Genoom-wijde moleculaire technologie toegepast in de genetische diagnostiek. Prof Maryse Bonduelle

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1 Genoom-wijde moleculaire technologie toegepast in de genetische diagnostiek Prof Maryse Bonduelle

2 Inleiding Fundamenteel doel van de genetica: ontrafelen van het genotype om het fenotype te verklaren 1977 Sanger sequencing 1 gen per analyse, base per base Combinatie van nieuwe instrumenten, databasen, bioinformatica en robotica exponentiele toename aan de mogelijkheden Next generation sequencing (NGS) of Massive parallel sequencing Enkele miljoenen of biljoenen sequencies in parallel lezen en in één enkele run analyseren 2 Genoomwijde Technologie Inleiding

3 Drastische toename van capaciteit en snelheid dalen van de kost Introductie van genoomwijde technologie in de dagelijkse diagnostiek Toename toepassingen met diagnostische doeleinden (vb NIPT, gene panels ) Nieuwe vragen en uitdagingen (begrijpen van het functionele genoom van van de variaties) Nieuwe bevindingen incidental findings (Informed Consent voor array, NIPT, NGS!) 3 Genoomwijde Technologie Inleiding

4 Genoomwijde technologiën in de kliniek Veranderingen in de klinische werking: Voor genoomwijde technologiën: klinische diagnostiek Sanger sequencing 1 gen volgende differentiaal diagnose Sanger sequencing volgend gen Met genoomwijde technologiën: Info over een panel van genen betrokken bij aandoening Info over varianten (polymorphismen) Info over meerdere genen (multicatoriële modellen?) Info over nieuwe genen (nog verder wetenschappelijk te staven via familiestudies en functionele studies) Info over andere niet betrokken genen bij de aandoening = incidental finding ongewenste info, gewenst? 4 Genoomwijde Technologie Inleiding

5 BRIGHT Platform : UZ Brussel-VUB-ULB BRIGHT : BRussels Interuniversity Genomics & High Throughput platform Nieuwe diagnostische en research noden!!! start zoektocht naar middelen toekennen middelen voor high troughput platform funding door VUB en UZ Brussel aankoop van high troughput toestellen, scanners Opstart platform deelname ULB in platform samenwerking in IB² bioinformatica platform (VUB-ULB ) 2014 organisatie en uitbreiding platform aankoop nieuwe toestellen 5 Genoomwijde Technologie Inleiding

6 Genoom-wijde moleculaire technologie toegepast in de genetische diagnostiek Overzicht van de nieuwe diagnostische tools Array technologie Ingevoerd op de werkvloer ter vervanging van de klassiek karyotypering Toepassingen in de genetische diagnostiek, postnataal, prenataal en preimplantatie NGS technologie Toelichting van verschillende technologieën Toepassing in niet-invasieve diagnostiek (NIPT), mitochondriaal genoom, erfelijke hartritmestoornissen 6 Genoomwijde Technologie Inleiding

7 Genoom-wijde moleculaire technologie toegepast in de genetische diagnostiek Algemene introductie en toepassingen in de kliniek Dr M. De Rademaeker & Dr Sci A. Van den Bogaert Array Technologie: Preimplantatie genetische diagnostiek Dr Sci C. Staessen & Prof M. De Rycke Nieuw Genomics platform op de campus UZ Brussel -VUB ir Ben Caljon Niet Invasieve Prenatale Test (NIPT) met genoomwijde analyse Dr Sci S. Van Dooren & Dr K. Van Berkel Mitochondriale genoom sequencing zoekt diagnostische bench. Prof S. Seneca Erfelijke hartritmestoornissen Dr Sci S. Van Dooren & Dr M. Meuwissen 7 Genoomwijde Technologie Inleiding

8 Algemene introductie en toepassingen in de kliniek: array technologie Ann Van Den Bogaert Marjan De Rademaeker

9 Array CGH Array gebaseerde vergelijkende genoomhybridisatie (array comparative genomic hybridisation) = array CGH In 1 test: onderzoek van het volledige genoom op kleine (submicroscopische) chromosomale afwijkingen ( kb) 2 Array CGH

10 Array CGH DNA 3 array CGH

11 Array CGH-Principe Referentie DNA Test DNA Log 2 test/referentie winst Labeling Cy 5 Cy Mix verlies Chromosomale positie Analyse Hybridisatie Scan 4 array CGH

12 Array CGH-in praktijk 4x44K arrays 4 keer unieke oligonucleotiden (probes/reporters) 60 basen lang over het gehele genoom verspreid Aantal oligo s per gebied 5 array CGH

13 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 6 array CGH

14 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 7 array CGH

15 Random Prime Labeling-Theorie Binding van korte primersequenties aan gedenatureerd DNA exo-klenow fragment van DNA polymerase 1: verlenging van de primers Tijdens elongatie: het merken van DNA, resp. met Cy3 en Cy5 ->inbouwen van gemerkte dntp s Ongeveer 10 maal geamplificeerd 8 array CGH

16 Random Prime Labeling-Theorie Genomisch DNA Denaturatie van dubbelstrengig DNA naar enkelstrengig DNA Binding van de random primers Exo-Klenow fragment bouwt nucleotiden in vanaf de random primers + binding van fluorescente nucleotiden Fluorescent nucleotide Exo-Klenow polymerase Random primer 9 array CGH

17 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 10 array CGH

18 Array-CGH hybridisatie-precipitatie Precipitatie Cy3 gemerkt patiënt DNA + Cy5 gemerkt referentie DNA NaAc 100% EtOH Precipitatie (30 min. bij -80 C) 11 array CGH

19 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 12 array CGH

20 Array-CGH hybridisatie-probebereiding Theorie Stap 1 Random Prime labeling Opzuiveren = verwijderen van niet ingebouwde nucleotiden 13 array CGH

21 Array-CGH hybridisatie-probebereiding Labo 14 array CGH

22 Array-CGH hybridisatie-probebereiding Theorie stap 2 Blokking reagent = blokkeert repetitieve sequenties Niet-specifieke binding : achtergrondsignaal 15 array CGH

23 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 16 array CGH

24 Array-CGH hybridisatie-hybridisatie Theorie Hybridisatie: = de mixen worden aangebracht op de slides Het gelabelde DNA bindt aan de probes die gespot zijn op de slide Vorming dubbelstrengig DNA: binding complementaire sequenties vanop het draagglaasje met het gelabelde DNA (mix patiëntreferentie) 17 array CGH

