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Paola Grammatico
Irene Bottillo
18-04-2015
Tecniche di nuova
generazione nella diagnostica
molecolare di patologie cardiac...
Sequence revolutions
Throughput
Kb
Gb
Mb
Lenght of Read (bp)
70030050
NGS
whole genome
whole transcriptome
whole exome
target seq
Sequence revo...
 Genome sequencing: intero genoma -> mutazioni puntiformi
+ riarrangiamenti cromosomici
 Exome sequencing: solo le regio...
NGS, applicazioni
RNA
 RNA-Seq
 studi di espressione
 Small-RNA
Regolazione
 metilazione
 analisi interazioni DNA-pro...
Molecular analysis of sarcomeric and non-sarcomeric genes
in patients with hypertrophic cardiomyopathy
Bottillo Irene1, D’...
Scopo del lavoro
Il test genetico permette di confermare o escludere la HCM in casi
atipici, e di identificare portatori i...
Principali proteine coinvolte nelle cardiomiopatie
HCM
DCM
ARVD
Materiali e Metodi
 44 pazienti HCM adulti, 23 ♂ e 21 ♀
 NGS (Ion Torrent PGM) di 62 geni (CDS, ss, 5’UTR, 3’UTR)
ABCC9,...
Esperimento Ampliseq su Ion Torrent PGM
ABCC9, ACTC1, ACTN2, ANKRD1, BAG3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTF1, DES, DM...
Risultati
 18.810 varianti/44 pazienti
~ 427 varianti/pazente
 Validazione del test NGS: confermate tutte le varianti id...
155 varianti
SANGER
VALIDATION
FILTERING
(MAF<0,01) +
PRIORITIZATION
(Ex ± 10bp)
18.810
varianti
Risultati
106 non-sinoni...
Risultati
 35/44 (82%) dei pazienti portatori di una o più varianti
non-sinonime
 4 pazienti: solo varianti sinonime
 4...
Numero varianti non-sinonime identificate per ciascun gene
Risultati
Risultati
Numero varianti non-sinonime identificate per ciascuna
categoria genica funzionale
“Other” includes the loci cod...
Risultati
Numero e zigosità dei cambi non-sinonimi identificati nei 44
pazienti HCM
Risultati
Numero dei pazienti mutate VS categoria genica
Sarcomeric
Other
Desmosomal
5 31
8
12 3
4
 Molecular Modelling
Risultati
Structural comparison of wild-type and mutant forms for (a)
FLH2 A37S; (b) LAMA4 E1646G; (...
Pt Gene Mutation NM Status Type
dbSNP138
clin ass
Referenc
e
chr start end
Re
f
Var UNIPROT Domain
PolyPh
en-2
SIFT
Conser...
Risultati
 Correlazioni genotipo-fenotipo
 Age at diagnosis
 Family History
 Heart Transplantation
 NYHA
 Maximal Wa...
Discussione
 Ruolo di 62 geni nell’HCM
Percorso sperimentale/bioinformatico applicabile
in un contesto clinico
 82% dei...
Discussione
3/44 pazienti sindromici
 Paz 24: ♀, 7 anni, sindrome di Cantù (AD, ipertricosi
congenita,osteocondrodisplas...
Prospettive future …
Toward a prenatal non-invasive diagnosis of Congenital Heart
Diseases (CHDs): identification of early...
Identificare miRNA come biomarcatori di CHD fetale in uno stadio precoce di
gravidanza
Scopo del progetto
Pazienti e Metod...
... Grazie Prof. Paola Grammatico
Dr. Lilia De Bernardo
Dr. Daniela D’Angelantonio
Dr. Martina Lipari
Dr. Marco Castori
Dr...
