension.

Purpose: To investigate the molecular pathogenesis of Leber's hereditary optic neuropathy (LHON) in Chinese families., Design: Six Han Chinese families who seem to have maternally transmitted LHON were studied by clinical, genetic, and molecular evaluations., Participants: One hundred twenty-seven subjects from 6 Chinese families with a wide range of age-at-onset and severity of visual impairment., Methods: All subjects underwent clinical examination, genetic evaluation, and molecular analysis of mitochondrial DNA (mtDNA)., Main Outcome Measures: The ophthalmologic examinations included visual acuity, visual field examination, visual evoked potentials, and fundus photography. The mtDNA analysis included the polymerase chain reaction (PCR) amplification of entire mtDNA and subsequent sequence determination., Results: Six families exhibited low penetrance of visual impairment, with an average of 10.8%. In particular, 9 (6 males/3 females) of 86 matrilineal relatives in these families exhibited variable severity and age at onset in visual dysfunction. The average age at onset of visual loss was 20 years. Molecular analysis of mtDNA in these families identified the homoplasmic ND5T12338C mutation and distinct set of variants belonging to the Asian haplogroup F2. The T12338C mutation is only present in the maternal lineage of those pedigrees and not in 178 Chinese controls. This mutation resulted in the replacement of the first amino acid, a translation-initiating methionine with a threonine, shortening 2 amino acids of ND5 polypeptide. The T12338C mutation is also located in 2 nucleotides adjacent to the 3' end of the tRNA(Leu(CUN)). Thus, this mutation may alter ND5 mRNA metabolism and the processing of RNA precursors. As a result, this mutation impairs respiratory function, leading to visual impairment., Conclusions: Several lines of evidence suggest that the mitochondrial ND5T12338C mutation is associated with LHON. The tissue specificity of this mutation is likely due to the involvement of nuclear modifier genes. The identification of nuclear modifiers is important for the elucidation of the pathogenic mechanism of LHON and an open avenue for therapeutic interventions. The T12338C mutation should be added to the list of inherited risk factors for future molecular diagnosis. Our findings are helpful for counseling families with LHON., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Leber's hereditary optic neuropathy (LHON) associated with mitochondrial DNA mutation is a maternally inherited eye disease. We reported here the clinical, genetic and molecular characterization of two Han Chinese families with Leber's hereditary optic neuropathy. Ophthalmologic examinations revealed that the variable severity and age-of-onset in visual impairment among probands and other matrilineal relatives of these families. Strikingly, there were extremely low penetrances of visual impairment in these families. Sequence analysis of complete mitochondrial genomes in these pedigrees identified the homoplasmic ND4 G11696A and ND5 T12338C mutation and distinct sets of polymorphism belonging to haplogroups F2. It is well known that mitochondrial DNA ND4 G11696A is associated with LHON. The ND5 T12338C mutation resulted in replacement of the first amino acid, translation-initiating methionine with a threonine, and shortening two amino acids of ND5. This mutation also locates in two nucleotides adjacent to the 3' end of the tRNALeu(Cun). Thus, this mutation may alter structural formation and stabilization of functional tRNA, thereby leading to a failure in protein synthesis and mitochondrial dysfunction involved in visual impairment. Therefore, the ND4 G11696A and ND5 T12338C mutation is likely associated with LHON in these two Chinese families. But these families exhibited extremely low penetrances of visual impairment. It suggests that other factors, such as nuclear modifier gene(s) or environmental factor(s), may play a role in the phenotypic expression of the LHON-associated ND4 G11696A and ND5 T12338C mutation.

Mutations in the mitochondrial DNA have been found to be one of the most important causes of sensorineural hearing loss. In particular, these mutations often occur in the mitochondrial 12S rRNA and tRNA genes. Of these, the homoplasmic A1555G and C1494T mutations in the 12S rRNA have been associated with both aminoglycoside induced and nonsyndromic hearing impairment in many families worldwide. Children carrying the A1555G or C1494T mutation are susceptible to the exposure of ototoxic drugs, thereby inducing or worsening hearing loss. Individuals harboring A1555G or C1494T mutation can also develop hearing loss even in the absence of aminoglycoside exposure. However, matrilineal relatives of intra-families or inter-families carrying the A1555G or C1494T mutation exhibit a wide range of severity, age-at-onset, and audiometric configuration of hearing impairment. These indicate that the A1555G or C1494T mutation is a primary factor underlying the development of deafness but insufficient to produce the clinical phenotype.Thus, other modifier factors, such as aminoglycoside(s), mitochondria l DNA haplotype(s) or nuclear modifier gene(s), play a role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA A1555G or C1494T mutation. In this review, we summarize the modifier factors for the phenotypic expression of deafness-associated 12S rRNA A1555G and C1494T mutations and propose the molecular pathogenetic mechanism of maternally inherited deafness.

Objective: Mitochondrial mutations have been shown to be responsible for syndromic as well as non-syndromic hearing loss. The G7444A mitochondrial DNA mutation affects COI/the precursor of tRNA(Ser(UCN)), encoding the first subunit of cytochrome oxidase. Here we report on the first Greek family with the G7444A mitochondrial DNA mutation., Methods: Clinical, cytogenetic, and molecular methods were employed in this study., Results: We describe the high variability of phenotypes among three family members harboring the G7444A mutation and also the frequent GJB2 c.35delG mutation of the nuclear genome in heterozygosity. Their phenotypes ranged from normal hearing to deafness, while the proband presented with several other symptoms., Conclusions: The G7444A mitochondrial DNA mutation has been reported in only a few cases worldwide, alone or in cosegregation with other mitochondrial DNA mutations, but to our knowledge, never before in coexistence with the GJB2 c.35delG mutation., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Purpose: The purpose of this study was to investigate the role of modifier factors in the expression of Leber's hereditary optic neuropathy (LHON)., Methods: Thirty-five subjects from two Han Chinese families with maternally transmitted LHON underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA., Results: Matrilineal relatives in the two Chinese families exhibited a wide range of severity in visual impairment, from blindness to nearly normal vision. Very strikingly, all nine affected individuals of 21 matrilineal relatives (13 females/8 males) were female, which translates to 33% and 57% of penetrance for optic neuropathy in the two families. The average age at onset was 22 and 25 years. These observations were in contrast with typical features in many LHON pedigrees that have a predominance of affected males. Molecular analysis of their mtDNAs identified the homoplasmic ND4 G11778A mutation and distinct sets of variants belonging to the Asian haplogroups M1 and M10a. Of other variants, the L175F variant in CO3; the I58V variant in ND6; and the I189V, L292R, and S297A variants in CYTB were located at highly conserved residues of polypeptides., Conclusions: Only female matrilineal relatives with a wide range of penetrance, severity, and age at onset of optic neuropathy in these two Chinese pedigrees showed the involvement of X-linked or autosomal recessive modifier genes in the phenotypic manifestation of the G11778A mutation. Furthermore, mitochondrial haplogroup-specific variants, together with epigenetic and environmental factors, may contribute to the phenotypic manifestation of the primary LHON-associated G11778A mutation in these pedigrees.

Mutations in mitochondrial DNA (mtDNA) have been found to be one of the most important causes of sensorineural hearing loss. We report here a clinical, genetic, molecular and biochemical characterization of a Han Chinese pedigree with maternally transmitted nonsyndromic hearing impairment. Seven of nine matrilineal relatives exhibited a variable severity and age-at-onset (8 years old) of hearing loss. Mutational analysis of mtDNA identified the novel homoplasmic tRNA(Ser(UCN)) 7505T>C mutation and other 37 variants belonging to haplogroup F1. The 7505T>C mutation, which is absent in 449 Chinese controls, is located at a highly conserved base-pairing (10A-20U) of tRNA(Ser(UCN)). The abolishment of 10A-20U base-pairing likely alters the tRNA(Ser(UCN)) metabolism. Functional significant of this mutation was supported by approximately 65% reductions in the level of tRNA(Ser(UCN)) observed in the lymphoblastoid cell lines carrying the 7505T>C mutation, compared with the wild-type cell lines. This reduced tRNA level is below the proposed threshold to support a normal respiration in lymphoblastoid cells. Furthermore, the highly conserved tRNA(Ala) 5587T>C and Cytb C93Y variants may have a modifying role of deafness expression associated with the 7505T>C mutation. However, genotyping analysis of nuclear modifier gene TRMU and the prominent deafness-cause gene GJB2 failed to detect any mutations in the member of this family. These data strongly indicate that the novel tRNA(Ser(UCN)) 7505T>C mutation is involved in maternally transmitted hearing loss. However, other genetic, epigenetic or environmental factors may contribute to the phenotypic variability of this family. Our findings will be helpful for counseling families of maternally inherited hearing loss., ((c) 2010 Elsevier Inc. All rights reserved.)

