Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases1. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof of concept studies involving abnormally fertilized human zygotes2 were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. Following optimisation, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.

[1]  S. Dimauro,et al.  Human mitochondrial DNA: roles of inherited and somatic mutations , 2012, Nature Reviews Genetics.

[2]  Cole Trapnell,et al.  TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions , 2013, Genome Biology.

[3]  D. Turnbull,et al.  The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations. , 2001, Nucleic acids research.

[4]  K. Niakan,et al.  Human pre-implantation embryo development , 2012, Development.

[5]  B. Levy,et al.  Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder. , 2012, Fertility and sterility.

[6]  Robert W. Taylor,et al.  Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation , 2002, Nature Genetics.

[7]  Laura C. Greaves,et al.  Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing , 2014, PLoS genetics.

[8]  Patrick F Chinnery,et al.  Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease , 2010, Nature.

[9]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[10]  N. Treff,et al.  Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection: a novel, paired randomized controlled trial using DNA fingerprinting. , 2012, Fertility and sterility.

[11]  Chris Mason,et al.  International community consensus standard for reporting derivation of human embryonic stem cell lines. , 2007, Regenerative medicine.

[12]  Geoffrey E. Hinton,et al.  Visualizing Data using t-SNE , 2008 .

[13]  L. Hyslop,et al.  Concise Reviews: Assisted Reproductive Technologies to Prevent Transmission of Mitochondrial DNA Disease , 2015, Stem cells.

[14]  D. Wallace,et al.  Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. , 2013, Cold Spring Harbor perspectives in biology.

[15]  I. D. de Coo,et al.  Preimplantation genetic diagnosis in mitochondrial DNA disorders: challenge and success , 2013, Journal of Medical Genetics.

[16]  Pgd,et al.  Preimplantation genetic diagnosis. , 2019, Fertility and sterility.

[17]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[18]  M. Herbert,et al.  Meiosis and maternal aging: insights from aneuploid oocytes and trisomy births. , 2015, Cold Spring Harbor perspectives in biology.

[19]  Marni J. Falk,et al.  Limitations of preimplantation genetic diagnosis for mitochondrial DNA diseases. , 2014, Cell reports.

[20]  L. Hyslop,et al.  Egg sharing for research: a successful outcome for patients and researchers. , 2012, Cell stem cell.

[21]  S. Mitalipov,et al.  Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck. , 2012, Cell reports.

[22]  P. Chinnery,et al.  Preventing the transmission of pathogenic mitochondrial DNA mutations: can we achieve long-term benefits from germ-line gene transfer? , 2013, Human reproduction.

[23]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[24]  Jenny C. Taylor,et al.  Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation , 2014, Journal of Medical Genetics.

[25]  Kay Elder,et al.  Defining the three cell lineages of the human blastocyst by single-cell RNA-seq , 2015, Development.

[26]  J. S. John,et al.  The Relationship Between Pluripotency and Mitochondrial DNA Proliferation During Early Embryo Development and Embryonic Stem Cell Differentiation , 2009, Stem Cell Reviews and Reports.

[27]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[28]  Hongkai Ji,et al.  Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells , 2015, Genes & development.

[29]  D. Egli,et al.  Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants , 2012, Nature.

[30]  D. Solter,et al.  Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. , 1983, Science.

[31]  I. D. de Coo,et al.  PGD and heteroplasmic mitochondrial DNA point mutations: a systematic review estimating the chance of healthy offspring. , 2012, Human reproduction update.

[32]  Stephen A. Roberts,et al.  Elective Single Embryo Transfer: Guidelines for Practice British Fertility Society and Association of Clinical Embryologists , 2008, Human fertility.

[33]  L. Hyslop,et al.  A Novel Isolator-Based System Promotes Viability of Human Embryos during Laboratory Processing , 2012, PloS one.

[34]  A. Suomalainen,et al.  Tissue- and cell-type–specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model , 2013, Proceedings of the National Academy of Sciences.

[35]  Iain G. Johnston,et al.  Mitochondrial DNA disease and developmental implications for reproductive strategies , 2014, Molecular human reproduction.

[36]  A. Munnich,et al.  Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis , 2005, Journal of Medical Genetics.

[37]  H. Schatten,et al.  The role of centrosomes in mammalian fertilization and its significance for ICSI. , 2009, Molecular human reproduction.

[38]  J. Fenwick,et al.  Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro. , 2002, Human reproduction.

[39]  Helen E White,et al.  Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing. , 2005, Genetic testing.

[40]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[41]  A. Munnich,et al.  Data from artificial models of mitochondrial DNA disorders are not always applicable to humans. , 2014, Cell reports.

[42]  S. Mitalipov,et al.  Towards germline gene therapy of inherited mitochondrial diseases , 2012, Nature.