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In 2015, the UK became the first country to approve the use of mitochondrial donation. This novel in vitro fertilisation treatment was developed to prevent transmission of mitochondrial DNA (mtDNA) disease and ultimately give more reproductive choice to women at risk of having severely affected offspring. The policy change was a major advance that surmounted many scientific, legislative and clinical challenges. Further challenges have since been addressed and there is now an NHS clinical service available to families with pathogenic mtDNA mutations that provides reproductive advice and options, and a research study to look at the outcome at 18 months of children born after mitochondrial donation.

Mitochondrial myopathies are progressive muscle conditions caused primarily by the impairment of oxidative phosphorylation (OXPHOS) in the mitochondria. This causes a deficit in energy production in the form of adenosine triphosphate (ATP), particularly in skeletal muscle. The diagnosis of mitochondrial myopathy is reliant on the combination of numerous techniques including traditional histochemical, immunohistochemical, and biochemical testing combined with the fast-emerging molecular genetic techniques, namely next-generation sequencing (NGS). This has allowed for the diagnosis to become more effective in terms of determining causative or novel genes. However, there are currently no effective or disease-modifying treatments available for the vast majority of patients with mitochondrial myopathies. Existing therapeutic options focus on the symptomatic management of disease manifestations. An increasing number of clinical trials have investigated the therapeutic effects of various vitamins, cofactors, and small molecules, though these trials have failed to show definitive outcome measures for clinical practice thus far. In addition, new molecular strategies, specifically mtZFNs and mtTALENs, that cause beneficial heteroplasmic shifts in cell lines harboring varying pathogenic mtDNA mutations offer hope for the future. Moreover, recent developments in the reproductive options for patients with mitochondrial myopathies mean that for some families, the possibility of preventing transmission of the mutation to the next generation is now possible. Electronic supplementary material The online version of this article (10.1007/s13311-018-00674-4) contains supplementary material, which is available to authorized users.

The development of any novel reproductive technology involving manipulation of human embryos is almost inevitably going to be controversial and evoke sincerely held, but diametrically opposing views. The plethora of scientific, ethical and legal issues that surround the clinical use of such techniques fuels this divergence of opinion. During the policy change that was required to allow the use of mitochondrial donation in the UK, many of these issues were intensely scrutinised by a variety of people and in multiple contexts. This extensive process resulted in the publication of several reports that informed the recommendations made to government. We have been intrinsically involved in the development of mitochondrial donation, from refining the basic technique for use in human embryos through to clinical service delivery, and have taken the opportunity in this article to offer our own perspective on the issues it raises.

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to

Mitochondrial diseases are common genetic disorders often associated with a high disease burden and significant mortality. There are currently few effective treatments and no known cures, meaning that women who have been diagnosed with a genetic mutation associated with mitochondrial disease often seek reproductive advice to minimise the risk of having a severely affected child. There are several reproductive options available, and it is important that patients receive specific advice to help them decide which option is appropriate for them. Until recently, the reproductive options available for women who harbour pathogenic mitochondrial DNA mutations were essentially the same as those for nuclear mutations, although the complex features of mitochondrial genetics mean that they will not always guarantee to prevent mitochondrial DNA disease. Furthermore, the options will not be suitable for all women who carry a mitochondrial DNA mutation and wish to contribute genetically to their offspring. In this regard, there have been major recent advances in the development of IVF techniques to prevent the transmission of mitochondrial DNA disease.

Mitochondrial disease is a challenging area of genetics because two distinct genomes can contribute to disease pathogenesis. It is also challenging clinically because of the myriad of different symptoms and, until recently, a lack of a genetic diagnosis in many patients. The last five years has brought remarkable progress in this area. We provide a brief overview of mitochondrial origin, function, and biology, which are key to understanding the genetic basis of mitochondrial disease. However, the primary purpose of this review is to describe the recent advances related to the diagnosis, genetic basis, and prevention of mitochondrial disease, highlighting the newly described disease genes and the evolving methodologies aimed at preventing mitochondrial DNA disease transmission.

