A 1-hour lecture to 4th-year undergraduate and/or MSc students in human genetics, focusing on exploring the role of DNA methylation in human complex disease.

1. The
role
of
DNA
methyla0on
in
complex
diseases
Jordana
Bell
Senior
Lecturer
Department
for
Twin
Research
and
Gene8c
Epidemiology
King’s
College
London
Dec
4th,
2014

2. The
epigene0c
landscape
Conrad
Waddington
Waddington
1942;
Waddington
1957

3. Epigene0c
Mechanisms
Reproduced
from
Nature
441,
143-­‐145
(11
May
2006)
DNA
methyla0on
Histone
modifica0ons
Regula0on
of
chroma0n
structure
and
gene
expression
‣
DNA
methyla0on
&
histone
modifica0ons
‣
Chroma0n-­‐remodeling
complexes
‣
Non-­‐coding
RNA-­‐mediated
gene-­‐silencing
‣
Transcrip0on-­‐factor
binding
‣
Mechanisms
involved
in
genera0ng
and
maintaining
heritable
chroma0n
structure
and
aTachment
to
the
nuclear
matrix.
Changes
to
the
genome
that
affect
gene
expression
without
changing
the
DNA
sequence

4. Gene
expression
Central
Dogma
A,C,T,G
A,C,U,G
Leu,
Pro,...
Transcrip0on
Start
Site
(TSS)
DNA
DNA
RNA
polymerase
pre-­‐mRNA
mRNA
5’UTR 3’UTRORF
5’UTR 3’UTR
pre-­‐mRNA
Transcrip0on
End
Site
(TES)
Transcrip0on

5. Epigene0c
regula0on
of
gene
expression
• Epigene0c
marks
regulate
gene
expression,
predominantly
by
changing
chroma0n
structure.
➡Euchroma0n?
➡Ac0ve
histone
marks?
➡No
promoter
DNA
methyla0on?
➡TF
promoter
binding?➡Enhancers
ac0ve?
➡No
RNA-­‐mediated
silencing?
Transcript
(mRNA)

6. DNA
methyla0on
• 5-­‐methylCytosine
(5mC)
• One
of
the
most
common
and
stable
epigene0c
marks
• Other
types:
5hmC,
5fC,
[4mC,
6mA
in
bacteria]
• CpG
dinucleo0des
&
CpG
islands
• Heritable
through
cell
division,
but
dynamic
or
REVERSIBLE
• Important
role
in
gene
regula0on,
XCI,
genome
stability
netic modifications. A methyl group is
n DNA is treated with bisulfite, unmethylated
thylated cytosines are protected.
Me
NH2
C
C
C
N
C
NO
NH2
C
C
C
N
C
NO
CH3
Unmethylated Methylated
C C
Bisulfite
Conversion
O
C
C
C
N
C
NO
U
NH2
C
C
C
N
C
NO
CH3
C
R A WIDE
ne of several epigenetic modifications. A methyl group is
cytosine C . When DNA is treated with bisulfite, unmethylated
d to uracil, but methylated cytosines are protected.
Me
Me
Me
Me
Me
Bisulfite
Conversion
O
C
C
C
N
C
NO
U
NH2
C
C
C
N
C
NO
CH3
C
R A WIDE
HYLATION
e of several epigenetic modifications. A methyl group is
cytosine C . When DNA is treated with bisulfite, unmethylated
d to uracil, but methylated cytosines are protected.
e
Me
Me
Me
Me
Me
NH2
C
C
C
N
C
NO
NH2
C
C
C
N
C
NO
CH3
Unmethylated Methylated
C C
Bisulfite
Conversion
O
C
C
C
N
C
NO
U
NH2
C
C
C
N
C
NO
CH3
C
SAM SAH
Methyltransferase

7. Origin
and
dynamics
of
epigene0c
varia0on?
Gene0c
influences
Reproduced
from
Mill
&
Heijmans
2013
Nature;
Reik
&
Kelsey
2014
Nature
-­‐
Guo
et
al
&
Smith
et
al
2014
Egg Sperm
Embryo
Demethyla)on
Rapid
Turnover
Direct
environmental
exposures
In
utero
effects
Ageing

8. The
importance
of
DNA
methyla0on
in
normal
development
and
throughout
life
‣ Gene
expression
regula0on
‣ Reprogramming:
cell
lineage
&
0ssue
differen0a0on
• All
cells
contain
the
same
genes
–
cell
iden00es
depend
on
which
genes
are
expressed
and
repressed.
This
process
is
in
part
regulated
by
DNA
methyla0on.
‣ Imprin0ng
• Preferen0al
DNA
methyla0on
of
the
promoter
of
a
parent-­‐of-­‐origin
specific
copy
of
the
allele
to
silence
its
expression.
‣ X-­‐chromosome
inac0va0on
‣ Genomic
stability

9. DNA
methyla0on
in
human
disease
• For
the
past
few
decades
DNA
methyla0on
changes
in
specific
genes
have
been
linked
to:
• Imprin0ng-­‐related
disorders
• X-­‐chromosome
abnormali0es
(eg
XO)
and
XCI
skewing
related
traits
• Cancers

10. Example
of
imprin0ng-­‐related
disorders:
Prader-­‐Willi
and
Angelman
syndrome
Imprinted
region
on
chromosome
15:
imprin0ng
is
not
the
cause
of
disease,
but
is
responsible
for
the
paTern
of
manifesta0on
of
the
disease
Reproduced
from
hTps://www.peds.ufl.edu

11. X-­‐chromosome
inac0va0on
skewing
• Mechanism
of
dosage
compensa0on
• XCI
skewing
can
occur
(eg
preferen0al
inac0va0on
of
maternal
X)
• A
recessive
muta0on
carried
on
the
ac0ve
X
chromosome
can
have
profound
adverse
phenotypic
effects.
• XCI
skewing
has
been
associated
with
haemophilia,
Fragile-­‐X
syndrome,
and
Duchenne
Muscular
Dystrophy.
• X-­‐chromosome
inac0va0on
(XCI)
is
the
random
silencing
of
one
X
chromosome
in
order
for
female
development
to
proceed
X-Chromosome Inactivation
E. Heard, March 17th 2014
Xa' Xa' X' Xi'
Xist""
RNA'
One of the two X chromosomes must be silenced during e
embryogenesis in order for female development to proc
RNA'Pol'II'
Ac' H3K4'
me2'
Ac' Ac' Ac'
H3K4'
me3'
PcH3K2
me3
Ac6ve'X'chromosome' Inac6
5V

