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Quality Improvement of Mitochondrial Respiratory Chain Complex Enzyme Assays Using Caenorhabditis Elegans Xiulian Chen, MD, Phd1, David R

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Quality Improvement of Mitochondrial Respiratory Chain Complex Enzyme Assays Using Caenorhabditis Elegans Xiulian Chen, MD, Phd1, David R ARTICLE Quality improvement of mitochondrial respiratory chain complex enzyme assays using Caenorhabditis elegans Xiulian Chen, MD, PhD1, David R. Thorburn, PhD2, Lee-Jun Wong, PhD3, Georgirene D. Vladutiu, PhD4, Richard H. Haas, MB, BChir5, Thuy Le, PhD5, Charles Hoppel, MD6, Margaret Sedensky, MD1,7, Philip Morgan, MD1,7, and Si Houn Hahn, MD, PhD1,8 Purpose: The diagnosis of a mitochondrial disorder relies heavily on the itochondrial diseases are the most common metabolic enzymatic analysis of mitochondrial respiratory chain complexes in muscle Mdiseases of childhood, with an estimated frequency of 1 in 1 or other tissues. However, considerable differences exist between clinical 5000 births. These often devastating disorders are clinically laboratories in the protocols or particular tests used for evaluation. In characterized by unexplained association of neuromuscular addition, laboratories can encounter difficulties in consistent technique, as and/or nonneuromuscular symptoms, an often rapidly progres- well as procurement of adequate positive or negative controls. Currently, sive course, and symptoms involving seemingly unrelated or- 2–4 there is no external quality assurance for respiratory chain complex assays. gans or tissues. The diagnosis of a mitochondrial disorder In this study, we explored the use of Caenorhabditis elegans mitochondria often relies on the enzymatic analysis of the respiratory chain as a potential aid to diagnostic centers that perform respiratory chain complexes (RCCs) in muscle homogenates or isolated muscle complex assays. Method: Five diagnostic test centers in the United States mitochondria. Muscle tissue assays require a relatively large and one from Australia comparatively analyzed enzyme activities of mito- sample, obtained by a costly, invasive, and potentially danger- chondria from C. elegans. The first survey consisted of three open-labeled ous biopsy procedure. The samples are sensitive to temperature samples including one normal control and two mutants; the second survey changes, prone to spurious results due to metabolite accumula- consisted of one open-labeled normal control and two blinded samples. tions or mishandling, and may be susceptible to anesthetics used Results: There was very good concordance among laboratories in detecting during the biopsy process. In addition, considerable differences the majority of the defects present in the mutant specimens. Despite the exist between clinical laboratories in the character, concentra- ability to detect respiratory chain complex defects, the scatter between tion, and composition of substrates used for RCC assays.5 centers for certain enzymatic assays, particularly I ϩ III, II, III, and IV, led Proficiency testing programs allow laboratories to regularly to different diagnostic interpretations between the centers. Conclusion: evaluate their performance and improve the accuracy of the The data strongly support the need for comparative testing of mitochondrial results they provide to patients. For example, the College of enzyme assays between multiple laboratories. Our overall results are en- American Pathology provides individual laboratories with un- couraging for the use of nematode mitochondria as a tool that might known specimens for testing, and each participating laboratory provide a virtually inexhaustible supply of mitochondria with defined receives a report of their performance.6–8 Currently, there is no defects for development of assays and as a potential source of control external quality assurance program for mitochondrial RCC as- specimens. Genet Med 2011:13(9):794–799. says or a method for comparison of results between laboratories. Key Words: quality improvement, quality assessment, mitochondria, It is challenging to identify and distribute adequate, identical respiratory chain complex, electron transport chain, enzyme assays, C. elegans human control specimens containing confirmed enzymatic de- fects to each test center. Frozen tissue from patients confirmed with mitochondrial disease is available only in a very limited From the 1Center for Developmental Therapeutics, Seattle Children’s Re- amount, insufficient to distribute to many diagnostic centers. search Institute, Seattle, Washington; 2The Murdoch Childrens Research Fibroblast lines from patients with known mitochondrial defects Institute and University of Melbourne Department of Pediatrics, Royal are not always available for each complex deficiency, but RCC 3 Children’s Hospital, Melbourne, Australia; Department of Molecular and activities in these fibroblasts are generally low, often not reflec- Human