mtDNA Disorders Research Articles

Index of Suspicion

The reader is encouraged to write possible diagnoses for each case before turning to the discussion. We invite readers to contribute case presentations and discussions. Please inquire first by contacting Dr. Deepak Kamat at DKamat@med.wayne.edu. A 2-year-old boy with a history of eczema and food allergies to nuts and soy presents to the emergency department with a 10-day history of generalized edema. The swelling began in the periorbital area but soon progressed to involve his entire body. There have been no fevers or any recent illnesses other than 1 day of cough and a few watery stools. He has had no new exposures to foods or other substances. His family history is remarkable for multiple allergies and eczema. Physical examination reveals a temperature of 98.8°F (37.1oC), heart rate of 98 beats per minute, respiratory rate of 36 breaths per minute, blood pressure of 103/65 mm Hg, and an oxygen saturation of 97% on room air. His weight is 12.6 kg (34th percentile). He is noted to have a moderate amount of periorbital edema, decreased breath sounds at the lung bases bilaterally, a protuberant abdomen, and 2+ pitting edema on his lower extremities. The rest of his physical examination findings are within normal limits. Laboratory evaluation reveals a white blood cell count of 23,800/μL (23.8 × 109/L), with 37% neutrophils, 49% lymphocytes, 4% bands, 7% monocytes, and 3% eosinophils; hemoglobin level of 13.3 g/dL (133 g/L); and platelet count of 427 × 103/μL (427 × 109/L). Serum electrolyte, blood urea nitrogen, and creatinine levels are normal. Sodium level is 132 mEq/L (132 mmol/L). Serum albumin level is 1.7 g/dL (17 g/L) (reference range, 3.4-5.4 g/dL [34-54 g/L]). Total protein level is 3.8 g/dL (38 g/L). Aspartate aminotransferase, alanine aminotransferase, and coagulation profiles …

Near-Identical Segregation of mtDNA Heteroplasmy in Blood, Muscle, Urinary Epithelium, and Hair Follicles in Twins With Optic Atrophy, Ptosis, and Intractable Epilepsy

Mitochondrial DNA (mtDNA) disorders have emerged as major causes of inherited neurologic disease. Despite being well recognized for more than 2 decades, the clinical presentation continues to broaden. The phenotypic heterogeneity is partly owing to different percentage levels of mutant mtDNA heteroplasmy in different tissues, but the factors influencing this are poorly understood. This case report describes monozygotic male twins with ptosis, optic atrophy, and recent-onset intractable myoclonic epilepsy. The assessment of respiratory chain enzyme activities in the muscle from 1 twin revealed a severe and isolated defect involving mitochondrial complex I. Mitochondrial DNA sequencing revealed a pathogenic m.14487T>C MTND6 mutation, which was present at very high levels of heteroplasmy in muscle (84%) and lower levels in blood (15%), urinary epithelium (75%), and buccal mucosa (58%). Of particular interest, his identical twin was found to harbor very similar levels of the m.14487T>C mutation in his blood, urine, buccal mucosa, and hair follicle DNA samples, while the presence of low levels in the mother's tissues confirmed maternal transmission. It was shown that m14487T>C can also cause the unusual combination of optic atrophy, ptosis, and encephalomyopathy leading to intractable seizures. Near-identical heteroplasmy levels in different tissues in both siblings support a nuclear genetic mechanism controlling the tissue segregation of mtDNA mutations.

Induced Pluripotent Stem Cells with a Mitochondrial DNA Deletion

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here, we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases.

Preimplantation genetic diagnosis in mitochondrial DNA disorders: challenge and success

BackgroundMitochondrial or oxidative phosphorylation diseases are relatively frequent, multisystem disorders; in about 15% of cases they are caused by maternally inherited mitochondrial DNA (mtDNA) mutations. Because of the possible severity...