12. Epigene0cs
of
cancer
• More
recently,
genome-­‐wide
scans
confirm
gross
epigene0c
abnormali0es
in
cancer
0ssue
• Cancer
is
defined
by
uncontrolled
division
of
abnormal
cells
Reproduced
from
Nephew
and
Huang
2003
Cancer
LeT
Progressive changes in promoter methylation at C
sites during cancer initiation and progression
Nephew"&,Huang,"Cancer"LeI."2003;190:125
• Progressive
epigene0c
changes
in
cancer
ini0a0on
and
progression:

13. DNA
methyla0on
in
human
disease
• For
the
past
few
decades
DNA
methyla0on
changes
in
specific
genes
have
been
linked
to:
• Imprin0ng-­‐related
disorders,
eg
Angelman
syndrome
and
Prader-­‐Willi
syndrome
(15q),
Beckwith-­‐Wiedemann
syndrome
(11)
• X-­‐chromosome
abnormali0es
(eg
XO)
and
XCI
skewing
related
traits
• Cancers
• Recent
technologies
allow
for
DNA
methyla0on
assays
throughout
genome,
and
have
the
poten0al
to
iden0fy
disease-­‐related
changes
of
modest
effects
across
a
wide
range
of
traits
[EWAS].

14. DNA
methyla0on
assays
Principles and challenges of genome-
wide DNA methylation analysis
Peter W. Laird
Abstract | Methylation of cytosine bases in DNA provides a layer of epigenetic control in
many eukaryotes that has important implications for normal biology and disease. Therefore,
profiling DNA methylation across the genome is vital to understanding the influence of
epigenetics. There has been a revolution in DNA methylation analysis technology over the
APPLICATIONS OF NEXT-GENERATION SEQUENCING
REVIEWS
Nature Reviews | Genetics
Numberofsamplesanalysed
Number of CpGs analysed per sample genome
1
Infinium
COBRA
Pyrosequencing
MSP
Southern blot
GoldenGate
Enzyme–chip
MeDIP–chip
Enzyme–seq
BSPP
BC–seq
WGSBS
RRBS
MeDIP–seq
Sanger BS
MethyLight
EpiTYPER
10 102
103
104
105
106
107
108
10
1
104
103
102
Figure 1 | Sample throughput versus genome coverage. A plot of sample throughput
against genome coverage for various DNA methylation techniques. Throughput is
determined by the number of samples that can be analysed per experiment, based on
large eukaryotic genomes. Coverage is determined by the number of CpGs in the
genome that can be analysed per experiment. BC–seq, bisulphite conversion followed
by capture and sequencing; BS, bisulphite sequencing; BSPP, bisulphite padlock
probes; –chip, followed by microarray; COBRA, combined bisulphite restriction
analysis; MeDIP, methylated DNA immunoprecipitation; MSP, methylation-specific
PCR; RRBS, reduced representation bisulphite sequencing; –seq, followed by
sequencing; WGSBS, whole-genome shotgun bisulphite sequencing.
reagent-intensive. The labour involved in many of the
current enzyme-based and affinity-enrichment meth-
ods precludes the processing of large numbers of samples.
Th
ods a
seque
on Hp
can a
NCB
huma
locus
MspI
the re
is A o
DNA
in the
cytos
in cis
betwe
The e
array
tribut
of the
resolu
distri
CpG)
seque
outpu
meth
attrib
comp
platfo
meas
Fu
cytos
and, as DNA methyltransferases are not present during
PCR or in biological cloning systems, DNA methyla-
tion information is erased during amplification. Some
investigators have suggested that it could be feasible to
maintain the pattern of methylation during PCR if an
appropriate DNA methyltransferase were present in the
tion–modification systems in bacteria and archaea is
sometimes overlooked. Each sequence-specific restric-
tion enzyme has an accompanying DNA methyl-
transferase that protects the endogenous DNA from
the restriction defence system by methylating bases in the
recognition site. Some restriction enzymes are inhibited
Table 1 | Main principles of DNA methylation analysis
Pretreatment Analytical step
Locus-specific analysis Gel-based analysis Array-based analysis NGS-based analysis
Enzyme
digestion
• HpaII-PCR • Southern blot
• RLGS
• MS-AP-PCR
• AIMS
• DMH
• MCAM
• HELP
• MethylScope
• CHARM
• MMASS
• Methyl–seq
• MCA–seq
• HELP–seq
• MSCC
Affinity
enrichment
• MeDIP-PCR • MeDIP
• mDIP
• mCIP
• MIRA
• MeDIP–seq
• MIRA–seq
Sodium
bisulphite
• MethyLight
• EpiTYPER
• Pyrosequencing
• Sanger BS
• MSP
• MS-SNuPE
• COBRA
• BiMP
• GoldenGate
• Infinium
• RRBS
• BC–seq
• BSPP
• WGSBS
AIMS, amplification of inter-methylated sites; BC–seq, bisulphite conversion followed by capture and sequencing; BiMP, bisulphite
methylation profiling; BS, bisulphite sequencing; BSPP, bisulphite padlock probes; CHARM, comprehensive high-throughput arrays
for relative methylation; COBRA, combined bisulphite restriction analysis; DMH, differential methylation hybridization; HELP, HpaII
tiny fragment enrichment by ligation-mediated PCR; MCA, methylated CpG island amplification; MCAM, MCA with microarray
hybridization; MeDIP, mDIP and mCIP, methylated DNA immunoprecipitation; MIRA, methylated CpG island recovery assay;
MMASS, microarray-based methylation assessment of single samples; MS-AP-PCR, methylation-sensitive arbitrarily primed PCR;
MSCC, methylation-sensitive cut counting; MSP, methylation-specific PCR; MS-SNuPE, methylation-sensitive single nucleotide
primer extension; NGS, next-generation sequencing; RLGS, restriction landmark genome scanning; RRBS, reduced representation
bisulphite sequencing; –seq, followed by sequencing; WGSBS, whole-genome shotgun bisulphite sequencing.
OLUME 11 www.nature.com/reviews/genetics
© 20 Macmillan Publishers Limited. All rights reserved10
dification systems in bacteria and archaea is
es overlooked. Each sequence-specific restric-
yme has an accompanying DNA methyl-
se that protects the endogenous DNA from
ction defence system by methylating bases in the
on site. Some restriction enzymes are inhibited
Array-based analysis NGS-based analysis
• DMH
• MCAM
• HELP
• MethylScope
• CHARM
• MMASS
• Methyl–seq
• MCA–seq
• HELP–seq
• MSCC
• MeDIP
• mDIP
• mCIP
• MIRA
• MeDIP–seq
• MIRA–seq
• BiMP
• GoldenGate
• Infinium
• RRBS
• BC–seq
• BSPP
• WGSBS
lowed by capture and sequencing; BiMP, bisulphite
es; CHARM, comprehensive high-throughput arrays
H, differential methylation hybridization; HELP, HpaII
and amplification; MCAM, MCA with microarray
MIRA, methylated CpG island recovery assay;
CR, methylation-sensitive arbitrarily primed PCR;
-SNuPE, methylation-sensitive single nucleotide
rk genome scanning; RRBS, reduced representation
hotgun bisulphite sequencing.
www.nature.com/reviews/genetics
and, as DNA methyltransferases are not present during
PCR or in biological cloning systems, DNA methyla-
tion information is erased during amplification. Some
investigators have suggested that it could be feasible to
maintain the pattern of methylation during PCR if an
appropriate DNA methyltransferase were present in the
tion–modification systems in bacteria and archaea is
sometimes overlooked. Each sequence-specific restric-
tion enzyme has an accompanying DNA methyl-
transferase that protects the endogenous DNA from
the restriction defence system by methylating bases in the
recognition site. Some restriction enzymes are inhibited
Table 1 | Main principles of DNA methylation analysis
Pretreatment Analytical step
Locus-specific analysis Gel-based analysis Array-based analysis NGS-based analysis
Enzyme
digestion
• HpaII-PCR • Southern blot
• RLGS
• MS-AP-PCR
• AIMS
• DMH
• MCAM
• HELP
• MethylScope
• CHARM
• MMASS
• Methyl–seq
• MCA–seq
• HELP–seq
• MSCC
Affinity
enrichment
• MeDIP-PCR • MeDIP
• mDIP
• mCIP
• MIRA
• MeDIP–seq
• MIRA–seq
Sodium
bisulphite
• MethyLight
• EpiTYPER
• Pyrosequencing
• Sanger BS
• MSP
• MS-SNuPE
• COBRA
• BiMP
• GoldenGate
• Infinium
• RRBS
• BC–seq
• BSPP
• WGSBS
AIMS, amplification of inter-methylated sites; BC–seq, bisulphite conversion followed by capture and sequencing; BiMP, bisulphite
methylation profiling; BS, bisulphite sequencing; BSPP, bisulphite padlock probes; CHARM, comprehensive high-throughput arrays
for relative methylation; COBRA, combined bisulphite restriction analysis; DMH, differential methylation hybridization; HELP, HpaII
tiny fragment enrichment by ligation-mediated PCR; MCA, methylated CpG island amplification; MCAM, MCA with microarray
hybridization; MeDIP, mDIP and mCIP, methylated DNA immunoprecipitation; MIRA, methylated CpG island recovery assay;
MMASS, microarray-based methylation assessment of single samples; MS-AP-PCR, methylation-sensitive arbitrarily primed PCR;
MSCC, methylation-sensitive cut counting; MSP, methylation-specific PCR; MS-SNuPE, methylation-sensitive single nucleotide
primer extension; NGS, next-generation sequencing; RLGS, restriction landmark genome scanning; RRBS, reduced representation
bisulphite sequencing; –seq, followed by sequencing; WGSBS, whole-genome shotgun bisulphite sequencing.
UME 11 www.nature.com/reviews/genetics
© 20 Macmillan Publishers Limited. All rights reserved10
• Further
reading:
450k
Laird
2010
Nat
Rev
Genet
Sequence-­‐based
assays:
Array-­‐based
assays:
Illumina
Infinium
450k,
etc

