CDKN1C mutations: Two sides of the same coin

Abstract

Cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) negatively regulates cellular proliferation and it has been shown that loss-of-function mutations in the imprinted CDKN1C gene (11p15.5) are associated with the overgrowth disorder Beckwith–Wiedemann syndrome (BWS). With recent reports of gain-of-function mutations of the PCNA domain of CDKN1C in growth-retarded patients with IMAGe syndrome or Silver–Russell syndrome (SRS), its key role for growth has been confirmed. Thereby, the last gap in the spectrum of molecular alterations in 11p15.5 in growth-retardation and overgrowth syndromes could be closed. Recent functional studies explain the strict association of CDKN1C mutations with clinically opposite phenotypes and thereby contribute to our understanding of the function and regulation of the gene in particular and epigenetic regulation in general.

Full text

CDKN1C
mutations:
two
sides
of
the
same
coin
Thomas
Eggermann
1
,
Gerhard
Binder
2
,
Fre
´de
´ric
Brioude
3
,
Eamonn
R.
Maher
4,5
,
Pablo
Lapunzina
6
,
Maria
Vittoria
Cubellis
7
,
Ignacio
Bergada
´
8
,
Dirk
Prawitt
9
,
and
Matthias
Begemann
1
1
Institute
of
Human
Genetics,
University
Hospital,
Technical
University
Aachen,
Aachen,
Germany
2
University
Children’s
Hospital,
Paediatric
Endocrinology,
University
of
Tu
¨bingen,
Tu
¨bingen,
Germany
3
AP-HP,
Hoˆ
pital
Armand
Trousseau,
Explorations
Fonctionnelles
Endocriniennes,
Paris,
France
4
Department
of
Medical
Genetics,
University
of
Cambridge,
Cambridge,
UK
5
NIHR
Cambridge
Biomedical
Research
Centre,
Cambridge,
UK
6
INGEMM,
Instituto
de
Gene´
tica
Me´
dica
y
Molecular,
Hospital
Universitario
La
Paz,
IdiPAZ,
CIBERER-ISCIII,
Madrid,
Spain
7
Dipartimento
di
Biologia,
Universita`
Federico
II,
Napoli,
Italy
8
Centro
de
Investigaciones
Endocrinolo´
gicas
‘Dr
Ce´
sar
Bergada´
’
(CEDIE),
CONICET–FEI–Divisio´
n
de
Endocrinologı
´a, Hospital
de
Nin
˜os
Ricardo
Gutie´
rrez,
Buenos
Aires,
Argentina
9
Molekulare
Pa
¨diatrie,
Zentrum
fu
¨r
Kinder-
und
Jugendmedizin,
Universita
¨tsmedizin
Mainz,
Mainz,
Germany
Cyclin-dependent
kinase
(CDK)-inhibitor
1C
(CDKN1C)
negatively
regulates
cellular
proliferation
and
it
has
been
shown
that
loss-of-function
mutations
in
the
imprinted
CDKN1C
gene
(11p15.5)
are
associated
with
the
over-
growth
disorder
Beckwith–Wiedemann
syndrome
(BWS).
With
recent
reports
of
gain-of-function
muta-
tions
of
the
PCNA
domain
of
CDKN1C
in
growth-retard-
ed
patients
with
IMAGe
syndrome
or
Silver–Russell
syndrome
(SRS),
its
key
role
for
growth
has
been
con-
firmed.
Thereby,
the
last
gap
in
the
spectrum
of
molec-
ular
alterations
in
11p15.5
in
growth-retardation
and
overgrowth
syndromes
could
be
closed.
Recent
func-
tional
studies
explain
the
strict
association
of
CDKN1C
mutations
with
clinically
opposite
phenotypes
and
thereby
contribute
to
our
understanding
of
the
function
and
regulation
of
the
gene
in
particular
and
epigenetic
regulation
in
general.
Imprinting
and
growth
Human
height
is
a
complex
trait,
with
contributions
from
both
heritable
and
environmental
factors.
Twin
studies
suggest
that
the
contribution
of
heritability
to
human
growth
and
height
accounts
for
more
than
80%
of
variability
[1].
In
addition
to
the
strong
impact
of
genomic
DNA
varia-
tion
on
phenotypic
features
such
as
growth,
the
role
of
epigenetic
(see
Glossary)
mechanisms
as
the
mediator
of
the
reversible
interactions
between
genes
and
the
environ-
ment
is
becoming
increasingly
apparent
[2,3].
These
epige-
netic
mechanisms,
mainly
DNA
methylation,
histone
modifications,
and
RNAi,
functionally
mediate
the
spatial
and
temporal
expression
of
genes.
This
interplay
of
a
large
number
of
intrinsic
and
extrinsic
factors
is
vulnerable
and
numerous
disturbances
have
been
reported
affecting
certain
epigenetically
regulated
chromosomal
regions
and/or
genes
that
are
expressed
in
a
parent-of-origin-specific
manner.
The
resulting
congenital
syndromes,
the
imprinting
disor-
ders
(IDs)
[4],
are
characterized
by
similar
molecular
altera-
tions
affecting
the
methylation
patterns
(epimutations)
of
differentially
methylated
regions
(DMRs)
or
the
genomic
structure
and
sequence
of
imprinted
regions
[chromosomal
duplications/deletions,
uniparental
disomies
(UPDs),
point
mutations
in
imprinted
genes].
In
addition
to
these
similar
molecular
alterations,
most
IDs
show
overlapping
clinical
features.
In
particular,
disturbed
growth
is
a
consistent
symptom
in
nearly
all
IDs,
an
observation
that
in
accor-
dance
with
the
‘parental-conflict
hypothesis’
[5].
Review
1471-4914/
ß
2014
Published
by
Elsevier
Ltd.
http://dx.doi.org/10.1016/j.molmed.2014.09.001
Corresponding
author:
Eggermann,
T.
(teggermann@ukaachen.de).
Keywords:
CDKN1C;
imprinting;
IMAGE
syndrome;
Beckwith–Wiedemann
syn-
drome;
Silver–Russell
syndrome;
point
mutations..
Glossary
Differentially
methylated
region
(DMR):
a
genomic
region
with
different
methylation
statuses
in
different
tissues,
resulting
in
differential
epigenetic
regulation
of
gene
expression.
Deletion:
an
aberration
in
which
part
of
a
chromosome
or
a
DNA
sequence
is
missing.
DNA
methylation:
a
molecular
modification
where
a
methyl
group
is
added
to
cytosine
residues.
Duplication:
an
aberration
in
which
part
of
a
chromosome
or
a
DNA
sequence
is
duplicated.
Epigenetics:
DNA
modifications
like
methylation
or
histone
modification
that
do
not
change
the
DNA
sequence
and
often
affect
gene
expression.
Epimutation:
aberrant
silencing/activation
of
gene
expression
without
a
change
in
the
DNA
sequence
but
due
to
an
epigenetic
change
(e.g.,
by
aberrant
DNA
methylation).
Imprinting
control
region
(ICR):
a
chromosomal
region
that
regulates
the
expression
or
silencing
of
imprinted
genes.
Imprinting
disorders
(IDs):
a
group
of
currently
eight
congenital
disorders
caused
by
molecular
alterations
of
imprinted
genes
(or
chromosomal
regions).
Imprinted
genes:
genes
that
are
expressed
in
a
parent-of-origin-specific
manner.
Molecular
karyotyping:
digital
analysis
of
data
obtained
from
the
analysis
of
short
DNA
sequences
from
loci
all
over
the
genome.
It
detects
genomic
copy
number
variations
at
a
higher
resolution
than
conventional
karyotyping.
Parental-conflict
hypothesis:
states
that
the
inequality
of
imprinting
patterns
between
parental
genomes
is
a
result
of
the
differing
interests
of
the
parents.
Translocations:
abnormalities
caused
by
the
rearrangement
of
parts
of
one
or
more
chromosomes.
Uniparental
disomy
(UPD):
the
unique
inheritance
of
both
chromosomes
of
a
pair
from
only
one
parent
rather
than
from
both
parents.
TRMOME-976;
No.
of
Pages
9
Trends
in
Molecular
Medicine
xx
(2014)
1–9
1

The
chromosomal
region
11p15.5
The
chromosomal
region
11p15.5
encodes
several
growth-
promoting
and
-inhibiting
factors
and
plays
a
key
role
in
human
growth
and
development.
The
region
spans
around
1
Mb
and
harbors
two
separate
imprinting
control
regions
(ICRs):
The
telomeric
ICR1
(H19
DMR)
is
methylated
on
the
paternal
allele,
whereas
the
centromeric
ICR2
(KvDMR1;
KCNQ1OT1
DMR)
is
maternally
methylated
(Figure
1).
In
addition
to
its
central
physiological
role,
it
has
been
postulated
that
the
11p15.5
region
is
the
central
element
of
a
network
of
imprinted
genes
[6].
Telomeric
ICR1
The
telomeric
ICR1
confers
differing
chromatin
architec-
tures
to
the
two
parental
alleles,
leading
to
reciprocal
ex-
pression
of
the
growth-suppressing
H19
noncoding
RNA
and
the
fetal
growth
factor
insulin-like
growth
factor
2
(IGF2).
These
two
genes
are
coexpressed
in
endoderm-
and
meso-
derm-derived
tissues
during
embryonic
development
and
compete
for
the
same
enhancers.
H19
is
expressed
from
the
unmethylated
maternal
allele
and
exerts
transcriptional
regulation
effects
on
other
genes
by
recruiting
methyl-
CpG-binding
domain
protein
1
(MBD1)
and
consequently
KCNQ1OT1
p15.4 p13 p12 q14.1 21 22.3 23.3 25
IGF2
chr11 (p15.5)
ICR1
ICR2
H19 IGF2
H19
KCNQ1
KCNQ1
KCNQ1OT1
CDKN1C
CDKN1C
CTCF
CTCF
Paternal
chromosome
Maternal
chromosome
KCNQ1OT1
KCNQ1
KCNQ1OT1
KCNQ1
Ex. 1
Ex. 1
Ex. 15
Ex. 15
CDKN1C
CTCF
–
Paternal
chromosome
Maternal
chromosome
+
CDKN1C
Candidate CDKN1C
enhancer regions
Methylated differenally
methylated region
CH3
CH3
CH3
CH3
Unmethylated differenally
methylated region
Key:
TRENDS in Molecular Medicine
Figure
1.
Schematic
of
imprinting
control
regions
(ICRs)
in
11p15.5.
Paternal
chromosomes
are
in
blue
and
maternal
chromosomes
in
red.
In
ICR1
the
paternal
allele
is
methylated,
leading
to
repression
of
the
paternal
H19
gene.
H19
is
expressed
from
the
unmethylated
maternal
allele.
The
maternal
unmethylated
ICR1
is
also
bound
by
CCCTC-binding
factor
(CTCF),
which
prevents
insulin-like
growth
factor
2
(IGF2)
expression,
while
the
methylated
paternal
allele
is
permissive
for
IGF2
expression.
In
ICR2,
the
noncoding
RNA
(ncRNA)
KCNQ1OT1
is
expressed
from
the
paternal
allele
(blue)
and
represses
the
paternal
CDKN1C
copy.
By
contrast,
the
maternal
CDKN1C
allele
is
transcribed
(red)
and
transcription
is
positively
enhanced
by
at
least
three
enhancer
motifs
that
have
been
delineated
from
families
with
different
deletions
within
ICR2.
Note
that
gene
sizes
and
distances
are
not
drawn
to
scale.
Review Trends
in
Molecular
Medicine
xxx
xxxx,
Vol.
xxx,
No.
x
TRMOME-976;
No.
of
Pages
9
2

increasing
methylation
at
histone
3
lysine
9
(H3K9me3)
at
targeted
loci
[7].
ICR1
on
the
unmethylated
maternal
allele
is
bound
by
CCCTC-binding
factor
(CTCF)
on
numerous
CTCF-binding
sites
(CTSs),
which
acts
as
an
insulator
for
the
interaction
of
enhancer
elements
and
the
IGF2
promoter
[8].
Methylation
of
the
paternal
allele
prevents
CTCF
bind-
ing
to
ICR1,
thereby
simultaneously
suppressing
H19
ex-
pression
and
triggering
IGF2
expression.
The
imprinted
expression
of
H19
and
suppression
of
IGF2
seems
to
require
predefined
CTCF
binding
to
the
maternal
allele.
Deletions
on
the
maternal
allele
that
reduce
the
number
of
CTSs
and
disturb
the
ICR
architecture
result
in
elevated
IGF2
levels
and
BWS
in
humans
[9].
Similar
deletions
in
mice
disrupt
imprinted
expression
due
to
loss
of
ICR1
insulator
activity
in
tissues
of
mesodermal
origin,
without
perturbing
DNA
methylation
at
the
ICR
[10].
Comparable
deletions
on
the
paternal
allele
have
not
been
associated
with
a
phenotype
in
humans
[11]
but
result
in
biallelic
expression
of
h19
in
the
corresponding
mouse
model
[10].
Centromeric
ICR2
The
centromeric
ICR2
regulates
the
expression
of
CDKN1C
and
potassium
channel
KQT-family
member
1
(KCNQ1),
as
well
as
other
genes
(e.g.,
PHLDA,
SLC22A18),
and
is
methylated
only
on
the
maternal
allele.
Overlapping
with
introns
9/10
and
exon
10
of
the
KCNQ1
gene
is
the
noncoding
RNA
KCNQ1OT1
(LIT1).
Methylation
of
the
maternal
allele
inhibits
the
expression
of
KCNQ1OT1
but
does
allow
expression
of
CDKN1C
and
KCNQ1.
The
pater-
nal
allele,
which
is
unmethylated,
expresses
KCNQ1OT1,
which
then
silences
the
flanking
imprinted
genes.
As
a
result,
CDKN1C
and
KCNQ1
are
expressed
only
from
the
maternal
gene
copy.
Molecular
defects
and
the
ICRs
Molecular
defects
affecting
the
ICRs
in
11p15.5
are
detect-
able
in
different
IDs
[12],
but
they
are
primarily
associated
with
BWS
and
SRS
(Box
1).
These
IDs
are
characterized
by
overgrowth
in
the
case
of
BWS
and
growth
retardation
in
SRS.
They
can
arise
due
to
genetic
alterations
(i.e.,
chro-
mosomal
duplications/deletions,
UPDs,
and
CDKN1C
point
mutations)
or
DNA
methylation
defects
(epimuta-
tions)
(Table
1).
More
than
40%
of
SRS
patients
show
DNA
hypomethylation
of
ICR1;
in
addition,
single
cases
of
ma-
ternal
duplications
of
the
whole
(or
parts)
of
11p15.5
have
been
reported
(for
a
review,
see
[13]).
Maternal
UPD
of
chromosome
11
(upd(11)mat)
has
been
observed
only
once
[14].
Opposite
epigenetic
or
genetic
changes
in
11p15.5
can
be
observed
in
nearly
70%
of
BWS
patients,
with
DNA
hypomethylation
at
ICR2
accounting
for
nearly
50%
of
all
BWS
cases.
Paternal
UPD
of
chromosome
11
(upd(11)pat)
is
the
second
most
common
alteration
(20%)
associated
with
BWS,
while
DNA
hypermethylation
at
ICR1
is
less
common
(5–10%)
[13].
In
several
patients
with
either
BWS
or
SRS,
CDKN1C
point
mutations
have
been
identified
as
disease
causing
[15].
CDKN1C:
gene
and
function
The
CDKN1C
gene
spans
1943
bp
and
contains
four
exons,
two
of
which
are
protein
coding
(Figure
2).
The
CDKN1C
protein
includes
three
functional
domains:
(i)
the
N-termi-
nal
CDK
inhibition
domain
(CdK);
(ii)
the
proline–alanine
repeat
(PAPA)
domain,
which
includes
a
hexanucleotide
repeat
encoding
a
proline–alanine
stretch
that
is
variable
in
length;
and
(iii)
the
C-terminal
proliferating
cell
nuclear
antigen
(PCNA)-binding
domain.
CDKN1C
negatively
reg-
ulates
cell
proliferation
by
inhibiting
cyclin/CdK
complexes
during
the
G1
phase
of
the
cell
cycle
via
the
N-terminal
CdK
[15–17].
The
PAPA
domain
is
involved
in
mitogen-activated
protein
kinase
(MAPK)
phosphorylation
[18,19]
and
the
C-terminal
PCNA-binding
domain
binds
to
PCNA,
a
cofac-
tor
of
DNA
polymerases
that
encircles
DNA
and
orches-
trates
the
recruitment
of
factors
to
the
replication
fork
[20].
CDKN1C
is
able
to
interact
with
transcription
factors
(such
as
MyoD)
and
proteins
of
the
cJun/stress-activated
kinase
pathway
[21,22].
Though
CDKN1C
is
a
cell
cycle
inhibitor,
disturbed
interactions
with
these
factors
due
to
Box
1.
The
11p15-associated
syndromes
BWS
BWS
was
initially
known
as
EMG
syndrome
due
to
its
three
cardinal
features:
exomphalos,
macroglossia,
and
(neonatal)
gigantism.
Addi-
tional
clinical
features
of
BWS
include
neonatal
hypoglycemia,
hemihypertrophy,
organomegaly,
earlobe
creases,
polyhydramnios,
facial
hemangioma,
renal
abnormalities,
and
cardiomyopathy.
In
5–
7%
of
BWS
patients,
embryonal
tumors
(most
commonly
Wilms’
tumor)
are
diagnosed
[39,40,44].
A
broad
spectrum
of
epigenetic
and
genomic
mutations
in
11p15.5
has
been
identified
and
accounts
for
about
80%
of
BWS
cases
[39,40,44].
A
correlation
between
the
molecular
subtype
and
the
risk
of
developing
neoplasia
has
been
described
[60];
therefore,
molecular
confirmation
of
BWS
is
required.
SRS
SRS
is
characterized
by
pre-
and
postnatal
growth
restriction
[58,61].
Patients
show
relative
macrocephaly
and
a
triangular-shaped
face
with
a
broad
forehead
and
a
pointed,
small
chin.
In
many
cases,
asymmetry
of
limbs
and
body
is
present
as
well
as
fifth-finger
clinodactyly.
Like
BWS,
the
diagnosis
of
SRS
is
hampered
by
the
unspecific
nature
of
several
of
these
clinical
symptoms.
The
clinical
heterogeneity
is
reflected
by
the
complex
molecular
findings
in
SRS
patients:
about
7–10%
carry
maternal
UPD
of
chromosome
7
(upd(7)mat),
while
hypomethylation
of
ICR1
in
11p15.5
is
present
in
more
than
40%
of
patients
[13].
Currently,
approximately
half
of
SRS
patients
do
not
have
a
molecular
diagnosis.
It
should
be
noted
that
some
patients
have
been
reported
who
were
initially
diagnosed
with
SRS
but
were
then
identified
as
carriers
of
other
microdeletions/
duplications
[62].
IMAGe
syndrome
IMAGe
syndrome
is
named
for
the
acronym
of
its
major
features:
intrauterine
growth
retardation
(IUGR),
metaphyseal
dysplasia,
adrenal
hypoplasia
congenita,
and
genital
anomalies
[63,64].
Adrenal
hypoplasia
congenita
is
the
most
common
feature
of
IMAGe
syndrome,
often
resulting
in
vomiting,
feeding
difficulties,
dehydra-
tion,
and
hypoglycemia.
Growth
retardation
persists
after
birth.
The
skeletal
findings
are
rather
mild
and
can
be
difficult
to
recognize
on
radiographs;
in
some
affected
individuals,
scoliosis
or
osteoporosis
has
been
observed.
The
genital
abnormalities
occur
only
in
affected
males
(micropenis,
cryptorchidism,
microorchidism,
and
in
some
cases
hypospadias).
Less
characteristic
features
include
distinctive
facial
features
(prominent
forehead,
low-set
ears,
short
nose
with
a
flat
nasal
bridge),
craniosynostosis,
cleft
or
bifid
uvula,
a
high-arched
palate,
and
micrognathia,
hypercalcemia,
and
hypercalciuria.
Single
IMAGe
patients
have
been
initially
diagnosed
as
having
SRS
[65].
The
only
molecular
defects
in
IMAGe
syndrome
reported
so
far
are
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CDKN1C
upregulation
could
inhibit
the
UV-
or
stress-
induced
apoptotic
pathway
and
thus
may
contribute
to
cancer
progression
or
resistance
to
therapy
in
some
tumors
[23].
This
concept
is
in
accordance
with
observations
of
CDKN1C
overexpression
in
several
tumors
including
hepatoblastoma
and
head
and
neck
cancers
[24,25].
How-
ever,
downregulation
of
CDKN1C
might
also
contribute
to
cancer
formation
–
for
example,
in
Wilms’
tumor
[26]
or
hepatocellular
carcinoma
[27]
–
by
reduced
ability
to
block
the
cell
cycle.
CDKN1C
is
expressed
in
heart,
lung,
brain,
kidney,
pancreas,
testis,
and
skeletal
muscle,
as
well
as
in
placen-
ta,
and
has
therefore
been
implicated
in
embryonic
devel-
opment.
While
in
most
tissues
cdkn1c/CDKN1C
[28,29]
is
expressed
from
the
maternal
chromosome
only
and
the
paternal
allele
is
silenced
in
cis
by
the
noncoding
RNA
KCNQ1OT1,
in
humans
the
imprint
seems
to
be
not
so
absolute.
The
paternal
allele
is
also
expressed
at
low
levels
in
most
tissues
and
at
levels
comparable
to
the
maternal
allele
in
fetal
brain
and
some
embryonal
tumors
[30].
The
paternally
expressed
CDKN1C
allele
contributes
between
10%
and
30%
of
transcripts
in
multiple
fetal
and
adult
tissues
[30,31].
This
may
explain
in
part
rare
cases
of
BWS
with
paternal
transmission
of
a
causative
CDKN1C
muta-
tion
[16].
Loss
of
methylation
of
the
maternal
ICR2
allele
corre-
lates
with
activation
of
KCNQ1OT1
and
a
decrease
of
CDKN1C
expression
[32,33]
and
is
associated
with
BWS.
By
contrast,
an
increase
in
CDKN1C
expression
has
been
reported
in
a
SRS
patient
carrying
dup(11p15.5)mat
[34]
and
in
two
fetuses
with
severe
growth
retardation
that
were
carrying
a
deletion
in
the
centromeric
domain
leading
to
Table
1.
Clinical
findings
in
the
three
congenital
disorders
associated
with
CDKN1C
mutations
BWS
SRS
IMAGe
syndrome
Frequency 1:13
700
1:100
000
25
patients
reported
worldwide
Clinical
findings
Major
clinical
findings Macrosomia
(>97th
centile)
Anterior
linear
earlobe
creases/
posterior
helical
ear
pits
Macroglossia
Omphalocele/umbilical
hernia
Visceromegaly
(liver,
spleen,
kidneys,
adrenal
glands,
pancreas)
Embryonal
tumors
(Wilms’
tumor,
hepatoblastoma,
neuroblastoma,
rhabdomyosarcoma,
adrenal
carcinomas)
Hemihyperplasia
Cytomegaly
of
the
fetal
adrenal
cortex
(pathognomonic)
Renal
abnormalities
including
structural
abnormalities,
nephromegaly,
nephrocalcinosis,
later
development
of
medullary
sponge
kidney
Placental
mesenchymal
dysplasia
Cardiomegaly
IUGR
(<10th
percentile)
Postnatal
growth
retardation
(<3rd
percentile)
Relative
macrocephaly
(normal
head
circumference;
3rd
to
97th
percentile)
Triangular
facies
Frontal
bossing/prominent
forehead
Hemihypoplasia
Limb,
body,
and/or
facial
asymmetry
Fifth-finger
clinodactyly
IUGR
Skeletal
abnormalities
(most
commonly
delayed
bone
age
and
short
stature;
occasionally,
metaphyseal
and
epiphyseal
dysplasia
of
varying
severity)
Adrenal
insufficiency
presenting
typically
in
the
first
month
of
life
as
an
adrenal
crisis
or,
rarely,
later
in
childhood
with
failure
to
thrive
and
recurrent
vomiting
Genital
abnormalities
in
males
(cryptorchidism,
micropenis,
and
hypospadias)
Minor/supportive
clinical
findings
Cleft
palate
Cardiomyopathy
Polyhydramnios
Neonatal
hypoglycemia
with
hyperinsulinism
Facial
nevus
flammeus,
other
vascular
malformations
Characteristic
facies
(mid-face
hypoplasia,
infraorbital
creases)
Structural
cardiac
anomalies
Diastasis
recti
Advanced
bone
age
Cafe´
au
lait
spots
or
skin
pigmentary
changes
Genitourinary
anomalies
(cryptorchidism,
hypospadias)
Motor,
speech,
and/or
cognitive
delays
Feeding
difficulties
Hypoglycemia
Molecular
alterations
Aberrant
ICR1
(H19/IGF2)
methylation
Hypermethylation:
5–7%
Hypomethylation:
40%
–
Aberrant
ICR2
(LIT1/KvDMR1)
methylation
Hypomethylation:
50–60%
–
–
Large
11p15
duplications
(including
ICR1
and
ICR2)
Paternal:
1%
Maternal:
1%
–
Small
ICR2
duplications
and
deletions
Single
cases;
the
clinical
outcome
depends
on
the
size
and
genomic
content
of
the
affected
segment
UPD
11p15 upd(11p15)pat:
20–25%
upd(11p15)mat:
single
cases
–
CDKN1C
point
mutations Loss-of-function
mutations
Sporadic
cases:
5%
Familial
cases:
50%
Gain-of-function
mutations
Sporadic
cases:
–
(n
=
128)
Familial
cases:
one
case
Gain-of-function
mutations
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overexpression
of
CDKN1C
[35].
Furthermore,
cis
elements
required
for
enhanced
expression
of
the
maternally
inher-
ited
Cdkn1c
have
been
postulated
to
be
in
close
proximity
in
11p15.5
(>25
kb
downstream
of
the
gene)
in
mouse
[36].
Cer-
rato
et
al.
[37]
recently
suggested
that
human
CDKN1C
expression
is
regulated
by
at
least
three
enhancer
