Gicquel, C., Rossignol, S., Cabrol, S., Houang, M., Steunou, V., Barbu, V. et al. Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome. Nat. Genet. 37, 1003-1007

Article (PDF Available)inNature Genetics 37(9):1003-7 · October 2005with28 Reads
DOI: 10.1038/ng1629 · Source: PubMed
Abstract
Silver-Russell syndrome (SRS, OMIM 180860) is a congenital disorder characterized by severe intrauterine and postnatal growth retardation, dysmorphic facial features and body asymmetry. SRS is genetically heterogenous with maternal uniparental disomy with respect to chromosome 7 occurring in approximately 10% of affected individuals. Given the crucial role of the 11p15 imprinted region in the control of fetal growth, we hypothesized that dysregulation of genes at 11p15 might be involved in syndromic intrauterine growth retardation. We identified an epimutation (demethylation) in the telomeric imprinting center region ICR1 of the 11p15 region in several individuals with clinically typical SRS. This epigenetic defect is associated with, and probably responsible for, relaxation of imprinting and biallelic expression of H19 and downregulation of IGF2. These findings provide new insight into the pathogenesis of SRS and strongly suggest that the 11p15 imprinted region, in addition to those of 7p11.2-p13 and 7q31-qter, is involved in SRS.

Figures

Epimutation of the telomeric imprinting center region
on chromosome 11p15 in Silver-Russell syndrome
Christine Gicquel
1
, Sylvie Rossignol
1
, Sylvie Cabrol
1
, Mur iel Houang
1
, Virginie Steunou
1
,Ve
´
ronique Barbu
2
,
Fabienne Danton
1
, Nathalie Thibaud
1
, Martine Le Merrer
3
, Lydie Burglen
4
, Anne-Marie Bertrand
5
,
Ire
`
ne Netchine
1
& Yves Le Bouc
1
Silver-Russell syndrome (SRS, OMIM 180860) is a congenital
disorder characterized by severe intrauterine and postnatal
growth retardation, dysmorphic facial features and body
asymmetry. SRS is genetically heterogenous with maternal
uniparental disomy with respect to chromosome 7 occurring
in B10% of affected individuals. Given the crucial role of the
11p15 imprinted region in the control of fetal growth, we
hypothesized that dysregulation of genes at 11p15 might be
involved in syndromic intrauterine growth retardation. We
identified an epimutation (demethylation) in the telomeric
imprinting center region ICR1 of the 11p15 region in several
individuals with clinically typical SRS. This epigenetic defect is
associated with, and probably responsible for, relaxation of
imprinting and biallelic expression of H19 and downregulation
of IGF2. These findings provide new insight into the
pathogenesis of SRS and strongly suggest that the 11p15
imprinted region, in addition to those of 7p11.2-p13 and
7q31-qter, is involved in SRS.
Human chromosome 11p15.5 contains a cluster of imprinted genes
that are crucial in the control of fetal growth. This cluster includes
paternally expressed (maternally imprinted) genes (such as IGF2 and
KCNQ1OT1) and maternally expressed (paternally imprinted) genes
(such as CDKN1C and H19). Studies of several transgenic or knockout
mouse models and of Beckwith-Wiedemann syndrome (BWS) in
humans indicate that this region has a key role in fetal development.
The main feature of mouse knockouts lacking either Ig f2 or Kcnq1ot1
is fetal growth retardation
1,2
. In contrast, disruption of H19 (resulting
in biallelic expression of Igf2) or transactivation of Igf2 in the
mouse leads to fetal overgrowth
3,4
. BWS is a model imprinting
disorder characterized by prenatal and postnatal overgrowth, macro-
glossia, abdominal wall defects, organomegaly, hemihyperplasia, hypo-
glycemia, ear abnormalities and an increased risk of childhood
tumors. BWS is caused by genetic or epigenetic alterations in the
imprinted 11p15 region
5
.
SRS is clinically and genetically heterogeneous, and several chro-
mosomal abnormalities involving chromosomes 7, 8, 15, 17 and 18
have been associated with SRS and SRS-like cases
6,7
. Only chromo-
somes 7 and 17 have been consistently implicated in individuals with a