15. Consequences
Genes
Gene
Expression
Complex
Phenotypes
DNA
methyla2on
varia2on
Causes
and
Consequences
of
DNA
methyla0on
varia0on
in
human
popula0ons
Time Environment
Causes

16. Consequences
Genes
Complex
Phenotypes
DNA
methyla2on
varia2on
Causes
and
Consequences
of
DNA
methyla0on
varia0on
in
human
popula0ons
Environment
Causes
1. Prior
to
disease
onset
(causal
&
biomarker)
2. Consequence
of
disease
(disease
progression)Gene
Expression
Time

17. Consequences
Complex
Phenotypes
DNA
methyla2on
varia2on
Causes
and
Consequences
of
DNA
methyla0on
varia0on
in
human
popula0ons
Causes
External
Environment
Technical
covariates
Cell
heterogeneity
Internal
Environment
Age
Sex Complex
Phenotypes
Germline
Soma0c
indels
etc
Hormones
Longitudinal
stability
Genes Environment
Gene
Expression
Time

18. Consequences
DNA
methyla2on
varia2on
Gene0c
and
environmental
impacts
on
DNA
methyla0on
variability,
and
their
consequences
Causes
External
Environment
Germline
Genes Environment
Complex
Phenotypes
Gene
Expression
1 2
3

19. TwinsUK
Cohort
Department
for
Twin
Research,
King’s
College
London
~13,000
volunteer
adult
twins:
6,000
monozygo0c
(MZ)
twins
www.twinsuk.ac.uk
Established
in
1992 Same-­‐sex
adult
(age
range:
16-­‐101)
twin
pairs

20. The
EpiTwin
Project1,2
Aim:
To
iden)fy
Differen)ally
Methylated
Regions
(DMRs)
in
common
complex
disease
www.epitwin.eu
Pain
sensi2vity
Type
2
Diabetes
Depression
Heart
Disease
IVF
Breast
Cancer
Telomeres
Allergy
Asthma
Osteoporosis
Hypertension
Osteoarthri2s
Muscle
mass
Lipids
Psoriasis
Colon
cancerEczema
Obesity
Alcohol
use
10M
methyla2on
sites,
5000
individuals
Differen2ally
Methylated
Regions
(DMRs)
in
Disease
Epigenome-­‐wide
Associa2on
Scan
(EWAS)
Bone
mineral
density
Autoimmune
disease
Epigene2c
profiles
of
5,000
UK
Twins
using
DNA
methyla2on
sequencing
and
Illumina
450k
profiles
in
whole
blood.
Select
disease-­‐discordant
MZ
twins
1Bell
et
al.
Nat
Commun
2014;
2Davies,
Krause,
Bell
et
al.
Genome
Bio
2014;

