Investigating the biology of mitochondrial DNA disease transmission to enable affected families to have healthy children

Lead Research Organisation: University of Oxford
Department Name: Obstetrics and Gynaecology

Abstract

Mitochondria are critically important for generating the energy that is essential for life. Despite the importance of mitochondria, one in 400 people has a maternally-inherited mutation in mitochondrial DNA (mtDNA), the blue print for some vital mitochondrial components. While most women transmitting these mtDNA mutations will have children who only develop mild symptoms, such as deafness in old age, they may be severely affected. The mtDNA mutations can cause a range of illnesses, including deafness, blindness, diabetes, loss of skills, heart and liver failure and there are no curative treatments.

Some centres are developing techniques for replacing disabled mitochondria with healthy ones. In this technique (so-called "nuclear transfer"), embryos of the couple at risk of having an affected child are generated in vitro. At an early stage, the nucleus that contains all of the genetic material apart from the mitochondria is removed from the egg (oocyte) and placed into a healthy egg or embryo from which the nucleus has been removed. The resulting embryo can then be placed in the mother's womb where it becomes a baby. While experiments on monkeys and mice suggest that such babies will probably be healthy, they have not been done in humans. Replacing the nucleus does not prevent development into a baby, but it causes damage that probably requires radical re-organisation of the cell. Because the embryo is already highly organised at this stage, such manipulations could have consequences later in life.

We aim to improve the genetic management of these diseases using less radical techniques. Current approaches are hampered by our poor understanding of the underlying mechanisms. MtDNA is inherited via the female line only. The chances that a woman carrying mtDNA mutations will pass them on to her child, and that child develops symptoms, are exceptionally difficult to predict. This is because both mutant and normal mtDNA are found in the same individual. The severity of the disease depends on the proportion of abnormal mtDNAs in particular cells of the body. However, this proportion varies from one generation to the next and cannot be predicted. The variability in the number of abnormal mtDNAs inherited is caused by an event known as the mitochondrial bottleneck which takes place in the female germline.

The biological processes that we plan to study determine the effectiveness of medical interventions. These treatments include preimplantation genetic diagnosis where eggs are fertilized in a test tube, tested, and low risk embryos selected to start a pregnancy. We will investigate existing cellular mechanisms that might be able to eliminate the unhealthy mitochondria, without such drastic interventions. In the long term we may determine whether nuclear transfer damages the cellular processes for maintaining healthy mitochondria. We may also develop less radical procedures, to generate embryos that have healthy mitochondria, without causing damage to the cell. Our ultimate aim is to enable affected families to have healthier children.

Technical Summary

Either mitophagy (recycling of mitochondria) or clonal proliferation of mtDNA subpopulations may underlie the unpredictable transmission of mutant mtDNA.

We propose that mtDNA turnover by mitophagy can be modulated to enhance purifying selection of normal mtDNA during early development.

We will determine whether:
1) mitochondrial function influences mtDNA content of:
a) "spare" human preimplantation embryos becoming available from families with mtDNA disease undergoing in vitro fertilisation
b) mouse oocytes with impaired mitochondrial function

2) mtDNA synthesis occurs in:
a) mouse and human oocytes maturating in vitro
b) mouse and donated human preimplantation embryos
using BrdU pulse labelling.

This may reveal any contribution of clonal proliferation of mtDNA to "purifying selection"

3) Modulators of mitochondrial recycling by mitophagy can be developed to enhance purifying selection. We will investigate and quantitate mitophagy in mouse pre-implantation embryos using transgenics with fluorescent organelles. We will cross mice with GFP targeted to LC3 in the autophagosomes against mice with DS-red targeted to mitochondria. Mitophagy is apparent by co-localisation of red and green signals in offspring (and this can be quantitated in cell lines using Imagestream, a technique combining flow cytometry and fluorescence microscopy, fig1).

We will use this model to determine whether mitophagy:
a) occurs in mouse oocytes and pre-implantation embryos
b) can be driven by exogenous treatments
c) is exacerbated in mouse models expected to modulate mitophagy, such as OPA1 knock down (required for mitochondrial fusion).

4) Purifying selection of wild type mtDNA in pluripotent cell cultures, heteroplasmic for pathogenic mtDNA mutants, can be driven by agents identified in (3).

