- Email: [email protected]
NADH fluorescence lifetime as an intrinsic biomarker of the metabolic change during osteogenic differentiation of human mesenchymal stem cells (hMSCs)
Abstracts
heterozygous nMut/VLM/VUS were identified in 1–3 genes but were insufficient to make a molecular diagnosis. The types of mutations identified include point mutations and insertions/deletions of 1–24 bps. The majority (~ 80%) of the disease-causing mutations and VLMs were identified in patients with a definitive/probable MtD or a discrete clinical syndrome. Some of the 24 cases with diagnostic mutations identified by this panel had either previously undergone extensive biochemical and/or molecular (single gene sequencing) evaluations without a definitive diagnosis, or mutation(s) were identified in a gene unexpected from the results of prior clinical, histo-pathological or electron transport chain (ETC) enzyme evaluations. Conclusion: The sensitivity of a targeted next generation sequencing panel of selected nuclear genes is significantly higher than single gene sequencing for the molecular diagnosis of MtD. The clinical sensitivity of this 24-nuclear gene panel is approximately 15% for patients with a definitive/probable MtD or with clinical features that fall into a discrete clinical syndrome.
doi:10.1016/j.mito.2012.07.097
110 PCR-based targeted enrichment and next-generation sequencing of 101 nuclear genes for the diagnosis of mitochondrial disorders Presenter: Renkui Bai Renkui Bai, Jaimie Higgs, Sharon Suchy, Federica Gibellini, Melanie Knight, Sabrina Buchholz, Sonia Benhamed, Dolores Arjona, Craig Chinault, Rhonda Brandon, Nizar Smaoui, Gabriele Richard, Sherri Bale GeneDx, Gaithersburg, MD 20877 Background: The majority of primary mitochondrial disorders (MtD) is caused by mutations in nuclear genes. To date, only about 100 nuclear genes have reported mutations associated with a primary MtD. About 25–30% of these genes have 1–35 pseudogenes or homologous sequences in other genes in the genome (www.pseudogene.org). Whole exome sequencing (WES), which uses hybridization-based capture as the targeted enrichment method making it unable to avoid pseudogenes or homologous sequences, creates many false positive and false negative results as well as many low/no coverage regions (in the exome data, usually about 7–10% of the exons of the genes of interest have insufficient sequence reads to make a variant call). By designing unique primers that selectively amplify only the gene of interest and using droplet-based multiplex PCR and massive parallel sequencing, we are able to simultaneously re-sequence 101 nuclear genes which account for over 95% of known nuclear genes/mutations associated with a primary MtD. This panel includes 976 exons with adjacent intronic sequences, and 25–50 bps into the promoters and 3′ UTR regions, as well as common mutations in deep intronic regions. Method: To validate this test, twelve DNA samples with mutations identified by single gene Sanger sequencing or by multiplex PCR-based targeted enrichment and next-generation sequencing of a Mito24 nuclear gene panel and confirmed by Sanger sequencing, were blinded and evaluated. Inter-run and intra-run variations were assessed for 3 of the samples. Results were compared to the original data. Results: Eight of the 12 samples had no low coverage, with 40×– 8000× read depth per nucleotide, resulting in average of approximately 2000× per nucleotide. The other four samples each had insufficient coverage for only 1–3 exons. 100% of all pathogenic mutations and previously detected sequence variants in the regions of interest were identified. Totally, each sample had 90–110 variants identified with 0–3 mutations or variants of unknown significance. Conclusion: This panel allows complete re-sequencing of all the exons and adjacent intronic sequences of 101 nuclear genes
589
simultaneously in a timely manner, with a technical sensitivity and specificity comparable to Sanger sequencing. In combination with exon level deletion/duplication testing of all these genes, it is an ideal panel for ruling out pathogenic mutations in genes known to be associated with a primary mitochondrial disorder before considering whole exome sequencing (WES). doi:10.1016/j.mito.2012.07.098