21. The
MuTHER
Study1,2
Mul2ple
Tissue
Human
Expression
Resource
TwinsUK(Resource!
!!
Adipose((subcutaneous!fat)!!
Whole(Skin((
Lymphoblastoid(cell(lines((LCL)!
Lymphocytes,!Skeletal!Muscle!
(
850!female!
!twins!
Punch!Biopsies!&!
!blood!samples!!
Mul$%centre+collabora$on+
Kings&College&London&
Wellcome&Trust&Sanger&Ins7tute&
University&of&Oxford&
University&of&Geneva&
University&of&Cambridge&
&&
Expression
Gene0cs
Epidemiology
DNA
methyla2on
Illumina
HT12
Array
Illumina
450k
Array3
~2mln
genotyped
and
imputed
SNPs
Clinical
&
Lifestyle
longitudinal
data
1Nica
et
al.
Plos
Gene0cs
2012
2Grundberg
et
al.
Nature
Gene0cs
2013;
3Grundberg
et
al.
American
Journal
of
Human
Gene0cs
2013;
Adipose
Skin
Lymphoblastoid
cell
lines
Lymphocytes,
Skeletal
Muscle
850 female
twins
Punch
biopsies
& blood
from
P
Deloukas

22. Consequences
DNA
methyla2on
varia2on
Gene0c
and
environmental
impacts
on
DNA
methyla0on
variability
Causes
External
Environment
Germline
Genes Environment
Complex
Phenotypes
Gene
Expression
1

23. DNA
methyla0on
heritability
MZ DZ
Genes
DNA
methyla0on
In
twins,
heritability
es0mates
compare
the
degree
of
phenotypic
similari0es
between
groups
of
MZ
and
DZ
twins.
Heritability
(H)
=
propor0on
of
the
phenotypic
variance
that
is
aTributable
to
gene0c
effects.
Here,
the
phenotype
is
DNA
methyla0on
at
1
CpG-­‐site
in
the
genome.
Twin-­‐based
studies
can
es0mate
the
narrow-­‐sense
heritability
(h2),
which
measures
the
propor0on
of
trait
variance
that
is
due
to
addi0ve
gene0c
effects.
h2
=
2(rMZ
-­‐
rDZ)

24. DNA
methyla0on
heritability
across
0ssues
1Grundberg
et
al.
American
Journal
of
Human
Gene0cs
2013;
2
Gordon
et
al.
2012Genome
Research;
3
Bell
et
al.
2012
PLoS
Gene0cs;
Genes
DNA
methyla0on
‣
Blood
samples
from
240
female
twins
‣
377,000
methyla0on
sites
‣
Assess
DNA
methyla0on
heritability
‣
Adipose
0ssue
from
518
female
twins1
‣
424,000
methyla0on
sites
‣
Assess
DNA
methyla0on
heritability
_
__
_
MZ DZ Singleton
0.980
0.985
0.990
0.995
Whole Blood Illumina 450k Adipose Tissue Illumina 450k
➡
Mean
CpG-­‐site
heritability
is
0.183
(blood)
and
0.191
(adipose).
10%
CpGs
with
>50%
heritability.
➡
Overlap
of
heritable
probes,
consistent
with
previous
blood(-­‐related)
es0mates
in
newborns2.
Correla0on
Zygosity Zygosity
Correla0on

25. DNA
methyla0on
QTLs
(meQTLs)
Genes
DNA
methyla0on
‣
Blood
samples
from
188
female
twins
‣
370,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
‣
Adipose
0ssue
from
649
female
twins1
‣
424,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
0 1
Methyla0on
Unmethylated Methylated
DNA
methyla2on
level
met-­‐QTLs
Individuals
AA
AG
GG
Genotype
1
Grundberg
et
al.
AJHG
2013;

26. DNA
methyla0on
QTLs
(meQTLs)
Genes
DNA
methyla0on
‣
Blood
samples
from
188
female
twins
‣
370,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
Distance from the CpG site (kb)
ProportionofmeQTL(1kbbins)
0.000.020.040.060.080.100.12
−100 −50 0 50 100
Distance
to
CpG
(kb)
ProbabilitythatSNPismeQTL
Genome-wide meQTLs
(FDR = 5%)
cis-meQTL (7e-5) 14,206
trans-meQTL (8e-10) 460
➡Cis
meQTLs
are
<5kb
away
from
CpGs
~3.9%
of
probes
‣
Adipose
0ssue
from
649
female
twins1
‣
424,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
Genome-wide meQTLs1
cis-meQTL 36,139
~10%
of
probes
from th
jects an
of metQ
was sign
restricte
or metQ
TVS me
and 52%
whole b
have be
among
metQTL
the rep
those e
sites and
is show
Distance from the probe (kb)
Frequency
−100 −80 −60 −40 −20 0 20 40 60 80 100
02,0004,0006,0008,00010,000
Figure 4. Distribution of Top SNPs Associated with the Probe
We performed metQTL analysis by associating methylation levels
with common sequence variants (MAF > 0.05) located close to the
Distance
to
CpG
(kb)
ProbabilitythatSNPismeQTL
➡Cis
meQTLs
are
<5kb
away
from
CpGs
1
Grundberg
et
al.
AJHG
2013;