Ultimately we will use the mouse with fluorescent autophagosomes to investigate mitophagy in:
a) nuclear and cytoplasmic transfer
b) multiple disease model

Planned Impact

One in 400 Caucasians carries a pathogenic mitochondrial DNA (mtDNA) mutation. Nevertheless, prenatal diagnosis to prevent transmission is poorly developed, because the unique segregation patterns resulting from heteroplasmy (co-existing normal and mutant mtDNA) during development are not understood. Even pre-implantation genetic diagnosis, where low risk embryos are preferentially transferred to the uterus, is poorly developed for mtDNA disease.

Immediate beneficiaries from this research are therefore those who suffer from these conditions, their progeny, and those who treat them. Longer-term beneficiaries include: both public-sector and commercial providers of fertility treatments; policymakers and regulators (including specifically the HFEA and its successor), who will gain information on the clinical possibilities; charities in the area of infertility treatment and counselling; and the general public whose understanding of science will be enlarged.

How will they benefit from this research?

The research will impact on the nation's health and wealth as follows. Understanding the biological mechanisms of mtDNA transmission will improve both prenatal and preimplantation diagnostic techniques for mtDNA diseases. As well as informing genetic management our study may also enable us to develop a new strategy for reducing transmission risks. Indeed, we intend to determine whether the existing cellular mechanisms for mitochondrial quality control can be used to destroy mutant mtDNA and reduce the transmission risk. This may be a safer alternative to genetic manipulation of mtDNA mutant load, or may be usable in conjunction with pre-implantation diagnosis. Furthermore the research required to develop safe nuclear transfer requires very many donated human oocytes. If our work superceeded nuclear transfer, oocytes would become available for other purposes. For instance, donor oocytes provide a simple alternative to affected women who want to reduce their transmission risk by being the birth mother but not the genetic mother. Available oocytes may also be donated for developing stem cell therapies.

Through this project, we will increase understanding of early embryo development by determining whether mitophagy (autophagy that specifically recycles spent mitochondria) occurs during preimplantation development, is important and/or affects segregation of mtDNA mutant populations. As nutrient availability affects autophagy, mitophagy could be an important determinant of metabolic programming. This will promote transfer of new, translational technology from laboratory to bedside and relieve the pressure on the donor oocytes needed for other applications.

The mouse model with red mitochondria and green autophagic vacuoles will facilitate future studies of mitophagy. A mouse model with down-regulated mitophagy would allow future studies of (i) potential ways to influence the mitochondrial transmission (ii) developmental programming of diabetes risks.

Public engagement
The principal applicant (JP) has experience explaining research to the general public. In 2009 she organised, chaired and lectured at an information evening for Oxford mitochondrial patients. This was highly rated by families attending ("Relevant to my family" 85%, "Overall satisfaction" 91). Patient support organisations presented posters at this meeting, including a woman who had a healthy child as a result of oocyte sampling (she described her experiences in JP's article Poulton et al, BMJ 338, 345 (2009)). A similar event is scheduled for 2011 and then every 1-2 years. In 2010 JP organised and chaired an international workshop on mtDNA transmission, with the European Neuromuscular Centre, to which she invited a patient representative. He presented lay views to the participating scientists and ethicists, including his experience of counselling, pre-implantation genetic diagnosis and the birth of his healthy child.

Publications

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Chapman TP (2014) Unexplained gastrointestinal symptoms: think mitochondrial disease. in Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver

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Diot A (2016) Mitophagy plays a central role in mitochondrial ageing. in Mammalian genome : official journal of the International Mammalian Genome Society

 
Description HFEA Review of the effectiveness and safety of methods to avoid mitochondria disease
Geographic Reach National 
Policy Influence Type Citation in other policy documents
Impact Invited to give evidence at Human Fertilisation and Embryology Authority (HFEA) review, which attracted international speakers (US and Netherlands). I was cited in the policy document arising from this.
URL http://www.hfea.gov.uk/docs/2011-04-18_Mitochondria_review_-_final_report.PDF
 
Description Invited to give 2 talks to HFEA committees
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact I suggested that HFEA should allow the Newcastle Mitochondrial research group working on nuclear transfer as a treatment for mitochondrial diseases should now be permitted to perform experiments on healthy single cell embryos rather than just abnormal ones.
 