We report here the clinical, genetic, and molecular characterization of five Han Chinese families with Leber's hereditary optic neuropathy (LHON). Strikingly, there were very low penetrances of visual impairment in these Chinese families, ranging from 4.2% to 22.2%, with an average of 10.2%. In particular, only 7 (4 males/3 females) of 106 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The age-at-onset for visual impairment in matrilineal relatives in these families, varied from 20 to 25 years, with an average of 21.8 years old. Molecular analysis of mitochondrial genomes identified the homoplasmic ND1 G3460A mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, C4a1, D5, F1, and R9, respectively. This suggests that the G3640A mutation occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of known secondary LHON-associated mtDNA mutations in these Chinese families. Very low penetrance of visual loss in these five Chinese pedigrees strongly indicated that the G3640A mutation was itself insufficient to develop the optic neuropathy. The absence of secondary LHON mtDNA mutations suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the G3640A mutation in those Chinese families with low penetrance of vision loss. However, nuclear modifier genes, epigenetic and environmental factors appear to be modifier factors for the phenotypic manifestation of the G3640A mutation in these Chinese families., (Copyright 2009 Elsevier Inc. All rights reserved.)

Background: Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families., Methods: One hundred fourteen subjects from ten Han Chinese families with LHON were studied by the clinical and genetic evaluation as well as molecular and biochemical analyses of mitochondrial DNA (mtDNA)., Results: Clinical evaluation revealed that ten families exhibited extremely low penetrance of visual impairment, with an average of 10%. In particular, ten (8 males/2 females) of 114 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The average age-of-onset of vision loss was 19 years old. Molecular analysis of mitochondrial DNA (mtDNA) identified the homoplasmic T14484C mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, D4, D4g1b, G3a2, R11, R11a and Z3, respectively. However, there was the absence of secondary LHON-associated mtDNA mutations in these ten Chinese families., Conclusion: The low penetrance of vision loss in these Chinese pedigrees strongly indicated that the T14484C mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the T14484C mutation in those Chinese families with low penentrace of vision loss. However, nuclear modifier genes and environmental factors appear to be modifier factors for the phenotypic manifestation of the T14484C mutation in these Chinese families., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Mutations in mitochondrial DNA have been associated with a wide spectrum of clinical abnormalities. We reported here the clinical, genetic and molecular characterization of a five-generation Han Chinese pedigree with Leber's Hereditary Optic Neuropathy (LHON) and limbs abnormity claudication. Of 27 matrilineal relatives, four exhibited only LHON, one suffered from only limbs abnormity claudication, and four had both LHON and limbs abnormity claudication. Sequence analysis of mitochondrial genome in this family identified the known T3866C mutation in ND1 gene and other 43 variants belonging to the Asian haplogroup D4a3. The T3866C (I187T) mutation resulted in the replacement of isoleucine at position 187 with theronine. The isoleucine at position 187 located at one of transmembrane domain in ND1 polypeptide. The isoleucine at position 187 was extremely conserved among 29 organisms, while other variants showed no evolutionarily conservation. Furthermore, the T3866C was absence in 135 Chinese control subjects. The T3866C mutation likely alters the complex I activity, which causes mitochondrial dysfunction associated with LHON and limbs abnormity claudication. Therefore, the T3866C mutation is likely associated with LHON and limbs abnormity claudication.

Mitochondrial 12S rRNA 1555A>G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. Our previous investigations showed that the A1555G mutation was a primary factor underlying the development of deafness but was insufficient to produce deafness phenotype. However, it has been proposed that mitochondrial haplotypes modulate the phenotypic manifestation of the 1555A>G mutation. Here, we performed systematic and extended mutational screening of 12S rRNA gene in a cohort of 1742 hearing-impaired Han Chinese pediatric subjects from Zhejiang Province, China. Among these, 69 subjects with aminoglycoside-induced and nonsyndromic deafness harbored the homoplasmic 1555A>G mutation. These translated to a frequency of approximately 3.96% for the 1555A>G mutation in this hearing-impaired population. Clinical and genetic characterizations of 69 Chinese families carrying the 1555A>G mutation exhibited a wide range of penetrance and expressivity of hearing impairment. The average penetrances of deafness were 29.5% and 17.6%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 5 and 30years old, with the average of 14.5years. Their mitochondrial genomes exhibited distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups A, B, C, D, F, G, M, N, R and Y, respectively. These indicated that the 1555A>G mutation occurred through recurrent origins and founder events. The haplogroup D accounted for 40.6% of the patient's mtDNA samples but only 25.8% of the Chinese control mtDNA samples. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, the mitochondrial haplogroup specific variants: 15927G>A of haplogroup B5b, 12338T>C of haplogroup F2, 7444G>A of haplogroup B4, 5802T>C, 10454T>C, 12224C>T and 11696G>A of D4 haplogroup, 5821G>A of haplogroup C, 14693A>G of haplogroups Y2 and F, and 15908T>C of Y2 may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in nuclear modifier gene TRMU suggested that TRMU may not be a modifier for the phenotypic expression of the 1555A>G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes modulate the variable penetrance and expressivity of deafness among these Chinese families.

