Open Access Research BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from Survival transcriptome in the coenzyme fi Q10 de ciency syndrome is acquired by epigenetic modifications: a modelling study for human fi coenzyme Q10 de ciencies Daniel J M Fernández-Ayala,1,2 Ignacio Guerra,1,2 Sandra Jiménez-Gancedo,1,2 Maria V Cascajo,1,2 Angela Gavilán,1,2 Salvatore DiMauro,3 Michio Hirano,3 Paz Briones,2,4 Rafael Artuch,2,5 Rafael De Cabo,6 Leonardo Salviati,7 Plácido Navas1,2 To cite: Fernández-Ayala DJM, ABSTRACT ARTICLE SUMMARY Guerra I, Jiménez-Gancedo S, Objectives: Coenzyme Q (CoQ ) deficiency et al 10 10 .Survivaltranscriptomein syndrome is a rare condition that causes mitochondrial the coenzyme Q deficiency Article focus 10 dysfunction and includes a variety of clinical syndrome is acquired ▪ To analyse the common gene expression profile by epigenetic modifications: presentations as encephalomyopathy, ataxia and renal in primary cell cultures of dermal fibroblasts a modelling study for human failure. First, we sought to set up what all have in from patients suffering any of the clinical presen- common, and then investigate why CoQ coenzyme Q10 deficiencies. 10 tation of the human syndrome of coenzyme Q10 BMJ Open 2013;3:e002524. supplementation reverses the bioenergetics alterations (CoQ10) deficiency (primary or secondary CoQ10 doi:10.1136/bmjopen-2012- in cultured cells but not all the cellular phenotypes. deficiency). 002524 Design Modelling study: This work models the ▪ To determine why CoQ10 treatment, the current transcriptome of human CoQ10 deficiency syndrome in therapy for all forms of CoQ10 deficiency, restored ▸ Prepublication history and primary fibroblast from patients and study the genetic respiration but not all the clinical phenotypes. http://bmjopen.bmj.com/ additional material for this response to CoQ10 treatment in these cells. ▪ To investigate the stable genetic cause respon- paper are available online. To Setting: Four hospitals and medical centres from sible for the survival adaptation to mitochondrial view these files please visit Spain, Italy and the USA, and two research laboratories dysfunction owing to CoQ10 deficiency. the journal online from Spain and the USA. (http://dx.doi.org/10.1136/ Participants: Primary cells were collected from Key messages bmjopen-2012-002524). ▪ The mitochondrial dysfunction owing to CoQ patients in the above centres. 10 deficiency induces a stable survival adaptation of Measurements: We characterised by microarray somatic cells in patients at early or postnatal analysis the expression profile of fibroblasts from Senior authorship is shared development by epigenetic modifications of chro- seven CoQ -deficient patients (three had primary on September 29, 2021 by guest. Protected copyright. 10 matin. Deficient cells unable to maintain this sur- by LS and PN. deficiency and four had a secondary form) and aged- vival state during differentiation would die matched controls, before and after CoQ Received 20 December 2012 10 contributing to the pathological phenotype. supplementation. Results were validated by Q-RT-PCR. Accepted 28 January 2013 ▪ Supplementation with CoQ restores respiration The profile of DNA (CpG) methylation was evaluated 10 through enhanced sugar rather than lipid metab- for a subset of gene with displayed altered expression. This final article is available olism; partially restores stress response, immun- for use under the terms of Results: CoQ10-deficient fibroblasts (independently ity, cell death and apoptotic pathways; and does the Creative Commons from the aetiology) showed a common transcriptomic not affect cell cycle, cell growth, and differenti- Attribution Non-Commercial profile that promotes cell survival by activating cell 2.0 Licence; see ation and development pathways. cycle and growth, cell stress responses and inhibiting ▪ http://bmjopen.bmj.com Survival transcriptome in the CoQ10 deficiency cell death and immune responses. Energy production syndrome is acquired by epigenetic modifications was supported mainly by glycolysis while CoQ10 of DNA: DNA-demethylated genes corresponded For numbered affiliations see supplementation restored oxidative phosphorylation. to unaffected genes by CoQ10 treatment, whereas end of article. Expression of genes involved in cell death pathways those with unchanged DNA-methylation pattern was partially restored by treatment, while genes corresponded to genes with responsive expres- involved in differentiation, cell cycle and growth were Correspondence to sion to CoQ10 supplementation. These results Dr Plácido Navas and not affected. Stably demethylated genes were would approach to explain the incomplete recov- Leonardo Salviati, unaffected by treatment whereas we observed restored ery