BACKGROUND: The use of nuclear transfer (NT) has been proposed as a novel reproductive treatment to overcome the transmission of maternally-inherited mitochondrial DNA (mtDNA) mutations. Pathogenic mutations in mtDNA can cause a wide-spectrum of lifelimiting disorders, collectively known as mtDNA disease, for which there are currently few effective treatments and no known cures. The many unique features of mtDNA make genetic counselling challenging for women harbouring pathogenic mtDNA mutations but reproductive options that involve medical intervention are available that will minimize the risk of mtDNA disease in their offspring. This includes PGD, which is currently offered as a clinical treatment but will not be suitable for all. The potential for NT to reduce transmission of mtDNA mutations has been demonstrated in both animal and human models, and has recently been clinically applied not only to prevent mtDNA disease but also for some infertility cases. In this review, we will interrogate the different NT techniques, including a discussion on the available safety and efficacy data of these technologies for mtDNA disease prevention. In addition, we appraise the evidence for the translational use of NT technologies in infertility. OBJECTIVE AND RATIONALE: We propose to review the current scientific evidence regarding the clinical use of NT to prevent mitochondrial disease. SEARCH METHODS: The scientific literature was investigated by searching PubMed database until Jan 2017. Relevant documents from Human Fertilisation and Embryology Authority as well as reports from both the scientific and popular media were also implemented. The above searches were based on the following key words: 'itochondria', 'mitochondrial DNA'; 'mitochondrial DNA disease', `fertility'; ` preimplantation genetic diagnosis', ` nuclear transfer', ` mitochondrial replacement' and ` mitochondrial donation'. OUTCOMES: While NT techniques have been shown to effectively reduce the transmission of heteroplasmic mtDNA variants in animal models, and increasing evidence supports their use to prevent the transmission of human mtDNA disease, the need for robust, long-term evaluation is still warranted. Moreover, prenatal screening would still be strongly advocated in combination with the use of these IVF-based technologies. Scientific evidence to support the use of NT and other novel reproductive techniques for infertility is currently lacking. WIDER IMPLICATIONS: It is mandatory that any new ART treatments are first adequately assessed in both animal and human models before the cautious implementation of these new therapeutic approaches is clinically undertaken. There is growing evidence to suggest that the translation of these innovative technologies into clinical practice should be cautiously adopted only in highly selected patients. Indeed, given the limited safety and efficacy data, close monitoring of any offspring remains paramount.

Very recently, two papers have presented intriguing data suggesting that prevention of transmission of human mitochondrial DNA (mtDNA) disease is possible. [Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N. et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 465, 82–85. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O. and Mitalipov, S. (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 461, 367–372.] These recent advances raise hopes for families with mtDNA disease; however, the successful translational of these techniques to clinical practice will require further research to test for safety and to maximize efficacy. Furthermore, in the UK, amendment to the current legislation will be required. Here, we discuss the clinical and scientific background, studies we believe are important to establish safety and efficacy of the techniques and some of the potential concerns about the use of these approaches.

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

The phenotypes associated with subtle deletions of the subtelomeric regions of many chromosomes have been reported. This is a detailed description of the clinical characteristics of two children with subtelomeric deletions of the long arm of chromosome 11 that were not apparent on the initial karyotype. We compare and contrast these with the clinical characteristics of a patient with a cytogenetically visible terminal 11q deletion, who shares similar craniofacial characteristics. All three suffered from moderate learning disability. Subtelomeric 11q deletions can be associated with mild-to-moderate learning difficulties and specific facial features, namely hypertelorism, down-slanting palpebral fissures and ptosis.

An extensive range of molecular defects have been identified in the human mitochondrial genome (mtDNA), causing a range of clinical phenotypes characterized by mitochondrial respiratory chain dysfunction. Sadly, given the complexities of mitochondrial genetics, there are no available cures for mtDNA disorders. In this review, we consider experimental, genetic-based strategies that have been or are being explored towards developing treatments, focussing on two specific areas which we are actively pursuing—assessing the benefit of exercise training for patients with mtDNA defects, and the prevention of mtDNA disease transmission.

Hypoplastic left heart is a severe human congenital heart defect characterized by left ventricular hypoplasiawith aortic and mitral valve atresia. A genetic etiology is indicated by an association of the hypoplastic left heart phenotype with terminal 11q deletions that span approximately 20 Mb (distal to FRA11B in 11q23). Here we define the breakpoints in four patients with heart defects in association with distal 11q monosomy and refine the critical region to an approximately 9-Mb region distal to D11S1351. Within this critical region we have identified JAM3, a member of the junction adhesion molecule family, as a strong candidate gene for the cardiac phenotype on the basis that it is expressed during human cardiogenesis in the structures principally affected in hypoplastic left heart.

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal.