sequences
localized
in
KCNQ1OT1.
This
suggestion
is
based
on
the
evolutionary
conservation
of
these
regions
and
by
functional
analyses
such
as
DNase
hypersensitivity,
transcription-fac-
tor
binding,
and
histone
modifications.
The
identification
of
patients
carrying
paternal
deletions
in
the
proposed
enhanc-
er
region
has
further
corroborated
this
work
(for
a
review,
see
[37]).
Congenital
disorders
associated
with
CDKN1C
mutations
Due
to
its
function
as
a
negative
regulator
of
cellular
proliferation,
alterations
in
CDKN1C
should
be
associat-
ed
with
aberrant
growth.
Indeed,
mutations
of
CDKN1C
contribute
to
the
mutational
spectrum
of
BWS
[38].
These
CDKN1C
mutations
belong
to
a
spectrum
of
genomic
and
epigenetic
disturbances
within
the
chromosomal
region
11p15.5
that
cause
BWS
[38–40].
Interestingly,
the
oppo-
site
statuses
of
these
11p15.5
alterations
are
detectable
in
SRS,
a
primordial
growth-retardation
disorder
(Box
1
and
Table
1).
For
example,
ICR1
is
hypermethylated
in
BWS
and
hypomethylated
in
SRS
and
paternal
duplica-
tions
have
been
observed
in
BWS
whereas
maternal
duplications
are
observed
in
SRS.
With
recent
reports
of
CDKN1C
mutations
in
growth-retarded
patients
with
IMAGe
syndrome
[41]
or
SRS
[15],
the
key
role
in
growth
of
factors
in
the
chromosomal
region
11p15.5
could
be
confirmed.
Mutations
of
CDKN1C:
types
and
functional
relevance
The
nature
of
molecular
alterations
affecting
the
sequence
and
copy
number
of
CDKN1C
and
its
neighboring
genomic
region
show
an
impressive
correlation
with
the
phenotype
of
their
carriers
and
allow
major
insights
into
the
function
and
role
of
this
cell
cycle-inhibiting
protein.
In
general,
three
types
of
mutation
can
be
distinguished:
(i)
loss-of-
function
CDKN1C
point
mutations
that
are
associated
with
BWS;
(ii)
gain-of-function
CDKN1C
point
mutations
that
cause
growth-retardation
syndromes
such
as
IMAGe
and
SRS
(currently
these
have
been
detected
only
in
the
PCNA-binding
domain);
and
(iii)
large
alterations
(such
as
deletions,
duplications,
and
UPDs)
in
11p15.5,
the
effect
of
which
on
the
expression
of
CDKN1C
depends
on
their
size
and
genomic
content.
Loss-of-function
mutations:
BWS
Maternally
inherited
CDKN1C
mutations
have
been
de-
scribed
in
up
to
5%
of
sporadic
and
50%
of
familial
cases
of
BWS
[42].
Most
often,
they
are
truncating
or
frameshift
mutations
and
distributed
along
the
entire
coding
region
(Figure
2),
whereas
missense
mutations
are
mainly
local-
ized
in
the
CdK.
In
rare
cases,
intronic
mutations
affecting
mRNA
splicing
have
been
described
[17,19,43].
Function-
ally,
CDKN1C
point
mutations
in
BWS
patients
have
been
classified
as
loss-of-function
variants
and
result
in
a
loss
of
cell
cycle
inhibition.
This
assumption
has
been
based
on
CDKN1C
inactivation
in
mouse
models,
which
exhibit
a
IMAGe(A)
(B)
(C)
(D)
(E)
SRS
BWS
p.Ala4Val
p.Leu33Arg
p.Leu42Pro
p.Gln47X
p.Leu50Pro
p.Tyr61Arg
p.Tyr63Asn
p.Pro70Leu
p.Gln78X
p.Tyr91His
c.821-2A>G
c.821-3C>G
p.Pro36Argfs*7
p.Asp62_Phe65delinsVal
p.Asp85_Valfs*39
p.Leu104Glyfs*168
p.Glu131Valfs*8
p.Glu134Glyfs*5
p.Leu154Trpfs*118
p.Leu154Argfs*118
p.Ala167Argfs*100
p.Pro172Ala176del
p.Pro194Glnfs*78
p.Gly234Alafs*38
p.Ala262Glyfs*23
p.Thr254Profs*18
p.Gln230X
p.Gln232X
p.Gln241X
p.Ser247X
p.Ser282X
p.Arg316Trp
Exon 3
Exon 2
Exon 1 Exon 4
p.Ile272Ser
p.Asp274Asn
p.Phe276Val
p.Phe276Ser
p.Lys278Glu
p.Arg279Pro
p.Arg281Ile
p.Arg279Leu
PCNA
(272-283)
p.Leu33His
NLS
Phosphorylaon
Ubiquinaon
CdK
(31-83)
PAPA
(169-189)
TRENDS in Molecular Medicine
Figure
2.
Functional
organization
of
the
CDKN1C
gene
and
protein.
The
organization
of
CDKN1C
(C)
across
four
exons.
Exons
2
and
3
encode
the
functional
protein,
represented
in
(D)
with
the
three
key
domains:
the
cyclin-dependent
kinase-inhibitor
domain
(CdK);
the
proline–alanine
repeat
(PAPA)
domain;
and
the
proliferating
cell
nuclear
antigen
(PCNA)
domain.
Numbers
under
domains
refer
to
amino
acids
(aa).
Also
depicted
are
the
ubiquitination
region
(aa
1–105),
nuclear
localization
signal
(NLS)
(aa
278–281),
and
phosphorylation
site
(aa
310).
The
figure
also
depicts
reported
mutations
in
IMAGe
patients
(A),
Silver–Russell
syndrome
(SRS)
(B),
and
Beckwith–
Wiedemann
syndrome
(BWS)
(E).
The
gain-of
function
mutations
in
(A)
IMAGe
patients
(indicated
by
red
circle)
and
(B)
the
SRS
family
(orange
circle)
are
all
localized
within
the
PCNA
domain;
however,
the
loss-of-function
mutations
in
(E)
BWS
(blue
circles)
are
distributed
throughout
the
gene.
Missense
mutations
are
indicated
by
hollow
circles,
nonsense
mutations
by
red
filled
circles,
frameshift
mutations
by
blue
filled
circles,
and
intronic/noncoding
mutations
by
gray
filled
circles.
Deletions/small
indels
are
represented
by
light-green
filled
circles.
Domains
are
not
drawn
to
scale.
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phenotype
that
closely
resembles
BWS
[39,44].
Further-
more,
downregulation
of
CDKN1C
leading
to
decreased
function
can
indirectly
be
achieved
by
multiple
mecha-
nisms,
including
KCNQ1OT1
overexpression
due
to
ICR2
hypomethylation
and
repressive
chromatin
structure
(for
reviews,
see
[32,33]).
BWS
patients
with
mutations
in
CDKN1C
nearly
al-
ways
show
abdominal
wall
defects
(i.e.,
exomphalos
or
at
least
umbilical
hernia,
although
these
are
rare
in
UPD
and
ICR1
hypermethylation)
and
seem
to
more
often
present
with
cleft
palate,
genital
abnormalities,
polydactyly,
and
extra
nipples
than
BWS
patients
with
other
molecular
defects
[19].
Loss
of
Cdkn1c
in
mice
results
in
developmen-
tal
abnormalities
also
observed
in
BWS,
including
abdom-
inal
wall
defects
and
cleft
palate,
gastrointestinal
abnormalities
ranging
from
an
inflated
gastrointestinal
tract
to
loss
of
the
jejunum
and
ileum
[45],
hypertrophic