Пути сохранения целостности митохондриальной ДНК и функций митохондрий в клетках, подвергшихся воздействию ионизирующей радиации

The analytical review deals with the results of studies devoted to mitochondrial DNA (mtDNA) disorders, the development of oxidative stress and possible pathways for the maintenance of mitochondrial functions in cells exposed to ionizing radiation (IR). Mitochondrial functions, which are closely related to the integrity of mtDNA, play a key role in many cellular processes. A wide range of degenerative diseases, carcinogenesis, and aging is associated with disturbances in mtDNA. MtDNA and the mitochondrion as a whole are increasingly considered as sensitive targets for cancer radio-chemotherapy. Knowledge of post-radiation processes in the mitochondria also facilitates creation of possible additional ways to reduce the radiation reaction of the organism. Injuries and mutations in mtDNA occur with a greater frequency than in the nuclear DNA (nDNA) in cells exposed to IR and other genotoxicants. On the other hand, functionally active copies of mtDNA can persist and survive in the cells exposed to clinically relevant doses of radiation. This safety is ensured by numerous copies of mtDNA in the cell, and due to their shielding from the effects of reactive oxygen (and nitrogen) species (ROS) by nucleoid proteins and by the operation of base excision repair in mitochondria. However, the generation of ROS increases in the mitochondria of cells exposed to IR. The increased generation of ROS in mitochondria can sometimes persist up to several days after the exposure of cells. The prolonged increased generation of ROS may be due to the involvement in the electron transport chain of the complexes of aberrant proteins expressed by the genes of mutated mtDNA copies. This may lead to the additional DNA damage, mitochondrial dysfunction, and instability of the nuclear genome. However, the development of oxidative stress can be restrained by antioxidant systems in the mitochondria. The key role here is played by activation of Mn-SOD2 and the protein p53. In addition, activation of mitochondrial biogenesis with the mtDNA synthesis, mitochondrial dynamics and mitophagy may be conjugated with the development of oxidative stress in mitochondria and mitochondrial dysfunction in irradiated cells. Thus, we can assume, that, although damage to mtDNA occurs at a high frequency and its repair is less efficient in mitochondria, the presence of multiple copies of mtDNA associated with proteins, the induction of antioxidant systems, the biogenesis of mitochondria with mtDNA synthesis, the activation of mitochondrial dynamics and mitophagy may contribute to the maintenance of mtDNA and functionally active mitochondria in the irradiated cells.

Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder

To evaluate the utility of trophectoderm biopsy for preimplantation genetic diagnosis (PGD) of mitochondrial (mt) DNA mutation load. A PGD case and analysis of blastocyst mosaicism. Academic center for reproductive medicine. A 30-year-old carrier of 35% 3243A>G mtDNA mutation load with a daughter affected by mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Blastocyst biopsy for PGD of mutation load and gender. Variation in mutation load within and among embryos, and newborn mutation load after PGD-based selection. Oocytes and embryos were found to possess a variety of 3243A>G mutation loads from 9% to 90% in oocytes and 7% to 91% in embryos, demonstrating that PGD would be a relevant procedure. Highly consistent results were obtained within multiple biopsies of both cleavage- and blastocyst-stage embryos. Importantly, mutation loads observed in trophectoderm were predictive of the inner cell mass (r(2) = 0.97). Transfer of a male embryo, predicted to possess 12% mutation load by analysis of a trophectoderm biopsy, resulted in the delivery of a boy with tissue-specific mutation loads ranging from undetectable to 15%. This study represents the first successful clinical application of PGD to reduce the transgenerational risk of transmitting an mtDNA disorder and supports the applicability of blastocyst trophectoderm PGD for carriers of mtDNA mutations attempting reproduction.