27. DNA
methyla0on
QTLs
(meQTLs)
across
0ssues
Genes
DNA
methyla0on
‣
Blood
samples
from
188
female
twins
‣
370,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
Distance from the CpG site (kb)
ProportionofmeQTL(1kbbins)
0.000.020.040.060.080.100.12
−100 −50 0 50 100
Distance
to
CpG
(kb)
ProbabilitythatSNPismeQTL
Genome-wide meQTLs
(FDR = 5%)
cis-meQTL (7e-5) 14,206
trans-meQTL (8e-10) 460
➡Cis
meQTLs
are
<5kb
away
from
CpGs
~3.9%
of
probes
‣
Adipose
0ssue
from
649
female
twins
‣
424,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
Genome-wide meQTLs1
cis-meQTL 36,139
~10%
of
probes
from th
jects an
of metQ
was sign
restricte
or metQ
TVS me
and 52%
whole b
have be
among
metQTL
the rep
those e
sites and
is show
Distance from the probe (kb)
Frequency
−100 −80 −60 −40 −20 0 20 40 60 80 100
02,0004,0006,0008,00010,000
Figure 4. Distribution of Top SNPs Associated with the Probe
We performed metQTL analysis by associating methylation levels
with common sequence variants (MAF > 0.05) located close to the
Distance
to
CpG
(kb)
ProbabilitythatSNPismeQTL
➡Cis
meQTLs
are
<5kb
away
from
CpGs
1
Grundberg
et
al.
AJHG
2013;
3
Shi
et
al.
2014
Nat
Commun
➡
30%
of
CpGs
with
me-­‐QTLs
overlap
across
0ssues
Blood
(14,206)
Adipose2 Lung3
1,722
5,7226,758
8,45171,116
4,255
20,131

28. DNA
methyla0on
QTLs
(meQTLs)
1
UK10K
project.
2
Grundberg
et
al.
2013
AJHG;
3
Shi
et
al.
2014
Nat
Commun
Genes
DNA
methyla0on
‣
Whole
blood
samples
from
188
female
twins
‣
370,000
methyla0on
sites
&
>3M
genotypes
‣
Genotype-­‐methyla0on
associa0ons
Distance from the CpG site (kb)
ProportionofmeQTL(1kbbins)
0.000.020.040.060.080.100.12
−100 −50 0 50 100
Distance
to
CpG
ProbabilitythatSNPismeQTL
Genome-wide meQTLs
(FDR = 5%)
cis-meQTL (7e-5) 14,206
trans-meQTL (8e-10) 460
➡Cis
meQTLs
are
located
near
CpGs
~3.9%
of
probes
➡
CpGs
with
me-­‐QTLs
overlap
across
0ssues
Blood
14,206
Adipose2 Lung3
1,722
5,7226,758
8,45171,116
4,255
20,131
A
propor0on
(4%-­‐10%)
of
CpG-­‐sites
exhibit
strongly
heritable
effects
with
evidence
for
meQTLs,
predominantly
in
cis.
At
many
of
these
regions
gene0c
effects
are
shared
across
0ssues
(30%
of
regions).
5-­‐25%
of
meQTLs
are
also
eQTLs,
depending
on
study.

29. Consequences
DNA
methyla2on
varia2on
Gene0c
and
environmental
impacts
on
DNA
methyla0on
variability
Causes
External
Environment
Germline
Genes Environment
Complex
Phenotypes
Gene
Expression
2

30. Environmental
Epigene0cs
in
Humans
Tobacco
smoking
Snuff
Diet
Stress
Alcohol
consump0on
Exercise
Pathogen
infec0on
UV
radia0on
Sunlight
Genome-­‐wide
studies Candidate-­‐gene
studies
Air
pollu0on
Lead
&
arsenic
Pes0cides
Benzene
PAHs
Organic
chemicals
Season
of
concep0on
…
1
Nat
Rev
Genet
13:97
2011;
Nat
Rev
Genet
8:
253
(2007);
Many
recent
references
(2014)

31. or# or#
MZ#twin#pair# Unrelated#individuals#
Common#
placenta#
Common#
amnion#
Shared#in#utero#environment#
+ 3#
Gene5cs#
In#utero#
environment#
Age#&#Sex#
Early3life#
environment#
Adult3life#
environment#
Similari5es# Differences#
MZ
twins:
Ideal
study
design
for
environmental
risk?

32. • Tobacco
Smoking
is
a
major
risk
factor
in
disease
• Mul0ple
EWAS
for
smoking
in
whole
blood:
>25
smoking
differen0ally
methylated
regions
(s-­‐DMRs)
iden0fied
and
replicated,
with
top
hits
in
AHRR,
F2RL3,
GFI1,
2q37
• S-­‐DMRs
also
observed
in
newborns
from
mothers
who
smoked
during
pregnancy
• Few
studies
in
samples
other
than
blood,
or
on
gene
expression
Environment
DNA
methyla0onSmoking
impacts
the
epigenome
➡Systemic
impacts
of
smoking
on
DNA
methyla0on
and
expression?

33. Environment
DNA
methyla0onSmoking
impacts
the
epigenome
Smoking influences DNA methylation and gene expression in
adipose tissue, and effects are conserved across tissues.
‣
Blood
samples
from
306
female
twins2
‣
26
current-­‐
,
94
ex-­‐,
186
non-­‐smokers
‣
377,000
methyla0on
sites
‣
smoking-­‐EWAS
‣117,000
exons
RNA-­‐seq
‣
smoking-­‐TWAS
(Transcriptome-­‐WAS)
‣
Analyses
account
for:
‣Methyla0on:
chip,
posi0on
on
chip,
BS
conversion
‣RNAseq:
mean
GC,
primer
index
‣age,
BMI,
family,
zygosity
‣no
probes
with
SNPs
&
mul0ple
alignments
1
Grundberg
et
al.
2013
AJHG;
2
Tsaprouni
et
al.
Epigene0cs,
in
press;
‣
Analyses
account
for:
‣Methyla0on:
chip,
posi0on
on
chip,
BS
conversion,
blood
cell
subtypes
(FACS)
‣RNAseq:
mean
GC,
primer
index
‣age,
BMI,
family,
zygosity
‣no
probes
with
SNPs
&
mul0ple
alignments
‣
Adipose
0ssue
from
349
female
twins1
‣
35
current-­‐,
128
ex-­‐,
186
non-­‐smokers
‣
396,000
methyla0on
sites
‣
smoking-­‐EWAS
‣119,000
exons
RNA-­‐seq
‣
smoking-­‐TWAS
(Transcriptome-­‐WAS)

34. GFI1
NOTCH1
LRP5
CYP1A1
C14orf43
F2RL3
CYP1B1
2q37.1
AHRR
Environment
DNA
methyla0on
39
CpGs
(23
genes
and
2
intergenic
regions)
at
FDR=5%
P=4.7e-­‐6Methyla2on
Markers
of
smoking
in
adipose
0ssue

35. Environment
DNA
methyla0on
39
CpGs
(23
genic
and
2
intergenic
regions)
at
FDR=5%
P=4.7e-­‐6
48
exons
(35
unique
genes)
at
FDR=5%
P=2.05e-­‐5
GFI1
NOTCH1
LRP5
CYP1A1
C14orf43
F2RL3
CYP1B1
2q37.1
AHRR
Methyla2on
RNAseq
Markers
of
smoking
in
adipose
0ssue

36. F2RL3
CYP1B1
AHRR
CYTL1
Environment
DNA
methyla0on
4 regions overlap: regulatory effects?
Hypo-­‐methylated
Up-­‐regulated
Methyla2on
RNAseq
Markers
of
smoking
in
adipose
0ssue

37. F2RL3
CYP1B1
AHRR
CYTL1
Environment
DNA
methyla0on
4 regions overlap: regulatory effects?
Hypo-­‐methylated
Up-­‐regulated
Methyla2on
RNAseq
Markers
of
smoking
in
adipose
0ssue
Methylation
Smoking
Expression
Smoking
Methylation
Expression
Methylation
Smoking
Expression
A: Smoking affects methylation which
modulates gene expression
C: Smoking affects methylation and gene
expression independently
B: Smoking affects gene expression
which modulates methylation

38. coMET:
a
regional
epigenome-­‐wide
associa2on
scan
viewer1
epigen.kcl.ac.uk/comet
Annotation
features
Co-methylation
patters
1Mar0n
et
al.,
submiTed
AHRR
Chromosome 5
301291 441406
301291 441406
0
1
2
3
4
5
6
7
8
9
−log(p−value)
●
●
● ●
●
●
●●●●
●
● ●●
●
●●●
●
●
●
●
●
●
●
●●
●
●
●
● ●●●
●
●
●
●●
●● ●●●
●
●
●
●●●●
●●
●
●
●
●
●
●
●
●● ● ●
●
●●●●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●
●●● ●
cg19405895
CpG
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
Correlation Matrix Map Type: spearman
Physical Distance: 140.1 kb
1 0.6 0.2 −0.2 −0.6 −1
AHRR
CTD−2228K2.2
PDCD6 CTD−2228K2.1 PDCD6
AHRR
CTD−2228K2.2
CTD−2228K2.1
Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
AHRR
Chromosome 5
301291 441406
301291 441406
0
1
2
3
4
5
6
7
8
9
−log(p−value)
●
●
● ●
●
●
●●●●
●
● ●●
●
●●●
●
●
●
●
●
●
●
●●
●
●
●
● ●●●
●
●
●
●●
●● ●●●
●
●
●
●●●●
●●
●
●
●
●
●
●
●
●● ● ●
●
●●●●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●
●●● ●
cg19405895
CpG
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
Correlation Matrix Map Type: spearman
Physical Distance: 140.1 kb
1 0.6 0.2 −0.2 −0.6 −1
AHRR
CTD−2228K2.2
PDCD6 CTD−2228K2.1 PDCD6
AHRR
CTD−2228K2.2
CTD−2228K2.1
Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
AHRR
Chromosome 5
301291 441406
301291 441406
0
1
2
3
4
5
6
7
8
9
−log(p−value)
●
●
● ●
●
●
●●●●
●
● ●●
●
●●●
●
●
●
●
●
●
●
●●
●
●
●
● ●●●
●
●
●
●●
●● ●●●
●
●
●
●●●●
●●
●
●
●
●
●
●
●
●● ● ●
●
●●●●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●
●●● ●
cg19405895
CpG
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
Correlation Matrix Map Type: spearman
Physical Distance: 140.1 kb
1 0.6 0.2 −0.2 −0.6 −1
AHRR
CTD−2228K2.2
PDCD6 CTD−2228K2.1 PDCD6
AHRR
CTD−2228K2.2
CTD−2228K2.1
Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
Fat data − GATA4 gene − 55 probes
Chromosome 8
11550345 11579113
11550345 11579113
0
3
6
9
12
15
18
21
−log(p−value)
●●
●● ● ●
●
●
●
●
●●
●
●
● ●
●●
●●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
● ● ●
cg25216696●
Discovery●
cg03579045
cg25945788
cg26020513
cg13434842
cg19172575
cg18119192
cg01546563
cg20279283
cg11981599
cg09626984
cg24646414
cg06704518
cg25216696
cg14487356
cg24738627
cg03007522
cg00090147
cg21073927
cg18283386
cg14666113
cg00397986
cg09951349
cg02726121
cg14379719
cg10866709
cg09130043
cg05412333
cg06707081
cg00595237
cg04233840
cg13166213
cg16795466
cg13680188
cg08379586
cg22320962
cg21227848
cg19971388
cg01182865
cg02146032
cg26006870
cg09098624
cg12117141
cg16441259
cg01098320
cg03024537
cg04167595
cg05237485
cg03333634
cg16844941
cg02119091
cg20908789
cg27625887
cg06558050
cg01509065
cg16015276
cg03579045
cg25945788
cg26020513
cg13434842
cg19172575
cg18119192
cg01546563
cg20279283
cg11981599
cg09626984
cg24646414
cg06704518
cg25216696
cg14487356
cg24738627
cg03007522
cg00090147
cg21073927
cg18283386
cg14666113
cg00397986
cg09951349
cg02726121
cg14379719
cg10866709
cg09130043
cg05412333
cg06707081
cg00595237
cg04233840
cg13166213
cg16795466
cg13680188
cg08379586
cg22320962
cg21227848
cg19971388
cg01182865
cg02146032
cg26006870
cg09098624
cg12117141
cg16441259
cg01098320
cg03024537
cg04167595
cg05237485
cg03333634
cg16844941
cg02119091
cg20908789
cg27625887
cg06558050
cg01509065
cg16015276
Correlation Matrix Map Type: spearman
Physical Distance: 28.6 kb
1 0.6 0.2 −0.2 −0.6 −1
1 2 3 4 5
GATA4Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
AHRR
Chromosome 5
301291 441406
301291 441406
0
1
2
3
4
5
6
7
8
9
−log(p−value)
●
●
● ●
●
●
●●●●
●
● ●●
●
●●●
●
●
●
●
●
●
●
●●
●
●
●
● ●●●
●
●
●
●●
●● ●●●
●
●
●
●●●●
●●
●
●
●
●
●
●
●
●● ● ●
●
●●●●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●
●●● ●
cg19405895
CpG
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
Correlation Matrix Map Type: spearman
Physical Distance: 140.1 kb
1 0.6 0.2 −0.2 −0.6 −1
AHRR
CTD−2228K2.2
PDCD6 CTD−2228K2.1 PDCD6
AHRR
CTD−2228K2.2
CTD−2228K2.1
Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
AHRR
Chromosome 5
301291 441406
301291 441406
0
1
2
3
4
5
6
7
8
9
−log(p−value)
●
●
● ●
●
●
●●●●
●
● ●●
●
●●●
●
●
●
●
●
●
●
●●
●
●
●
● ●●●
●
●
●
●●
●● ●●●
●
●
●
●●●●
●●
●
●
●
●
●
●
●
●● ● ●
●
●●●●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●
●●● ●
cg19405895
CpG
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
Correlation Matrix Map Type: spearman
Physical Distance: 140.1 kb
1 0.6 0.2 −0.2 −0.6 −1
AHRR
CTD−2228K2.2
PDCD6 CTD−2228K2.1 PDCD6
AHRR
CTD−2228K2.2
CTD−2228K2.1
Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
AHRR
Chromosome 5
301291 441406
301291 441406
0
1
2
3
4
5
6
7
8
9
−log(p−value)
●
●
● ●
●
●
●●●●
●
● ●●
●
●●●
●
●
●
●
●
●
●
●●
●
●
●
● ●●●
●
●
●
●●
●● ●●●
●
●
●
●●●●
●●
●
●
●
●
●
●
●
●● ● ●
●
●●●●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●
●●● ●
cg19405895
CpG
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
cg05842815
cg26954197
cg21972741
cg16336872
cg14448919
cg22951524
cg10841124
cg11610050
cg18541609
cg08556107
cg26850624
cg16172278
cg16577724
cg05516328
cg07448928
cg24891125
cg14684960
cg00976097
cg12251573
cg09478603
cg26076054
cg08385200
cg07599136
cg24955955
cg19405895
cg00401753
cg22937882
cg01141993
cg11557553
cg24064903
cg02385153
cg08238319
cg08491376
cg05597431
cg19039843
cg05527650
cg14647125
cg17401179
cg22698028
cg03604011
cg17248487
cg21161138
cg16219322
cg11894422
cg19442702
cg25648203
cg04551776
cg17287155
cg14817490
cg18615970
cg15168497
cg26320890
cg06678548
cg04021706
cg05601199
cg18205372
cg01097768
cg26703534
cg24130459
cg04141806
cg08714121
cg22103736
cg05575921
cg23576855
cg14744022
cg04202140
cg01899089
cg12202185
cg11902777
cg23916896
cg03991871
cg12806681
cg05521499
cg15179499
cg23953254
cg17048538
cg00629928
cg02088390
cg05655106
cg24980413
cg04135110
cg24688690
cg16371648
cg09338136
cg14714797
cg09078014
cg03891523
cg16995193
cg02356223
cg25004427
cg08606254
cg23067299
cg17924476
cg01970407
cg12961784
cg06802630
cg07137034
cg16896326
cg09470163
cg11554391
cg14453201
cg00300637
cg13404472
cg02527419
cg18584368
cg11148817
cg12845747
cg09854184
cg08519949
cg03561637
cg13707777
cg16081854
cg06605558
cg17386114
cg03569073
cg06047773
cg20554397
cg07780979
Correlation Matrix Map Type: spearman
Physical Distance: 140.1 kb
1 0.6 0.2 −0.2 −0.6 −1
AHRR
CTD−2228K2.2
PDCD6 CTD−2228K2.1 PDCD6
AHRR
CTD−2228K2.2
CTD−2228K2.1
Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
Fat data − GATA4 gene − 55 probes
Chromosome 8
11550345 11579113
11550345 11579113
0
3
6
9
12
15
18
21
−log(p−value)
●●
●● ● ●
●
●
●
●
●●
●
●
● ●
●●
●●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
● ● ●
cg25216696●
Discovery●
cg03579045
cg25945788
cg26020513
cg13434842
cg19172575
cg18119192
cg01546563
cg20279283
cg11981599
cg09626984
cg24646414
cg06704518
cg25216696
cg14487356
cg24738627
cg03007522
cg00090147
cg21073927
cg18283386
cg14666113
cg00397986
cg09951349
cg02726121
cg14379719
cg10866709
cg09130043
cg05412333
cg06707081
cg00595237
cg04233840
cg13166213
cg16795466
cg13680188
cg08379586
cg22320962
cg21227848
cg19971388
cg01182865
cg02146032
cg26006870
cg09098624
cg12117141
cg16441259
cg01098320
cg03024537
cg04167595
cg05237485
cg03333634
cg16844941
cg02119091
cg20908789
cg27625887
cg06558050
cg01509065
cg16015276
cg03579045
cg25945788
cg26020513
cg13434842
cg19172575
cg18119192
cg01546563
cg20279283
cg11981599
cg09626984
cg24646414
cg06704518
cg25216696
cg14487356
cg24738627
cg03007522
cg00090147
cg21073927
cg18283386
cg14666113
cg00397986
cg09951349
cg02726121
cg14379719
cg10866709
cg09130043
cg05412333
cg06707081
cg00595237
cg04233840
cg13166213
cg16795466
cg13680188
cg08379586
cg22320962
cg21227848
cg19971388
cg01182865
cg02146032
cg26006870
cg09098624
cg12117141
cg16441259
cg01098320
cg03024537
cg04167595
cg05237485
cg03333634
cg16844941
cg02119091
cg20908789
cg27625887
cg06558050
cg01509065
cg16015276
Correlation Matrix Map Type: spearman
Physical Distance: 28.6 kb
1 0.6 0.2 −0.2 −0.6 −1
1 2 3 4 5
GATA4Genes ENSEMBL
CG Island
Broad ChromHMM
DNase Clusters
SNP UCSC
AHRR

39. F2RL3
1
Tsaprouni
et
al.
Epigene0cs,
in
press;
Environment
DNA
methyla0on
GFI1
2q37.1
AHRR
4 regions overlap: tissue shared effects
Adipose
Blood1
Robust
markers
of
smoking
in
blood
and
fat

