Selective elimination of mitochondria mutations in the germline by genome editing
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Karen Montgomery
PROVIDER: E-GEOD-67371 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Currently there is no treatment for mitochondrial disease, a group of devastating inherited disorders caused by mutations in mitochondrial DNA (mtDNA). Here we report a strategy to prevent the germline transmission of mitochondrial diseases. This technique is based on the specific elimination of mutated mtDNA through the use of mitochondria targeted nucleases. Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA.
2015-04-23 | GSE67371 | GEO
Project description:Purpose: To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1). Methods: Next-generation sequencing was performed on DNA samples from 4 affected and 4 unaffected members of a previously reported family with PPCD1 linked to chromosome 20 between D20S182 and D20S195. Custom capture probes were utilized for targeted region capture of the linked interval. Single nucleotide variants (SNVs) and insertions/deletions (indels) were identified using two bioinformatics pipelines and two annotation databases. Candidate variants met the following criteria: quality score â¥20, read depth â¥5X, heterozygous, novel or rare (minor allele frequency (MAF) ⤠0.05), present in each affected individual and absent in each unaffected individual. Structural variants were detected with two different microarray platforms to identify indels of varying sizes. Results: Sequencing reads aligned to the linked region on chromosome 20, and high coverage was obtained across the sequenced region. The majority of identified variants were detected with both pipelines and annotation databases, although unique variants were identified. Twelve SNVs in 10 genes (2 synonymous variants and 10 noncoding variants) and 9 indels in 7 genes met the filtering criteria and were considered candidate variants for PPCD1. Conclusions: Next-generation sequencing of the PPCD1 interval has identified 17 genes containing novel or rare SNVs and indels that segregate with the affected phenotype in an affected family previously mapped to the PPCD1 locus. We anticipate that screening of these candidate genes in other families previously mapped to the PPCD1 locus will result in the identification of the genetic basis of PPCD1. Four affected and 4 unaffected individuals from a single family were analyzed for copy number variation within the PPCD1 disease locus. Array design and analysis is based on genome build hg19.
2016-06-30 | E-GEOD-72617 | biostudies-arrayexpress
Project description:Inherited mitochondrial DNA (mtDNA) diseases transmit maternally and cause severe phenotypes. Since no effective treatment or genetic screening is available, nuclear genome transfer between patients’ and healthy eggs to replace mutant mtDNAs holds promises. Since polar body contains very few mitochondria and share same genomic material as oocyte, here we perform polar body transfer to prevent the transmission of inherited mtDNA variants. We compare the value of different germline genome transfer (spindle-chromosome, pronuclear, first and second polar body) in a mouse model. Reconstructed embryos support normal fertilization and produce live offspring. Strikingly, genetic analysis confirms F1 generation after polar body transfer possesses minimal donor mtDNA carry-over compared with spindle-chromosome (low/medium carry-over) and pronuclear (medium/high carry-over) transfer. Moreover, mtDNA genotype remains stable in F2 generation of progeny after polar body transfer. Our preclinical model demonstrates polar body transfer holds great potential in preventing the transmission of inherited mtDNA diseases. The objective of the present study was to detect genomic aberrations between PB1 and its counterpart, spindle-chromosome complex in human MII oocyte, PB2 and female pronucleus in human zygote at a single-cell level.
2014-06-19 | E-GEOD-56676 | biostudies-arrayexpress
Project description:It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether MSCs transfer mitochondria to the cells under several different conditions of mitochondrial dysfunction, including human pathogenic mitochondrial DNA (mtDNA) mutations. Using biochemical selection methods, we found that exponentially growing cells in restrictive media (uridine and bromodeoxyuridine [BrdU]+) after coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mtDNA identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B 0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. A chemotaxis inhibitory agent blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential “cell therapy-based mitochondrial restoration or mitochondrial gene therapy” for human diseases caused by mitochondrial dysfunction. time series
2011-11-22 | E-GEOD-33831 | biostudies-arrayexpress
Project description:Inherited mitochondrial DNA (mtDNA) diseases transmit maternally and cause severe phenotypes. Since no effective treatment or genetic screening is available, nuclear genome transfer between patients’ and healthy eggs to replace mutant mtDNAs holds promises. Since polar body contains very few mitochondria and share same genomic material as oocyte, here we perform polar body transfer to prevent the transmission of inherited mtDNA variants. We compare the value of different germline genome transfer (spindle-chromosome, pronuclear, first and second polar body) in a mouse model. Reconstructed embryos support normal fertilization and produce live offspring. Strikingly, genetic analysis confirms F1 generation after polar body transfer possesses minimal donor mtDNA carry-over compared with spindle-chromosome (low/medium carry-over) and pronuclear (medium/high carry-over) transfer. Moreover, mtDNA genotype remains stable in F2 generation of progeny after polar body transfer. Our preclinical model demonstrates polar body transfer holds great potential in preventing the transmission of inherited mtDNA diseases.