Human Induced Pluripotent Stem Cells Harbor Homoplasmic and Heteroplasmic Mitochondrial DNA Mutations While Maintaining Human Embryonic Stem Cell–like Metabolic Reprogramming

Human induced pluripotent stem cells (iPSCs) have been recently found to harbor genomic alterations. However, the integrity of mitochondrial DNA (mtDNA) within reprogrammed cells has yet to be investigated. mtDNA mutations occur at a high rate and contribute to the pathology of a number of human disorders. Furthermore, the lack of mtDNA integrity may alter cellular bioenergetics and limit efficient differentiation. We demonstrated previously that the derivation of iPSCs is associated with mitochondrial remodeling and a metabolic switch towards glycolysis. Here, we have discovered that alterations of mtDNA can occur upon the induction of pluripotency. Massively parallel pyrosequencing of mtDNA revealed that human iPSCs derived from young healthy donors harbored single base mtDNA mutations (substitutions, insertions, and deletions), both homoplasmic (in all mtDNA molecules) and heteroplasmic (in a fraction of mtDNAs), not present in the parental cells. mtDNA modifications were mostly common variants and not disease related. Moreover, iPSC lines bearing different mtDNA mutational loads maintained a consistent human embryonic stem cell-like reprogramming of energy metabolism. This involved the upregulation of glycolytic enzymes, increased glucose-6-phosphate levels, and the over-expression of pyruvate dehydrogenase kinase 1 protein, which reroutes the bioenergetic flux toward glycolysis. Hence, mtDNA mutations within iPSCs may not necessarily impair the correct establishment of pluripotency and the associated metabolic reprogramming. Nonetheless, the occurrence of pathogenic mtDNA modifications might be an important aspect to monitor when characterizing iPSC lines. Finally, we speculate that this random rearrangement of mtDNA molecules might prove beneficial for the derivation of mutation-free iPSCs from patients with mtDNA disorders.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Poor Correlations in the Levels of Pathogenic Mitochondrial DNA Mutations in Polar Bodies versus Oocytes and Blastomeres in Humans

Because the mtDNA amount remains stable in the early embryo until uterine implantation, early human development is completely dependent on the mtDNA pool of the mature oocyte. Both quantitative and qualitative mtDNA defects therefore may negatively impact oocyte competence or early embryonic development. However, nothing is known about segregation of mutant and wild-type mtDNA molecules during human meiosis. To investigate this point, we compared the mutant levels in 51 first polar bodies (PBs) and their counterpart (oocytes, blastomeres, or whole embryos), at risk of having (1) the "MELAS" m.3243A>G mutation in MT-TL1 (n = 30), (2) the "MERRF" m.8344A>G mutation in MT-TK (n = 15), and (3) the m.9185T>G mutation located in MT-ATP6 (n = 6). Seven out of 51 of the PBs were mutation free and had homoplasmic wild-type counterparts. In the heteroplasmic PBs, measurement of the mutant load was a rough estimate of the counterpart mutation level (R(2) = 0.52), and high mutant-load differentials between the two populations were occasionally observed (ranging from -34% to +34%). The mutant-load differentials between the PB and its counterpart were higher in highly mutated PBs, suggestive of a selection process acting against highly mutated cells during gametogenesis or early embryonic development. Finally, individual discrepancies in mutant loads between PBs and their counterparts make PB-based preconception diagnosis unreliable for the prevention of mtDNA disorder transmission. Such differences were not observed in animal models, and they emphasize the need to conduct thorough studies on mtDNA segregation in humans.

Derivation of Disease-Free Induced Pluripotent Stem Cells From Patients with Pearson Marrow Pancreas Syndrome

