Serum Profiling Identifies Novel Muscle miRNA and Cardiomyopathy-Related miRNA Biomarkers in Golden Retriever Muscular Dystrophy Dogs and Duchenne Muscular Dystrophy Patients
2014; Elsevier BV; Volume: 184; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2014.07.021
ISSN1525-2191
AutoresLaurence Jeanson-Leh, Julie Lameth, Soraya Krimi, Julien Buisset, Fatima Amor, Caroline Le Guiner, Inès Barthélémy, Laurent Servais, Stéphane Blot, Thomas Voit, David Israeli,
Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoDuchenne muscular dystrophy (DMD) is a fatal, X-linked neuromuscular disease that affects 1 boy in 3500 to 5000 boys. The golden retriever muscular dystrophy dog is the best clinically relevant DMD animal model. Here, we used a high-thoughput miRNA sequencing screening for identification of candidate serum miRNA biomarkers in golden retriever muscular dystrophy dogs. We confirmed the dysregulation of the previously described muscle miRNAs, miR-1, miR-133, miR-206, and miR-378, and identified a new candidate muscle miRNA, miR-95. We identified two other classes of dysregulated serum miRNAs in muscular dystrophy: miRNAs belonging to the largest known miRNA cluster that resides in the imprinting DLK1-DIO3 genomic region and miRNAs associated with cardiac disease, including miR-208a, miR-208b, and miR-499. No simple correlation was identified between serum levels of cardiac miRNAs and cardiac functional parameters in golden retriever muscular dystrophy dogs. Finally, we confirmed a dysregulation of miR-95, miR-208a, miR-208b, miR-499, and miR-539 in a small cohort of DMD patients. Given the interspecies conservation of miRNAs and preliminary data in DMD patients, these newly identified dysregulated miRNAs are strong candidate biomarkers for DMD patients. Duchenne muscular dystrophy (DMD) is a fatal, X-linked neuromuscular disease that affects 1 boy in 3500 to 5000 boys. The golden retriever muscular dystrophy dog is the best clinically relevant DMD animal model. Here, we used a high-thoughput miRNA sequencing screening for identification of candidate serum miRNA biomarkers in golden retriever muscular dystrophy dogs. We confirmed the dysregulation of the previously described muscle miRNAs, miR-1, miR-133, miR-206, and miR-378, and identified a new candidate muscle miRNA, miR-95. We identified two other classes of dysregulated serum miRNAs in muscular dystrophy: miRNAs belonging to the largest known miRNA cluster that resides in the imprinting DLK1-DIO3 genomic region and miRNAs associated with cardiac disease, including miR-208a, miR-208b, and miR-499. No simple correlation was identified between serum levels of cardiac miRNAs and cardiac functional parameters in golden retriever muscular dystrophy dogs. Finally, we confirmed a dysregulation of miR-95, miR-208a, miR-208b, miR-499, and miR-539 in a small cohort of DMD patients. Given the interspecies conservation of miRNAs and preliminary data in DMD patients, these newly identified dysregulated miRNAs are strong candidate biomarkers for DMD patients. Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease that affects 1 boy in 3500 to 5000 boys. This recessive lethal disorder is the most frequent form of muscular dystrophy in children. DMD is caused by a deficiency of the dystrophin protein that leads to progressive degeneration of skeletal and cardiac muscle tissues that is eventually replaced by fat and connective tissues. Death is provoked almost exclusively by respiratory or cardiac failure, with a growing proportion of cardiac failure in recent years.1Passamano L. Taglia A. Palladino A. Viggiano E. D'Ambrosio P. Scutifero M. Rosaria Cecio M. Torre V. De Luca F. Picillo E. Paciello O. Piluso G. Nigro