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

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.Medical Research Counci

COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency.

BACKGROUND: Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. METHODS: Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. RESULTS: We found compound heterozygous mutations in COA7: a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. CONCLUSIONS: We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested.Supported by Telethon-Italy grant GGP15041 (to DG); Telethon-Italy Network of Genetic Biobank grant GTB12001J; ERC advanced grant ERC FP7-322424 (to MZ). MRC QQR grant MC_UP_1002/1

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing

Background: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. / Methods: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. / Findings: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. / Interpretation: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. / Funding: Foundation for Liver Research, London

In vivo modeling of patient genetic heterogeneity identifies new ways to target cholangiocarcinoma.

L. Boulter was funded by The Wellcome Trust (207793/Z/17/Z), AMMF (2016/108, 2017/115), and Cancer Research UK (C52499/A27948). L. Boulter is also supported by an MRC university grant to the MRC Human Genetics Unit

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy