mtDNA mutations variously impact mtDNA maintenance throughout the human embryofetal development. S. RONDEAU1, S. MONNOT1, P. VACHIN1, E. HERZOG1, B. BESSIERES1, N. GIGAREL1, D. SAMUELS2, L. HESTERS3, N. FRYDMAN3, G. CHALOUHI4, S. GOBIN LIMBALLE1, M. RIO1, A. ROTIG1, A.-S. LEBRE1, A. BENACHI5, L. SALOMON4, A. MUNNICH1, J.-P. BONNEFONT1, J. STEFFANN1 1) Genetics, INSERM U781, Necker-Enfants Malades Hospital, Paris, France; 2) Center for Human Genetics Research, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, USA; 3) Reproductive Genetics, Antoine-Beclere Hospital, Clamart, France; 4) Obstetrics, Necker-Enfants Malades Hospital, Paris, France; 5) Obstetrics, Antoine-Beclere Hospital, Clamart, France.
Mitochondrial DNA (mtDNA) mutations cause serious disorders with maternal inheritance and a high transmission risk, resulting in common requests for preimplantation or prenatal diagnoses. These procedures are hampered by the lack of data on the pathophysiology of mtDNA mutations during human development. Specifically, it is not known whether and how mtDNA mutations impact the overall mtDNA content throughout human embryofetogenesis. We collected oocytes, preimplantation embryos, whole placentas and fetal tissues at various stages of development, from controls and carriers of m.3243A>G (MTTL1), m.8344A>G (MTTK) and m.8993T>G (MTATP6), responsible for MELAS, MERRF, and NARP syndromes, respectively. We devised a test assessing simultaneously mtDNA copy number (CN) and mutant load in single cells. mtDNA CN i)was identical in control oocytes and embryos, in agreement with mtDNA replication silencing during the first embryonic cleavages; ii)gradually increased from the germinal vesicle to the blastocyst stage in m.3243A>G cells (p<0.01), suggestive of a mutation-dependent induction of mtDNA replication; iii)correlated with the m.3243A>G mutant load (p<0.001), suggesting some compensation for the respiratory chain dysfunction; iv)was identical in m.8344A>G vs control embryos, indicating that modulation of the mtDNA CN depends on the mutation type. Analyses of placentas (multiple biopsies) showed that mtDNA CN i)did not vary within a placenta, ii)significantly increased in m.3243A>G vs control at 11-GW (p <0.01), iii)decreased thereafter, becoming identical to controls at delivery. These data could be accounted for by a placental energy demand maximal at the end of the 1st trimester. Analyses of 7 fetal tissues (12-22 GW) showed that mtDNA CN was i)similar in all control tissues apart from the heart (p<0.01); ii)similar in m.3243A>G vs control tissues apart from the lung (p<0.01); iii)lower in m.8993T>G muscle, heart, and liver vs control (p=0.01, 0.01, 0.001, respectively). Mutant loads were identical in all tissues from a given fetus, indicating an absence of mutant load/mtDNA CN correlation. These data highlight the complex relationships between mtDNA mutations and mtDNA content, depending on mutation types, mutant loads, cell types and development stages. Transcriptome studies and measurement of mtDNA replication should help us in unravelling the molecular bases of these observations, of particular relevance for therapeutic approaches in mtDNA disorders.
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