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 defects 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 designed to reveal potential differences in freeze-thaws on mitochondrial RCC enzyme activities were also diagnoses between centers. investigated. The study design is illustrated in Figure 1. Briefly, the mitochondria isolated from N2 were aliquoted and stored at Ϫ80°C. After a week, aliquots of mitochondria were taken out of MATERIALS AND METHODS the freezer and thawed on ice. Part of the thawed aliquot was used C. elegans for RCC testing, and this sample was labeled Sample A. The rest Ϫ All C. elegans strains were obtained from the Caenorhabditis of the aliquot was returned to the 80°C freezer and was labeled Genetics Center (Minneapolis, MN). Wild type (N2) and mu- Sample B. After another month, Sample B was thawed, and part of tants, including a Complex I mutant (gas-1(fc21)), a Complex II the aliquot was used for testing RCC enzyme activities. The rest of mutant (mev-1(kn1)), and a Complex III mutant (isp-1(qm150)), the aliquot was returned to the freezer and labeled as Sample D. In were used in this study. All are canonical missense alleles in a similar fashion, Samples C and E–K were generated for up to 3 nuclear DNA. Worms were grown according to standard pro- months storage and four freeze-thaws. The RCC profile was tested tocols, and mitochondrial fractions were prepared from wild- at each step. type and mutant animals using established techniques.13 Mitochondria sample preparation and distribution RCC assays Nematode mitochondria were isolated as previously pub- The RCC profile is a collection of spectrophotometric en- lished13 at the Seattle Children’s Research Institute, which also zyme assays measuring rotenone-sensitive NADH CoQ reduc- processed and mailed samples to each center. Mitochondria tase (Complex I), NADH ferricyanide reductase (first four sub- extracted from multiple cultures of each genotype were pooled units of Complex I), succinate dehydrogenase (part of Complex and realiquoted at 200 ␮g protein per tube. The protein con- II), thenoyltriflouroacetone-inhibited rate of succinate-dichloro- centration was determined using the Bradford assay. Mitochon- phenolindophenol reductase with and without duroquinone, an- drial aliquots were stored at Ϫ80°C until shipped to participat-

Fig. 1. Study design for C. elegans mitochondrial sample stability study. The samples were set up in the fashion described in “Materials and Methods.” The storage time in Ϫ80°C and freeze-thaw conditions are summarized in the right panel. RCC, respiratory chain complex.

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Fig. 2. C. elegans mitochondrial stability study. The RCC was measured by the Hahn laboratory after multiple cycles of freeze-thaw over time. The panel on the right summarizes the number of cycles of freeze-thaws and length of storage. Complex IV activity was missing in Sample J due to spectrophotometer technical issue. Complex IV activity marginally declined over time with successive cycles of freeze-thaw but not at a magnitude that would affect interpretation, when compared with mutants. Overall, the entire RCC activities remained stable over 3 months after up to four freeze and thaws. CS, citrate synthase; I ϩ III, Complexes I ϩ III; I, Complex I; II ϩ III, Complexes II ϩ III; II, Complex II; III, Complex III; IV, Complex IV. ing centers on dry ice. In the first survey, we provided open- known that the Complex III deficiency mutant has significantly labeled samples to establish the range of enzymatic activities for decreased Complex III enzyme activity and also reduced Complex control and mutants at each test center. Each test center received I, Complexes I ϩ III, and Complexes II ϩ III activity.25 Only two three strains, wild type (N2), Complex III mutant (isp-1), and centers reported Complex III activity, with the average ratio to Complex I mutant (gas-1). The ratios of the RCC activities of normal control of 0.02. All test centers were able to detect the defective mitochondria over the normal control were used for reduced enzyme activities of Complex I and Complexes I ϩ III in comparison. In the second survey, each test center received the Complex-I-deficient mutant. The average ratio of Complex I three strains, N2, the wild-type control, and two blinded mu- enzyme activity was 0.56 for NADH dehydrogenase and 0.26 for tants, a Complex III mutant (isp-1) and a Complex II mutant NADH CoQ reductase. Complexes I ϩ III enzyme activity mea- (mev-1). For wild-type normal controls, 800 ␮g of mitochon- sured in three centers showed an average ratio to normal control of drial protein was provided in four tubes. For each of the mu- 0.39, an expected range from previous study.13 tants, 600 ␮g of mitochondrial protein was provided in three tubes. Samples were shipped on dry ice and arrived frozen. All The second survey results samples were kept at Ϫ80°C until the assay was done. The The second survey contained three samples, including one guidelines for sample preparation were provided to each center wild-type control (N2), one Complex-III-deficiency mutant with instructions to send results back to the Seattle Children’s (isp-1), and one Complex-II-deficiency mutant (mev-1). The Hospital Research Institute within 3 months. samples were stored by different centers for time periods varying from 10 days to 2.5 months. The RCC data from each Survey of RCC protocols diagnostic center are summarized in Figure 4. RCC enzyme Each test center completed a survey to compare the methods activities in mutants were normalized to normal controls, and used for clinical testing. The survey included sample type, the ratios are presented. Two centers reported Complex III sample preparation, methods of calculations, units of enzymatic activity with a mean of 0.08. The Complex II mutant had activity, and methods for each enzymatic activity assay. reduced Complex II and Complexes II ϩ III activities. All six test centers were able to show reduced Complex II and Com- RESULTS plexes II ϩ III activities. Five test centers were able to detect the significantly reduced Complex II activity, whereas one test The sample stability center reported the ratio to normal control as 0.86 (the average All RCC enzyme activities were stable over 3 months, even ratio to normal control was 0.24). All test centers were able to after four freeze-thaw cycles. The data are shown in Figure 2. find significantly reduced Complexes II ϩ III activity (average Complex IV activity marginally declined over time with suc- ratio to normal control is 0.07). However, as the activities were cessive cycles of freeze-thaw but not at a magnitude that would scattered for various enzymes, the exact interpretation was affect interpretation when compared with mutants. Complex IV significantly different among centers (Table 1). activity was missing in Sample J due to a technical error. RCC protocol survey results The first survey results We sent out a survey to collect basic information about methods The first survey samples were kept at Ϫ80°C before the analysis and sample requirements used for clinical assay at each center. The at each center for a period ranging from 2 weeks to 3 months. The results are summarized in Table 2. It was notable that some centers RCC data from each center are summarized in Figure 3. It is use crude homogenates, whereas others use isolated mitochondria