25 Array-CGH hybridisatie-hybridisatie Theorie Het array glaasje met 4 keer unieke oligonucleotiden (probes/reporters) Het gelabelde DNA (mix patiënt/referentie) op het oppervlak van het array glaasje + dekglaasje Hybridisatie: competitie tussen verschillend gelabeld patiënt en referentie DNA voor binding met oligonucleotiden op array glaasje 18 array CGH

26 Array-CGH hybridisatie-hybridisatie Labo 65 C, 24 uur 19 array CGH

27 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 20 array CGH

28 Array-CGH hybridisatie-wassen Theorie Wassen Enkel de probes die specifiek gebonden zijn aan het gelabelde DNA kunnen een signaal geven 21 array CGH

29 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 22 array CGH

30 Scannen (Agilent microarray scanner) Laser -> excitatie Cy3 en Cy5 23 array CGH

31 Scannen (Agilent microarray scanner) Theorie Na het scannen Beelden: Feature Extraction Software Vindt en plaatst microarrayrooster De gemeten intensiteiten~gespot stukje van het genoom (probes) De intensiteit van één spot en de gemiddelde waarden van het achtergrondsignaal rond de spots worden gemeten 24 array CGH

32 Feature Extraction Software-Labo Groen signaal Geel signaal Rood signaal 25 array CGH

33 Feature Extraction Software-Labo Duplicatie: het gespot DNA op het glaasje bevat meer patiënten DNA (Cy3; groen) dan referentie DNA (Cy5; rood) => Groen signaal in de rooster Deletie: het gespot DNA op het glaasje bevat minder patiënten DNA (Cy3; groen) dan referentie DNA (Cy5; rood) => Rood signaal in de rooster Normaal: het gespot DNA op het glaasje bevat evenveel patiënten DNA (Cy3; groen) dan referentie DNA (Cy5; rood) => Geel signaal in de rooster 26 array CGH

34 Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen Scannen Analyse 27 array CGH

35 Analyse-Theorie Verwerking en visualisatie: arraycghbase (Menten et al., 2005) Ruwe data wordt geconverteerd en gevisualiseerd -> interpretatie Log2-ratio per probe/reporter uitgezet t.o.v. zijn chromosomale positie 28 array CGH

36 Analyse in praktijk 29 array CGH

37 Analyse array CGH Cartagenia: Labo: array resultaten Artsen: kliniek Labo: koppeling tussen genotype/fenotype Interpretatie onafhankelijk en daarna overleg tussen wetenschappelijke medewerker en arts Verschil postnatale-prenatale arrays 30 array CGH

38 Copy Number Variants-theorie CNVs=DNA fragmenten >1Kb Goedaardig/beninge Normaal Copy Number Variants (CNVs) 15%-25% van het humane genoom is polymorf Array CGH = de term CNP (Copy Number Polymorphisms) Effect op! Genen Genetische aandoeningen/pathogeen 31 array CGH

39 Copy Number Variants-in praktijk Uitdaging: Het verschil tussen CNVs die wel of niet bijdragen tot de kliniek Publieke databanken CNVs van gezonde personen Databank van genomische varianten (DGV) 32 array CGH

40 Array CGH in de kliniek Prenatale diagnose Indicaties/ Interpretatie Casuistiek Postnatale diagnose Indicaties Casuistiek Conclusie 33 array CGH

41 Prenatale diagnose Verhoogd risico op chromosomale afwijking (leeftijd, abnormale niet invasieve screening) Verhoogd risico monogene aandoening Echografische afwijkingen Psychosociale redenen 34 array CGH

42 Prenatale diagnose België sinds 2013: moleculair karyotype/ array CGH Nationale consensus Centra Medische Genetica België 1 : Pre en post counseling Interpretatie resultaten Protocoleren resultaten 1 Implementation of genomic arrays in prenatal diagnosis: The Belgian approach to meet the challenges, Eur J Med Genet Mar;57(4): array CGH

43 Prenatale diagnose Benign Pathogeen Unclassified Toevallige bevinding Eur J Med Genet Mar;57(4): array CGH

44 Casus 24 weken Echografische afwijking: duodenale atresie 37 array CGH

45 Casus 38 array CGH

46 Casus 39 array CGH

47 Casus Williams syndroom 40 array CGH

48 Casus 25 weken Echografie: cerebellaire atrofie, gedilateerd pyelum, polyhydramnios, normale groei 41 array CGH

49 Casus Trisomie 18/ Edwards syndroom Cave: geen laaggradige mozaïcisme! 42 array CGH

50 Casus 20 weken Echografie: afwezigheid neusbeentje 43 array CGH

51 Casus 2080,2kb duplicatie 1q21.1-q21.2, 33 genen, GJA5 gen 44 array CGH

52 Casus 1q21 duplicatie risico factor Macrocefalie Aangeboren afwijkingen Ontwikkelingsstoornissen (autisme, leerstoornissen) Hartafwijkingen (VSD/ASD/PVS/ TOF,..) GJA5 gen 45 array CGH

53 Casus 24 weken Echografie: cardiopathie 46 array CGH

54 Casus 9,4MB duplicatie16p13.13p genen, 75 proteine coderende genen (NDE1, MYH11, ABCC1, ABCC6,...) 47 array CGH

55 Casus 16 p13.11 duplicatie risico factor neurologische problemen (ADHD, autisme, ) Cardiovasculaire problemen (aorta dilatatie,bicuspide aortaklep) MYH11 gen Variabele penetrantie/ expressie Consortium: Ouders: overgëerfd Rapporteren Cardiopathie / grotere duplicatie (meer genen) 48 array CGH

56 Casus Zwangerschap 16 weken Indicatie prenatale diagnose: post PGD voor metabole aandoening 49 array CGH

57 Casus 339 kb duplicatie van chromosomenband 6q22.3, PLN gen PLN gen puntmutaties of deleties phospholamban associatie met cardiomyopathie duplicatie: slechts 1 casus doch associatie met cardiomyopathie Consortium: Ouders: overgeërfd rapporteren,opvolging mogelijk 50 array CGH

58 Postnatale diagnosis Verstandelijke beperking, neuropsychiatrische aandoeningen dysmorfismen, aangeboren afwijkingen Ouders van individu met chromosomale afwijking Abnormaal karyotype verfijnen 51 array CGH

59 Casus Jongen Pinealoblastoma 52 array CGH

60 Casus Array CGH: 2,4 Mb deletie 22q11 geen deletie van tumorgen SMARCB1 Geen deletie van tumor gen INI1 22q11 deletie syndroom (velocardiofaciaal syndroom), geen verklaring tumor 53 array CGH