New advances in medicine often raise challenges, and none more so than those involving the manipulation of human oocytes and embryos. Issues around clinical need and ethical considerations must be taken into account, as well as the safety of the proposed technique. The discussion around the proposed mitochondrial replacement techniques to prevent the transmission of mitochondrial DNA disease has perhaps raised more challenges than most [1]. Mitochondrial DNA diseases are both common and, in their severest forms, devastating [2]. There are limited treatments available for these patients, and those that are successful are focused on treating complications such as epilepsy and cardiac disease [3]. Mitochondrial DNA diseases are transmitted maternally, and for families carrying these mutations, a major, and justifiable, desire is to have unaffected children. For some women, preimplantation or prenatal diagnosis may be helpful [4], [5], but for other women, these techniques will not result in disease-free offspring and the only options available are either oocyte donation or mitochondrial replacement at the oocyte or zygote stage. The need for this technique for these families is well established, as are the experimental methods that are required for mitochondrial replacement [6]–[8]. The major scientific concerns for those of us working in the field revolve around safety and efficacy. In the United Kingdom, the Human Fertilisation and Embryology Authority (HFEA) recently considered the safety issues after extensive expert and public consultation [9]. This independent group of scientists reviewed all the evidence and concluded that mitochondrial replacement techniques have the potential to be used for patients with mitochondrial DNA disease, although further experiments are required before introduction into clinical practice, to provide further reassurance with respect to efficiency and safety. Recently [10], it has been suggested that the possibility of a harmful interaction between the mitochondrial and nuclear genomes has not been given due weight. Should we therefore stop further clinical developments in this area with immediate effect? The authors raise an interesting evolutionary argument that the human mitochondrial genome co-evolves with the nuclear genome in females, raising the possibility of a conflict with the paternal nuclear genome. They suggest Leber's hereditary optic neuropathy (LHON) and male infertility could be potential examples of this in humans [10]. Firstly, LHON is not a male-limited disease as they suggest [11]. The disorder affects ∼10% of women carrying specific mtDNA mutations, and although there is increased penetrance in males, strenuous efforts have failed to identify a nuclear modifier gene to date, and the increased penetrance in men could simply reflect the absence of oestrogens [12]. As regards male infertility, there is no convincing evidence in man that inherited variants of mtDNA are at all relevant in the general population [13], [14]. Indeed it is interesting that even in male patients with pathogenic mitochondrial DNA mutations, such as LHON, reduced fertility has not been reported to be a major clinical feature. The studies in macaques are also highly relevant to the risks proposed in humans associated with mitochondrial replacement. There are now multiple reports of the health status of the offspring born after mitochondrial replacement, and all have shown no difference between these offspring and controls [6], [7], [15]. As highlighted in the reports, the macaques used for these experiments were not, as suggested by the authors of the recent commentary [10], highly genetically related, but some were from divergent subspecies with extensive differences in the rhesus macaque genome [6]. Thus, the experiments using the animal model closest to man have not shown any adverse effects from mitochondrial transfer. Some studies in laboratory mice have proposed a nuclear DNA–mitochondrial DNA interaction, but there are others that have reported no defect despite the use of very divergent genomes [16]–[18]. It is important to recognise that these studies, and those in invertebrates, have been performed on highly inbred species (often inbred over thousands of generations) and the relevance to human populations must be questioned. Most human populations are outbred with considerable mixing of the genome over recent generations. In these populations the mixing of alleles will inevitably dilute the effect of potentially harmful nuclear DNA-mitochondrial DNA interactions. There has never been any direct evidence of a “mismatch” between the two in humans—either on an evolutionary scale or in the context of disease. This is even the case for couples from divergent haplogroups, where potential nuclear-mitochondrial mismatches are at their most extreme. Thus, from the mitochondrial DNA perspective, any mitochondrial transfer experiment is just recapitulating what is happening every day all around the world—and without any known adverse effects. Whilst we accept that any new technique is associated with risk, we think the lack of any reliable evidence of mitochondrial-nuclear interaction as a cause of disease in human outbred populations provides the necessary reassurance to proceed. The recent studies in macaques after mitochondrial replacement are also supportive that the possible harmful interactions are unlikely to occur in man [6], [7]. Human preimplantation embryos and embryonic stem cells generated with “unmatched” mtDNA replacement demonstrated normal development and differentiation potential [7], [8]. As suggested by the HFEA [9], it is possible to match mitochondrial haplotype between the mother and the mitochondrial donor to avoid any concern, even though the evidence says it should not be needed. We do not believe this important development should be delayed—for families carrying mtDNA mutations, the clock is ticking, and the desire to have children free of mitochondrial DNA disease is entirely justified. Ultimately, we believe those that carry mitochondrial DNA mutations must be fully informed of the potential risks, and that they will decide which option to take.

Corrigendum: Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease

87 Defects of mitochondrial function are increasingly recognised as important causes of disease. The clinical phenotype of mitochondrial diseases is extremely variable, aff ecting patients at any age and in a wide variety of tissues. 1 Patients are referred to and cared for by doctors from a range of specialties. Mitochondria are under the genetic control of both the mitochondrial and nuclear genomes, with defects of either genome resulting in mitochondrial dysfunction. Many adults and children with mitochondrial disease carry inheritable defects of the mitochondrial genome, with at least one in 8500 of the population carrying a pathogenic mitochondrial DNA (mtDNA) mutation. 2 This means that at least 3500 females in the UK—a large number of whom are of childbearing age—are carrying an mtDNA mutation. Over the past 17 years since defects of mtDNA were fi rst described, 3,4 we have become more effi cient at diagnosing patients, but still have little to off er in the way of treatment. 1

Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Although the mutant protein is widely expressed, the earliest and most striking neuropathological changes are observed in the striatum. Here we show dramatic mutation length increases (gains of up to 1000 CAG repeats) in human striatal cells early in the disease course, most likely before the onset of pathological cell loss. Studies of knock-in HD mouse models indicate that the size of the initial CAG repeat mutation may influence both onset and tissue-specific patterns of age-dependent, expansion-biased mutation length variability. Given that CAG repeat length strongly correlates with clinical severity, we suggest that somatic increases of mutation length may play a major role in the progressive nature and cell-selective aspects of both adult-onset and juvenile-onset HD pathogenesis and we discuss the implications of this interpretation of the data presented.