Transmission of mitochondrial DNA following assisted reproduction and nuclear transfer

Review of the articleMitochondria are the organelles responsible for producing the majority of a cell's ATP and also play an essential role in gamete maturation and embryo development. ATP production within the mitochondria is dependent on proteins encoded by both the nuclear and the mitochondrial genomes, therefore co-ordination between the two genomes is vital for cell survival. To assist with this co-ordination, cells normally contain only one type of mitochondrial DNA (mtDNA) termed homoplasmy. Occasionally, however, two or more types of mtDNA are present termed heteroplasmy. This can result from a combination of mutant and wild-type mtDNA molecules or from a combination of wild-type mtDNA variants. As heteroplasmy can result in mitochondrial disease, various mechanisms exist in the natural fertilization process to ensure the maternal-only transmission of mtDNA and the maintenance of homoplasmy in future generations. However, there is now an increasing use of invasive oocyte reconstruction protocols, which tend to bypass mechanisms for the maintenance of homoplasmy, potentially resulting in the transmission of either form of mtDNA heteroplasmy. Indeed, heteroplasmy caused by combinations of wild-type variants has been reported following cytoplasmic transfer (CT) in the human and following nuclear transfer (NT) in various animal species. Other techniques, such as germinal vesicle transfer and pronuclei transfer, have been proposed as methods of preventing transmission of mitochondrial diseases to future generations. However, resulting embryos and offspring may contain mtDNA heteroplasmy, which itself could result in mitochondrial disease. It is therefore essential that uniparental transmission of mtDNA is ensured before these techniques are used therapeutically

Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns

Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic die

Neck muscle cross-sectional area, brain volume and cognition in healthy older men; A cohort study

BACKGROUND: Two important consequences of the normal ageing process are sarcopenia (the age-related loss of muscle mass and function) and age-related cognitive decline. Existing data support positive relationships between muscle function, cognition and brain structure. However, studies investigating these relationships at older ages are lacking and rarely include a measure of muscle size. Here we test whether neck muscle size is positively associated with cognition and brain structure in older men. METHODS: We studied 51 healthy older men with mean age 73.8 (sd 1.5) years. Neck muscle cross-sectional area (CSA) was measured from T1-weighted MR-brain scans using a validated technique. We measured multiple cognitive domains including verbal and visuospatial memory, executive functioning and estimated prior cognitive ability. Whole brain, ventricular, hippocampal and cerebellar volumes were measured with MRI. General linear models (ANCOVA) were performed. RESULTS: Larger neck muscle CSA was associated with less whole brain atrophy (t = 2.86, p = 0.01, partial eta squared 17%). Neck muscle CSA was not associated with other neuroimaging variables or current cognitive ability. Smaller neck muscle CSA was unexpectedly associated with higher prior cognition (t = −2.12, p < 0.05, partial eta squared 10%). CONCLUSIONS: In healthy older men, preservation of whole brain volume (i.e. less atrophy) is associated with larger muscle size. Longitudinal ageing studies are now required to investigate these relationships further

Clinical pharmacology of nurses

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The evolution and outcome of cavitating periventricular leukomalacia in infancy. A study of 46 cases

Peer Reviewe

Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase &gamma; catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (&lt;10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (&gt;40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (&gt;20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization

Flow cytometry in the study of mitochondrial respiratory chain disorders

We have developed a flow cytometric assay to measure the oxidative capacity of cultured lymphoblasts as a possible screening test for patients suspected of having a defect of the mitochondrial respiratory chain. Cells were incubated overnight in serum free media, followed by incubation with dihydroethidium with and without rotenone, and then analysed using flow cytometry to measure fluorescence. Inhibition with rotenone gave an increase in fluorescence compared to uninhibited cells. The change in fluorescence was significantly lower in four of the six patient cell lines, with a correlation between the activity of complex I and change in fluorescence. This method may be applicable to cell lines with defects in other complexes of the respiratory chain.9 page(s

The Effects of Nuclear Reprogramming on Mitochondrial DNA Replication

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers