Università degli Studi di Torino
Dipartimento di Genetica Biologia e
Biochimica
Mario De Marchi
Alfredo Brusco
Silvia Saviozzi
Alessandro Saluto Nicola Migone
Via Santena 19, 10126 Torino (Tel. 011.6706662)
Torta Margherita
200 g fecola di patate
200 g zucchero
4 uova
succo di mezzo limone
1 bustina zucchero vanigliato
1 bustina lievito
1 pizzico sale
Sbattere bene i tuorli d'uovo con lo zucchero e,
sempre mescolando piano piano, aggiungere
gradatamente la fecola di patate, il limone, lo
zucchero vanigliato, gli albumi montati a neve e, da
ultimo, il lievito. Disporre l'impasto in una teglia
imburrata e passare in forno appena acceso per
circa 25 minuti a temperatura moderata.
Il DNA è un codice per la
sintesi di proteine
La “ricetta” (=sequenza) del gene ATM
GCGAGAGGAGTCGGGATCTGCGCTGCAGCCACCGCCGCGGTTGA
TACTACTTTGACCTTCCGAGTGCAGTGAGGCATACATCACAATTTGG
AATTATGCATTGGTTTATCAATTTACTTGTTTATTGTCACCCTGCTGCCCAGAT
ATGACTTCATGAGGACAGTGATGTGTGTTCTGAAATTGTGAACCATGAGTCTAGTACTTAATGATCT
GCTTATCTGCTGCCGTCAACTAGAACATGATAGAGCTACAGAACGAAAGAAAGAAGTTGAGAAATTTAAGCGCCTGATTCGAGATCCT
GAAACAATTAAACATCTAGAATTGGGATGCTGTTTTTAGATTTTTACAGAAATATATTCAGAAAGAAACAGAATGTCTGAGAATAGCAAAACCAAATGTATCAGCCTCAACACATCGGCATTCA
GATTCCAAACAAGGAAAATATTTGAAGCCTCCAGGCAGAAAAAGATGCAGGAAATCAGTAGTTTGGTCAAATACTTCATCAAATGTGCAAACAGAAGAGCACCTAGGCTAAAATGTCAAGAACTCTTAAATTATATCATGGATACAGTGAAAGATTCATCTAATGGT
GCTATTTACGGAGCTGATTGTAGCAACATACTACTCAAAGACATTCTTTCTGTGAGAAAATACTGGTGTGAAATATCTCAGCAACAGTGGTTAGAATTGTTCTCTGTGTACTTCAGGCTCTATCTGAAACCTTCACAAGATGTTCATAGAGTTTTAGTGGCTAGAATAATTCATGCTG
TTACCAAAGGATGCTGTTCTCAGACTGACGGATTAAATTCCAAATTTTTGGACTTTTTTTCCAAGGCTATTCAGTGTGCGAGACAAGAAAAGAGCTCTTCAGGTCTAAATCATATCTTAGCAGCTCTTACTATCTTCCTCAAGACTTTGGCTGTCAACTTTCGAATTCGAGTGTGTGA
ATTAGGAGATGAAATTCTTCCCACTTTGCTTTATATTTGGACTCAACATAGGCTTAATGATTCTTTAAAAGAAGTCATTATTGAATTATTTCAACTGCAAATTTATATCCATCATCCGAAAGGAGCCAAAACCCAAGAAAAAGGTGCTTATGAATCAACAAAATGGAGAAGTATTTTA
TACAACTTATATGATCTGCTAGTGAATGAGATAAGTCATATAGGAAGTAGAGGAAAGTATTCTTCAGGATTTCGTAATATTGCCGTCAAAGAAAATTTGATTGAATTGATGGCAGATATCTGTCACCAGGTTTTTAATGAAGATACCAGATCCTTGGAGATTTCTCAATCTTACACTA
