DNA Methylation and Application in Forensic Sciences

Abstract

DNA methylation of cytosine residues is a stable epigenetic alteration, beginning as early as foetal development in the uterus and continuously evolving throughout life. DNA methylation as well as other epigenetic modifications such as chromatin remodelling and histone modifications are indispensable in mammalian development. Methylation is to a large extent influenced by the ageing process, diets and lifestyle choices. Our understanding of this crucial modification may even contribute to the treatment and prevention of age-related illnesses in the very near future. Genome-wide methylation analysis using high throughput DNA technologies has discovered numerous differentially methylated regions (tDMRs) which differ in levels of methylation in various cell types and tissues. TDMRs have been useful in various applications, particularly medicine and forensic sciences. Forensic scientists are constantly seeking exciting and novel methods to aid in the reconstruction of crime scenes, and the analysis of tDMRs represents a new and reliable technique to identify biological fluids and tissues found at the scene of a violent act. Not only has research been able to unequivocally identify various fluids and tissues, but methods to determine the sex, age and phenotype of donors has been developed. New tDMRs in genes are being searched for consistently to serve as novel markers in forensic DNA analysis.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Full text

Review
Article
DNA
methylation
and
application
in
forensic
sciences
Farzeen
Kader,
Meenu
Ghai *
Department
of
Genetics,
School
of
Life
Sciences,
University
of
KwaZulu
Natal
–
Westville
Campus,
Private
Bag
X
54001,
Durban,
KwaZulu
Natal,
South
Africa
Contents
1.
Epigenetics
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256
2.
DNA
methylation
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256
2.1.
DNA
methylation
and
gene
expression
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256
3.
Environmental
influences
and
DNA
methylation
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3.1.
Age
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3.2.
Nutrition
and
diets
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3.3.
Life
experiences
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258
4.
Differential
DNA
methylation
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4.1.
CpG
island
methylation
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5.
Differential
DNA
methylation:
application
in
forensic
sciences.
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5.1.
Verification
of
DNA
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259
5.2.
Identification
of
biological
fluids/tissues
by
analysis
of
tDMRs
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259
5.3.
Sex
determination
by
analysis
of
DNA
methylation
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261
5.4.
Predicting
age
of
body
fluids
and
tissues;
association
with
disease.
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261
5.5.
Ancestry
informative
markers
of
fluid/tissue
donor
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262
5.6.
Distinguishing
between
monozygotic
twins:
associations
with
sex,
age
and
phenotype
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6.
Conclusion
and
future
outlook
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263
References
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263
Forensic
Science
International
249
(2015)
255–265
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
18
July
2014
Received
in
revised
form
22
January
2015
Accepted
29
January
2015
Available
online
11
February
2015
Keywords:
DNA
methylation
tDMRs
Forensic
science
Body
fluid
identification
A
B
S
T
R
A
C
T
DNA
methylation
of
cytosine
residues
is
a
stable
epigenetic
alteration,
beginning
as
early
as
foetal
development
in
the
uterus
and
continuously
evolving
throughout
life.
DNA
methylation
as
well
as
other
epigenetic
modifications
such
as
chromatin
remodelling
and
histone
modifications
are
indispensable
in
mammalian
development.
Methylation
is
to
a
large
extent
influenced
by
the
ageing
process,
diets
and
lifestyle
choices.
Our
understanding
of
this
crucial
modification
may
even
contribute
to
the
treatment
and
prevention
of
age-related
illnesses
in
the
very
near
future.
Genome-wide
methylation
analysis
using
high
throughput
DNA
technologies
has
discovered
numerous
differentially
methylated
regions
(tDMRs)
which
differ
in
levels
of
methylation
in
various
cell
types
and
tissues.
TDMRs
have
been
useful
in
various
applications,
particularly
medicine
and
forensic
sciences.
Forensic
scientists
are
constantly
seeking
exciting
and
novel
methods
to
aid
in
the
reconstruction
of
crime
scenes,
and
the
analysis
of
tDMRs
represents
a
new
and
reliable
technique
to
identify
biological
fluids
and
tissues
found
at
the
scene
of
a
violent
act.
Not
only
has
research
been
able
to
unequivocally
identify
various
fluids
and
tissues,
but
methods
to
determine
the
sex,
age
and
phenotype
of
donors
has
been
developed.
New
tDMRs
in
genes
are
being
searched
for
consistently
to
serve
as
novel
markers
in
forensic
DNA
analysis.
ß
2015
Elsevier
Ireland
Ltd.
All
rights
reserved.
*Corresponding
author.
Tel.:
+27
31
260
8617.
E-mail
addresses:
farzeenkader68@gmail.com
(F.
Kader),
ghai@ukzn.ac.za
(M.
Ghai).
Contents
lists
available
at
ScienceDirect
Forensic
Science
International
jou
r
nal
h
o
mep
age:
w
ww.els
evier
.co
m/lo
c
ate/fo
r
sc
iin
t
http://dx.doi.org/10.1016/j.forsciint.2015.01.037
0379-0738/ß
2015
Elsevier
Ireland
Ltd.
All
rights
reserved.

1.
Epigenetics
Epigenetics
is
a
broad
term
that
is
used
to
describe
various
reversible
modifications
to
the
genome.
The
precise
definition
of
epigenetics
has
baffled
scientists
for
several
years.
On
top
of
the
genetic
code,
the
epigenetic
code
comprises
an
additional
layer
of
information.
Whereas
the
former
provides
a
framework
for
RNA
and
structure
of
protein;
the
epigenetic
code
controls
packaging
of
DNA
as
well
as
gene
regulation
[79,88].
The
term
epigenetics
was
first
introduced
by
Conrad
Waddington
in
early
1940,
who
defined
epigenetics
as
‘‘the
branch
of
biology
which
studies
the
causal
interactions
between
genes
and
their
products
which
bring
the
phenotype
into
being’’
[93,94].
The
definition
of
epigenetics
has
modified
since
then,
with
the
development
of
genetics
research.
According
to
Riggs
et
al.
[74],
the
definition
of
epigenetics
was
‘‘the
study
of
mitotically
and/or
miotically
heritable
changes
in
gene
function
that
cannot
be
explained
by
changes
in
DNA
sequence’’.
Currently,
a
widely
acknowledged
definition
is
the
‘‘study
of
processes
that
produce
a
heritable
phenotype
that
does
not
strictly
depend
on
the
DNA
sequence’’
[54].
Epigenetic
modifications
include
histone
modifications,
DNA
methylation,
chromatin
remodelling
and
non-coding
RNAs;
all
of
which
play
a
pertinent
role
in
regulation
of
gene
expression
devoid
of
changes
in
DNA
sequence
[91,78].
The
molecular
basis
of
epigenetics
is
multiface-
ted
and
principally
involves
alterations
in
the
activation
of
specific
genes.
Furthermore,
chromatin
proteins
in
association
with
DNA
may
be
silenced
or
activated,
thus
ensuring
that
cells
express
only
necessary
genes
required
for
an
activity.
Epigenetic
programming
is
believed
to
begin
as
early
as
foetal
development
in
the
uterus.
As
DNA
is
inherited
from
one
generation
to
the
next,
so
too
are
epigenetic
patterns
preserved
during
cell
division,
yet
modifica-
tions
have
been
observed
over
an
individual’s
lifetime.
These
changes
have
been
found
to
occur
in
response
to
environmental
exposure
and
various
factors
such
as
smoking
and
diet.
Epigenetic
processes
include
imprinting,
reprogramming,
gene
silencing,
X-
chromosome
inactivation
and
carcinogenesis.
In
mammals,
a
vital
cell
function
regulated
by
epigenetic
processes
is
cell
differentia-
tion
wherein
during
embryogenesis,
stem
cells
are
completely
differentiated
[33,88,91].
2.
DNA
methylation
DNA
methylation
is
an
epigenetic
mark
of
paramount
impor-
tance
for
normal
development
in
the
human
genome.
The
loss
of
DNA
methylation
leads
to
apoptosis
or
growth
arrest
in
normal
cells.
DNA
methylation
involves
the
addition
of
a
methyl
group
(–
CH
3
)
at
the
5
0
position
of
cytosine
residues.
Most
DNA
methylation
occurs
in
CpG
dinucleotides,
although
methylation
outside
of
these
dinucleotides
has
been
reported
in
human
DNA
in
recent
years
[56,103].
Methyl-cytosine
was
thought
to
be
as
the
only
chemical
modification
of
the
mammalian
genomic
DNA.
However,
the
existence
of
hydroxymethyl-cytosine
in
mammalian
cells
was
proven
by
Kriaucionis
and
Heintz
[50]
and
Tahiliani
et
al.
[87].
5
0
-
Hydroxymethyl-cytosine
is
an
oxidation
product
of
5
0
-methyl-
cytosine
and
the
conversion
of
5
0
-methyl-cytosine
into
5
0
-
hydroxymethyl-cytosine
could
be
the
first
step
in
a
pathway
leading
towards
DNA
demethylation.
Due
to
its
probable
regulatory
role
in
gene
transcription,
not
unlike
methyl-cytosine,
hydroxymethyl-cytosine
has
been
termed
the
‘sixth
base’
[62,88].
The
entire
human
genome
contains
about
30
million
CpG
dinucleotides;
these
may
exist
in
an
unmethylated
or
methylated
state.