G. Politano L. Improvement of survival in Duchenne Muscular Dystrophy: retrospective analysis of 835 patients.Acta Myol. 2012; 31: 121-125PubMed Google Scholar, 2Kieny P. Chollet S. Delalande P. Le Fort M. Magot A. Pereon Y. Perrouin Verbe B. Evolution of life expectancy of patients with Duchenne muscular dystrophy at AFM Yolaine de Kepper centre between 1981 and 2011.Ann Phys Rehabil Med. 2013; 56: 443-454Crossref PubMed Scopus (116) Google Scholar The best clinically relevant animal model for DMD is the golden retriever muscular dystrophy (GRMD) dog.3Banks G.B. Chamberlain J.S. The value of mammalian models for duchenne muscular dystrophy in developing therapeutic strategies.Curr Top Dev Biol. 2008; 84: 431-453Crossref PubMed Scopus (103) Google Scholar The GRMD dog has a mutation in the sixth intron of the canine dystrophin gene, causing an aberrant splicing event, resulting in a premature stop codon and the absence of dystrophin protein. Clinically, symptoms in dogs are close to those in DMD patients: abnormal posture, locomotion defects, progressive muscle degeneration, fibrosis, adipogenesis, respiratory insufficiency, and dilated cardiomyopathy.4Kornegay J.N. Bogan J.R. Bogan D.J. Childers M.K. Li J. Nghiem P. Detwiler D.A. Larsen C.A. Grange R.W. Bhavaraju-Sanka R.K. Tou S. Keene B.P. Howard Jr., J.F. Wang J. Fan Z. Schatzberg S.J. Styner M.A. Flanigan K.M. Xiao X. Hoffman E.P. Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies.Mamm Genome. 2012; 23: 85-108Crossref PubMed Scopus (122) Google Scholar Moreover, the GRMD model size is comparable with that of young DMD boys, providing an attractive advantage when translating treatments from animals to patients. Classically, analytical methods for diagnosis and monitoring of DMD patients are based on muscle histology, quantification of serum creatine kinase (CK), electromyography, electrocardiography, and DNA mutation analysis.5Emery A.E. The muscular dystrophies.Lancet. 2002; 359: 687-695Abstract Full Text Full Text PDF PubMed Scopus (1146) Google Scholar Disease monitoring from muscle biopsies provides important indications but remains an invasive medical act that provides variable results, depending on the biopsy sites and the timing of sampling. The muscle isoform of CK is expressed in skeletal muscle and released into the circulation after muscle fiber membrane leakiness and/or breakdown. However, CK lacks specificity because it also rises after exercise.6Gasper M.C. Gilchrist J.M. Creatine kinase: a review of its use in the diagnosis of muscle disease.Med Health R I. 2005; 88 (400–404): 398PubMed Google Scholar Moreover, in the GRMD model, the serum CK level might be affected by immunosuppressive treatments irrespective of therapeutic benefit and muscle phenotype,7Rouger K. Larcher T. Dubreil L. Deschamps J.Y. Le Guiner C. Jouvion G. Delorme B. Lieubeau B. Carlus M. Fornasari B. Theret M. Orlando P. Ledevin M. Zuber C. Leroux I. Deleau S. Guigand L. Testault I. Le Rumeur E. Fiszman M. Chérel Y. Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs.Am J Pathol. 2011; 179: 2501-2518Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Barthélémy I. Uriarte A. Drougard C. Unterfinger Y. Thibaud J.L. Blot S. Effects of an immunosuppressive treatment in the GRMD dog model of Duchenne muscular dystrophy.PLoS One. 2012; 7: e48478Crossref PubMed Scopus (22) Google Scholar further limiting its utility. Several recent phase 2 and 3 clinical trials in DMD patients were aimed at the restoration of dystrophin expression. The evaluations of results in these clinical trials were based principally on the quantification of dystrophin expression in muscle biopsies and on a functional assessment of muscle capacity by