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Human Molecular Genetics, 2011, Vol. 20, Review Issue 2
doi:10.1093/hmg/ddr373
Advance Access published on August 18, 2011
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Mitochondrial DNA disease: new options
for prevention
Lyndsey Craven 1, Joanna L. Elson 1, Laura Irving 1, Helen A. Tuppen 1,2, Lisa M. Lister 3,4,5,
Gareth D. Greggains 3,4,5, Samantha Byerley 3, Alison P. Murdoch 3,5, Mary Herbert 3,4,5
and Doug Turnbull 1,2,5,∗
1
Mitochondrial Research Group and 2Newcastle University Centre for Brain Ageing and Vitality, Institute for Ageing
and Health, Newcastle University, Newcastle upon Tyne NE2 4HH, UK, 3Newcastle Fertility Centre at Life,
International Centre for Life, Newcastle upon Tyne NE1 4EP, UK and 4Institute for Ageing and Health and 5North East
England Stem Cell Institute (NESCI), Bioscience Centre, Centre for Life, Newcastle University, Newcastle upon Tyne
NE1 4EP, UK
Received July 20, 2011; Revised and Accepted August 16, 2011
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.
MITOCHONDRIAL GENETICS AND DISEASE
Mitochondrial DNA (mtDNA) is the only constitutive extra
chromosomal DNA within human cells (1). It is a small
genome (16.6 kb) with a very limited number of genes. All 37
genes (13 protein encoding and 24 RNA genes) are involved
with the synthesis of proteins which form crucial subunits of
the mitochondrial respiratory chain. The mitochondrial genome
is also present in multiple copies in all cells. Defects of the mitochondrial genome take the form of point mutations and deletions,
and many hundreds of different mutations associated with disease
have been described. These mtDNA mutations may be either
homoplasmic, in which all copies are mutated, or heteroplasmic,
with a mixture of mutated and wild-type mtDNA present. The
majority of mtDNA mutations described are heteroplasmic and
in patients there is a clear link between the levels of mutated
mtDNA versus wild-type mtDNA; patients with large amounts
of mutated mtDNA (associated with lower amount of wild-type)
have the most severe symptoms (2).
The clinical features of mtDNA disease are very variable and
relate in part to the severity and amount of the mtDNA mutation
(3). The clinical features usually affect tissues in which there is a
high metabolic demand such as central nervous system, skeletal
muscle or heart. However, other tissues are frequently involved
such as the b-cells of the pancreas leading to diabetes, the inner
hair cells of the cochlear causing deafness or the renal tubules
leading to kidney dysfunction. There are a number of welldefined clinical syndromes but the majority of patients do not
fall into easily defined clinical groups, and definition by a genotype is often helpful (4,5).
∗
To whom correspondence should be addressed at: Mitochondrial Research Group, Centre for Brain Ageing and Vitality, Institute for Ageing and
Health, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Tel: +44 1912228565; Fax: +44 1912824373; Email: doug.turnbull@newca
stle.ac.uk
# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
Human Molecular Genetics, 2011, Vol. 20, Review Issue 2
The incidence of mtDNA disease is not known but there have
been a number of epidemiological studies either looking for the
presence of specific mutations or the incidence of mtDNA
disease within a limited population. The frequency of pathogenic
mtDNA mutations in the population is high (1 in 200 people)
(6–8). The majority of these individuals will not be affected by
disease because the mtDNA mutation is present at low levels of
heteroplasmy or mutation detected is only pathogenic in the presence of a specific insult. The other epidemiological approach
which focuses solely on known cases is inevitably an underestimate but suggests that the incidence of mtDNA disease is at
least 1 in 10 000 affected with many more at risk (9).
mtDNA in mammals is strictly maternally inherited with all
homoplasmic mtDNA mutations transmitted to offspring (2).
The transmission of heteroplasmic mtDNA mutations is complicated by both a selection process and a genetic bottleneck
present during development of the female germline. Mice
expressing an error-prone mtDNA polymerase provide an opportunity to study the changes able to pass though the bottleneck and reveal a very strong selection against mutations in
the protein-encoding genes of mtDNA, which were eliminated
rapidly (10). There is also marked variation between the offspring of heteroplasmic mothers due to an extreme reduction
in mtDNA copy number during germ cell development and/
or selective genome replication (11– 13).
TREATMENT OF MTDNA DISEASE
Unfortunately, advances in treatment for patients with mtDNA
disease have not advanced at the same pace as the ability to establish a diagnosis. For the vast majority of patients with
mtDNA disease, therapy is limited to symptomatic relief and
early detection of treatable symptoms such as epilepsy,
cardiac disease and diabetes. There have been limited
numbers of clinical trials despite numerous reports of studies
in the literature (14). However, a recent randomized study has
shown that idebenone, a ubiquinone analogue, is helpful in
patients with Leber’s Hereditary Optic Neuropathy
(Chinnery, P.F. et al., unpublished data). Evidence from
animal models and from clinical studies indicates that increasing mitochondrial biogenesis by exercise and drugs also holds
promise (15,16). Such treatments are most likely to be effective
in those patients with milder mtDNA disease, where small
increases in wild-type mtDNA will improve function. More
fundamental treatments to either remove mutated mtDNA by
specifically targeting restriction endonucleases (17) or inhibiting replication of mutated mtDNA (18) have either worked in
vitro or in animal models, but are some way from use in
patients. Other approaches of bypassing the biochemical
defect using alternative oxidases, for example, also hold
promise for the future (19).
CURRENT REPRODUCTIVE OPTIONS FOR
WOMEN CARRYING MTDNA MUTATION
There are now a number of options available (20). Genetic
counselling is important to explain risks involved, but must
be given carefully, taking account of the specific mutation
and the number of affected family members. More recently,
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pre-natal and pre-implantation genetic diagnoses have
become an option particularly relevant to those mothers carrying mutations which segregate widely between offspring such
as the m.8993T.C NARP mutation. With both techniques,
there is a concern that cells taken for genetic analysis may
not reflect the level of heteroplasmy in the embryo or fetus.
However, recent studies suggest that, at least during early
stages of development, there is relatively little variation in heteroplasmy between cells (21). Nonetheless, for women with
homoplasmic or high levels of heteroplasmic mtDNA mutations, the only option at present to ensure an unaffected
child is oocyte donation. These oocytes tend to be in short
supply and have the limitation of not being genetically
related to the mother.
PREVENTING TRANSMISSION OF MTDNA
DISEASE
In view of the lack of curative treatment and the extremely
disabling phenotype in many patients, preventing transmission
of mtDNA defects is an important goal for families. One
approach to achieving this is to transplant the nuclear
genome from the oocyte or embryo of an affected woman to
those from a healthy donor (Fig. 1). This has the potential to
enable women to have a genetically related child without
transmitting mutated mtDNA. Early experiments in mouse,
using fertilized eggs (1 cell zygote), indicated that transplantation of the male and female pronuclei between zygotes was
compatible with onward development and birth of normal offspring (22,23). It was later demonstrated that pronuclear transfer is effective in preventing transmission of an mtDNA
rearrangement in a mouse model (24). More recently, proof
of principle experiments have been conducted using abnormally fertilized human zygotes (25) (Fig. 2). A related technique
involving transplantation of the metaphase II spindle between
unfertilized oocytes (Fig. 1) has resulted in live born rhesus
monkeys (26).
The efficacy of these techniques in preventing transmission
of mtDNA disease is highly dependent on minimizing the
transfer of mitochondria during transplantation of the
nuclear genome. Both metaphase II spindle transfer and pronuclear transfer involve fusion of a karyoplast containing the
nuclear material surrounded by a small amount of cytoplasm,
which may well contain mitochondria. Thus, there is the
potential for transmission of the affected mitochondria to the
offspring. Techniques employed to minimize the size of the
karyoplast have been successful in limiting this transmission
such that there is no detectable carryover of mtDNA or very
low levels detected in the developing embryo (,2%). In the
presence of heteroplasmy, there is a threshold of mutated to
wild-type mtDNA which must be reached before clinical features are observed. In the vast majority of transmitted mtDNA
mutations, this threshold is high and thus low levels of
mutated mtDNA transferred during nuclear transplantation
are highly unlikely to generate a phenotype. There is a slight
risk that the mutated genotype may segregate to specific
tissues, but marked tissue-specific segregation of mtDNA
mutations seems to be confined to mutations which are
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Figure 1. Nuclear transfer techniques to prevent transmission of mtDNA disease. (A) Pronuclear transfer technique using a mitochondrial donor zygote.
(B) Metaphase II spindle transfer technique using a mitochondrial donor oocyte followed by intracytoplasmic sperm injection fertilization. Both techniques
will involve monitoring embryo development following transfer.
rarely passed through the germline (such as large-scale
mtDNA deletions) and thus unlikely to be a major concern.
It will also be important to determine whether the manipulations associated with nuclear genome transplantation are detrimental to embryo development. While the wealth of
evidence from mouse studies indicates that pronuclear transfer
is compatible with normal development, evidence from human
studies indicates a 50% reduction in the proportion of embryos
developing to the blastocyst stage (25). However, it should be
noted that the human studies were conducted on abnormally
fertilized zygotes containing either one or three pronuclei,
which show considerable variation in chromosome constitution and have reduced developmental capacity. While the experimental design included restoration of diploidy, the
paternal and maternal pronuclei of human zygotes are morphologically indistinguishable. Thus, the parent of origin of the
two pronuclei contained in the reconstituted zygote was
unknown. It is therefore likely that the presence of either
two paternal or two maternal genomes contributed to the
reduced development of human pronuclear transfer embryos.
It will be important to repeat these studies using normally fertilized human zygotes.
Evidence on the effect of metaphase II spindle transfer on
embryo development is largely confined to studies in rhesus
monkey with a report that the technique of spindle transfer
is compatible with the birth of normal offspring (26). There
has also been one report of blastocyst development following
metaphase II spindle transfer in human oocytes (27).
Comparison between metaphase II spindle transfer and
pronuclear transfer
These two techniques have separately been shown to be technically feasible and lead to low levels of carryover mtDNA,
but the question of which is the better technique for patients
with mtDNA disease remains unresolved. Metaphase II
spindle transfer is performed at an earlier stage and the karyoplast is smaller than with pronuclear transfer, with the potential for less carryover of mtDNA. However, recent studies in
human embryos show equivalent levels of carryover (unpublished data), providing further evidence that the risks of offspring developing disease are likely to be minimal.
Unlike pronuclear transfer, the chromosomes of the metaphase II oocyte are not enclosed within a nuclear membrane.
Instead, they are attached to the spindle, which can be visualized by polarized light birefringence. However, it is not possible to see the chromosomes without the use of a fluorescent
dye, which may affect onward development. While correctly
aligned chromosomes are positioned on the spindle equator,
evidence from studies on human oocytes indicate that chromosome scattering can occur. This has also been reported in
human oocytes from older women (28) and in response to
exposure to ambient conditions (29). Therefore, it will be
important to perform the manipulations in tightly controlled
environmental conditions and to develop techniques to minimize the risk of inducing aneuploidy during transplantation of
the metaphase II spindle.
While pronuclear transfer offers the advantage of having the
maternal and paternal genomes enclosed in clearly visible pronuclei, the ability to faithfully transmit chromosomes to
daughter cells during division to the two-cell stage depends
on the assembly of a normal bipolar spindle during the first
mitosis. It will therefore be important to ensure that reconstituted zygotes contain only two centrioles, which in humans are
derived from the sperm and are tightly apposed to the pronuclear membrane (30). Thus, in reducing the size of the karyoplast to minimize mtDNA carryover, it will be important to
ensure that there is sufficient cytoplasm to include the centrioles and, possibly other spindle assembly components, in
the karyoplast.