Within
the
genome,
it
is
estimated
that
60–90%
of
CpGs
are
methylated
in
mammals;
unmethylated
CpGs
are
frequently
found
grouped
in
regions
referred
to
as
CpG
islands.
Such
islands
are
300–3000
bp
long
and
have
>55%
GC
content.
They
are
located
at
the
5
0
end
(regulatory
region)
of
human
genes
[67,79,88,91].
The
prominent
property
of
CpG
islands
is
that
they
are
unmethylated
in
germ-line,
and
most
somatic
tissues.
CpG
islands
are
believed
to
be
protected
from
methylation
by
cis-acting
elements;
this
ensures
continued
existence
in
the
presence
of
strong
mutagenic
pressure
of
methyl-cytosine
deamination.
CpG
islands
generally
act
as
strong
promoters
and
are
also
thought
to
serve
as
replication
origins.
Rather
than
on
regions
in
which
majority
of
methylation
is
frequently
found,
most
investigations
on
the
role
of
DNA
methylation
in
mammals
have
focussed
on
CpG
islands
[45,79].
DNA
methyltransferases
are
enzymes
responsible
for
de
novo
methylation
and
maintenance
of
methylation.
(For
details,
refer
to
Supplementary
Information
Section
1.)
2.1.
DNA
methylation
and
gene
expression
A
key
role
of
DNA
methylation
is
to
control
gene
expression;
it
is
long
believed
that
there
is
an
indirect
correlation
between
gene
expression
and
DNA
methylation
of
CpG
island
promoter
regions.
DNA
methylation
has
been
associated
with
silencing
of
gene
expression
and
condensed
nuclease-resistant
heterochromatin.
Majority
of
the
CpG
islands
of
the
inactive
X
chromosome
display
methylation,
and
mono-allelic
methylation
of
imprinted
genes
is
linked
with
mono-allelic
gene
expression.
DNA
methylation
status
of
the
CpG-rich
promoters
of
the
GATA2
(gene
encoding
GATA
binding
protein
2;
transcription
factor)
and
MASPIN
(mammary
serine
protease
inhibitor)
genes
correlates
well
with
gene
silencing
[84,70,37,85,91].
Conversely,
there
have
been
instances
in
which
DNA
methyl-
ation
showed
no
correlation
with
transcriptional
regulation
and
gene
expression,
or
was
even
associated
with
gene
activation.
Straussman
et
al.
[85]
identified
50
loci
where
host
genes
were
expressed
in
the
same
cell-type
only
when
the
regions
were
methylated.
This
was
mainly
observed
in
non-CpG
islands,
possibly
a
result
of
selective
demethylation
of
inactive
genes
[70,37,73,85,91].
Research
by
Walsh
and
Bestor
[95]
to
investigate
the
methylation
status
of
seven
genes
found
no
relationship
with
gene
expression
and
studies
by
Warnecke
and
Clark
[97]
also
found
that
expression
of
skeletal
a
-actin
genes
in
adult
mice
did
not
correlate
with
methylation
status
of
the
promoter.
There
are
various
explanations
for
the
lack
of
correlation
observed.
Several
genes
are
able
to
engender
numerous
transcripts
by
using
other
transcription
start
sites.
Despite
the
high
degree
of
methylation
displayed
by
the
promoter
of
the
PARP12
gene
which
encodes
poly
(ADP-ribose)
polymerase
12,
gene
expression
was
observed,
as
demonstrated
in
the
study
by
Rauch
et
al.
[73],
yet
rapid
amplification
of
cDNA
ends
revealed
transcription
initiation
from
downstream
of
the
methylated
CpG
island,
via
an
intragenic
promoter.
It
has
been
speculated
that
when
intragenic
islands
are
not
associated
with
a
known
transcription
start
site,
the
methylation
status
could
hinder
spurious
body
transcription
which
may
obstruct
correct
expression
of
parent
genes
[37].
Studies
by
Sleutels
et
al.
[82]
provided
evidence
that
intragenic
CpG
islands
are
able
to
localise
to
sites
of
antisense
ncRNA
transcription
initiation;
this
results
in
negative
regulation
of
the
sense
transcripts.
Both
Tsix
and
Air
ncRNA
are
derived
from
CpG
islands
and
partake
in
the
regulation
of
the
sense
transcripts
[82,85].
A
ncRNA
that
is
transcribed
from
the
HOXC
locus
(of
the
Homeobox
super
family),
HOTAIR,
represses
the
HOXD
cluster
in
trans
[75].
In
all
cases
described,
methylation
of
CpG
islands
led
to
the
derepression
of
genes
that
were
silenced
by
ncRNA.
Numerous
hypermethylated
CpG
islands
are
known
to
have
no
regulatory
roles
in
gene
transcription
as
they
are
located
outside
coding
sequences,
in
intergenic
DNA.
Yet,
monoallelic
expression
of
the
H19/IGF2
imprinted
locus
is
determined
by
methylation
of
an
intergenic
CpG
island
that
is
situated
upstream
of
the
H19
gene
that
encodes
a
2.3
kb
spliced,
capped,
and
polyadenylated
long
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
256

noncoding
RNA
(lncRNA)
[10].
The
IGF2
gene
cluster
has
a
gametic
DMR
located
2
kb
upstream
of
the
H19
lncRNA
promoter
that
is
methylated
only
in
the
paternal
gamete
and
is
maintained
thereafter
in
all
somatic
tissues
[7].
The
high
methylation
status
of
the
CpG
island
enhances
expression
of
IGF2
from
the
paternal
allele
[7,28],
whilst
preventing
interaction
with
CTCF,
the
insulator
element.
This
demonstrates
a
possible
mechanism
by
which
the
high
degree
of
methylation
in
an
intergenic
CpG
island
may
induce
transcriptional
effects
[19,85].
These
are
merely
a
few
examples
that
represent
the
high
degree
of
complexity
involved
in
determining
the
transcriptional
effects
that
the
DNA
methylation
status
of
a
CpG
island
has.
Further
information
on
methylation
patterns
of
germ
cells,
embryonic
stem
cells
and
somatic
cells
are
discussed
in
Supplementary
Section
2.
3.
Environmental
influences
and
DNA
methylation
Although
considered
a
stable
epigenetic
modification,
DNA
methylation
is
largely
influenced
by
various
exogenous
and
endogenous
factors
such
as
nutrition
and
diets,
early
life
experiences,
ageing,
exposure
to
pollutants
as
well
as
social
environments.
3.1.
Age
The
relationship
between
environmental
signals
and
epige-
netics
is
not
well
defined
in
mammals;
however
hypo-
and
hypermethylation
have
been
associated
with
ageing
[41,24,17,88].
Studies
that
focus
on
monozygotic
twins
have
defined
links
between
environment
or
ageing
and
long-term
epigenetic
effects
on
phenotype.
Due
to
sharing
the
same
genetic
basis,
monozygotic
twins
serve
as
the
perfect
system
to
study
epigenetics.
During
early
years,
twins
display
similar
methylation
patterns;
however
later
in
life
they
demonstrate
different
amounts
and
patterns
of
methylation
[23,91].
Studies
of
this
particular
locus
by
Pirazzini
et
al.
[69]
led
to
the
discovery
of
2
regions
in
which,
after
60
years
of
age,
twins
displayed
considerable
increase
in
intra-couple
variation.
It
was
observed
that
the
range
of
methylation
values
increased
only
in
IGF2
shore
region
thus
emphasizing
that
the
range
of
variation
in
methylation
depends
on
the
genomic
location
[83].
With
increase
in
age,
DNA
methylation
machinery
tends
to
lose
its
ability
to
maintain
methylation
patterns
across
cellular
divisions
[29].
Variation
in
methylation
of
imprinted
genes,
such
as
the
H19/IGF2
locus
has
also
been
previously
observed
by
Woodfine
et
al.
[101].
Furthermore,
methylation
of
CpG
islands
linked
with
various
genes
such
as
that
encoding
oestrogen
receptor,
MYOD
and
IGF2
was
untraceable
in
young
individuals,
however,
with
age
became
detectable
in
normal
tissue.
The
correlation
between
age
and
DNA
methylation
spurs
questions
regarding
how
epigenetic
altera-
tions
influence
various
tissue
types
over
time.
It
has
been
proposed
that
such
variation
in
DNA
methylation
patterns
could
have
been
a
stochastic
process
of
random
epigenetic
drift
[41,14,9].
Studies
by
Bjornsson
et
al.
[6],
Calvanese
et
al.
[11]
and
Zhang
et
al.
[104]
have
demonstrated
that
with
age,
comes
a
general
genomic
decrease
in
DNA
methylation
with
hypermethy-
lation
of
certain
gene
promoters
within
the
genome.
The
study
by
Zhang
et
al.
[104]
found
that
in
a
population
of
individuals
between
the
ages
of
45
and
75,
ageing
tissues
do
in
fact
demonstrate
a
progressive
decrease
in
methyl-cytosine,
however
although
the
age-related
reduction
in
methylation
was
not
too
significant;
this
could
possibly
have
been
due
to
the
limited
population
size
and
age
range.
Day
and
colleagues
provided
a
few
of
their
own
speculations
to
explain
changes
in
DNA
methylation
with
age.
CpG
methylation
changes
were
observed
in
blood
and
various
other
tissues.
