Principi ed applicazioni del sequenziamento automatico nella pratica clinica Eliana Disabella

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1 Principi ed applicazioni del sequenziamento automatico nella pratica clinica Eliana Disabella IRCCS Policlinico San Matteo di Pavia Laboratorio di Genetica Molecolare, Patologia Cardiovascolare e dei Trapianti (direttore Dr.ssa E. Arbustini) elianadisabella@hotmail.com

2 Some definitions Sensitivity (truly positives/true positives + false negative) Specificity (true negatives/true negatives + false positives) Throughput Costs

3 Sample Preparation System Nucleic acid isolation from whole blood and tissue

4 Aims of nucleic acid purification technologies Isolation of highly pure nucleic acids for gene expression research genotyping sequencing pathogen detection plus many others

5 Major Extraction Methods Purity and Stability Caothropic reagents (Phenol/Chloroform, Rnazol etc) Spin columns Silica Based membranes Magnetic beads

6 Workflow Whole Fresh or Frozen Blood 1. 85uL PK Digestion Buffer 2. 15uL ProK incubation C 3. Add 500uL DNA Purification Solution Blood Lysate 1. Vacuum Filtration in 96 well format DNA trapped on 96-well tray Purified DNA 1. 3 wash steps 2. Elution of DNA requires 3 minute incubation at room temperature

7 How do you process tissue? Pulverize/Macerate/H omogenize tissue in lysis buffer using Rotor/Stator Homogenizors Bead Beaters Pass homogenate across purification tray in 6700

8 Workflow Before After Tissue Tissue Lysate 1. Tissue-treat with ProK and Digestion Buffer for 1 hour at 65 C 1. Tissue-Add DNA Purification Solution Pre Filter to remove the biggest fragments DNA trapped on 96-well tray 1. Wash/Vacuum multiple times 2. Elution of DNA Purified DNA

9 PCR inhibitors Sample related: Heparine Hemoglobin > 0,15 mg/ml > 1 mg/ml Melanin, humidic acids, chlorophyll, polysaccharides Extraction related: SDS > 0,01% (w/v) Phenole > 0,2% (v/v) Ethanol > 1% Sod. acetate > 5 mm PCR additives: DTT > 1 mm DMSO > 5 %

10 What is pure RNA & DNA? Quality and Quantity Measured by UV Absorbance (OD) Nucleic Acid Proteins A260/280 Ratio 260 nm 280 nm Agarose Gel to determine degradation/gdna levels 2.0 Pure RNA 1.8 Pure DNA

11 Techniques Mutation Detection TaqMan VNTR RFLPs PCR MicroArray Sequencing DHPLC Mutation Detection + Mutation Discovery

12 DNA Amplification with the Polymerase Chain Reaction (PCR) Starting DNA Template Separate strands (denaturation) Forward primer Reverse primer

13 PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle In In cycles at at 100% efficiency, 1.07 billion copies of of targeted DNA region are created

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15 Turnaround time roughly 4 hrs

16 Sequencing

17 Sanger method : Chain Termination Sequencing : Dye Terminator Sequencing Extension Product Matrix DNA deoxycytidine triphosphate (dctp) HO dideoxycytidine triphosphate (ddctp)

18 Sanger method : Dye Terminator Sequencing Extension Product Matrix DNA dctp HO ddctp 5 phosphate group Elongation can go on

19 Sanger method : Dye Terminator Sequencing Extension Product Matrix DNA dctp HO ddctp NO possible elongation

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22 Names of Parts 18 to 65 C

23 Workflow Diagram Instrument Setup Prepare samples Load plates Schedule runs Polymer Filling Automatic Data Analysis Start run complete automation Results Electrokinetic Injection & Electrophoresis Automatic Data Extraction

24 Electrokinetic Injection Electrode (cathode) Capillary Capillary and electrode (cathode) are placed into the sample Voltage is applied Negatively-charged DNA enters the capillary as it migrates toward the positively-charged electrode (anode) at the other end of the capillary

25 Capillary Electrophoresis

26 Principle of Capillary Electrophoresis + - H + H + H + H + H + H + H + H + H + H +

27 Fluorescent Signal Detection - Electrophoresis Laser beam generated by single argon laser split to form dual pathway Split beam allows simultaneous illumination of 16 (4) capillaries from both sides of array at detection cell Fluorescence signal emitted from DNA fragments at the detection cell are collected on CCD camera Laser Energy dye CCD +

