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

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

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

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Genoomwijde Technologie Inleiding

21-5-2014



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 !)

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21-5-2014

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? 

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…


21-5-2014

BRIGHT Platform : UZ Brussel-VUB-ULB BRIGHT : BRussels Interuniversity Genomics & High Throughput platform  Nieuwe diagnostische en research noden!!!  2012  

start zoektocht naar middelen

2013  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 

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aankoop nieuwe toestellen


21-5-2014

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  

6

Toelichting van verschillende technologieën Toepassing in niet-invasieve diagnostiek (NIPT), mitochondriaal genoom, erfelijke hartritmestoornissen


21-5-2014

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

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21-5-2014

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

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 (200-400 kb)

2

Array CGH

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Array CGH

DNA 3

array CGH

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Array CGH-Principe Referentie DNA

Test DNA

Log 2 test/referentie winst

0.3

Labeling

0

Cy 5

Cy 3

-0.3

verlies

Mix

Chromosomale positie

Analyse

Hybridisatie

Scan

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array CGH

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Array CGH-in praktijk 4x44K arrays 4 keer 44000 unieke oligonucleotiden (probes/reporters) 60 basen lang over het gehele genoom verspreid Aantal oligo’s per gebied ≠

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array CGH

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Procedure Random Prime Labeling Array-CGH hybridisatie Precipitatie Probebereiding Hybridisatie Wassen

Scannen Analyse

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Scannen Analyse

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array CGH

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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

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array CGH

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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

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Scannen Analyse

10

array CGH

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Array-CGH hybridisatie-Precipitatie Precipitatie Cy3 gemerkt patiënt DNA + Cy5 gemerkt referentie DNA

NaAc 100% EtOH Precipitatie (30 min. bij -80°C)

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array CGH

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Scannen Analyse

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Array-CGH hybridisatie-Probebereiding Theorie Stap 1

Random Prime labeling

Opzuiveren = verwijderen van niet ingebouwde nucleotiden

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array CGH

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Array-CGH hybridisatie-Probebereiding Labo

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array CGH

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Array-CGH hybridisatie-Probebereiding Theorie stap 2

Blokking reagent = blokkeert repetitieve sequenties Niet-specifieke binding : achtergrondsignaal

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array CGH

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Scannen Analyse

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array CGH

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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)

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array CGH

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Array-CGH hybridisatie-Hybridisatie Theorie Het array glaasje met 4 keer 44000 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

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Array-CGH hybridisatie-Hybridisatie Labo

65°C, 24 uur 19

array CGH

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Scannen Analyse

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Array-CGH hybridisatie-Wassen Theorie

Wassen Enkel de probes die specifiek gebonden zijn aan het gelabelde DNA kunnen een signaal geven

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Scannen Analyse

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Scannen (Agilent microarray scanner)

Laser -> excitatie Cy3 en Cy5

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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

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Feature Extraction Software-Labo

Groen signaal

25

array CGH

Geel signaal

Rood signaal

22-5-2014

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

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Scannen Analyse

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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

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Analyse in praktijk

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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

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Copy Number Variants-theorie CNVs=DNA fragmenten >1Kb

Copy Number Variants (CNVs) = de term CNP (Copy Number Polymorphisms)

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array CGH

“Goedaardig/beninge”

Normaal 15%-25% van het humane genoom is polymorf

Array CGH

Effect op ! Genen Genetische aandoeningen/pathogeen

22-5-2014

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)

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array CGH

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Array CGH in de kliniek Prenatale diagnose Indicaties/ Interpretatie Casuistiek

Postnatale diagnose Indicaties Casuistiek

Conclusie

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

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Prenatale diagnose België sinds 2013: moleculair karyotype/ array CGH Nationale consensus Centra Medische Genetica België1: Pre en post counseling Interpretatie resultaten Protocoleren resultaten 1Implementation

of genomic arrays in prenatal diagnosis: The Belgian approach to meet the challenges, Eur J Med Genet. 2014 Mar;57(4):151-156

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array CGH

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Prenatale diagnose

Benign Pathogeen “Unclassified” Toevallige bevinding

Eur J Med Genet. 2014 Mar;57(4):151-156 36

array CGH

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Casus 24 weken

Echografische afwijking: duodenale atresie

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Casus

38

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Casus

39

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Casus

Williams syndroom 40

array CGH

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Casus 25 weken

Echografie: cerebellaire atrofie, gedilateerd pyelum, polyhydramnios, normale groei

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array CGH

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Casus

Trisomie 18/ Edwards syndroom Cave: geen laaggradige mozaïcisme!

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array CGH

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Casus 20 weken

Echografie: afwezigheid neusbeentje

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array CGH

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Casus 2080,2kb duplicatie 1q21.1-q21.2,

33 genen, GJA5 gen

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array CGH

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

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array CGH

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Casus 24 weken

Echografie: cardiopathie

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array CGH

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Casus 9,4MB duplicatie16p13.13p12.2

211 genen, 75 proteine coderende genen (NDE1, MYH11, ABCC1, ABCC6,...)

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array CGH

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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)

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Casus Zwangerschap 16 weken

Indicatie prenatale diagnose: post PGD voor metabole aandoening

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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

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

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Casus Jongen

Pinealoblastoma

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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

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array CGH

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Casus Jongen Microftalmie en hypospadias

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array CGH

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

55

array CGH

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Casus Meisje, pasgeborene

Epileptische encephalopathy

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array CGH

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

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Casus Risico factor → Gekend deletie syndroom

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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

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

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

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

PGS

*Hook EB. Cross PK.

Schreinemachers DM. (1983)

Mat Age

Risk at birth

35

0.5%

38

0.98%

40

1.5%

45

4.8%

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%)

Analytical methods for chromosomal abnormalities (numerical – structural)  

FISH-based PGD protocols for chromosomal abnormalities Comparative genome hybridization (aCGH)based PGD for chromosomal abnormalities

FISH:principle

Multi - color FISH 1 → 3 consecutive FISH procedures

Y

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

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, 38:197-208, 1986.

Reciprocal translocation: probe design 46,XX,t(6;11)(q21.1;q22)

CEP 6 SA Tel 6q SO

CEP 11 SG Tel 11q SO

Validation of the probe mixture: metaphase interphase CEP 6 Aqua

Der 6

CEP 11 Green Der 11

Tel 11q Orange

6

11

Efficiency of probe mixture: at least 85% Carrier/partner

PGD- FISH cycle: day 3 biopsy BIOPSY

ROUND 1

ROUND 2 • Sex determination FISH

PROCEDURE

(x-linked disorder)

• Chromosomal aberrations (numerical and structural)

FIXATION

• Aneuploidy screening

PGD-FISH: reciprocal translocation

CEP 6 aqua

CEP 6 aqua

CEP 11 green

CEP 11 green

Tel 11q orange

Tel 11q orange

Normal/balanced embryo

Unbalanced embryo

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

The causes of misdiagnosis and adverse outcomes in PGD: data collection I - VIII 0.1% misdiagnosis rate

Wilton et al., Hum. Reprod., 24(5), 1221-28, 2009

Misdiagnosis: possible reasons 

Technical: failure FISH signals, overlapping signals, splitted spots



Human errors: inadequate probe design



Biological: mosaicism

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)

Comparative genome hybridization (aCGH)based PGD for chromosomal abnormalities

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)

Electrophoresis gel picture after a successfully WGA experiment

1

2

3

4

5

6 7

8

9

Lines 1,2,3,5,6,7 = amplified DNA Line 4 = ladder Line 8 = negative control (PBS) Line 9 = positive control (genomic DNA)

Array-CGH cytochip BlueGnome (~ 12-24h) 24 sure (1Mb) 24 sure + (0.5 - 0.25 Mb for telomeric regions)

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)

carrier 46,XX,t(2;5)(p11;q34) PGD-FISH: dup(2)(ptel), del(5)(qtel)

3X tel2p 2X tel2q 88 Mb

2X 5p15.2 1X 5q35

24 sure + cytochip (bluegnome)

