van criekinge next_generation_epigenetic_profling_vuppsala
DESCRIPTION
Epigenetic - PhD Programme UppsalaTRANSCRIPT
PhD programme
Slides available www.bioinformatics.be
Lab for Bioinformatics and computational genomics
10 “genome hackers” mostly engineers (statistics)
42 scientiststechnicians, geneticists, clinicians
>100 people hardware engineers,
mathematicians, molecular biologists
Next Generation Epigenetic Profiling
Wim Van Criekinge12th december, Uppsala (SE)
OverviewEpigenetics
– Introduction– Methylation & Oncology– Biomarkers
MDxHealth–NEXT-GENeration Epigenetic Biomarkers–PharmacoDX: Methylation Based Biomarkers
for Predictive and Prognostic Use
The genome is just the start …
Defining Epigenetics
Reversible changes in gene expression/function
Without changes in DNA sequence
Can be inherited from precursor cells
Allows to integrate intrinsic with environmental signals (including diet)
Genome
DNA
Gene Expression
Epigenome
Chromatin
Phenotype
DNA Methylation Differentiates Totipotent Embryonic Stem Cells from Unipotent Adult Stem Cells
Alex Meissner, Henry Stewart Talks
Paula Vertino, Henry Stewart Talks
Reprogramming the DNA methylome
Transgenerational inheritence
ActionableEpigenome
Epigenetic (meta)information = stem cells
250 different cell types
Epigenetic (meta)information = stem cells
Cellular programming
OutsideOncology ?
Historically, Cancer Was Considered to be Driven Mostly by Genetic Changes
Example: Replication errors
GENETIC
Altered DNA/mRNA/proteins
Altered DNA sequence
X X
Oncogenesis
Tumor
Mutations in p53
Activating mutations in RAS
Mutations or amplifications of the
HER-2 gene
Chromosomal translocations in
myeloid cells and the generation of
the BCR-ABL fusion protein
Epigenetic Changes are Important in Causing Cancer
Example: Replication errors
GENETIC EPIGENETIC
Example: Chromatin modification errors
Altered DNA/mRNA/proteins
Altered DNA sequence
Altered levels ofmRNA/proteins
Alteredchromatin structure
X X
Oncogenesis
Tumor
Source: Schuebel et al 2007
SYNE1
APC2
GPNMB
MM
P2EVL
STARD8
PTPRDCD109
LGR6
RETCHD%
RNF182
ICAM5
0
20
40
60
80
100
120
Methylated Mutated
76-100 51-75 21-50 1-20
Dx
CDx
Example of Methylation vs Mutation: Colon & Breast Cancer
MGMT BiologyO6 Methyl-Guanine Methyl Transferase
Essential DNA Repair Enzyme
Removes alkyl groups from damaged guanine bases
Healthy individual: - MGMT is an essential DNA repair enzymeLoss of MGMT activity makes individuals susceptible to DNA damage and prone to tumor development
Glioblastoma patient on alkylator chemotherapy: - Patients with MGMT promoter methylation show have longer PFS and OS with the use of alkylating agents as chemotherapy
MGMT Promoter Methylation Predicts Benefit form DNA-Alkylating Chemotherapy
Post-hoc subgroup analysis of Temozolomide Clinical trial with primary glioblastoma patients show benefit for patients with MGMT promoter methylation
Median Overall Survival
21.7 months
12.7 months
radiotherapy
plus temozolomide
Methylated MGMT Gene
Non-Methylated MGMT Gene
radiotherapy
Adapted from Hegi et al.NEJM 2005352(10):1036-8.Study with 207 patients
OverviewEpigenetics
– Introduction– Methylation & Oncology– Biomarkers
MDxHealth–NEXT-GENeration Epigenetic Biomarkers
Can we rediscover MGMT ? What does the epigenome look like ?….
MBD_Seq
DNA Sheared
Immobilized Methyl Binding Domain
Condensed Chromatin
DNA Sheared
Immobilized Methyl binding domain
MgCl2
Next Gen SequencingGA Illumina: 100 million reads
MBD_Seq
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
Quality evaluation of Methyl Binding Domain based kits for enrichment DNA-methylation sequencing
De Meyer et al (2013) Plos One
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
Quality evaluation of Methyl Binding Domain based kits for enrichment DNA-methylation sequencing
MBD_SeqMGMT = dual core
# samples
# markers
MBD_Seq
Genome-wide methylation …. by next generation sequencing
Discovery
1-2 millionmethylation
cores
BRCA1,a bidirectional promoter
27
28
Splice variants
Zooming into Exon 1
29
Zooming into Exon 1
30
Zooming into Exon 1
31
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Where is the mC ?
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25% 50% 25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25% 50% 25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense methylated needed for transcriptional silencingAre there alleles with all three positions methylated ?
GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT
unmethylated alleles
less methylationmethylated alleles
more methylation
Deep Sequencing
Deep Sequencing MGMT Heterogenic complexity
Data integration with
DEEP Sequencing, Infinium, Reactivation, (directional) Expression …
Data integrationCorrelation tracks
methylation methylation
expression expression
Corr =-1 Corr = 1
Correlation trackin GBM @ MGMT
+1
-1
BRCA1
41
1 T47D2 BT5493 MCF74 MDAMB2315 HS578T6 NORMAL7 NORMAL8 NORMAL
9 BI T21 (BRCA1 MUT)10 BI T22 (BRCA1 MUT)11 BLC12 BLC13 BLC14 BLC15 BLC16 BLC17 BLC
13 BLC14 BLC15 BLC16 BLC17 BLC18 BLC
19 IVM20 DKO
# samples
# markers
MBD_Seq
BT_Seq
MSP
Genome-wide methylation …. by next generation sequencing
Discovery
Verification
Validation
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
Genetics
107 106 105 104 103 102 101 1108109
Full genome bp
Whole-genomeBisulphite seq
MSPProbes
(450-27K)Enrichment
Targeted Panels
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
Genetics
107 106 105 104 103 102 101 1108109
Full genome bp
GENETIC
Whole-genomesequencing
Enrichment seq(Exome)
PCREnrichment
Targeted Panels
Instrument and Assay providers
CLIA Lab service providers
Sequencing
DeepSeq
Molecular Unification
107 106 105 104 103 102 101 1108109
Full genome bp
Whole-genomeBisulphite seq
RUO Clinical
EPI
GENETIC
Whole-genomesequencing
Enrichment seq(MBD, RRBS)
Enrichment seq(Exome)
Probes(450-27K)
Enrichment Targeted Panels
Enrichment Targeted Panels
UltraDeep
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
236 cancer-related genes (3,769 exons) plus 47 introns from 19 genes often rearranged or altered in cancer. These genes are known to be somatically altered in human solid cancers based on recent scientific and clinical literature.
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
PubMeth.org Reviewed methylation database in cancer
Epigenetic Alterations Associated with Cancer Therapy Response
PCFT – folate transport (MTX)
WRN
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
440 cancer-related genes genes are known to be epigenetically altered in human solid cancers based on recent scientific and clinical literature.
Sequencing
DeepSeq
Molecular Unification
107 106 105 104 103 102 101 1108109
Full genome bp
Whole-genomeBisulphite seq
RUO Clinical
EPI
GENETIC
Whole-genomesequencing
Enrichment seq(MBD, RRBS)
Enrichment seq(Exome)
Probes(450-27K)
Enrichment Targeted Panels
Enrichment Targeted Panels
UltraDeep
53
Acknowledgments
Geert TrooskensSimon DenilKlaas MensaertJean-Pierre RenardSarah De KeulenaerEllen De MeesterTim De Meyer
Olivier Thas
Wim van den Berghe
Manon Van Engeland Jurgen Veeck
Monika HegiPierre BadySebastian Kurscheid
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