111 NADH fluorescence lifetime as an intrinsic biomarker of the metabolic change during osteogenic differentiation of human mesenchymal stem cells (hMSCs) Presenter: Hsing-Wen Wang Han Wen Guoa, Jia Sin Yua, Shu Han Hsub, Yau Huei Weib,c, Oscar K. Leea,d, Hsing Wen Wanga a Institute of Biophotonics, National Yang Ming University, Taipei 112, Taiwan b Department of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan c Department of Medicine, Mackay Medical College, San-Jhih, Taipei County 252, Taiwan d Institute of Clinical Medicine, National Yang Ming University, Taipei 112, Taiwan Fluorescence lifetime of NADH had been proposed to be used as an intrinsic biomarker for monitoring cellular metabolism. In our previous studies, we have demonstrated that NADH fluorescence lifetime of hMSCs increase gradually with time of osteogenic differentiation [1]. However, the cause of NADH fluorescence lifetime change is not clear. In this study, we performed NADH lifetime measurement of hMSCs from a different donor to study its association with several metabolic indices such as ATP level, oxygen consumption, and lactate release. We also measured the quantity of Complexes I, III, IV and V during hMSC differentiation. NADH fluorescence lifetime images were performed as our previous studies (Guo et al., 2008) that treated hMSC cells were imaged with a two-photon laser scanning microscope by a 60 × 1.45 NA PlanApochromat oil objective lens (Olympus Corp., Japan). NADH fluorescence was excited at 740 nm by a Verdi pumped modelocked femtosecond Ti:sapphire laser (Coherent, Inc., Santa Clara, California) at 76 MHz and the emitted fluorescent light was detected at 450 ± 40 nm by a bandpass filter (Edmund Optics, Inc., Barrington, New Jersey). Fluorescence photons were detected by a photon-counting photomultiplier H7422P-40 (Hamamatsu Photonics K.K., Hamamatsu, Japan). Timeresolved detection was conducted by the single-photoncounting SPC830 printed circuit board (Becker & Hickl GmbH, Berlin, Germany). Data were analyzed with the commercially available SPCImage v2.8 software (Becker & Hickl GmbH, Berlin, Germany) via a convolution of the two component exponential decay function and the instrument response function (IRF), and then the convolved result were fitted to the actual data to derive lifetime parameters t1 (NADH short lifetime component), t2 (NADH long lifetime component), a1 (amplitude related to t1), a2 (amplitude related to t2), and tm. Mean lifetime tm is defined as (a1t1 + a2t2)/(a1 + a2). IRF was measured using a second-harmonic generated signal from a periodically poled lithium niobate crystal. The cell respiration rate was measured by a 782 Oxygen Meter as previously reported (Chen et al., 2008) and intercellular ATP level was measured by the bioluminescent somatic cell ATP assay kit (Sigma-Aldrich, St. Louis, Missouri). The results show more oxygen consumption, higher ATP level expressed and less lactate released during differentiation. Similar to our previous study, NADH fluorescence lifetime increased gradually up to 4 weeks after osteogenic differentiation. We observed a good
590
Abstracts
correlation between the increase of NADH fluorescence lifetime and ATP level and oxygen consumption (R2 = 0.88 and 0.95 respectively). Significant higher expression of the total Complex protein than controls was observed at 3 and 4 weeks after differentiation. However, Complex I expression, which was directly related to NADH, did not show a significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime has potential as an intrinsic biomarker to monitor the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria during differentiation. References Guo, H.W., Chen, C.T., Wei, Y.H., Lee, O.K., Gukassyan, V., Kao, F.J., Wang, H.W*., 2008. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies. J. Biomed. Opt. (Letters) 13 (5), 050505. Chen, C.T., Shih, Y.R., Kuo, T.K., Lee, O.K., Wei, Y.H., 2008. Coordinated changes of mitochondrial biogenesis and antioxidant enzymes during osteogenic differentiation of human mesenchymal stem cells. Stem Cells 26 (4), 960–968.