12,6 Mb

CHR 2

 47,XX, dup(2)(ptel-p11.2), del(5)(q34-qtel), +22

CHR 5

Result: succesful WGA – aCGH (D3) Total Number of cycles

24

Embryos biopsied

104

Succesful WGA

99 (95.2%)

Result a-CGH

99 (100%)

Preliminary: aCGH - genetic result Indication

Translocations (8 cycles)

PGD enumeration (8 cycles)

PGS (8 cycles)

Total (24 cycles)

N embryos with diagnosis

N normal

29

4 (13.8%)

38

2 (5.3%)

32

10 (31.3%)

99

16 (16.2%)

Preliminary: aCGH - clinical outcome Indication

Translocations (8 cycles)

PGD enumeration (8 cycles)

PGS (8 cycles)

Total (24 cycles)

N of ET

3

N of + HCG

2 +1 too early

2

2

5

2

10

6 (60%)

Summary PGD a-CGH Total Cycles with pick-up

29

Cycles with biopsy

24 (82.8%)

Age

37.6  5.2

COC

9.4  4.6

2PN Biopsied Result WGA Result a-CGH Normal

Total N Abnormal

83

5.7  2.7

Detected FISH Not detected with FISH

64 19

104 (4.32.6)

n ET

10

99 (95.2%) 99 16 (16.2%)

N +HCG

N +FHB Outcome

Titel van de presentatie | pag. 25

(22.6%)

6 + 1 too early

4 1 delivered rest ongoing

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 log2 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

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

PGD: multidisciplinary team work Center Medical Genetics

Accurate genetic diagnosis

Fertilisation in vitro Center Reproductive Medicine (IVF or ICSI) OPU – fertilisation in vitro

Appropriate genetic counselling Genetic Diagnosis

Embryo biopsy

Transfer 2 unaffected embryos

Preimplantation genetic diagnosis Martine De Rycke [email protected]

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

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titel

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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)

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titel

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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 Yspecific 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 uterus1. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and Xlinked 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

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History of PGD at UZ Brussel 700 600

500 400 300 200

100 0

PGD-PCR

5

titel

PGD-FISH

PGD-AS

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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

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titel

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PGD clinical cycle 10 oocytes day 0

ICSI

8 normally fertilised oocytes day 1 6 embryos for biopsy day 3 genetic testing day 3/4 transfer day 5

no diagnosis unaffected affected affected unaffected

transfer cryo, if good morphology

unaffected bad morphology

no transfer

PGD clinical cycle

amplification

embryo biopsy with laser (day 3)

FISH

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titel

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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



9

requires extensive optimisation and validation of PCR conditions

titel

20-5-2014

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

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titel

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Single cell amplification

universal single cell Whole Genome Amplification

several µg of DNA haplotyping: regular PCR of STR downstream analyses

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

20-5-2014

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

20-5-2014

SNP bead array preparation

13

titel

20-5-2014

SNP bead array: workflow MDA based

14

titel

20-5-2014

Whole genome amplification: MDA 

Multiple Displacement Amplification, (MDA) isothermal amplification (30°C) => DNA fragments up to 70 kb, low error rates

Dean et al., 2002 15

titel

20-5-2014

SNP array: principle target

denaturation and hybridisation on beadChip

probe

single base extension

LaFramboise T , 2009

16

titel

20-5-2014

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

17

titel

20-5-2014

SNP array: interpretation genotype information 1) identify informative SNPs in region of interest 2) phase SNPs in embryo vs reference

aff

18

titel

wt

aff

aff

unaff

aff

20-5-2014

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

Infrastructure + applications Ir Ben Caljon

Available Sequencers VUB/UZ BRUSSEL

CMG ULB

Ion Torrent PGM MiSeq

GS Junior

HiSeq 1500 3

Nieuw genomics platform

22-05-2014

System Comparison

Run mode Output range Run time Reads per flowcell Maximum read length Quality 1x400 bp

Run mode Output range Run time Reads per flowcell Maximum read length Quality 2x50 bp Quality 2x75 bp Quality 2x100 bp Quality 2x125 bp Quality 2x150 bp Quality 2x250 bp Quality 2x300 bp 4

Roche GS Junior PicoTiterPlate 40 Mb 10h 100 thousand 400 bp (average) >99% > Q20

MiSeq Nano 500 Mb 4-39h 1 million 2x250 bp

Ion Torrent PGM 314 chip 30-50 Mb 2,3h 400-550 thousand 1x200 bp (400 bp)

Micro 1,2 Gb 4-24h 4 million 2x150 bp

316 chip 300-600 Mb 3,0h 2-3 million 1x200 bp (400 bp)

Standard 15 Gb 4-65h 15-25 million 2x300 bp

318 chip 600 Mb-1 Gb 4,4h 4-4,5 million 1x200 bp (400 bp)

HiSeq 1500 Rapid Run 5-90 Gb 7-40h 300 million 2x150 bp >85% > Q30

High Output v3 47-300 Gb 2-11 days 1,5 billion 2x100 bp >85% > Q30

High Output v4 64-500 Gb 1-6 days 2 billion 2x125 bp >85% > Q30

>80% > Q30

>80% > Q30

>80% > Q30 >80% > Q30

>85% >Q30

>80% > Q30 >75% > Q30


>80% > Q30

>75% > Q30 >75% > Q30 22-05-2014

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 2014

 2x 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


22-05-2014

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


22-05-2014


Whole exome sequencing (WES) Shear DNA

(get appropriately sized DNA fragments)


Enrich targets (capture specific regions/exons with probes)


7


22-05-2014


Non-Invasive Prenatal Testing (NIPT)

1. Phlebotomy

5. Cluster generation

8


2. Plasma isolation

6. Sequencing

3. cfDNA extraction

7. Data-analysis

4. Library preparation

8. Reporting

22-05-2014


Bisulphite sequencing

Bisulphite treatment + PCR (convert unmethylated C to U)



9


22-05-2014


Mitochondrial resequencing

Amplify mtDNA - lrPCR (select for mtDNA copies)

Shear lrPCR product (get appropriately sized DNA fragments)


Sequence (HiSeq for complex, MiSeq for small genomes) 10


22-05-2014


11

mRNA sequencing


22-05-2014

Future prospects    

12

Small RNA sequencing (miRNA) ChIP sequencing rRNA typing (metagenomics) Molecular Inversion Probe (MIP) assays


22-05-2014

Questions?

13


22-05-2014

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

What is NIPT ? 1. 2. 3. 4. 5. 6.

7.

Definition Introduction NIPT technology Indications, contra-indications and limitations Practical Future Conclusions

2013

Definition 

NIPT = non-invasive prenatal test



Prenatal screening for aneuploidy



Risk calculation

Introduction 

Screening for trisomy 21  Ultrasound  PAPP-A/combination test (1T)  Triple Test (2T)



Invasive prenatal diagnosis  Chorion villi sampling  Amniotic fluid punction

Screening for trisomy 21 

Ultrasound  1st trimester:   

nuchal translucency (NT) ductus venosus (DV) tricuspidalis valve (TV)

 2nd trimester: soft markers  sensitivity max 70%

Screening for trisomy 21 NT

Screening for trisomy 21

DV


TV


PAPP-A/combination test  1st trimester US + biochemical markers in maternal bloed (ßhCG and PAPP-A)


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


TT  AFP, ßhCG and oestriol

Screenen naar trisomie 21 

TT  AFP, ßhCG and oestriol



Second trimester soft-markers      

NF, ventriculomegaly Femur, humerus Echogene focus Dense intestines Pyelectasy SUA


Invasive screening for trisomy 21 

Chorionic Villi Sampling (11-13w)



Punction of amniotic fluid (>15w)


Conventional karyotyping



Molecular karyotyping

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 11-12 weeks on

Overview NIPT technique 1. Phlebotomy


18

2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

20-5-2014

NIPT - sampling

19


20-5-2014

NIPT methodologies NIPT cfDNA based

cfRNA based

Clinical utility SNP based approaches

Digital PCR

t-MPS

qPCR

(targeted massive parallel Sequencing)

s-MPS

(shotgun massive parallel sequencing)

(abs quant chr21 vs chr 1)

(diff methylated regions)

RNA expression

(trophoblast vs maternal )


cfRNA based


Digital PCR

t-MPS

qPCR


s-MPS




RNA expression


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. 2007 Aug 7;104(32):13116-21.

