Genoom-wijde moleculaire technologie toegepast in de...

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, bio-

informatica 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

Genoomwijde Technologie Inleiding 2 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 !)


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?

…


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

aankoop nieuwe toestellen


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



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

Algemene introductie en toepassingen in de kliniek: array technologie

Ann Van Den BogaertMarjan De Rademaeker

Array CGH2 22-05-2014

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)

array CGH3

Array CGH

22-5-2014

DNA

array CGH4 22-5-2014

Array CGH-Principe

Referentie DNA Test DNA

Labeling

Cy 5 Cy 3

Hybridisatie

Scan

Analyse

Chromosomale positie

Log 2 test/referentie

winst

verlies

0.3

0

-0.3

Mix


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 ≠


Procedure

� Random Prime Labeling� Array-CGH hybridisatie

� Precipitatie� Probebereiding� Hybridisatie� Wassen

� Scannen� Analyse


Procedure





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


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 nucleotideExo-Klenow polymerase

Random primer

array CGH10 22-5-2014

Procedure




array CGH11 22-5-2014

Array-CGH hybridisatie-Precipitatie

� Precipitatie� Cy3 gemerkt patiënt DNA + Cy5 gemerkt referentie

DNA

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

array CGH12 22-5-2014

Procedure




array CGH13 22-5-2014

Array-CGH hybridisatie-Probebereiding Theorie Stap 1

Opzuiveren= verwijderen van niet

ingebouwde nucleotiden

Random Prime labeling

array CGH14 22-5-2014

Array-CGH hybridisatie-Probebereiding Labo

array CGH15 22-5-2014

Array-CGH hybridisatie-Probebereiding Theorie stap 2

� Blokking reagent

= blokkeert repetitieve sequenties

� Niet-specifieke binding : achtergrondsignaal

array CGH16 22-5-2014

Procedure




array CGH17 22-5-2014

Array-CGH hybridisatie-HybridisatieTheorie

� 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 patiënt-referentie)

array CGH18 22-5-2014

Array-CGH hybridisatie-HybridisatieTheorie

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

array CGH19 22-5-2014

Array-CGH hybridisatie-HybridisatieLabo

65°C, 24 uur

array CGH20 22-5-2014

Procedure




array CGH21 22-5-2014

Array-CGH hybridisatie-WassenTheorie

� Wassen

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

array CGH22 22-5-2014

Procedure




array CGH23 22-5-2014

Scannen (Agilent microarray scanner)

Laser -> excitatie Cy3 en Cy5

array CGH24 22-5-2014

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

array CGH25 22-5-2014

Feature Extraction Software-Labo

Groen signaal Geel signaal Rood signaal

array CGH26 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

array CGH27 22-5-2014

Procedure




array CGH28 22-5-2014

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

array CGH29 22-5-2014

Analyse in praktijk

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

array CGH30 22-5-2014

Copy Number Variants-theorie

array CGH31 22-5-2014

Array CGH

Copy

Number

Variants

(CNVs)

Effect op ! Genen

Genetische aandoeningen/pathogeen

“Goedaardig/beninge”

Normaal

15%-25% van het

humane genoomis polymorf

CNVs=DNA fragmenten>1Kb

= de termCNP (Copy NumberPolymorphisms)

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)

22-5-2014array CGH32

Array CGH in de kliniek

� Prenatale diagnose� Indicaties/ Interpretatie� Casuistiek

� Postnatale diagnose� Indicaties� Casuistiek

� Conclusie

array CGH33 22-5-2014

Prenatale diagnose

� Verhoogd risico op chromosomale afwijking (leeftijd, abnormale niet invasieve screening)

� Verhoogd risico monogene aandoening

� Echografische afwijkingen

� Psychosociale redenen

array CGH34 22-5-2014

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

array CGH35 22-5-2014

Prenatale diagnose

array CGH36 22-5-2014

� Benign

� Pathogeen

�“Unclassified”

�Toevallige bevinding

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

Casus

� 24 weken

� Echografische afwijking: duodenale atresie

array CGH37 22-5-2014

Casus

array CGH38 22-5-2014

Casus

array CGH39 22-5-2014

Casus

array CGH40 22-5-2014

Williams syndroom

Casus

� 25 weken

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

array CGH41 22-5-2014

Casus

array CGH42 22-5-2014

Trisomie 18/ Edwards syndroom

Cave: geen laaggradige mozaïcisme!