One
possible
explanation
was
that
variation
with
age
may
be
due
to
adult
stem
cell
activity;
stability
of
DNA
methylation
in
mice
was
found
to
be
necessary
for
self-renewal
of
hematopoietic
stem
cells
and
demethylation
of
CpG
sites
was
observed
near
myeloid-specific
genes
associated
with
differenti-
ation.
Another
explanation
involved
changes
in
chromatin
structure
over
time
since
enrichment
of
methylation
with
age
has
been
noted
within
bivalent
or
repressive
chromatin
and
DNTM1
was
found
to
reside
in
hypermethylated
regions
of
transcribed
genes.
3.2.
Nutrition
and
diets
Possibly
ensuing
an
‘epigenetic
diet’,
various
dietary
bioactive
food
components
have
been
observed
to
alter
gene
expression
via
changes
in
DNA
methylation
[32,68].
The
availability
of
the
methyl-donor,
S-adenosylmethionine
(SAM)
is
determined
by
one-
carbon
metabolism.
This
is
a
pathway
that
involves
vitamins
B
6
and
B
12
,
betaine,
folate
and
choline
as
well
as
various
amino
acids
such
as
glycine,
methionine,
serine
and
cysteine.
If
a
component
of
the
pathway
is
missing,
such
as
deficiency
in
B
vitamins,
DNA
methylation
is
altered
[65].
In
addition
to
altering
availability
of
B
vitamins,
alcohol
consumption
causes
wastage
of
choline
and
methionine,
reducing
the
amount
of
SAM
available
and
thereby
altering
DNA
methylation
[59,88].
The
diet
is
found
to
be
an
imperative
determinant
in
the
manifestation
of
late-onset
disease.
Vitamins
and
folates
influence
activity
of
enzymes
that
partake
in
cellular
methylation
processes
and
very
much
influence
the
rate
of
disease
symptoms.
Genomic
instability
and
hypomethylation
is
allied
with
reduced
amounts
of
folates
[40,41].
In
mice,
increased
intake
of
folic
acid
increased
DNA
methylation
of
an
allele
in
the
coat
colour
agouti
locus,
resulting
in
gene
silencing
and
phenotypic
modifications
[99,90].
Additionally,
methyl-deficient
diets
induce
liver
cancers
associated
with
hypomethylation
and
enhanced
expression
of
oncogenes
such
as
c-fos
and
c-ras
[25,41].
Other
bioactive
ingredients
may
have
the
opposite
effects
on
DNA
methylation.
Epigallocatechin-3-gallate,
which
is
a
primary
polyphenol
found
in
green
tea
was
found
to
reduce
global
DNA
methylation
in
cancer
cell
lines
via
competitive
inhibition
of
DNA
methyltransferases.
This
reverses
repression
of
tumour
suppressor
genes
such
as
p16,
O-6-methylguanine-DNA
methyltransferase
and
reversion-inducing-cysteine
rich
protein
with
kazal
motifs
[22,46].
The
red
carotenoid,
lycopene
was
observed
to
have
demethylating
capabilities
in
a
breast
cancer
cell
line
[47].
Seleni-
um,
found
in
some
vegetables
and
grains
alter
DNA
methylation,
as
a
reduction
in
this
mineral
leads
to
decrease
in
global
DNA
methylation
with
reduced
expression
of
DNMT1
(DNA
methyl-
transferase
1)
in
colon
and
prostate
cancer
cell
lines,
and
rat
colon
and
liver
tissue
[106,88].
Maternal
diets
also
affect
the
offspring
phenotypes
and
disease-risks.
Intake
of
B-vitamins
is
linked
to
change
in
the
susceptibility
of
offspring
to
breast
and
colon
cancers
[77,88].
Pro-
tein-restriction
in
rats
has
been
found
to
epigenetically
program
the
metabolism
of
offspring.
With
mothers
that
were
fed
low-
protein
diets,
decreased
methylation
along
with
increased
expression
of
peroxisome
proliferator-activated
receptor
a
in
the
liver
of
pups
has
been
demonstrated
[55].
Although
a
similar
trend
was
observed
for
the
glucocorticoid
receptor
gene,
the
effect
was
lost
in
pups
of
mothers
fed
a
low-protein
high-folate
diet,
demonstrating
influences
of
the
maternal
diet
on
offspring
carbohydrate
and
fat
metabolism.
Effects
on
global
DNA
methylation
in
genomes
of
pigs
has
been
demonstrated;
with
changes
in
expression
of
DNA
methyltransferases
in
liver
and
skeletal
muscles
of
newborn
offspring
being
observed
during
high
and
low
protein
diets
of
maternal
pigs.
Clearly,
instead
of
using
the
term,
‘you
are
what
you
eat’,
the
phrase
‘you
are
what
your
parents
ate’
seems
more
suitable
[1,42,88].
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
257

3.3.
Life
experiences
Adult
risk
factors
such
as
tobacco
smoking
have
also
been
related
with
DNA
methylation
patterns
in
tumour
tissues.
Links
between
psycho-social
factors
such
as
cortisol
output
and
perceived
stress
and
DNA
methylation
have
been
established,
as
was
early
life
socio-economic
status
[89,14,51].
Exposure
to
chemical
and
environmental
pollutants
induces
changes
in
DNA
methylation
without
altering
the
genetic
sequence,
resulting
in
epimutation-associated
phenotypes.
The
anti-androgenic
fungi-
cide
vinclozolin,
which
is
an
endocrine
disruptor,
alters
methylation
patterns
in
sperm;
effects
have
been
shown
to
persist
for
at
least
four
generations
[4].
During
comparative
studies
of
epigenetic
patterns
of
suicide
victims
without
a
history
of
childhood
abuse
to
suicide
victims
with
a
history
of
abuse,
increased
methylation
of
the
promoter
of
nuclear
receptor
subfamily
3
genes
was
found.
This
gene
encodes
neuron-specific
glucocorticoid
receptor,
which
when
stimulated
inhibits
the
hypothalamic–pituitary–adrenal
stress
response.
These
results
were
also
found
in
a
study
of
rats,
where
pups
raised
with
less
grooming
and
licking,
as
well
as
less
arched-
back
nursing
also
demonstrated
altered
stress
response.
‘Cellular
memory’
mechanisms
cause
cells
to
remember
and
maintain
their
chosen
fates,
even
long
after
the
stimulus
is
gone
and
hence
perturbations
at
early
stages
have
long-lasting
effects
[57,90,88].
4.
Differential
DNA
methylation
Despite
the
magnitude
and
purpose
originally
postulated
for
this
vital
epigenetic
mark,
there
is
still
an
immense
lack
of
understanding
of
exact
characteristics
of
DNA
methylation
particularly
in
individual
human
tissues;
necessitating
detailed
analysis
of
tissue-specific
methylation
of
individual
tissues
[36].
Genome-wide
studies
have
revealed
that
DNA
methylation
profiles
are
tissue-specific
and
there
are
several
chromosome
segments
called
tissue-specific
differentially
methylated
regions
(tDMRs)
that
are
known
to
show
varying
methylation
patterns
according
to
tissue
or
cell
type
[72,52,2,13].
4.1.
CpG
island
methylation
As
previously
mentioned,
comprehensive
mapping
of
genomes
revealed
a
lack
of
methylation
in
the
bulk
of
CpG
islands.
However,
studies
have
revealed
that
4–17%
of
CpG
sites
vary
in
methylation
amongst
tissues
and
cellular
processes
[15].
Patterns
of
methyl-
ation
differences
in
CpG
islands
may
be
explained
by
two
not-
necessarily
conflicting
models;
the
hindrance
of
access
to
methylation
sites
caused
by
proteins
that
are
bound
to
particular
DNA
regions
and
sequence-specific
binding
proteins
which
confer
a
methyl-targeting
mechanism.
Evidence
for
hindrance
to
access
of
methylation
sites
has
been
derived
from
research
showing
removal
of
Sp1
binding
sites
flanking
a
CpG
island,
leading
to
its
de
novo
methylation
during
development.
This
might
imply
that
if
the
Sp1
sites
were
occupied
by
the
Sp1
transcription
factors,
DNA
methyltransferases
could
not
gain
access
to
the
relevant
CpG
island.
Furthermore,
modulation
of
DNA
binding
protein
affinity
may
have
a
direct
effect
on
methylation
states
of
sites
to
which
the
proteins
bind
[45].
Several
tDMRs
containing
genes
are
relatively
poor
in
CpG
content.
In
mammals,
the
Sry
gene,
encoding
a
transcription
factor
of
the
major
protein
that
initiates
testis
differentiation,
contains
a
tDMR
with
not
many
CpG
sites.
In
the
mouse,
the
upstream
region
of
the
linear
form
of
the
Sry
gene
contains
a
tDMR
with
merely
8
CpG
sites.
Such
studies
determined
that
demethylation
of
only
minimal
CpG
sites
is
essential
for
spatio-temporal
expression
of
Sry
whilst
these
particular
tDMRs
are
hypermethylated
in
other
tissues
[12,66,67].
TDMRs
are
enriched
at
the
margins
of
CpG
islands
and
both
CpG
and
G/C
content
is
lower
than
that
of
surrounding
regions.
CpG
islands
are
generally
assumed
to
play
the
role
of
developmental
switches;
providing
cells
with
epigenetic
memory
by
generating
cell-type-specific
hypo-
and
hypermethylation
patterns.