using the 6-minute walk test. However, dystrophin expression varies, depending on the different muscles and biopsies, its correct quantification is technically not completely resolved, and its correlation to clinical benefit has not been established. Moreover, the measured performance of DMD boys in the 6-minute walk test does not depend merely on muscle capacity, and interpreting results is therefore another unresolved issue.9Wood M.J. To skip or not to skip: that is the question for duchenne muscular dystrophy.Mol Ther. 2013; 21: 2131-2132Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 10Hoffman E.P. McNally E.M. Exon-skipping therapy: a roadblock, detour, or bump in the road?.Sci Transl Med. 2014; 6: 230fs14Crossref PubMed Scopus (28) Google Scholar, 11Wilton S.D. Fletcher S. Flanigan K.M. Dystrophin as a therapeutic biomarker: are we ignoring data from the past?.Neuromuscul Disord. 2014; 24: 463-466Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 12Hoffman E.P. Connor E.M. Orphan drug development in muscular dystrophy: update on two large clinical trials of dystrophin rescue therapies.Discov Med. 2013; 16: 233-239PubMed Google Scholar Cardiac troponin (Tpn) is the gold standard serum biomarker for acute coronary syndrome and heart failure, although its elevation can also be associated with other conditions such as respiratory diseases or infections.13Kelley W.E. Januzzi J.L. Christenson R.H. Increases of cardiac troponin in conditions other than acute coronary syndrome and heart failure.Clin Chem. 2009; 55: 2098-2112Crossref PubMed Scopus (167) Google Scholar Cardiac Tpn was found to correlate with cardiac pathology in DMD patients,14Matsumura T. Saito T. Fujimura H. Shinno S. Cardiac troponin I for accurate evaluation of cardiac status in myopathic patients.Brain Dev. 2007; 29: 496-501Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar which, however, could not be confirmed in another study.15Castro-Gago M. Gómez-Lado C. Eirís-Puñal J. Cardiac troponin I for accurate evaluation of cardiac status in myopathic patients.BrainDev. 2009; 31 (letter to the editor): 184Scopus (5) Google Scholar The N-terminal pro-brain natriuretic peptide, a hormone produced mainly by the cardiomyocyte of the ventricular wall, has also been evaluated previously as a biomarker in DMD patients. Ergul et al16Ergul Y. Ekici B. Nisli K. Tatli B. Binboga F. Acar G. Ozmen M. Omeroglu R.E. Evaluation of the North Star Ambulatory Assessment scale and cardiac abnormalities in ambulant boys with Duchenne muscular dystrophy.J Paediatr Child Health. 2012; 48: 610-616Crossref PubMed Scopus (11) Google Scholar have identified significant elevated levels of both cardiac Tpn and N-terminal pro-brain natriuretic peptide in the serum of DMD patients with reduced left ventricular ejection fraction (<55%) and increased risk of development of dilated cardiomyopathy.16Ergul Y. Ekici B. Nisli K. Tatli B. Binboga F. Acar G. Ozmen M. Omeroglu R.E. Evaluation of the North Star Ambulatory Assessment scale and cardiac abnormalities in ambulant boys with Duchenne muscular dystrophy.J Paediatr Child Health. 2012; 48: 610-616Crossref PubMed Scopus (11) Google Scholar They therefore suggested the routine quantification of these two enzymes along with ECG/echocardiographic tests in DMD patients. In contrast, according to Schade van Westrum et al,17Schade van Westrum S. Dekker L. de Haan R. Endert E. Ginjaar I. de Visser M. van der Kooi A. Brain natriuretic peptide is not predictive of dilated cardiomyopathy in Becker and Duchenne muscular dystrophy patients and carriers.BMC Neurol. 