40. Methyla2on
markers
of
smoking
in
3
previously
iden2fied2
genes.
Current
smoker
Never
smoked
12 MZ pairs
‣
Illumina
450k
profiles
(1.7%
CpGs)
‣
Blood
(correc0on1
for
cell
heterogeneity)
Chromosome
ATAD3B
2q37.1 AHRR
GNG12 KLHL29
PCGF3
AHRR INTS1
KCNQ1 ETS2
1""""""""""""""""""""""""""""2"""""""""""""""""""""""""3"""""""""""""""""""""4"""""""""""""""""""""5"""""""""""""""""""6""""""""""""""""""7"""""""""""""""""8""""""""""""""""9""""""""""""""10"""""""""""""11""""""""""""12"""""""""""13""""""""""14"""""""""15""""""""16""""""17"""""18"""""19""""20""""21"""22"
-­‐log(P-­‐value)
FDR
25%
P=2e-­‐6
FDR
90%
P=1e-­‐4
1
Houseman
et
al.
BMC
Bioinforma0cs
2012;
2
Shenker
et
al.
HMG
2012;
Besingi&Johansson
HMG
2013;
Philibert
et
al.
Clin
Epigenet
2013;
Sun
et
al.
Hum
Genet
2013;
Zeilinger
et
al.
PLoS
One
2013;
EllioT
et
al.
Clin
Epigenet
2014;
(ALPPL2)
Markers
iden0fied
in
12
smoking-­‐discordant
MZ
pairs
Environment
DNA
methyla0on

41. Methyla2on
markers
of
smoking
in
3
previously
iden2fied2
genes.
Current
smoker
Never
smoked
12 MZ pairs
‣
Illumina
450k
profiles
(1.7%
CpGs)
‣
Blood
(correc0on1
for
cell
heterogeneity)
Chromosome
ATAD3B
2q37.1 AHRR
GNG12 KLHL29
PCGF3
AHRR INTS1
KCNQ1 ETS2
1""""""""""""""""""""""""""""2"""""""""""""""""""""""""3"""""""""""""""""""""4"""""""""""""""""""""5"""""""""""""""""""6""""""""""""""""""7"""""""""""""""""8""""""""""""""""9""""""""""""""10"""""""""""""11""""""""""""12"""""""""""13""""""""""14"""""""""15""""""""16""""""17"""""18"""""19""""20""""21"""22"
-­‐log(P-­‐value)
FDR
50%
P=2e-­‐6
FDR
90%
P=1e-­‐4
1
Houseman
et
al.
BMC
Bioinforma0cs
2012;
2
Shenker
et
al.
HMG
2012;
Besingi&Johansson
HMG
2013;
Philibert
et
al.
Clin
Epigenet
2013;
Sun
et
al.
Hum
Genet
2013;
Zeilinger
et
al.
PLoS
One
2013;
EllioT
et
al.
Clin
Epigenet
2014;
(ALPPL2)
Markers
iden0fied
in
12
smoking-­‐discordant
MZ
pairs
Environment
DNA
methyla0on
Smoking-­‐DMRs
in
adipose
0ssue:
adipose-­‐specific
and
0ssue-­‐shared
effects
Smoking
influences
not
only
methyla0on,
but
also
gene
expression
Smoking
should
be
included
as
a
covariate
in
EWAS
Use
of
methyla0on
markers
as
a
proxy
of
smoking
in
large-­‐scale
epidemiological
studies

42. Consequences
DNA
methyla2on
varia2on
Epigenome-­‐wide
Associa0on
Scans
(EWAS)
Causes
External
Environment
Germline
Genes Environment
Complex
Phenotypes
Gene
Expression 3

43. Consequences
DNA
methyla2on
varia2on
Epigenome-­‐wide
Associa0on
Scans
(EWAS)
Considera0ons
Causes
External
Environment
Germline
Genes Environment
Complex
Phenotypes
Gene
Expression
Functional annotation
EWAS study design
& power
Methylation assay:
coverage vs sensitivity
Time
Cause or Consequence?
Validation & ReplicationCell and Tissue
specificity

44. Consequences
DNA
methyla2on
varia2on
Epigenome-­‐wide
Associa0on
Scans
(EWAS)
Considera0ons:
Study
design
Causes
External
Environment
Germline
Genes Environment
Complex
Phenotypes
Gene
Expression
vs
vs
Case-­‐control
T0 T1 T2
Discordant
MZ
twins
Longitudinal
samples

45. EWAS
Methods
and
considera0ons
• EWAS
Study
designs:
tailored
to
research
ques0on
• Tissue
specificity:
disease
relevance/cell
heterogeneity
• Bias
and
covariates:
approaches
to
correct
for
artefacts
• Sta0s0cal
analysis:
test
sta0s0c/signal
distribu0on
• Mul0ple
tes0ng
correc0on
• Valida0on
using
independent
assay
• Replica0on
in
an
independent
sample
• Longitudinal
follow
up
to
establish
reversibility
• Func0onal
follow
up
to
establish
func0onal
relevance

46. EWAS
Examples
• Type
1
Diabetes,
SLE
• Type
2
Diabetes
• Obesity
• Alzheimer’s
disease
• Bipolar
disorder
• Depression
• Au0sm
• Pain
sensi0vity
See
further
reading
list

47. Conclusions
• Epigene0cs
represents
a
set
of
regulatory
mechanisms
that
can
be
stable,
heritable,
yet
also
reversible
• Epigene0c
changes
can
be
under
gene0c
control,
but
also
influenced
by
environmental
factors
• Epigene0c
changes
have
been
linked
to
complex
disease
• Epigene0cs
can
complement
and
extend
previous
genome-­‐wide
gene0c
studies
to
help
us
understand
molecular
mechanisms
in
complex
disease.

48. Further
Reading
• DNA
methyla0on
assays
• Laird
Nat
Rev
Genet.
2010
Mar;11(3):191-­‐203.
doi:
10.1038/nrg2732
• Heritability
• Visscher
et
al
Nature
Reviews
Gene0cs
12,
529-­‐541
doi:10.1038/nrg3000
• EWAS
overviews
• Rakyan
et
al
Nature
Reviews
Gene0cs
12,
529-­‐541
doi:10.1038/nrg3000
• Tsai
et
al.
Epigenomics.
2012
Oct;4(5):511-­‐26.
doi:
10.2217/epi.12.45.
• Some
EWAS
examples:
• Bell
et
al.
Nature
Commun
2014;
Liu
et
al.
Nature
Biotech
2013;
Rakyan
et
al.
PLoS
Gene0cs
2011;
Bell
et
al.
PLoS
Gene0cs
2012;
Dempster
et
al.
Hum
Molec
Genet
2011;