chondrocytes,
renal
medullary
dysplasia,
adrenal
cortical
hyperplasia
and
cytomegaly
and
lens
cell
hyperprolifera-
tion
[46],
placentomegaly
[47],
and
maternal
preeclampsia
[48].
Surprisingly,
no
somatic
overgrowth
at
birth
was
observed
in
these
mice;
however,
the
embryos
of
Cdkn1c
knockout
mice
are
nearly
20%
heavier
until
E18.5
than
corresponding
wild
type
embryos
[49].
Tunster
and
collea-
gues
[49]
argue
that
loss
of
function
of
Cdkn1c
results
in
somatic
overgrowth;
however,
in
multiparous
mice,
this
overgrowth
is
attenuated
by
intrauterine
competition,
un-
like
singleton
human
pregnancies.
Gain-of-function
mutations:
IMAGe
syndrome
and
SRS
In
contrast
to
the
mutations
observed
in
BWS,
CDKN1C
mutations
associated
with
the
growth-retardation
syn-
dromes
represent
gain-of-function
alterations.
These
mutations
are
generally
located
in
the
PCNA-binding
do-
main
of
the
gene.
This
domain
is
required
for
PCNA-
dependent
and
CRL4Cdt2-mediated
ubiquitination
(for
a
review,
see
[43]),
but
compared
with
the
PCNA
motif
in
the
closely
related
CDKN1A,
the
CDKN1C
motif
is
imperfect.
As
a
result,
low-affinity
binding
to
PCNA
has
been
sug-
gested,
which
would
be
sufficient
for
monoubiquitination
but
not
for
the
polyubiquitination
needed
for
degradation
[50].
Three
potential
sites
can
be
predicted
in
the
consen-
sus
sequence
for
PCNA-dependent
and
CRL4Cdt2-medi-
ated
ubiquitination:
Lys278,
Lys280,
and
Lys
286.
In
addition,
the
consensus
overlaps
a
bipartite
signal
for
nuclear
localization.
The
first
evidence
for
a
role
of
CDKN1C
mutations
in
growth
retardation
was
published
in
2012,
based
on
a
five-
generation
family
with
seven
siblings
and
four
unrelated
patients
affected
by
IMAGe
syndrome.
Arboleda
et
al.
[41]
identified
five
different
gain-of
function
mutations.
The
group
demonstrated
that
the
targeted
expression
of
some
of
these
mutations
in
Drosophila
caused
severe
eye-growth
defects
and
therefore
they
postulated
that
the
IMAGe
syndrome-associated
CDKN1C
mutations
lead
to
aggra-
vated
inhibition
of
growth
and
differentiation.
One
year
later,
Brioude
et
al.
[15]
reported
on
the
first
CDKN1C
mutation
in
a
SRS
family,
p.Arg279Leu.
The
mutation
segregated
with
the
SRS
phenotype
in
a
four-generation
family
with
nine
female
SRS
patients.
In
all
affected
members,
the
mutation
and
the
phenotype
were
inherited
by
the
mothers.
The
clinical
diagnosis
of
SRS
was
based
on
three
or
four
positive
criteria
from
the
score
published
by
Netchine
et
al.
[51];
however,
there
was
no
evidence
for
IMAGe
syndrome.
Body
asymmetry,
one
of
the
major
features
of
SRS,
was
also
not
observed.
Despite
this
excit-
ing
finding,
further
carriers
of
CDKN1C
mutations
could
not
be
detected
in
either
sporadic
(n
=
128)
or
familial
(n
=
29)
SRS
patients
[15,52].
Interestingly,
gain-of-function
mutations
affecting
the
same
amino
residue
have
been
reported
for
both
IMAGe
syndrome
and
SRS
(p.Arg279Pro
and
p.Arg279Leu)
[15,41].
The
Arg279
residue
has
been
strongly
conserved
in
evolution,
but
in
flow
cytometry
studies
the
SRS-specific
variant
p.Arg279Leu
did
not
affect
the
cell
cycle
[15].
In
the
same
experiment,
the
IMAGe
syndrome
mutation
p.Arg279Pro
demonstrated
a
positive
effect
on
the
cell
cycle.
Further
analysis
revealed
that
p.Arg279Leu
was
associated
with
increased
stability
of
the
protein,
an
ob-
servation
in
agreement
with
that
of
Hamajima
et
al.
[53].
Furthermore,
it
can
be
speculated
that
the
two
muta-
tions
differently
affect
monoubiquitination
and
thereby
the
regulatory
properties
of
the
domain.
From
the
clinical
viewpoint,
Brioude
et
al.
[15]
explained
the
lack
of
adrenal
hypoplasia
in
their
SRS
family
by
the
less
severe
effect
of
p.Arg279Leu
observable
in
their
functional
assays,
which
might
not
affect
development
of
the
adrenal
cortex.
Another
growth-retarded
family
with
a
gain-of-function
mutation
(p.Arg281Ile)
in
the
PCNA
domain
has
been
reported
recently;
however,
this
family
showed
a
non-
IMAGe
phenotype
[Kerns,
S.L.
et
al.
(2014)
Novel
variant
in
CDKN1C
associated
with
intrauterine
growth
restric-
tion,
short
stature,
and
early-adulthood
onset
diabetes.
ICE/ENDO
2014
(https://www.endocrine.org/endo-2014/
program-and-events/scientific-program)].
Similar
to
the
SRS
case
described
above
[15],
there
was
no
evidence
for
adrenal
insufficiency.
This
mutation
may
affect
PCNA-
dependent
ubiquitination
or
nuclear
localization.
Large
imbalances
(deletions/duplications)
and
UPD
The
activating
or
silencing
nature
of
point
mutations
in
the
CDKN1C
gene
is
also
reflected
by
larger
duplications
or
deletions
within
11p15.5
including
CDKN1C.
However,
two
molecular
properties
of
11p15.5
must
be
considered
to
delineate
the
functional
consequence
of
genomic
imbal-
ance:
(i)
the
parent
of
origin;
and
(ii)
the
size
of
the
deletion/
duplication.
CDKN1C
is
expressed
from
the
maternal
allele;
there-
fore,
duplications
of
the
maternal
CDKN1C
copy
cause
increased
expression
that
results
in
growth
retardation
[34].
However,
one
BWS
patient
has
been
described
with
a
160-kb
inverted
maternal
microduplication
of
the
centro-
meric
imprinting
cluster.
This
particular
duplication
leads
to
the
expression
of
a
truncated
KCNQ1OT1
transcript
and
to
CDKN1C
silencing
in
the
case
of
maternal
transmission
[54].
Duplications
of
the
paternal
copy
can
cause
BWS,
although
these
rarely
affect
only
the
centromeric
imprinting
cluster
and
do
not
always
include
the
CDKN1C
gene.
There-
fore
the
phenotype
has
been
postulated
to
be
caused
by
the
disturbed
expression
of
other
11p15.5
encoded
factors
like
KCNQ1OT1
or
other
ICR2-regulated
transcripts
that
also
can
have
a
modifying
effect
on
CDKN1C
expression
[55].
Review Trends
in
Molecular
Medicine
xxx
xxxx,
Vol.
xxx,
No.
x
TRMOME-976;
No.
of
Pages
9
6