CTACACAAAGAGAATCTAGTGATTACAGTGTCCCTTGCAAAAGGAAGAAAATAGAACTAGGCTGGGAAGTAATAAAAGATCACCTTCAGAAGTCACAGAATGATTTTGATCTTGTGCCTTGGCTACAGATTGCAACCCAATTAATATCAAAGTATCCTGCAAGTTTACCTAACTGTGA
GCTGTCTCCATTACTGATGATACTATCTCAGCTTCTACCCCAACAGCGACATGGGGAACGTACACCATATGTGTTACGATGCCTTACGGAAGTTGCATTGTGTCAAGACAAGAGGTCAAACCTAGAAAGCTCACAAAAGTCAGATTTATTAAAACTCTGGAATAAAATTTGGTGTATT
ACCTTTCGTGGTATAAGTTCTGAGCAAATACAAGCTGAAAACTTTGGCTTACTTGGAGCCATAATTCAGGGTAGTTTAGTTGAGGTTGACAGAGAATTCTGGAAGTTATTTACTGGGTCAGCCTGCAGACCTTCATGTCCTGCAGTATGCTGTTTGACTTTGGCACTGACCACCAGTA
TAGTTCCAGGAGCGGTAAAAATGGGAATAGAGCAAAATATGTGTGAAGTAAATAGAAGCTTTTCTTTAAAGGAATCAATAATGAAATGGCTCTTATTCTATCAGTTAGAGGGTGACTTAGAAAATAGCACAGAAGTGCCTCCAATTCTTCACAGTAATTTTCCTCATCTTGTACTGGA
GAAAATTCTTGTGAGTCTCACTATGAAAAACTGTAAAGCTGCAATGAATTTTTTCCAAAGCGTGCCAGAATGTGAACACCACCAAAAAGATAAAGAAGAACTTTCATTCTCAGAAGTAGAAGAACTATTTCTTCAGACAACTTTTGACAAGATGGACTTTTTAACCATTGTGAGAGAA
TGTGGTATAGAAAAGCACCAGTCCAGTATTGGCTTCTCTGTCCACCAGAATCTCAAGGAATCACTGGATCGCTGTCTTCTGGGATTATCAGAACAGCTTCTGAATAATTACTCATCTGAGATTACAAATTCAGAAACTCTTGTCCGGTGTTCACGTCTTTTGGTGGGTGTCCTTGGCT
GCTACTGTTACATGGGTGTAATAGCTGAAGAGGAAGCATATAAGTCAGAATTATTCCAGAAAGCCAACTCTCTAATGCAATGTGCAGGAGAAAGTATCACTCTGTTTAAAAATAAGACAAATGAGGAATTCAGAATTGGTTCCTTGAGAAATATGATGCAGCTATGTACACGTTGCTT
GAGCAACTGTACCAAGAAGAGTCCAAATAAGATTGCATCTGGCTTTTTCCTGCGATTGTTAACATCAAAGCTAATGAATGACATTGCAGATATTTGTAAAAGTTTAGCATCCTTCATCAAAAAGCCATTTGACCGTGGAGAAGTAGAATCAATGGAAGATGATACTAATGGAAATCTA
ATGGAGGTGGAGGATCAGTCATCCATGAATCTATTTAACGATTACCCTGATAGTAGTGTTAGTGATGCAAACGAACCTGGAGAGAGCCAAAGTACCATAGGTGCCATTAATCCTTTAGCTGAAGAATATCTGTCAAAGCAAGATCTACTTTTCTTAGACATGCTCAAGTTCTTGTGTT
TGTGTGTAACTACTGCTCAGACCAATACTGTGTCCTTTAGGGCAGCTGATATTCGGAGGAAATTGTTAATGTTAATTGATTCTAGCACGCTAGAACCTACCAAATCCCTCCACCTGCATATGTATCTAATGCTTTTAAAGGAGCTTCCTGGAGAAGAGTACCCCTTGCCAATGGAAGA
TGTTCTTGAACTTCTGAAACCACTATCCAATGTGTGTTCTTTGTATCGTCGTGACCAAGATGTTTGTAAAACTATTTTAAACCATGTCCTTCATGTAGTGAAAAACCTAGGTCAAAGCAATATGGACTCTGAGAACACAAGGGATGCTCAAGGACAGTTTCTTACAGTAATTGGAGCA
TTTTGGCATCTAACAAAGGAGAGGAAATATATATTCTCTGTAAGAATGGCCCTAGTAAATTGCCTTAAAACTTTGCTTGAGGCTGATCCTTATTCAAAATGGGCCATTCTTAATGTAATGGGAAAAGACTTTCCTGTAAATGAAGTATTTACACAATTTCTTGCTGACAATCATCACC
AAGTTCGCATGTTGGCTGCAGAGTCAATCAATAGATTGTTCCAGGACACGAAGGGAGATTCTTCCAGGTTACTGAAAGCACTTCCTTTGAAGCTTCAGCAAACAGCTTTTGAAAATGCATACTTGAAAGCTCAGGAAGGAATGAGAGAAATGTCCCATAGTGCTGAGAACCCTGAAAC
TTTGGATGAAATTTATAATAGAAAATCTGTTTTACTGACGTTGATAGCTGTGGTTTTATCCTGTAGCCCTATCTGCGAAAAACAGGCTTTGTTTGCCCTGTGTAAATCTGTGAAAGAGAATGGATTAGAACCTCACCTTGTGAAAAAGGTTTTAGAGAAAGTTTCTGAAACTTTTGGA
TATAGACGTTTAGAAGACTTTATGGCATCTCATTTAGATTATCTGGTTTTGGAATGGCTAAATCTTCAAGATACTGAATACAACTTATCTTCTTTTCCTTTTATTTTATTAAACTACACAAATATTGAGGATTTCTATAGATCTTGTTATAAGGTTTTGATTCCACATCTGGTGATTA
GAAGTCATTTTGATGAGGTGAAGTCCATTGCTAATCAGATTCAAGAGGACTGGAAAAGTCTTCTAACAGACTGCTTTCCAAAGATTCTTGTAAATATTCTTCCTTATTTTGCCTATGAGGGTACCAGAGACAGTGGGATGGCACAGCAAAGAGAGACTGCTACCAAGGTCTATGATAT
GCTTAAAAGTGAAAACTTATTGGGAAAACAGATTGATCACTTATTCATTAGTAATTTACCAGAGATTGTGGTGGAGTTATTGATGACGTTACATGAGCCAGCAAATTCTAGTGCCAGTCAGAGCACTGACCTCTGTGACTTTTCAGGGGATTTGGATCCTGCTCCTAATCCACCTCAT
TTTCCATCGCATGTGATTAAAGCAACATTTGCCTATATCAGCAATTGTCATAAAACCAAGTTAAAAAGCATTTTAGAAATTCTTTCCAAAAGCCCTGATTCCTATCAGAAAATTCTTCTTGCCATATGTGAGCAAGCAGCTGAAACAAATAATGTTTATAAGAAGCACAGAATTCTTA
AAATATATCACCTGTTTGTTAGTTTATTACTGAAAGATATAAAAAGTGGCTTAGGAGGAGCTTGGGCCTTTGTTCTTCGAGACGTTATTTATACTTTGATTCACTATATCAACCAAAGGCCTTCTTGTATCATGGATGTGTCATTACGTAGCTTCTCCCTTTGTTGTGACTTATTAAG
TCAGGTTTGCCAGACAGCCGTGACTTACTGTAAGGATGCTCTAGAAAACCATCTTCATGTTATTGTTGGTACACTTATACCCCTTGTGTATGAGCAGGTGGAGGTTCAGAAACAGGTATTGGACTTGTTGAAATACTTAGTGATAGATAACAAGGATAATGAAAACCTCTATATCACG
ATTAAGCTTTTAGATCCTTTTCCTGACCATGTTGTTTTTAAGGATTTGCGTATTACTCAGCAAAAAATCAAATACAGTAGAGGACCCTTTTCACTCTTGGAGGAAATTAACCATTTTCTCTCAGTAAGTGTTTATGATGCACTTCCATTGACAAGACTTGAAGGACTAAAGGATCTTC
GAAGACAACTGGAACTACATAAAGATCAGATGGTGGACATTATGAGAGCTTCTCAGGATAATCCGCAAGATGGGATTATGGTGAAACTAGTTGTCAATTTGTTGCAGTTATCCAAGATGGCAATAAACCACACTGGTGAAAAAGAAGTTCTAGAGGCTGTTGGAAGCTGCTTGGGAGA
AGTGGGTCCTATAGATTTCTCTACCATAGCTATACAACATAGTAAAGATGCATCTTATACCAAGGCCCTTAAGTTATTTGAAGATAAAGAACTTCAGTGGACCTTCATAATGCTGACCTACCTGAATAACACACTGGTAGAAGATTGTGTCAAAGTTCGATCAGCAGCTGTTACCTGT
TTGAAAAACATTTTAGCCACAAAGACTGGACATAGTTTCTGGGAGATTTATAAGATGACAACAGATCCAATGCTGGCCTATCTACAGCCTTTTAGAACATCAAGAAAAAAGTTTTTAGAAGTACCCAGATTTGACAAAGAAAACCCTTTTGAAGGCCTGGATGATATAAATCTGTGGA
