BMJ Open BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from Survival transcriptome in coenzyme Q10 deficiency syndrome is acquired by epigenetic modifications: a modelling study for human coenzyme Q10 deficiencies. For peer review only Journal: BMJ Open Manuscript ID: bmjopen-2012-002524 Article Type: Research Date Submitted by the Author: 20-Dec-2012 Complete List of Authors: Fernández-Ayala, Daniel; University Pablo Olavide, Centro Andaluz de Biología del Desarrollo (CABD-CSIC/UPO); CIBERER, Instituto de Salud Carlos III, Guerra, Ignacio; University Pablo Olavide, Centro Andaluz de Biología del Desarrollo (CABD-CSIC/UPO); CIBERER, Instituto de Salud Carlos III, Jiménez-Gancedo, Sandra; University Pablo Olavide, Centro Andaluz de Biología del Desarrollo (CABD-CSIC/UPO); CIBERER, Instituto de Salud Carlos III, Cascajo, Maria; University Pablo Olavide, Centro Andaluz de Biología del Desarrollo (CABD-CSIC/UPO); CIBERER, Instituto de Salud Carlos III, Gavilán, Ángela; University Pablo Olavide, Centro Andaluz de Biología del Desarrollo (CABD-CSIC/UPO); CIBERER, Instituto de Salud Carlos III, DiMauro, Salvatore; Columbia University Medical Center, Department of http://bmjopen.bmj.com/ Neurology Hirano, Michio; Columbia University Medical Center, Department of Neurology Briones, Paz; Instituto de Bioquímica Clínica, Corporació Sanitaria Clínic, ; CIBERER, Instituto de Salud Carlos III, Artuch, Rafael; Hospital Sant Joan de Déu, Department of Clinical Biochemistry; CIBERER, Instituto de Salud Carlos III, deCabo, Rafael; National Institute on Aging, NIH, Laboratory of Experimental Gerontology Salviati, Leonardo; University of Padova, Clinical Genetics Unit, Department on September 28, 2021 by guest. Protected copyright. of Woman and Child Health Navas, Placido; University Pablo Olavide, Centro Andaluz de Biología del Desarrollo (CABD-CSIC/UPO); CIBERER, Instituto de Salud Carlos III, <b>Primary Subject Genetics and genomics Heading</b>: Secondary Subject Heading: Genetics and genomics, Diagnostics Keywords: Neuromuscular disease < NEUROLOGY, BASIC SCIENCES, GENETICS For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 1 of 84 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40 41 42 on September 28, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 84 BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from 1 2 Survival transcriptome in the coenzyme Q10 deficiency syndrome is acquired by epigenetic 3 4 modifications: a modelling study for human coenzyme Q10 deficiencies. 5 6 7 8 Daniel J. M. Fernández-Ayala1,5, Ignacio Guerra1,5, Sandra Jiménez-Gancedo1,5, Maria V. Cascajo1,5, 9 10 1,5 2 2 3,5 4,5 11 Angela Gavilán , Salvatore DiMauro , Michio Hirano , Paz Briones , Rafael Artuch , Rafael de 12 6 7,8 1,5,8 13 Cabo , Leonardo Salviati and Plácido Navas 14 15 For peer review only 16 17 1Centro Andaluz de Biología del Desarrollo (CABD-CSIC), Universidad Pablo Olavide, Seville, Spain 18 19 2 20 Department of Neurology, Columbia University Medical Center, New York, NY, USA 21 3 22 Instituto de Bioquímica Clínica, Corporació Sanitaria Clínic, Barcelona, Spain 23 24 4Department of Clinical Biochemistry, Hospital Sant Joan de Déu, Barcelona, Spain 25 26 5CIBERER, Instituto de Salud Carlos III, Spain 27 28 6Laboratory of Experimental Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, 29 30 31 USA 32 7 33 Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Italy http://bmjopen.bmj.com/ 34 35 8 Senior authorship is shared by LS and PN 36 37 38 39 40 Corresponding authors 41 42 Plácido Navas and Leonardo Salviati on September 28, 2021 by guest. Protected copyright. 43 44 Tel (+34)954349381 and (+39) 3471207364 45 46 e-mail: [email protected] and [email protected] 47 48 49 50 51 52 53 Word count (abstract): 285 54 Word count (summary) 295 55 Word count (introduction, methods, results and discussion): 2947 56 Tables and Illustrations: 4 57 References: 34 58 59 60 1 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 3 of 84 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from 1 2 ABSTRACT 3 4 5 6 Objectives 7 Coenzyme Q10 (CoQ10) deficiency syndrome is a rare condition that causes a mitochondrial 8 dysfunction and includes a variety of clinical presentations, as encephalomyopathy, ataxia and renal 9 10 failure. We sought to set up what all have in common and to investigate why CoQ10 supplementation 11 reverses the bioenergetics alterations in cultured cells but not all the cellular phenotypes. 