2013; 13: 88Crossref PubMed Scopus (15) Google Scholar the measurement of N-terminal pro-brain natriuretic peptide was not helpful for the diagnosis of dilated cardiomyopathy in DMD patients in any phase of the disease. These contradictory reports indicate the urgent necessity in the identification of reliable cardiomyopathy biomarkers for diagnosis, prognosis, and monitoring in DMD patients. miRNAs are small-sized RNA molecules involved in post-transcriptional control of gene expression. Although these molecules are normally present and function inside cells, it was recently discovered that they are also secreted and can be detected in every body fluid. miRNAs are particularly stable in blood samples, and their abundance varies in correlation to the pathophysiological state of the tissue(s) of origin.18Weber J.A. Baxter D.H. Zhang S. Huang D.Y. Huang K.H. Lee M.J. Galas D.J. Wang K. The microRNA spectrum in 12 body fluids.Clin Chem. 2010; 56: 1733-1741Crossref PubMed Scopus (1980) Google Scholar, 19Mitchell P.S. Parkin R.K. Kroh E.M. Fritz B.R. Wyman S.K. Pogosova-Agadjanyan E.L. Peterson A. Noteboom J. O'Briant K.C. Allen A. Lin D.W. Urban N. Drescher C.W. Knudsen B.S. Stirewalt D.L. Gentleman R. Vessella R.L. Nelson P.S. Martin D.B. Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection.Proc Natl Acad Sci U S A. 2008; 105: 10513-10518Crossref PubMed Scopus (6419) Google Scholar, 20Chen X. Ba Y. Ma L. Cai X. Yin Y. Wang K. Guo J. Zhang Y. Chen J. Guo X. Li Q. Li X. Wang W. Zhang Y. Wang J. Jiang X. Xiang Y. Xu C. Zheng P. Zhang J. Li R. Zhang H. Shang X. Gong T. Ning G. Wang J. Zen K. Zhang J. Zhang C.Y. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.Cell Res. 2008; 18: 997-1006Crossref PubMed Scopus (3753) Google Scholar Thus, serum miRNAs have the potential to be used as specific and reliable biomarkers for disease diagnosis, prognosis, and treatment monitoring.21Cortez M.A. Bueso-Ramos C. Ferdin J. Lopez-Berestein G. Sood A.K. Calin G.A. MicroRNAs in body fluids–the mix of hormones and biomarkers.Nat Rev Clin Oncol. 2011; 8: 467-477Crossref PubMed Scopus (1126) Google Scholar Recently, it was found that circulating miRNAs were dysregulated in the mdx mouse, in the CXMDj (canine X-linked muscular dystrophy in Japan) dog (beagle dogs harboring the GRMD mutation), and in human DMD and Becker muscular dystrophyBMD patients (K. Wahbi, F. Amor, L. Jeanson-Leh, N. Vignier, A. Béhin, T. Stojkovic, G. Bonne, T. Voit, D. Israeli, unpublished data).22Vignier N. Amor F. Fogel P. Duvallet A. Poupiot J. Charrier S. Arock M. Montus M. Nelson I. Richard I. Carrier L. Servais L. Voit T. Bonne G. Israeli D. Distinctive serum miRNA profile in mouse models of striated muscular pathologies.PLoS One. 2013; 8: e55281Crossref PubMed Scopus (78) Google Scholar, 23Cacchiarelli D. Legnini I. Martone J. Cazzella V. D'Amico A. Bertini E. Bozzoni I. miRNAs as serum biomarkers for Duchenne muscular dystrophy.EMBO Mol Med. 2011; 3: 258-265Crossref PubMed Scopus (207) Google Scholar, 24Mizuno H. Nakamura A. Aoki Y. Ito N. Kishi S. Yamamoto K. Sekiguchi M. Takeda S. Hashido K. Identification of muscle-specific microRNAs in serum of muscular dystrophy animal models: promising novel blood-based markers for muscular dystrophy.PLoS One. 2011; 6: e18388Crossref PubMed Scopus (151) Google Scholar, 25Roberts T.C. Blomberg K.E. McClorey G. Andaloussi S.E. Godfrey C. Betts C. Coursindel T. Gait M.J. Smith C.I. Wood M.J. Expression analysis in multiple muscle groups and serum reveals complexity in the microRNA transcriptome of the mdx mouse with implications for therapy.Mol Ther Nucleic Acids. 