These
tDMRs
have
been
implicated
in
indispensible
involvement
of
mammalian
development
and
tissue
differentiation.
Differentially
methylated
regions
are
believed
to
function
by
either
preventing
or
attracting
the
binding
of
specific
factors
in
a
methyl-dependent
manner
[36,67,37,15,91].
Numerous
studies
have
characterised
a
large
number
of
tDMRs
via
comparison
of
DNA
methylation
profiles
amongst
various
cell
lines
and
tissues
[72,14,52,2,17,35,13].
These
tDMRs
are
cate-
gorised
based
on
collective
behaviour
(hypo-
or
hypermethylation)
of
a
group
of
spatially
clustered
CpGs.
Despite
the
fact
that
correlations
of
tDMR
methylation
levels
with
transcriptional
state
have
been
documented,
the
precise
active
regulatory
role
of
methylation
in
tDMRs
is
not
quite
clear.
However,
major
links
between
gene
silencing
and
tDMRs
have
been
established
[67,15,35].
Methods
employed
in
studying
DNA
methylation
are
discussed
in
Supplementary
Section
3.
5.
Differential
DNA
methylation:
application
in
forensic
sciences
DNA
is
most
frequently
used
as
a
biological
source
for
personal
identification
profiling.
Often
merely
a
minute
quantity
of
biological
fluid
is
enough
to
break
a
case
and
ability
to
identify
evidence
at
a
crime
scene
in
a
non-destructive
manner
is
imperative
in
order
to
preserve
the
sample
and
DNA
evidence
for
further
use.
Existing
presumptive
and
confirmatory
tests
are
applicable
to
only
specific
biological
fluids.
The
investigator
would
be
required
to
make
a
choice
as
to
which
test
to
perform,
based
on
which
fluid
is
most
likely
to
be
identified;
necessitating
a
precise
method
in
which
the
fluid
may
be
identified
irrespective
of
the
nature
[92].
PCR-based
techniques
are
used
to
analyse
DNA
from
biological
material.
DNA
databases
are
then
scanned
for
matches
to
DNA
profiles
found
at
crime
scenes.
There
are
countless
commercially
manufactured
DNA
systems
that
have
been
ap-
proved
for
forensic
applications
[76,91,30].
In
particular,
body
fluids
such
as
blood,
saliva,
semen
and
vaginal
fluids
are
often
found
at
crime
scenes;
however
others
such
as
sweat
and
urine
also
serve
as
informative
sources
of
evidence
[64,92,71,100].
Each
of
these
fluids
contains
DNA
and
thus
it
is
imperative
that
great
precautionary
measures
are
implemented
when
handling
such
crucial
aspects
of
investigations.
Several
presumptive
and
confirmatory
tests
have
been
developed
for
body
fluid
identification
[92].
Presumptive
tests
are
used
as
screening
tests
but
tend
to
have
specificity
limitations.
Confirmatory
tests
are
used
for
absolute
tissue
identification
and
can
be
useful
in
reconstructing
the
events
of
a
crime.
Confirmatory
tests
based
on
proteins
and
mRNAs
are
associated
with
lack
of
stability
issues
[107,31].
Since
DNA
methylation
is
the
most
accessible
and
characterised
component
of
the
numerous
chromatin
marks
that
comprise
the
epigenome
and
is
a
specific
chemical
modification
of
an
extremely
stable
biological
macromolecule,
the
DNA
methyl-
ation
status
of
a
chosen
gene
is
an
attractive
detection
and
diagnostic
biomarker;
serving
as
a
perfect
target
for
epigenetic
studies
in
human
populations.
Body
fluid
identification
based
on
DNA
methylation
shows
great
potential,
especially
in
forensic
sciences
where
detection
and
identification
of
body
fluids
present
at
crime
scenes
are
crucial
aspects
of
forensic
investigations.
Establishing
the
identity
and
origin
of
the
body
fluid
helps
to
reconstruct
the
crime
scene
[61,51,52,2,13].
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
258

From
a
forensics
perspective,
analysis
of
methylation
profiles
provides
clues
on
circumstances
leading
to
injury
or
death,
pathological
states
as
well
as
tissue-identification.
Current
super-
ior
technologies
allow
for
methylation
profiles
of
DNA
to
reveal
information
about
how
or
when
biological
fluids
were
deposited
during
crime
scenes,
provide
identification
of
exactly
what
biological
tissue
or
fluid
source
the
sample
originated
from
as
well
as
provide
estimates
of
age,
gender
and
phenotypic
characteristics
of
perpetrators
[91,30].
Analysing
the
methylation
status
of
DNA
is
favourable
as
in
addition
to
demonstrating
high
sensitivity
and
specificity,
simple
extraction
and
purification
methods
may
be
attuned
with
novel
nucleic
acid
technologies.
Since
the
methods
target
only
extracted
DNA,
there
is
minimal
consumption
or
destruction
of
physical
material
which
is
essential
in
criminal
cases,
as
when
sample
quantities
are
limited
forensic
scientists
are
not
faced
with
decisions
of
which
assays
to
perform
for
fear
of
loss
of
crucial
evidence
of
crime
scenes.
Special
training
is
not
required
to
perform
tests
and
irrespective
of
which
samples
or
biological
fluids
are
being
assayed;
standard
protocols
may
be
applied.
A
major
advantage
of
DNA
methylation-based
assays
in
forensic
sciences
is
efficiency
and
convenience;
the
analysis
of
multiple
tissues
in
a
single
assay
without
prior
knowledge
of,
or
decision
making
regarding
the
nature
of
sample
[27,58,2,13,30].
5.1.
Verification
of
DNA
samples
It
is
generally
believed
that
every
trace
of
DNA
found
at
a
crime
scene
is
of
biological
origin,
although
this
is
not
always
the
case.
This
is
quite
disturbing,
especially
since
DNA
evidence
holds
such
heavy
weight
in
the
courtroom.
Frumkin
et
al.
[26]
considered
the
ease
of
fabricating
DNA
evidence
by
criminals,
since
only
basic
equipment,
minimal
financial
expense
and
no
particular
expertise
would
be
necessary.
There
is
an
alarming
possibility
that
DNA
planted
at
crime
scenes
may
be
overlooked
during
forensic
investigations.
By
artificially
synthesizing
blood,
saliva
and
skin,
mock
forensic
samples
were
generated.
A
selection
of
tDMRs,
which
were
known
to
be
methylated
or
unmethylated
at
particular
regions,
were
analysed
in
an
authentication
assay.
Methylation
profiles
of
artificial
DNA
were
generated
using
bisulfite
conversion
followed
by
PCR
amplification,
and
compared
to
natural
human
DNA
samples.
The
research
revealed
that
artificially
synthesised
DNA
is
consistently
unmethylated
in
all
loci
whereas
biological
DNA
displays
methylation
in
some
loci
and
unmethylation
in
others.
This
study,
using
differential
methylation,
provided
a
mechanism
that
enables
forensic
scientists
and
judicial
law
to
convict
the
correct
criminal,
rather
than
one
wrongfully
accused.
Methylation
patterns
of
tDMRs
were
used
to
uphold
the
credibility
of
trace
DNA
located
at
crime
scenes,
which
is
unquestionably
of
extreme
importance
in
forensic
investigations.
5.2.
Identification
of
biological
fluids/tissues
by
analysis
of
tDMRs
It
is
assumed
that
evolutionary
dynamics
of
CpGs,
that
actively
functional
tDMRs
are
found
in,
provide
indications
for
a
selective
signature
[15,35].
Once
established,
DNA
methyltransferases
ensure
that
methylation
patterns
of
tDMRs
remain
fixed.
This
phenomenon
was
indeed
proved
by
Eckhardt
et
al.
[18]
in
a
study
that
described
methylation
profiles
at
tDMRs
to
be
specific
and
stable,
thus
rendering
them
excellent
markers
for
body
tissue
identification.
Hence
it
is
possible
to
resolve
the
identification
of
extracted
DNA
samples
using
epigenetic
markers.
Much
effort
has
been
placed
on
researching
tDMRs
for
forensic
science
applications
by
various
scientists
worldwide.
For
the
purpose
of
this
review,
studies
based
on
only
a
few
frequently
found
biological
fluids
will
be
discussed
in
great
detail.
TDMRs
demonstrate
variation
in
methylation
patterns
accord-
ing
to
tissue/cell/fluid
type.
Such
regions
may
be
unmethylated
in
particular
tissues,
yet
display
varying
degrees
of
methylation
in
others,
hence
providing
distinguishing
characteristics
between
the
tissues
[86,13].
One
of
the
first
fruitful
attempts
at
DNA
methylation-based
tissue
identification
was
by
Frumkin
et
al.
[27]
leading
to
successful
differentiation
between
blood,
saliva,
semen
and
skin
samples.
The
authors
selected
16
loci
that
displayed
considerable
differential
amplification
patterns
and
subjected
a
mere
1
ng
of
DNA
from
samples
to
methylation
sensitive
restriction
enzyme
PCR
(MSRE-
PCR)
followed
by
capillary
electrophoresis.
The
tissue
identification
assay
was
designed
such
that
loci
with
low
degrees
of
methylation
were
amplified
with
low
efficiency
and
demonstrated
weak
signals
on
the
resulting
electropherogram,
loci
with
high
degrees
of
methylation
conversely
displayed
strong
signals.