doi:10.1016/j.mito.2012.07.099
112 Mitochondrial DNA depleted mouse B cells are defective in the response to antigen-stimulation Presenter: Hyongi Chon Hyongi Chon, Lina A. Gugliotti, Kiran Sakhuja, Mariya London, Susana M. Cerritelli, Robert J. Crouch Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Dr. Bethesda, MD 20892, USA RNase H1 is one of the enzymes that degrade RNA of RNA/DNA hybrids. Two different forms of the protein, localizing to nuclei and mitochondria, are translated from a single mRNA. RNase H1 knockout mice are embryonic lethal, resulting from loss of mitochondrial DNA (mtDNA) in the knockout mice, indicating that RNase H1 is essential for mtDNA replication. To examine the importance of this gene in later stages of development, specifically in B cells, we constructed a mouse whose Rnaseh1 gene is conditionally knocked-out (KO) by a Cre recombinase uniquely expressed in B cells (mb1-Cre), which has been used to efficiently delete conditional alleles at early stage of B cell development (Hobeika et al., 2006). To respond to diverse antigens, large varieties of B cells are constantly produced from hematopoietic stem cells. Some of these B cells undergo developmentally determined apoptosis, for which mitochondria are essential. Purified resting B cells from the conditional knockout mice showed more than 90% depletion of both RNase H1 protein and mtDNA levels. Surprisingly, B cell development in the bone marrow and spleen from the conditional Rnaseh1-KO mice was nearly normal, although antibodies in the blood serum were significantly decreased. In agreement with the blood serum antibody level, Rnaseh1-KO B cells were defective in the response to LPS/IL4 stimulation in the in vitro cultures. Additionally, stimulation did not occur even when supplements (high glucose, pyruvate and uridine) that allow the growth of cells in the absence of mtDNA were included in the medium. Analysis of the membrane potential of the purified B cells from WT and the conditional knockout did not show a difference prior to stimulation. However, a clear difference was observed following the 1 day
stimulation. This variation in membrane potential may correspond to the defect of Ca2 + signaling essential to B cell response to antigens. (1) Hobeika E. et al., PNAS (2006), 103, 13789–13794. doi:10.1016/j.mito.2012.07.100
113 Ablation of the gene coding for complex I subunit NDUFA5 in CNS results in a mild phenotype Presenter: Susana Peralta S. Peralta, A. Torraco, T. Wenz, C.T. Moraes Department of Neurology, University of Miami Miller School of Medicine, United States Mitochondrial complex I deficiencies are associated with severe pathologies which are often lethal in early childhood. To design new therapies, animal models are needed for the study of the pathophysiology and adaptive responses in complex I defects. The NADHubiquinone oxidoreductase 1a subcomplex subunit 5 (NDUFA5) is a nuclear encoded structural subunit of complex I. This 13 kDa protein is located in the peripheral arm of complex I. We have created a conditional NDUFA5 knock-out ablating the endogenous alleles and including a rescuing transgene flanked by loxP sites. The floxed transgene was deleted selectively in neurons by expressing cre driven by the CamK-IIa promoter. NDUFA5-CMK-IIa conditional knock-out (cKO) mice presented lethargy and loss of motor skills at the age of 12 months and death by the 16th month. These mice presented high levels of lactate levels in the blood at the age of 12 months. Blue Native gel electrophoresis showed that the amount of intact complex I was greatly reduced in the cortex and no accumulation of complex I subassemblies was observed. Accordingly, complex I proteins levels were markedly reduced in the NDUFA5-CMK-IIa knock-out mice. Biochemical measurement showed that complex I activity was reduced to 60% of control levels in the cortex at the same age. The activity levels of the other OXPHOS complexes and citrate synthase activity remained unchanged. Our data suggest that NDUFA5-CMK-IIa cKO is a useful mouse model to study the molecular mechanism of mild mitochondrial encephalopathies. doi:10.1016/j.mito.2012.07.101
114 Multiple mitochondrial electron transport chain enzyme deficiencies associated with a decrease in skeletal muscle coenzyme Q10 status Presenter: Iain Hargreaves Iain Hargreaves, John Land Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK Coenzyme Q10 (CoQ10) functions as an electron carrier in the mitochondrial electron transport chain (ETC) as well as serving as a potent lipid soluble antioxidant. Diagnosis of a CoQ10 deficiency normally follows a finding of decreased activity of the linked ETC complex II + III and/or I–III which utilize endogenous CoQ10 and normal activities of complexes I, II and III. Diagnosis is confirmed by HPLC analysis of muscle CoQ10. However, since all ETC complexes are susceptible to free radical induced oxidative damage a deficit in CoQ10 status may in theory result in multiple ETC deficiencies. In order to investigate this hypothesis we assessed the muscle CoQ10 status of 12 patients with combined ETC I (0.075 ± 0.006; ref interval:
heterozygous nMut/VLM/VUS were identified in 1–3 genes but were insufficient to make a molecular diagnosis. The types of mutations identified include point mutations and insertions/deletions of 1–24 bps. The majority (~ 80%) of the disease-causing mutations and VLMs were identified in patients with a definitive/probable MtD or a discrete clinical syndrome. Some of the 24 cases with diagnostic mutations identified by this panel had either previously undergone extensive biochemical and/or molecular (single gene sequencing) evaluations without a definitive diagnosis, or mutation(s) were identified in a gene unexpected from the results of prior clinical, histo-pathological or electron transport chain (ETC) enzyme evaluations. Conclusion: The sensitivity of a targeted next generation sequencing panel of selected nuclear genes is significantly higher than single gene sequencing for the molecular diagnosis of MtD. The clinical sensitivity of this 24-nuclear gene panel is approximately 15% for patients with a definitive/probable MtD or with clinical features that fall into a discrete clinical syndrome.