Casus

� 20 weken

� Echografie: afwezigheid neusbeentje

array CGH43 22-5-2014

Casus

� 2080,2kb duplicatie 1q21.1-q21.2,

� 33 genen, GJA5 gen

array CGH44 22-5-2014

Casus

� 1q21 duplicatie risico factor� Macrocefalie� Aangeboren afwijkingen � Ontwikkelingsstoornissen (autisme,

leerstoornissen)� Hartafwijkingen (VSD/ASD/PVS/ TOF,..) GJA5

gen

array CGH45 22-5-2014

Casus

� 24 weken

� Echografie: cardiopathie

array CGH46 22-5-2014

Casus

� 9,4MB duplicatie16p13.13p12.2

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

array CGH47 22-5-2014

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)

array CGH48 22-5-2014

Casus

� Zwangerschap 16 weken

� Indicatie prenatale diagnose: post PGD voor metabole aandoening

array CGH49 22-5-2014

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

array CGH50 22-5-2014

Postnatale diagnosis

� Verstandelijke beperking, neuropsychiatrische aandoeningen dysmorfismen, aangeboren afwijkingen

� Ouders van individu met chromosomale afwijking

� Abnormaal karyotype verfijnen

array CGH51 22-5-2014

array CGH52 22-5-2014

Casus

� Jongen

� Pinealoblastoma

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

array CGH53 22-5-2014

Casus

� Jongen� Microftalmie en hypospadias

array CGH54 22-5-2014

Casus

� 1.1 Mb deletion 2q23 ZEB2 gen

� De novo

� ZEB2 gen mutaties/ exon deleties/

� Mowat Wilson syndroom

array CGH55 22-5-2014

Casus

� Meisje, pasgeborene

� Epileptische encephalopathy

array CGH56 22-5-2014

Casus

� Array CGH: 2235,9kb deletion 15q11.2

� Overgeërfd van de moeder

� Risico factor postnataal!� Neuropsychiatrische aandoeningen (epilepsie,

autisme, gedrags en taalproblemen, verstandelijke beperking)

array CGH57 22-5-2014

Casus

� Risico factor → Gekend deletie syndroom

array CGH58 22-5-2014

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

array CGH59 22-5-2014

Conclusie array CGH

� Uitdaging� Voor elke CNV de relatie tot fenotype bepalen� Counseling

array CGH60 22-5-2014

PGD for chromosomal abnormalities

Catherine Staessen, PhD

Centre of Medical Genetics

The main causes of chromosomal anomalies

Inheritance of the parental pathology

- true inheritance: e.g.parental translocation

Meiotic nondisjunction

80-85% related to oocytes

10-15% related to spermatozoa

Postzygotic mitotic non-disjunction

5-15% of cases of trisomies

High-

genetic risk

Low- genetic

risk

PGD

PGS

Mat

Age

Risk at

birth

35 0.5%

38 0.98%

40 1.5%

45 4.8%

*Hook EB. Cross PK. Schreinemachers DM. (1983)

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

Y

Multi - color FISH

1 → 3 consecutive FISH procedures

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

11

Der 6

Der 11

6

CEP 6 Aqua

CEP 11 Green

Tel 11q Orange

Efficiency of probe mixture: at least 85%

Carrier/partner

Validation of the probe mixture: metaphase - interphase

BIOPSY

FIXATION

ROUND 1 ROUND 2

FISH

PROCEDURE

PGD- FISH cycle: day 3 biopsy

• Sex determination

(x-linked disorder)

• Chromosomal

aberrations

(numerical and structural)

• Aneuploidy screening

PGD-FISH: reciprocal translocation

Normal/balanced embryo Unbalanced embryo

CEP 6 aqua

CEP 11 green

Tel 11q orange

CEP 6 aqua

CEP 11 green

Tel 11q orange

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

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

0.1% misdiagnosis rate

Misdiagnosis: possible reasons

Technical: failure FISH signals,

overlapping signals, splitted spots

Human errors: inadequate probe design

Biological: mosaicism

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)