Description Nuffield Council on Bioethics
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact I was invited to comment on the utility of nuclear transfer for preventing mtDNA diseases at a consultation meeting at the House of Lords when the findings of the council were launched.
URL http://www.hfea.gov.uk/docs/Mito-Annex_VIII-science_review_update.pdf
 
Description Submissions to the HFEA open consultation on new techniques to prevent transmission of serious mitochondrial disease
Geographic Reach National 
Policy Influence Type Citation in other policy documents
Impact Our collaborative paper (with Drs Joerg Burgstaller and Iain Johnston) on mitochondrial DNA segregation suggests that an additional restriction on the practice of mitochondrial donation for preventing mtDNA disease. We made a presentation to the review, suggesting that there should be an acceptably close mitochondrial genetic distance between donors of permitted embryos and of permitted eggs. When two mtDNA haplotypes are present in a cell (mtDNA heteroplasmy), it is usually assumed that segregation (the proliferation of one haplotype over another) is negligible and best explained by random drift. An intensively studied mouse model showing tissue-specific mtDNA segregation is currently seen as an exception to this assumption. However, existing studies of mtDNA dynamics have focused on laboratory mouse strains with very limited genetic diversity, thus ignoring the fact that genetic differences between haplotypes may lead to proliferative differences. We have data showing that in the mouse, segregation between two mtDNA haplotypes is the rule rather than the exception and strongly depends on the genetic distance. Segregation is much less when mitochondrial haplotypes are closely related than when they are diverse. The dataset on which this is based is substantially larger than in any previous such studies. Hence, the mtDNA of the permitted embryo might increase to disease-causing levels in later generations. We cannot exclude the possibility that there might be segregation to significant levels during development in foetuses developing from embryos generated by nuclear transfer. The risk would be substantially reduced by matching the mtDNA haplotype of donors. These data were quoted in the HFEA report on the consultation.
URL http://www.hfea.gov.uk/docs/Third_Mitochondrial_replacement_scientific_review.pdf
 
Description Small grant
Amount £14,000 (GBP)
Funding ID SG/12-13/01 
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2012 
End 12/2013
 
Description Studentship obtained from Lily Foundation @ £100K from 2015-2018
Amount £100,000 (GBP)
Organisation The Lily Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Title Developed high throughput imaging for detecting mitophagy 
Description INCell 1000 being used to quantify transient low frequency events 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Paper under review Human Molecular Genetics 
 
Title Developing ImageStream to quantify mitophagy 
Description Developing ImageStream (fluorescence mitoscopy combined with cytometry in a single platfrom) to quantify mitophagy 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Successful grant application (Wellcome) and oral presentation at Biochemical Society meeting 
 
Title RedMIT mefs 
Description Cell line with DS red targeted to mitochondria for studying mitophagy 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact Biochemical society meeting oral presentation 
 
Title RedMIT/GFP-LC3 mouse 
Description Mice with fluorescent organelles for investigating mitophagy 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Liver stem cells with fluorescent organelles 
 
Description Collaboration with Dr K Simon 
Organisation University of Oxford
Department Nuffield Department of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Research activity
Collaborator Contribution Access to ImageStream technology and Expertise Wellcome Trust Funding
Impact Submitted publications Abstracts accepted for Biochemical Society meeting, Mitochondrial Dynamics meeting, EUROMIT
Start Year 2009
 
Description Collaboration with Helsinki 
Organisation University of Helsinki
Department Institute of Biomedicine
Country Finland 
Sector Academic/University 
PI Contribution Cell lines have been shared with us
Collaborator Contribution They sent cell lines from their mouse model
Impact ongoing research project
Start Year 2013
 
Description Collaboration with Mitochondrial Biology Unit 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Public 
PI Contribution I provide him with ideas and patient materials.
Collaborator Contribution Ian has provided reagents advice and expertise for many years
Impact We co-chaired an international workshop (163rd ENMC International Workshop: Nucleoid and nucleotide biology in syndromes of mitochondrial DNA depletion myopathy. 12-14 December 2008, Naarden, The Netherlands) resulting in 2 publications. Cross-ferlisation of ideas result in a PLOS-genetics paper in 2010 and a Nucleic Acids Research paper in 2009.
 
