Dominant optic atrophy (DOA)1,2 and axonal peripheral neuropathy (Charcot-Marie-Tooth Type 2 or CMT2)3 are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively4. In yeast, homologs of OPA1(Mgm1) and MFN2(Fzo1) work in concert with Ugo15,6, which has no human equivalent to date7. By whole exome sequencing patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein, mitofilin(Fcj1). Loss-of-function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2, while exemplifying a novel class of modified solute transporters linked to mitochondrial dynamics.

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families shows biparental mtDNA transmission with an autosomal dominantlike inheritance mode. Our results suggest that, although the central dogma of maternal inheritance of mtDNA remains valid, there are some exceptional cases where paternal mtDNA could be passed to the offspring. Elucidating the molecular mechanism for this unusual mode of inheritance will provide new insights into how mtDNA is passed on from parent to offspring and may even lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells, we showed ∼70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

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