22


20-5-2014

NIPT – Digital PCR (2)

23


20-5-2014


cfRNA based


Digital PCR

t-MPS

qPCR


s-MPS




RNA expression


NIPT – DMR: MeDIP PCR or qMSP(1)

L. Osherovich, Chromosome triple play, 25


20-5-2014

NIPT – DMR technology (2) 

Chromosome 21(MeDIP PCR)

Papageorgiou et al. Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21. Nat Med. 2011 Apr;17(4):510-3.

26




Chromosome 18 (qMSP)

Lee et al. Non-Invasive Prenatal Testing of Trisomy 18 by an Epigenetic Marker in First Trimester Maternal Plasma. PLOSOne 2013 Nov; 8(11)

20-5-2014


cfRNA based


Digital PCR

t-MPS

qPCR


s-MPS




RNA expression


NIPT – SNP based approaches

28


20-5-2014


cfRNA based


Digital PCR

t-MPS

qPCR


s-MPS




RNA expression


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. 2012 Apr;206(4):319.e1-9.

30


20-5-2014


cfRNA based


Digital PCR

t-MPS

qPCR


s-MPS




RNA expression


NIPT – sMPS technology 1. Library preparation


3. Sequencing

32


20-5-2014

NIPT – sMPS data analysis Millions

4. Coverage: # of reads/sample

5. Aligning raw data

10 8 6 unmapped reads

4

6. GC correction

NIPT23

NIPT21

NIPT19

NIPT17

NIPT15

NIPT13

NIPT11

NIPT9

NIPT7

NIPT5

NIPT3

0

NIPT1

2 mapped reads

7. Data normalisation

8. Counting statistics: Z-score calculation

Binning Loess correction

33

# of data


20-5-2014

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. 2012 Dec;32(13):1233-41.

34


20-5-2014

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. 2013 Dec;52(4):470-4.

35


20-5-2014

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. 2013 Dec;52(4):470-4.

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. 2013 Jun;22(3):291-5

36


20-5-2014

False positive rates and predictive values

Bianchi et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 2014 Feb 27;370(9):799-808.

Indications for NIPT    



Combination test with higher risk Previous pregnacy with trisomy 21 35 years or older Psycho social Other

Contra-indications  



Dizygotic twin or multiple pregnancy Prior blood transfusion, stem cell therapy, immuno therapy, transplantation Chromosomal abberations  Preferably combination test

Limitations    



Mozaicism Small abberations of chromosome 21 Monogenic disorder Obesitas Ultrasound abnormalities

Practical 1st trimester US 1112w

abnormalities

ao abnormalities

Counseling options

Option1: Option combination 2: NIPT test

high risk low risk: US 20w

Nl: US 20w

higher risk

Option3: PND CVS (11-13w) AF (>15w)

Future 

Current reporting :  trisomy 21, 18, 13, gender



Future reporting:  Other chromosomes  Small chromosomal abberations  Monogenic disorders?



Reimbursment

Future

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

Acknowledgements Medical genetics UZ Brussel 

Clinic   





Prof .Dr. Maryse Bonduelle Dr. Kim Van Berkel Dr. Martine Biervliet

Dr. Dr. Dr. Dr.

Sci. Sci. Sci. Sci.

Sonia Van Dooren Catherine Staessen Ann Van de Bogaert Alexander Gheldof

NGS platform BRIGHT  

Ir. Ben Caljon Dr. Sci. Didier Croes

Clinic  

Lab    



Gynaecology Dr. Anniek Vorsselmans Dr. Kim Van Berkel

Scientific partner 

Clinic 



Prof. Dr. Eric Legius

Lab  

Dr. Sci. Joris Vermeesch Dr. Sci. Nathalie Brison

MT GENOOM SEQUENCING ZOEKT DIAGNOSTISCHE BENCH mtDNA analyze

Prof. Sara Seneca

mt genoom zoekt diagnostische bench

Mitochondriale genoom sekwensing

Wat ? Waarom ?

