7. Defining
Epigene*cs
Genome
DNA
§ Reversible
changes
in
gene
expression/func5on
§ Without
changes
in
DNA
Chroma*n
sequence
Epigenome
§ Can
be
inherited
from
precursor
cells
Gene
Expression
§ Allows
to
integrate
intrinsic
with
environmental
signals
Phenotype
(including
diet)
8. DNA Methylation Differentiates Totipotent Embryonic
Stem Cells from Unipotent Adult Stem Cells!
Alex Meissner, Henry Stewart Talks
15. Epigene*c
Changes
are
Important
in
Causing
Cancer
GENETIC
EPIGENETIC
Example:
Example:
Replica*on
errors
Chroma*n
modifica*on
errors
X
X
Altered
Altered
DNA
sequence
chroma*n
structure
Oncogenesis
Altered
Altered
levels
of
DNA/mRNA/proteins
mRNA/proteins
Tumor
16. Example
of
Methyla*on
vs
Muta*on:
Colon
&
Breast
Cancer
120
100
80
60
Dx
40
20
CDx
0
Methylated
Mutated
Source:
Schuebel
et
al
2007
76-‐100
51-‐75
21-‐50
1-‐20
17. MGMT
Biology
O6
Methyl-‐Guanine
Methyl
Transferase
Essen5al
DNA
Repair
Enzyme
Removes
alkyl
groups
from
damaged
guanine
bases
Healthy
individual:
-‐
MGMT
is
an
essen5al
DNA
repair
enzyme
Loss
of
MGMT
ac5vity
makes
individuals
suscep5ble
to
DNA
damage
and
prone
to
tumor
development
Glioblastoma
pa*ent
on
alkylator
chemotherapy:
-‐
Pa5ents
with
MGMT
promoter
methyla5on
show
have
longer
PFS
and
OS
with
the
use
of
alkyla5ng
agents
as
chemotherapy
18. MGMT
Promoter
Methyla*on
Predicts
Benefit
form
DNA-‐Alkyla*ng
Chemotherapy
Post-‐hoc
subgroup
analysis
of
Temozolomide
Clinical
trial
with
primary
glioblastoma
pa5ents
show
benefit
for
pa5ents
with
MGMT
promoter
methyla5on
Median
Overall
Survival
25
21.7 months
20 plus
temozolomide
15
12.7 months
radiotherapy
10
radiotherapy
5
Adapted
from
Hegi
et
al.
NEJM
2005
0 352(10):1036-‐8.
Non-‐Methylated
Methylated
Study
with
207
pa5ents
MGMT
Gene
MGMT
Gene
32. Next_next
miRNA,
(l)ncRNA,
CIS/TRANS
splicing,
SV,
fusion
loci
,
bidirec*onal
promoters
?
RNA_seq:
sequence
RNA
molecules
Next
Gen
Pla`orm
Total
RNA_seq:
all
RNA
molecules
(normalisa*on
procedure)
Direc*onal
Total
RNA_seq:
before
amplifica*on
use
different
5’
and
3’
adaptors
Integrated
Direc*onal
Total
RNA_seq:
Combine
with
other
datasets
eg.
enrichment
sequencing
data,
visualise
and
query
in
genome
browser
32
38. GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25%
50%
25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense
methylated
needed
for
transcrip5onal
silencing
Are
there
alleles
with
all
three
posi.ons
methylated
?
39. Deep
Sequencing
unmethylated
alleles
methylated
alleles
less
methyla5on
more
methyla5on
GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT!
41. Conclusion
Combina5on
of
different
sequencing
techniques
is
emerging
as
best
prac5ce
Bioinforma5cs
is
challenging
§ Methods
for
normalisa5on
under
construc5on
§ Reference
databases
are
generated
Data
visualiza5on
and
integra5on
is
key
41