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Transmission of mitochondrial DNA disorders: possibilities for the future
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The Lancet, Current Issue, Volume 368, Number 9529, 01 July 2006
DT Brown PhD a, M Herbert PhD b, VK Lamb PhD b, Prof PF Chinnery MRCP a, RW Taylor PhD a, Prof RN Lightowlers PhD a, L Craven BSc a, L Cree PhD a, JL Gardner BSc a and Prof DM Turnbull FMed Sci
a. Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, The Medical School, University of Newcastle upon Tyne, Newcastle NE2 4HH, UK
b. Reproductive Medicine, Bioscience Centre, International Centre for Life, Newcastle upon Tyne, UK
Defects of mitochondrial function are increasingly recognised as important causes of disease. The clinical phenotype of mitochondrial diseases is extremely variable, affecting 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 first described,3,4 we have become more efficient at diagnosing patients, but still have little to offer in the way of treatment.1
The mitochondrial genome is small (16-6 kb) and encodes 13 essential subunits of the respiratory chain, as well as 24 RNA molecules needed for intra-mitochondrial protein synthesis. The mitochondrial genome is present in multiple copies within the cell. In healthy individuals, all copies of the mitochondrial genome are identical-called homoplasmy.1 In patients with genetic defects of the mitochondrial genome, a mutation can be present in all copies of the genome (homoplasmic mutation) or in only some copies (heteroplasmic mutation).
Current approaches
Genetic counselling
Mitochondrial DNA is usually maternally inherited. Although there has been one report of paternal mtDNA in the muscle of a patient with a pathogenic 2bp deletion,6 the mutation was not detected in the father's tissues. It could have been present in his germline. Male patients with mtDNA disease should be reassured that there is no evidence of male-to-child transmission of an mtDNA genetic defect to their offspring.
Numerous mtDNA point mutations and rearrangements have been described. Family studies have shown that mutations can arise spontaneously or can be inherited to affect many family members. Mutations differ considerably in the risk of transmission-for example, single mtDNA deletions are often sporadic,7 whereas the mutations that cause Leber's hereditary optic neuropathy are usually transmitted.4 Identifying specific mutations and investigating other family members can guide clinicians on the likelihood of transmission through the germline. Detailed studies of large cohorts of patients also provide valuable information on risk of transmission. For example, an analysis of 226 families in which an mtDNA deletion had been identified in the proband showed that the risk of recurrence in the offspring of an affected mother was 4-11%. If the mother was unaffected, then there was no record of affected siblings of the proband, suggesting the risk of recurrence is low.7
Although maternal transmission of some mtDNA defects is well established, the outcome for specific pregnancies remains unpredictable. For heteroplasmic disorders, this is largely due to the genetic bottleneck that occurs during development, which results in considerable variation in the mutated DNA load to different offspring.1 Since many of the clinical features correlate with the ratio of mutated to wild-type (or the absolute amount of wild-type) mtDNA, the outcome for each pregnancy is difficult to predict.8
In homoplasmic mtDNA disorders, the mutation will be transmitted to all offspring. Even in these patients, however, the development of clinical features is difficult to predict since there is variable penetrance,9,10 presumably due to a combination of environmental and nuclear genetic factors. Although progress has been made with the most common homoplasmic mutations presenting with Leber's hereditary optic neuropathy,9,10 genetic counselling remains challenging.
Oocyte donation
Oocyte donation from an unrelated individual would prevent transmission of an mtDNA mutation to an offspring. However, although the oocyte would be fertilised using the father's sperm, the child would have a nuclear genotypic contribution from the donor female. Some potential mothers have said they would be concerned by this (Turnbull DM, unpublished data). In addition, there is considerable delay in many countries in obtaining donor oocytes.
Chorionic villus sampling and amniocentesis
Amniocentesis and chorionic villus sampling are widely used to diagnose autosomal and chromosomal abnormalities, and could be used in mtDNA disorders. When these techniques have been used for patients with specific mtDNA mutations (8993T>G, 8993T>C), the results have influenced the clinical management.11,12 In many heteroplasmic mtDNA disorders, there are often marked tissue-specific differences in the level of heteroplasmy, resulting in a reasonable concern about whether a prenatal sample would accurately predict the outcome for the fetus. However, evidence suggests this is not likely to be a problem.13,14 Although we might be able to predict the outcome for either very high or very low levels of heteroplasmy for some mutations, prediction is difficult for intermediate levels.15
Preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD) allows either analysis of mtDNA from the polar body of unfertilised oocytes, or one or two single cells taken from 6-8 cell embryos, followed by replacement implantation of any healthy embryos into the uterus. Preliminary experiments done by our group (Lamb, Craven, Cree, Gardner) and others (Lin and colleagues1) to determine the mtDNA copy number in individual human blastomeres have shown that each individual cell has on average around 10 - 100,000 copies of mtDNA. Although copy number can vary between cells from different embryos, the high copy number suggests that PGD should be feasible for mtDNA disease. There is a concern that either polar bodies or individual cells might not be representative of total mutational load, although experiments in heteroplasmic mice and human blastomeres suggest homogeneous distribution in the early embryo.16,17
PGD has been successfully used in a family with the 8993T>G NARP mutation,17 but for other mtDNA mutations there remains concern about the level of heteroplasmy and clinical outcome, even though there is evidence from studies in adults in which a link between the level of heteroplasmy and severity of disease for the two common mtDNA point mutations (3243A>G and 8344A>G).18
Development of new techniques
Genetic counselling, PGD, and prenatal diagnosis might be useful for some patients with heteroplasmic mtDNA mutations, but these techniques will be of little value to patients with homoplasmic mtDNA mutations. For these families, it is important to consider the development of techniques to prevent transmission of mtDNA disease.