The mitochondrial 12S rRNA A1555G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we employed an RNA-directed chemical-modification approach to understanding the pathogenesis of aminoglycoside-induced hearing loss. The patterns of chemical modification of RNA oligonucleotides carrying the A1555G mutation by dimethyl sulfate (DMS) were distinct from those of the RNA oligonucleotides carrying wild-type sequence in the presence of aminoglycosides. In the RNA analogue carrying the A1555G mutation, reduced reactivity to DMS occurred in base G1555 as well as in bases C1556 and A1553 in the presence of paromomycin, neomycin, gentamicin, kanamycin, tobramycin, or streptomycin. In particular, base G1555 exhibited marked but similar levels of protection in the presence of 0.1 microM to 100 microM neomycin, gentamicin, or kanamycin. In contrast, the levels of protection in base G1555 appeared to be correlated with the concentration of paromycin, tobramycin, or streptomycin. Furthermore, increasing reactivities to DMS in the presence of these aminoglycosides were observed for bases A1492, C1493, C1494, and A1557 in the RNA analogue carrying the A1555G mutation. These data suggested that the A1555G mutation altered the binding properties of aminoglycosides at the A site of 12S rRNA and led to local conformational changes in 12S rRNA carrying the A1555G mutation. The interaction between aminoglycosides and 12S rRNA carrying the A1555G mutation provides new insight into the pathogenesis of aminoglycoside ototoxicity.

We report here the clinical, genetic and molecular characterization of four Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age-of-onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T3394C (Y30H) mutation, which localized at a highly conserved tyrosine at position 30 of ND1, and distinct sets of mtDNA polymorphisms belonging to haplogroups D4b and M9a. The occurrence of T3394C mutation in these several genetically unrelated subjects affected by visual impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. However, there was the absence of functionally significant mtDNA mutations in these four Chinese pedigrees carrying the T3394C mutation. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated T3394C mutation.

Mitochondrial DNA mutations have been associated with cardiovascular disease. We report here the clinical, genetic, and molecular characterization of 1 Han Chinese family with suggestively maternally transmitted hypertension. Matrilineal relatives in this family exhibited the variable degree of hypertension at the age at onset of 44 to 55 years old. Sequence analysis of entire mitochondrial DNA in this pedigree identified the known homoplasmic 4435A>G mutation, which is located immediately at the 3 prime end to the anticodon, corresponding with the conventional position 37 of tRNA(Met), and 35 other variants belonging to the Asian haplogroup B5a. The adenine (A37) at this position of tRNA(Met) is extraordinarily conserved from bacteria to human mitochondria. This modified A37 was shown to contribute to the high fidelity of codon recognition, the structural formation, and stabilization of functional tRNAs. In fact, a 40% reduction in the levels of tRNA(Met) was observed in cells carrying the 4435A>G mutation. As a result, a failure in mitochondrial tRNA metabolism, caused by the 4435A>G mutation, led to approximately 30% reduction in the rate of mitochondrial translation. However, the homoplasmic form, mild biochemical defect, and late onset of hypertension in subjects carrying the 4435A>G mutation suggest that the 4435A>G mutation itself is insufficient to produce a clinical phenotype. The other modifier factors, such as nuclear modifier genes, environmental, and personal factors may also contribute to the development of hypertension in the subjects carrying this mutation. Our findings imply that the 4435A>G mutation may act as an inherited risk factor for the development of hypertension in this Chinese pedigree.

