Genome-wide and targeted analysis of DNA methylation patterns
Devin Absher, Ph.D. HudsonAlpha Institute for Biotechnology Genetic Association Studies
oBipolar Disorder o Pritzker Consortium oCardiovascular Disease o Stanford, HudsonAlpha, Kaiser Clinics oAutoimmune Diseases o Rheumatoid Arthritis (Lou Bridges, UAB) o Systemic Lupus Erythematosus (Bob Kimberly, UAB)
Genetic Association Studies
oADVANCE: Multi-ethnic study of cardiovascular disease o Two-stage design: 1500 cases / 1500 controls; 7000 cases / 7000 controls o Meta-analysis of other phenotypes: smoking, BMI, height, weight, MetS oCARDIOGRAM: Meta-analysis of CAD o >87,000 individuals Genetic Association Studies Genetic Association Studies
oCardiovascular Disease o 25 loci o <5% of genetic risk explained oType 2 Diabetes o 16 loci o <10% of genetic risk explaned
Why Epigenetics?
oHeritability (?) oConfounding of genetic effects oMolecular readout of environmental variance
Complexities and Questions
oTissue specific epigenetic marks o How much complexity can we handle? oGenetics of epigenetics o What are the genetic influences on epigenetics? o How diverse are human populations? oAging o How does the epigenome change with age? oEnvironment o Smoking, Diet oDiseases o Cause or effect? Aging and DNA Methylation
o4 human tissues (kidney, brain, blood, muscle); 50-100 each
oBroad age range
oIllumina Methylation27 microarrays (>27,000 CpGs)
oLinear Regression Model
%DNAm = b1Age + b2Gender + e Aging and DNA Methylation Aging and DNA Methylation
Kidney Brain Blood
1499 614 1172 Significant CpGs (FDR<0.01) Aging and DNA Methylation
Brain
269
127 55
110
881 197 638
Kidney Blood Process GO Terms
Cell development 22 Regulation of transcription 21 Cell differentiation 20 Developmental process 19 Multicellular organismal process18 Multicellular organismal dev 17
Embryonic limb development16
Neurogenesis 15 kidney Limb morphogenesis 14 Embryonic development 13 blood Anatomical struct morphogenesis12 brain Organ morphogenesis 11
Neuron differentiation 10
Limb development 9
Embryonic morphogenesis 8
Nervous system development7
Neuron generation 6
Pattern specification 5
Cell fate commitment 4
Anatomical struct development3
Organ development 2
System development 1
0 2 4 6 8 10 12 14 -log (Corrected P-value) Targeted Bisulfite Sequencing
Multiple genes, small regions Targeted Bisulfite Sequencing
96 1-2kb PCR Products (96 genes)
T7
T7
T7
T7
T7 Bisulfite
GAIIx Targeted Bisulfite Sequencing
o 24 sample multiplexing in each 36bp PE lane
o >150X mean coverage per sample
o 60-70% reads on target Targeted Bisulfite Sequencing
10 oldest v. 11 youngest kidney tissues
NEUROD1 SOX14
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182547000 182547500 182548000 182548500 137482500 137483000 137483500 137484000 Targeted Bisulfite Sequencing
10 oldest v. 11 youngest kidney tissues
ELOVL3
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103985600 103985800 103986000 103986200 103986400 103986600 Targeted Bisulfite Sequencing
Large, contiguous regions Cardiovascular Disease Cardiovascular Disease Cardiovascular Disease
ANRIL 9p21 CDKN2A CDKN2B Cardiovascular Disease
ANRIL 9p21 CDKN2A CDKN2B Methylation 450
o >480,000 CpGs
o >95% of RefSeq genes
o CpG Island, gene bodies, promoters, 3’ ends, predicted regulatory elements 4 Brain Samples – Methyl450 and RRBS
Methyl450 RRBS Pearson Correlation Coeff.
0.935 0.940 0.945 0.950 0.955 0.960 0.965 All CpGs
MspI
Sites
NonMspI 42,595 RRBS
Same Strand CpGs v . Methyl450.
in Common
Opp. Strand
Inf. Inf. I
Inf. Inf. II
Controls – Unmethylated, Hemi-methylated, Methylated 0 2 Inf. I
Inf. II
5
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5 0
0.0 0.2 0.4 0.6 0.8 1.0
Beta Methylation 450
o Lupus
o Triglycerides / Diet
o Cardiovascular Disease
o Bipolar Disorder
Twins – Discordant, Autoimmune
RA JRA RA JDM JDM
JDM SLE SLE SLE SLE Acknowledgments
Absher Lab Collaborators Kenny Day Jan Dumanski, Uppsala Lindsay Jones Fred Miller, NIH Kimberly Hobbs Uli Broeckel, MCW Krista Thibeault Andy West, UAB Kevin Roberts Donna Arnett, UAB Matt Lewis Jim Brooks, Stanford Tom Quertermous, Stanford