Drawback of FISH - based PGD

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)

45,XY, der(13;14)(q10;q10) Result PGD FISH :

XX Abnormal

(1x LSI 13 Red,

3x LSI 14q32)

13: 114 Mb; 14: 106 Mb

24 sure cytochip bluegnome

carrier 46,XX,t(2;5)(p11;q34)

3X tel2p

2X tel2q

2X 5p15.2

1X 5q35

88 Mb

12,6 Mb CHR 2 CHR 5

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

PGD-FISH: dup(2)(ptel), del(5)(qtel)

24 sure + cytochip (bluegnome)

Result: succesful WGA – aCGH (D3)

Total

Number of cycles 24

Embryos biopsied

104

Succesful WGA

99 (95.2%)

Result a-CGH

99 (100%)

Indication N embryos with

diagnosis

N normal

Translocations

(8 cycles)

29 4 (13.8%)

PGD enumeration

(8 cycles)

38 2 (5.3%)

PGS

(8 cycles)

32 10 (31.3%)

Total

(24 cycles)

99 16 (16.2%)

Preliminary: aCGH - genetic result

Indication N of ET

N of + HCG

Translocations

(8 cycles)

3 2

+1 too early

PGD enumeration

(8 cycles)

2 2

PGS

(8 cycles)

5 2

Total

(24 cycles)

10 6 (60%)

Preliminary: aCGH - clinical outcome

Titel van de presentatie

| pag. 25

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 5.7 2.7

Biopsied

104

(4.32.6)

Result WGA 99 (95.2%)

Result a-CGH 99

Normal 16 (16.2%)

Total

N Abnormal

Detected FISH

Not detected with FISH

83

64

19 (22.6%)

n ET 10

N +HCG 6

+ 1 too early

N +FHB 4

Outcome 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

Fertilisation in vitro (IVF or ICSI)

Center Reproductive Medicine

OPU – fertilisation in vitro

Embryo biopsy

Center Medical Genetics Accurate genetic diagnosis

Appropriate genetic counselling

Genetic Diagnosis

Transfer 2 unaffected embryos

Preimplantation genetic diagnosis

Martine De Rycke

[email protected]

mailto:[email protected]

titel 2 20-5-2014

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

titel 3 20-5-2014

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)

titel 4 20-5-2014

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 uterus1. 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.

titel 5 20-5-2014

History of PGD at UZ Brussel

0

100

200

300

400

500

600

700

PGD-PCR PGD-FISH PGD-AS

titel 6 20-5-2014

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

PGD clinical cycle

10 oocytes day 0

8 normally fertilised oocytes

day 1

6 embryos for biopsy

day 3

genetic testing

day 3/4

transfer

day 5

unaffected affected unaffected bad morphology

affected unaffected

transfer no transfer

no diagnosis

cryo, if good morphology

ICSI

titel 8 20-5-2014

PGD clinical cycle

embryo biopsy

with laser (day 3)

amplification

FISH

titel 9 20-5-2014

Single cell amplification

targeted (2 copies of the region of interest)

=> single cell multiplex PCR (monogenic diseases)