61 Casus Jongen Microftalmie en hypospadias 54 array CGH

62 Casus 1.1 Mb deletion 2q23 ZEB2 gen De novo ZEB2 gen mutaties/ exon deleties/ Mowat Wilson syndroom 55 array CGH

63 Casus Meisje, pasgeborene Epileptische encephalopathy 56 array CGH

64 Casus Array CGH: 2235,9kb deletion 15q11.2 Overgeërfd van de moeder Risico factor postnataal! Neuropsychiatrische aandoeningen (epilepsie, autisme, gedrags en taalproblemen, verstandelijke beperking) 57 array CGH

65 Casus Risico factor Gekend deletie syndroom 58 array CGH

66 Conclusie array CGH Genoomwijd onderzoek van hoge resolutie voor opsporing deleties/duplicaties specifieke postnatale indicaties alle invasieve prenatale diagnoses Cave Beperkte detectie mozaïcisme /geen detectie gebalanceerde afwijkingen Detectie afwijkingen van onduidelijke klinische relevantie 59 array CGH

67 Conclusie array CGH Uitdaging Voor elke CNV de relatie tot fenotype bepalen Counseling 60 array CGH

68 PGD for chromosomal abnormalities Catherine Staessen, PhD Centre of Medical Genetics

69 The main causes of chromosomal anomalies Highgenetic risk Inheritance of the parental pathology - true inheritance: e.g.parental translocation PGD Low- genetic risk Meiotic nondisjunction 80-85% related to oocytes 10-15% related to spermatozoa Postzygotic mitotic non-disjunction 5-15% of cases of trisomies Mat Age Risk at birth % % % PGS *Hook EB. Cross PK. Schreinemachers DM. (1983) %

70 Carriers of balanced structural chromosomal abnormalities Have a greater chance of being infertile, producing chromosomally abnormal offspring and having multiple spontaneous abortions Incidence 0.2% in neonatal population Higher incidence (Stern et al., 1999) Infertile couples (0.6%) RA couples (9.2%) ICSI population (2-3.2%)

71 Analytical methods for chromosomal abnormalities (numerical structural) FISH-based PGD protocols for chromosomal abnormalities Comparative genome hybridization (acgh)- based PGD for chromosomal abnormalities

72 FISH:principle Multi - color FISH 1 3 consecutive FISH procedures Y

73 FISH-based PGD protocols for structural chromosomal abnormalities: pre PGD work-up Determination of meiotic segregation for the specific structural abnormality Karyotype: confirmation chromosomal abnormality Design of probe mixture Lymphocyte FISH work-up: validation of the probe mixture

74 Meiotic segregation reciprocal translocation Alternate: normal/balanced Adjacent 1 Adjacent 2 3:1 segregation 4:0 segregation With/without recombination Anaphase 2 non-disjunction Brandriff et al; AJHG, 38: , 1986.

75 Reciprocal translocation: probe design 46,XX,t(6;11)(q21.1;q22) CEP 6 SA Tel 6q SO CEP 11 SG Tel 11q SO

76 Validation of the probe mixture: metaphase - interphase Der 6 Der 11 CEP 6 Aqua CEP 11 Green Tel 11q Orange 6 11 Efficiency of probe mixture: at least 85% Carrier/partner

77 PGD- FISH cycle: day 3 biopsy BIOPSY FIXATION ROUND 1 ROUND 2 FISH PROCEDURE Sex determination (x-linked disorder) Chromosomal aberrations (numerical and structural) Aneuploidy screening

78 PGD-FISH: reciprocal translocation CEP 6 aqua CEP 11 green Tel 11q orange CEP 6 aqua CEP 11 green Tel 11q orange Normal/balanced embryo Unbalanced embryo

79 PGD Round 2 : 16 q11.2 Orange 22 q11.2 Green Round 1 : X p11.1-q11.1 Blue Y p11.1-q11.1 Gold 13 q14 Red 18 p11.1-q11.1 Aqua 21 q22.13-q22.2 Green

80 The causes of misdiagnosis and adverse outcomes in PGD: data collection I - VIII 0.1% misdiagnosis rate Wilton et al., Hum. Reprod., 24(5), , 2009

81 Misdiagnosis: possible reasons Technical: failure FISH signals, overlapping signals, splitted spots Human errors: inadequate probe design Biological: mosaicism

82 Drawback of FISH - based PGD FISH technique related limitations Development of a patient specific protocol = time consuming Fixation of the cell: critical step (possible loss of micronuclei, chromosomes) Subjective analysis of the signals and compromised by weak, splitted or overlapping signals Only chromosomes involved in rearrangement are investigated Development of genome-wide techniques Comparative Genomic hybridization (a-cgh; micro-array)

83 Comparative genome hybridization (acgh)- based PGD for chromosomal abnormalities

84 Procedure: tubing & Whole Genome Amplification (WGA) Tubing of single cell (D3) or multiple cells (D5) WGA Amplification (SurePlex kit BlueGnome) Lysis of the cell(s) Extraction of the DNA Random fragmentation to form a library of DNA Amplification of DNA by PCR Electrophoresis (1.5% agarose)

85 Electrophoresis gel picture after a successfully WGA experiment Lines 1,2,3,5,6,7 = amplified DNA Line 4 = ladder Line 8 = negative control (PBS) Line 9 = positive control (genomic DNA)

86 Array-CGH cytochip BlueGnome (~ 12-24h) 24 sure (1Mb) 24 sure + ( Mb for telomeric regions)

87 24 sure cytochip bluegnome 13: 114 Mb; 14: 106 Mb 45,XY, der(13;14)(q10;q10) Result PGD FISH : XX Abnormal (1x LSI 13 Red, 3x LSI 14q32)

88 carrier 46,XX,t(2;5)(p11;q34) PGD-FISH: dup(2)(ptel), del(5)(qtel) 3X tel2p 2X tel2q 2X 5p15.2 1X 5q35 88 Mb 24 sure + cytochip (bluegnome) 12,6 Mb CHR 2 CHR 5 47,XX, dup(2)(ptel-p11.2), del(5)(q34-qtel), +22

89 Result: succesful WGA acgh (D3) Total Number of cycles 24 Embryos biopsied 104 Succesful WGA 99 (95.2%) Result a-cgh 99 (100%)

90 Preliminary: acgh - genetic result Indication N embryos with diagnosis N normal Translocations (8 cycles) PGD enumeration (8 cycles) PGS (8 cycles) Total (24 cycles) 29 4 (13.8%) 38 2 (5.3%) (31.3%) (16.2%)