03 p grammatico tecniche di nuova generazione
03 p grammatico tecniche di nuova generazione
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03 p grammatico tecniche di nuova generazione

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03 p grammatico tecniche di nuova generazione

  1. 1. Paola Grammatico Irene Bottillo 18-04-2015 Tecniche di nuova generazione nella diagnostica molecolare di patologie cardiache
  2. 2. Sequence revolutions
  3. 3. Throughput Kb Gb Mb Lenght of Read (bp) 70030050 NGS whole genome whole transcriptome whole exome target seq Sequence revolutions
  4. 4.  Genome sequencing: intero genoma -> mutazioni puntiformi + riarrangiamenti cromosomici  Exome sequencing: solo le regioni codificanti di un genoma  Trascriptome sequencing: sequenziamento e quantificazione degli RNA, sia codificanti che non- codificanti -> fusioni intrageniche + mutazioni puntiformi + isoforme + profili di espressione  Target sequencing: diverse regioni/geni contemporaneamente
  5. 5. NGS, applicazioni RNA  RNA-Seq  studi di espressione  Small-RNA Regolazione  metilazione  analisi interazioni DNA-proteine DNA  resequencing geni/genomi noti  sequenziamento de novo  sequenziamento di regioni target  identificazione SNP/mutazioni  identificazione riarrangiamenti strutturali e CNV transcriptome sequencing whole genome sequencing - target sequencing - exome sequencing ChIP-Seq
  6. 6. Molecular analysis of sarcomeric and non-sarcomeric genes in patients with hypertrophic cardiomyopathy Bottillo Irene1, D’Angelantonio Daniela, Caputo Viviana2, Paiardini Alessandro3 , Lipari Martina1, De Bernardo Carmelilia1, Giannarelli Diana4, Pizzuti Antonio2, Majore Silvia1, Castori Marco1, Zachara Elisabetta5, Re Federica5, Grammatico Paola1 1Medical Genetics, Department of Molecular Medicine, Sapienza University, San Camillo-Forlanini Hospital, Rome, Italy; 2Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy; 3Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy; 4Biostatistic Unit, Regina Elena National Cancer Institute, Rome, Italy; 5Cardiomyopathies Unit, Division of Cardiology and Cardiac Arrhythmias, San Camillo-Forlanini Hospital, Rome, Italy
  7. 7. Scopo del lavoro Il test genetico permette di confermare o escludere la HCM in casi atipici, e di identificare portatori in fase pre-clinica attraverso screening familiare Analisi genetiche mediante Sanger si limitano a 4-5 geni e richiedono tempi/costi impegnativi Assenza di chiare correlazioni genotipo-fenotipo NGS di 62 geni cardiomiopatia-associati per un test molecolare altamente sensibile e per determinare il contributo di ogni gene nell’insorgenza ed espressione della HCM
  8. 8. Principali proteine coinvolte nelle cardiomiopatie HCM DCM ARVD
  9. 9. Materiali e Metodi  44 pazienti HCM adulti, 23 ♂ e 21 ♀  NGS (Ion Torrent PGM) di 62 geni (CDS, ss, 5’UTR, 3’UTR) ABCC9, ACTC1, ACTN2, ANKRD1, BAG3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTF1, DES, DMD, DSC2, DSG2, DSP, DTNA, EMD, EYA4, FHL2, FXN, GATAD1, GLA, ILK, JPH2, JUP, LAMA4, LAMP2, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOM1, MYOZ2, MYPN, NEBL, NEXN, PDLIM3, PKP2, PLN, PRKAG2, PTPN11, RAF1, RBM20, RYR2, SCN5A, SGCD, TAZ, TCAP, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TTR, TXNRD2 e VCL
  10. 10. Esperimento Ampliseq su Ion Torrent PGM ABCC9, ACTC1, ACTN2, ANKRD1, BAG3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTF1, DES, DMD, DSC2, DSG2, DSP, DTNA, EMD, EYA4, FHL2, FXN, GATAD1, GLA, ILK, JPH2, JUP, LAMA4, LAMP2, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOM1, MYOZ2, MYPN, NEBL, NEXN, PDLIM3, PKP2, PLN, PRKAG2, PTPN11, RAF1, RBM20, RYR2, SCN5A, SGCD, TAZ, TCAP, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TTR, TXNRD2 e VCL Analisi dei dati (Torrent Suite 4.0)  Alignment on hg19  Coverage/Read depth analyses  varianti’ calling Annotazione delle varianti (wAnnovar2 WEB SERVER) Filtraggio delle varianti  MAF < 0,01 Prioritizzazione delle varianti  Exonic  ±10bp from splicing junction Validazione delle varianti (Sanger) Materiali e Metodi Predizioni funzionali in silico Molecular Modelling Correlazioni genotipo- fenotipo