TTCCTCTAAGTGAAAATCATGACATTTGGATAAAGACACTGACTTGTGCTTTTTTGGACAGTGGAGGCACAAAATGTGAAATTCTTCAATTATTAAAGCCAATGTGTGAAGTGAAAACTGACTTTTGTCAGACTGTACTTCCATACTTGATTCATGATATTTTACTCCAAGATACAAA
TGAATCATGGAGAAATCTGCTTTCTACACATGTTCAGGGATTTTTCACCAGCTGTCTTCGACACTTCTCGCAAACGAGCCGATCCACAACCCCTGCAAACTTGGATTCAGAGTCAGAGCACTTTTTCCGATGCTGTTTGGATAAAAAATCACAAAGAACAATGCTTGCTGTTGTGGAC
TACATGAGAAGACAAAAGAGACCTTCTTCAGGAACAATTTTTAATGATGCTTTCTGGCTGGATTTAAATTATCTAGAAGTTGCCAAGGTAGCTCAGTCTTGTGCTGCTCACTTTACAGCTTTACTCTATGCAGAAATCTATGCAGATAAGAAAAGTATGGATGATCAAGAGAAAAGAA
GTCTTGCATTTGAAGAAGGAAGCCAGAGTACAACTATTTCTAGCTTGAGTGAAAAAAGTAAAGAAGAAACTGGAATAAGTTTACAGGATCTTCTCTTAGAAATCTACAGAAGTATAGGGGAGCCAGATAGTTTGTATGGCTGTGGTGGAGGGAAGATGTTACAACCCATTACTAGACT
ACGAACATATGAACACGAAGCAATGTGGGGCAAAGCCCTAGTAACATATGACCTCGAAACAGCAATCCCCTCATCAACACGCCAGGCAGGAATCATTCAGGCCTTGCAGAATTTGGGACTCTGCCATATTCTTTCCGTCTATTTAAAAGGATTGGATTATGAAAATAAAGACTGGTGT
CCTGAACTAGAAGAACTTCATTACCAAGCAGCATGGAGGAATATGCAGTGGGACCATTGCACTTCCGTCAGCAAAGAAGTAGAAGGAACCAGTTACCATGAATCATTGTACAATGCTCTACAATCTCTAAGAGACAGAGAATTCTCTACATTTTATGAAAGTCTCAAATATGCCAGAG
TAAAAGAAGTGGAAGAGATGTGTAAGCGCAGCCTTGAGTCTGTGTATTCGCTCTATCCCACACTTAGCAGGTTGCAGGCCATTGGAGAGCTGGAAAGCATTGGGGAGCTTTTCTCAAGATCAGTCACACATAGACAACTCTCTGAAGTATATATTAAGTGGCAGAAACACTCCCAGCT
TCTCAAGGACAGTGATTTTAGTTTTCAGGAGCCTATCATGGCTCTACGCACAGTCATTTTGGAGATCCTGATGGAAAAGGAAATGGACAACTCACAAAGAGAATGTATTAAGGACATTCTCACCAAACACCTTGTAGAACTCTCTATACTGGCCAGAACTTTCAAGAACACTCAGCTC
CCTGAAAGGGCAATATTTCAAATTAAACAGTACAATTCAGTTAGCTGTGGAGTCTCTGAGTGGCAGCTGGAAGAAGCACAAGTATTCTGGGCAAAAAAGGAGCAGAGTCTTGCCCTGAGTATTCTCAAGCAAATGATCAAGAAGTTGGATGCCAGCTGTGCAGCGAACAATCCCAGCC
TAAAACTTACATACACAGAATGTCTGAGGGTTTGTGGCAACTGGTTAGCAGAAACGTGCTTAGAAAATCCTGCGGTCATCATGCAGACCTATCTAGAAAAGGCAGTAGAAGTTGCTGGAAATTATGATGGAGAAAGTAGTGATGAGCTAAGAAATGGAAAAATGAAGGCATTTCTCTC
ATTAGCCCGGTTTTCAGATACTCAATACCAAAGAATTGAAAACTACATGAAATCATCGGAATTTGAAAACAAGCAAGCTCTCCTGAAAAGAGCCAAAGAGGAAGTAGGTCTCCTTAGGGAACATAAAATTCAGACAAACAGATACACAGTAAAGGTTCAGCGAGAGCTGGAGTTGGAT
GAATTAGCCCTGCGTGCACTGAAAGAGGATCGTAAACGCTTCTTATGTAAAGCAGTTGAAAATTATATCAACTGCTTATTAAGTGGAGAAGAACATGATATGTGGGTATTCCGGCTTTGTTCCCTCTGGCTTGAAAATTCTGGAGTTTCTGAAGTCAATGGCATGATGAAGAGAGACG