requires extensive optimisation and validation of PCR

conditions

* 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

titel 10 20-5-2014


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

request for mutation/gene/locus 1 => develop single cell PCR 1

request for mutation/gene/locus n => develop single cell PCR n

titel 11 20-5-2014


universal single cell Whole Genome Amplification

several µg of DNA

downstream analyses

optimisation and validation of single cell whole genome amplification

(WGA): only 1 time! => pre-PGD workup labour, time and costs are reduced

genome-wide tests

haplotyping: regular PCR of STR

titel 12 20-5-2014

PGD: emerging genetic tests

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

single-cell WGA and SNP arrays

- mutation analysis by haplotyping

- full chromosomal constitution

- Single Nucleotide Polymorphism

titel 13 20-5-2014

SNP bead array preparation

titel 14 20-5-2014

SNP bead array: workflow

MDA based

titel 15 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

titel 16 20-5-2014

target

probe

single base

extension

LaFramboise T , 2009

denaturation and

hybridisation on beadChip

SNP array: principle

titel 17 20-5-2014

SNP bead array

A = A/T base

B = G/C base

NC = no call

titel 18 20-5-2014

SNP array: interpretation

genotype information

1) identify informative SNPs

in region of interest

aff wt aff aff unaff aff

2) phase SNPs in embryo

vs reference

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

Nieuw genomics platform 3 22-05-2014

VUB/UZ BRUSSEL CMG ULB

HiSeq 1500

GS Junior

Ion Torrent PGM

MiSeq

System Comparison

Roche GS Junior Ion Torrent PGM

Run mode PicoTiterPlate 314 chip 316 chip 318 chip

Output range 40 Mb 30-50 Mb 300-600 Mb 600 Mb-1 Gb

Run time 10h 2,3h 3,0h 4,4h

Reads per flowcell 100 thousand 400-550 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 47-300 Gb 64-500 Gb

Run time 4-39h 4-24h 4-65h 7-40h 2-11 days 1-6 days

Reads per flowcell 1 million 4 million 15-25 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 2x150 bp >80% > Q30 >80% > Q30 >75% > Q30



https://www.google.be/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=N05pZLx6u45Y3M&tbnid=zMv2gZRR1oZ5nM:&ved=0CAUQjRw&url=https://www.ksre.ksu.edu/igenomics/p.aspx?tabid=40&ei=xSf7Uoa0Hq3s0gX_jYHYCw&bvm=bv.61190604,d.d2k&psig=AFQjCNEhNgrYBTx-M0PzSW5B_yDzbn_wzw&ust=1392277815587046

http://www.google.be/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=pnlJxx7uIRbRRM&tbnid=8vHNdhAFu0GVwM:&ved=&url=http://454.com/products/what-were-working-on/&ei=vv96U4jjE8avOeW1gagC&bvm=bv.67229260,d.ZWU&psig=AFQjCNHqY9YagpRed3n4wrg_KRJ4wCeOKA&ust=1400656190751954

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

22-05-2014 Nieuw genomics platform 5

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)

Applications (2)

Whole exome sequencing (WES)


Shear DNA (get appropriately sized DNA fragments)




Enrich targets (capture specific regions/exons with probes)

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

Applications (4)

Bisulphite sequencing





Bisulphite treatment +

PCR (convert unmethylated C to U)

http://www.google.be/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=9dW80huZEjxBoM&tbnid=amDRPN9DMK1VsM:&ved=0CAUQjRw&url=http://en.wikipedia.org/wiki/Bisulfite_sequencing&ei=Ggx7U9bAHfL40gWuyIF4&bvm=bv.67229260,d.d2k&psig=AFQjCNGXC_7oL02HzABZ4t7AdgPtwqQ42A&ust=1400659337604078

Applications (5)

Mitochondrial resequencing


Shear lrPCR product (get appropriately sized DNA fragments)




Amplify mtDNA - lrPCR (select for mtDNA copies)

http://www.google.be/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=rG4c6mNd5nw0pM&tbnid=fvzLxqryJQQ7zM:&ved=0CAUQjRw&url=http://en.wikipedia.org/wiki/Gel_electrophoresis&ei=twx7U7ibE8SW0AWqzoDQCw&bvm=bv.67229260,d.d2k&psig=AFQjCNE1sgrP7W_L_mMVGMVpDynqU7EE-g&ust=1400659495625920

Applications (6)

mRNA sequencing


Future prospects

Small RNA sequencing (miRNA)

ChIP sequencing

rRNA typing (metagenomics)

Molecular Inversion Probe (MIP) assays


Questions?


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. Definition

2. Introduction

3. NIPT technology

4. Indications, contra-indications and

limitations

5. Practical

6. Future

7. Conclusions

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


Ultrasound

1st trimester:

nuchal translucency (NT)

ductus venosus (DV)

tricuspidalis valve (TV)

2nd trimester: soft markers

sensitivity max 70%


NT


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

NIPT - Non-invasive prenatal testing 18 20-5-2014

1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation

5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting

NIPT - sampling


NIPT methodologies

NIPT

s-MPS (shotgun massive

parallel sequencing)

Digital PCR (abs quant

chr21 vs chr 1)

qPCR (diff methylated

regions)

t-MPS (targeted massive

parallel Sequencing)

RNA expression

(trophoblast vs maternal )

cfDNA based cfRNA based

SNP based approaches

Clinical utility

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.

