title: Capture of somatic mtDNA point mutations with severe effects on oxidative phosphorylation in synaptosome cybrid clones from human brain.
abstract: Mitochondrial DNA (mtDNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low-level mtDNA somatic mutants in cancerous and normal human tissues. To capture somatic mtDNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mtDNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953delCTATT mutation in MT-RNR2 (which encodes mitochondrial 16S rRNA) revealed a severe disruption of mtDNA-encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT-ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mtDNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mtDNA mutants in vitro to study effects on mitochondrial respiratory chain function.
authors.affiliation: Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, Clayton, Victoria, 3168, Australia; Monash University, Clayton, Victoria, 3168, Australia.
authors.forename: Matthew
authors.initials: M
authors.lastname: McKenzie
authors.forename: Maria
authors.initials: M
authors.lastname: Chiotis
authors.forename: Jana
authors.initials: J
authors.lastname: Hroudová
authors.forename: Maria I G
authors.initials: MI
authors.lastname: Lopez Sanchez
authors.forename: Sze Chern
authors.initials: SC
authors.lastname: Lim
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Cook
authors.forename: Penny
authors.initials: P
authors.lastname: McKelvie
authors.forename: Richard G H
authors.initials: RG
authors.lastname: Cotton
authors.forename: Michael
authors.initials: M
authors.lastname: Murphy
authors.forename: Justin C
authors.initials: JC
authors.lastname: St John
authors.forename: Ian A
authors.initials: IA
authors.lastname: Trounce
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001921:Brain / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010085:Oxidative Phosphorylation; D017354:Point Mutation; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: D004272:DNA, Mitochondrial; D000255:Adenosine Triphosphate
keywords: mitochondrial DNA; oxidative phosphorylation; somatic mutation; synaptosome cybrid