GAATGAAGATTCCAACATATAAATTTTTGCCTCTTATGTACCAATTGGCTGCTAGAATGGGGACCAAGATGATGGGAGGCCTAGGATTTCATGAAGTCCTCAATAATCTAATCTCTAGAATTTCAATGGATCACCCCCATCACACTTTGTTTATTATACTGGCCTTAGCAAATGCAAA
CAGAGATGAATTTCTGACTAAACCAGAGGTAGCCAGAAGAAGCAGAATAACTAAAAATGTGCCTAAACAAAGCTCTCAGCTTGATGAGGATCGAACAGAGGCTGCAAATAGAATAATATGTACTATCAGAAGTAGGAGACCTCAGATGGTCAGAAGTGTTGAGGCACTTTGTGATGCT
TATATTATATTAGCAAACTTAGATGCCACTCAGTGGAAGACTCAGAGAAAAGGCATAAATATTCCAGCAGACCAGCCAATTACTAAACTTAAGAATTTAGAAGATGTTGTTGTCCCTACTATGGAAATTAAGGTGGACCACACAGGAGAATATGGAAATCTGGTGACTATACAGTCAT
TTAAAGCAGAATTTCGCTTAGCAGGAGGTGTAAATTTACCAAAAATAATAGATTGTGTAGGTTCCGATGGCAAGGAGAGGAGACAGCTTGTTAAGGGCCGTGATGACCTGAGACAAGATGCTGTCATGCAACAGGTCTTCCAGATGTGTAATACATTACTGCAGAGAAACACGGAAAC
TAGGAAGAGGAAATTAACTATCTGTACTTATAAGGTGGTTCCCCTCTCTCAGCGAAGTGGTGTTCTTGAATGGTGCACAGGAACTGTCCCCATTGGTGAATTTCTTGTTAACAATGAAGATGGTGCTCATAAAAGATACAGGCCAAATGATTTCAGTGCCTTTCAGTGCCAAAAGAAA
ATGATGGAGGTGCAAAAAAAGTCTTTTGAAGAGAAATATGAAGTCTTCATGGATGTTTGCCAAAATTTTCAACCAGTTTTCCGTTACTTCTGCATGGAAAAATTCTTGGATCCAGCTATTTGGTTTGAGAAGCGATTGGCTTATACGCGCAGTGTAGCTACTTCTTCTATTGTTGGTT
ACATACTTGGACTTGGTGATAGACATGTACAGAATATCTTGATAAATGAGCAGTCAGCAGAACTTGTACATATAGATCTAGGTGTTGCTTTTGAACAGGGCAAAATCCTTCCTACTCCTGAGACAGTTCCTTTTAGACTCACCAGAGATATTGTGGATGGCATGGGCATTACGGGTGT
TGAAGGTGTCTTCAGAAGATGCTGTGAGAAAACCATGGAAGTGATGAGAAACTCTCAGGAAACTCTGTTAACCATTGTAGAGGTCCTTCTATATGATCCACTCTTTGACTGGACCATGAATCCTTTGAAAGCTTTGTATTTACAGCAGAGGCCGGAAGATGAAACTGAGCTTCACCCT
ACTCTGAATGCAGATGACCAAGAATGCAAACGAAATCTCAGTGATATTGACCAGAGTTTCGACAAAGTAGCTGAACGTGTCTTAATGAGACTACAAGAGAAACTGAAAGGAGTGGAAGAAGGCACTGTGCTCAGTGTTGGTGGACAGGTGAATTTGCTCATACAGCAGGCCATAGACC
CCAAAAATCTCAGCCGACTTTTCCCAGGATGGAAAGCTTGGGTGTGATCTTCAGTATATGAATTACCCTTTC
e la proteina ATM
ATM
• E’ presente in tutti i tessuti nei soggetti
sani
• E’ assente o fortemente ridotta nei
malati
Che cos’è una mutazione:
un cambiamento nella sequenza del DNA
•Le mutazioni sono alla base della variabilità
genetica, ad es. l’altezza, la pressione
sanguigna, il colore della pelle.
• In alcuni casi la variabilità genetica porta ad
una malattia genetica