NIPT – Digital PCR (2)


NIPT methodologies

NIPT




chr21 vs chr 1)


regions)



RNA expression




Clinical utility

NIPT – DMR: MeDIP PCR or qMSP(1)


L. Osherovich, Chromosome triple play,

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.

Lee et al. Non-Invasive Prenatal Testing of Trisomy 18 by an Epigenetic

Marker in First Trimester Maternal Plasma. PLOSOne 2013 Nov; 8(11)

Chromosome 18 (qMSP)

NIPT methodologies

NIPT




chr21 vs chr 1)


regions)



RNA expression




Clinical utility

NIPT – SNP based approaches


NIPT methodologies

NIPT




chr21 vs chr 1)


regions)



RNA expression




Clinical utility

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.

NIPT methodologies

NIPT




chr21 vs chr 1)


regions)



RNA expression




Clinical utility

NIPT – sMPS technology


1. Library preparation

3. Sequencing

2. Cluster generation

NIPT – sMPS data analysis


4. Coverage: # of reads/sample 5. Aligning raw data

6. GC correction Binning Loess correction

7. Data normalisation

# of data

8. Counting statistics: Z-score calculation

0

2

4

6

8

10

NIPT1

NIPT3

NIPT5

NIPT7

NIPT9

NIPT11

NIPT13

NIPT15

NIPT17

NIPT19

NIPT21

NIPT23

Mill

ion

s

unmappedreads

mappedreads

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.

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.

Claimed accuracy per chromosome


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

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.

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 11-

12w

ao abnormalities abnormalities

Counseling

options

Option1:

combination

test

Option

2: NIPT

Option3: PND

CVS (11-13w)

AF (>15w)

Nl: US 20w

higher risk

high risk

low risk:

US 20w

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

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

Gynaecology

Clinic

Dr. Anniek Vorsselmans

Dr. Kim Van Berkel

Clinic

Prof. Dr. Eric Legius

Lab

Dr. Sci. Joris Vermeesch

Dr. Sci. Nathalie Brison

Scientific partner

MT GENOOM SEQUENCING ZOEKT DIAGNOSTISCHE BENCH

mtDNA analyze Prof. Sara Seneca

mt genoom zoekt diagnostische bench

Mitochondriale genoom sekwensing

Wat ? Waarom ?

20/05/2014mt genoom zoekt diagnostische bench2

overzicht

� Introductie

� mt aandoening

� mtDNA

� MPS

� Data analyse & resultaten

� platform 1

� platform2

� Conclusies

mt genoom zoekt diagnostische bench 20/05/20143

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 ademhalings-

keten ( of OXPHOS systeem)


illustratie klinisch beeld


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

mtDNA map 16, 5 kb (1)

� kleine circulaire dubbel strenige molecule

� 37 genen� 13 protein

� 22 tRNA

� 2 rRNA

� polymorf


mtDNA map 16, 5 kb (2)

� maternele overerving

� polyploid

� homoplasmie

� heteroplasmie� range 0-100%

� drempel effect� afhankelijk mutatie

� afhankelijk weefsel/orgaan

� afhankelijk leeftijd

drempeleffect


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


moleculaire diagnostiek (1)

� OXPHOS systeem

� duale genetische controle

�nucleair DNA

�mtDNA

� hier: focus op analyse mtDNA



� mtDNA testing : stapsgewijs proces

� frekwente punt mutaties

� PCR gebaseerde screeningstechniek

� Sanger sekwensing varianten

� kwantificatie van heteroplasmie

� deleties : Southern blot of LR-PCR



� hot spot regio’s en hot spot posities

� melas, merrf, narp, LHON, …

� verspreid over ganse genoom

analyse vanvolledig mtDNAnodig


Massieve Parallel Sekwensing (MPS)


MPS van mtDNA


32 stalen : piloot studie

28 patiënten + 4 Cs

6/32 stalen3 patiënten + 3 Cs

LR-PCR library :3 overlappende of 1 groot amplicon

Ion Torrent PGM systeem Illumina MiSeq systeem

pH verandering fluorescentie

14

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

mt genoom zoekt diagnostische bench15 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 %


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


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 %


Massieve parallel sekwensing


data analyseIon Torrent PGM versus

MiSeq

fastqTorrent

suite v3.6

VCF-file

coverage analysis(samtools)

AnnovarMitomap

rapport

varianten + coverage

in-house pipeline

(BWA; GATK;…)

VCF-file

19

Begrip ‘coverage’


Integrative Genomics Viewer (IGV) beeld

20

MPS resultaten

‘non-deleted’ template

‘multiple’ deleties‘single large scale’ deleties


Coverage profiel (1)

mt genoom zoekt diagnostische bench

0

0,5

1

1,5

2

2,5

3

3,5

11

89

377

565

753

941

112

91

31

71

50

51

69

31

88

12

06

92

25

72

44

52

63

32

82

1

relative coverage

mtDNA position

+

-

20/05/2014

biased

22


� onafhankelijk vh DNA staal

� onafhankelijk vd primerset in LR-PCR

� onafhankelijk vd shearing methodologie

� ook zonder 1ste PCR amplificatie


lacZα

ori

amp pUC19

23



MiSeq systeemIon Torrent PGM

24

Variant calling – stap 1 - deleties

‘non-deleted’ template

‘multiple’ deleties‘single large scale’ deleties


Variant calling – stap 2 - varianten

� VCF annotatie van varianten


Variant calling – stap 3 – Q_filtering

� detectie limiet

� Ion Torent PGM : < 5%

� MiSeq : < 2%


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


resultaten van de piloot studie

Ion Torrent

MiSeq


Variant calling (1)

vals negatieven

Sanger sekwensing MPS sekwensing

< detectie limietSanger sekwensing

20/05/2014

1282834

mt genoom zoekt diagnostische bench30

Variant calling (2)

Sanger Ion Torrent PGM

# varianten 862 828

vals negatieven 34

extra 12

Sanger versus Ion Torrent PGM sekwensing

piloot studie van 32 DNA stalen

variant # stalen

m.302-316 30

m.16183A>C 3

m.7402delC 1


Variant calling (3)

� Sanger sekwensing vs Ion Torrent sekwensing vsMiSeq

� piloot studie van 6 DNA stalen


Sangersekwensing

Ion TorrentPGM

MiSeq

# varianten 214 208 214

vals negatieven 7 0

extra 4 6

variant AF

m.5609T>C 4.5%

m.8207C>T 2%

32

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%


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** 2de techniek nodig voor kwantificatie

34

Met dank aan alle medewerkers

REGE VUB

CMG UZ Brussel


Challenges in cardiogenetics

research, diagnostics and

prevention

Sonia Van Dooren

Marije Meuwissen

Inherited cardiac arrhythmias

Cardiac

arrhythmia

Primary

cardiac

arrhythmia

Secondary

cardiac

arrhythmia

electrical disease

no structural abnormalities

cardiomyopathy

structural

abnormalities

LQT

HCM

DCM

SQT

BrS

ARVD

CPVT

Brugada syndrome (BrS)

20-5-2014

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

BrS - clinical diagnosis

20-5-2014

ECG morphology

spontaneous – drug-induced

(ajmaline)

type: saddle back - coved

Symptoms:

syncopes

palpitations

ventricular arrhythmias

sudden cardiac death

Family history

EPS: electrophysiology studies

WF

WG

2012

: BrS

cardi

omic

s

rese

arch

Mizusawa Y , and Wilde A A Circ Arrhythm Electrophysiol 2012;5:606-616

http://www.google.be/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=zpHjgvNAoY9-3M&tbnid=lMNJTCoeJHjS6M:&ved=0CAUQjRw&url=http://www.brugadadrugs.org/druglist_diagnostic/&ei=9h9BUpSUJcKStQaA6IHoBw&bvm=bv.52434380,d.Yms&psig=AFQjCNHo3AggV5CpuQ0Mb1fdWdFwRbI9nw&ust=1380085911452630

http://www.google.be/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=zpHjgvNAoY9-3M&tbnid=s6Ngxd39KdkmQM:&ved=0CAUQjRw&url=http://www.brugadadrugs.org/druglist_diagnostic/&ei=Cy9BUs2DEcaohAfF14HgBA&psig=AFQjCNEa03lNlI-QEUIJAbAQvmkxhmuKmg&ust=1380089977291061