2012; 1: e39Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 26Zaharieva I.T. Calissano M. Scoto M. Preston M. Cirak S. Feng L. Collins J. Kole R. Guglieri M. Straub V. Bushby K. Ferlini A. Morgan J.E. Muntoni F. Dystromirs as serum biomarkers for monitoring the disease severity in Duchenne muscular dystrophy.PLoS One. 2013; 8: e80263Crossref PubMed Scopus (110) Google Scholar These circulating miRNAs included miR-1, miR-133a, miR-133b, miR-206, and miR-378 (muscle miRNA, designated dystromiRs), which are all up-regulated in the serum of affected animals and patients with dystrophin deficiency, probably because of their leakage into the circulation after muscle fiber damage, similar to CK. Oversecretion of exosomes and microparticles from dystrophin-deficient tissues are another potential source of miRNA release into the circulation.27Duguez S. Duddy W. Johnston H. Laine J. Le Bihan M.C. Brown K.J. Bigot A. Hathout Y. Butler-Browne G. Partridge T. Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion.Cell Mol Life Sci. 2013; 70: 2159-2174Crossref PubMed Scopus (49) Google Scholar, 28Le Bihan M.C. Bigot A. Jensen S.S. Dennis J.L. Rogowska-Wrzesinska A. Laine J. Gache V. Furling D. Jensen O.N. Voit T. Mouly V. Coulton G.R. Butler-Browne G. In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts.J Proteomics. 2012; 77: 344-356Crossref PubMed Scopus (98) Google Scholar Studies on various cardiac pathologies previously identified circulating miRNA biomarkers for cardiac dysfunction,29Creemers E.E. Tijsen A.J. Pinto Y.M. Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease?.Circ Res. 2012; 110: 483-495Crossref PubMed Scopus (794) Google Scholar including miR-208b and miR-499, which are expressed in both heart and skeletal muscles, and the heart-specific miR-208a.30Corsten M.F. Dennert R. Jochems S. Kuznetsova T. Devaux Y. Hofstra L. Wagner D.R. Staessen J.A. Heymans S. Schroen B. Circulating MicroRNA-208b and MicroRNA-499 reflect myocardial damage in cardiovascular disease.Circ Cardiovasc Genet. 2010; 3: 499-506Crossref PubMed Scopus (630) Google Scholar, 31Oliveira-Carvalho V. Carvalho V.O. Bocchi E.A. The emerging role of miR-208a in the heart.DNA Cell Biol. 2013; 32: 8-12Crossref PubMed Scopus (71) Google Scholar, 32Wang G.K. Zhu J.Q. Zhang J.T. Li Q. Li Y. He J. Qin Y.W. Jing Q. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans.Eur Heart J. 2010; 31: 659-666Crossref PubMed Scopus (976) Google Scholar However, an evaluation of serum miRNA biomarkers for cardiac pathology in dystrophic animals and patients has not yet been reported. In the first phase of this study we used a miRNA high-throughput sequencing (HTS) technology for a comprehensive identification of dysregulated miRNAs in the serum in GRMD dogs. In a second study phase, a subselection of the identified dysregulated miRNAs was characterized in details in a large-scale (using hundreds of serum samples) longitudinal study. Finally, the dysregulation of cardiac-enriched miRNAs was evaluated in a functional investigation in GRMD dogs, and their dysregulation was confirmed in a small cohort of DMD patients. All dog and mouse procedures were performed in accordance with local ethics committees [Ethical Committee of the Région des Pays de la Loire, University of Angers, France, the common Ethical Committee of ANSES (Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail)/ UPEC (Université Paris-Est Créteil)/ENVA (Ecole Nationale Vétérinaire d'Alfort), and Ethical Committee of Généthon, Evry, France]. Animals were handled according to A1 biosafety requirements and in accordance with the European guidelines for use of experimental animals (L358-86/609/EEC). All experiments were performed to minimize animal discomfort. The human study (DMD patients and controls) was conducted according the principles of the Declaration of Helsinki ethical principles for medical research and was specifically approved by the Ethical