Novel reproductive technologies to prevent mitochondrial disease.

Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases

The challenges of mitochondrial replacement.

BackgroundMitochondrial disease are multi-system disorders affecting virtually any organ and cause significant morbidity. The prevalence of mitochondrial DNA (mtDNA) disease is estimated at 1 in 5000, but epidemiological studies suggest...

While the fertilized egg inherits its nuclear DNA from both parents, the mitochondrial DNA is strictly maternally inherited. Cells contain multiple copies of mtDNA, each of which encodes 37 genes, which are essential for energy production by oxidative phosphorylation. Mutations can be present in all, or only in some copies of mtDNA. If present above a certain threshold, pathogenic mtDNA mutations can cause a range of debilitating and fatal diseases. Here, we provide an update of currently available options and new techniques under development to reduce the risk of transmitting mtDNA disease from mother to child. Preimplantation genetic diagnosis (PGD), a commonly used technique to detect mutations in nuclear DNA, is currently being offered to determine the mutation load of embryos produced by women who carry mtDNA mutations. The available evidence indicates that cells removed from an eight-cell embryo are predictive of the mutation load in the entire embryo, indicating that PGD provides an effective risk reduction strategy for women who produce embryos with low mutation loads. For those who do not, research is now focused on meiotic nuclear transplantation techniques to uncouple the inheritance of nuclear and mtDNA. These approaches include transplantation of any one of the products or female meiosis (meiosis II spindle, or either of the polar bodies) between oocytes, or the transplantation of pronuclei between fertilized eggs. In all cases, the transferred genetic material arises from a normal meiosis and should therefore, not be confused with cloning. The scientific progress and associated regulatory issues are discussed. Stem Cells2015;33:639–645

Mutations of mitochondrial (mt)DNA are a major cause of morbidity and mortality in humans, accounting for approximately two thirds of diagnosed mitochondrial disease. However, despite significant advances in technology since the discovery of the first disease-causing mtDNA mutations in 1988, the comprehensive diagnosis and treatment of mtDNA disease remains challenging. This is partly due to the highly variable clinical presentation linked to tissue-specific vulnerability that determines which organs are affected. Organ involvement can vary between different mtDNA mutations, and also between patients carrying the same disease-causing variant. The clinical features frequently overlap with other non-mitochondrial diseases, both rare and common, adding to the diagnostic challenge. Building on previous findings, recent technological advances have cast further light on the mechanisms which underpin the organ vulnerability in mtDNA diseases, but our understanding is far from complete. In this review we explore the origins, current knowledge, and future directions of research in this area.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

A menage a trois (French for “household of three”) is a domestic arrangement in which three people having romantic and/or sexual relations with each other occupy the same household—From Wikipedia, the Free Encyclopedia Inherited diseases caused by mitochondrial gene (mtDNA) mutations affect at least one in 5000–10,000 children and are associated with severe clinical symptoms [1]. Mitochondrial disease mainly affects children, but is also common in adults due to deteriorating mitochondrial function with age [1]. Novel reproductive techniques designed to replace mutated mtDNA in oocytes or early embryos have been proposed to prevent transmission of disease from parents to their children [2, 3]. The aim is ultimately to prevent children from inheriting genetic diseases caused by mutations in DNA housed by their mitochondria—components of cells, which produce energy. Mitochondrial DNA (mtDNA) mutations are relatively a common cause of progressive disorders that can be severe or even life threatening [4]. There is currently no cure for these disorders; therefore, recent research has been focused on attempting to prevent the transmission of these maternally inherited mutations. These research techniques have been referred to with several terms, including “mitochondria replacement,” “mitochondrial manipulation,” “oocyte modification,” “three-person embryos,” “three-parent babies,” and “nuclear genome transfer.” The “mitochondria replacement” is a new approach that is being researched with the goal of allowing a woman who has mutations in her mtDNA to lessen the risk of passing on inherited mitochondrial disease to her child. The techniques being developed are variations on combining the nuclear DNA from an egg of an affected woman with the mtDNA of an unaffected woman’s egg. A resulting child would possess genes from three adults, and this altered genome would be passed on to succeeding generations.

BackgroundFamilies with a child who died of severe, maternally inherited mitochondrial DNA (mtDNA) disease need information on recurrence risk. Estimating this risk is difficult because of (a) heteroplasmy—the coexistence of...

