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6000 muscle, 240 nerve, 122 thymus, 18 heart, 54 skin, 9 chorion villi NEUROMUSCULAR BANK OF TISSUES AND DNA SAMPLES CORRADO ANGELINI - Dept. Neurosciences,

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Presentazione sul tema: "6000 muscle, 240 nerve, 122 thymus, 18 heart, 54 skin, 9 chorion villi NEUROMUSCULAR BANK OF TISSUES AND DNA SAMPLES CORRADO ANGELINI - Dept. Neurosciences,"— Transcript della presentazione:

1 6000 muscle, 240 nerve, 122 thymus, 18 heart, 54 skin, 9 chorion villi NEUROMUSCULAR BANK OF TISSUES AND DNA SAMPLES CORRADO ANGELINI - Dept. Neurosciences, University of Padova, via Giustiniani 5, and Venetian Institute of Molecular Medicine, TELETHON project # GTF02009 via Orus 2, Padova. TEL FAX TELETHON project # UNIVERSITA’ DI PADOVA Sample collection and storage The bank collects about 6500 tissue samples stored frozen (6000 skeletal muscle, 240 nerve, 122 thymus, 18 heart, 54 skin, 9 chorion villi) and about 300 new samples are collected every year. The bank stores the slides of stained sections of about 7000 muscle biopsies, and 2400 muscle specimens embedded in epoxy resin for electron microscopy. About 2000 DNA samples from patients and relatives with inherited neuromuscular disorders are stored. A standard protocol for collection of biopsies is followed: at the arrival in our laboratory, each sample is subdivided in several fragments, stored both in a bank of frozen tissues for morphological and biochemical studies, and in a bank for tissue culture, where more than 2400 muscle biopsy samples are kept in "vital freezing" in liquid nitrogen.Since most of muscle diseases are inherited, in many of them the genetic or protein defect has been identified. However, the parallel clinical follow- up of the patients enables to assess a more precise diagnosis and implies occasional revision and updating of diagnoses. A computer database has been implemented, in which are recorded the data of patients who underwent muscle biopsy and DNA sample collection (clinical report, histopathological and molecular diagnosis, sample code number, storage location). A new web site page has been implemented to describe the presence and activity of the bank. Activity of the last year, utilization of the bank During the last year, 213 specimens of muscle biopsy, 4 samples of muscle biopsy embedded in epoxy resin, and 30 DNA samples have been transferred to other italian and foreign laboratories, to analyse muscle specific proteins and transcripts, and to identify gene mutations in rare muscle disorders. The availability of muscle tissues stored in our bank has allowed both scientific collaborations of great importance for a better comprehension of the pathogenetic mechanisms in muscle disorders, and to obtain a molecular, biochemical and pathological diagnosis in a large number of neuromuscular patients. ABSTRACT ORGANIZATION OF THE BANK The bank includes tissues from patients affected with a variety of neuromuscular disorders (Tab.1). A parallel patients’ clinical follow-up and appropriate molecular analyses enable to assess more precise diagnoses. A standard protocol for collection of biopsies is followed. At the arrival in laboratory, each sample is subdivided in several fragments, stored both in a bank of frozen tissues and in a bank for tissue culture kept in "vital freezing" in liquid nitrogen (Tab.2). The bank stores 2000 DNA samples from patients and relatives with inherited neuromuscular disorders (Tab.3). An informatic database records the clinical data, diagnosis, muscle biopsy code number and location of each sample. A web site describing the activity of our bank is available at: TANK OF LIQUID NITROGEN ULTRA FREEZER -80° C DATA BASE COMPUTER CELL CULTURES MYOBLAST FIBROBLAST CYBRYD FROZEN TISSUES MORPHOLOGY BIOCHEMISTRY PROTEIN ANALYSIS PRENATAL DIAGNOSIS STORAGE AND ACCESS TO THE BANK FREEZER -20° C DNA/RNA MOLECULAR BIOLOGY SAMPLE REQUEST APPROVAL SAMPLES PROVIDED TO LABORATORIES MUSCLE BIOPSYDNA SAMPLES INCOMING MUSCLE BIOPSY SAMPLES TANK OF LIQUID N 2 ULTRA FREEZER –80°C DATABASE COMPUTER CELL CULTURES MUSCLE MORPHOLOGY, IMMUNOHISTOCHEMISTRY BIOCHEMISTRY, PROTEIN ANALYSIS PRENATAL DIAGNOSIS FROZEN TISSUES ? Myoblasts, myotubes Myotubes INCOMING DNA SAMPLES MUTATION DETECTION MOLECULAR DIAGNOSIS FREEZER –20°C COMPUTER DATA BANK RECEPTION, TREATMENT, STORAGE DNA/RNA EXTRACTION * OUTGOING SAMPLES WRITTEN REQUEST ON TELETHON FORMS COMMITTEE APPROVAL DELIVERY OF SAMPLES EXPRESS COURIER Proper collection and storage of frozen tissues, samples for cell cultures, and DNA samples; check samples’ safety by continuous supervision of liquid nitrogen, freezer’s temperature, etc. Keep an updated database of collected samples, and record the material provided to other laboratories. Obtain accurate and updated diagnoses by appropriate protein and gene analyses involved in new disorders. STAFF EFFORT EQUIPMENT 3 ultra-deep freezers for storage of about 5000 frozen tissues. 2 vial-tanks for liquid N 2 for tissue storage for cell culture 1 freezer (-20°C) for DNA samples storage general laboratory equipment for microscopy, morpohology, biochemistry, electrophoresis, cell culture, molecular biology. Bank of muscle samples kept in vital freezing in liquid nitrogen for cell culture PICTURES OF THE BANK Bank of frozen muscle samples and slides DISEASE N.DISEASE N. Duchenne dystrophy 114Polymyositis 22 Becker dystrophy 85Dermatomyositis 20 DMD/BMD carrier 23Inclusion body myositis 9 Sarcoglycanopathy 22Myasthenia gravis 9 Calpainopathy 19Congenital myasthenia 1 Limb-girdle dystrophy 61Acid maltase deficiency 5 Facio-scap-humeral 54McArdle glycogenosis 14 Congenital dystrophy 25Other glycogenoses 5 Myotonic dystrophy 44Respiratory chain defect 8 Congenital myotonic 8Mitochondrial enceph. 20 Thomsen disease 13PEO/KSS 27 Scapulo-peroneal 16Mitochondrial myopathy 23 Oculo-pharyngeal 5Metabolic myopathy 14 Distal myopathy 12 Lipid storage myopathy 46 Nemaline myopathy 7 Myasthenic syndrome 2 Centralcore myopathy 1 Periodic paralysis 5 Minicore myopathy 18 Malignant hyperthermia 24 Myotubular myopathy 5 Werdnig-Hoffmann 36 Tubular aggregates 9 Kugelberg-Welander 36 Fiber type disproportion 33 Amyotrophic lateral sclerosis 118 Charcot-Marie-Tooth 13 Chronic neurogenic atrophy 38 SAMPLES STORED IN VITAL FREEZING FOR CELL CULTURE Muscle biopsies: 2320, fibroblasts/myoblasts: 102, cell lines: 6 DNA SAMPLES OF DIFFERENT PATHOLOGY Blood:1550, muscle:414, cells: 7 TOTAL = 2000 DISEASE N. of samples Dystrophinopathies 58 Sarcoglycanopathies 84 Limb-girdle dystrophy 431 Myotonic dystrophy 114 Facio-scapulo-humeral 103 Mitochondrial - metabolic myopathy 330 Channelopathies 74 Spinal muscular atrophies 79 Lipomatosis 26 ACTIVITY OF THE BANK DURING LAST YEAR During the last year, 213 specimens of muscle biopsy, 4 samples of muscle biopsy embedded in epoxy resin, and 30 DNA samples have been transferred to other italian and foreign laboratories, to analyse muscle specific proteins and transcripts, and to identify gene mutations in rare muscle disorders. The availability of muscle tissues stored in our bank has allowed both scientific collaborations of great importance for a better comprehension of the pathogenetic mechanisms in muscle disorders, and to obtain a molecular, biochemical and pathological diagnosis in a large number of neuromuscular patients. UTILIZATION OF THE BANK BY OTHER RESEARCHERS RIASSUNTO La banca ha raccolto finora circa 6500 campioni di tessuti congelati da pazienti affetti da una varietà di malattie neuromuscolari (6000 muscolo, 240 nervo, 122 timo, 18 cuore, 54 cute, 9 villi coriali) ed ogni anni vengono raccolti circa 300 nuovi campioni. Sono conservati anche i vetrini delle sezioni colorate di 6500 biopsie muscolari, e 2400 campioni di biopsie muscolari incluse in resina per microscopia elettronica. La banca di DNA conserva oltre 2000 campioni di pazienti (e familiari) con malattie neuromuscolari ereditarie. Per la raccolta delle biopsie e del DNA viene seguito un protocollo standard: ogni campione viene suddiviso in vari frammenti, destinati parallelamente alla banca di tessuti congelati e alla banca di tessuti per colture cellulari, dove oltre 2400 campioni vengono mantenuti in congelamento vitale in azoto liquido. La maggior parte delle malattie muscolari sono ereditarie, e in molte di queste un difetto genetico o proteico è già stato identificato. Tuttavia, il continuo aggiornamento delle diagnosi è reso possibile da una parallela rivalutazione clinica dei pazienti, da nuovi ed appropriati studi molecolari sul DNA, da studi biochimici sulle proteine muscolari e sui deficit metabolici. E’ stato allestito un database computerizzato contenente i dati clinici, la diagnosi istopatologica, le indagini molecolari, immunoistochimiche e biochimiche, la collocazione di stoccaggio dei campioni, ed è stata edita una pagina web per presentare l'attività della banca. Durante l'ultimo anno, 213 campioni di biopsie muscolari, 4 campioni di tessuto muscolare incluso per microscopia elettronica, e 30 campioni di DNA sono stati trasferiti a vari laboratori italiani o esteri, per l’analisi di proteine e trascritti muscolo-specifici e per la ricerca di mutazioni genetiche. La disponibilità dei tessuti muscolari conservati nella nostra banca ha permesso sia delle collaborazioni scientifiche di grande importanza per una migliore comprensione dei meccansimi patogenetici nelle malattie muscolari, sia di ottenere una diagnosi molecolare, biochimica e istopatologica in un gran numero di pazienti con malattie neuromuscolari. Disease Disease code MIM # N. of cases Duchenne dystrophy DMD * Becker dystrophy BMD * DMD/BMD carrier DMD/BMD , * Sarcoglycanopathy LGMD2C-2F , , , * Calpainopathy LGMD2A * Disferlinopathy LGMD2B * Caveolinopathy LGMD1C * Limb-girdle dystrophy LGMD 199 Limb-girdle dystrophy type 2I LGMD2I * Merosinopathy LAMA * Congenital dystrophy CMD 41 Facio-scapulo-humeral FSHD * Myotonic dystrophy DM * Cong. myotonic dystrophy DM * Thomsen disease TD Scapulo-peroneal 33 Oculo-pharyngeal OPMD Distal myopathy 10 Nemaline myopathy NEM-1, NEM , Central-core myopathy CCD Minicore myopathy Myotubular myopathy MTM , , Fiber type disproportion Tubular aggregates Pompe disease * Mc Ardle disease * Respiratory chain defects 15 * Mitochondrial encephalomyop. 27 * PEO, Kearns-Sayre syndrome KSS Mitochondrial myopathy 33 Metabolic myopathy 39 Lipid storage myopathy LSM 82 CPT deficiency 12 Polymyositis PM 69 Dermatomyositis DM 50 Inclusion body myositis IBM 20 Disendocrine myopathy 64 Multiple lipomatosis LMS Myasthenia gravis MG 119 Congenital myastenia CMG 3 Myastenic syndrome 5 Periodic paralysis HP , , Malignant hyperthermia MH , * Werdnig-Hoffmann SMA-1, SMA * Kugelberg-Welander SMA * Amyotrophic lateral sclerosis ALS 292 Charcot-Marie-Tooth disease CMT-1A Chronic neuropathy 37 TISSUE SAMPLES STORED FROZEN * MOLECULARLY OR BIOCHEMICALLY PROVEN Claude DesnuelleHospital de l'Archet, Nice, France Giovanna CenacchiUniversity of Bologna, Italy Elena PegoraroUniversity of Padova, Italy Carlo Foresta University of Padova, Italy Aaron Russell Clinique de readaptation, Sion, Switzerland Elena Pagoraro University of Padova, Italy Marco Carrozzi Burlo Garofolo Hospital, Trieste, Italy Luigi Fulizio University of Padova, Italy Alessandra FerliniUniversity of Ferrara, Italy Leonardo SalviatiUniversity of Padova, Italy Andrea MartinuzziMedea Hospital, Conegliano, Italy Botta Annalisa Tor Vergata University Rome, Italy Gerolamo LanfranchiCRIBI, University of Padova, Italy Raffaella Di LisiUniversity of Padova, Italy Peter Van der BerghLab. Neuropathologie, Brussels, Belgium Leonardo SalviatiUniversity of Padova, Italy Marina Fanin University of Padova, Italy Roberto Massa Tor Vergata University Rome, Italy Cinzia Gellera Besta Neurologic Institute Milan Publications with acknowledgments of this project: 1. Angelini C, Pegoraro E, Zambito Marsala S, Vergani L, Nascimbeni AC, Fulizio L, Fanin M. Adult acid maltase deficiency: an open trial with albuterol and brached-chain aminoacids. Basic Appl. Myol. 14: 71-78; Fanin M, Fulizio L, Nascimbeni AC, Spinazzi M, Piluso G, Ventriglia VG, Ruzza G, Siciliano G, Trevisan CP, Politano L, Nigro V, Angelini C. Molecular diagnosis of LGMD2A: gene mutation analysis or protein testing? Hum. Mut. 24: 52-62; Prandini P, Berardinelli A, Fanin M, Morello F, Zardini E, Pichiecchio A, Uggetti C, Lanzi G, Angelini C, Pegoraro E. Laminin  -2 negative congenital muscular dystrophy (MDC1A) presenting with a mild phenotype. Neurology 63: ; Fulizio L, Nascimbeni AC, Fanin M, Piluso G, Politano L, Nigro V, Angelini C. Molecular and muscle pathology in a series of caveolinopathy patients. Hum. Mut. 25: 82-89; Cenacchi G, Fanin M, Badiali De Giorgi L, Angelini C. Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism. J. Clin. Pathol (in press) 6. Fanin M, Nascimbeni AC, Fulizio L, Angelini C. The frequency of limb girdle muscular dystrophy 2A in northeastern Italy. Neuromusc. Disord (in press) 7. Piluso G, Politano L, Aurino S, Fanin M, Ricci E, Ventriglia VM, Belsito A, Totaro A, Saccone V, Topaloglu H, Nascimbeni AC, Fulizio L, Broccolini A, Canki-Klain N, Comi LI, Nigro G, Angelini C, Nigro V. The extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes. J. Med. Genet (in press)


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