12 13 Design: modelling study 14 This work models the transcriptome of human CoQ10 deficiency syndrome in primary fibroblast from 15 patients and studyFor the genetic peer response to CoQreview10 treatment in these only cells. 16 17 Setting 18 Four hospitals and medical centres from Spain, Italy and USA, and two research laboratories from 19 20 Spain and USA. 21 22 Participants 23 Primary cells were collected from patients in the above centres. 24 25 Measurements 26 We characterized by microarray analysis the expression profile of fibroblasts from seven CoQ10- 27 deficient patients (three had primary deficiency, four had a secondary form) and aged-matched 28 controls, before and after CoQ supplementation. Results were validated by Q-RT-PCR. The profile 29 10 30 of DNA (CpG) methylation was evaluated for a subset of gene with displayed altered expression. 31 32 Results 33 CoQ10 deficient fibroblasts (independently from the etiology) showed a common transcriptomic profile http://bmjopen.bmj.com/ 34 that promotes cell survival by activating cell cycle and growth, cell stress responses, and inhibiting cell 35 death and immune responses. Energy production was supported mainly by glycolysis while CoQ10 36 supplementation restored oxidative phosphorylation. Expression of genes involved in cell death 37 pathways was partially restored by treatment, while genes involved in differentiation, cell cycle and 38 growth were not affected. Stably demethylated genes were unaffected by treatment whereas we 39 40 observed restored gene expression in either non-methylated genes or those with an unchanged 41 methylation patterns. 42 on September 28, 2021 by guest. Protected copyright. 43 Conclusions 44 CoQ10 deficiency induces a specific transcriptomic profile that promotes cell survival, which is only 45 partially rescued by CoQ10 supplementation. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 84 BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from 1 2 SUMMARY 3 4 5 Article Focus 6 • To analyse the common gene expression profile in primary cell cultures of dermal fibroblasts 7 from patients suffering any of the clinical presentation of the human syndrome of coenzyme 8 Q (CoQ ) deficiency (primary or secondary CoQ deficiency). 9 10 10 10 10 • To exanimate why CoQ10 treatment, the current therapy for all forms of CoQ10 deficiency, 11 restored respiration but not all the clinical phenotypes. 12 • To investigate the stable genetic cause responsible for the survival adaptation to mitochondrial 13 dysfunction due to CoQ10 deficiency. 14 15 For peer review only 16 Key Messages 17 18 • The mitochondrial dysfunction due to CoQ10 deficiency induces a stable survival adaptation of 19 somatic cells in patients at early or postnatal development by epigenetic modifications of 20 chromatin. Deficient cells unable to maintain this survival state during differentiation would 21 death contributing to the pathological phenotype. 22 • Supplementation with CoQ10 restores respiration through enhanced sugar rather than lipid 23 metabolism; partially restores stress response, immunity, cell death and apoptotic pathways; 24 and does not affect cell cycle, cell growth, and differentiation and development pathways. 25 26 • Unaffected genes by CoQ10 treatment corresponded to DNA-demethylated genes, whereas 27 those responsive to CoQ10 corresponded to genes with unchanged DNA-methylation pattern. 28 These results would approach to explain the incomplete recovery of clinical symptoms after 29 CoQ10 treatment, at least in some patients. 30 31 32 Strengths and Limitations 33 • Human CoQ10 deficiencies are considered rare diseases with low prevalence, which limits the http://bmjopen.bmj.com/ 34 sample size. 35 36 • The genetic heterogeneity of this disease is due to mutations in any of the 11 genes directly 37 involved in the synthesis of CoQ10 inside mitochondria, or other mutations altering somehow 38 the mitochondria and its metabolism, affecting their inner CoQ10 synthesis as a side effect, will 39 course with CoQ10 deficiency. 40 • Among this genetic heterogeneity, all cells showed a common transcriptomic profile that 41 justified their pathological phenotype, responded equally to CoQ10 treatment, and presented the on September 28, 2021 by guest. Protected copyright. 42 same DNA methylation pattern. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 84 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from 1 2 KEY WORDS 3 4 Coenzyme Q, CoQ10-deficiency, transcriptome, epigenetic, human fibroblast 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40 41 42 on September 28, 2021 by guest.