genotyping-single-nucleotide-polymorphisms-mitochondrial-genome

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

University of Cambridge, Cambridge Biomedical Campus

Enrichment of desired mitochondrial DNA (mtDNA) haplotypes, in both experimental systems and the clinic, is an end sought by many. Through use of a designer nuclease platform optimized for delivery to mitochondria-the mitochondrially targeted zinc finger-nuclease (mtZFN)-it is possible to discriminate between mtDNA haplotypes with specificity to the order of a single nucleotide substitution. Site-specific cleavage of DNA produces a shift in the heteroplasmic ratio in favor of the untargeted haplotype. Here, we describe protocols for assembly of paired, conventional tail-tail mtZFN constructs and experimental approaches to assess mtZFN activity in mammalian cell cultures.

Engineered mtZFNs for Manipulation of Human Mitochondrial DNA Heteroplasmy.

Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.

Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.

The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNA compared with the porcine use of mt-tRNA We have explored this observation further. Our studies examine whether a range of mt-tRNAs are used by different mammals, or whether the mt-tRNA selection is strictly limited to only these two species of the 22 tRNAs encoded by the mitochondrial genome (mtDNA); whether there is tissue-specific variation within a single organism; and what happens to the human mitoribosome when levels of the mt-tRNA are depleted. Our data demonstrate that only mt-tRNA or mt-tRNA are found in the mitoribosomes of five different mammals, each mammal favors the same mt-tRNA in all tissue types, and strikingly, when steady-state levels of mt-tRNA are reduced, human mitoribosome biogenesis displays an adaptive response by switching to the incorporation of mt-tRNA to generate translationally competent machinery.

Article	Year
Engineered mtZFNs for Manipulation of Human Mitochondrial DNA Heteroplasmy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26530680	2016
Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic acids research\| PubMed ID: 27466392	2016
Human mitochondrial ribosomes can switch their structural RNA composition. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27729525	2016

Michal A. Minczuk

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