A homozygous mutation of TWNK identified in premature ovarian insufficiency warns of late-onset perrault syndrome.

BACKGROUND: Primary ovarian insufficiency (POI) is defined as cessation of ovarian function before the age of 40 years, which is characterized by amenorrhoea, infertility, elevated gonadotrophin level and sex-steroid deficiency. The phenotypes of POI are heterogeneous, including isolated and syndromic forms. Perrault syndrome (PS), characterized by sensorineural hearing loss (SNHL) and ovarian dysfunction before 40 years in females, is one type of syndromic POI. Genetic defects play a vital role in the pathogenesis of POI. METHODS AND RESULTS: To illustrate the genetic causation of Perrault syndrome, we performed whole exome sequencing (WES) in one pedigree with the disease, and identified a novel homozygous mutation in TWNK (c.1388G > A, p.R463Q). TWNK encodes a hexameric DNA helicase in mitochondria and plays a critical role in mtDNA replication. In order to determine the effect of the novel mutation on the mitochondrial function, we generated immortalized cell lines by infecting lymphocytes from the family members with EB virus in vitro. Functional studies found that TWNK p.R463Q impaired mtDNA replication and the respiration potential of mitochondria, while the ROS level remains unaffected. CONCLUSION: Our study provided evidence that TWNK mutation impaired the ovarian function by dysfunctional mitochondria. Moreover, considering the patients here presented POI onset earlier than SNHL, specific variants localizing in different locus of TWNK might induce heterogeneous phenotypes, indicating that the genetic screening of patients with POI would be useful for early recognition of other disease or other phenotypes of syndromic POI.

Clinical and genetic analysis of essential hypertension with mitochondrial tRNAMet 4435A>G and YARS2 mutation. / 携带线粒体tRNAMet 4435A>Gå’Œæ ¸åŸºå› YARS2çªå˜çš„åŽŸå‘æ€§é«˜è¡€åŽ‹å®¶ç³»é—ä¼ å­¦åˆ†æž.

OBJECTIVES: To investigate the role of m.4435A>G and YARS2 c.572G>T (p.G191V) mutations in the development of essential hypertension. METHODS: A hypertensive patient with m.4435A>G and YARS2 p.G191V mutations was identified from previously collected mitochondrial genome and exon sequencing data. Clinical data were collected, and a molecular genetic study was conducted in the proband and his family members. Peripheral venous blood was collected, and immortalized lymphocyte lines constructed. The mitochondrial transfer RNA (tRNA), mitochondrial protein, adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) in the constructed lymphocyte cell lines were measured. RESULTS: Mitochondrial genome sequencing showed that all maternal members carried a highly conserved m.4435A>G mutation. The m.4435A>G mutation might affect the secondary structure and folding free energy of mitochondrial tRNA and change its stability, which may influence the anticodon ring structure. Compared with the control group, the cell lines carrying m.4435A>G and YARS2 p.G191V mutations had decreased mitochondrial tRNA homeostasis, mitochondrial protein expression, ATP production and MMP levels, as well as increased ROS levels (all P<0.05). CONCLUSIONS: The YARS2 p.G191V mutation aggravates the changes in mitochondrial translation and mitochondrial function caused by m.4435A>G through affecting the steady-state level of mitochondrial tRNA and further leads to cell dysfunction, indicating that YARS2 p.G191V and m.4435A>G mutations have a synergistic effect in this family and jointly participate in the occurrence and development of essential hypertension.

Deafness-associated tRNAPhe mutation impaired mitochondrial and cellular integrity.

Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAPhe 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNAPhe structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fission with abnormal mitochondrial morphology. Excessive fission impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our findings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity.

Deficient tRNA posttranscription modification dysregulated the mitochondrial quality controls and apoptosis.

Mitochondria are dynamic organelles in cellular metabolism and physiology. Mitochondrial DNA (mtDNA) mutations are associated with a broad spectrum of clinical abnormalities. However, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we demonstrated that mt-tRNAMet 4435A>G mutation-induced nucleotide modification deficiency dysregulated the expression of nuclear genes involved in cytosolic proteins involved in oxidative phosphorylation system (OXPHOS) and impaired the assemble and integrity of OXPHOS complexes. These dysfunctions caused mitochondrial dynamic imbalance, thereby increasing fission and decreasing fusion. Excessive fission impaired the process of autophagy including initiation phase, formation, and maturation of autophagosome. Strikingly, the m.4435A>G mutation upregulated the PARKIN dependent mitophagy pathways but downregulated the ubiquitination-independent mitophagy. These alterations promoted intrinsic apoptotic process for the removal of damaged cells. Our findings provide new insights into mechanism underlying deficient tRNA posttranscription modification regulated intracellular signaling related to the mitochondrial and cellular integrity.

Induced pluripotent stem cells: ex vivo models for human diseases due to mitochondrial DNA mutations.

Mitochondria are essential organelles for cellular metabolism and physiology in eukaryotic cells. Human mitochondria have their own genome (mtDNA), which is maternally inherited with 37 genes, encoding 13 polypeptides for oxidative phosphorylation, and 22 tRNAs and 2 rRNAs for translation. mtDNA mutations are associated with a wide spectrum of degenerative and neuromuscular diseases. However, the pathophysiology of mitochondrial diseases, especially for threshold effect and tissue specificity, is not well understood and there is no effective treatment for these disorders. Especially, the lack of appropriate cell and animal disease models has been significant obstacles for deep elucidating the pathophysiology of maternally transmitted diseases and developing the effective therapy approach. The use of human induced pluripotent stem cells (iPSCs) derived from patients to obtain terminally differentiated specific lineages such as inner ear hair cells is a revolutionary approach to deeply understand pathogenic mechanisms and develop the therapeutic interventions of mitochondrial disorders. Here, we review the recent advances in patients-derived iPSCs as ex vivo models for mitochondrial diseases. Those patients-derived iPSCs have been differentiated into specific targeting cells such as retinal ganglion cells and eventually organoid for the disease modeling. These disease models have advanced our understanding of the pathophysiology of maternally inherited diseases and stepped toward therapeutic interventions for these diseases.

Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation. METHODS: Nucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays. RESULTS: The transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function. CONCLUSION: We demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases.

Clinical and genetic analysis of essential hypertension with CYB gene m.15024G>A mutation. / 携带CYB m.15024G>Açªå˜çš„åŽŸå‘æ€§é«˜è¡€åŽ‹å®¶ç³»é—ä¼ å­¦åˆ†æž.

OBJECTIVES: To explore the role of mitochondrial CYB 15024G>A mutation in the development of essential hypertension. METHODS: Mitochondrial genome sequences of hypertensive patients were obtained from previous studies. Clinical and genetic data of a hypertensive patient with mitochondrial CYB 15024G>A mutation and its pedigree were analyzed. Lymphocytes derived from patient and family members were transformed into immortalized lymphoblastoid cell lines, and the levels of adenosine triphosphate (ATP), mitochondrial membrane potential and intracellular reactive oxygen species (ROS) were detected. RESULTS: The penetrance of this essential hypertension family was 42.9%, and the age of onset was 46-68 years old. Mitochondrial genome sequencing results showed that all maternal members carried a highly conserved mitochondrial CYB 15024G>A mutation. This mutation could affect the free energy of mitochondrial CYB for secondary and tertiary structure and protein folding, thereby changing its structural stability and the structure of the electron transfer function area around the mutation site. Compared with the control, the cell line carrying the mitochondrial CYB 15024G>A mutation showed significantly decreased levels of mitochondrial CYB, ATP and mitochondrial membrane potential, and increased levels of ROS (P<0.01). CONCLUSIONS: Mitochondrial CYB 15024G>A mutation may affect the structure of respiratory chain subunits and mitochondrial function, leading to cell dysfunction, which suggests that the mutation may play a synergistic role in essential hypertension.

cdh23 affects congenital hearing loss through regulating purine metabolism.

Introduction: The pathogenic gene CDH23 plays a pivotal role in tip links, which is indispensable for mechanoelectrical transduction in the hair cells. However, the underlying molecular mechanism and signal regulatory networks that influence deafness is still largely unknown. Methods: In this study, a congenital deafness family, whole exome sequencing revealed a new mutation in the pathogenic gene CDH23, subsequently; the mutation has been validated using Sanger sequencing method. Then CRISPR/Cas9 technology was employed to knockout zebrafish cdh23 gene. Startle response experiment was used to compare with wide-type, the response to sound stimulation between wide-type and cdh23-/-. To further illustrate the molecular mechanisms underlying congenital deafness, comparative transcriptomic profiling and multiple bioinformatics analyses were performed. Results: The YO-PRO-1 assay result showed that in cdh23 deficient embryos, the YO-PRO-1 signal in inner ear and lateral line neuromast hair cells were completely lost. Startle response experiment showed that compared with wide-type, the response to sound stimulation decreased significantly in cdh23 mutant larvae. Comparative transcriptomic showed that the candidate genes such as atp1b2b and myof could affect hearing by regulating ATP production and purine metabolism in a synergetic way with cdh23. RT-qPCR results further confirmed the transcriptomics results. Further compensatory experiment showed that ATP treated cdh23-/- embryos can partially recover the mutant phenotype. Conclusion: In conclusion, our study may shed light on deciphering the principal mechanism and provide a potential therapeutic method for congenital hearing loss under the condition of CDH23 mutation.

