J.L. Murphy

Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease

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.

Resistance training in patients with single, large-scale deletions of mitochondrial DNA

Dramatic tissue variation in mitochondrial heteroplasmy has been found to exist in patients with sporadic mitochondrial DNA (mtDNA) mutations. Despite high abundance in mature skeletal muscle, levels of the causative mutation are low or undetectable in satellite cells. The activation of these typically quiescent mitotic cells and subsequent shifting of wild-type mtDNA templates to mature muscle have been proposed as a means of restoring a more normal mitochondrial genotype and function in these patients. Because resistance exercise is known to serve as a stimulus for satellite cell induction within active skeletal muscle, this study sought to assess the therapeutic potential of resistance training in eight patients with single, large-scale mtDNA deletions by assessing: physiological determinants of peak muscle strength and oxidative capacity and muscle biopsy-derived measures of damage, mtDNA mutation load, level of oxidative impairment and satellite cell numbers. Our results show that 12 weeks of progressive overload leg resistance training led to: (i) increased muscle strength; (ii) myofibre damage and regeneration; (iii) increased proportion of neural cell adhesion molecule (NCAM)-positive satellite cells; (iv) improved muscle oxidative capacity. Taken together, we believe these findings support the hypothesis of resistance exercise-induced mitochondrial gene-shifting in muscle containing satellite cells which have low or absent levels of deleted mtDNA. Further investigation is warranted to refine parameters of the exercise training protocol in order to maximize the training effect on mitochondrial genotype and treatment potential for patients with selected, sporadic mutations of mtDNA in skeletal muscle.

Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance

Progressive external ophthalmoplegia (PEO) is a canonical feature of mitochondrial disease, but in many patients its genetic basis is unknown. Using exome sequencing, Pfeffer et al. identify mutations in SPG7 as an important cause of PEO associated with spasticity and ataxia, and uncover evidence of disordered mtDNA maintenance in patients.

Cytochrome c oxidase-intermediate fibres: Importance in understanding the pathogenesis and treatment of mitochondrial myopathy

An important diagnostic muscle biopsy finding in patients with mitochondrial DNA disease is the presence of respiratory-chain deficient fibres. These fibres are detected as cytochrome c oxidase-deficient following a sequential cytochrome c oxidase-succinate dehydrogenase reaction, often in a mosaic pattern within a population of cytochrome c oxidase-normal fibres. Detailed analysis of muscle biopsies from patients with various mitochondrial DNA defects shows that a spectrum of deficiency exists, as there are a large number of fibres which do not correspond to being either completely cytochrome c oxidase-normal (brown staining) or cytochrome c oxidase-deficient (blue staining). We have used a combination of histochemical and immunocytochemical techniques to show that a population of cytochrome c oxidase-intermediate reacting fibres are a gradation between normal and deficient fibres. We show that cytochrome c oxidase-intermediate fibres also have different genetic characteristics in terms of amount of mutated and wild-type mtDNA, and as such, may represent an important transition between respiratory normal and deficient fibres. Assessing changes in intermediate fibres will be crucial to evaluating the responses to treatment and in particular to exercise training regimes in patients with mitochondrial DNA disease.

Detection of cytochrome c oxidase activity and mitochondrial proteins in single cells.

Cytochrome c oxidase or mitochondrial respiratory chain complex IV is where over 90% of oxygen is consumed. The relationship between complex IV activity and mitochondrial proteins, which provides a guide to understanding the mechanisms in primary mitochondrial disorders, has been determined by histochemistry (activity) and immunohistochemistry in serial sections. In the central nervous system (CNS), mitochondrial activity and immunoreactivity have been determined in populations of cells in serial sections as capturing cells in more than one section is difficult. In this report we describe a method to determine complex IV activity in relation to mitochondrial proteins at a single cell level in the CNS. We performed complex IV histochemistry and immunohistochemistry consecutively in snap frozen sections. Although the product of complex IV histochemistry reduces the sensitivity of standard immunohistochemistry (secondary antibody and ABC method) the biotin-free Menapath polymer detection system (A. Menarini Diagnostics, Wokingham, UK) enables mitochondrial proteins to be detected following complex IV histochemistry. The co-occurring chromogens may then be separately visualised and analysed using multi-spectral imaging (Nuance system CRi, Woburn, MA). Our technique is applicable for exploring mitochondrial defects within single cells in a variety of CNS disorders and animal models of those diseases.

Accurate Measurement of Mitochondrial DNA Deletion Level and Copy Number Differences in Human Skeletal Muscle

Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied MT-ND1/MT-ND4 multiplex mtDNA deletion assay and a recently developed MT-ND1/B2M singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.

Mitochondrial DNA deletions in muscle satellite cells: implications for therapies

Progressive myopathy is a major clinical feature of patients with mitochondrial DNA (mtDNA) disease. There is limited treatment available for these patients although exercise and other approaches to activate muscle stem cells (satellite cells) have been proposed. The majority of mtDNA defects are heteroplasmic (a mixture of mutated and wild-type mtDNA present within the muscle) with high levels of mutated mtDNA and low levels of wild-type mtDNA associated with more severe disease. The culture of satellite cell-derived myoblasts often reveals no evidence of the original mtDNA mutation although it is not known if this is lost by selection or simply not present in these cells. We have explored if the mtDNA mutation is present in the satellite cells in one of the commonest genotypes associated with mitochondrial myopathies (patients with single, large-scale mtDNA deletions). Analysis of satellite cells from eight patients showed that the level of mtDNA mutation in the satellite cells is the same as in the mature muscle but is most often subsequently lost during culture. We show that there are two periods of selection against the mutated form, one early on possibly during satellite cell activation and the other during the rapid replication phase of myoblast culture. Our data suggest that the mutations are also lost during rapid replication in vivo, implying that strategies to activate satellite cells remain a viable treatment for mitochondrial myopathies in specific patient groups.

