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, 35128 Padova. TEL. 049.8211943 FAX. 049.8751770 e-mail: [email protected] TELETHON project # GTF02009
ABSTRACT
ORGANIZATION OF THE BANK
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 followup 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.
INCOMING DNA SAMPLES
FREEZER –20°C
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:
http://morgan.imag.fr:9443/eurobiobank/en/partners/partners.htm
STORAGE AND ACCESS TO THE BANK
MUSCLE BIOPSY
TANK OF
LIQUID
NITROGEN
ULTRA
FREEZER
-80° C
FREEZER
-20° C
FROZEN TISSUES CELL CULTURES
MORPHOLOGY
MYOBLAST
BIOCHEMISTRY
FIBROBLAST
PROTEIN ANALYSIS
CYBRYD
PRENATAL DIAGNOSIS
• 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.
PICTURES OF THE BANK
SAMPLE
REQUEST
DNA SAMPLES
REQUEST
APPROVAL
COMPUTER
DATA BASE
DNA/RNA
MOLECULAR
BIOLOGY
SAMPLES PROVIDED
TO LABORATORIES
INCOMING MUSCLE BIOPSY SAMPLES
COMPUTER
STAFF EFFORT
ULTRA FREEZER –80°C
TANK OF LIQUID N2
FROZEN TISSUES
COMPUTER
CELL CULTURES
Bank of frozen muscle samples and slides
OUTGOING SAMPLES
WRITTEN REQUEST ON TELETHON FORMS
DELIVERY OF SAMPLES
DATABASE
MUSCLE MORPHOLOGY, IMMUNOHISTOCHEMISTRY
DATA BANK
RECEPTION, TREATMENT, STORAGE
BIOCHEMISTRY, PROTEIN ANALYSIS
DNA/RNA EXTRACTION
Myoblasts, myotubes
*
PRENATAL DIAGNOSIS
MUTATION DETECTION
TISSUE SAMPLES STORED FROZEN
6000 muscle, 240 nerve, 122 thymus, 18 heart, 54 skin, 9 chorion villi
Disease
Disease code
MIM #
N. of cases
Duchenne dystrophy
DMD
310200
199 *
Becker dystrophy
BMD
310100
165 *
DMD/BMD carrier
DMD/BMD
Sarcoglycanopathy
LGMD2C-2F
Calpainopathy
LGMD2A
253600
57 *
Disferlinopathy
LGMD2B
253601
13 *
Caveolinopathy
LGMD1C
607801
8*
Limb-girdle dystrophy
LGMD
Limb-girdle dystrophy type 2I
LGMD2I
607155
13 *
Merosinopathy
LAMA-2
156225
22 *
Congenital dystrophy
CMD
Facio-scapulo-humeral
FSHD
158900
29 *
Myotonic dystrophy
DM-1
160900
57 *
Cong. myotonic dystrophy
DM-1
160900
14 *
Thomsen disease
TD
160800
18
310200, 310100
253700, 600119, 604286, 601287
Oculo-pharyngeal
52 *
34 *
199
41
Scapulo-peroneal
33
OPMD
164300
Distal myopathy
13
10
Nemaline myopathy
NEM-1, NEM-2
Central-core myopathy
CCD
Minicore myopathy
Myotubular myopathy
MTM-1
161800, 256030
14
117000
25
157550
45
310400, 160150, 255200
29
Fiber type disproportion
255310
55
Tubular aggregates
160565
26
Pompe disease
232300
15 *
Mc Ardle disease
232600
36 *
Respiratory chain defects
15 *
Mitochondrial encephalomyop.
27 *
PEO, Kearns-Sayre syndrome
KSS
530000
64
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
151900
Multiple lipomatosis
LMS
Myasthenia gravis
MG
119
Congenital myastenia
CMG
3
Myastenic syndrome
11
5
Periodic paralysis
HP
170400, 170500, 170600
5
Malignant hyperthermia
MH
145600, 154275
59 *
Werdnig-Hoffmann
SMA-1, SMA-2
253300
45 *
Kugelberg-Welander
SMA-3
253400
31 *
Amyotrophic lateral sclerosis
ALS
Charcot-Marie-Tooth disease
CMT-1A
292
118220
Chronic neuropathy
* MOLECULARLY OR BIOCHEMICALLY PROVEN
Myotubes
?
MOLECULAR DIAGNOSIS
54
37
COMMITTEE APPROVAL
SAMPLES STORED IN VITAL
FREEZING FOR CELL CULTURE
Muscle biopsies: 2320, fibroblasts/myoblasts: 102, cell lines: 6
DISEASE
Duchenne dystrophy
Becker dystrophy
DMD/BMD carrier
Sarcoglycanopathy
Calpainopathy
Limb-girdle dystrophy
Facio-scap-humeral
Congenital dystrophy
Myotonic dystrophy
Congenital myotonic
Thomsen disease
Scapulo-peroneal
Oculo-pharyngeal
Distal myopathy
Nemaline myopathy
Centralcore myopathy
Minicore myopathy
Myotubular myopathy
Tubular aggregates
Fiber type disproportion
Charcot-Marie-Tooth
N.
114
85
23
22
19
61
54
25
44
8
13
16
5
12
7
1
18
5
9
33
13
DISEASE
N.
Polymyositis
22
Dermatomyositis
20
Inclusion body myositis
9
Myasthenia gravis
9
Congenital myasthenia
1
Acid maltase deficiency
5
McArdle glycogenosis
14
Other glycogenoses
5
Respiratory chain defect
8
Mitochondrial enceph.
20
PEO/KSS
27
Mitochondrial myopathy
23
Metabolic myopathy
14
Lipid storage myopathy
46
Myasthenic syndrome
2
Periodic paralysis
5
Malignant hyperthermia
24
Werdnig-Hoffmann
36
Kugelberg-Welander
36
Amyotrophic lateral sclerosis 118
Chronic neurogenic atrophy 38
DNA SAMPLES OF DIFFERENT PATHOLOGY
Blood:1550, muscle:414, cells: 7
DISEASE
TOTAL = 2000
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
EXPRESS COURIER
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
Claude Desnuelle
Giovanna Cenacchi
Elena Pegoraro
Carlo Foresta
Aaron Russell
Elena Pagoraro
Marco Carrozzi
Luigi Fulizio
Alessandra Ferlini
Leonardo Salviati
Andrea Martinuzzi
Botta Annalisa
Gerolamo Lanfranchi
Raffaella Di Lisi
Peter Van der Bergh
Leonardo Salviati
Marina Fanin
Roberto Massa
Cinzia Gellera
Hospital de l'Archet, Nice, France
University of Bologna, Italy
University of Padova, Italy
University of Padova, Italy
Clinique de readaptation, Sion, Switzerland
University of Padova, Italy
Burlo Garofolo Hospital, Trieste, Italy
University of Padova, Italy
University of Ferrara, Italy
University of Padova, Italy
Medea Hospital, Conegliano, Italy
Tor Vergata University Rome, Italy
CRIBI, University of Padova, Italy
University of Padova, Italy
Lab. Neuropathologie, Brussels, Belgium
University of Padova, Italy
University of Padova, Italy
Tor Vergata University Rome, Italy
Besta Neurologic Institute Milan
Bank of muscle samples kept in vital
freezing in liquid nitrogen 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.
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: 1118-21; 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. 2005 (in press)
6. Fanin M, Nascimbeni AC, Fulizio L, Angelini C. The frequency of limb girdle muscular dystrophy 2A in northeastern Italy. Neuromusc. Disord. 2005 (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. 2005 (in press)