Committee CPP Ile de France VI, July 20, 2010, and the Comité d'Ethique (412) du CHR La Citadelle (Liège, Belgium) January 26, 2011. The mouse strains included were the dystrophin-null mdx CV4 and its genetic background control C57BL/6J. Before blood extraction, mice were anesthetized by intraperitoneal injection of ketamine/xylazine. Anesthetized mice were sacrificed by cervical elongation at the end of the experiments. Blood samples were collected into nonheparinized tubes in the absence of anticoagulation treatment. Fresh blood samples were allowed to coagulate 30 minutes at room temperature and spun down at 1800 × g for 10 minutes. Supernatant fluid was collected into fresh tubes and stored at −80°C until further processing. Sera of dogs 60 days old were obtained from the GRMD colonies maintained in the Boisbonne Center for Gene Therapy of the National Veterinary School of Nantes (Atlantic Gene Therapy, ONIRIS, Nantes, France) or from the National Veterinary School of Alfort (Maisons-Alfort, France). Dog cardiac and skeletal muscle (ulnar lateral muscle) tissues were taken from 6- to 7-month-old GRMD dogs and controls. Cardiac TpnI was measured in dog serum samples by using the dedicated kit for the Immulite 2000 analyzer (reference L2KTI; Siemens Healthcare, Malvern, PA). The analytical sensitivity of this test was 0.2 ng/mL, and the maximum measurable concentration was 180 ng/mL. For some dogs with elevated values, a dilution of the serum samples was made by using serum from a healthy dog with confirmed undetectable TpnI. For the cardiac function study, 17 GRMD dogs aged 9.6 to 13.7 months old (mean age, 11.6 ± 1.5 months) underwent a blood sample (miRNA and TpnI levels) assessment synchronized with an echocardiographic examination combined with two-dimensional color Doppler tissue imaging, as previously described.33Chetboul V. Athanassiadis N. Carlos C. Nicolle A. Zilberstein L. Pouchelon J.L. Lefebvre H.P. Concordet D. Assessment of repeatability, reproducibility, and effect of anesthesia on determination of radial and longitudinal left ventricular velocities via tissue Doppler imaging in dogs.Am J Vet Res. 2004; 65: 909-915Crossref PubMed Scopus (55) Google Scholar Briefly, the following parameters were calculated: end-systolic and end-diastolic left ventricular diameters, left ventricular free wall and septum thicknesses; left ventricular fractional shortening (FS) was calculated as[leftventricularend-diastolicdiameter−leftventricularend-systolicdiameter]/leftventricularend-diastolicdiameter×100%.(1) Pulsed-wave Doppler velocities were also measured, notably the mitral E wave-to-A wave ratio was calculated. Doppler tissue imaging examination provided radial left ventricular free wall motion velocities and endoepicardial systolic gradients. The correlation between miRNA levels, TpnI, and all the obtained echocardiographic parameters were first assessed. In a second time the 17 GRMD dogs were divided in two groups according to their FS value, as a quantitative measure of their left ventricular contractile dysfunction. The threshold was set at 30% (normal values obtained in healthy littermates range from 37% to 42.5%34Si J.B. Carzorla O. Blot S. Blanchard-Gulton N. Ait Mou Y. Barthélémy I. Sambin L. Sampedrano C.C. Gouni V. Unterfinger Y. Aguilar P. Thibaud J.L. Bizé A. Pouchelon J.L. Dabiré H. Ghaleh B. Berdeaux A. Chetboul V. Lacampagne A. Hittinger L. Bradykinin restores left ventricular function, sarcomeric protein phosphorylation, and e/nNOS levels in dogs with Duchenne muscular dystrophy cardiomyopathy.Cardiovasc Res. 2012; 95: 86-96Crossref PubMed Scopus (26) Google Scholar), 12 of 17 GRMD dogs had a FS 30% (ranging from 30.6% to 36.4%; mean age, 11.3 ± 1.5 months). A group effect on the level of TpnI and candidate cardiac miRNAs was assessed. Blood samples were obtained after informed consent at CPP Ile de France VI on July 20, 2010, and at the Comité d'Ethique (412) du CHR La Citadelle (Liège, Belgium) on January 26, 2011. Patient inclusion criterion was genetically proven DMD diagnosis. According to the ethical requirements, human blood samples were collected from male subjects >3 years old weighing at least 15 kg for control and for genetically confirmed DMD patients. Peripheral blood samples were collected into 5-mL K3EDTA tubes (Greiner Bio-One, Longwood, FL). Plasma was separated from buffy coat and red blood cells after 10 minutes of centrifugation at 1800 × g and stored at −80°C until further processing. Human skeletal muscle tissues were obtained from the Myobank, the tissue bank of the Association Francaise contre les Myopathies. Open skeletal muscle biopsies were performed, after informed consent, according to the Declaration of Helsinki. Muscle biopsies included in this study were derived from the paravertebral muscle (three controls and two DMD donors) and the dorsal muscles (one DMD donor). In preliminary experiments (data not shown), the miRNeasy (Qiagen, Valencia, CA) and miRVana PARIS (Ambion, Austin, TX) RNA extraction kits were identified as suitable for our experimental system for dog serum and human plasma, respectively. Total RNA extraction was performed from 300 μL of dog serum or 600 μL of human plasma. RNA was eluted in 100 μL of RNase-free water, precipitated overnight, and resuspended in 10 μL of RNase-free water. Total RNA was quantified by using a Nanodrop spectrophotometer (ND8000; Labtech, Wilmington, DE) and analyzed with the Agilent small and pico RNA kit in the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Other RNA samples used in this study are dog total RNA from skeletal muscle, heart, and brain (Zyagen, San Diego, CA) and human total RNA from skeletal muscle, heart, and brain (Ambion). miRNA sequencing was performed by Integragen (Evry, France). Libraries were constructed as described,35Vigneault F. Ter-Ovanesyan D. Alon S. Eminaga S. Christodoulou D.C. Seidman J.G. Eisenberg E. Church G.M. High-throughput multiplex sequencing of miRNA.Curr Protoc Hum Genet. 2012; (Chapter 11:Unit 11.12.1–10)PubMed Google Scholar with some modifications for efficiency improvement in small samples. Briefly, a 3′ adenylated DNA adaptor was ligated in the presence of 12% polyethylene glycol and the absence of ATP, avoiding miRNAs self-ligation. A 5′ RNA adaptor was ligated in the presence of ATP. Reverse transcription primer complementary to the 3′ adaptor was added, forming a duplex to reduce adapter dimer formation. Reverse transcription reaction was done with 1.75 pmol adaptors (3′ adaptor/5′ adaptor/reverse transcription primer), and cDNAs were amplified by 13 PCR cycles with primers complementary to the 3′ and 5′ adaptors. During this PCR step, a specific barcode was incorporated for individual sample recognition. PCR sample band quantification was done with Fragment Analyzer (Advanced Analytical Technologies, Inc., Ames, IA). An equimolar pool of 10 different samples migrated on PAGE, and the miRNA band was extracted (MinElute column; Qiagen). Libraries were quantified by real-time quantitative PCR (qPCR), to load precisely 7 pmol/L pool per line of HiSeq Flow-Cell. The HiSeq 36b and index (barcode) sequencing was done as instructed (Illumina, San Diego, CA) with a SBS V3 kit leading on 150 million passing filter clones. Unique miRNA reads and their copy numbers were analyzed with miRanalyzer online software (http://bioinfo2.ugr.es/miRanalyzer/miRanalyzer.php),36Hackenberg