91 Preliminary: acgh - clinical outcome Indication N of ET N of + HCG Translocations (8 cycles) PGD enumeration (8 cycles) PGS (8 cycles) Total (24 cycles) too early (60%)

92 Summary PGD a-cgh Total Cycles with pick-up 29 Cycles with biopsy 24 (82.8%) Age N Abnormal Total 83 COC PN Biopsied 104 ( ) Result WGA 99 (95.2%) Result a-cgh 99 Normal 16 (16.2%) Detected FISH Not detected with FISH 64 n ET 10 N +HCG 19 (22.6%) too early N +FHB 4 Outcome 1 delivered rest ongoing Titel van de presentatie pag. 25

93 acgh Reliability and feasibility demonstrated for detection of chromosomal imbalances in embryos (Gutiérrez- Mateo et al., 2011; Colls et al., 2012) In comparison with FISH: - Not dependent of critical step of cell fixation - Evaluating multiple loci along the length of each chromosome region - Data analysis performed by computerized analysis of signal intensities (based on a log 2 ratio and quality criteria (SD, signal-to-noise ratio)) instead of subjective signal scoring In the future: automated workstations - increase of number of samples - reduces the risk of errors

94 acgh: Allows screening for all chromosomes in addition to the unbalanced derivatives associated with the specific structural abnormality No development of a patient specific probe-mixture and preclinical validation - Detection limits: the probability of detecting an unbalanced translocation, and therefore the success of the array-cgh based analysis, is dependent upon the location of the translocation breakpoints in the chromosomes and the size of the unbalanced region(s) Limitations: - acgh cannot detect haploidy and some triploidies (69,XXX) - cannot differentiate normal versus balanced translocation carrier acgh represents at this time an expensive option for embryo testing compared to the FISH technology

95 PGD: multidisciplinary team work Center Medical Genetics Accurate genetic diagnosis Fertilisation in vitro (IVF or ICSI) Center Reproductive Medicine OPU fertilisation in vitro Appropriate genetic counselling Genetic Diagnosis Embryo biopsy Transfer 2 unaffected embryos

96 Preimplantation genetic diagnosis Martine De Rycke

97 Preimplantation Genetic Diagnosis an alternative to prenatal diagnosis and TOP involves genetic testing of cells biopsied from in vitro obtained oocytes and/or in vitro fertilised embryos and selective transfer of unaffected embryos for couples at high risk of transmitting a genetic condition to their children 2 titel

98 Preimplantation Genetic Screening PGS or aneuploidy screening involves selection of euploid embryos to improve IVF results and reduce miscarriage rates for specific IVF patients groups at low risk (advanced maternal age, recurrent IVF failure or repeated miscarriages) 3 titel

99 History of PGD 1990: Handyside et al.: first PGD for X-linked disease 1992: Handyside et al.: baby after PGD for Cystic Fibrosis Pregnancies from biopsied human preimplantation embryos sexed by Y- specific DNA amplification A. H. Handyside, E. H. Kontogianni, K. Hardy & R. M. L. Winston Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK OVER 200 recessive X chromosome-linked diseases, typically affecting only hemizygous males, have been identified. In many of these, prenatal diagnosis is possible by chorion villus sampling (CVS) or amniocentesis, followed by cytogenetic, biochemical or molecular analysis of the cells recovered from the conceptus. In others, the only alternative is to determine the sex of the fetus. If the fetus is affected by the defect or is male, abortion can be offered. Diagnosis of genetic defects in preimplantation embryos would allow those unaffected to be identified and transferred to the uterus 1. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and X- linked mental retardation. Two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six- to eight-cell stage, and sexing by DNA amplification of a Y chromosome-specific repeat sequence. Both women are confirmed as carrying normal female twins. 4 titel

100 History of PGD at UZ Brussel PGD-PCR PGD-FISH PGD-AS 5 titel

101 PGD/PGS: indications for chromosomal aberrations (numerical and structural) PGD-FISH/aCGH sex determination (X-linked disorders) PGD-FISH/aCGH or PGD-PCR (mutation identified) for monogenic diseases (X-linked, autosomal dominant/recessive) and HLA typing PGD-PCR for aneuploidy screening PGS-aCGH 6 titel

102 PGD clinical cycle 10 oocytes day 0 8 normally fertilised oocytes day 1 6 embryos for biopsy day 3 genetic testing day 3/4 ICSI no diagnosis unaffected affected unaffected affected unaffected bad morphology transfer day 5 transfer no transfer cryo, if good morphology

103 PGD clinical cycle amplification embryo biopsy with laser (day 3) FISH 8 titel

104 Single cell amplification targeted (2 copies of the region of interest) => single cell multiplex PCR (monogenic diseases) * simultaneous amplification of multiple loci per cell = flanking Short Tandem Repeat markers +/- mutation locus * more accurate: allows diagnosis AND reveals contamination & ADO * fluorescent: allows fragment length detection via capillary electrophoresis on automated sequencers requires extensive optimisation and validation of PCR conditions 9 titel

105 Single cell amplification request for mutation/gene/locus 1 => develop single cell PCR 1 request for mutation/gene/locus n => develop single cell PCR n customised protocols: optimisation and validation at the single cell level has to be repeated each time => pre-pgd workup is labour-intensive and time-consuming and yields high costs 10 titel

106 Single cell amplification universal single cell Whole Genome Amplification several µg of DNA downstream analyses haplotyping: regular PCR of STR genome-wide tests optimisation and validation of single cell whole genome amplification (WGA): only 1 time! => pre-pgd workup labour, time and costs are reduced 11 titel

107 PGD: emerging genetic tests single-cell WGA and SNP arrays - mutation analysis by haplotyping - full chromosomal constitution - Single Nucleotide Polymorphism single-cell WGA and NGS - reveal also point mutations balanced chrom. rearrangements - high cost, still under validation emerging platforms are genome-wide and allow standardisation and automation 12 titel

108 SNP bead array preparation 13 titel

109 SNP bead array: workflow MDA based 14 titel

110 Whole genome amplification: MDA Multiple Displacement Amplification, (MDA) isothermal amplification (30 C) => DNA fragments up to 70 kb, low error rates Dean et al., titel

111 SNP array: principle target probe denaturation and hybridisation on beadchip single base extension LaFramboise T, titel

112 SNP bead array A = A/T base B = G/C base NC = no call 17 titel

113 SNP array: interpretation genotype information 1) identify informative SNPs in region of interest 2) phase SNPs in embryo vs reference aff wt aff aff unaff aff 18 titel