2


20/05/2014

overzicht

Introductie mt aandoening mtDNA MPS

Data analyse & resultaten platform 1 platform2

3

Conclusies


20/05/2014

mitochondriale aandoeningen

4

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)


20/05/2014

illustratie klinisch beeld

5


20/05/2014

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 2013

6


20/05/2014

mtDNA map 16, 5 kb (1)

kleine circulaire dubbel strenige molecule 37 genen 13 protein 22 tRNA 2 rRNA

7


polymorf

20/05/2014

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


20/05/2014

diagnostiek mt aandoening

diagnose

studies

patiënt anamnese

klinische onderzoeken

familie historiek

microscopie, enzymologie, histologie, immunohistochemie, …

stamboom

genetische test

mtDNA nucleair DNA

verschillende weefsels (bloed, epitheelcel, fibro’s, spier, lever, …)

9


20/05/2014

moleculaire diagnostiek (1)

OXPHOS systeem

duale genetische controle nucleair DNA mtDNA hier: focus op analyse mtDNA

10


20/05/2014


mtDNA testing : stapsgewijs proces

11

frekwente punt mutaties PCR gebaseerde screeningstechniek Sanger sekwensing varianten kwantificatie van heteroplasmie

deleties : Southern blot of LR-PCR


20/05/2014


hot spot regio’s en hot spot posities melas, merrf, narp, LHON, …

verspreid over ganse genoom analyse van volledig mtDNA nodig

12


20/05/2014

Massieve Parallel Sekwensing (MPS)

13


20/05/2014

MPS van mtDNA 6/32 stalen 3 patiënten + 3 Cs

32 stalen : piloot studie 28 patiënten + 4 Cs LR-PCR library : 3 overlappende of 1 groot amplicon

Ion Torrent PGM systeem

pH verandering 14


Illumina MiSeq systeem

fluorescentie 20/05/2014

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


20/05/2014

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


20/05/2014

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


20/05/2014

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


20/05/2014

Massieve parallel sekwensing data analyse Ion Torrent PGM versus MiSeq fastq Torrent suite v3.6 VCFfile

in-house pipeline (BWA; GATK;…) coverage analysis (samtools)

VCFfile

Annovar Mitomap rapport varianten + coverage 19


20/05/2014

Begrip ‘coverage’ Integrative Genomics Viewer (IGV) beeld

20


20/05/2014

MPS resultaten ‘non-deleted’ template

‘single large scale’ deleties

21


‘multiple’ deleties

20/05/2014

Coverage profiel (1) relative coverage

3,5 3 2,5 2 1,5

+

1

-

0,5 1 189 377 565 753 941 1129 1317 1505 1693 1881 2069 2257 2445 2633 2821

0

mtDNA position

biased

22


20/05/2014

Coverage profiel (2)

onafhankelijk vh DNA staal onafhankelijk vd primerset in LR-PCR onafhankelijk vd shearing methodologie ook zonder 1ste PCR amplificatie lacZα

amp

pUC19

ori

23


20/05/2014

Coverage profiel (3) Ion Torrent PGM

24


MiSeq systeem

20/05/2014

Variant calling – stap 1 - deleties ‘non-deleted’ template

‘single large scale’ deleties

25


‘multiple’ deleties

20/05/2014

Variant calling – stap 2 - varianten

26

VCF annotatie van varianten


20/05/2014

Variant calling – stap 3 – Q_filtering

detectie limiet Ion Torent PGM : < 5% MiSeq : < 2%

27


20/05/2014

Variant calling – stap 3 – Q_filtering

detectie limiet Ion Torent PGM : < 5% MiSeq : < 2%

28

QC : heteroplasmie vs gemiddelde systeem fout vgl. mtDNA MPS data set


20/05/2014

resultaten van de piloot studie Ion Torrent MiSeq

29


20/05/2014

Variant calling (1)

Sanger sekwensing

34

vals negatieven

30


MPS sekwensing

828

12

< detectie limiet Sanger sekwensing

20/05/2014

Variant calling (2) Sanger versus Ion Torrent PGM sekwensing piloot studie van 32 DNA stalen # varianten

