Analysis of DNA methylation patterns at the CNG

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Analysis of DNA methylation patterns at the CNG
Jörg Tost, Ph.D. Group Leader Epigenetics

• Paris, 12.5.2005

tost_at_cng.fr
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DNA based variations
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Biological significance of DNA methylation

• X inactivation
• Imprinting
• Essential for embryogenesis and development
• Genome integrity
• Association with transcriptional activity (in
  some cases)

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DNA methylation in disease

• Cancer
• Complex disorders
• - Type 2 Diabetes
• Imprinting disorders
• - Beckwith-Wiedemann syndrome
• - Silver Russel syndrome
• - Prader-Willi syndrome
• - Angelman syndrome
• Neurodevelopmental disorders
• - Fragile X
• - ICF
• - Rett syndrome

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Aberrant methylation pattern
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but no black and white situation
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Methylation acts as allele inactivating hit, but
is reversible
Balmain et al., Nature Genetics  33, 238 - 244
(2003)
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Methods for DNA methylation analysis

• Global methylation levels
• In situ detection of methylated cytosines
• Methylation sensitive restriction endonucleases
• Bisulphite conversion

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Bisulphite conversion of DNA
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Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry(MALDI ToF MS)
3-hydroxypicolinic acid
This picture was kindly provided by Ole Brandt.
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GOOD assay for the analysis and quantification
of DNA methylation patterns

• Freeze methylation status by treatment with
  sodium bisulphite

Tost et al., NAR, 2003, e50
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High-throughput epigenotyping in the MHC
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Tissue specific methylation patterns
Promoter BF
DAXX Exon 1
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DNA methylation based clustering for GSTP1 in
breast cancer samples
Ronneberg, Tost et al., manuscript under revision
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The Pyrosequencing technology
Pictures reproduced from the application note A
new light on DNA, A. Sylvan, 2000
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Workflow for pyrosequencing based DNA methylation
analysis
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Methylation profiles of the GSTP1 promoter in
cancerous prostate biopsies
Four sequencing primers analyse 15 out of 17 CpGs
within the amplified fragment
Tost et al., BioTechniques, 2003, e50
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Serial pyrosequencing for quantitative DNA
methylation analysis

• Cost reduction
• Accellerates workflow
• Saves precious DNA resources

Tost et al., BioTechniques, 2006, June Issue
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The IGF2/H19 locus
Picture taken from the website of the Biology
Department - University of Nevada, Reno
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11p15.5
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IGF2 variable methylation degree at consecutive
CpGs
a.Maternal Blood Cells (MBC, n17)
Methylation level ()
Methylation level ()
b. Early Term Placentas (ETP, n6)
0 2 4 6 8
10 12 14 16
18 20
0 2 4 6 8 10

Methylation level ()
Methylation level ()
0 2 4 6 8
10 12 14 16
18 20
0 2 4 6 8 10

c. Full Term Placentas (FTP, n16)
Methylation level ()
Methylation level ()
0 2 4 6 8
10 12 14 16
18 20
0 2 4 6 8 10
Position of analysed CpG
Position analysed CpG
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Cancer DNA methylation projects at CNG

• Breast Cancer (Human)
• in collab. with Prof. Vessela Kristensen (Radium
  Hospital, Oslo, Norway) 450 samples, 15 genes,
  Status analysis of the first 96 samples
• Lung Cancer (Human)
• in collab. with Dr. Zdenko Herceg (IACR, Lyon,
  France) pilot study 96 samples, 4 genes, Status
  analysis of the samples
• Liver Cancer (Human)
• in collab. with Dr. Catherine Cavard and Dr.
  Benoit Terris (Cochin Hospital, Paris, France) 96
  samples, 11 genes, Status analysis of the
  samples
• Pancreatic Cancer (Mouse Model)
• in collab. with Dr. Chang Zhang (University of
  Lyon, France) 13 mice (5 organs of each), 6
  genes, Status first round finished, reproduction
  of results

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hMlh1 methylation in microsatellite instable
colon cancers
DNA extracted from tumor
DNA extracted from normal adjacent tissue
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Pyrosequencing based methylation assays developed
at CNG
Cancer assays
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Pyrosequencing based methylation assays developed
at CNG
All assays
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Pipeline for epigenetic analysis
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Acknowledgments

• Centre National de Génotypage, Evry
• The Epigenetics Laboratory
• Hafida El abdalaoui
• Florence Busato
• Emelyne Dejeux
• Virginie Audard (Hopital Cochin)
• Ivo Gut
• Mark Lathrop
• Collaborators
• Jean-Michel Dupont (University Paris V, Hopital
  Cochin)
• Hélène Jammes, Daniel Vaiman (INSERM U709,
  Hopital Cochin, Paris)
• Catherine Cavard and Benoit Terris (Hopital
  Cochin, Paris)
• Vessela Kristensen (Radium Hospital Oslo, Norway)
• Zdenko Herceg (IACR, Lyon)
• Tim Frayling and Andrew Hattersley (Peninsular
  Medical School, Exeter, UK)
• Chang Zhang (University of Lyon)