A
methylation
ratio
for
each
pair
of
differentially
methylated
loci
was
calculated,
allowing
for
discrimination
between
the
biological
fluids.
Precise
identification
of
semen
was
achieved
by
low
methylation
levels
of
L91762
corresponding
with
hypermethylation
of
L68346.
Methyl-
ation
levels
of
L91762
were
much
higher
in
blood,
saliva
and
skin.
A
low
ratio
of
L91762/L68346
was
again
used
to
differentiate
semen
samples
from
other
tissues.
The
methylation
ratios
of
L76138/
L26688
were
higher
in
semen
and
skin
than
in
blood
and
saliva.
Identification
of
skin
epidermis
was
confirmed
by
high
ratios
of
methylation
in
L91762/L68346
as
well
as
L76138/L26688.
The
assay
positively
identified
tissues
from
a
single
source,
as
well
as
those
in
mixtures
as
a
combined
semen
assay
was
also
performed.
Semen
was
mixed
in
varying
ratios
with
urine
and
saliva,
and
the
absence
or
presence
of
semen
was
detected.
In
a
study
by
Madi
et
al.
[58]
bisulfite
modification
and
pyrosequencing
procedures
were
employed
to
identify
new
tDMRs.
Focus
was
directed
at
particular
CpG
sites
in
tandem
as
flanking
sites
may
display
differing
methylation
levels
and
the
methylation
levels
at
single
CpG
sites
were
compared
between
samples.
Various
CpG
sites
of
ZC3H12D,
FGF7,
C20orf117
and
BCAS4
genes
were
analysed
to
display
differential
methylation
profiles
of
semen,
blood,
and
saliva
and
skin
tissue.
ZC3H12D
and
FGF7
successfully
differentiated
semen
from
other
fluids.
All
5
CpG
sites
tested
in
ZC3H12D
displayed
high
methylation
levels
ranging
from
82
to
100%
in
blood,
saliva
and
skin,
but
hypomethylation,
below
12%,
in
sperm.
Presumed
initially
to
distinguish
skin
samples
from
others;
the
FGF7
marker
enabled
identification
of
semen,
with
hypermethylation
relative
to
blood,
saliva
and
skin.
Blood,
in
particular,
white
blood
cells,
was
positively
identified
by
the
C20orf117
locus
due
to
hypermethylation.
High
levels
of
methylation
of
CD4
+
and
CD8
+
lymphocytes
relative
to
skin
and
sperm
were
demonstrated
by
Eckhardt
et
al.
[18].
The
C20orf117
marker
shows
great
prospective
for
the
differentiation
of
blood
from
other
tissues
however,
one
must
consider
that
accurate
differentiation
from
skin
was
inconclusive
in
the
experiment
as
only
one
skin
sample
could
be
amplified
due
to
errors
in
bisulfite
conversions
or
primer
hybridization.
Also
with
this
marker
the
methylation
of
sperm
was
particularly
low
compared
to
other
fluids.
Based
on
the
outcome
of
the
report
by
Eckhardt
and
colleagues,
a
high
level
of
methylation
for
semen
was
expected
in
the
BCAS4
marker.
However
this
result
was
not
reproduced
as
sperm
displayed
less
than
20%
methylation
and
saliva
displayed
highest
methylation;
thus
this
marker
can
be
used
for
saliva
identification
[58].
The
same
group
of
researchers
conducted
a
study
in
2013,
testing
the
same
tDMRs
and
body
fluids.
Analogous
results
were
obtained
for
the
markers;
and
in
addition
the
study
determined
mean
percent
methylation
of
9
year
old
blood
samples,
and
20
year
old
blood
and
semen
samples.
Interestingly,
methylation
patterns
were
observed
to
be
unwavering
over
such
long
periods
of
time;
percent
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
259

methylation
was
the
same
as
samples
collected
at
the
time
of
the
study
[3].
In
a
study
by
Lee
et
al.
[52]
DNA
methylation
profiles
of
blood,
saliva,
semen,
vaginal
fluid
and
menstrual
blood
were
generated
by
a
selection
of
five
tDMRs.
TDMRs
for
USP49
and
DACT1
genes
were
selected
as
semen-specific
markers,
and
tDMRs
for
PFN3,
PRMT2
and
HOXA4
genes
were
chosen
as
blood-specific
markers
as
different
methylation
patterns
in
blood,
spleen
and
brain
tissues
were
observed
in
previous
reports
by
Illingworth
and
colleagues
[38].
Bisulfite
conversion
of
DNA
followed
by
PCR
and
sequencing,
demonstrated
differential
methylation
patterns
in
all
tested
tissues
and
fluids.
DACT1
and
USP49
were
unmethylated
in
over
90%
of
clones
from
semen
and
hypermethylated
in
almost
all
blood,
saliva,
vaginal
fluid
and
menstrual
blood
clones.
The
methylation
profiles
of
these
latter
tissues
and
fluids
did
not
significantly
differ
in
these
tDMRs,
thus
they
may
be
used
to
positively
identify
semen
which
will
aid
terrifically
in
sexual
assault
cases
[52].
In
addition,
the
VASA
tDMR
may
also
be
used
to
identify
semen,
as
it
is
hypomethylated
in
testis
and
demonstrates
high
degrees
of
methylation
in
other
tissues.
Furthermore,
it
is
expressed
solely
in
germ
cells
[86].
The
HOXA4
tDMR
displayed
high
degrees
of
methylation
in
blood
and
female
saliva,
but
there
was
no
considerable
difference
in
these
methylation
patterns
to
allow
accurate
differentiation.
The
HOXA4
tDMR
was
hypo-
methylated
in
vaginal
fluid
and
menstrual
blood,
and
the
PFN3
gene
displayed
methylation
of
65%
of
loci
in
vaginal
fluid
whereas
more
than
80%
methylation
was
observed
in
other
tissues
and
fluids.
PRMT2
was
hypermethylated
in
vaginal
fluid
and
menstrual
blood,
and
demonstrated
great
differences
between
semen
and
vaginal
fluid,
and
semen
and
menstrual
blood.
From
this
data,
the
authors
suggested
that
low
methylation
of
HOXA4,
and
high
methylation
of
PRMT2,
USP49
and
DACT1
may
be
used
to
confirm
the
presence
of
vaginal
fluid
and
menstrual
blood.
For
testing
pooled
DNA
of
semen
and
vaginal
fluid,
the
combined
use
of
DACT1,
USP49,
PRMT2
and
PFN3
genes
may
be
employed.
However
any
issues
regarding
mixtures
of
samples,
which
are
often
the
case
in
real-life
situations,
may
be
alleviated
by
USP49
and
DACT1,
as
methylation
patterns
are
quite
distinct
in
semen
compared
to
the
other
tissues.
The
same
study
also
tested
aged
samples;
all
tissues
and
fluids
were
left
at
ambient
temperatures
for
30
days.
A
methylation-specific
PCR
procedure
revealed
that
all
unmethylation
and
methylation
patterns
remained
consistent
over
this
period
[52].
An
et
al.
[2]
and
Choi
et
al.
[13]
employed
some
of
the
same
tDMRs
for
body
fluid
identification.
An
and
colleagues
demon-
strated
age
related
changes
in
methylation
(discussed
later)
and
methylation
profiles
of
USP49,
DACT1,
PFN3
and
PRMT2
genes
within
blood,
saliva,
semen,
vaginal
fluid
and
menstrual
blood.
In
addition
to
MSRE-PCR,
the
study
also
determined
methylation
profiles
using
a
less
common
method
known
as
Methylation
SNaPshot,
which
is
a
multiplex
individual-base
extension,
designed
using
in
silico
bisulfite
converted
genomic
reference
sequences
for
specific
genes,
with
subsequent
PCR
amplification.
Blood
and
saliva
displayed
highest
methylation
in
PFN3,
interme-
diate
in
PRMT2
and
lowest
in
DACT1.
Levels
of
methylation
in
DACT1
and
USP49
were
significantly
higher
than
PRMT2
and
PFN3
for
vaginal
fluid
and
menstrual
blood.
Analogous
results
for
methylation
SNaPshot
were
observed.
Blood
and
saliva
displayed
over
90%
methylation
in
USP49,
DACT1
and
PFN3.
Similar
results
to
Lee
et
al.
[52]
were
achieved
for
vaginal
fluid
and
menstrual
blood
as
high
methylation
was
observed
at
USP49
and
DACT1,
and
low
methylation
at
PFN3.
Yet,
in
contrast
to
the
same
study,
semen
was
found
to
display
complete
unmethylation
at
the
tDMR
of
PFN3
using
both
methods
[52,13].
Choi
and
colleagues
employed
MSRE-
PCR
and
tested
the
same
fluids
using
tDMRs
USP49,
DACT1,
PFN3
and
L81528.
Vaginal
fluid
and
menstrual
blood,
analogous
to
the
two
previously
described
reports
by
Lee
et
al.
[52]
and
An
et
al.
[2],
displayed
low
methylation
levels
at
PFN3.
The
L81528
tDMR
was
selected
as
a
semen-specific
methylation
marker;
and
10
out
of
the
18
non-vasectomised
semen
samples
generated
amplicons
only
at
this
tDMR.
Sensitivity
tests
were
conducted
by
testing
varying
quantities
of
DNA
produced
by
serial
dilutions.
Methylation
patterns
could
be
generated
for
saliva
and
semen
with
just
500
pg
or
more
of
DNA
and
a
measly
250
pg
of
DNA
from
vaginal
fluid
was
sufficient.
Aged
samples
generated
identical
results
to
An
et
al.
[2]
as
all
fluids
were
analysed
except
for
saliva.
Mixtures
of
saliva
and
semen,
in
1:1
and
1:2
ratios
were
clearly
distinguished
by
amplification
of
L81528.
Mixtures
of
semen
and
vaginal
fluid
was
also
distinguishable
as
profiles
were
comparable
to
that
of
vaginal
fluid
when
using
a
general
DNA
extraction
method,
and
a
differential
extraction
method
facilitated
identification
of
vaginal
fluid
from
the
resultant
supernatant,
and
semen
from
the
pellet.
Testing
serial
dilutions
of
DNA
and
mixtures
of
samples
are
quite
beneficial;
in
actual
crime
scenarios
only
minute
quantities
of
DNA
in
mixtures
may
be
located
at
the
scene
of
a
violent
act.
A
single
post-coital
penile
sample
and
three
post-coital
vaginal
samples
were
tested
for
an
artificial
sexual
assault
case.
General
DNA
extraction
methods
resulted
in
two
of
the
vaginal
fluid
and
the
penile
samples
showing
a
mixed
sample
profile
by
generating
low
peaks
at
PFN3,
and
a
semen-specific
L81528
amplicon.
Again,
the
differential
extraction
method
facilitated
a
display
of
a
profile
consistent
with
vaginal
fluid,
for
the
third
post-coital
sample
[13].
From
the
above
studies
it
is
clear
that
additional
tDMRs
are
required
for
precise
discrimination
between
blood
and
saliva,
and
vaginal
fluid
and
menstrual
blood.
Wasserstrom
et
al.
[98]
introduced
a
kit
known
as
DNA-source
identifier
(DSI-semen)
for
confirmation
of
semen.
Blood,
saliva,
semen,
vaginal
fluid,
and
menstrual
blood
and
urine
samples
were
tested
by
analysis
of
methylation
of
five
genomic
loci.
All
samples
that
were
tested
were
correctly
identified
with
confidence
levels
above
0.9999.
Real
forensic
casework
samples
were
also
analysed
and
all
were
in
complete
concordance
except
for
one
discrepancy;
a
single
sample
displayed
semen
and
non-semen
components.
However
further
investigation
revealed
that
the
assay
was
more
accurate
than
even
the
much-relied
microscopic
analysis
which
did
not
detect
semen
at
all.
Mock
casework
samples
were
also
correctly
recognised;
vaginal
swabs
were
mixed
with
varying
quantities
of
semen.
In
each
sample
the
pellet
was
classified
as
semen
and
supernatant
as
non-semen.
This
will
enable
scientists
to
apply
the
assay
for
analysis
of
internal
vaginal
fluid
samples
obtained
from
victims
of
assault.
The
DSI-semen
assay
correctly
discriminated
between
semen
and
male
urine,
rendering
it
quite
a
reliable
tool.
The
assay
is
user-independent,
fully
automatable
and
operating
the
software
would
not
require
any
special
training.
Furthermore,
there
is
now
potential
for
the
development
of
analogous
assays
for
identification
other
common
fluids
and
tissues
found
at
crime
scenes,
as
the
only
variable
would
be
primers;
the
same
setup,
reagents
and
computer
software
would
be
employed.
In
a
study
unrelated
to
forensic
sciences,
additional
tDMRs
were
discovered
that
distinguish
semen
from
other
tissues.
Testes
DNA
of
eight
individuals
were
tested
for
methylation
patterns.
Quantitative
analysis
of
DNA
methylation
was
conducted
by
Sequenom
Mass
Array
using
base-specific
cleavage
and
matrix-
assisted
laser
desorption/ionization
time
of
flight
mass
spectros-
copy
(MALDI-TOF
MS).
Other
internal
body
tissues
were
analysed
as
well,
but
these
are
not
frequently
found
at
crime
scenes.
Percent
methylation
for
tDMRs
of
13
genes
were
measured
and
the
study
revealed
that
the
tDMRs
tested
(precise
regions
stated
in
report)
for
MBNL2
(muscle
blind-like
2),
ZNF755,
SPESP1
(sperm
equatorial
segment
protein
1),
ZFPM1
and
PHLDB3
(Pleckstrin
homology-like
domain
family
B
member
3)
genes
displayed
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
260

variable
levels
of
hypomethylation
[36].
These
markers,
in
addition
to
USP49,
DACT1,
ZC3H12D
and
FGF7
may
be
considered
during
forensic
investigations
for
sperm
identification.
5.3.
Sex
determination
by
analysis
of
DNA
methylation
Even
though
DNA
methylation
profiling
is
relatively
new,
it
has
been
previously
reported
in
1993
by
Naito
and
colleagues.
The
study
introduced
DNA
methylation
into
forensic
sciences
by
developing
a
simple
procedure
for
female
sex
typing
based
on
varying
methylation
pattern
of
DXZ4,
an
X
chromosome-specific
region.
The
DXZ4
sequence
showed
low
degrees
of
methylation
on
the
inactive
X,
but
hypermethylation
on
the
active
X
chromosome.
The
protocol
was
quite
sensitive
as
due
to
the
high
copy
number
of
DXZ4
in
the
genome,
only
a
minute
amount
of
DNA
was
necessary
for
accurate
sex
typing.
The
researchers
also
suggested
application
for
detecting
sex-reversed
patients
[63,91].
It
may
be
possible
to
apply
the
HOXA4
tDMR
to
differentiate
between
male
and
female
saliva.
In
the
research
study
by
Lee
et
al.
[52],
differences
in
methylation
of
male
and
female
samples
were
quite
evident
by
this
marker.
The
tDMR
for
HOXA4
was
hypermethylated
(above
90%)
in
male
saliva,
yet
displayed
below
53%
methylation
in
female
saliva.
However,
cross-reactivity
with
other
fluids
such
as
blood
may
interfere
with
results
and
hence
this
must
be
considered
during
application
in
forensics.
Perhaps
other
confirmatory
methods,
such
as
use
of
one
or
more
additional
markers,
may
be
employed
in
conjunction.
The
relationships
between
DNA
methylation
and
gender,
amongst
other
factors,
were
explored
by
Boks
et
al.
[9]
using
Illumina
GoldenGate
bead
array
followed
by
statistical
analysis
and
sequencing.
Up
to
1505
CpG
sites
of
just
over
800
gene
regions
were
examined
from
DNA
of
peripheral
blood
of
46
males,
46
females
and
96
controls.
The
study
revealed
independent
links
of
DNA
methylation
with
gender;
all
X-chromosomal
probes
were
extensively
more
methylated
in
females
than
males.
According
to
Shen
et
al.
[81]
this
pattern
of
methylation
loci
on
X
chromosomes
could
primarily
be
due
to
the
X
inactivation
mechanism
in
females.
Numerous
autosomal
loci
of
CpGs
were
also
found
to
display
differential
methylation
in
females
[9],
and
this
was
also
demonstrated
previously
[21]
signifying
that
gender-dependent
methylation
may
not
be
an
uncommon
occurrence.
Overall,
the
variation
in
levels
of
methylation
between
the
two
sexes
were
not
too
high,
but
still
considerable,
although
these
particular
CpG
loci
have
not
been
employed
for
methylation-based
forensic
sex-typing
[9].
A
previous
report
also
examined
blood
of
96
males
and
96
females
[21].
Alu
and
LINE
1
repetitive
DNA
elements
were
considered,
single
loci
were
explored
for
three
tDMRs
at
imprinted
genes,
namely
H19,
NESP55
and
PEG3.
Also
examined
were
2
loci;
19q13.4
which
is
located
between
ubiquitin-specific
protease
29
and
PEG3,
and
Xq28
(F8
gene).
The
mean
methylation
difference
between
males
and
females
in
LINE
1
was
reported
to
be
between
1.61
and
5.80%,
all
lower
in
females.
Majority
of
the
loci
that
were
studied
displayed
greater
degrees
of
methylation
patterns
in
males
with
the
only
exceptions
being
tDMRs
at
the
imprinted
genes
which
demonstrated
similar
methylation
levels
in
both
males
and
females,
yet
still
a
slight
inclination
towards
lower
methylation
in
females
was
observed.
Later
in
2011,
Zhang
and
colleagues
also
examined
LINE-1
of
peripheral
blood
of
males
and
females.
Again,
this
study
reported
a
1.8%
difference
in
global
methylation
between
the
two
sexes,
with
females
being
lower.
This
is
a
notable
pattern;
Hsiung
et
al.
[34],
and
Zhu
et
al.
[105]
found
a
1.17%
and
0.8%
methylation
difference,
respectively.
In
addition
to
the
proposed
idea
that
X
chromosome
inactivation
mechanisms
diminish
the
resources
necessary
for
methylation
of
autosomal
loci,
it
is
also
believed
that
low
degrees
of
total
methylation
in
females
may
be
due
to
consumption
of
varying
amounts
of
one-carbon
nutrients
such
as
methionine
and
B
vitamins,
or
dietary
folate
[81,104].