2014-06-19 | GSE56676 | GEO
Project description:Characterization of copy number alterations and unbalanced breakpoints in human esophageal squamous cell carcinoma cell lines by array-based comparative genomic hybridization. Six cell lines
2013-02-28 | E-GEOD-36115 | biostudies-arrayexpress
Project description:Damage to the mitochondrial genome (mtDNA) severely affects the cell and causes disease. Mutations to mtDNA also accumulate throughout the lifespan of many organisms and may be a proximal cause of aging. There is no effective treatment for ailments caused by mtDNA mutation. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. Experiments using the tractable eukaryote Saccharomyces cerevisiae allow us to investigate the connection between nutrient-sensing and mitochondrial function. We have focused our current studies on the protein kinase A (PKA) pathway, which controls S. cerevisiae behavior according to glucose availability. We found that reduced PKA signaling can lead, in a background-dependent manner, to improved fitness after mtDNA loss. Specifically, over-expression of the cyclic AMP phosphodiesterase Pde2p, removal of PKA isoform Tpk3p, or ablation of other proteins promoting PKA activity leads to improved proliferation of cells deleted of the mitochondrial genome. Over-expression of Pde2p also suppresses the inviability of several mutants that normally cannot survive mtDNA loss. Moreover, Pde2p over-expression diminishes the nuclear transcriptional response to mtDNA damage, further supporting the idea that glucose sensation is harmful for cells lacking the mitochondrial genome. These findings are heavily dependent upon yeast genetic background. Interestingly, robust import of mitochondrial polytopic membrane proteins may be required in order for cells with no mtDNA to receive the full benefits of PKA reduction. Our findings support the idea that perturbation of nutrient-sensing pathways, and specifically the sensation of glucose, may benefit cells with dysfunctional mitochondria. Four experimental conditions were used: BY4743 (WT) cells containing empty pRS426 vector and containing mtDNA because EtBr was not used, BY4743 (WT) cells containing empty pRS426 vector and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide, BY4743 (WT) cells overexpressing TIP41 from plasmid pRS426 and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide, and BY4743 (WT) cells overexpressing PDE2 from plasmid pRS426 and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide. Two replicates were performed for each sample type.
2015-08-07 | E-GEOD-71252 | biostudies-arrayexpress
Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).
2013-02-12 | E-GEOD-27545 | biostudies-arrayexpress
Project description:Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Assisted reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable women who carry mtDNA mutations to have a genetically related child with a greatly reduced risk of disease. Here we report for the first time that pronuclear transplantation (PNT) between normally fertilised human zygotes provides an effective approach to preventing transmission of mtDNA disease. We found that the procedures previously used to perform PNT between abnormally fertilized human zygotes are highly inefficient when applied to those that undergo normal fertilization. We have therefore developed an alternative approach based on transplanting PN shortly after completion of the second meiotic division rather than shortly before onset of the first mitosis. This approach promotes highly efficient development to the blastocyst stage without affecting nuclear genome integrity. Furthermore, the expression profile of genes encoded by the nuclear and mitochondrial genomes was indistinguishable from unmanipulated control embryos. Importantly, levels of mtDNA transferred with the nuclear genome are below the threshold for mtDNA disease. Together these data indicate that transplantation of pronuclei early in the first cell cycle holds promise as a safe and effective approach to preventing transmission of mtDNA disease.
2016-06-03 | GSE76284 | GEO
Project description:Chromosomal structural variation can cause alterations in gene dosage and gene regulation between genomes. Structural variants producing a change in the number of copies of a genomic region are termed copy number variants (CNVs). CNVs have been demonstrated to have causative effects on both Mendelian and complex traits, including susceptibility to infectious diseases. We are interested in mapping CNVs to domesticated chicken breeds to help determine structural variation between genomes that influences economically important traits. For this study, Fayoumi, Leghorn, Line A broiler and Line B broiler chicken were chosen. Fayoumi and Leghorn chickens were selected as these two breeds harbor different responses certain pathogens like Avian Influenza Virus and coccidiosis; Broiler Line A and Line B indivduals were chosen as they harbor different intestinal colonization loads to the bacterium Campylobacter jejuni. Campylobacter genetic Line A and genetic Line B are from a commercial producer have been previously described as either resistant (Line A) or susceptible (Line B). Highly inbred chicken lines Fayoumi M15.2 (n=6) and Leghorn GHs6 (n=6) and broilers from Line A (n=24 individuals in pools of 4) and Line B (n=24 individuals in pools of 4)were subjected to array Comparative Genomic Hybridization (aCGH). Each sample was normalized to a Red Jungle Fowl reference. CNVs for each individual and between lines were determined. The major goal of this study was to discover and characterize CNVs in chickens to further narrow in on Quantitative Trait Loci (QTLs) affecting disease response. For the test DNA in Fayoumi and Leghorn, samples from 6 inbred Fayoumi and 6 inbred Leghorn individuals were used; For the test DNA in the Campylobacter genetic lines, samples from 24 individual broilers of Line A (in pools of 4) and 24 individual broilers of Line B (in pools of 4) were used. For the reference DNA, Red Jungle Fowl line UCD001 was used from a self-self hybridization.
2016-02-11 | E-GEOD-44440 | biostudies-arrayexpress