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

http://www.google.be/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Cyv4uqqKDR5VBM&tbnid=j_QWRKdgtUKKkM:&ved=0CAUQjRw&url=http://cardiovascres.oxfordjournals.org/content/67/3/367/F4.expansion.html&ei=HyhBUqjlBpCrhQf5mICQDA&psig=AFQjCNHii5tJtlQMNgGB4-7WVmBHWMi7VQ&ust=1380088179229918

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

up to 30%

+10%

Diagnostic yield

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

association SCN5A variant ECG BrS

probands

122

BL type 1: 29 (23,8%) +: 10 (34,5%)

BrS: 8 (27,6%)

Likely pathogenic: 2 (6,9%)

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

proband: BrS +

family member: conduction abnormality

family member: BrS - ?

Ajm + ST segment elevation > 2mm

Ajm doubtfull ST segment elevation <

2mm

Ajm - widening of QRS complex

Revision of ECGs

ECG Baseline ECG after Ajmaline testing

SCN5A segregation analysis

SCN5A+ families

18

4 variants

14 mutations

24 SCN5A+ probands

Incomplete segregation of SCN5A mutations and variants

Is the identified mutant/variant the MAJOR causal one?

Incomplete penetrance and variable expression

Segregation

12 BrS

2 arrhythmia

4 novel

1 complete 6%

3 incomplete 17%

0 complete

2 half 11%

8 complete 44%

4 major 22%

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. 2013 – Nature Genetics

NGS approach

Reference: Clark et al. 2011 – Nature Biotechnology - Performance

comparison of exome DNA sequencing technologies

‘Exome’ (all exons of a genome)

‘All’ coding sequences

of a human genome

(>180,000 exons),

sequenced and analyzed

in one experiment

± 1 % of the whole human genome

‘Single gene’ (all exons of a gene)

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

SCN5A

16 BrS genes

all genes

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

16 BrS + / SCN5A - patients (8 families)

2 novel variant in known BrS genes

2 novel candidate genes

4 genetically ‘unresolved’ families Sequencing extra clinically

+ or – family members

Functional investigations

TARGETED EXON

RESEQUENCING

Gene panel for primary arrhythmias ( ± 70 genes)

Gene panel for structural cardiopathies (± 70 genes)

15 patients with structural cardiopathies

4 known confirmed variants

6 novel variants Validated by Sanger

+ genetic diagnosis ?

OR

functional studies required?

Known pathogenic SCN5A

mutation

Complete segregation with

phenotype

Brugada syndrome: Family 1

child wish


kinderwen

s

?

SCN5A variant

Incomplete

segregation

Gene panel in

progress


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

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

gene # requests # work-ups # cycles for

couples (total # of

cycles)

# pregnancies

Cardiomyopathies

MYBPC3 6 5 3 (4) 1

MYH7 6 5 3 (5) -

TNNT2 1 1 1 1

Primary arrhythmias

KCNQ1 6 6 3 (5) 3

SCN5A 5 5 1 BrS (2)

2 BrS + Steinert

(10)

1 BrS+ Bartter: (4)

-

2

1

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

Lab

Sonia Van Dooren, Dr Sci

Dorien Daneels

Uschi Peeters

NGS platform BRIGHT

Ben Caljon

Didier Croes

Cardiology UZ Brussel

Staff

Prof. Dr. Pedro Brugada

Prof. Dr. Carlo De Asmundis

Dr. Sophie Van Malderen

Research nurse

Gudrun Pappaert

Prof. Dr. Ramon Brugada

Research partner

Wetenschappelijk fonds Willy Gepts 2010/2012

WOK Prof. P. Brugada

Basis financing RGRG cluster

IB² (Interuniversity Brussels Bioinformatics Institute)

Funding

Innoviris (BridgeIris)

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