AbstractAbstract 3Mutations in mitochondrial DNA (mtDNA) cause several incurable diseases. In congenital mtDNA disorders, a mixture of normal and mutated mtDNA termed heteroplasmy exists at varying levels in different tissues, which determines the severity and phenotypic expression of the disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The clinical hallmarks of PS include sideroblastic anemia and other cytopenias, pancreatic insufficiency, metabolic acidosis, and other systemic organ dysfunction. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Somatic cells can be directly reprogrammed using defined genetic and chemical factors to yield “induced pluripotent stem” (iPS) cells, which have the capacity to differentiate into any tissue. iPS cells are particularly amenable for modeling mtDNA disorders, as cytoplasmic genetic material is retained during reprogramming. We sought to generate iPS cells from patients with PS and related mtDNA disorders to investigate the effects of mitochondrial dysfunction on stem cells and hematopoiesis. From a patient with PS, we generated bone marrow-derived fibroblasts carrying a high heteroplasmic burden of mutant mtDNA, and reprogrammed them into iPS cells. Although reprogramming efficiency was very low and kinetics of iPS colony emergence delayed, PS-iPS cells carrying the pathogenic mutation could be generated and displayed all hallmarks of pluripotency. We observed that PS-iPS cells initially demonstrated slow growth and a propensity for differentiation, but with ongoing passage in tissue culture, these characteristics improved. Unexpectedly, we found that the proportion of mutant mtDNA decreased rapidly in the PS-iPS lines as a function of passage. By subcloning, we were able to generate iPS cell lines with virtually undetectable amounts of mutant mtDNA, but which retained a viral integration pattern confirming their nuclear genetic identity to the original, highly heteroplasmic iPS clone. From “purged” PS-iPS cells, we generated hematopoietic progenitors free of detectable mutant mtDNA, thus yielding genetically identical, disease-free iPS cells and blood cells from a patient with Pearson syndrome. Disease-free iPS cells were readily obtained from the skin-derived fibroblasts of two other patients that carried a lower burden of mutant mtDNA. Our results suggest that mitochondrial dysfunction drives the segregation or elimination of mutant mtDNA in iPS cells as a function of passage, implying that maintenance of self-renewal and pluripotency are highly dependent on intact mitochondrial function. Importantly, a similar depletion of mutant mtDNA can be observed over time in vivo in certain tissues, such as hematopoietic cells, of patients with PS and other mtDNA disorders. This work provides a unique set of in vitro cellular models carrying varying degrees of mtDNA heteroplasmy to interrogate the effects of mitochondrial dysfunction on hematopoiesis. PS-iPS cells also provide a valuable opportunity to determine the factors driving changes in mtDNA heteroplasmy in stem cells, which holds important therapeutic implications for PS and a variety of congenital and acquired mtDNA disorders. Disclosures:No relevant conflicts of interest to declare.

Nuclear transfer to prevent mitochondrial DNA disorders: revisiting the debate on reproductive cloning

Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount to reproductive cloning, one theoretical variant, blastomere transfer does. This seems the most challenging both technically and ethically. It is prohibited by many jurisdictions and also the scientific community seems to avoid it. Nevertheless, this paper examines the moral acceptability of blastomere transfer as a method to prevent mtDNA disorders. The reason for doing so is that most objections against reproductive cloning refer to reproductive adult cloning, while blastomere transfer would amount to reproductive embryo cloning. After clarifying this conceptual difference, this paper examines whether the main non-safety objections brought forward against reproductive cloning also apply in the context of blastomere transfer. The conclusion is that if this variant were to become safe and effective, dismissing it because it would involve reproductive cloning is unjustified. Nevertheless, as it may lead to more complex ethical appraisals than the other variants, researchers should initially focus on the development of the other types of nuclear transfer to prevent mtDNA disorders.

Ethics of modifying the mitochondrial genome

Recent preclinical studies have shown the feasibility of specific variants of nuclear transfer to prevent mitochondrial DNA disorders. Nuclear transfer could be a valuable reproductive option for carriers of mitochondrial...