  11. 11. Risultati  18.810 varianti/44 pazienti ~ 427 varianti/pazente  Validazione del test NGS: confermate tutte le varianti identificate precedentemente mediante Sanger di MYBPC3, MYH7, TNNT2, TNNI3 in 23/40 pazienti
  12. 12. 155 varianti SANGER VALIDATION FILTERING (MAF<0,01) + PRIORITIZATION (Ex ± 10bp) 18.810 varianti Risultati 106 non-sinonime (nsSNV)  81 missense  15 intronic (±10 bp)  4 nonsense  4 frameshift  1 stop-loss  1 in frame deletion 49 sinonime in Htz
  13. 13. Risultati  35/44 (82%) dei pazienti portatori di una o più varianti non-sinonime  4 pazienti: solo varianti sinonime  4 pazienti negativi  24/62 geni negativi ACTC1, ANKRD1, CASQ2, CRYAB, CTF1, DES, EMD, EYA4, FXN, LDB3, MYL3, MYLK2, MYOZ2, NEXN, PLN, PRKAG2, PTPN11, SGCD, TAZ, TCAP, TMEM43, TNNC1, TNNI3 and TTR
  14. 14. Numero varianti non-sinonime identificate per ciascun gene Risultati
  15. 15. Risultati Numero varianti non-sinonime identificate per ciascuna categoria genica funzionale “Other” includes the loci coding for lysosomal, thioredoxin reductase, anti-apoptotic and membrane proteins
  16. 16. Risultati Numero e zigosità dei cambi non-sinonimi identificati nei 44 pazienti HCM
  17. 17. Risultati Numero dei pazienti mutate VS categoria genica Sarcomeric Other Desmosomal 5 31 8 12 3 4
  18. 18.  Molecular Modelling Risultati Structural comparison of wild-type and mutant forms for (a) FLH2 A37S; (b) LAMA4 E1646G; (c) MYH6 R23H; (d) MYH7 A226T; (e) MYH7 R143Q; (f) MYOM1 R711H; (g) PKP2 R767S; (h) RYR2 E1127G; (i) RYR2 R485Q. The mutation is indicated in white. The predicted structural effects of mutations are: (a, d) steric hindrance (red circles); (b) local misfolding of linker domain (orange); (c, e, f, g) loss of important inter-residues contacts; (h) loss of a π-anion interaction; (i) loss of a π-cation interaction. Effects of nsSNVs for (a) the cadherin domain of DSC2; (b) the melibiase domain of GLA; (c) the FGF13 interaction domain of SCN5; (d) the Na Channel of SCN5 (the approximate position of the negatively charged Asp872 residue is shown in red, in each of the four protein subunits forming the channel).
  19. 19. Pt Gene Mutation NM Status Type dbSNP138 clin ass Referenc e chr start end Re f Var UNIPROT Domain PolyPh en-2 SIFT Conservation- GERP score Comment 1 MYBPC3 c.T1664C, p.M555T 000256 Htz missense - Girolami et al. 2006 chr11 47363668 47363668 A G Q14896 Ig-like C2- type 4 B (0) T (1) 4.22 Mol Mod: No predicted deleterious effect RyR2 c.A3380G, p.E1127G 001035 Htz missense rs200525962 - chr1 237730032 237730032 A G Q92736 B30.2/SPR Y 2 D (0.979) DLT (0) 5.29 Mol Mod: Probably destructuring the tertiary structure (loss of π-anion interaction with Trp1145) 3 TMPO c.A1037G, p.H346R 003276 Htz missense - - chr12 98927072 98927072 A G P42166 disordered region B (0.001) T (0.32) -1.03 Mol Mod: No possible prediction 5 MYH6 c.643-5C>T 002471 Htz intronic rs199859986 - chr14 23873602 23873602 G A - - - - -1.15 Branch-point site broken 8 RBM20 c.G3373A, p.E1125K 001134 363 Htz missense rs116908219 - chr10 112583294 112583294 G A Q5T481 - PD (0.911) T (0.34) 6.16 Probably not pathogenic MYH6 c.2928+5G >A 002471 Htz intronic rs28730772 - chr14 23862870 23862870 C T - - 4.37 Donor splice site broken CSRP3 c.T10C, p.W4R 003476 Htz missense rs45550635 Knoll et al. 2002; Geier et al. 2008 chr11 19213986 19213986 A G P50461 LIM zinc- binding 1 PD (0.888) DLT (0.01) 6.02 Previously reported alone in CMD1M pazienti and in conjunction with a sarcomeric mutation in HCM pazienti Risultati … … … Dall’analisi della tipologia delle varianti, revisione della letteratura, analisi in silico e molecular modeling, tutti i pazienti sono risultati portatori di almeno una nsSNV patogenetica, o già descritta come un modificatrice del fenotipo
  20. 20. Risultati  Correlazioni genotipo-fenotipo  Age at diagnosis  Family History  Heart Transplantation  NYHA  Maximal Wall Thickness  Left Ventricular Mass  Cardiac magnetic resonance areas of contrast  Left Ventricular Ejection Fraction VS  total number of nsSNVs  number of sarcomeric nsSNVs  number desmosomal nsSNVs  number of nsSNVs in genes for K+ and Na+ channels and interacting proteins  number of nsSNVs in mRNA splicing, cellular enzymes and nuclear genes  number of cytoskeletal nsSNVs  number of Z-disk nsSNVs  number of intracellular calcium homeostasis nsSNVs  ↑ num. nsSNVs: ↓ età di esordio  nsSNVS in geni per Ca++ homeostasis, per canali K+ e Na+, e per proteine del citoscheletro possono modulare l’espressione di HCM  Sudden Cardiac Death  Obstructive Cardiomyopathy  Supraventricular Arrhythmias  Non-Sustained Ventricular Tachycardia  Syncope/Presyncope  VO2  Maximal oxygen uptake  Diastolic Dysfunction  Thoracic pains  ECG Hypertrophy criteria  Conduction abnormalities  Hypertension  Intracardiac Defibrillator  Pacemaker  Myectomy  Coronary-Ventriculography  AMI/NSTEMI  Coronary Bypass
  21. 21. Discussione  Ruolo di 62 geni nell’HCM Percorso sperimentale/bioinformatico applicabile in un contesto clinico  82% dei casi: almeno una nsSNV rara
  22. 22. Discussione 3/44 pazienti sindromici  Paz 24: ♀, 7 anni, sindrome di Cantù (AD, ipertricosi congenita,osteocondrodisplasia, cardiomegalia, HCM e dismorfismi) ABCC9 c.C3460T,p.R1154W (Htz): descritta in 3 pazienti con S. di Cantù (vanBon et al 2012)  Paz 27: ♀, 23 anni, malattia di Danon o glicogenosi da deficit di LAMP2 (Lysosomal-Associated Membrane Protein 2), X-linked recessiva (cardiomiopatia , debolezza muscolare, ritardo mentale) LAMP2 c.453delT,p.F151fs mai descritta prima  Paz 10: ♀, 65 anni GLA c.A644G,p.N215S (alfa-galattosidasi A, malattia di Fabry, X-linked recessiva, segni neurologici, angiocheratoma, insufficienza renale, cardiomiopatia) p.N215S già associata a forma con coinvolgimento solo cardiaco (Davies et, al, Eng et al 1993)
  23. 23. Prospettive future … Toward a prenatal non-invasive diagnosis of Congenital Heart Diseases (CHDs): identification of early plasmatic markers (miRNAs) Non-Invasive Prenatal Diagnosis (NIPD) targets cell-free DNA andRNA (fetal + maternal) circulating in maternal plasma miRNA vengono rilasciati dalla placenta e poi esportati nella circolazione materna, qualificandosi come BIOMARKER PRENATALI DI PATOLOGIA miR-19b, miR-22, miR-29c, miR-375 sono up-regolati in gravidanze di feti CHD
  24. 24. Identificare miRNA come biomarcatori di CHD fetale in uno stadio precoce di gravidanza Scopo del progetto Pazienti e Metodi  Gravidanze di feti non-cromosomici  Gravidanze di feti cromosomici (trisomia 13, 18, 21 e monosomia X)  Gravidanze di controllo (non crososomici, no CHD) Studio del miRNoma sia da plasma materno che da liquid amniotico
  25. 25. ... Grazie Prof. Paola Grammatico Dr. Lilia De Bernardo Dr. Daniela D’Angelantonio Dr. Martina Lipari Dr. Marco Castori Dr. Silvia Majore Dr. Elisabetta Zachara Dr. Federica Re Dr. Flavia Ventriglia Dr. Antonella Giancotti Dr. Angela Caiaro Dr. Viviana Caputo Dr. Alessandro Paiardini

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