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Capture of Somatic mt DNA Point Mutations with Severe Effects on Oxidative Phosphorylation in Synaptosome Cybrid Clones from Human Brain
Author(s) -
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.
Publication year - 2014
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22694
Subject(s) - biology , mitochondrial dna , point mutation , somatic cell , oxidative phosphorylation , microbiology and biotechnology , mitochondrion , genetics , mutation , mutant , dna , gene , biochemistry
Mitochondrial DNA (mt DNA ) 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 mt DNA somatic mutants in cancerous and normal human tissues. To capture somatic mt DNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mt DNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and S anger 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_2953del CTATT mutation in MT ‐ RNR 2 (which encodes mitochondrial 16 S r RNA ) revealed a severe disruption of mt DNA ‐encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT ‐ ND 1 , previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mt DNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mt DNA mutants in vitro to study effects on mitochondrial respiratory chain function.