Description Collaboration with Prof M Votruba, Cardiff 
Organisation Cardiff University
Department School of Optometry and Vision Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are drafting grant applications together and have
Collaborator Contribution Marcela has provided us with mice, we provide her with samples from our mouse models
Impact submitted a joint publication
Start Year 2008
 
Description Investigation of patients with mitochondrial disease 
Organisation University of Oulu
Department Department of Paediatric Neurology
Country Finland 
Sector Academic/University 
PI Contribution JP collected the original cohort of patients, conceived the first proposal which was funded by The Sigrid Juselius Foundation, Finland (Co-applicant on Senior Research Fellowship for Dr J Uusimaa) 50,000 Eur and The Paediatric Foundation, Finlandand 20,000 Eur. JP and KM both co-authored the current application (Academy of Finland) on which both are collaborators.
Collaborator Contribution JP flew to Oulu for collaborative meeting which enabled discussions with several other groups
Impact Paper submitted to Epilepsia Prospective study of POLG1 mutations presenting in children with intractable epilepsy-prevalence and clinical features J Uusimaa, V Gowda, A McShane, C Smith, J Evans, A Shrier, M Narasimhan, A O'Rourke, Y Rajabally, F Cowan, C Fratter, J Poulton Paper in draft for submission to J Med Genet Benign Cytochrome Oxidase Deficiency or Reversible Infantile Respiratory Chain Deficiency: Evidence for genetic heterogeneity Johanna Uusimaa, Heinz Jungbluth, G Crisponi, Lucy Feng, Massimo Zeviani, Imelda Hughes, Eileen Treacy, Jacqueline Birks, Caroline Sewry, Francesco Muntoni, and Joanna Poulton
Start Year 2008
 
Description Mathematics of mitochondrial disease 
Organisation Imperial College London
Department Department of Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide the biology
Collaborator Contribution They do the mathematics on our dataset
Impact Papers in draft/submission
Start Year 2011
 
Description Invited speaker, Murdoch Institute of Medical Research, Melbourne Australia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, Murdoch Institute of Medical Research, Melbourne Australia
Year(s) Of Engagement Activity 2017
 
Description BPNA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary talk

Increased patient referrals
Year(s) Of Engagement Activity 2007,2012
 
Description European society for Human Reproduction and Embryology (Geneva) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact European society for Human Reproduction and Embryology (Geneva) workshop on mitochondrial replacement therapy
Year(s) Of Engagement Activity 2017
 
Description European society for Human Reproduction and Embryology (Sofia) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a teaching session on the novel Mitochondrial Replacement Therapy which is licensed and active in the UK but not in most of the rest of the world. This ESHRE meeting and the last one in Geneva led to a successful application to the European Neuromuscular Conrsortium (ENMC) for a workshop on this topic convened by myself
Year(s) Of Engagement Activity 2018
URL https://www.enmc.org/workshops/upcoming-workshops/
 
Description Invited speaker, Hudson Institute of Medical Research, Monash, Australia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, Hudson Institute of Medical Research, Monash, Australia
Year(s) Of Engagement Activity 2017
 
Description Invited speaker, Kolling Institute of Medical Research, Sidney, Australia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker, Kolling Institute of Medical Research, Sidney, Australia
Year(s) Of Engagement Activity 2017
 
Description Invited talk University of Manchester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact Invited talk to 50 researchers

collaborations
Year(s) Of Engagement Activity 2013
 
Description Invited talk to Australian Mitochondrial Disease Federation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was invited to update the audience on mitochondrial replacement therapy at the annual meeting of this patient organisation. Because this is a hot topic in the media, available in the UK but not Australia I was in a good position to give up to the minute information. The local press reported the meeting
Year(s) Of Engagement Activity 2017
URL https://www.amdf.org.au/
 
Description Invited talk, Czech Academy of Science, Prague 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I gave an invited talk to scientists working on the basic science that directly impacts on our understanding of the diseases in my patients. This progressed our analysis of a group of 3 patients and increased their enthusiasm for and interaction with clinical problems. It increased their understanding of the cutting edge therapies available for these patients and progress made through this award.
Year(s) Of Engagement Activity 2016
 
Description Invited talk, University of Oulu, Finland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Health professionals
Results and Impact 20 people attended seminar

Ongoing collaboration
Year(s) Of Engagement Activity 2013
 
Description Invited talk, University of Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact 50 people attended seminar

Ongoing collaboration
Year(s) Of Engagement Activity 2013
 
Description Invited talk, University of Tampere 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Health professionals
Results and Impact 50 people attended seminar

Ongoing collaboration
Year(s) Of Engagement Activity 2013
 
Description Presented a Poster at the Biochemical Soc Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation by Karl Morten at a Biochemical Society Meeting

None
Year(s) Of Engagement Activity 2006