Fig. 3. Summary of the RCC data on Complexes I and III deficiency mutant mitochondria in the first survey. The ratios of RCC enzyme activities in Complex I deficiency Fig. 4. Summary of the RCC data on Complexes II and III mutant (top panel) and Complex III deficiency mutant deficiency mutant mitochondria in the second survey. The (bottom panel) to normal control (N2) were compared. ratios of RCC enzyme activities in Complex II deficiency Each column group represents an RCC complex: Com- mutant (top panel) and Complex III deficiency mutant plexes I, I ϩ III, II, II ϩ III, III, IV, and CS. Each color (bottom panel) to normal control (N2) were compared. represents a different test center. The first column of each Each column group represents an RCC complex: Com- ϩ ϩ group represents the mean of different test centers, and plexes I, I III, II, II III, III, IV, and CS. Each color standard deviations are shown as bar. The column labeled represents a different test center. The first column of each as “A” represents the result from Seattle Children’s Re- group represents the mean of different test centers results, search Institute. *I denotes NADH dehydrogenase activity. and standard deviations are shown. The column labeled as **I denotes measurements of NADH CoQ Reductase. “A” represents the result from Seattle Children’s Research Institute. *I was measured as NADH dehydrogenase. **I was measured as NADH CoQ reductase. for sample preparation. There are variations among protocols, in particular for Complexes I, II ϩ III, III, and IV. For Complexes III Table 1 Summary of interpretations for Complex-III- and IV, some test centers use initial rates, whereas others use a deficient blind sample first-order rate constant. The units for reporting are also variable among centers, from grams of wet tissue to milligrams of protein Interpretations No. centers for normalization of enzymatic activity. To assay Complex I ac- ϩ ϩ tivity, four test centers measure NADH CoQ Reductase activity, Complexes I, I III, and II III deficiency with 1 partial reduction in Complexes II and IV while three test centers defined NADH Dehydrogenase as repre- senting Complex I. Combined Complexes I ϩ III deficiency with partial 1 reduction in Complex II DISCUSSION Primary Complex III deficiency with secondary 1 Complex I deficiency or combined Complexes I ϩ Enzyme assays of muscle or other tissues are a critical III deficiency component for diagnosing patients with suspected mitochondrial disorders. The clinician caring for the mystifying patient Partial reduction in Complex I and Complexes I ϩ III 1 must process potentially very complicated RCC data with other activity laboratory studies, as well as the clinical picture. It is unfortu- nate that there is no external proficiency testing program for these clinical assays given that muscle biopsy is a relatively frequently performed procedure. Multiple issues exist concern- the specimen before assay may be different, as is the language ing assays of mitochondrial function that have led to this situ- used in patient reports. The identification of normal controls is ation. There is no standard assay that is agreed on by clinical also extremely problematic. In addition, it is easily conceivable laboratories for individual electron transport chain enzymes as that regardless of the choice of a standard, the assay would have the correct assay. Even if the same assay is used, treatment of intrinsic limitations. For example, it is doubtful that any one