Sanger

Ion Torrent PGM

862

828

vals negatieven

34

extra

12

variant m.302-316

31


# stalen 30

m.16183A>C

3

m.7402delC

1 20/05/2014

Variant calling (3)

Sanger sekwensing vs Ion Torrent sekwensing vs MiSeq piloot studie van 6 DNA stalen Sanger sekwensing

Ion Torrent PGM

MiSeq

214

208

214

vals negatieven

7

0

extra

4

6

# varianten

variant

32


AF

m.5609T>C

4.5%

m.8207C>T

2%

20/05/2014

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


20/05/2014

Conclusies (2)

Sanger stalen/run

1

MiSeq

tot 12

tot 145

problematisch

uitstekend

neen

+*

+*

+** AF>15-20%

+ AF>5%

+ AF>2%

neen

problematisch

-

coverage deleties punt mutaties

Ion Torrent

homopolymeren

* met bepaling van breekpunten ** 2de techniek nodig voor kwantificatie

34


20/05/2014

Met dank aan alle medewerkers REGE VUB CMG UZ Brussel

35


20/05/2014

Challenges in cardiogenetics research, diagnostics and prevention

Sonia Van Dooren Marije Meuwissen

Inherited cardiac arrhythmias LQT SQT

Cardiac arrhythmia

Primary cardiac arrhythmia

BrS

electrical disease no structural abnormalities

ARVD CPVT

Secondary cardiac arrhythmia cardiomyopathy structural abnormalities

HCM DCM

Brugada syndrome (BrS) 

Incidence:

Lo et al. 2004

 0.05 to 0.6 % in adults  0.0006 % in children 

Congenital primary cardiac arrhythmia  autosomal dominant  incomplete penetrance & variable expression

WF WG 2012 : BrS cardi omic s rese arch 3

20-5-2014

BrS - clinical diagnosis 

ECG morphology  



Symptoms:    

 WF WG 2012 :BrS cardi omic s rese arch

spontaneous – drug-induced (ajmaline) type: saddle back - coved

syncopes palpitations ventricular arrhythmias sudden cardiac death

Family history EPS: electrophysiology studies Mizusawa Y , and Wilde A A Circ Arrhythm Electrophysiol 2012;5:606616

20-5-2014

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. 2010 May;63(5):620

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

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

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

GPD1-L

London, 2007

MOG1

Kattygnarath, 2011

SLMAP

Ishikawa, 2012

TRPM4

Liu, 2013

Potassium channels

Sodium channel trafficking

Diagnostic yield up to 30%

+10%

60% remains genetically undiagnosed

CMG / UZBrussel experience 

Clinical diagnostics @ HRMC   



400 BrS families 45 new families/year 150 family screenings/year

Genetic diagnostics @ CMG    

SCN5A: ~165 probands SCN1B-4B: ~83 probands targeted resequencing: gene panels whole exome sequencing

SCN5A genetic diagnosis & ECG BrS probands

ECG

SCN5A variant

association BrS: 8 (27,6%)

122

BL type 1: 29 (23,8%)

+: 10 (34,5%)

BL type 2: 27 (22,1%)

+: 4 (14,8%)

Likely pathogenic: 2 (6,9%) BrS: 2 (7,4%) Disease ass SNP: 2 (7,4%) BrS: 6 (9,1%)

Ajm +: 66 (54,1%)

+: 9 (7,4%)

Arrhythmia: 1 (1,5%) Likely pathogenic: 2 (3,0%)

Baseline (BL) type 1: diagnostic yield ~ literature Baseline (BL) type 2 and ajm +: added value

Revision of ECGs ECG Baseline

ECG after Ajmaline testing



proband: BrS +



Ajm +  ST segment elevation > 2mm



family member: conduction abnormality



Ajm doubtfull  ST segment elevation < 2mm



family member: BrS - ? 