114 Genoom-wijde moleculaire technologie toegepast in de genetische diagnostiek Nieuw genomics platform op campus UZ Brussel / VUB 22/5/2014

115 Infrastructure + applications Ir Ben Caljon

116 Available Sequencers VUB/UZ BRUSSEL CMG ULB Ion Torrent PGM MiSeq GS Junior HiSeq Nieuw genomics platform

117 System Comparison Roche GS Junior Ion Torrent PGM Run mode PicoTiterPlate 314 chip 316 chip 318 chip Output range 40 Mb Mb Mb 600 Mb-1 Gb Run time 10h 2,3h 3,0h 4,4h Reads per flowcell 100 thousand thousand 2-3 million 4-4,5 million Maximum read length 400 bp (average) 1x200 bp (400 bp) 1x200 bp (400 bp) 1x200 bp (400 bp) Quality 1x400 bp >99% > Q20 MiSeq HiSeq 1500 Run mode Nano Micro Standard Rapid Run High Output v3 High Output v4 Output range 500 Mb 1,2 Gb 15 Gb 5-90 Gb Gb Gb Run time 4-39h 4-24h 4-65h 7-40h 2-11 days 1-6 days Reads per flowcell 1 million 4 million million 300 million 1,5 billion 2 billion Maximum read length 2x250 bp 2x150 bp 2x300 bp 2x150 bp 2x100 bp 2x125 bp Quality 2x50 bp >85% > Q30 >85% > Q30 >85% > Q30 Quality 2x75 bp >85% >Q30 Quality 2x100 bp >80% > Q30 >80% > Q30 >80% > Q30 Quality 2x125 bp >80% > Q30 Quality 2x150 bp >80% > Q30 >80% > Q30 >75% > Q30 Quality 2x250 bp >75% > Q30 Quality 2x300 bp >75% > Q30 4 Nieuw genomics platform

118 IT infrastructure IT Infrastructure (UZ Brussel) 5 servers installed with Opensuse 12.2 (linux) 5 x (16cpu,192Gb Ram, 1.6 Tb HD) 40 Tb Shared Network drive (backuped) Sever capacity will be doubled in x HP Z600 workstation 24 virtual cores (Intel Xeon E5645 2,4 GHz) 2x2Tb (RAID1) 24 Gb RAM 1x Opensuse 12.2 (linux) + 1x Win7 Grid management System: Open Grid Scheduler (ogs/sge) (IB)²: interuniversity bioinformatics unit Collaboration ULB/VUB/UZ Brussel 5 Nieuw genomics platform

119 Applications (1) Whole genome sequencing (WGS) Shear DNA (get appropriately sized DNA fragments) Ligate adapters (modify DNA fragments to be compatible with sequencing instruments) Sequence (HiSeq for complex, MiSeq for small genomes) 6 Nieuw genomics platform

120 Applications (2) Whole exome sequencing (WES) Shear DNA (get appropriately sized DNA fragments) Ligate adapters (modify DNA fragments to be compatible with sequencing instruments) Enrich targets (capture specific regions/exons with probes) Sequence (HiSeq for complex, MiSeq for small genomes) 7 Nieuw genomics platform

121 Applications (3) Non-Invasive Prenatal Testing (NIPT) 1. Phlebotomy 2. Plasma isolation 3. cfdna extraction 4. Library preparation 5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting 8 Nieuw genomics platform

122 Applications (4) Bisulphite sequencing Bisulphite treatment + PCR (convert unmethylated C to U) Ligate adapters (modify DNA fragments to be compatible with sequencing instruments) Sequence (HiSeq for complex, MiSeq for small genomes) 9 Nieuw genomics platform

123 Applications (5) Mitochondrial resequencing Amplify mtdna - lrpcr (select for mtdna copies) Shear lrpcr product (get appropriately sized DNA fragments) Ligate adapters (modify DNA fragments to be compatible with sequencing instruments) Sequence (HiSeq for complex, MiSeq for small genomes) 10 Nieuw genomics platform

124 Applications (6) mrna sequencing 11 Nieuw genomics platform

125 Future prospects Small RNA sequencing (mirna) ChIP sequencing rrna typing (metagenomics) Molecular Inversion Probe (MIP) assays 12 Nieuw genomics platform

126 Questions? 13 Nieuw genomics platform

127 Non-invasive prenatal testing 22/05/2014 Dr. Kim van Berkel Dep. Gynaecology Centre for Medical Genetics Dr. Sci. Sonia Van Dooren Centre for Medical Genetics

128 What is NIPT? 1. Definition 2. Introduction 3. NIPT technology 4. Indications, contra-indications and limitations 5. Practical 6. Future 7. Conclusions

129 2013

130 Definition NIPT = non-invasive prenatal test Prenatal screening for aneuploidy Risk calculation

131 Introduction Screening for trisomy 21 Ultrasound PAPP-A/combination test (1T) Triple Test (2T) Invasive prenatal diagnosis Chorion villi sampling Amniotic fluid punction

132 Screening for trisomy 21 Ultrasound 1st trimester: nuchal translucency (NT) ductus venosus (DV) tricuspidalis valve (TV) 2nd trimester: soft markers sensitivity max 70%

133 Screening for trisomy 21 NT

134 Screening for trisomy 21 DV

135 Screening for trisomy 21 TV

136 Screening for trisomy 21 PAPP-A/combination test 1st trimester US + biochemical markers in maternal bloed (ßhCG and PAPP-A)

137 Screening for trisomy 21 PAPP-A/combination test 1st trimester echo + biochemical markers in maternal blood (ßhCG and PAPP-A) Combined risk calculation for Down Cutoff 1/250 Sensitivity 80-85% 5% false positive

138 Screening for trisomy 21 TT AFP, ßhCG and oestriol

139 Screenen naar trisomie 21 TT AFP, ßhCG and oestriol Second trimester soft-markers NF, ventriculomegaly Femur, humerus Echogene focus Dense intestines Pyelectasy SUA

140 Screening for trisomy 21

141 Invasive screening for trisomy 21 Chorionic Villi Sampling (11-13w) Punction of amniotic fluid (>15w)

142 Screening for trisomy 21 Conventional karyotyping Molecular karyotyping

143 Screening for trisomy 21: non-invasive prenatal testing (NIPT) NIPT: cell-free fetal DNA (cffdna) in maternal plasma shedding of trophoblast cells short half life (2 h clearance) 3% to 20% of total cfdna reliable detection from weeks on

144 Overview NIPT technique 1. Phlebotomy 2. Plasma isolation 3. cfdna extraction 4. Library preparation 5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting 18 NIPT - Non-invasive prenatal testing

