Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations

Abstract

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.

Assisted reproductive technologies to prevent human mitochondrial disease transmission

“…Likewise, fertilization and blastulation rates were similar among human MST embryos with differing degrees of divergence between the karyoplast and cytoplast mtDNA haplotypes, ranging from close (six SNPs), to medium (33 SNPs), to distant (57 SNPs), supporting the conclusion that MST does not adversely affect mito-nuclear DNA interactions during human embryonic development in vitro 38 . These and other findings lead to the conclusion that switching nuclear genomes does not detectably affect mitochondrial gene expression [38][39][40] .…”

“…These and other findings lead to the conclusion that switching nuclear genomes does not detectably affect mitochondrial gene expression [38][39][40] .…”

“…However, because mitochondria are thought to be quiescent during pre-implantation stages of development, the panel recommended carefully examining the behavior of carried-over mtDNA in a context where mtDNA replication occurs, namely in ES cells derived from the embryos and in differentiated cell types obtained from these (http://hfeaarchive.uksouth.cloudapp.azure.com/www.hfea.gov.uk/docs/Third_ Mitochondrial_replacement_scientific_review.pdf). All three studies summarized above [38][39][40] derived ES cells from blastocysts generated by MST or PNT and, in the case of Kang et al 38 and Yamada et al 42 , also by somatic cell nuclear transfer. In most cases, these lines were passaged extensively in order to determine whether the initial low levels of carried-over mtDNA were maintained after multiple rounds of cell division in vitro.…”

“…It has also been suggested that mtDNA haplotypes with specific Dloop polymorphisms might be amplified preferentially compared to others following MST, leading to reversion 38 . This could be addressed in MRT by selecting oocyte recipients with D-loop sequences similar to those of spindle-associated mtDNA, or with a known replicative advantage.…”

Prévenir la transmission des mutations de l’ADN mitochondrial par don de cytoplasme : où en est-on ?

“…Le transfert nucléaire entre ovocytes en métaphase de deuxième division méiotique semble une alternative efficace (Figure 1B), une équipe ayant obtenu la naissance de deux singes rhésus porteurs de moins de 3 % d'ADNmt du karyoplaste [10]. Très récem-ment, cette procédure a été appliquée à des ovocytes humains porteurs de mutations de l'ADNmt [11]. Les embryons résultant de ce transfert au stade préim-plantatoire portaient des taux d'ADNmt contaminant très faibles (moins de 1 %).…”