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29 Malattia di Fabry La malattia di Fabry: una patologia a gestione interdisciplinare 1898 Anderson-Fabry 1967 Brady identificazione difetto enzimatico 1989 Sequenza del gene

30 FABRY DISEASE FABRY DISEASE Alternative titles; symbols ANGIOKERATOMA, DIFFUSE ANDERSON-FABRY DISEASE HEREDITARY DYSTOPIC LIPIDOSIS ALPHA-GALACTOSIDASE A DEFICIENCY GLA DEFICIENCY CERAMIDE TRIHEXOSIDASE DEFICIENCY GALACTOSIDASE, ALPHA; GLA ALPHA-GALACTOSIDASE A Gene map locus Xq22.1 PubMed query: 1,437 references

31 Fabry disease

32 Patologia legata all X : ereditarietà XY XX XY XY XX XX

33 Incidenza 0.2* - 0.8** per 100,000 newborns *Meikle PJ et al. JAMA 281: , 1999 **Poorthuis BJ et al Hum Genet 105:151-6, 1999

34 Glycosphingolipids (GSLs): plasma membrane components Hydrolysing enzymes + Cofactors (sphingolipid activated proteins) Lysosome Hydrolysing enzymes + Cofactors (sphingolipid activated proteins)

35 alpha-galactosidase Endothelial and vascular cells Parenchimal cells Stromal cells

36 Fabry disease : baseline clinical manifestations of 366 pts in the Fabry Outcome Survey Eur J Clin Invest 2004 (34), Età all esordio M 10.9 ± 7.1 aa; F 22.6 ± 16.2 aa Dall esordio dei sintomi alla diagnosi: 13.7 ±12.9 anni nei maschi 26.3 ±14.7anni nelle femmine (rara; all esordio sintomi solo soggettivi, difficilmente quantificabili) Nella maggior parte dei pts coinvolgimento multisitemico (il numero di organi e apparati coinvolti aumenta con l età) Una minoranza di pz presenta coinvolgimento di un singolo apparato (su 23: cute 3, occhio 3, disturbi generali 2, neurologico 12, renale 3) Mortalità: età media M:45.5 ± 12.6 aa; F 55.4 ±14.9 aa Causa più frequente nei M: ictus (54.5%) nelle F: patologia cardiaca (26.7%)

37 Criteri per lo screening Setting cardiologico adulto o pediatrico HCM in cui non siano presenti difetti dei geni proteine sarcomeriche HCM che non siano a trasmissione maschio-maschio HCM associate a segni extracardiaci tipici di M. di Fabry Setting nefrologico Proteinurie idiopatiche Setting neurologico Stroke criptico Setting dermatologico Angiocheratomi Setting oculo-visivo Cornea verticillata Setting gastroenterologico Ambito pediatrico acroparestesie

38 Gruppo Interdisciplinare per la Malattia di Anderson-Fabry - GIMAF Diagnostica molecolare (routinariamente eseguita da circa in pz con HCM e/o con alterazioni ultrastrutturali suggestive nella biopsia endomiocardica) Attività α-gal Dosaggio Gb3 Consulenza genetica Valutazione multidisciplinare Collaborazioni: Centri nazionali per diagnostica molecolare Centro regionale nefrologia pediatrica (De Marchi MI - Dr. GL. Ardissino) Bambini con proteinuria ad eziologia non nota dopo valutazione specialistica IRCCS- Fondazione Maugeri (Dr. Febo) HCM e stroke criptici in pazienti <50 anni IRCCS Policlinico San Matteo - PV

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41 Angiocheratoma: M53%, F30% Eruzioni micropapuloemorragiche rilevate colore rosso scuro Età pediatrica: --> divengono più larghe e numerose con l età

42 Coinvolgimento oculare: M 73% F70% Cornea verticillata (lampada a fessura) I depositi di GB3 si formano nello strato basale dell epitelio corneale

43 Descrizione di un caso campione DNA PCR purif marcatura purif risultato

44 Risultato indagine molecolare Arg wt His R112H emizigosi His R112H eterozigosi

45 Grazie per l attenzione!!