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enzymatic assay could interrogate all possible functions of Table 2 Survey summary of the RCC protocol used for complicated entity similar to a 45-subunit protein complex. clinical diagnosis in different test centers Given all these variables, it is perhaps not surprising that, to Survey contents No. centers date, there has not been any comparison study for the assays that are currently performed in the United States. Sample type Our supply of purified nematode mitochondria of course Frozen muscle 6 bypassed many issues that will need to be addressed by true proficiency testing of RCC assays. Our nematode mitochondria Fresh muscle 2 were harvested from the whole organism; all laboratories re- Skin fibroblasts 3 ceived a purified sample initially processed by a central laboratory. Not all centers isolate mitochondria for their RCC as- Other tissues: liver, heart 2 says. In addition, procurement of human tissue is a very large Chorionic villi sample 1 concern and a very different clinical situation. Tissue harvest- ing, transport, and treatment on arrival in the laboratory are just Preparation a few of the steps that we have completely sidestepped in this Crude homogenate 6 study. Also, mitochondria from liver may need to be assayed differently than mitochondria from muscle or fibroblasts. This is Isolated mitochondria 3 not a concern in worms. In addition, although we have been Denomination very impressed with the similarity between human and nematode mitochondria, differences do exist. On the one hand, we Protein concentration 4 can note that virtually every mitochondrial protein listed in Wet tissue weight 3 MitoCarta can be found within the worm genome. Moreover, our laboratory did not find it necessary to modify any protocols Complex I, rotenone sensitive originally developed for mammalian mitochondria in their RCC NADH CoQ reductase 4 assays (Morgan and Sedensky, Seattle Children’s Hospital, per- sonal communication, 2009). NADH dehydrogenase 3 Nevertheless, our results from two surveys of RCC activities are Oxidation of NADH 4 quite encouraging, in that most centers were able to detect electron transport chain defects in mutant samples. For example, all centers Reduction of DCPIP 1 that report Complex III activity detected the severely reduced Complexes I ϩ III, rotenone sensitive activity in isp-1, and Complex I activity was reported as reduced by all centers for gas-1. This was precisely the result we had envi- NADH cytochrome c reductase 5 sioned. Nevertheless, there was significant variability between cen- No assay 1 ters for absolute amounts of activity for certain enzymatic assays, particularly I ϩ III, II, III, and IV. Not all centers performed the Complex II same RCC assays. For example, some infer Complex III activity ϩ ϩ Succinate dehydrogenase 5 by analyzing overlapping Complexes I III and II III. Ulti- mately, this difference in enzyme assays led to significantly dif- Thenoyltriflouroacetone (TTFA)-inhibited 1 ferent interpretations of identical samples between centers. This is rate of succinate-DCPIP reductase seen in Table 1, where it appears that four centers arrived at very with and without duroquinone different diagnoses of isp-1. This variation does not reflect widely Succinate CoQ1 oxidoreductase at 280 nm 1 varying abilities to measure Complex III activity but rather reveals the complexity of interpreting intertwined data, albeit data from a Complex III simple animal, that ultimately leads to a diagnosis. This difference Antimycin A-sensitive decylubiquinol cytochrome 5 in interpretation is probably not surprising, as significant variations c reductase of RCC test has been previously reported by different groups.5,10 However, it is of concern as it could potentially affect patient care. First-order rate constant 3 The reasons for the discrepant results in specific enzyme activities Initial rate 1 are probably due to the fact that the diagnostic centers use different protocols in the tissue preparation, kinetic determination, assay No assay 1 buffers, temperatures, and substrate concentrations.5 For Complex Complex IV I, different subsets of the complex’s enzymatic activities can be reported simply as Complex I. We confirmed that the assay meth- First-order rate constant 3 ods including sample preparation are variable among participating Initial rate 2 centers. It is conceivable that these many differences can each potentially contribute to the different RCC assay results. Citrate synthase The French network of mitochondrial disease diagnostic centers 5-thio-2-nitrobenzoate anion at 412 nm 6 undertook a monumental task to standardize the RCC protocols 26 The basic methods for testing Complexes I, I ϩ III, II, II ϩ III, III, IV, and CS used among French test centers. The results are not only encouraging by six test centers are included. The numbers of centers that have the same answers but also limited because of a lack of proper quality control. With- to the survey questions are counted and listed under the number of centers. out proper quality controls, it is difficult to evaluate whether the NADH, nicotinamide adenine dinucleotide; DCPIP, dichlorophenolindophenol. present French standardization is sufficient to obtain comparable data in all centers. The muscle samples from true mitochondrial patients would be ideal for proficiency testing, but it is challenging to obtain enough

798 © 2011 Lippincott Williams & Wilkins Genetics IN Medicine • Volume 13, Number 9, September 2011 Mitochondrial RCC enzyme assays tissue to distribute the same sample to many testing centers at one ACKNOWLEDGMENTS time. A set of cell lines with diverse enzymatic defect can be This project was supported from American College of Med- another possible option to serve as blinded test samples. However, ical Genetics/Luminex Grant Award, 2009. The authors thank culturing cell lines with defects in electron transport chains in large Dr. Beatrice Predoi for her support during the sample prepara- quantities is challenging due to very slow growth. Many diagnostic tion. They also thank Dr. Valeria Vasta for her tremendous help centers do not have a protocol for skin fibroblast samples. Al- in preparing the manuscript. though imperfect, nematode mitochondria may be useful as a quality improvement scheme until true proficiency testing via a consortium of banked patient tissues is available. 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