145 NIPT - sampling 19 NIPT - Non-invasive prenatal testing

146 NIPT methodologies NIPT Clinical utility cfdna based cfrna based SNP based approaches Digital PCR (abs quant chr21 vs chr 1) RNA expression (trophoblast vs maternal ) t-mps (targeted massive parallel Sequencing) qpcr (diff methylated regions) s-mps (shotgun massive parallel sequencing)

147 NIPT methodologies NIPT Clinical utility cfdna based cfrna based SNP based approaches Digital PCR (abs quant chr21 vs chr 1) RNA expression (trophoblast vs maternal ) t-mps (targeted massive parallel Sequencing) qpcr (diff methylated regions) s-mps (shotgun massive parallel sequencing)

148 NIPT Digital PCR (1) Lo YM, et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci U S A Aug 7;104(32): NIPT - Non-invasive prenatal testing

149 NIPT Digital PCR (2) 23 NIPT - Non-invasive prenatal testing

150 NIPT methodologies NIPT Clinical utility cfdna based cfrna based SNP based approaches Digital PCR (abs quant chr21 vs chr 1) RNA expression (trophoblast vs maternal ) t-mps (targeted massive parallel Sequencing) qpcr (diff methylated regions) s-mps (shotgun massive parallel sequencing)

151 NIPT DMR: MeDIP PCR or qmsp(1) L. Osherovich, Chromosome triple play, 25 NIPT - Non-invasive prenatal testing

152 NIPT DMR technology (2) Chromosome 21(MeDIP PCR) Chromosome 18 (qmsp) Papageorgiou et al. Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21. Nat Med Apr;17(4): Lee et al. Non-Invasive Prenatal Testing of Trisomy 18 by an Epigenetic Marker in First Trimester Maternal Plasma. PLOSOne 2013 Nov; 8(11) 26 NIPT - Non-invasive prenatal testing

153 NIPT methodologies NIPT Clinical utility cfdna based cfrna based SNP based approaches Digital PCR (abs quant chr21 vs chr 1) RNA expression (trophoblast vs maternal ) t-mps (targeted massive parallel Sequencing) qpcr (diff methylated regions) s-mps (shotgun massive parallel sequencing)

154 NIPT SNP based approaches 28 NIPT - Non-invasive prenatal testing

155 NIPT methodologies NIPT Clinical utility cfdna based cfrna based SNP based approaches Digital PCR (abs quant chr21 vs chr 1) RNA expression (trophoblast vs maternal ) t-mps (targeted massive parallel Sequencing) qpcr (diff methylated regions) s-mps (shotgun massive parallel sequencing)

156 NIPT tmps (1) Sparks AB, et al.. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol Apr;206(4):319.e NIPT - Non-invasive prenatal testing

157 NIPT methodologies NIPT Clinical utility cfdna based cfrna based SNP based approaches Digital PCR (abs quant chr21 vs chr 1) RNA expression (trophoblast vs maternal ) t-mps (targeted massive parallel Sequencing) qpcr (diff methylated regions) s-mps (shotgun massive parallel sequencing)

158 NIPT smps technology 1. Library preparation 2. Cluster generation 3. Sequencing 32 NIPT - Non-invasive prenatal testing

159 NIPT smps data analysis Millions Coverage: # of reads/sample 5. Aligning raw data NIPT1 NIPT3 NIPT5 NIPT7 NIPT9 NIPT11 NIPT13 NIPT15 NIPT17 NIPT19 NIPT21 NIPT23 unmapped reads mapped reads 6. GC correction Binning Loess correction 7. Data normalisation # of data 8. Counting statistics: Z-score calculation 33 NIPT - Non-invasive prenatal testing

160 Test performance - targeted NIPT Zimmermann B, et al.. Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci. Prenat Diagn Dec;32(13): NIPT - Non-invasive prenatal testing

161 Test performance - genome-wide NIPT Shaw SW, et al. From Down syndrome screening to noninvasive prenatal testing: 20 years' experience in Taiwan. Taiwan J Obstet Gynecol Dec;52(4): NIPT - Non-invasive prenatal testing

162 Claimed accuracy per chromosome Shaw SW, et al. From Down syndrome screening to noninvasive prenatal testing: 20 years' experience in Taiwan. Taiwan J Obstet Gynecol Dec;52(4): Devers PL, Cronister A, Ormond KE, Facio F, Brasington CK, Flodman P. Noninvasive prenatal testing/noninvasive prenatal diagnosis: the position of the National Society of Genetic Counselors. J Genet Couns Jun;22(3): NIPT - Non-invasive prenatal testing

163 False positive rates and predictive values Bianchi et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med Feb 27;370(9):

164 Indications for NIPT Combination test with higher risk Previous pregnacy with trisomy years or older Psycho social Other

165 Contra-indications Dizygotic twin or multiple pregnancy Prior blood transfusion, stem cell therapy, immuno therapy, transplantation Chromosomal abberations Preferably combination test

166 Limitations Mozaicism Small abberations of chromosome 21 Monogenic disorder Obesitas Ultrasound abnormalities

167 Practical 1 st trimester US 11-12w ao abnormalities abnormalities Counseling options Option1: combination test Option 2: NIPT high risk low risk: US 20w Option3: PND CVS (11-13w) AF (>15w) Nl: US 20w higher risk

168 Future Current reporting : trisomy 21, 18, 13, gender Future reporting: Other chromosomes Small chromosomal abberations Monogenic disorders? Reimbursment

169 Future

170 Conclusion NIPT is an intermediate screening test currently mainly for trisomy 21, 18 and 13 risk calculation: HIGH or EQUAL or LOW high sensitivity and specificity (false pos. rate 1%) Preferentially for high-risk pregnancies Confirmation of abnormal result by invasive test array CGH on chorion villi or amniotic fluid Evolution towards diagnostic test in the future

171 Acknowledgements Medical genetics UZ Brussel Clinic Prof.Dr. Maryse Bonduelle Dr. Kim Van Berkel Dr. Martine Biervliet Gynaecology Clinic Dr. Anniek Vorsselmans Dr. Kim Van Berkel Lab Dr. Sci. Sonia Van Dooren Dr. Sci. Catherine Staessen Dr. Sci. Ann Van de Bogaert Dr. Sci. Alexander Gheldof NGS platform BRIGHT Ir. Ben Caljon Dr. Sci. Didier Croes Scientific partner Clinic Prof. Dr. Eric Legius Lab Dr. Sci. Joris Vermeesch Dr. Sci. Nathalie Brison