Nuclear modifier YARS2 allele correction restored retinal ganglion cells-specific deficiencies in Leber's hereditary optic neuropathy.

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.

Abnormal morphology and function in retinal ganglion cells derived from patients-specific iPSCs generated from individuals with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease that results from degeneration of retinal ganglion cells (RGC). Mitochondrial ND4 11778G > A mutation, which affects structural components of complex I, is the most prevalent LHON-associated mitochondrial DNA (mtDNA) mutation worldwide. The m.11778G > A mutation is the primary contributor underlying the development of LHON and X-linked PRICKLE3 allele (c.157C > T, p.Arg53Trp) linked to biogenesis of ATPase interacts with m.11778G > A mutation to cause LHON. However, the lack of appropriate cell and animal models of LHON has been significant obstacles for deep elucidation of disease pathophysiology, specifically the tissue-specific effects. Using RGC-like cells differentiated from induced pluripotent stem cells (iPSCs) from members of one Chinese family (asymptomatic subjects carrying only m.11778G > A mutation or PRICKLE3 p.Arg53Trp mutation, symptomatic individuals bearing both m.11778G > A and PRICKLE3 p.Arg53Trp mutations and control lacking these mutations), we demonstrated the deleterious effects of mitochondrial dysfunctions on the morphology and functions of RGCs. Notably, iPSCs bearing only m.11778G > A or p.Arg53Trp mutation exhibited mild defects in differentiation to RGC-like cells. The RGC-like cells carrying only m.11778G > A or p.Arg53Trp mutation displayed mild defects in RGC morphology, including the area of soma and numbers of neurites, electrophysiological properties, ATP contents and apoptosis. Strikingly, those RGC-like cells derived from symptomatic individuals harboring both m.11778G > A and p.Arg53Trp mutations displayed greater defects in the development, morphology and functions than those in cells bearing single mutation. These findings provide new insights into pathophysiology of LHON arising from RGC deficiencies caused by synergy between m.11778G > A and PRICKLE3 p.Arg53Trp mutation.

Emerging functions of mitochondria-encoded noncoding RNAs.

Mitochondria, organelles that harbor their own circular genomes, are critical for energy production and homeostasis maintenance in eukaryotic cells. Recent studies discovered hundreds of mitochondria-encoded noncoding RNAs (mt-ncRNAs), including novel subtypes of mitochondria-encoded circular RNAs (mecciRNAs) and mitochondria-encoded double-stranded RNAs (mt-dsRNAs). Here, we discuss the emerging field of mt-ncRNAs by reviewing their expression patterns, biogenesis, metabolism, regulatory roles, and functional mechanisms. Many mt-ncRNAs have regulatory roles in cellular physiology, and some are associated with, or even act as, causal factors in human diseases. We also highlight developments in technologies and methodologies and further insights into future perspectives and challenges in studying these noncoding RNAs, as well as their potential biomedical applications.

Exportin 4 depletion leads to nuclear accumulation of a subset of circular RNAs.

Numerous RNAs are exported from the nucleus, abnormalities of which lead to cellular complications and diseases. How thousands of circular RNAs (circRNAs) are exported from the nucleus remains elusive. Here, we provide lines of evidence to demonstrate a link between the conserved Exportin 4 (XPO4) and nuclear export of a subset of circRNAs in metazoans. Exonic circRNAs (ecircRNAs) with higher expression levels, larger length, and lower GC content are more sensitive to XPO4 deficiency. Cellular insufficiency of XPO4 leads to nuclear circRNA accumulation, circRNA:DNA (ciR-loop) formation, linear RNA:DNA (liR-loop) buildup, and DNA damage. DDX39 known to modulate circRNA export can resolve ciR-loop, and splicing factors involved in the biogenesis of circRNAs can also affect the levels of ciR-loop. Testis and brain are two organs with high abundance of circRNAs, and insufficient XPO4 levels are detrimental, as Xpo4 heterozygous mice display male infertility and neural phenotypes. Increased levels of ciR-loop, R-loop, and DNA damage along with decreased cell numbers are observed in testis and hippocampus of Xpo4 heterozygotes. This study sheds light on the understandings of mechanism of circRNA export and reveals the significance of efficient nuclear export of circRNAs in cellular physiology.

