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The Molecular Anatomy of Mitochondrial Disease: Identification and Characterisation of Novel Nuclear-Encoded Mitochondrial Disease Genes


Type

Thesis

Change log

Authors

Martinez Lyons, Anabel 

Abstract

Mitochondrial diseases are a group of clinically and genetically heterogeneous disorders typically associated with abnormal oxidative phosphorylation (OXPHOS). In recent years, next generation sequencing technologies have allowed for accurate genetic diagnoses of inherited mitochondrial diseases directly from patient DNA by facilitating the identification of potential candidate genetic defects in either nuclear or mitochondrial genomes. In such a manner, two compound heterozygous sequence variants in COA7, which encodes a putative cytochrome c oxidase (Complex IV, COX) assembly factor, and homozygous recessive sequence variants in TMCO6, which encodes an uncharacterised protein, were identified in patients presenting with classical clinical and biochemical hallmarks of mitochondrial disease. The aim of this work was to 1) assess the pathogenicity of the COA7 and TMCO6 mutant variants in causing mitochondrial disease, and 2) to investigate the association of the resulting proteins in the assembly pathways of complexes of the mitochondrial respiratory chain: Complex IV (CIV, COX) for COA7 and Complex I (CI) for TMCO6.

Compound heterozygous mutant variants in COA7 (NM_023077.3:c.410A>G;c.287+1G>T) led to total disappearance of its gene product in patient skin fibroblasts, owing to two aberrant mRNA transcripts. Loss of COA7 steady-state level correlated with low abundance of certain COX subunits, intermediates, monomeric and supercomplex COX species, as well as isolated COX enzymatic deficiency. Stable expression of COA7WT in patient fibroblasts by lentivirus-mediated complementation rescued COX abundance, the quantities of its affected subunits, subassembly and supercomplex species, and its activity, to normal levels, confirming the pathogenicity of the compound heterozygous COA7 mutant variants. A combination of super-resolution microscopy and subcellular fractionation and protease digestion studies confirmed the intracellular localisation of COA7 to be the mitochondrial intermembrane space.

Secondly, a novel homozygous recessive variant in TMCO6 (NM_018502.5: c.271C>T) was identified in a paediatric proband presenting with severe developmental delay, generalised hypotonia and progressive cerebral and cerebellar atrophy. Biochemical measurement of a skeletal muscle biopsy revealed CI enzymatic deficiency, and patient-derived skin fibroblasts showed destabilisation of CI-containing supercomplexes. TMCO6 was found to co-localise with the CI holocomplex by 2D-BNGE, and this result was further corroborated by immunoprecipitation experiments. Additionally, cellular models for TMCO6 gene silencing and overexpression were characterised, and an attempted generation of a knockout cellular model is described. Subcellular fractionation and protease treatment experiments determined TMCO6 to localise to the mitochondrial inner membrane. A Tmco6-knockout murine model was characterised, which exhibited several neurological, physiological and motor debilities, isolated CI deficiency in heart and skeletal muscle, and abnormal cardiac electrophysiology. Recombinant AAV-mediated expression of wildtype, human TMCO6 rescued the CI deficiency and electrophysiological function of 3 month-old knockout mice hearts. In contrast, stable expression of the patient mutant protein variant failed to recover the isolated CI deficiency, and additionally resulted in severe cardiac fibrosis. Together, these findings causally link ablation of, or mutations in, TMCO6 with mitochondrial dysfunction and disease.

Description

Date

2019-10-30

Advisors

Zeviani, Massimo

Keywords

mitochondrial medicine, mitochondrial disease, mitochondrial genetics, mitochondrial disease genes, OXPHOS assembly factors, electron transport chain, electron transport chain assembly factors, Complex I assembly factor, Complex IV assembly factors, COA7, TMCO6, whole exome sequencing

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
MRC (1659341)
Medical Research Council