Large
11p15.5
duplications
affecting
both
ICR1
and
ICR2
are
generally
associated
either
with
BWS
(in
the
case
of
duplications
of
the
paternal
copy)
or
with
SRS
(in
the
case
of
the
maternal
copy;
Figure
3
and
Table
1).
Smaller
duplications
or
deletions
within
ICR2
require
careful
characterization.
As
CDKN1C
expression
is
regu-
lated
by
the
CDKN1C
promoter,
enhancer
sequences,
and
the
noncoding
RNA
KCNQ1OT1,
the
functional
conse-
quences
of
small
deletions
or
duplications
depend
on
the
precise
content
and
the
parental
origin
of
the
affected
genomic
region.
As
a
result,
overgrowth,
growth
retarda-
tion,
or
a
normal
phenotype
can
result
(for
a
review,
see
[56]).
Relevance
for
genetic
counseling
and
clinical
management
Mutations
within
the
CDKN1C
coding
sequence
can
lead
to
opposite
phenotypes
and
this
association
appears
to
be
very
strict;
therefore,
the
phenotype
in
families
with
CDKN1C
point
mutations
should
be
uniform:
either
growth
retardation/SRS
or
BWS
should
occur
in
the
same
family.
However,
only
maternal
inheritance
of
the
CDKN1C
variant
is
associated
with
an
aberrant
phenotype
(Figure
3A).
Due
to
the
silencing
of
the
paternal
CDKN1C
copy,
males
carrying
a
CDKN1C
mutation
are
not
at
risk
of
having
children
with
one
of
these
disorders,
but
50%
of
their
children
will
be
heterozygous
carriers
of
the
muta-
tion.
A
heterozygous
daughter
would
mean
that
the
grand-
children
of
these
unaffected
male
carriers
have
an
increased
risk
(50%).
In
the
case
of
larger
deletions/duplications
affecting
the
CDKN1C
gene
and
larger
parts
of
the
11p15.5
region,
both
the
parental
origin
of
the
affected
chromosome
and
the
size
of
the
imbalance
require
careful
analysis.
Any
diag-
nostic
algorithm
should
comprise
quantitative
approaches
and
molecular
karyotyping
[e.g.,
array-based
comparative
genome
hybridization
(aCGH),
single
nucleotide
polymor-
phism
(SNP)
array
typing]
as
well
as
cytogenetic
and
molecular
cytogenetic
[fluorescence
in
situ
hybridization
(FISH)]
analyses
of
whole
families
to
identify
familial
translocations
(Figure
3B).
For
whole
11p15.5
duplica-
tions
that
include
both
ICRs,
a
SRS
phenotype
can
be
expected
when
the
maternal
chromosome
is
duplicated;
by
contrast,
a
BWS
phenotype
is
expected
for
the
paternal
duplication
(for
a
review,
see
[56]).
However,
it
should
be
noted
that
in
these
cases
the
clinical
outcome
might
also
be
influenced
by
other
genes
affected
by
the
aberration.
Whole
11p15.5
deletions
have
not
been
reported
and
are
probably
not
viable.
In
cases
of
smaller
deletions/duplica-
tions
in
11p15.5
that
affect
CDKN1C
itself
and/or
regula-
tory
elements
(e.g.,
enhancers),
the
extent
and
the
parent
of
origin
of
the
affected
fragment
has
to
be
precisely
determined,
as
both
clinically
affected
and
unaffected
individuals
have
been
reported
(for
reviews,
see
[37,56]),
and
prediction
of
the
clinical
outcome
might
be
difficult.
Based
on
these
divergent
possibilities,
we
propose
that
appropriate
molecular
tests
should
be
considered
in
the
following
situations.
(i)
Sequencing
of
the
CDKN1C
gene
should
be
performed
in
patients
with
a
family
history
of
BWS.
Further-
more,
CDKN1C
sequencing
should
be
considered
in
BWS
patients
born
to
preeclamptic
women
or
with
cleft
palate,
hypospadias,
and/or
supernumerary
nipples
[57].
In
cases
of
sporadic
BWS,
CDKN1C
testing
should
be
performed
in
patients
with
umbili-
cal
hernia
or
exomphalos
[8],
as
mutations
have
not
yet
been
reported
to
be
associated
with
macroglossia
or
in
cases
of
isolated
hemihyperplasia.
Systematic
screening
studies
and
comprehensive
genotype–
phenotype
studies
will
have
to
address
this
issue
in
the
near
future.
BWS: Loss-of funcon mutaons
IMAGe/SRS: Gain-of funcon mutaons
Unaffected, non-carrier
Affected, mutaon carrier
Unaffected, mutaon carrier
Unaffected, balanced 11p15 translocaon carrier
Key: Key:
BWS, paternal 11p15 duplicaon carrier
SRS, maternal 11p15 duplicaon carrier
BWS/SRS: 11p15 translocaon/duplicaons(A)
(B)
TRENDS in Molecular Medicine
Figure
3.
Clinical
outcome
of
11p15
mutations
in
families.
Family
pedigrees
are
shown
with
inheritance
of
(A)
point
mutations
(both
loss
and
gain
of
function)
and
(B)
large
11p15.5
duplications
including
both
imprinting
control
region
(ICR)
1
and
ICR2
(it
should
be
noted
that
the
phenotype
of
carriers
of
smaller
imbalances
is
influenced
by
the
size
and
content
of
the
aberration).
In
these
cases,
further
analysis
of
the
parents
and
family
is
needed
to
identify
balanced
chromosomal
aberrations
resulting
in
increased
recurrence
risks.
Review Trends
in
Molecular
Medicine
xxx
xxxx,
Vol.
xxx,
No.
x
TRMOME-976;
No.
of
Pages
9
7

(ii)
The
PCNA
region
(30end
of
exon
2
and
exon
3)
of
CDKN1C
should
be
sequenced
in
patients
in
whom
IMAGe
syndrome
is
suspected.
(iii)
In
SRS,
the
PCNA
region
of
CDKN1C
should
be
analyzed
only
in
familial
cases,
as
screening
of
a
large
cohort
of
sporadic
patients
as
well
as
of
29
patients
with
a
positive
family
history
of
growth
retardation
[15]
did
not
reveal
any
pathogenic
variant.
From
a
clinical
viewpoint,
the
identification
of
a
CDKN1C
mutation
can
confirm
the
diagnosis.
However,
for
IMAGe
syndrome
and
SRS
it
is
too
early
for
a
geno-
type–phenotype
correlation
as
a
basis
for
personalized
management,
due
to
the
low
number
of
cases.
In
SRS,
slightly
differing
responses
to
growth
hormone
treatment
in
11p15.5
and
upd(7)mat
patients
have
been
observed
[58]
and
follow-up
of
CDKN1C-associated
patients
will
thus
be
needed
to
confirm
this
observation.
As
both
syndromes
have
not
yet
been
associated
with
increased
tumor
risk,
a
tumorigenic
nature
of
the
respective
CDKN1C
mutations
is
not
probable
and
a
specific
tumor
surveillance
program
is
therefore
not
required.
For
BWS
patients
the
situation
is
different
as
there
is
a
significant
risk
of
embryonal
tumors,
but
it
is
difficult
to
precisely
define
this
risk
for
CDKN1C
mutation
carriers
as
the
number
of
patients
is
currently
too
small
and
the
types
of
tumor
might
differ
from
those
in
the
general
BWS
cohort
[39,59].
However,
after
exclusion
of
the
currently
known
11p15.5
disturbances
in
BWS
and
SRS,
and
CDKN1C
mutations
in
all
three
syndromes,
there
is
a
gap
of
patients
without
molecular
confirmation
of
the
clinical
diagnosis,
ranging
from
20%
in
BWS
to
40%
in
SRS
and
unknown
for
IMAGe.
Therefore,
permanent
follow-up
on
the
spectrum
of
molecular
alterations
within
the
known
genomic
regions
for
these
syndromes
is
required
and
must
be
implemented
in
the
routine
diagnostic
workup,
and
a
careful
clinical
diagnosis
is
needed
to
consider
differential
diagnoses
for
all
three
disorders
(e.g.,
Simpson–Golabi–Behmel
syn-
drome,
Sotos
syndrome,
and
Perlman
syndrome
in
the
case
of
BWS,
3
M
syndrome
in
the
case
of
SRS).
Concluding
remarks
Loss-of-function
mutations
of
the
CDKN1C
gene
have
been
known
to
cause
overgrowth,
but
with
recent
reports
of
gain-
of-function
mutations
in
CDKN1C
in
growth-retarded
patients
the
last
gap
in
the
impressive
strictly
opposite
molecular
spectrum
of
molecular
alterations
in
11p15.5
in
growth-retardation
and
overgrowth
syndromes
could
be
closed.
Based
on
the
current
data,
we
suggest
that
testing
of
CDKN1C
should
be
offered
to
patients
with
BWS
or
IMAGe
syndrome,
whereas
the
clinical
diagnosis
of
SRS
is
primarily
not
an
indication
for
CDKN1C
analysis.
We
especially
recommend
geneticists
and
pediatric
endocrinol-
ogists
to
be
aware
of
the
possible
occurrence
of
life-threat-
ening
conditions
in
the
offspring
of
women
carriers
related
to
a
family
member
with
IMAGe
syndrome.
The
recent
identification
of
gain-of-function
mutations
in
CDKN1C
generally
illustrates
how
mutational
diversity
in
humans
provides
us
with
fundamental
insights
into
basic
biological
mechanisms
and
their
dysregulation.
For
CDKN1C,
further
investigations
are
needed
to
fully
un-
derstand
the
mechanisms
responsible
for
the
pathogenici-
ty
of
its
mutations
and
to
illuminate
the
general
impact
of
11p15.5
disturbances
on
the
expression
of
its
subordinated
genes
(Box
2).
Acknowledgments
T.E.,
D.P.,
and
M.B.
are
supported
by
the
Bundesministerium
fu¨
r
Bildung
und
Forschung
(Network
‘Imprinting
Diseases’
01GM1114C
to
T.E.
and
01GM114D
to
D.P.)
and
Ipsen
Pharma.
The
authors
are
members
of
COST
Action
BM1208
and
the
European
Congenital
Imprinting
Dis-
orders
Network
(EUCID.net)
(http://www.imprinting-disorders.eu).
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Box
2.
Outstanding
questions

What
are
the
molecular
consequences
of
CDKN1C
gain-of-
function
mutations?
Does
monoubiquitination
represent
a
mod-
ification
that
affects
its
interaction
with
other
partners?

What
are
the
interaction
partners
influenced
by
CDKN1C
mono-
ubiquitination?

What
is
the
precise
molecular
basis
for
CDKN1C
regulation
and
are
the
predicted
enhancers
the
true
regulatory
elements?

Which
features
can
be
observed
in
CDKN1C
mutation
carriers
and
what
are
the
long-term
consequences
of
these
profound
genetic
defects?

Does
the
unraveling
of
CDKN1C
regulation
provide
us
with
new
targets
for
therapeutic
intervention
in
CDKN1C
disorders?

Do
other
molecular
defects
in
11p15.5
(e.g.,
epimutations,
UPDs)
affect
the
expression
of
CDKN1C
and
is
CDKN1C
dysregulation
a
common
feature
of
11p15.5-associated
disorders?
Review Trends
in
Molecular
Medicine
xxx
xxxx,
Vol.
xxx,
No.
x
TRMOME-976;
No.
of
Pages
9
8

15
Brioude,
F.
et
al.
(2013)
CDKN1C
mutation
affecting
the
PCNA-
binding
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