Diagnostic and therapeutic approaches to prevent the transmission of heritable mitochondrial diseases

Mitochondria are the energy producing organelles in a cell, possessing their own genetic content, the mitochondrial DNA (mtDNA). Defects in the mtDNA or in the chromosomal DNA may cause mitochondrial diseases. Normally, all the copies of mtDNA in a cell are identical, also known as ‘homoplasmy’. However, more than one type of mtDNA variants may exist within the same cell, a state called ‘heteroplasmy’. Most of the pathogenic mtDNA mutations are heteroplasmic in nature with cells containing both wild type (WT) and mutant mtDNAs. Unlike nuclear DNA, mtDNAs are transmitted through the maternal lineage only. No therapeutic remedy is yet available to cure mtDNA diseases. Therefore, we attempted to optimize the diagnostic and therapeutic approaches to avoid the transmission of pathogenic mtDNA. First, we investigated the reliability and suitability of the technique of pre-implantation genetic diagnosis (PGD) at different stages of embryonic development using a heteroplasmic BALB/cOlaHsd mouse model, containing two polymorphic mtDNA sequence variants coming from homoplasmic BALB/cByJ and homoplasmic NZB/OlaHsd mice. We observed that the level of mtDNA heteroplasmy in polar bodies (PBs) was poorly correlated with their corresponding oocytes and zygotes, whereas trophectoderm (TE) cells were strongly correlated with their corresponding blastocysts. In spite of the strong correlation between individual blastomeres within an embryo, occasional interblastomere variation made it risky to rely on single blastomere biopsy only for the accurate diagnosis. Next, we carried out PGD in a patient who was a carrier of the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (m.3243A>G; MELAS mutation) with the aim to avoid the transmission of mutant mtDNA to the offspring. We showed that the transfer of a blastocyst with undetectable mutant load resulted in the birth of a healthy baby boy, without detectable mutation in the peripheral blood, buccal swab and urine cells. When no embryos are suitable for embryo transfer because of the high mutation loads, mitochondrial replacement therapy (MRT) could be an option to avoid the transmission of mtDNA disease to the offspring. Therefore, we investigated the technology of germ line nuclear genome transfer (NT) in unfertilized and fertilized oocytes as a potential therapeutic approach in a mouse model. The mtDNA carry-over and embryonic development potential was determined and compared between various NT techniques. We demonstrated that metaphase-II spindle transfer (MST) and pronuclear transfer (PNT) were equally potent to minimize the mtDNA carry-over without compromising embryonic developmental competence. In contrast, in spite of the undetectable mtDNA carry-over after germinal vesicle nuclear-transfer (GVT), no blastocysts were formed. Finally, we investigated the mtDNA heterogeneity in embryonic stem cells (mESCs) derived from the heteroplasmic BALB/cOlaHsd mice before and after differentiation, both in colonies and at single cell level. In addition, the level of mtDNA heteroplasmy was compared between the pluripotent stem cells (PSCs) and their embryonic counterparts they originated from. The mtDNA heteroplasmy was heterogeneously distributed at the cellular level, both in pluripotent and in differentiated mouse ESCs. Level of mtDNA heteroplasmy in TE cells was more informative about the level of heteroplasmy in the subsequently derived ESCs compared to their corresponding second PBs. We found that the single cell heterogeneity displayed homoplasmic condition for BALB mtDNA and NZB mtDNA haplotypes. To conclude, our results in the mouse model and in human show that the level of mtDNA heteroplasmy in the TE cells is corresponding with the mtDNA heteroplasmy in the whole blastocyst. Our data also show that preventing the transmission of heritable mtDNA disorders is possible by performing PGD on blastocysts in a MELAS mutation carrier. Our results also showed that performing MST or PNT could minimize the mtDNA carry-over without compromising embryonic development competence in mice. These results demonstrate that PGD and NT are potential approaches to avoid the transmission of heritable mtDNA diseases. However, the offspring born after these interventions must be followed throughout life to assess the safety and efficacy of the approaches. Our newfound data in ESCs showed a large degree of heterogeneity in the level of mtDNA heteroplasmy in mouse ESCs, which could be linked to the inter-cellular variation in individuals with heteroplasmic mtDNA. We also found the passage-associated increase in heteroplasmy, indicating the possible proliferation of mtDNA haplotype upon progressive passages in culture