NEUROMUSCULAR BANK OF TISSUES AND DNA SAMPLES

Presentazione sul tema: "NEUROMUSCULAR BANK OF TISSUES AND DNA SAMPLES"— Transcript della presentazione:

1 NEUROMUSCULAR BANK OF TISSUES AND DNA SAMPLES
UNIVERSITA’ DI PADOVA CORRADO ANGELINI - Dept. Neurosciences, University of Padova, via Giustiniani 5, and Venetian Institute of Molecular Medicine, via Orus 2, Padova. TEL FAX TELETHON project # GTF02009 ABSTRACT ORGANIZATION OF THE BANK STAFF EFFORT 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. 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: 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. EQUIPMENT 3 ultra-deep freezers for storage of about 5000 frozen tissues. 2 vial-tanks for liquid N2 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. STORAGE AND ACCESS TO THE BANK PICTURES OF THE BANK MUSCLE BIOPSY DNA SAMPLES SAMPLE REQUEST ULTRA TANK OF FREEZER REQUEST FREEZER LIQUID COMPUTER -20° C APPROVAL -80° C NITROGEN FROZEN TISSUES CELL CULTURES DNA/RNA DATA BASE MORPHOLOGY MYOBLAST MOLECULAR BIOCHEMISTRY FIBROBLAST BIOLOGY PROTEIN ANALYSIS CYBRYD PRENATAL DIAGNOSIS SAMPLES PROVIDED TO LABORATORIES Bank of frozen muscle samples and slides INCOMING DNA SAMPLES INCOMING MUSCLE BIOPSY SAMPLES OUTGOING SAMPLES MUTATION DETECTION MOLECULAR DIAGNOSIS FREEZER –20°C COMPUTER DATA BANK RECEPTION, TREATMENT, STORAGE DNA/RNA EXTRACTION * WRITTEN REQUEST ON TELETHON FORMS COMMITTEE APPROVAL DELIVERY OF SAMPLES EXPRESS COURIER ULTRA FREEZER –80°C TANK OF LIQUID N2 COMPUTER FROZEN TISSUES CELL CULTURES DATABASE MUSCLE MORPHOLOGY, IMMUNOHISTOCHEMISTRY BIOCHEMISTRY, PROTEIN ANALYSIS Myoblasts, myotubes PRENATAL DIAGNOSIS ? Bank of muscle samples kept in vital freezing in liquid nitrogen for cell culture Myotubes ACTIVITY OF THE BANK DURING LAST YEAR TISSUE SAMPLES STORED FROZEN SAMPLES STORED IN VITAL FREEZING FOR CELL CULTURE 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. 6000 muscle, 240 nerve, 122 thymus, 18 heart, 54 skin, 9 chorion villi 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. Muscle biopsies: 2320, fibroblasts/myoblasts: 102, cell lines: 6 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 Limb-girdle dystrophy type 2I LGMD2I * Merosinopathy LAMA * Congenital dystrophy CMD Facio-scapulo-humeral FSHD * Myotonic dystrophy DM * Cong. myotonic dystrophy DM * Thomsen disease TD Scapulo-peroneal Oculo-pharyngeal OPMD Distal myopathy 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 * Mitochondrial encephalomyop * PEO, Kearns-Sayre syndrome KSS Mitochondrial myopathy Metabolic myopathy Lipid storage myopathy LSM CPT deficiency Polymyositis PM Dermatomyositis DM Inclusion body myositis IBM Disendocrine myopathy Multiple lipomatosis LMS Myasthenia gravis MG Congenital myastenia CMG Myastenic syndrome Periodic paralysis HP , , Malignant hyperthermia MH , * Werdnig-Hoffmann SMA-1, SMA * Kugelberg-Welander SMA * Amyotrophic lateral sclerosis ALS Charcot-Marie-Tooth disease CMT-1A Chronic neuropathy DISEASE N. DISEASE N. Duchenne dystrophy Polymyositis Becker dystrophy Dermatomyositis DMD/BMD carrier Inclusion body myositis Sarcoglycanopathy Myasthenia gravis Calpainopathy Congenital myasthenia Limb-girdle dystrophy Acid maltase deficiency Facio-scap-humeral McArdle glycogenosis Congenital dystrophy Other glycogenoses Myotonic dystrophy Respiratory chain defect Congenital myotonic Mitochondrial enceph Thomsen disease PEO/KSS Scapulo-peroneal Mitochondrial myopathy Oculo-pharyngeal Metabolic myopathy Distal myopathy Lipid storage myopathy Nemaline myopathy Myasthenic syndrome Centralcore myopathy Periodic paralysis Minicore myopathy Malignant hyperthermia Myotubular myopathy Werdnig-Hoffmann Tubular aggregates Kugelberg-Welander Fiber type disproportion Amyotrophic lateral sclerosis 118 Charcot-Marie-Tooth Chronic neurogenic atrophy UTILIZATION OF THE BANK BY OTHER RESEARCHERS Claude Desnuelle Hospital de l'Archet, Nice, France Giovanna Cenacchi University of Bologna, Italy Elena Pegoraro University 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 Ferlini University of Ferrara, Italy Leonardo Salviati University of Padova, Italy Andrea Martinuzzi Medea Hospital, Conegliano, Italy Botta Annalisa Tor Vergata University Rome, Italy Gerolamo Lanfranchi CRIBI, University of Padova, Italy Raffaella Di Lisi University of Padova, Italy Peter Van der Bergh Lab. Neuropathologie, Brussels, Belgium Marina Fanin University of Padova, Italy Roberto Massa Tor Vergata University Rome, Italy Cinzia Gellera Besta Neurologic Institute Milan DNA SAMPLES OF DIFFERENT PATHOLOGY Blood:1550, muscle:414, cells: 7 TOTAL = 2000 DISEASE N. of samples Dystrophinopathies Sarcoglycanopathies Limb-girdle dystrophy Myotonic dystrophy Facio-scapulo-humeral Mitochondrial - metabolic myopathy Channelopathies Spinal muscular atrophies Lipomatosis * MOLECULARLY OR BIOCHEMICALLY PROVEN 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; 2004 2.  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; 2004 3. Prandini P, Berardinelli A, Fanin M, Morello F, Zardini E, Pichiecchio A, Uggetti C, Lanzi G, Angelini C, Pegoraro E. Laminin a-2 negative congenital muscular dystrophy (MDC1A) presenting with a mild phenotype. Neurology 63: ; 2004 4.  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; 2005 5.  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)