Tipi di mutazione
Cambia il senso
Interrompe il codice
ATM
20-40%
60-80%
Il gene ATM è uno dei più grandi geni noti
ATM
Gene medio
10.000 basi
2500 basi
Come si cercano le mutazioni
• Prelievo di sangue
• Estrazione del DNA
• Analisi con tecniche di biologia molecolare
(individuano la regione colpita)
• Lettura della “sequenza” del DNA
Perché si cercano le mutazioni
• Diagnosi certa
• Identificazione dei portatori
• Diagnosi prenatale
• Scopi di ricerca
I nostri risultati
Registro italiano pazienti A-T
(RIAT)- Roma-Prof. L.Chessa
Pazienti analizzati: 29
Analisi di mutazioni di ATM mediante PTT
T7-eu-ATG
4-13
cDNA
12-24
22-32
cDNA
31-44
Reverse
transcription and
nested PCR
PCR fragments
41-53
52-66
cCC
1
2
in vitro transcription
mRNA
in vitro translation
protein
PAGE-SDS
3
Alcune mutazioni di ATM
Exon 1
10
20
30
Interrompono il codice
1240 C T
2413 C T
5229 A T
5574 A T
6913 C T
7327 C T
7792 C T
8545 C T
8977 C T
9139 C T
Q414X Gilad 1996
R805X
K1743X Gatti
W1858XGilad 1998
Q2304X
R245X
R2598X Wright 1996
K2848X Gatti el al.
R2993X
R3065X Gilad 1998
40
50
60
Cambiano il senso
128 T C L43P Shiloh
7408 TG Y2470D
8708 C T P2903L
8711 A G E2904G Gilad
Distribuzione delle mutazioni ATM
*
Exon 1
10
20
*
30
40
50
Inserzioni
Non senso
Difetti di splicing
Interrompono il codice
Delezioni
Cambiano il senso
*
Mutazioni presenti in più famiglie
60
Conclusioni
• Sono state individuate il 70% delle mutazioni
• La proteina può essere colpita in qualunque
posizione
• Sono rare le mutazioni che ricorrono in più
famiglie
• Non è possibile predire l’andamento della
patologia sulla base delle mutazioni
Nuovo progetto
Messa a punto di sistemi di analisi di
mutazioni mediante DHPLC
Analisi di ATM mediante DHPLC
Per ogni paziente si
eseguono 66 reazioni
indipendenti che la
macchina analizza in
automatico.
Se in una reazione si
ottiene una possibile
mutazione si sequenzia
il DNA corrispondente
Prospettive
• Sistema rapido per analisi di mutazioni
• Ricerca di tutti i tipi di mutazione
• Fornire un test per le famiglie che lo richiedano
Ringraziamenti
Associazione ONLUS
Gli Amici di Valentina