Chapter 14 - Reproductive options in mitochondrial disease

Pathogenic mutations in mitochondrial DNA (mtDNA) result in a wide spectrum of disorders with a prevalence of over 1 in 5,000. The thousands of mtDNA copies that are present in every cell are virtually identical in unaffected individuals, but in individuals with mtDNA-related disease there may be a mixture of normal and mutated mtDNA molecules (heteroplasmy) which can vary among tissues. Prenatal (PND) and preimplantation (PGD) diagnosis of many types of mtDNA diseases are unreliable, since it is hard to predict with certainty the way in which heteroplasmic mtDNA mutations segregate within tissues. This makes the prediction of recurrence risk immensely difficult. Monnot et al. (Hum Mutat 32:116–125, 2011) perform an elegant study examining the segregation of one of the most common mtDNA mutations (m.3243A>G, causing MELAS) throughout the entire embryofetal development period. To determine whether m.3243A>G segregation is governed by random drift in somatic tissues during early development, the authors quantified the levels of mutant to wild-type mtDNA. Their results demonstrated a uniform tissue distribution of mtDNA mutant load in somatic tissues from the developing fetus sampled between 12–25 weeks, indicating a lack of selection. The authors also measured mtDNA mutant load in individual blastomeres in a large number of samples from embryos of a single patient and showed that there is very little inter-blastomere variability. Understanding this segregation pattern is of critical importance to the success of PGD, whereby the biopsied blastomere has to be representative of the genetic composition of the embryo as a whole. Should this distribution not be uniform, then there is the possibility of misdiagnosis, resulting in either eligible embryos being unnecessarily discarded or the birth of a severely affected individual. Given the uncertainties in reproductive counselling for mitochondrial disorders, such studies as this are vital for improving the reliability of prenatal and preimplantation diagnosis.

SummaryThe diagnosis of mitochondrial DNA (mtDNA) diseases remains challenging with next-generation sequencing, where bioinformatic analysis is usually more focused on the nuclear genome. We developed a workflow for the evaluation of mtDNA diseases and applied it in a large European rare disease cohort (Solve-RD). A semi-automated bioinformatic pipeline with MToolBox was used to filter the unsolved Solve-RD cohort for rare mtDNA variants after validating this pipeline on exome datasets of 42 individuals previously diagnosed with mtDNA variants. Variants were filtered based on blood heteroplasmy levels (≥1%) and reported association with disease. Overall, 10,157 exome and genome datasets from 9,923 affected individuals from 9,483 families within Solve-RD met the quality inclusion criteria. 136 mtDNA variants in 135 undiagnosed individuals were prioritized using the filtering approach. A focused MitoPhen-based phenotype similarity scoring method was tested in a separate genetically diagnosed “phenotype test cohort” consisting of nuclear gene and mtDNA diseases using a receiving operator characteristic evaluation. We applied the MitoPhen-based phenotype similarity score of >0.3, which was highly sensitive for detecting mtDNA diseases in the phenotype test cohort, to the filtered cohort of 135 undiagnosed individuals. This aided the prioritization of 34 out of 37 (92%) individuals who received confirmed and likely causative mtDNA disease diagnoses. The phenotypic evaluation was limited by the quality of input data in some individuals. The overall pipeline led to an additional diagnostic yield of 0.4% in a cohort where mitochondrial disease was not initially suspected. This highlights the value of our mtDNA analysis pipeline in diverse datasets.

In the recent past, human genetics and in vitro fertilization (IVF) have undergone various advances to combat with several congenital and developmental disorders. These advances are a boon for the families and patients who were restricted from having a child due to one or the other reasons. One such reason is the mitochondrial DNA (mtDNA) mutations, which are definitely transmitted from the mother to the child due to uniparental/maternal inheritance of mitochondria. Depending upon the range of the mutation (mutation loads) present, the mtDNA mutation leads to various devitalizing to fatal disorders, all of which are incurable. Scientists and researchers developed a technique known as mitochondrial donation technique or mitochondrial replacement therapy (MRT) to combat with the mtDNA mutations. The technique relies on the replacement of faulty mitochondria in the mother's egg with the normal wild-type from a donor female resulting in a "three-parent baby." On the other side, forensic scientists and anthropologists continuously explore the mtDNA in various medicolegal cases and in uncoupling the mystery of human origin and migration respectively. In this regard, we explored the genetic, forensic and ethical aspects of a "three-parent baby." The present communication also attempts to highlight the importance and limitations of the MRT technique/three-parent baby in a medicolegal context.

Expanding DdCBE-mediated targeting scope to aC motif preference in rat

Editor—Mitochondrial DNA (MtDNA) is highly polymorphic. Each person is estimated to differ from another on average at about 25 base pairs among the 16 569 that comprise the mitochondrial genome.1...

Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder