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Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases (1). 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 zygotes (2) 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. After optimization, mtDNA carryover was reduced to, Predicting the risk of serious disease in children of women who carry mtDNA mutations is complicated by a number of factors. Mutations in mtDNA can be either homoplasmic (all copies [...]

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births (1-3) and at least 1 in 10,000 adults in the UK are affected by mtDNA disease (4). Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease (5,6). Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans., Mitochondrial DNA is present in all cells in multiple copies. Mutations in it are maternally transmitted7. In patients with mtDNA disease, either all mtDNA copies are mutated (homoplasmy) or there [...]

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 [micro]M azide resulted in 48 [+ or -] 3% and 56 [+ or -] 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 [+ or -] 39 pmol/min/mg (mean [+ or -] SE) in untreated cells. This increased to 669 [+ or -] 69 and 823 [+ or -] 83 pmol/min/mg in cells treated with 50 and 75 [micro]M azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.

A heterozygous dominant mutation in POLG2 is described, the gene encoding the p55 accessory subunit of pol gama, that causes progressive external opthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase-(COX) deficient muscle fibres. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibres and results in the clinical phenotype.

The understanding of the fixation of mutations within human tissues and their subsequent clonal expansion is a considerable problem, of which little is known. We have previously shown that nononcogenic mutations in the mitochondrial genome occur in one of a number of morphologically normal colonic crypt stem cells, the progeny of which later occupy the whole crypt. We propose that these wholly mutated crypts then clonally expand by crypt fission, where each crypt divides into two mutated daughter crypts. Here we show that (i) mutated crypts in the process of fission share the same mutated mitochondrial genotype not present in neighboring cytochrome c oxidase-positive crypts (the odds of this being a random event are [greater than or equal to] 2.48 x [10.sup.9]:1); (ii) neighboring mutated crypts have the same genotype, which is different from adjacent cytochrome c oxidase-positive crypts; (iii) mutated crypts are clustered together throughout the colon; and (iv) patches of cytochrome c oxidase-deficient crypts increase in size with age. We thus demonstrate definitively that crypt fission is the mechanism by which mutations spread in the normal human colon. This has important implications for the biology of the normal adult human colon and possibly for the growth and spread of colorectal neoplasms. fission | mitochondria

A family with mitochondrial disease was found to have an mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II (COX II) gene. The mutation brings lower levels of the protein and a mitochondrial encephalomyopathy. It is predicted that the T-->C transition changes the initiating methionine to threonine. The mutation load was at 67% in muscle from the index case and at 91% in muscle from the clinically affected son of the patient. An isolated COX deficiency was seen in muscle biopsy samples. The mutation is a new mechanism through which mtDNA mutations can cause disease-defective translation initiation.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.07.002 Byline: Robert W. Taylor (1)(2), Douglass M. Turnbull (1)(2)(3) Abstract: In this issue of Cell, describe the nuclear encoded protein MTERF3 as a negative regulator of mitochondrial DNA transcription initiation. This study highlights a mechanism by which mitochondrial DNA transcription (and therefore oxidative phosphorylation) may be regulated in response to alterations in the cell's physiological and metabolic demands. Author Affiliation: (1) Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, The Medical School, Newcastle University, Newcastle upon Tyne, UK (2) Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, UK (3) Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK

Author(s): Louise A. Hyslop; Paul Blakeley; Lyndsey Craven; Jessica Richardson; Norah M. E. Fogarty; Elpida Fragouli; Mahdi Lamb; Sissy E. Wamaitha; Nilendran Prathalingam; Qi Zhang; Hannah OKeefe; Yuko Takeda; Lucia [...]

Author(s): Douglass M. Turnbull [1]; Robert N. Lightowlers [1] The mitochondrial DNA (mtDNA) diseases are now recognized as an important group of genetic disorders. Their clinical manifestations are highly variable [...]