Mutations in mitochondrial DNA have been associated with a wide spectrum of clinical abnormalities. We reported here the clinical and genetic evaluations as well as mutational analysis of mitochondrial DNA(mtDNA) in a three-generation Chinese Han family with maternally transmitted hearing loss and epilepsy. Of 14 matrilineal relatives, three suffered from hearing loss, three had epilepsy, and other did not have significant clinical abnormalities. Sequence analysis of mitochondrial genome in this family identified the novel 7472delC in tRNASer(UCN) and 33 variants belonging to Asian haplogroup B4b1a2. The 7472delC locates at the highly conserved residue of T-arm of this tRNA. In fact, the 7472insC at the same position of this tRNA has been associated with hearing loss and epilepsy in several genetically unrelated families. The 7472insC has been shown to lead to a failure in tRNA metabolism and mitochondrial dysfunction. Thus, 7472delC mutation, similar to 7472insC mutation, may result in the mitochondrial dysfunctions responsible for the hearing loss and epilepsy. Furthermore, none of mutation in deafness-associated GJB2 gene was detected in this Chinese family. Therefore, the 7472delC is likely a new mitochondrial mutation with hearing loss and epilepsy.

We report here the clinical, genetic and molecular characterization of three Chinese pedigrees with nonsyndromic bilateral hearing loss. Clinical and genetic evaluations revealed the variable severity and age-of-onset in hearing impairment in these families. Strikingly, there were extremely low penetrances of hearing impairment in these Chinese families. Sequence analysis of the complete mitochondrial DNA (mtDNA) showed the known A7445C mutation in two pedigrees and the novel A7445T mutation in another pedigree, in addition to distinct sets of mtDNA polymorphisms belong to Asian haplogroups D4j and F4. Indeed, the A7445C or A7445T mutation in the CO1 and the precursor of tRNA(Ser(UCN)) genes was present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families and 164 Chinese controls. In fact, the A7445C or A7445T mutation results in a read-through of the stop condon AGA of the CO1 message on the H strand of mtDNA, thereby adding three amino acids (Ser-Gln-Lys) to the C-terminal of the polypeptide. However, the mutated polypeptide may retain a partial function. Alternatively, the A7445C or A7445T mutation is adjacent to the site of 3' end endonucleolytic processing of L-strand RNA precursor, spanning tRNA(Ser(UCN)) and ND6 mRNA. Thus, the A7445C or A7445T mutation may also cause a defect in the processing of the L-strand RNA precursor, thus causing mitochondrial dysfunctions. Furthermore, the occurrence of the mutations at position 7445 in these genetically unrelated subjects affected by hearing impairment strongly indicates that mutations at the position 7445 are involved in the pathogenesis of hearing impairment.

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age-at-onset, audiometric configuration in these subjects. The penetrance of hearing loss in WZD8, WZD9, and WZD10 pedigrees were 46%, 46%, and 50%, respectively, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrance of hearing loss in these pedigrees were 23%, 31%, and 37.5%, respectively. Mutational analysis of the complete mitochondrial genomes showed the homoplasmic A1555G mutation and distinct sets of mitochondrial DNA variants belonging to haplogroups D4b2b, B5b1, and F2, respectively. Of these, the tRNA(Cys) T5802C, tRNA(Thr) A15924C, and ND5 T12338C variants are of special interest as these variants occur at positions which are highly evolutionarily conserved nucleotides of tRNAs or amino acid of polypeptide. These homoplasmic mtDNA variants were absent among 156 unrelated Chinese controls. The T5802C and G15927A variants disrupted a highly conserved A-U or C-G base-pairing at the anticodon-stem of tRNA(Cys) or tRNA(Thr), while the ND5 T12338C mutation resulted in the replacement of the translation-initiating methionine with a threonine, and also located in two nucleotides adjacent to the 3' end of the tRNA(Leu(CUN)). Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA variants. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees., (2008 Wiley-Liss, Inc.)

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder leading to rapid, painless, bilateral and usually permanent central vision loss in young adults, males are preferentially affected. The maternal transmission of this visual dysfunction in LHON families suggested that mutations in the mitochondrial DNA (mtDNA) are the molecular bases of the disorder. The ND1 G3460A, ND4 G11778A and ND6 T14484C mutations in the genes encoding the subunits of respiratory chain complex I, account for more than 50% of LHON families worldwide. These three mutations are designated to be primary mutations because they impart a high risk for LHON expression. However, matrilineal relatives within and among families, despite carrying the same LHON-associated mtDNA mutation(s), exhibit a wide range of onset, severity, and the progression of visual impairment. These findings strongly indicated that the LHON-associated primary mutation(s) are the primary factors underlying the development of vision loss, but they themselves are insufficient to produce a clinic phenotype. The prone to male, incomplete penetrance, and phenotypic variability of vision loss suggest that other modifier factors including personal factors, environmental factors, nuclear modifier genes and mitochondrial haplotypes contribute to the phenotypic expression of these mtDNA mutations. In particular, the mitochondrial haplotypes may play a synergic role in the development of vision loss in the families carrying the LHON-associated primary mtDNA mutation(s).