Genetics, Medical Genetics Laboratories, Baylor College of Medicine, Houston, Texas; 4Departments of Pediatrics, Neurology and Pathology & tive of values obtained from fresh muscle, and assays in fibro- 9 Anatomical Sciences, Robert Guthrie Biochemical and Molecular Genetics blasts are only available in a few diagnostic laboratories. Laboratory, State University of New York at Buffalo, New York; 5Department Consequently, comparing results from different centers, or even of Neurosciences and Pediatrics, The Mitochondrial and Metabolic Disease assuring rigorous standardization and controls within a center, Center, University of California San Diego, La Jolla, California; 6Departments have been long-standing hindrances to our ability to adequately of Pharmacology and Medicine, Center for Mitochondrial Disease, Case West- diagnose children with mitochondrial disorders.5,10 ern Reserve University School of Medicine, Cleveland, Ohio; 7Department of Anesthesiology, and 8Division of Genetic Medicine, Department of Pediatrics, Caenorhabditis elegans is an aerobic nematode that is 1 mm University of Washington School of Medicine, Seattle, Washington. long and easy to grow inexpensively. The genome of the nem- Ͼ Sihoun Hahn, MD, PhD, Division of Genetic Medicine, Department of atode has been fully sequenced and shares 83% identifiable Pediatrics, University of Washington School of Medicine, Seattle Children’s homology with human genes.11 There is one common wild-type Hospital, 4800 Sand Point Way, B6594, Seattle, WA 98105. E-mail: strain with a low forward mutation rate. Animals can be frozen [email protected]. indefinitely, are archived by the Caenorhabditis Genetics Cen- Disclosure: The authors declare no conflict of interest. ter, and stocks travel easily in the regular mail. Mitochondrial Submitted for publication November 9, 2010. mutants have been identified and well characterized in the nematode.12–16 These mutants manifest clearly defined defects Accepted for publication March 15, 2011. in RCC enzyme activities. They are a readily renewable, inex- Published online ahead of print May 30, 2011. pensive source of isogenic animals which carry defects within DOI: 10.1097/GIM.0b013e31821afca5 defined steps of electron transport. Available nuclear encoded 794 Genetics IN Medicine • Volume 13, Number 9, September 2011 Genetics IN Medicine • Volume 13, Number 9, September 2011 Mitochondrial RCC enzyme assays mutants encoding mitochondrial proteins include gas-1, a mu- timycin-sensitive decylubiquinol cytochrome c reductase (Com- tation in the 49 kD (NDUFS2) subunit of Complex I; mev-1, a plex III), rotenone-sensitive NADH-cytochrome c reductase mutation in the SDHC subunit of Complex II; and isp-1, a (Complexes I ϩ III), antimycin A-inhibited succinate cyto- mutation in the Rieske iron sulfur protein subunit of Complex chrome c reductase (Complexes II ϩ III), cytochrome c oxidase III.12–16 Reproducible knockdown of Complex IV subunits has (Complex IV), and citrate synthase as a mitochondrial internal also been established using interference RNA (RNAi).17 The marker enzyme.18–24 Each laboratory used their usual muscle RCC profile has been characterized for each of these muta- tissue protocol for patient diagnosis. Five laboratories in the tions.13,17 Worm mitochondria can be a virtually limitless United States and one in Australia received worm mitochondria source of invariable positive controls for mitochondrial RCC samples. The results of the Hahn laboratory are designated as A enzyme deficiencies, as well as a genetically invariant normal in all the figures comparing results between centers. control. Thus, C. elegans is a powerful translational model for human mitochondrial disease. It is clear that nematode mito- Determination of optimal conditions for large-scale chondria are not identical to human mitochondria and that the preparation, storage, and distribution of ideal solution to problems in proficiency testing for RCC assays mitochondria from C. elegans would be a bank of human tissue with defined defects leading to The stability of RCC enzyme activities, including Complexes I, mitochondrial dysfunction. In addition to this goal, we proposed ϩ ϩ to develop a supply of mitochondria which can be used for II, III, IV, I III, II III, and citrate synthase, was studied using quality improvement of mitochondrial RCC enzyme assays mitochondria from wild-type C. elegans, N2. This study was using C. elegans mutants that carry invariant, defined defects in undertaken only by the Hahn laboratory. The original sample is electron transport. As a first step, we collected basic information from the same preparation as that which provided laboratory A comparison data of RCC enzyme activities between laboratories. about the assay method of each center. We then compared the Ϫ results of RCC activities from open-labeled samples, followed The effects of storage at 80°C for up to 3 months and four by blinded samples
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