doi:10.1016/j.mito.2012.07.097
110 PCR-based targeted enrichment and next-generation sequencing of 101 nuclear genes for the diagnosis of mitochondrial disorders Presenter: Renkui Bai Renkui Bai, Jaimie Higgs, Sharon Suchy, Federica Gibellini, Melanie Knight, Sabrina Buchholz, Sonia Benhamed, Dolores Arjona, Craig Chinault, Rhonda Brandon, Nizar Smaoui, Gabriele Richard, Sherri Bale GeneDx, Gaithersburg, MD 20877 Background: The majority of primary mitochondrial disorders (MtD) is caused by mutations in nuclear genes. To date, only about 100 nuclear genes have reported mutations associated with a primary MtD. About 25–30% of these genes have 1–35 pseudogenes or homologous sequences in other genes in the genome (www.pseudogene.org). Whole exome sequencing (WES), which uses hybridization-based capture as the targeted enrichment method making it unable to avoid pseudogenes or homologous sequences, creates many false positive and false negative results as well as many low/no coverage regions (in the exome data, usually about 7–10% of the exons of the genes of interest have insufficient sequence reads to make a variant call). By designing unique primers that selectively amplify only the gene of interest and using droplet-based multiplex PCR and massive parallel sequencing, we are able to simultaneously re-sequence 101 nuclear genes which account for over 95% of known nuclear genes/mutations associated with a primary MtD. This panel includes 976 exons with adjacent intronic sequences, and 25–50 bps into the promoters and 3′ UTR regions, as well as common mutations in deep intronic regions. Method: To validate this test, twelve DNA samples with mutations identified by single gene Sanger sequencing or by multiplex PCR-based targeted enrichment and next-generation sequencing of a Mito24 nuclear gene panel and confirmed by Sanger sequencing, were blinded and evaluated. Inter-run and intra-run variations were assessed for 3 of the samples. Results were compared to the original data. Results: Eight of the 12 samples had no low coverage, with 40×– 8000× read depth per nucleotide, resulting in average of approximately 2000× per nucleotide. The other four samples each had insufficient coverage for only 1–3 exons. 100% of all pathogenic mutations and previously detected sequence variants in the regions of interest were identified. Totally, each sample had 90–110 variants identified with 0–3 mutations or variants of unknown significance. Conclusion: This panel allows complete re-sequencing of all the exons and adjacent intronic sequences of 101 nuclear genes
589
simultaneously in a timely manner, with a technical sensitivity and specificity comparable to Sanger sequencing. In combination with exon level deletion/duplication testing of all these genes, it is an ideal panel for ruling out pathogenic mutations in genes known to be associated with a primary mitochondrial disorder before considering whole exome sequencing (WES). doi:10.1016/j.mito.2012.07.098
111 NADH fluorescence lifetime as an intrinsic biomarker of the metabolic change during osteogenic differentiation of human mesenchymal stem cells (hMSCs) Presenter: Hsing-Wen Wang Han Wen Guoa, Jia Sin Yua, Shu Han Hsub, Yau Huei Weib,c, Oscar K. Leea,d, Hsing Wen Wanga a Institute of Biophotonics, National Yang Ming University, Taipei 112, Taiwan b Department of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan c Department of Medicine, Mackay Medical College, San-Jhih, Taipei County 252, Taiwan d Institute of Clinical Medicine, National Yang Ming