Tuesday, January 10, 2017

Mitochondrial replacement 2

Kang E, Wu J, Gutierrez NM, Koski A, Tippner-Hedges R, Agaronyan K, Platero-Luengo A, Martinez-Redondo P, Ma H, Lee Y, Hayama T, Van Dyken C, Wang X, Luo S, Ahmed R, Li Y, Ji D, Kayali R, Cinnioglu C, Olson S, Jensen J, Battaglia D, Lee D, Wu D, Huang T, Wolf DP, Temiakov D, Belmonte JC, Amato P, Mitalipov S. Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations. Nature. 2016 Dec 8;540(7632):270-275.

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.

In the current study, Dr. Mitalipov and colleagues collected the oocytes from four women who have children with Leigh syndrome and MELAS and donor eggs from 11 healthy women, screened to confirm they did not carry inherited pathogenic mutations in their mtDNA. Subsequent genetic testing of blood, skin fibroblasts, and urine revealed that the mitochondrial disease of one of the families was not maternally inherited, so only three families were eligible for the replacement therapy…

“When we replace mtDNA we try to do it thoroughly, but because we're dealing with about a half-million copies of mtDNA in the egg, moving it all and replacing with the donor's is not a simple task,” explained Dr. Mitalipov. “That 1 percent [maternal mtDNA] was always kind of out there, but we thought it would stay at 1 percent. And 1 percent mutation, as far as we know, does not cause problems for cells, or the muscles or other vital organs and tissues, so there would be no disease. It was always thought that you could disregard it.

”But while the majority maintained greater than 99 percent donor mtDNA at 10 weeks, four of 26 blastocyst-derived embryonic stem cell lines reverted back to the maternal mtDNA haplotype.
To try to learn more about why the reversal occurred, the researchers zeroed in on a portion of the mtDNA known as the non-coding D-loop region, which initiates replication of the entire genetic sequence. They found that two of the four embryonic stem lines that reverted back to the diseased maternal mtDNA contained guanosine additions in the region of the maternal D-loops, while the healthy donor lines carried guanosine deletions.

The phenotype with the guanosine deletions replicated slower, while the phenotype with the guanosine additions replicated faster. The goal is to have the healthy mitochondrial DNA replicate faster than any contaminating diseased mitochondrial DNA.

Dr. Mitalipov and colleagues concluded that recipient eggs should always be checked to avoid the guanosine deletions; otherwise the  diseased mtDNA will out replicate the healthy DNA.
“Even though we don't yet have a thorough answer for every type of combination, the beginning is there,” Dr. Mitalipov explained. “We can say that there is a phenomenon of reversal, and it looks like to avoid it you would have to somehow match not [necessarily] the entire mtDNA molecule. [We have learned that the] regulatory region is more important than anything else.”


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