Research into Policy: A Brief History of Mitochondrial Donation

Mitochondrial DNA disorders are a group of common genetic diseases which affect both children and adults. They lead to progressive multisystem disease for which there is no curative treatment. Inherited mitochondrial DNA mutations are transmitted maternally, and preventing transmission of these diseases is a priority for families. An important new approach is a novel in vitro fertilisation (IVF) technique called mitochondrial donation using either maternal spindle transfer or pronuclear transfer [1]. The 4th March 2015 saw an historic event—the legislation was put in place to make mitochondrial donation legal in the UK. Critics of the technique have claimed that the new law was rushed through and that there had been insufficient time to debate the issues. Here we provide a brief account of the past 17 years to show that this is not the case. The complete human mitochondrial genome, made up of only 16,569 base pairs, was first sequenced in 1981 [2]. Around the same time, in an entirely unrelated area of research, the technique of nuclear transplantation between mouse embryos was described [3, 4]. The first human pathogenic mitochondrial DNA mutations were identified in 1988 [5, 6] and by 1995, the possibility of preventing the transmission of mitochondrial DNA disease by nuclear transplantation was already being considered [7]. Since then, although the name for the technique we now know as mitochondrial donation has changed several times, the scientific, ethical, and legal issues have been examined in detail by a number of independent groups and committees over many years. The need to regulate the use of human embryos in both fertility treatment and scientific research was recognized in the UK following the birth of the first IVF baby in 1978. This major breakthrough led to concerns about the social and legal implications of such advances in human assisted reproduction. To address these issues, the Government established the Committee of Inquiry into Human Fertilisation and Embryology chaired by the now Baroness Mary Warnock. The report [8], published in 1984, set out a blue-print for the regulation of both IVF and embryo research, and with admirable foresight, included a chapter describing possible future developments in embryo research. This report was followed by the White Paper “Human Fertilisation and Embryology: A Framework for Legislation” which was published in 1987 and formed the basis for the Human Fertilisation and Embryology Act 1990 (“the 1990 Act”) [9]. There were rapid and significant advances in non-human embryo research over subsequent years, including somatic cell nuclear transfer and the birth of Dolly the sheep in 1996 [10]. These developments generated much public interest and highlighted the need for a clarification of the legislation regarding the implications for human clinical embryology. A report by the Human Fertilisation and Embryology Authority (HFEA) and Human Genetics Advisory Commission (HGAC) [11], published in 1998, acknowledged that some of the scientific possibilities being discussed at this time had not been envisaged when the 1990 Act was drafted. Specifically, the Report recommended that the purposes for which human embryos could be used in research should be extended to allow the development of methods of therapy for mitochondrial diseases. Following this Report, the Government established an expert group chaired by the Chief Medical Officer (Professor Sir Liam Donaldson) to examine the potential benefits of a number of new areas of human embryo research, including methods to prevent mitochondrial disease. The report [12] made several recommendations Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, and Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom; Newcastle Fertility Centre, Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom; Hempsons, Hempsons House, Villiers Street, London, United Kingdom

Scientific and Ethical Issues in Mitochondrial Donation

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.

C.O.2 DNM2 mutations cause multiple mtDNA deletions in muscle: A novel disorder of mtDNA maintenance

Abstract Centronuclear myopathy (CNM) is a congenital myopathy characterised by delayed motor milestones, progressive facial weakness, ptosis, ophthalmoplegia and centrally-located myonuclei caused by autosomal dominant mutations in the dynamin-2 gene, DNM2. Dnm2 is a large GTPase protein, one of three classical dynamins that is ubiquitously expressed, and key to regulating cytoskeleton and membrane trafficking within cells. Recently, a DNM2 (KI-Dnm2R465W) animal model was shown to develop phenotypic and morphological abnormalities similar to those observed in the human disease, with abnormal central accumulation of mitochondria in the muscle fibres of the heterozygous mice. Furthermore, mutations in OPA1 and MFN2 – also dynamin-like GTPase proteins which are involved in mitochondrial membrane fusion – have recently been shown to be associated with impaired mitochondrial DNA (mtDNA) stability, inferring a crucial role for these proteins in regulating mitochondrial networks and mtDNA maintenance. We investigated five patients with dominant DNM2 mutations. In addition to the typical features of CNM, the muscle biopsies demonstrated variable amount of cytochrome c oxidase (COX)-deficient muscle fibres indicative of mitochondrial dysfunction. We show here that these focal mitochondrial biochemical defects are due to clonally-expanded mtDNA deletions, characteristic of mtDNA maintenance abnormality. Confocal microscopy studies of patient fibroblasts reveal a quantitative disruption of the dynamic mitochondrial network associated with the p.R369W DNM2 mutation, which interestingly corresponded with the most severe mitochondrial defect in muscle. Similar results were observed in HeLa cells following Dnm2 siRNA knockdown, whilst transfection of NIH3T3 cells with mutant p.R369W Dnm2 led to a significant decrease in mitochondrial objects compared to controls. Together, our data suggest an important and emerging role for Dnm2 in mtDNA maintenance and stability.