University, Taipei 112, Taiwan Fluorescence lifetime of NADH had been proposed to be used as an intrinsic biomarker for monitoring cellular metabolism. In our previous studies, we have demonstrated that NADH fluorescence lifetime of hMSCs increase gradually with time of osteogenic differentiation [1]. However, the cause of NADH fluorescence lifetime change is not clear. In this study, we performed NADH lifetime measurement of hMSCs from a different donor to study its association with several metabolic indices such as ATP level, oxygen consumption, and lactate release. We also measured the quantity of Complexes I, III, IV and V during hMSC differentiation. NADH fluorescence lifetime images were performed as our previous studies (Guo et al., 2008) that treated hMSC cells were imaged with a two-photon laser scanning microscope by a 60 × 1.45 NA PlanApochromat oil objective lens (Olympus Corp., Japan). NADH fluorescence was excited at 740 nm by a Verdi pumped modelocked femtosecond Ti:sapphire laser (Coherent, Inc., Santa Clara, California) at 76 MHz and the emitted fluorescent light was detected at 450 ± 40 nm by a bandpass filter (Edmund Optics, Inc., Barrington, New Jersey). Fluorescence photons were detected by a photon-counting photomultiplier H7422P-40 (Hamamatsu Photonics K.K., Hamamatsu, Japan). Timeresolved detection was conducted by the single-photoncounting SPC830 printed circuit board (Becker & Hickl GmbH, Berlin, Germany). Data were analyzed with the commercially available SPCImage v2.8 software (Becker & Hickl GmbH, Berlin, Germany) via a convolution of the two component exponential decay function and the instrument response function (IRF), and then the convolved result were fitted to the actual data to derive lifetime parameters t1 (NADH short lifetime component), t2 (NADH long lifetime component), a1 (amplitude related to t1), a2 (amplitude related to t2), and tm. Mean lifetime tm is defined as (a1t1 + a2t2)/(a1 + a2). IRF was measured using a second-harmonic generated signal from a periodically poled lithium niobate crystal. The cell respiration rate was measured by a 782 Oxygen Meter as previously reported (Chen et al., 2008) and intercellular ATP level was measured by the bioluminescent somatic cell ATP assay kit (Sigma-Aldrich, St. Louis, Missouri). The results show more oxygen consumption, higher ATP level expressed and less lactate released during differentiation. Similar to our previous study, NADH fluorescence lifetime increased gradually up to 4 weeks after osteogenic differentiation. We observed a good
590
Abstracts
correlation between the increase of NADH fluorescence lifetime and ATP level and oxygen consumption (R2 = 0.88 and 0.95 respectively). Significant higher expression of the total Complex protein than controls was observed at 3 and 4 weeks after differentiation. However, Complex I expression, which was directly related to NADH, did not show a significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime has potential as an intrinsic biomarker to monitor the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria during differentiation. References Guo, H.W., Chen, C.T., Wei, Y.H., Lee, O.K., Gukassyan, V., Kao, F.J., Wang, H.W*., 2008. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies. J. Biomed. Opt. (Letters) 13 (5), 050505. Chen, C.T., Shih, Y.R., Kuo, T.K., Lee, O.K., Wei, Y.H., 2008. Coordinated changes of mitochondrial biogenesis and antioxidant enzymes during osteogenic differentiation of human mesenchymal stem cells. Stem Cells 26 (4), 960–968.