Human Molecular Genetics, 2011, Vol. 20, Review Issue 2
Figure 2. Pronuclei removal from an abnormally fertilized human embryo and transfer to an enucleated abnormally fertilized human embryo. (A) The tripronucleate human embryo (two pronuclei can be
seen, arrowheads). A hole has been made in the zona pellucida using a microsurgical laser (arrow). (B) The biopsy pipette is inserted into the embryo and a pronucleus aspirated within a karyoplast
(arrowhead). (C) The pronuclear karyoplast can be seen within the biopsy pipette (arrowhead). (D) A single pronuclear karyoplast removed from the embryo. (E) The pronuclear karyoplasts can be
seen within the biopsy pipette (arrowheads). (F) The biopsy pipette is inserted through the hole in the zona pellucida. (G) The pronuclear karyoplasts are transferred into the enucleated embryo along
with a small amount of HVJ-E. (H) The pronuclear karyoplasts (arrowheads) are allowed to fuse with the enucleated embryo.
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Nuclear transplantation also requires the use of reversible
cytoskeletal inhibitors, the effect of which has not been rigorously tested in human oocytes or zygotes. Metaphase II
spindle transfer requires inhibition of the actin cytoskeleton
only, whereas pronuclear transfer requires inhibition of
actin and tubulin polymerization. While both treatments are
reversible, further studies are required to determine whether
they have a detrimental effect on subsequent embryonic
development.
In addition to the potential impact of the manipulations on
embryo development, logistical considerations will also have
an impact on the choice between metaphase II spindle transfer
and pronuclear transfer. For example, clinical application will
involve ovarian stimulation of the oocyte donor and the
affected woman. However, due to biological variation in
response to ovarian stimulation, it will not be possible to guarantee that both the donor and the recipient will be ready for
oocyte collection on the same day. It may therefore be necessary to cryopreserve oocytes or zygotes until nuclear genome
transplantation can be performed. Thus, the efficacy of cryopreservation and/or vitrification techniques at the different
stages of development will also need to be considered. In
this regard, cryopreservation of zygotes is a long-established
procedure in assisted conception; however, vitrification techniques for the successful storage of metaphase II human oocytes
are a relatively recent development in clinical assisted reproduction (31).
POTENTIAL DISADVANTAGES OF NUCLEAR
GENOME TRANSPLANTATION TECHNIQUES TO
PREVENT TRANSMISSION OF MTDNA DISEASE
In addition to the risk of aneuploidy and other effects of the
technical procedures, concern has been raised about the implications of possible incompatibilities between the nuclear genotype of the parents and the donor mitochondrial genomes. The
potential biological significance of this stems from the fact
that the majority of proteins involved in mitochondrial metabolism are encoded by the nuclear genome (32,33). However, in
the vast majority of non-consanguineous births, the paternal
nuclear genome comes from a different mitochondrial background. As there is no evidence of parental imprinting of
nuclear-encoded mitochondrial proteins, it is assumed that
paternally encoded genes contribute to mitochondrial function.
Thus, in the process of fertilization, biology appears to make
provision for ‘incompatibilities’ between the nuclear and mitochondrial genomes. This may be explained by the relative sequence similarity between different mitochondrial genotypes.
For example, only a small number of amino acid substitutions
(ranging from just a few to 20) are observed when two human
mtDNAs are compared. It is also worth noting that the largest
incompatibility between nuclear and mitochondrial backgrounds will be between people with heritages rooted in
different continents and we are not aware of any evidence
of increased mitochondrial disease in children with mixed
heritage.
Assisted reproduction techniques, particularly invasive
techniques such as intracytoplasmic sperm injection, have
been reported to be associated with an increased incidence
of epigenetic abnormalities such as Angelman syndrome and
Beckwith – Wiedemann syndrome (34). The question of
whether the manipulations associated with nuclear genome
transplantation might induce epigenetic anomalies remains to
be resolved. One study in mouse following transfer of the
female pronucleus showed transcriptional repression and
methylation of liver proteins associated with impaired
growth (35). However, a recent report describing intra-strain
maternal pronuclear transfers, metaphase-II spindle transfers
and ooplasm transfers between C57BL/6 and DBA/2 mice
found no major, long-term growth defects or epigenetic abnormalities, in either males or females, associated with intergenotype transfers (36). Furthermore, ongoing studies of the rhesus
monkey offspring born following metaphase-II spindle transfer (26) indicate that they are growing normally (unpublished
data).
FURTHER STUDIES REQUIRED TO EVALUATE
SAFETY AND EFFICACY ON NUCLEAR
TRANSFER TECHNIQUES
Legislation relating to embryo research varies considerably
between different countries—from an outright ban on all
research to no legislation at all. The UK has been at the vanguard
of permissive legislation combined with strict regulation of research in this area; however, translation to clinical treatment
to prevent the transmission of mtDNA disease will require
that the UK Parliament introduces ‘regulations’ so that an
embryo is considered a ‘permitted’ embryo if it has undergone
manipulation using material provided by two women. A ‘permitted’ embryo could then be transferred to the uterus for fertility treatment. As a first step, the Secretary of State for Health
recently commissioned the Human Fertilisation and Embryology Authority (HFEA) to convene an expert scientific panel
to review methods to prevent mitochondrial disease. The
expert panel has now completed their report (http://www.hfea.
gov.uk/6372.html). This report states, ‘Although optimistic
about the potential of these techniques, the panel recommends
a cautious approach and advises that this research is carried
out, and the results taken into account, before the techniques
can be considered safe and effective for clinical use’. In other
countries that regulate human embryo research, there is very
little legislation that refers specifically to nuclear transfer techniques to prevent transmission of mtDNA disease. However,
other countries are considering these issues and, for example,
a legislative review of the law related to embryo research is
being undertaken in Australia and recommendations have
been made to allow nuclear genome transfer between embryos
for mitochondrial studies (The Australian Academy of
Science, 2011). Further detail of legislation in different countries is available in a recent background paper prepared for the
Nuffield Council on Bioethics www.nuffieldbioethics.org/.../
Germline_therapies_background_paper.pdf.
All nuclear genome transplantation studies so far have either
used animal (including non-human primate) oocytes or zygotes
or abnormally fertilized human zygotes. To assess safety and efficacy, it will be important to perform a systematic analysis of
metaphase-II spindle transfer and pronuclear transfer in
normal human oocytes and zygotes. This should include
Human Molecular Genetics, 2011, Vol. 20, Review Issue 2
optimizing the manipulations to limit the size of the karyoplast
and the fusion techniques between the karyoplast and the recipient oocyte/zygote. In addition, it will be important to optimize
vitrification and cryopreservation procedures in order to minimize loss of oocyte/zygote viability during cryostorage and subsequent transplantation of the nuclear genome. The efficacy of the
two techniques should also be evaluated by studying embryo development, and epigenetic and genome stability after karyoplast
transfer. Finally, since the goal is to minimize mtDNA carryover
during karyoplast transfer, this will have to be evaluated as well
as establishing whether residual karyoplast-associated mtDNA
partitions equally between cells during embryo development.
CONCLUSIONS
This is an exciting time in terms of research to provide treatments and prevent disease transmission for patients with
mtDNA disease. It is important that the field moves from
simply documenting new diseases and mutations to effectively
helping patients with new therapeutic approaches. Most recent
progress has been made in the area of disease prevention but
further work in this area is essential before such interventions
are made available to patients. However, whilst these studies
are ongoing, it is crucial that there is continued debate about
the ethical and regulatory issues surrounding these innovative
IVF-based techniques so that any advances can rapidly be converted to improved patient care.
Conflict of Interest statement. None declared.
FUNDING
Our work in this area has been funded by the Muscular Dystrophy Campaign, The Wellcome Trust (074454/Z/04/Z),
Medical Research Council (G0601157, G0601943), One
North East, UK NIHR Biomedical Research Centre for
Ageing and Age-Related Disease and the Newcastle University Centre for Brain Ageing and Vitality supported by
BBSRC, EPSRC, ESRC and MRC as part of the Cross-council
Lifelong Health and Wellbeing Initiative (G0700718).
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