Details

Autor(en) / Beteiligte

• McKenzie, Matthew
• Chiotis, Maria
• Hroudová, Jana
• Lopez Sanchez, Maria I.G.
• Lim, Sze Chern
• Cook, Mark J.
• McKelvie, Penny
• Cotton, Richard G. H.
• Murphy, Michael
• St John, Justin C.
• Trounce, Ian A.

Titel

Capture of Somatic mtDNA Point Mutations with Severe Effects on Oxidative Phosphorylation in Synaptosome Cybrid Clones from Human Brain

Ist Teil von

• Human mutation, 2014-12, Vol.35 (12), p.1476-1484

Ort / Verlag

United States: Blackwell Publishing Ltd

Erscheinungsjahr

2014

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. By fusion of freshly isolated synaptic endings with mtDNA‐less cells, synaptosome cybrid clones can be produced in large numbers. We use mutation detection to identify a range of mtDNA mutations captured in such cybrids, and demonstrate severe OXPHOS phenotypes consequent to some of these. In the absence of methods to engineer mtDNA mutants, this approach allows novel structure‐function studies of OXPHOS complexes with mutated or absent mtDNA‐encoded subunits.