5.4.
Predicting
age
of
body
fluids
and
tissues;
association
with
disease
Extensive
research
has
been
done
on
the
effects
of
age
upon
DNA
methylation
(briefly
discussed
in
Section
3.1)
and
hence
forensic
scientists
have
resorted
to
analysis
of
DNA
methylation
patterns
to
estimate
the
age
of
individuals
involved
in
criminal
investigations,
or
possible
biological
age
of
tissues
at
time
of
death
for
cadavers
[91].
By
use
of
simple
DNA
extraction
and
Illumina
HumanMethylation27
arrays,
a
group
of
researchers
developed
a
regression
model
to
calculate
an
estimate
of
age
of
biological
sample.
Saliva
samples
collected
from
participants
between
the
ages
of
21
and
55
were
examined.
The
study
revealed
that
the
methylation
patterns
in
three
sites
of
the
promoters
of
TOM1L1
(target
of
myb1
chicken-like
1,
coding
for
a
signalling
molecule)
and
EDARADD
genes
decreased
with
age,
and
NPTX2
demonstrated
a
progressive
increase.
The
regression
model
to
estimate
the
age
of
the
fluids
was
built
by
using
a
few
cytosines
from
these
loci.
Such
a
model
would
predict
the
age
of
the
biological
sample
with
an
accuracy
of
approximately
5
years
[8].
Koch
and
Wagner
[48]
also
employed
HumanMethylation27
arrays
and
acknowledged
an
epigenetic-ageing-signature
which
predicts
age
of
various
tissues
including
skin,
epithelial
cells
of
the
cervix
and
CD4
+
T
cells
and
CD14
+
monocytes
of
peripheral
blood.
Irrespective
of
gene
function,
5
CpG
sites
that
demon-
strated
differential
methylation,
namely
TRIM58
(tripartite
motif
containing),
GRIA2,
KCNQ1DN,
NPXT2
and
BIRC4P,
were
selected.
When
all
5
CpG
sites
were
tested
for
age,
the
predictions
of
the
signature
identified
correlated
with
precision
of
approximately
9
years,
and
when
only
KCNQ1DN,
GRIA2
and
NPXT2
were
examined
(these
demonstrated
most
apparent
age-associated
methylation)
precision
of
approximately
10
years
was
achieved.
In
the
testes
tissues
that
were
examined
by
Igarashi
et
al.
[36],
an
age-related
linear
correlation
of
DNA
methylation
was
also
found.
Often
studies
that
determine
the
effects
of
age
on
methylation
find
a
link
with
the
onset
of
disease
[41,80,14,90,8,88].
This
trend
was
observed
by
Boks
et
al.
[9]
who
analysed
genomic
DNA
of
peripheral
blood.
Differential
methylation
of
loci
studied
in
the
IL6
gene
was
implicated
in
metabolic
syndromes
of
elderly
males.
Change
in
DNA
methylation
with
time,
in
the
AXINI
locus
is
linked
to
caudal
duplication
anomaly.
The
parathyroid
hormone
receptor
1
gene
(PTHR1)
displayed
variable
methylation
in
females
and
is
associated
with
bone
mineral
density
which
deteriorates
with
age.
In
the
study
by
Bockland
et
al.
[8],
an
association
of
DNA
methylation
with
age-related
illnesses
was
also
found.
Of
the
genes
tested,
mutations
of
EDARADD,
which
is
the
Edar
associated
death
domain,
can
reduce
speed
of
wound
healing
resulting
in
loss
of
sweat
glands,
hair
and
teeth.
Methylation
of
NPTX2,
neuronal
pentraxtin
II,
is
found
to
be
upregulated
in
pancreatic
cancer
and
increased
expression
during
Parkinson’s
disease
has
also
been
observed.
A
correlation
between
methylation
of
NPTX2
has
also
been
demonstrated
with
age
and
blood
as
well.
Instead
of
chronological
age,
biological
age
may
be
used
and
treatments
for
individuals
who
are
ill
may
be
tailored
to
suit
specific
needs
based
on
the
interaction
of
genes
and
time
[96,8,33].
Whilst
applying
DNA
methylation
and
its
association
to
disease
in
order
to
determine
age
of
body
tissues
and
fluids
is
still
in
infant
stages;
it
shows
great
potential.
An
individual
of
a
certain
age,
who
has
been
diagnosed
with
a
disease,
may
either
be
convicted
or
exonerated
by
the
findings
of
the
methylation
patterns
of
tissues.
This
prospect
requires
much
more
research.
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
261

5.5.
Ancestry
informative
markers
of
fluid/tissue
donor
Other
than
differentiating
between
individuals
based
on
gender,
great
progress
has
been
made
in
determining
ones
ancestry
informative
characteristics
using
differential
methyla-
tion.
Fraser
et
al.
[24]
demonstrated
population
specificity
of
DNA
methylation
in
lymphoblastoid
cell
lines
(LCLs)
of
blood.
Human-
Methylation27
BeadChip
arrays
followed
by
pyrosequencing
determined
methylation
of
27,578
CpG
sites
near
transcription
start
sites
of
14,495
genes.
The
study
population
comprised
of
30
trios
(consisting
of
father,
mother
and
offspring)
of
Northern
European
ancestry
and
30
trios
of
families
of
West
African
ancestry.
Methylation
profiles
of
approximately
30%
of
CpG
sites,
which
is
over
a
third
of
the
genes
examined,
differed
between
the
populations.
Specifically,
14%
of
sites
differed
in
methylation
by
an
average
of
over
5%,
and
3.9%
of
sites
differed
by
over
10%.
Although
the
differences
were
small,
they
were
still
quite
significant.
In
a
study
also
concentrated
on
LCLs
of
blood,
Heyn
et
al.
[33]
attempted
to
identify
methylation
differences
between
three
human
populations,
and
provided
a
link
between
the
methylation
and
the
possible
natural
phenotypic
variation
that
occurs.
Research
involved
LCLs
and
naı
¨ve
blood
of
unrelated,
healthy
African
Americans,
Caucasian
Americans
and
Han-Chinese
Americans.
In
total,
439
CpG
sites
were
found
to
display
differential
methylation
between
the
three
populations.
The
study
associated
this
differential
methylation
with
defining
characteristics
such
as
response
to
various
stimuli,
disease
frequencies,
sensory
percep-
tion
and
appearances.
Analysis
of
differential
DNA
methylation
was
incorporated
into
genome-wide
association
studies;
and
resulted
in
a
direct
relationship
between
differential
methylation
of
CpG
sites
of
the
HLA-DPA1
locus.
This
locus
is
strongly
correlated
with
chronic
hepatitis
B
(HBV)
infection.
HBV
infection
risk
alleles
are
usually
enriched
in
populations
such
as
Africans
or
Asians
as
they
have
higher
frequency
of
diseases.
Ten
HBV-infection
SNPs
displayed
high
correlation
with
17
CpG
sites
in
HLA-DPA1.
Results
revealed
that
the
risk
alleles
were
linked
to
variation
in
DNA
methylation,
showing
high
incidence
in
the
African
and
Asian
populations.
Risk
alleles
were
related
to
the
high
degree
of
methylation
of
the
HLA-DPA1
promoter
and
hypomethylation
of
the
gene
body,
and
this
is
associated
with
low
levels
of
gene
expression
[44,33].
Gene
repression
of
HLA-DPA1
was
associated
with
DNA
methylation
in
these
two
populations
by
identifying
the
risk
alleles
that
mediate
this
phenomenon
which
spurs
variation
in
presentation
of
cell
surface
receptors,
altered
binding
of
HBV
and
risk
of
infection
[33].
This
warrants
further
research
into
these
particular
CpG
sites;
it
may
be
possible
that
they
appear
specifically
in
other
forensically
relevant
fluids
and
tissues.
A
study
quite
relevant
to
discriminating
ancestry
informative
markers
which
is
highly
applicable
to
forensic
sciences
observed
differences
in
Blacks,
Whites
and
Hispanics
in
New
York
City
birth
cohort
until
mid-life.
Blood
samples
were
collected
and
analysed
by
[
3
H]-methyl
acceptance
assay
which
involves,
in
short,
incubating
DNA
with
[
3
H]
SAM
in
the
presence
of
SssI
prokaryotic
methylases.
This
will
methylate
unmethylated
CpG
sites,
and
is
quantified
followed
by
statistical
analysis
relevant
to
the
study.
In
the
birth
record,
Blacks
were
found
to
display
lower
levels
of
methylation
than
Whites
and
Hispanics.
This
association
between
DNA
methylation
and
the
groups
did
not
alter
after
adjusting
life
course
variables.
Other
factors
such
as
smoking,
passive
smoking,
and
family
socio-economic
status
were
considered
in
the
study
as
well,
but
overall,
Blacks
demonstrated
lowest
levels
of
methylation
[89].
Non-Hispanic
Blacks,
Non-Hispanic
Whites
and
Hispanics
were
also
studied
by
Zhang
et
al.
[104]
who
examined
LINE-1
of
peripheral
blood.
Similar
results
were
obtained
as
Blacks
displayed
lowest
percentage
methylation
of
73.1%,
Hispanics
74%
and
Whites
75.3%.
Although
using
differential
methylation
to
distinguish
between
certain
populations
seems
quite
promising,
it
is
a
tool
of
major
distress.
Due
to
legal
and
ethical
issues
of
such
applications,
using
these
ancestry
informative
markers
may
be
problematic.
Predom-
inantly,
the
possibility
for
misapplication
as
an
ancestry
informa-
tive
marking
method
remains
an
extensive
concern
as
discussed
by
Koops
and
Maurice
[49]
and
M’charek
et
al.
[60].
5.6.
Distinguishing
between
monozygotic
twins:
associations
with
sex,
age
and
phenotype
Since
monozygotic
twins
theoretically
possess
the
same
genomic
DNA
sequences,
discrimination
of
twins
in
forensic
casework
has
always
been
taxing
using
conventional
DNA
typing.
One
in
300
individuals
has
a
twin.
If
evidence
is
found
at
a
crime
scene,
scientists
are
baffled
as
to
which
twin
may
be
responsible
for
the
crime
[43,53].
Various
researchers
have
proposed
that
DNA
methylation
might
be
employed
to
solve
this
forensic
mystery,
owing
to
monozygotic
twins
displaying
varying
degrees
of
phenotypic
differences
[9,5,69,53].
However,
only
a
few
studies
focus
on
distinguishing
between
twins,
such
as
Li
et
al.
[53]
who
examined
peripheral
blood
samples
of
13
pairs
of
female
twins
and
9
pairs
of
male
twins,
between
the
ages
of
17
and
74.
3616
CpG
sites
with
differential
methylation
were
identified
and
of
these,
92
sites
were
selected.
These
were
located
on
all
chromosomes
except
18
and
22,
and
each
of
the
twin
pairs
was
successfully
distinguished.
The
authors
hypothesise
that
since
only
peripheral
blood
was
analysed
in
this
study,
the
identified
CpG
sites
may
only
be
applicable
to
blood
and
thus
more
research
is
required
for
other
fluids
such
as
saliva
or
semen.
Fraga
et
al.
[23]
performed
a
broad
and
quite
informative
study
on
monozygotic
twins.
The
study
population
consisted
of
30
male
pairs
and
50
female
pairs,
all
Caucasians
of
ages
3
to
74.
Initially,
peripheral
lymphocytes
were
examined,
however
due
to
the
discordances
that
were
observed,
other
tissues
were
analysed
including
epithelial
mouth
cells.
First,
using
MSRE-PCR,
X
chromosome
inactivation
was
analysed
in
females.
19%
of
all
female
twins
displayed
a
skewed
methylation
pattern
which
varied
between
siblings.
Surprisingly,
in
35%
of
the
twin
pairs,
considerable
differences
in
methyl-cytosine
were
observed
be-
tween
siblings.
To
establish
where
exactly
in
the
genome
these
epigenetic
differences
transpire,
amplification
of
inter-methylated
sites
(AIMS),
which
is
a
global
methylation
fingerprinting
method,
was
employed.
Roughly
600
bands
were
present
on
the
AIMS
gels;
0.5–35%
of
which
was
dissimilar
between
the
twin
pairs.
Twins
displaying
most
varied
bands
were
the
same
as
those
with
highest
variability
in
methyl-cytosine
content.
For
additional
characteri-
zation,
some
of
the
AIMS
bands
were
examined
further
by
cloning
and
sequencing.
Nearly
half
of
the
clones
matched
Alu
sequences,
some
corresponded
to
single
copy
genes
and
a
few
matched
repetitive
sequences
such
as
LINES,
MIR
and
MER.
Bisulfite
treatment
and
sequencing
of
one
of
the
twins
revealed
hyper-
methylation
while
the
other
had
hypomethylation
at
those
sites.
Analysis
of
chromosome
regions
with
different
methylation
levels
revealed
distinct
methylation
patterns
in
metaphase
chromo-
somes;
predominantly,
majority
of
telomeres
and
some
selected
gene-rich
regions
such
as
1p36,
3p21
and
8q21,
amongst
others.
When
the
same
methods
were
employed
for
epithelial
mouth
cells,
similar
results
were
obtained.
Interestingly,
the
same
pattern
was
observed
in
all
the
above-mentioned
results
for
each
different
method
used;
those
twins
who
had
spent
the
major
part
of
their
lives
apart
or
had
more
dissimilar
natural
health
medical
history
demonstrated
the
greatest
differences.
In
addition
to
victorious
discrimination
of
monozygotic
twins
by
differential
methylation,
which
is
indispensable
in
forensic
casework,
Fraga
and
colleagues
were
also
able
to
conclude
that
epigenetic
markers
were
much
F.
Kader,
M.
Ghai
/
Forensic
Science
International
249
(2015)
255–265
262

more
diverse
in
older
monozygotic
twins.
Since
those
who
were
younger
and
lived
analogous
lifestyles
with
more
time
together
demonstrated
minimal
variation
in
methylation,
observed
differ-
ences
between
genetically
identical
siblings
may
be
due
to
both
internal
and
external
stimuli
such
as
diet,
smoking,
alcohol
consumption,
exposure
to
pollutants,
as
discussed
earlier.
This
highlights
the
noteworthy
impact
of
environmental
factors
in
translation
of
a
common
genotype
into
a
very
different
phenotype.
6.
Conclusion
and
future
outlook
Epigenetics
is
swiftly
expanding
to
contain
numerous
altera-
tions
to
the
genome
devoid
of
changes
in
DNA
sequence.
Since
these
mechanisms
boggle
the
minds
of
many
researchers,
constant
studies
are
being
conducted
to
clarify
the
mechanisms
by
which
these
changes
occur,
the
factors
that
influence
these
changes
and
the
effects
that
they
may
render
upon
an
individual’s
genotype
and
phenotype.
However,
akin
to
all
research,
the
field
of
epigenetics
does
not
come
without
hurdles
and
challenges.
Most
research
focuses
only
on
specific
tissues
and
cells,
owing
to
epigenetic
traits
being
specific
to
these
and
thus
this
prevents
extrapolation
of
information
across
the
whole
genome
of
an
organism
[88].
Furthermore,
one
must
consider
the
internal
and
external
influences
that
alter
patterns
of
one
individual,
family
or
population
but
not
the
to,
as
pointed
out
in
the
above-mentioned
studies
(Section
3).
Much
attention
from
those
involved
in
the
medical
field
and
medical
diagnosis
is
directed
upon
epigenetics
and
DNA
methyl-
ation
due
to
age-associated
diseases.
Since
in
tumours,
global
hypomethylation
has
been
detected
in
genomes
on
countless
occasions
and
is
thought
to
induce
oncogenesis
[20],
cancer
is
the
most
researched
disease.
DNA
methylation
serves
as
the
ideal
biomarker
for
clinical
management
of
those
suffering
with
cancer.
During
tumorigenesis
as
well
as
other
diseases,
thorough
mapping
and
analysis
of
variations
in
methylation
will
significantly
aid
in
enhancing
our
understanding
of
the
great
value
of
epigenetics
in
development
and
on-set
of
diseases.
Only
the
future
will
reveal
if
such
knowledge
will
improve
current
attempts
at
clinical
treatments,
heal
unhealthy
individuals
as
well
as
provide
novel
methods
to
reduce
the
rate
of
disease
manifestation
[41,90].
As
is
plainly
evident,
the
analysis
of
DNA
methylation
and
tDMRs
in
forensic
sciences
is
quite
favourable.
Various
body
fluids
and
tissues
have
been
unequivocally
identified
by
employing
DNA
methylation
assays,
but
there
are
a
few
factors
to
consider
first.
When
selecting
a
particular
CpG
site
to
analyse,
it
is
vital
to
examine
several
samples
to
certify
that
the
site
does
not
demonstrate
low
levels
of
inter-individual
variation.
Also,
it
must
be
established
that
the
site
is
not
influenced
by
external
stimuli
or
is
age-dependent.
It
is
necessary
to
select
sites
in
which
high
variation
of
methylation
is
apparent
when
quantification
is
required.
For
example,
to
link
a
selected
site
with
a
specific
cause
of
death,
when
compared
to
a
normal
corresponding
tissue,
it
must
display
about
65%
variability.
False
results
and
estimations
resulting
from
inter-individual
differences
may
also
be
avoided
this
way
[91].
Given
that
high
amounts
of
sound
quality,
fresh
DNA
are
rarely,
if
ever,
found
at
a
crime
scene,
the
method
of
analysis,
amplifications
and
design
of
assays
must
be
given
much
thought
and
deliberation.
There
is
no
doubt
that
each
method
will
present
its
own
rewards
and
limitations,
and
these
must
be
critically
weighed
against
each
other
prior
to
decision-making.
Forensic
experts
are
knowledgeable
in
methods
of
profiling
DNA
methyl-
ation
and
tDMRs
are
plentiful
in
the
mammalian
genome,
thus
there
should
not
be
much
hindrance
in
the
expansion
of
novel
and
exciting
scientific
and
technological
procedures
for
efficient,
reliable
tissue
and
body
fluid
identification
for
forensic
applica-
tions.
Present
methods
are
quite
dependable
but
in
order
to
cope
with
the
tricky
nature
of
specimens
located
at
crime
scenes,
improvements
are
imperative.
The
future
of
DNA
methylation
analysis
in
forensics
will
surely
be
constantly
evolving
and
will
most
likely
be
poles
apart
from
what
we
see
today
[52,76].
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.forsciint.2015.01.
037.
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