Cytoplasmic transfer
In this technique, normal mitochondria (in cytoplasts) would be transferred into the oocyte, and thus dilute the effect of any mtDNA defect. Cytoplasmic transfer between human oocytes has been done to try and improve the outcome of assisted reproduction methods.19 Nevertheless, some of the children born were heteroplasmic with low levels of mtDNA from the donor oocyte.20 However, despite the observation that changes in heteroplasmy can occur, it is likely that this technique will have little value in patients with mtDNA disease. Experiments in mice suggest that the amount of mtDNA that can be transferred is less than a third of total mtDNA,5 and thus the relative proportion of mutated to wild-type mtDNA will change little. In addition, there are concerns that cytoplasmic transfer could cause major epigenetic modifications,21 and two of the first 16 pregnancies involving cytoplasmic transfer had chromosomal abnormalities.22
Nuclear or pronuclear transfer
An alternative strategy would be to transfer the nuclear DNA from a mother with mtDNA disease to an enucleated oocyte or embryo from a healthy female.23
Nuclear transfer at the mature oocyte stage
Transfer of nuclear DNA between mature (metaphase II arrested oocytes) with similar techniques to those used in producing cloned embryos is theoretically possible. However, unlike in cloning, the viability and integrity of the oocyte nuclear DNA would need to be maintained. This would be difficult because mature mammalian oocytes do not have a nuclear membrane, hence there would be a large risk of chromosome loss during transfer between oocytes. Furthermore, in the absence of nuclear membrane, visualisation of the DNA would require use of fluorophores, which may well have an adverse effect on subsequent embryonic development.
Germinal vesicle stage karyoplast transfer
Another possibility would be to transfer DNA between immature oocytes in which chromosomes are enclosed in a clearly visible germinal vesicle. Work in mouse oocytes indicates that this approach rescues the effects of induced mitochondrial damage in mouse oocytes.24 However, the efficacy of germinal vesicle transfer is limited by the poor developmental competence of in-vitro matured mouse and human oocytes.25
Pronuclear transfer between single-cell embryos
This involves removal of a karyoplast containing the male and female pronuclei, which are distinct structures that become apparent after fertilisation. Smith and colleagues have shown that in mice the reconstructed embryos contained on average 19% mtDNA of karyoplast origin, although most progeny had a lower percentage and produced exclusively homoplasmic mtDNA of donor oocyte in the offspring.26,27 They measured tissue heteroplasmy levels in a founder female and recorded values that varied with tissue type, from 6% in the lung to 69% in the heart. The progeny were all heteroplasmic, but some in the second and third generation were homoplasmic. Heteroplasmy was detected up to the fifth generation, but with lessened tissue and litter variability, indicating a low but stable, and persistent transmission of both mtDNA species through the maternal line. This approach is effective in reducing the transmission of a pathogenic mtDNA mutation28 in mice. Most importantly, none of the mice generated by pronuclear transfer developed any clinical symptoms associated with the mtDNA mutation.
In September 2005, the UK Human Fertilisation and Embryology Authority approved a research licence application to determine whether pronuclear transfer would be a feasible option for the prevention of transmission of mtDNA disease in human embryos. Approved studies will determine the feasibility of this approach by using abnormally fertilised embryos (tripronuclear) to determine if pronuclear transfer is possible in human embryos. In embryos in which transfer is successful, the percentage of mtDNA of karyoplast origin will be determined to assess the potential carryover from a mother with mtDNA disease. These embryos will also be examined for the presence of either cytogenetic or epigenetic abnormalities.
Conclusion
Genetic advice for families with mtDNA disease is important, since the risks of transmission are different for different mtDNA mutations. Until there is a better general understanding of these conditions, we suggest that clinicians with experience in this area provide the advice, or are at least contacted for advice about the relative risk within a family by the clinical team involved. There has been little experience as regards techniques such as chorionic villus sampling for mtDNA diseases and it will be important that any results of this or other techniques (such as PGD) are made widely available so we can provide appropriate advice for all affected mothers. Developing techniques to improve the chances of a mother with mtDNA disease having a healthy child is a priority for families with mtDNA defects. Experiments in mice suggest that pronuclear transfer could prevent transmission of the mutation, and the recent granting of a research licence by the UK Human Fertilisation and Embryology Authority to enable initial studies on human embryos is a bold decision that enables scientists to assess the feasibility of such an approach in human embryos.
In view of the difficulties in treating mtDNA disease, a priority is to provide families with appropriate genetic advice5 and to consider ways in which we might prevent transmission of mtDNA disease. Here, we discuss current clinical practice, and outline future possibilities for preventing disease transmission, including the use of pronuclear transfer between embryos, an approach which has already been shown to be successful in animal models.
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