174th ENMC International Workshop: Applying pre-implantation genetic diagnosis to mtDNA diseases: Implications of scientific advances 19–21 March 2010, Naarden, The Netherlands

Ten years and 100 workshops after the first ENMC International Workshop on genetic management for mitochondrial DNA (mtDNA) disorders [4], 18 participants, including clinicians, basic scientists, an ethicist and a patient representative from 9 countries gathered on 19–21 March, 2010, in Naarden, The Netherlands. This was in order to (1) provide a consensus statement on the role and application of pre-implantation genetic diagnosis (PGD) to mtDNA disease and (2) review new data on the biology of mtDNA in the germline and assess its implications for genetic management.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double strand breaks (DSB) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In the present work we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results demonstrate the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Reproductive decision‐making in the context of mitochondrial DNA disorders: views and experiences of professionals

Although a scientific and ethical debate about the possible reproductive options for carriers of mitochondrial DNA (mtDNA) mutations is developing, not much information regarding the views and experiences of professionals exists. This paper explores the attitudes and experiences of professionals involved on a daily basis with their patients' reproductive decision-making in the context of mtDNA disease. Qualitative international multicenter design using in-depth semi-structured interviews with 20 professionals has been utilized. We identified four main themes emerging from the interviews. Firstly, we illustrate the discussion among professionals as to what extent mitochondrial genetics differs from other areas in genetics, both technically and ethically. Secondly, we show the discomfort and doubts of professionals when an mtDNA mutation is involved, because of the uncertainty remaining after testing. Thirdly, we discuss how professionals struggle with the tension between, on the one hand, the ideal of reproductive autonomy and, on the other hand, the reality of their professional responsibility and complex clinical decision-making. Fourthly, we delineate the strategies used by professionals in order to make attempts to control uncertainty. This paper illustrates the impact on professionals of reproductive decision-making in the context of mtDNA disease. It shows their feelings of discomfort when interpreting and explaining uncertain or ambiguous data and may be perceived as an example of how professionals deal with the inherent limitations in genetic knowledge representing the state of the art. Insight into the experiences of professionals may contribute to a further improvement of reproductive genetic counseling in the context of mtDNA disorders.

Information for genetic management of mtDNA disease: sampling pathogenic mtDNA mutants in the human germline and in placenta

BackgroundFamilies with a child who died of severe, maternally inherited mitochondrial DNA (mtDNA) disease need information on recurrence risk. Estimating this risk is difficult because of (a) heteroplasmy—the coexistence of...

Expression and localization of augmenter of liver regeneration in human muscle tissue

Mitochondrial DNA (mt-DNA) disorders and abnormal regulation of nuclear-derived proteins devoted to the cross-talk between the two cellular genomes have recently interested researchers in the field of neuromuscular diseases. We have identified, isolated and sequenced a new gene, augmenter of liver regeneration (ALR) that stimulates in vivo hepatocyte proliferation and up-regulates mt-DNA expression and ATP production. ALR protein (Alrp) is mainly located, in rat, in the mitochondrial inter-membrane space and its mRNA is particularly abundant in brain, muscle, testis and liver, tissues whose activity is mostly dependent on mitochondrial metabolism. Studies on rat Alrp sequence revealed the presence of homologous amino-acid sections into proteins derived from mouse, human, Drosophyla, plants and even DNA viruses. In this article, we evaluated ALR expression in normal human muscular tissues, both as protein and as mRNA. The data, obtained by molecular biology, immunohistochemistry and electron microscopy, demonstrated that: (i) Alrp and ALR mRNA are present in human muscular tissue; (ii) Alrp is particularly expressed in muscular fibres rich in mitochondria; (iii) Alrp is localized in the mitochondrial inter-membrane space or associated to mitochondrial cristae; and (iv) in subjects younger then 35 years of age, ALR mRNA expression is different between male and female subjects. In conclusion, the present data set Alrp, as a factor associated with mitochondria also in human tissue, call for future studies aimed at establishing Alrp as an important factor involved in the molecular events that trigger neuromuscular diseases.