Objective: To report the clinical, genetic, and molecular characterization of two Chinese families with Leber's hereditary optic neuropathy (LHON)., Methods: Ophthalmological examinations showed that only probands in two families exhibited visual loss at the age of 10 and 17 years respectively. The entire mitochondrial genome of two probands was PCR amplified in 24 overlapping fragments using sets of oligonucleotide primers., Results: Mutational analysis of mitochondrial DNA (mtDNA) in these pedigrees revealed the absence of three common LHON associated G11778A, G3460A and T144484 mutations but the presence of homoplastic LHON associated ND4 G11696A mutation, which was present in one out of 167 Chinese healthy controls., Conclusion: Sequence analysis of the complete mitochondrial genomes in two pedigrees showed the distinct sets of mtDNA polymorphisms, belonging to Eastern Asian haplogroup D4. The incomplete penetrance of visual loss and the presence of one in 167 controls suggested that this mutation itself is insufficient to produce a clinical phenotype and other modifier factors play a role in the phenotypic manifestation. The lack of functional mtDNA variants in these pedigrees ruled out the role of mitochondrial background in the phenotypic expression of visual loss. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated G11696A mutation in two Chinese pedigrees.

We report here the clinical, genetic and molecular characterization of one three-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON) and hearing loss. Four of 14 matrilineal relatives exhibited the moderate central vision loss at the average age of 12.5 years. Of these, one subject exhibited both LHON and mild hearing impairment. Sequence analysis of the complete mitochondrial genomes in the pedigree showed the presence of homoplasmic LHON-associated ND6 T14484C mutation, deafness-associated 12S rRNA A1555 mutation and 47 other variants belonging to Eastern Asian haplogroup H2. None of other mitochondrial variants was evolutionarily conserved and functional significance. Therefore, the coexistence of the A1555G mutation and T14484C mutations in this Chinese family indicate that the A1555G mutation may play a synergistic role in the phenotypic manifestation of LHON associated ND6 T14484C mutation. However, the incomplete penetrance of vision and hearing loss suggests the involvement of nuclear modifier genes and environmental factors in the phenotypic expression of these mtDNA mutations.

We report here the clinical, genetic, and molecular characterization of one Han Chinese family with maternally transmitted Leber's hereditary optic neuropathy (LHON). Three of seven matrilineal relatives in this family exhibited the variable degree of central vision loss at the age of 12, 14, and 16 years old, respectively. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the ND1 G3460A mutation and 47 other variants, belonging to the Asian haplogroup M7b2. The G3460A mutation is present at homoplasmy in matrilineal relatives of this Chinese family. Of other variants, the homoplasmic A14693G mutation is of special interest as it was implicated to be associated with other mitochondrial disorders. This mutation is located at the TpsiC-loop, at conventional position 54 of tRNA(Glu). The uridine at this position (U54), which is highly conserved from bacteria to human mitochondria, has been implicated to be important for tRNA structure and function. Thus, the A14693G mutation may alter the tertiary structure of this tRNA, cause a failure in this tRNA metabolism, thereby worsening the mitochondrial dysfunction associated with the primary G3460A mutation. Therefore, the tRNA(Glu) A14693G mutation may have a potential modifier role in the phenotypic manifestation of the primary LHON-associated G3460A mutation in this Chinese family.

We report here the characterization of a four-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON). This Chinese family exhibited a variable severity and age-at-onset of visual loss. Notably, the average age-at-onset of vision impairment changed from 26 years (generation III) to 14 years (generation IV), with the average of 18 years in this family. In addition, 30% and 50% of matrilineal relatives in generation III and IV of this family developed visual loss with a variability of severity, ranging from blindness to normal vision. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the homoplasmic ND4 G11778A mutation and 33 other variants, belonging to the Asian haplogroup D4. Of other variants, the homoplasmic G11696A mutation in the ND4 gene is of special interest as it was implicated to be associated with LHON in a large Dutch family and five Chinese pedigrees with extremely penetrance of visual loss. In fact, the G11696A mutation caused the substitution of an isoleucine for valine at amino acid position 313, located in a predicted transmembrane region of ND4. These imply that the G11696A mutation may act in synergy with the primary LHON-associated G11778A mutation in this Chinese pedigree.