Ajm -  widening of QRS complex

SCN5A segregation analysis SCN5A+ probands

24

SCN5A+ families

18 14 mutations

Segregation

4 variants

12 BrS

2 arrhythmia

4 novel

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

Recent technological progress 

Single gene analysis



GWAS

Sanger sequencing

SNP array

(Genome-wide association study)

Reference: Bezzina et al. 2013 – Nature Genetics



NGS

(Next Generation Sequencing)

Gene panels/whole exome/whole genome

NGS approach SCN5A

‘Single gene’ (all exons of a gene)

16 BrS genes

‘Gene panel’ (all exons of a package of genes)

all genes

‘Exome’ (all exons of a genome) ± 1 % of the whole human genome

‘All’ coding sequences of a human genome (>180,000 exons), sequenced and analyzed in one experiment Reference: Clark et al. 2011 – Nature Biotechnology - Performance comparison of exome DNA sequencing technologies

Genome-wide technologies: impact on BrS ? 

In general: rare disease diagnostics exome sequencing  resolution of cases : ~5%  25%



Heterogeneous genetic disorders: more complex



Effect on BrS diagnostic yield?

Cardiac arrhythmias: next generation sequencing WHOLE EXOME SEQUENCING

TARGETED EXON RESEQUENCING

16 BrS + / SCN5A - patients (8 families)

Gene panel for primary arrhythmias ( ± 70 genes) Gene panel for structural cardiopathies (± 70 genes)

2 novel variant in known BrS genes 2 novel candidate genes

4 genetically ‘unresolved’ families

Functional investigations

15 patients with structural cardiopathies

Sequencing extra clinically + or – family members 4 known confirmed variants Validated by Sanger

6 novel variants

+ genetic diagnosis ? OR functional studies required?

Brugada syndrome: Family 1

child wish

Known pathogenic SCN5A mutation Complete segregation with phenotype

Brugada syndrome: Family 2 SCN5A variant Incomplete segregation Gene panel in progress

?

kinderwen s

Brugada syndrome: Family 3 SCN5A no mutation

Exome sequencing: mutation in candidate gene Complete segregation with phenotype

Challenges in cardiogenetics diagnostics 

Power of Ajmaline testing in clinical diagnosis of BrS    

  

Helpful in genetic diagnosis Discordancies Diagnostic criteria too strict? Genotype-phenotype revision needed? Appropriate patient selection for NGS

Incomplete segregation Incomplete penetrance and variable expression Every novel and validated variant  functional studies? Brugada syndrome

monogenic

oligogenic

polygenic

Impact on BrS cardiogenetics prevention  

Prenatal diagnosis Pre-implantation genetic diagnosis  20 years of experience  ~ 500 PGD cycles/year  >1600 PGD children born

gene

# requests

# work-ups

# cycles for couples (total # of cycles)

# pregnancies

MYBPC3

6

5

3 (4)

1

MYH7

6

5

3 (5)

-

TNNT2

1

1

1

1

KCNQ1

6

6

3 (5)

3

SCN5A

5

5

1 BrS (2) 2 BrS + Steinert (10) 1 BrS+ Bartter: (4)

2 1

Cardiomyopathies

Primary arrhythmias

!!! caution !!! : monogenic ?  oligogenic ?  complex ?

Conclusions 



In order to improve cardiogenetics prevention invest in genome-wide BrS genetic research & diagnostics

Given oligogenic to complex nature  large amounts of genome-wide data required 

extra 5 to 10 to … years of further scientific cardiogenetic progress are needed to resolve questions & current challenges

Brugada team + acknowledgements Medical genetics UZ Brussel 

Clinic  





Prof .Dr. Maryse Bonduelle Dr. Marije Meuwissen

Staff   

Prof. Dr. Pedro Brugada Prof. Dr. Carlo De Asmundis Dr. Sophie Van Malderen

Lab   



Cardiology UZ Brussel

Sonia Van Dooren, Dr Sci Dorien Daneels Uschi Peeters

NGS platform BRIGHT  

Ben Caljon Didier Croes



Research nurse 

Gudrun Pappaert

Research partner 

Prof. Dr. Ramon Brugada Funding Wetenschappelijk fonds Willy Gepts 2010/2012 WOK Prof. P. Brugada Basis financing RGRG cluster IB² (Interuniversity Brussels Bioinformatics Institute) Innoviris (BridgeIris)

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

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