of clinical symptoms after CoQ10 treatment, at [email protected] and gene expression in either non-methylated genes or least in some patients. [email protected] those with an unchanged methylation pattern. Fernández-Ayala DJM, Guerra I, Jiménez-Gancedo S, et al. BMJ Open 2013;3:e002524. doi:10.1136/bmjopen-2012-002524 1 Survival transcriptome in coenzyme Q10 deficiency syndrome BMJ Open: first published as 10.1136/bmjopen-2012-002524 on 25 March 2013. Downloaded from Co, St Louis, Missouri) and 20% fetal bovine serum (FBS, ARTICLE SUMMARY Linus). When required, CoQ10 prediluted in FBS was fi Strengths and limitations of this study added to the plates at a nal concentration of 30 µM ▪ Human CoQ deficiencies are considered rare diseases with (CoQ10, Synthetic Minimum 98%, high-performance 10 fi low prevalence, which limits the sample size. liquid chromatography, Sigma). We studied ve patients fi ▪ The genetic heterogeneity of this disease is owing to mutations with primary CoQ10 de ciency: two siblings harboured a in any of the 11 genes directly involved in the synthesis of homozygous p.Y297C mutation in the COQ2 gene,5 other CoQ10 inside mitochondria, or other mutations altering with a pathogenic mutation (c.483G>C) in the COQ4 gene somehow the mitochondria and its metabolism, affecting their (this paper), and another one with haploinsufficiency of inner CoQ synthesis as a side effect, will course with CoQ COQ4 18 fi 10 10 . Patients with secondary CoQ10 de ciency deficiency. included: a mitochondrial encephalopathy, lactic acidosis ▪ Among this genetic heterogeneity, all cells showed a common and stroke-like episodes patient harbouring the transcriptomic profile that justified their pathological pheno- m.3243A>G in the mitochondrial tRNALeu(UUR) with 43% type, responded equally to CoQ10 treatment and presented the 8 same DNA methylation pattern. heteroplasmy level, a patient with mtDNA depletion syn- drome12 and a third patient with ataxia of unknown origin.4 Table 1 summarises the clinical phenotype and biochemical studies of these patients. Conclusions: CoQ deficiency induces a specific transcriptomic 10 Transcriptome analysis profile that promotes cell survival, which is only partially rescued by RNA extraction, probe synthesis and hybridisation with CoQ10 supplementation. two independent expression arrays (GeneChip Human Genome U133 Plus 2.0 and GeneChip Human Gene 1.0 ST, Affymetrix) were used as described.19 Gene expres- INTRODUCTION sion was validated by the MyiQ Single Color Real Time Coenzyme Q10 (CoQ10) is a small electron carrier which PCR Detection System (Biorad). See supplementary is an essential cofactor for several mitochondrial bio- methods for full description. chemical pathways such as oxidative phosphorylation, Data had been deposited with the NCBI-GEO data- β-oxidation and pyrimidine nucleotide biosynthesis. base, at http://www.ncbi.nlm.nih.gov/geo/, accession 1 CoQ10 biosynthesis depends on a multienzyme complex number GSE33941 (this SuperSeries is composed of two that involves at least 11 proteins encoded by COQ genes. subset Series, see online supplementary table S7 for an Mutations in any of these genes cause primary CoQ10 explanation). deficiencies, which are clinically heterogeneous mito- Statistical analyses were performed comparing each http://bmjopen.bmj.com/ chondrial diseases.2 Clinical presentations include ence- signal of patient’s fibroblasts RNA with the correspond- phalomyopathy with lipid storage myopathy and ing signal of control RNA by two different approaches. myoglobinuria,3 ataxia and cerebellar atrophy,4 severe The main statistical analysis for both GeneChip Human infantile encephalomyopathy with renal failure,5 isolated Genome U133 Plus 2.0 Array and GeneChip Human 6 7 myopathy, and nephrotic syndrome. Secondary CoQ10 Gene 1.0 ST Array was achieved as previously deficiency has also been associated with diverse mito- described,19 which selects the most significant genes 8–13 chondrial diseases. In all of these conditions, CoQ10 commonly and equally regulated in all samples using supplementation partially improves symptoms14 15 and very stringent parameters. In a few special cases, other on September 29, 2021 by guest. Protected copyright. usually induces a return to normal growth and respir- unselected but regulated genes were studied because of fi fi 81617 fi ation in CoQ10-de cient broblasts. Adaptation of their role in speci c processes and pathways. They were fi somatic cells to CoQ10 de ciency may affect both onset equally described in table 2. The second statistical ana- and course of the disease. We document common tran- lysis approach for the Gene Ontology (GO) study was scriptomic profile alterations in somatic cells of performed as previously described20