doi:10.1016/j.mito.2012.07.099
112 Mitochondrial DNA depleted mouse B cells are defective in the response to antigen-stimulation Presenter: Hyongi Chon Hyongi Chon, Lina A. Gugliotti, Kiran Sakhuja, Mariya London, Susana M. Cerritelli, Robert J. Crouch Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Dr. Bethesda, MD 20892, USA RNase H1 is one of the enzymes that degrade RNA of RNA/DNA hybrids. Two different forms of the protein, localizing to nuclei and mitochondria, are translated from a single mRNA. RNase H1 knockout mice are embryonic lethal, resulting from loss of mitochondrial DNA (mtDNA) in the knockout mice, indicating that RNase H1 is essential for mtDNA replication. To examine the importance of this gene in later stages of development, specifically in B cells, we constructed a mouse whose Rnaseh1 gene is conditionally knocked-out (KO) by a Cre recombinase uniquely expressed in B cells (mb1-Cre), which has been used to efficiently delete conditional alleles at early stage of B cell development (Hobeika et al., 2006). To respond to diverse antigens, large varieties of B cells are constantly produced from hematopoietic stem cells. Some of these B cells undergo developmentally determined apoptosis, for which mitochondria are essential. Purified resting B cells from the conditional knockout mice showed more than 90% depletion of both RNase H1 protein and mtDNA levels. Surprisingly, B cell development in the bone marrow and spleen from the conditional Rnaseh1-KO mice was nearly normal, although antibodies in the blood serum were significantly decreased. In agreement with the blood serum antibody level, Rnaseh1-KO B cells were defective in the response to LPS/IL4 stimulation in the in vitro cultures. Additionally, stimulation did not occur even when supplements (high glucose, pyruvate and uridine) that allow the growth of cells in the absence of mtDNA were included in the medium. Analysis of the membrane potential of the purified B cells from WT and the conditional knockout did not show a difference prior to stimulation. However, a clear difference was observed following the 1 day
stimulation. This variation in membrane potential may correspond to the defect of Ca2 + signaling essential to B cell response to antigens. (1) Hobeika E. et al., PNAS (2006), 103, 13789–13794. doi:10.1016/j.mito.2012.07.100
113 Ablation of the gene coding for complex I subunit NDUFA5 in CNS results in a mild phenotype Presenter: Susana Peralta S. Peralta, A. Torraco, T. Wenz, C.T. Moraes Department of Neurology, University of Miami Miller School of Medicine, United States Mitochondrial complex I deficiencies are associated with severe pathologies which are often lethal in early childhood. To design new therapies, animal models are needed for the study of the pathophysiology and adaptive responses in complex I defects. The NADHubiquinone oxidoreductase 1a subcomplex subunit 5 (NDUFA5) is a nuclear encoded structural subunit of complex I. This 13 kDa protein is located in the peripheral arm of complex I. We have created a conditional NDUFA5 knock-out ablating the endogenous alleles and including a rescuing transgene flanked by loxP sites. The floxed transgene was deleted selectively in neurons by expressing cre driven by the CamK-IIa promoter. NDUFA5-CMK-IIa conditional knock-out (cKO) mice presented lethargy and loss of motor skills at the age of 12 months and death by the 16th month. These mice presented high levels of lactate levels in the blood at the age of 12 months. Blue Native gel electrophoresis showed that the amount of intact complex I was greatly reduced in the cortex and no accumulation of complex I subassemblies was observed. Accordingly, complex I proteins levels were markedly reduced in the NDUFA5-CMK-IIa knock-out mice. Biochemical measurement showed that complex I activity was reduced to 60% of control levels in the cortex at the same age. The activity levels of the other OXPHOS complexes and citrate synthase activity remained unchanged. Our data suggest that NDUFA5-CMK-IIa cKO is a useful mouse model to study the molecular mechanism of mild mitochondrial encephalopathies. doi:10.1016/j.mito.2012.07.101
114 Multiple mitochondrial electron transport chain enzyme deficiencies associated with a decrease in skeletal muscle coenzyme Q10 status Presenter: Iain Hargreaves Iain Hargreaves, John Land Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK Coenzyme Q10 (CoQ10) functions as an electron carrier in the mitochondrial electron transport chain (ETC) as well as serving as a potent lipid soluble antioxidant. Diagnosis of a CoQ10 deficiency normally follows a finding of decreased activity of the linked ETC complex II + III and/or I–III which utilize endogenous CoQ10 and normal activities of complexes I, II and III. Diagnosis is confirmed by HPLC analysis of muscle CoQ10. However, since all ETC complexes are susceptible to free radical induced oxidative damage a deficit in CoQ10 status may in theory result in multiple ETC deficiencies. In order to investigate this hypothesis we assessed the muscle CoQ10 status of 12 patients with combined ETC I (0.075 ± 0.006; ref interval: