Università degli Studi di Torino Dipartimento di Genetica Biologia e Biochimica

1. 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)

2. 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.

3. Il DNA è un codice per la sintesi di proteine

4. La “ricetta” (=sequenza) del gene ATM GCGAGAGGAGTCGGGATCTGCGCTGCAGCCACCGCCGCGGTTGATACTACTTTGACCTTCCGAGTGCAGTGAGGCATACATCACAATTTGGAATTATGCATTGGTTTATCAATTTACTTGTTTATTGTCACCCTGCTGCCCAGATATGACTTCATGAGGACAGTGATGTGTGTTCTGAAATTGTGAACCATGAGTCTAGTACTTAATGATCTGCTTATCTGCTGCCGTCAACTAGAACATGATAGAGCTACAGAACGAAAGAAAGAAGTTGAGAAATTTAAGCGCCTGATTCGAGATCCTGAAACAATTAAACATCTAGAATTGGGATGCTGTTTTTAGATTTTTACAGAAATATATTCAGAAAGAAACAGAATGTCTGAGAATAGCAAAACCAAATGTATCAGCCTCAACACATCGGCATTCAGATTCCAAACAAGGAAAATATTTGAAGCCTCCAGGCAGAAAAAGATGCAGGAAATCAGTAGTTTGGTCAAATACTTCATCAAATGTGCAAACAGAAGAGCACCTAGGCTAAAATGTCAAGAACTCTTAAATTATATCATGGATACAGTGAAAGATTCATCTAATGGTGCTATTTACGGAGCTGATTGTAGCAACATACTACTCAAAGACATTCTTTCTGTGAGAAAATACTGGTGTGAAATATCTCAGCAACAGTGGTTAGAATTGTTCTCTGTGTACTTCAGGCTCTATCTGAAACCTTCACAAGATGTTCATAGAGTTTTAGTGGCTAGAATAATTCATGCTGTTACCAAAGGATGCTGTTCTCAGACTGACGGATTAAATTCCAAATTTTTGGACTTTTTTTCCAAGGCTATTCAGTGTGCGAGACAAGAAAAGAGCTCTTCAGGTCTAAATCATATCTTAGCAGCTCTTACTATCTTCCTCAAGACTTTGGCTGTCAACTTTCGAATTCGAGTGTGTGAATTAGGAGATGAAATTCTTCCCACTTTGCTTTATATTTGGACTCAACATAGGCTTAATGATTCTTTAAAAGAAGTCATTATTGAATTATTTCAACTGCAAATTTATATCCATCATCCGAAAGGAGCCAAAACCCAAGAAAAAGGTGCTTATGAATCAACAAAATGGAGAAGTATTTTATACAACTTATATGATCTGCTAGTGAATGAGATAAGTCATATAGGAAGTAGAGGAAAGTATTCTTCAGGATTTCGTAATATTGCCGTCAAAGAAAATTTGATTGAATTGATGGCAGATATCTGTCACCAGGTTTTTAATGAAGATACCAGATCCTTGGAGATTTCTCAATCTTACACTACTACACAAAGAGAATCTAGTGATTACAGTGTCCCTTGCAAAAGGAAGAAAATAGAACTAGGCTGGGAAGTAATAAAAGATCACCTTCAGAAGTCACAGAATGATTTTGATCTTGTGCCTTGGCTACAGATTGCAACCCAATTAATATCAAAGTATCCTGCAAGTTTACCTAACTGTGAGCTGTCTCCATTACTGATGATACTATCTCAGCTTCTACCCCAACAGCGACATGGGGAACGTACACCATATGTGTTACGATGCCTTACGGAAGTTGCATTGTGTCAAGACAAGAGGTCAAACCTAGAAAGCTCACAAAAGTCAGATTTATTAAAACTCTGGAATAAAATTTGGTGTATTACCTTTCGTGGTATAAGTTCTGAGCAAATACAAGCTGAAAACTTTGGCTTACTTGGAGCCATAATTCAGGGTAGTTTAGTTGAGGTTGACAGAGAATTCTGGAAGTTATTTACTGGGTCAGCCTGCAGACCTTCATGTCCTGCAGTATGCTGTTTGACTTTGGCACTGACCACCAGTATAGTTCCAGGAGCGGTAAAAATGGGAATAGAGCAAAATATGTGTGAAGTAAATAGAAGCTTTTCTTTAAAGGAATCAATAATGAAATGGCTCTTATTCTATCAGTTAGAGGGTGACTTAGAAAATAGCACAGAAGTGCCTCCAATTCTTCACAGTAATTTTCCTCATCTTGTACTGGAGAAAATTCTTGTGAGTCTCACTATGAAAAACTGTAAAGCTGCAATGAATTTTTTCCAAAGCGTGCCAGAATGTGAACACCACCAAAAAGATAAAGAAGAACTTTCATTCTCAGAAGTAGAAGAACTATTTCTTCAGACAACTTTTGACAAGATGGACTTTTTAACCATTGTGAGAGAATGTGGTATAGAAAAGCACCAGTCCAGTATTGGCTTCTCTGTCCACCAGAATCTCAAGGAATCACTGGATCGCTGTCTTCTGGGATTATCAGAACAGCTTCTGAATAATTACTCATCTGAGATTACAAATTCAGAAACTCTTGTCCGGTGTTCACGTCTTTTGGTGGGTGTCCTTGGCTGCTACTGTTACATGGGTGTAATAGCTGAAGAGGAAGCATATAAGTCAGAATTATTCCAGAAAGCCAACTCTCTAATGCAATGTGCAGGAGAAAGTATCACTCTGTTTAAAAATAAGACAAATGAGGAATTCAGAATTGGTTCCTTGAGAAATATGATGCAGCTATGTACACGTTGCTTGAGCAACTGTACCAAGAAGAGTCCAAATAAGATTGCATCTGGCTTTTTCCTGCGATTGTTAACATCAAAGCTAATGAATGACATTGCAGATATTTGTAAAAGTTTAGCATCCTTCATCAAAAAGCCATTTGACCGTGGAGAAGTAGAATCAATGGAAGATGATACTAATGGAAATCTAATGGAGGTGGAGGATCAGTCATCCATGAATCTATTTAACGATTACCCTGATAGTAGTGTTAGTGATGCAAACGAACCTGGAGAGAGCCAAAGTACCATAGGTGCCATTAATCCTTTAGCTGAAGAATATCTGTCAAAGCAAGATCTACTTTTCTTAGACATGCTCAAGTTCTTGTGTTTGTGTGTAACTACTGCTCAGACCAATACTGTGTCCTTTAGGGCAGCTGATATTCGGAGGAAATTGTTAATGTTAATTGATTCTAGCACGCTAGAACCTACCAAATCCCTCCACCTGCATATGTATCTAATGCTTTTAAAGGAGCTTCCTGGAGAAGAGTACCCCTTGCCAATGGAAGATGTTCTTGAACTTCTGAAACCACTATCCAATGTGTGTTCTTTGTATCGTCGTGACCAAGATGTTTGTAAAACTATTTTAAACCATGTCCTTCATGTAGTGAAAAACCTAGGTCAAAGCAATATGGACTCTGAGAACACAAGGGATGCTCAAGGACAGTTTCTTACAGTAATTGGAGCATTTTGGCATCTAACAAAGGAGAGGAAATATATATTCTCTGTAAGAATGGCCCTAGTAAATTGCCTTAAAACTTTGCTTGAGGCTGATCCTTATTCAAAATGGGCCATTCTTAATGTAATGGGAAAAGACTTTCCTGTAAATGAAGTATTTACACAATTTCTTGCTGACAATCATCACCAAGTTCGCATGTTGGCTGCAGAGTCAATCAATAGATTGTTCCAGGACACGAAGGGAGATTCTTCCAGGTTACTGAAAGCACTTCCTTTGAAGCTTCAGCAAACAGCTTTTGAAAATGCATACTTGAAAGCTCAGGAAGGAATGAGAGAAATGTCCCATAGTGCTGAGAACCCTGAAACTTTGGATGAAATTTATAATAGAAAATCTGTTTTACTGACGTTGATAGCTGTGGTTTTATCCTGTAGCCCTATCTGCGAAAAACAGGCTTTGTTTGCCCTGTGTAAATCTGTGAAAGAGAATGGATTAGAACCTCACCTTGTGAAAAAGGTTTTAGAGAAAGTTTCTGAAACTTTTGGATATAGACGTTTAGAAGACTTTATGGCATCTCATTTAGATTATCTGGTTTTGGAATGGCTAAATCTTCAAGATACTGAATACAACTTATCTTCTTTTCCTTTTATTTTATTAAACTACACAAATATTGAGGATTTCTATAGATCTTGTTATAAGGTTTTGATTCCACATCTGGTGATTAGAAGTCATTTTGATGAGGTGAAGTCCATTGCTAATCAGATTCAAGAGGACTGGAAAAGTCTTCTAACAGACTGCTTTCCAAAGATTCTTGTAAATATTCTTCCTTATTTTGCCTATGAGGGTACCAGAGACAGTGGGATGGCACAGCAAAGAGAGACTGCTACCAAGGTCTATGATATGCTTAAAAGTGAAAACTTATTGGGAAAACAGATTGATCACTTATTCATTAGTAATTTACCAGAGATTGTGGTGGAGTTATTGATGACGTTACATGAGCCAGCAAATTCTAGTGCCAGTCAGAGCACTGACCTCTGTGACTTTTCAGGGGATTTGGATCCTGCTCCTAATCCACCTCATTTTCCATCGCATGTGATTAAAGCAACATTTGCCTATATCAGCAATTGTCATAAAACCAAGTTAAAAAGCATTTTAGAAATTCTTTCCAAAAGCCCTGATTCCTATCAGAAAATTCTTCTTGCCATATGTGAGCAAGCAGCTGAAACAAATAATGTTTATAAGAAGCACAGAATTCTTAAAATATATCACCTGTTTGTTAGTTTATTACTGAAAGATATAAAAAGTGGCTTAGGAGGAGCTTGGGCCTTTGTTCTTCGAGACGTTATTTATACTTTGATTCACTATATCAACCAAAGGCCTTCTTGTATCATGGATGTGTCATTACGTAGCTTCTCCCTTTGTTGTGACTTATTAAGTCAGGTTTGCCAGACAGCCGTGACTTACTGTAAGGATGCTCTAGAAAACCATCTTCATGTTATTGTTGGTACACTTATACCCCTTGTGTATGAGCAGGTGGAGGTTCAGAAACAGGTATTGGACTTGTTGAAATACTTAGTGATAGATAACAAGGATAATGAAAACCTCTATATCACGATTAAGCTTTTAGATCCTTTTCCTGACCATGTTGTTTTTAAGGATTTGCGTATTACTCAGCAAAAAATCAAATACAGTAGAGGACCCTTTTCACTCTTGGAGGAAATTAACCATTTTCTCTCAGTAAGTGTTTATGATGCACTTCCATTGACAAGACTTGAAGGACTAAAGGATCTTCGAAGACAACTGGAACTACATAAAGATCAGATGGTGGACATTATGAGAGCTTCTCAGGATAATCCGCAAGATGGGATTATGGTGAAACTAGTTGTCAATTTGTTGCAGTTATCCAAGATGGCAATAAACCACACTGGTGAAAAAGAAGTTCTAGAGGCTGTTGGAAGCTGCTTGGGAGAAGTGGGTCCTATAGATTTCTCTACCATAGCTATACAACATAGTAAAGATGCATCTTATACCAAGGCCCTTAAGTTATTTGAAGATAAAGAACTTCAGTGGACCTTCATAATGCTGACCTACCTGAATAACACACTGGTAGAAGATTGTGTCAAAGTTCGATCAGCAGCTGTTACCTGTTTGAAAAACATTTTAGCCACAAAGACTGGACATAGTTTCTGGGAGATTTATAAGATGACAACAGATCCAATGCTGGCCTATCTACAGCCTTTTAGAACATCAAGAAAAAAGTTTTTAGAAGTACCCAGATTTGACAAAGAAAACCCTTTTGAAGGCCTGGATGATATAAATCTGTGGATTCCTCTAAGTGAAAATCATGACATTTGGATAAAGACACTGACTTGTGCTTTTTTGGACAGTGGAGGCACAAAATGTGAAATTCTTCAATTATTAAAGCCAATGTGTGAAGTGAAAACTGACTTTTGTCAGACTGTACTTCCATACTTGATTCATGATATTTTACTCCAAGATACAAATGAATCATGGAGAAATCTGCTTTCTACACATGTTCAGGGATTTTTCACCAGCTGTCTTCGACACTTCTCGCAAACGAGCCGATCCACAACCCCTGCAAACTTGGATTCAGAGTCAGAGCACTTTTTCCGATGCTGTTTGGATAAAAAATCACAAAGAACAATGCTTGCTGTTGTGGACTACATGAGAAGACAAAAGAGACCTTCTTCAGGAACAATTTTTAATGATGCTTTCTGGCTGGATTTAAATTATCTAGAAGTTGCCAAGGTAGCTCAGTCTTGTGCTGCTCACTTTACAGCTTTACTCTATGCAGAAATCTATGCAGATAAGAAAAGTATGGATGATCAAGAGAAAAGAAGTCTTGCATTTGAAGAAGGAAGCCAGAGTACAACTATTTCTAGCTTGAGTGAAAAAAGTAAAGAAGAAACTGGAATAAGTTTACAGGATCTTCTCTTAGAAATCTACAGAAGTATAGGGGAGCCAGATAGTTTGTATGGCTGTGGTGGAGGGAAGATGTTACAACCCATTACTAGACTACGAACATATGAACACGAAGCAATGTGGGGCAAAGCCCTAGTAACATATGACCTCGAAACAGCAATCCCCTCATCAACACGCCAGGCAGGAATCATTCAGGCCTTGCAGAATTTGGGACTCTGCCATATTCTTTCCGTCTATTTAAAAGGATTGGATTATGAAAATAAAGACTGGTGTCCTGAACTAGAAGAACTTCATTACCAAGCAGCATGGAGGAATATGCAGTGGGACCATTGCACTTCCGTCAGCAAAGAAGTAGAAGGAACCAGTTACCATGAATCATTGTACAATGCTCTACAATCTCTAAGAGACAGAGAATTCTCTACATTTTATGAAAGTCTCAAATATGCCAGAGTAAAAGAAGTGGAAGAGATGTGTAAGCGCAGCCTTGAGTCTGTGTATTCGCTCTATCCCACACTTAGCAGGTTGCAGGCCATTGGAGAGCTGGAAAGCATTGGGGAGCTTTTCTCAAGATCAGTCACACATAGACAACTCTCTGAAGTATATATTAAGTGGCAGAAACACTCCCAGCTTCTCAAGGACAGTGATTTTAGTTTTCAGGAGCCTATCATGGCTCTACGCACAGTCATTTTGGAGATCCTGATGGAAAAGGAAATGGACAACTCACAAAGAGAATGTATTAAGGACATTCTCACCAAACACCTTGTAGAACTCTCTATACTGGCCAGAACTTTCAAGAACACTCAGCTCCCTGAAAGGGCAATATTTCAAATTAAACAGTACAATTCAGTTAGCTGTGGAGTCTCTGAGTGGCAGCTGGAAGAAGCACAAGTATTCTGGGCAAAAAAGGAGCAGAGTCTTGCCCTGAGTATTCTCAAGCAAATGATCAAGAAGTTGGATGCCAGCTGTGCAGCGAACAATCCCAGCCTAAAACTTACATACACAGAATGTCTGAGGGTTTGTGGCAACTGGTTAGCAGAAACGTGCTTAGAAAATCCTGCGGTCATCATGCAGACCTATCTAGAAAAGGCAGTAGAAGTTGCTGGAAATTATGATGGAGAAAGTAGTGATGAGCTAAGAAATGGAAAAATGAAGGCATTTCTCTCATTAGCCCGGTTTTCAGATACTCAATACCAAAGAATTGAAAACTACATGAAATCATCGGAATTTGAAAACAAGCAAGCTCTCCTGAAAAGAGCCAAAGAGGAAGTAGGTCTCCTTAGGGAACATAAAATTCAGACAAACAGATACACAGTAAAGGTTCAGCGAGAGCTGGAGTTGGATGAATTAGCCCTGCGTGCACTGAAAGAGGATCGTAAACGCTTCTTATGTAAAGCAGTTGAAAATTATATCAACTGCTTATTAAGTGGAGAAGAACATGATATGTGGGTATTCCGGCTTTGTTCCCTCTGGCTTGAAAATTCTGGAGTTTCTGAAGTCAATGGCATGATGAAGAGAGACGGAATGAAGATTCCAACATATAAATTTTTGCCTCTTATGTACCAATTGGCTGCTAGAATGGGGACCAAGATGATGGGAGGCCTAGGATTTCATGAAGTCCTCAATAATCTAATCTCTAGAATTTCAATGGATCACCCCCATCACACTTTGTTTATTATACTGGCCTTAGCAAATGCAAACAGAGATGAATTTCTGACTAAACCAGAGGTAGCCAGAAGAAGCAGAATAACTAAAAATGTGCCTAAACAAAGCTCTCAGCTTGATGAGGATCGAACAGAGGCTGCAAATAGAATAATATGTACTATCAGAAGTAGGAGACCTCAGATGGTCAGAAGTGTTGAGGCACTTTGTGATGCTTATATTATATTAGCAAACTTAGATGCCACTCAGTGGAAGACTCAGAGAAAAGGCATAAATATTCCAGCAGACCAGCCAATTACTAAACTTAAGAATTTAGAAGATGTTGTTGTCCCTACTATGGAAATTAAGGTGGACCACACAGGAGAATATGGAAATCTGGTGACTATACAGTCATTTAAAGCAGAATTTCGCTTAGCAGGAGGTGTAAATTTACCAAAAATAATAGATTGTGTAGGTTCCGATGGCAAGGAGAGGAGACAGCTTGTTAAGGGCCGTGATGACCTGAGACAAGATGCTGTCATGCAACAGGTCTTCCAGATGTGTAATACATTACTGCAGAGAAACACGGAAACTAGGAAGAGGAAATTAACTATCTGTACTTATAAGGTGGTTCCCCTCTCTCAGCGAAGTGGTGTTCTTGAATGGTGCACAGGAACTGTCCCCATTGGTGAATTTCTTGTTAACAATGAAGATGGTGCTCATAAAAGATACAGGCCAAATGATTTCAGTGCCTTTCAGTGCCAAAAGAAAATGATGGAGGTGCAAAAAAAGTCTTTTGAAGAGAAATATGAAGTCTTCATGGATGTTTGCCAAAATTTTCAACCAGTTTTCCGTTACTTCTGCATGGAAAAATTCTTGGATCCAGCTATTTGGTTTGAGAAGCGATTGGCTTATACGCGCAGTGTAGCTACTTCTTCTATTGTTGGTTACATACTTGGACTTGGTGATAGACATGTACAGAATATCTTGATAAATGAGCAGTCAGCAGAACTTGTACATATAGATCTAGGTGTTGCTTTTGAACAGGGCAAAATCCTTCCTACTCCTGAGACAGTTCCTTTTAGACTCACCAGAGATATTGTGGATGGCATGGGCATTACGGGTGTTGAAGGTGTCTTCAGAAGATGCTGTGAGAAAACCATGGAAGTGATGAGAAACTCTCAGGAAACTCTGTTAACCATTGTAGAGGTCCTTCTATATGATCCACTCTTTGACTGGACCATGAATCCTTTGAAAGCTTTGTATTTACAGCAGAGGCCGGAAGATGAAACTGAGCTTCACCCTACTCTGAATGCAGATGACCAAGAATGCAAACGAAATCTCAGTGATATTGACCAGAGTTTCGACAAAGTAGCTGAACGTGTCTTAATGAGACTACAAGAGAAACTGAAAGGAGTGGAAGAAGGCACTGTGCTCAGTGTTGGTGGACAGGTGAATTTGCTCATACAGCAGGCCATAGACCCCAAAAATCTCAGCCGACTTTTCCCAGGATGGAAAGCTTGGGTGTGATCTTCAGTATATGAATTACCCTTTC

5. e la proteina ATM ATM • E’ presente in tutti i tessuti nei soggetti sani • E’ assente o fortemente ridotta nei malati

6. 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

7. Tipi di mutazione ATM 20-40% Cambia il senso 60-80% Interrompe il codice

8. Il gene ATM è uno dei più grandi geni noti 10.000 basi ATM 2500 basi Gene medio

9. 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

10. Perché si cercano le mutazioni • Diagnosi certa • Identificazione dei portatori • Diagnosi prenatale • Scopi di ricerca

11. Registro italiano pazienti A-T(RIAT)- Roma-Prof. L.Chessa I nostri risultati Pazienti analizzati: 29

12. c 1 2 3 C C PAGE-SDS Analisi di mutazioni di ATM mediante PTT 4-13 T7-eu-ATG cDNA 12-24 22-32 31-44 cDNA 41-53 Reverse transcription and nested PCR 52-66 PCR fragments in vitro transcription mRNA in vitro translation protein

13. Alcune mutazioni di ATM Exon 10 20 30 40 50 60 1 Cambiano il senso Interrompono il codice 1240 C T Q414X Gilad 1996 2413 C T R805X 5229 A T K1743X Gatti 5574 A T W1858XGilad 1998 6913 C T Q2304X 7327 C T R245X 7792 C T R2598X Wright 1996 8545 C T K2848X Gatti el al. 8977 C T R2993X 9139 C T R3065X Gilad 1998 128 T C L43P Shiloh 7408 TG Y2470D 8708 C T P2903L 8711 A G E2904G Gilad

14. Distribuzione delle mutazioni ATM * * Exon 10 20 30 40 50 60 1 Inserzioni Non senso Interrompono il codice Difetti di splicing Delezioni Cambiano il senso * Mutazioni presenti in più famiglie

15. 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

16. Nuovo progetto Messa a punto di sistemi di analisi di mutazioni mediante DHPLC

17. 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

18. Prospettive • Sistema rapido per analisi di mutazioni • Ricerca di tutti i tipi di mutazione • Fornire un test per le famiglie che lo richiedano

19. Ringraziamenti Associazione ONLUS Gli Amici di Valentina