Human TRUB1 is a highly conserved pseudouridine synthase responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

Pseudouridine (Ψ) at position 55 in tRNAs plays an important role in their structure and function. This modification is catalyzed by TruB/Pus4/Cbf5 family of pseudouridine synthases in bacteria and yeast. However, the mechanism of TRUB family underlying the formation of Ψ55 in the mammalian tRNAs is largely unknown. In this report, the CMC/reverse transcription assays demonstrated the presence of Ψ55 in the human mitochondrial tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). TRUB1 knockout (KO) cell lines generated by CRISPR/Cas9 technology exhibited the loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro but did not affect other 18 mitochondrial tRNAs. An in vitro assay revealed that recombinant TRUB1 protein can catalyze the efficient formation of Ψ55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient Ψ55 modifications in these tRNAs in TRUB1KO HeLa cells. TRUB1 deficiency affected the base-pairing (18A/G-Ψ55), conformation and stability but not aminoacylation capacity of these tRNAs. Furthermore, TRUB1 deficiency impacted mitochondrial translation and biogenesis of oxidative phosphorylation system. Our findings demonstrated that human TRUB1 is a highly conserved mitochondrial pseudouridine synthase responsible for the Ψ55 modification in the mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism.

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.

Leber's hereditary optic neuropathy-associated ND6 14484T > C mutation caused pleiotropic effects on the complex I, RNA homeostasis, apoptosis and mitophagy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14484T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484T > C mutation in the pathogenesis of LHON.

Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier.

The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.

Mitochondrial tRNA variants in 811 Chinese probands with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is the maternal inheritance of eye disorder. LHON-linked mitochondrial DNA (mtDNA) mutations affect the ND1, ND4 or ND6 genes encoding essential subunits of complex I. However, the role of mitochondrial tRNA defects in the pathogenesis of LHON is poorly understood. In this report, Sanger sequence analysis of 22 mitochondrial tRNA genes identified 139 variants in a cohort of 811 Han Chinese probands and 485 control Chinese subjects. Among these, 32 (4 known and 28 novel/putative) tRNA variants in 71 probands may contribute to pathogenesis of LHON, as these exhibited (1) present in < 1% of controls; (2) evolutionary conservation; (3) potential and significance of structural and functional modifications. Such variants may have potentially compromised structural and functional aspects in the processing of tRNAs, structure stability, tRNA charging, or codon-anticodon interactions during translation. These 32 variants presented either singly or with multiple mutations, with the primary LHON-linked ND1 3640G > A, ND4 11778G > A or ND6 14484 T > C mutations in the probands. The thirty-eight pedigrees carrying only one of tRNA variants exhibited relatively low penetrances of LHON, ranging from 5.7% to 42.9%, with an average of 19%. Strikingly, the average penetrances of optic neuropathy among 33 Chinese families carrying both a known/putative tRNA variant and a primary LHON-associated mtDNA mutation were 40.1%. These findings suggested that mitochondrial tRNA variants represent a significant causative factor for LHON, accounting for 8.75% cases in this cohort. These new insights may lead to beneficial applications in the pathophysiology, disease management, and genetic counseling of LHON.

Heteroplasmic and homoplasmic m.616T>C in mitochondria tRNAPhe promote isolated chronic kidney disease and hyperuricemia.

Inherited kidney diseases are the fifth most common cause of end-stage renal disease (ESRD). Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mitochondrial-transfer RNA (mt-tRNA) variants and their pathogenic contributions to kidney disease remain largely unclear. In this study, we identified the pathogenic mt-tRNAPhe 616T>C mutation in 3 families and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, 1 proband with homoplamic m.616T>C presented ESRD as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m.616T>C exhibited swollen mitochondria, underwent active mitophagy, and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial ROS. Pathogenic m.616T>C abolished a highly conserved base pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mtDNA-encoded polypeptides. Collectively, these findings provide potentially new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.

Genetic correction of TRMU allele restored the mitochondrial dysfunction-induced deficiencies in iPSCs-derived hair cells of hearing-impaired patients.

Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A>G mutation has been associated with hearing loss in many families. The m.1555A>G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G>T, p.Ala10Sser) encoding tRNA thiouridylase interact with m.1555A>G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A>G mutation, symptomatic individual bearing both m.1555A>G and c.28G>T mutations, and control subject). The c.28G>T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A>G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A>G and TRMU c.28G>T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A>G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G>T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease.

Verification of an iPSC line (LZUi002-A) from a patient with a novel mutation in the TBL1X gene.

More than 120 genes have been reported to be associated with deafness, and deletion of the TBL1X gene may cause deafness in humans. In this study, we generated an induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a 34-year-old deaf person with a novel variant c.342_343insGCGGCG in the TBL1X gene. The induced patient-specific iPSC line with a normal karyotype and expressed pluripotent markers, it also shows differentiation totipotency and tridermogenesis in vivo. It may be a good model for studying hearing loss in vitro and it will benefit to the development of new therapies for deafness.