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      Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

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          Abstract

          Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

          Author Summary

          Mitochondrial complex III deficiency is a devastating disorder that impairs energy generation, and leads to variable symptoms such as developmental regression, seizures, kidney dysfunction and frequently death. The genetic basis of complex III deficiency is not fully understood, with around half of cases having no known cause. This lack of genetic diagnosis is partly due to an incomplete understanding of the genes required for complex III assembly and function. We have identified two key proteins required for complex III, UQCC1 and UQCC2, and have elucidated the role of these inter-dependent proteins in the biogenesis of cytochrome b, the only complex III subunit that is encoded by mitochondrial DNA. We have shown that mutations in UQCC2 cause human complex III deficiency in a patient with neonatal lactic acidosis and renal tubulopathy. This work contributes to an improved understanding of complex III biogenesis, and will aid future molecular diagnoses of complex III deficiency.

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          Most cited references51

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          Expression analysis of G Protein-Coupled Receptors in mouse macrophages

          Background Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.
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            Respiratory complex III is required to maintain complex I in mammalian mitochondria.

            A puzzling observation in patients with oxidative phosphorylation (OXPHOS) deficiencies is the presence of combined enzyme complex defects associated with a genetic alteration in only one protein-coding gene. In particular, mutations in the mtDNA encoded cytochrome b gene are associated either with combined complex I+III deficiency or with only complex III deficiency. We have reproduced the combined complex I+III defect in mouse and human cultured cell models harboring cytochrome b mutations. In both, complex III assembly is impeded and causes a severe reduction in the amount of complex I, not observed when complex III activity was pharmacologically inhibited. Metabolic labeling in mouse cells revealed that complex I was assembled, although its stability was severely hampered. Conversely, complex III stability was not influenced by the absence of complex I. This structural dependence among complexes I and III was confirmed in a muscle biopsy of a patient harboring a nonsense cytochrome b mutation.
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              Common variants in the GDF5-UQCC region are associated with variation in human height.

              Identifying genetic variants that influence human height will advance our understanding of skeletal growth and development. Several rare genetic variants have been convincingly and reproducibly associated with height in mendelian syndromes, and common variants in the transcription factor gene HMGA2 are associated with variation in height in the general population. Here we report genome-wide association analyses, using genotyped and imputed markers, of 6,669 individuals from Finland and Sardinia, and follow-up analyses in an additional 28,801 individuals. We show that common variants in the osteoarthritis-associated locus GDF5-UQCC contribute to variation in height with an estimated additive effect of 0.44 cm (overall P < 10(-15)). Our results indicate that there may be a link between the genetic basis of height and osteoarthritis, potentially mediated through alterations in bone growth and development.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                December 2013
                December 2013
                26 December 2013
                : 9
                : 12
                : e1004034
                Affiliations
                [1 ]Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
                [2 ]Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
                [3 ]Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands
                [4 ]Nijmegen Center for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands
                [5 ]Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
                [6 ]Victorian Clinical Genetics Services, Royal Children's Hospital, Melbourne, Victoria, Australia
                [7 ]Genetic Metabolic Disorders Research Unit, Children's Hospital at Westmead, Westmead, New South Wales, Australia
                [8 ]Disciplines of Paediatrics & Child Health and Genetic Medicine, University of Sydney, Sydney, New South Wales, Australia
                [9 ]Department of Biochemistry, Jichi Medical University, Tochigi, Japan
                [10 ]ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne, Australia
                University of Miami, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: EJT BFJW RS HSM AGC MTR LGN MAH DRT. Performed the experiments: EJT BFJW RS HSM XWW MAMvdB AML RJR BR AGC AEF DLB. Analyzed the data: EJT BFJW RS HSM RJR AGC DLB MTR LGN MAH DRT. Contributed reagents/materials/analysis tools: EJT BFJW RS HSM RJR AGC DLB JC HE MTR LGN MAH DRT. Wrote the paper: EJT BFJW RS HSM MTR LGN MAH DRT.

                [¤]

                Current address: Department of Clinical Genetics, Unit Clinical Genomics, Maastricht University Medical Centre, Maastricht, The Netherlands.

                Article
                PGENETICS-D-13-01737
                10.1371/journal.pgen.1004034
                3873243
                24385928
                a294d347-fa45-46b8-af8e-30c79a27a218
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 June 2013
                : 29 October 2013
                Page count
                Pages: 15
                Funding
                This work was supported by grants (1023619), a Principal Research Fellowship (D1022896, DRT) and a Peter Doherty Early Career Fellowship (1054432, EJT) from the Australian National Health and Medical Research Council, the Victorian Government's Operational Infrastructure Support Program (DRT), the Netherlands Genomics Initiative (Horizon Programme) and the Centre for Systems Biology and Bioenergetics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article

                Genetics
                Genetics

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