Preimplantation genetic diagnosis for mitochondrial DNA disorders: ethical guidance for clinical practice

Although morally acceptable in theory, preimplantation genetic diagnosis (PGD) for mitochondrial DNA (mtDNA) disorders raises several ethical questions in clinical practice. This paper discusses the major conditions for good clinical practice. Our starting point is that PGD for mtDNA mutations should as far as possible be embedded in a scientific research protocol. For every clinical application of PGD for mtDNA disorders, it is not only important to avoid a 'high risk of serious harm' to the future child, but also to consider to what extent it would be possible, desirable and proportional to try to reduce the health risks and minimize harm. The first issue we discuss is oocyte sampling, which may point out whether PGD is feasible for a specific couple. The second issue is whether one blastomere represents the genetic composition of the embryo as a whole -- and how this could (or should) be investigated. The third issue regards the cutoff points below which embryos are considered to be eligible for transfer. We scrutinize how to determine these cutoff points and how to use these cutoff points in clinical practice -- for example, when parents ask to take more or less risks. The fourth issue regards the number of cycles that can (or should) justifiably be carried out to find the best possible embryo. Fifth, we discuss whether follow-up studies should be conducted, particularly the genetic testing of children born after IVF/PGD. Finally, we offer the main information that is required to obtain a truly informed consent.

Ooplasmic and nuclear transfer to prevent mitochondrial DNA disorders: conceptual and normative issues

BACKGROUND; Mitochondrial DNA (mtDNA) disorders are an important cause of human diseases. In view of the limitations of prenatal diagnosis and preimplantation genetic diagnoses, alternatives such as ooplasmic transfer (OT) and nuclear transfer (NT) have been proposed to prevent the transmission of mtDNA mutations. Both OT and NT are radical in the sense that they do not entail genetic selection, but genetic intervention to correct the genetic cause of the disease. After interviews with experts in the field, the relevant literature was searched and analyzed. Bioethical issues were divided into conceptual and normative points. OT is the transfer of normal mitochondria to a carrier's oocyte containing mutant mtDNA. In case of NT, a donated oocyte is enucleated and replaced with the nuclear DNA from a woman carrying a mtDNA mutation. NT can be performed both before and after in vitro fertilization, respectively, with the nucleus of an unfertilized oocyte, with the pronuclei of the zygote, or with the nucleus of a blastomere of an embryo. Conceptual questions regard whether these techniques amount to germ-line modification and human cloning. Normative questions concern, among others, the significance of intervening in the mtDNA, the implications of having 'three genetic parents', the ethics of oocyte donation and the health and safety risks for children conceived as a result of one of these techniques. Further interdisciplinary debate and research is needed to determine whether a clinical application of OT and NT can be morally justified, and if so, under which conditions.

Mitochondrial Gene Therapy: An Evaluation of Strategies for the Treatment of Mitochondrial DNA Disorders

Mitochondrial DNA (mtDNA) disorders include a vast range of pathological conditions, despite each sharing a mutual inability to produce ATP efficiently as a result of defective oxidative phosphorylation. There is no clear consensus regarding an effective therapeutic approach, and consequently the current treatment strategies are largely supportive rather than curative. This is almost certainly the result of there being virtually no defined genotype-phenotype relationships among the mtDNA disorders; hence an identical mutation may be responsible for multiple phenotypes, or the same phenotype may be produced by different mutations. In light of this, the development of gene therapy to treat mtDNA disorders offers a promising approach, as it potentially circumvents the complication of the aforementioned genotype-phenotype inconsistency and ultimately the current inability to treat individual disorders with sufficient efficacy. Such an approach will ultimately require the combination of efficient mitochondrial targeting, and an effective therapeutic molecule. Although promising proof-of-principle developments in this field have been demonstrated, the realization of a successful therapeutic mitochondrial gene therapy strategy has not come to fruition. This review critiques the key approaches under development by discussing the theory underlying each strategy, and detailing the current progress made. We also emphasize the potential hurdles that must be acknowledged and overcome if the potential of a therapeutic gene therapy to treat mitochondrial DNA disorders is to be realized.