M. Rodríguez-Ezpeleta N. Aransay A.M. miRanalyzer: an update on the detection and analysis of microRNAs in high-throughput sequencing experiments.Nucleic Acids Res. 2011; 39: W132-W138Crossref PubMed Scopus (211) Google Scholar using dog, human, and mouse miRbases as references (19th miRbase, accessed August 2012). Only miRNA with >10 reads in at least half of the animals were considered as expressed. miRNAs were quantified by quantitative RT-PCR (RT-qPCR) with technologies from both Applied Biosystems (AB; Foster City, CA) and Exiqon (Woburn, MA). Total RNA (350 ng) was reverse-transcribed with the Megaplex Primer Pools A and B (human version 3), and miRNAs were quantified with TaqMan Array MicroRNA Cards A and B (human version 3) on the 7900HT Real-Time PCR System (AB) according to the manufacturer's guidelines. Quantification cycle (Cq) values were calculated with the SDS software version 2.3 (AB) by using automatic baseline with a threshold fixed at 0.1. Total RNA (10 ng or 50 ng in the case of miR-208a) was converted to cDNA via miRNA-specific stem loop reverse transcription primers (Applied Biosystems miRNA assays). cDNA was diluted 8× or 4× in the case of miR-208a and quantified with miRNA-specific primers and Taqman probes by using the 7900HT Real-Time PCR system (AB). Cq values were calculated with the SDS software version 2.3 (AB) by using automatic baseline with a threshold fixed at 0.2. Total RNA (20 ng) was converted into poly-A–primed universal cDNA, and miRNAs were quantified with miRNA-specific locked nucleic acid primers on the 7900HT Real-Time PCR System (AB) according to the manufacturer's guidelines. Cq values were calculated with the SDS software version 2.3 (AB) by using automatic baseline with a threshold fixed at 0.2. miRNA abundances were calculated from the HTS data by normalizing unique miRNA read counts relative to the total read count per serum sample. miRNA expression RT-qPCR results, expressed as raw Cq with a threshold Cq ≤ 35, were normalized in the large-scale screenings to the calculated mean Cq of the sample (21) and to miR-16 for individual assays. Differential expression was calculated with the 2-ΔCt method, and miRNAs were considered differentially expressed beyond a threshold of a two-fold change. The HTS technology was used to profile and compare miRNA expression in the serum of 6-week-old GRMD dogs with control dogs (n = 5). Mean read count was approximately 1.2 million miRNAs per GRMD and 0.8 million miRNAs per control dog sample. Recovered sequences were analyzed by miRAnalyzer.36Hackenberg M. Rodríguez-Ezpeleta N. Aransay A.M. miRanalyzer: an update on the detection and analysis of microRNAs in high-throughput sequencing experiments.Nucleic Acids Res. 2011; 39: W132-W138Crossref PubMed Scopus (211) Google Scholar We took advantage of the strong homology between human and dog and between mouse and dog complete miRNA content (perfect identity of 57% and 47%, respectively; 19th miRbase, August 2012) to match the obtained sequences with the dog, human, and mouse miRbases. Among a total of 430 expressed miRNA species, 198 (46%) were referenced in the dog miRbase and 232 (54%) were not, but they were identified by identity to their human and mouse orthologs. A list of all detected miRNAs in dog serum is presented in Supplemental Table S1. Dysregulated miRNAs in GRMD dogs are presented graphically in Figure 1 and Supplemental Tables S2 and S3 for the up- and down-regulated miRNAs in GRMD dog serum, respectively. Among the up-regulated miRNAs we found the previously identified dystromiRs miR-1, miR-133a, miR-133b, miR-206, and miR-378,22Vignier N. Amor F. Fogel P. Duvallet A. Poup
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