172 MT GENOOM SEQUENCING ZOEKT DIAGNOSTISCHE BENCH mtdna analyze Prof. Sara Seneca

173 mt genoom zoekt diagnostische bench Mitochondriale genoom sekwensing Wat? Waarom? 2 mt genoom zoekt diagnostische bench 20/05/2014

174 overzicht Introductie mt aandoening mtdna MPS Data analyse & resultaten platform 1 platform2 Conclusies 3 mt genoom zoekt diagnostische bench 20/05/2014

175 mitochondriale aandoeningen zeer heterogene groep aandoeningen multi-systeem ziekte waarbij vele weefsels en organen betrokken (kunnen) zijn incidentie 1/5000 geen genezing, noch therapie vage genotype-fenotype relatie diagnose is complex defect vd ademhalingsketen ( of OXPHOS systeem) 4 mt genoom zoekt diagnostische bench 20/05/2014

176 illustratie klinisch beeld 5 mt genoom zoekt diagnostische bench 20/05/2014

177 OXPHOS system energie (ATP) genererend systeem, in mitochondria duale genetische controle voor structurele subeenheden + vele nucleair gecodeerde genproducten direct & indirect defecten van genproducten van OXPHOS systeem mt ziekte Schon mt genoom zoekt diagnostische bench 20/05/2014

178 mtdna map 16, 5 kb (1) kleine circulaire dubbel strenige molecule 37 genen 13 protein 22 trna 2 rrna polymorf 7 mt genoom zoekt diagnostische bench 20/05/2014

179 mtdna map 16, 5 kb (2) maternele overerving polyploid homoplasmie heteroplasmie range 0-100% drempel effect afhankelijk mutatie afhankelijk weefsel/orgaan afhankelijk leeftijd drempel effect 8 mt genoom zoekt diagnostische bench 20/05/2014

180 diagnostiek mt aandoening diagnose studies genetische test patiënt anamnese familie historiek stamboom klinische onderzoeken microscopie, enzymologie, histologie, immunohistochemie, verschillende weefsels (bloed, epitheelcel, fibro s, spier, lever, ) mtdna nucleair DNA 9 mt genoom zoekt diagnostische bench 20/05/2014

181 moleculaire diagnostiek (1) OXPHOS systeem duale genetische controle nucleair DNA mtdna hier: focus op analyse mtdna 10 mt genoom zoekt diagnostische bench 20/05/2014

182 moleculaire diagnostiek (2) mtdna testing : stapsgewijs proces frekwente punt mutaties PCR gebaseerde screeningstechniek Sanger sekwensing varianten kwantificatie van heteroplasmie deleties : Southern blot of LR-PCR 11 mt genoom zoekt diagnostische bench 20/05/2014

183 moleculaire diagnostiek (3) hot spot regio s en hot spot posities melas, merrf, narp, LHON, verspreid over ganse genoom analyse van volledig mtdna nodig 12 mt genoom zoekt diagnostische bench 20/05/2014

184 Massieve Parallel Sekwensing (MPS) 13 mt genoom zoekt diagnostische bench 20/05/2014

185 MPS van mtdna 32 stalen : piloot studie 28 patiënten + 4 Cs 6/32 stalen 3 patiënten + 3 Cs LR-PCR library : 3 overlappende of 1 groot amplicon Ion Torrent PGM systeem Illumina MiSeq systeem ph verandering fluorescentie 14 mt genoom zoekt diagnostische bench 20/05/2014

186 Target enrichment aanrijking van mtdna NUMTs proove geen amplificatie van nucleaire mt sekwenties PCR based methodologie controle van de primerkoppels op amplificatie Long Range-PCR 3 amplicons 1 amplicon 15 mt genoom zoekt diagnostische bench 20/05/2014

187 Massieve Parallel Sekwensing bepaling van systeem s detectie drempel onderscheid ts heteroplasmie en systeemfout puc19 plasmide DNA sekwentie Ion Torrent PGM : ± 0.8% drempel 5% veelvuldige homopolymeer fouten (gekend probleem) drempel 5% MiSeq drempel : ± 0.5% drempel 2 % 16 mt genoom zoekt diagnostische bench 20/05/2014

188 puc19 analyse bepaling van detectie drempel systeem foutenmarge : ratio van # niet referentie basen met totaal # basen op eenzelfde specifieke positie wordt bepaald voor elke positie in genoom gemid. systeem fout wordt berekend 17 mt genoom zoekt diagnostische bench 20/05/2014

189 Massieve Parallel Sekwensing bepaling van systeem s detectie drempel onderscheid ts heteroplasmie en systeemfout puc19 plasmide DNA sekwentie Ion Torrent PGM : ± 0.8% drempel 2% veelvuldige homopolymeer fouten (gekend probleem) drempel 5% MiSeq drempel : ± 0.5% drempel 2 % 18 mt genoom zoekt diagnostische bench 20/05/2014

190 Massieve parallel sekwensing data analyse Ion Torrent PGM versus MiSeq fastq Torrent suite v3.6 coverage analysis (samtools) Annovar Mitomap in-house pipeline (BWA; GATK; ) VCFfile VCFfile rapport varianten + coverage 19 mt genoom zoekt diagnostische bench 20/05/2014

191 Begrip coverage Integrative Genomics Viewer (IGV) beeld 20 mt genoom zoekt diagnostische bench 20/05/2014

192 MPS resultaten non-deleted template single large scale deleties multiple deleties 21 mt genoom zoekt diagnostische bench 20/05/2014

193 Coverage profiel (1) mt genoom zoekt diagnostische bench 3,5 3 2,5 2 1,5 1 0,5 0 relative coverage mtdna position biased 20/05/2014

194 Coverage profiel (2) onafhankelijk vh DNA staal onafhankelijk vd primerset in LR-PCR onafhankelijk vd shearing methodologie ook zonder 1 ste PCR amplificatie laczα amp puc19 ori 23 mt genoom zoekt diagnostische bench 20/05/2014

195 Coverage profiel (3) Ion Torrent PGM MiSeq systeem 24 mt genoom zoekt diagnostische bench 20/05/2014

196 Variant calling stap 1 - deleties non-deleted template single large scale deleties multiple deleties 25 mt genoom zoekt diagnostische bench 20/05/2014

197 Variant calling stap 2 - varianten VCF annotatie van varianten 26 mt genoom zoekt diagnostische bench 20/05/2014

198 Variant calling stap 3 Q_filtering detectie limiet Ion Torent PGM : < 5% MiSeq : < 2% 27 mt genoom zoekt diagnostische bench 20/05/2014

199 Variant calling stap 3 Q_filtering detectie limiet Ion Torent PGM : < 5% MiSeq : < 2% QC : heteroplasmie vs gemiddelde systeem fout vgl. mtdna MPS data set 28 mt genoom zoekt diagnostische bench 20/05/2014

200 resultaten van de piloot studie Ion Torrent MiSeq 29 mt genoom zoekt diagnostische bench 20/05/2014

201 Variant calling (1) Sanger sekwensing MPS sekwensing vals negatieven < detectie limiet Sanger sekwensing 30 mt genoom zoekt diagnostische bench 20/05/2014

202 Variant calling (2) Sanger versus Ion Torrent PGM sekwensing piloot studie van 32 DNA stalen Sanger Ion Torrent PGM # varianten vals negatieven 34 extra 12 variant # stalen m m.16183a>c 3 m.7402delc 1 31 mt genoom zoekt diagnostische bench 20/05/2014

203 Variant calling (3) Sanger sekwensing vs Ion Torrent sekwensing vs MiSeq piloot studie van 6 DNA stalen Sanger sekwensing Ion Torrent PGM MiSeq # varianten vals negatieven 7 0 extra 4 6 variant AF m.5609t>c 4.5% m.8207c>t 2% 32 mt genoom zoekt diagnostische bench 20/05/2014

204 Conclusies (1) complete re-sequencing van 28 patiënten stalen nieuwe (pathogene) varianten variant gen weefsel % heteroplasmie m.14721g>a MT-TE spier 48% m.7402delc MT-COI p.(pro500hisfs*12) spier 80% m.15453t>c MT-CYB p.(leu236pro) bloed 100% 33 mt genoom zoekt diagnostische bench 20/05/2014

205 Conclusies (2) Sanger Ion Torrent MiSeq stalen/run 1 tot 12 tot 145 coverage problematisch uitstekend deleties neen +* +* punt mutaties +** AF>15-20% + AF>5% + AF>2% homopolymeren neen problematisch - * met bepaling van breekpunten ** 2 de techniek nodig voor kwantificatie 34 mt genoom zoekt diagnostische bench 20/05/2014

206 Met dank aan alle medewerkers REGE VUB CMG UZ Brussel 35 mt genoom zoekt diagnostische bench 20/05/2014

207 Challenges in cardiogenetics research, diagnostics and prevention Sonia Van Dooren Marije Meuwissen

208 Inherited cardiac arrhythmias LQT SQT Cardiac arrhythmia Primary cardiac arrhythmia electrical disease no structural abnormalities BrS ARVD CPVT Secondary cardiac arrhythmia cardiomyopathy structural abnormalities HCM DCM

209 Brugada syndrome (BrS) Incidence: Lo et al to 0.6 % in adults % in children Congenital primary cardiac arrhythmia autosomal dominant incomplete penetrance & variable expression WF WG 2012 : BrS cardi omic s rese arch

210 BrS - clinical diagnosis ECG morphology spontaneous drug-induced (ajmaline) type: saddle back - coved Symptoms: syncopes palpitations ventricular arrhythmias sudden cardiac death Family history WF WG 2012 : BrS EPS: electrophysiology studies cardi omic s rese arch Mizusawa Y, and Wilde A A Circ Arrhythm Electrophysiol 2012;5:

211 Molecular basis of BrS BrS = Channelopathy Purely electrophysical disease No structural problems Altered function of ion channels in the heart To date NaCN, CaCN & KCN Accessory proteins Imbalance between inward and outward ion currents Rev Esp Cardiol May;63(5):620

212 BrS etiopathogenisis Basic arrhythmogenic mechanisms Principle arrhythmogenic site: RVOT Hypotheses: depolarization hypothesis: slow conduction repolarization hypothesis developmental abnormalities in cardiac neural crest embryonic cells in heart development

213 BrS genetic diagnosis type gene reference Sodium channel α-subunit SCN5A MAJOR gene Kapplinger, 2010 (compendium) Sodium channel β-subunits SCN1B Watanabe, 2008 SCN3B Hu, 2009 Potassium channels KCND3 Giudicessi, 2011 KCNH2 Verkerk, 2005 KCNE3 Delpón, 2008 KCNE5 Ohno, 2011 KCNJ8 Medeiros-Domingo, 2010 Pacemaker channel HCN4 Ueda, 2009 L-type calcium channels CACNA1C Antzelevitch, 2007 CACNB2B Antzelevitch, 2007 CACNA2D1 Burashnikov, 2010 Sodium channel trafficking GPD1-L London, 2007 MOG1 Kattygnarath, 2011 SLMAP Ishikawa, 2012 TRPM4 Liu, 2013 Diagnostic yield up to 30% +10% 60% remains genetically undiagnosed

214 CMG / UZBrussel experience Clinical HRMC 400 BrS families 45 new families/year 150 family screenings/year Genetic CMG SCN5A: ~165 probands SCN1B-4B: ~83 probands targeted resequencing: gene panels whole exome sequencing

215 SCN5A genetic diagnosis & ECG BrS probands ECG SCN5A variant association BL type 1: 29 (23,8%) +: 10 (34,5%) BrS: 8 (27,6%) Likely pathogenic: 2 (6,9%) 122 BL type 2: 27 (22,1%) +: 4 (14,8%) BrS: 2 (7,4%) Disease ass SNP: 2 (7,4%) Ajm +: 66 (54,1%) +: 9 (7,4%) BrS: 6 (9,1%) Arrhythmia: 1 (1,5%) Likely pathogenic: 2 (3,0%) Baseline (BL) type 1: diagnostic yield ~ literature Baseline (BL) type 2 and ajm +: added value

216 Revision of ECGs proband: BrS + ECG Baseline ECG after Ajmaline testing Ajm + ST segment elevation > 2mm family member: conduction abnormality Ajm doubtfull ST segment elevation < 2mm family member: BrS -? Ajm - widening of QRS complex

217 SCN5A segregation analysis SCN5A+ probands 24 SCN5A+ families mutations 4 variants 12 BrS 2 arrhythmia 4 novel Segregation 8 complete 44% 0 complete 1 complete 6% 4 major 22% 2 half 11% 3 incomplete 17% Incomplete segregation of SCN5A mutations and variants Is the identified mutant/variant the MAJOR causal one? Incomplete penetrance and variable expression

218 Recent technological progress Single gene analysis Sanger sequencing GWAS SNP array (Genome-wide association study) NGS Gene panels/whole exome/whole genome (Next Generation Sequencing) Reference: Bezzina et al Nature Genetics