strict diagnosis of SRS. Maternal uniparental disomy with respect to
chromosome 7 occurs in B10% of SRS cases; these individuals
generally have a milder phenotype
8
.
There are no reports concerning the epigenetic status of the 11p15
region in individuals with intrauterine growth retardation. Duplica-
tions of chromosome 11p15 of maternal origin in individuals with
phenotypes consistent with SRS have been identified
9,10
,butno
pathogenic mutations in IGF2, CDKN1C or KCNQ1OT1 were
found in individuals with SRS
11,12
.
The strong involvement of the 11p15 region in fetal growth, the
severe growth retardation in individuals with maternally derived
11p15 duplications and the frequent occurrence of body asym-
metry in individuals with SRS led us to investigate whether indivi-
duals with SRS who do not have maternal uniparental disomy
with respect to chromosome 7 had epigenetic defects in the
11p15 region.
We first examined the methylation status of KvDMR1 in
KCNQ1OT1 (centromeric domain) and the H19 promoter (telomeric
domain) in the 11p15 imprinted region in leukocyte DNA from nine
individuals with SRS (Table 1 and Supplementary Table 1 online).
Five individuals (individuals 1, 3, 4, 6 and 8; Table 1 and Fig. 1)
showed partial loss of H19 promoter methylation (methylation indices
(MIs) between 8.5 and 32.3%; normal MI (mean ± s.d.) ¼ 53.3 ±
3.1%; ref. 5) but had normal methylation of KCNQ1OT1. Individual 6
has a twin, who also showed partial loss of H19 promoter methylation
with a MI of 27% (Fig. 1).
We assessed the methylation status of H19-IGF2 imprinting center
region 1 (ICR1, also called H19 differentially methylated region
(DMR)), located upstream of H19, by Southern blotting (Fig. 2a).
The five individuals with hypomethylation of the H19 promoter
and individual 6’s twin showed a partial loss of H19-IGF2 ICR1
Published online 7 August 2005; doi:10.1038/ng1629
1
Laboratoire d’Explorations Fonctionnelles Endocriniennes, Inserm U515 et UPMC Paris 6, Ho
ˆ
pital Armand Trousseau, AP-HP, 26 avenue Arnold Netter, 75012 Paris,
France.
2
Inserm U680, Faculte
´
Saint-Antoine, 27 rue Chaligny 75012 Paris, France.
3
De
´
partement de Ge
´
ne
´
tique, Ho
ˆ
pital Necker-Enfants Malades, 149 rue de Se
`
vres,
75015 Paris, France.
4
Unite
´
de Ge
´
ne
´
tique, Ho
ˆ
pital Armand Trousseau, 26 avenue Arnold Netter, 75012 Paris, France.
5
Service d’Endocrinologie Pe
´
diatrique, Ho
ˆ
pital
de Besanc¸on, 2 Place Saint-Jacques, 25030 Besanc¸on, France. Correspondence should be addressed to C.G. (christine.gicquel@trs.aphp.fr).
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methylation (Ta ble 1 and Fig. 2b,c). In skin fibroblasts, individual 6
showed a loss of H19-IGF2 ICR1 methylation (MI ¼ 10%) but her
twin had a normal methylation pattern (MI ¼ 50%; Fig. 2b).
We then carried out bisulfite genomic sequencing to determine the
methylation status of repeat B1 containing CCCTC-binding factor
(CTCF) site 6 (Fig. 2a) in individuals 3 and 8. We observed intense
demethylation of CTCF site 6 in these individuals relative to control
subjects (Ta ble 1 and Fig. 2d,e).
Analysis of ICR1 by ApaI digestion and Southern blotting showed a
normal restriction pattern, with a 7.7-kb fragment detected in all
individuals with SRS and control subjects (data not shown), ruling out
the possibility of microdeletion.
IGF2 DMR2 is located in the ninth exon of IGF2 and is differentially
methylated, with the maternal allele hypomethylated. All five individu-
als with partial loss of methylation at ICR1 and the H19 promoter
also showed partial loss of methylation of IGF2 DMR2 (Ta b le 1 and
Fig. 3a,b). Individual 6 and her twin were informative for the ApaI-
AvaII polymorphism in the ninth exon of IGF2, making possible
the analysis of parental allele-specific methylation. Analysis after
digestion of leukocyte DNA with AvaII and HpaII confirmed partial
loss of methylation of the normally methylated 0.9-kb paternal allele
of IGF2 (Fig. 3a).
We examined the effect of the methylation defect on expression
of IGF2. We assessed total IGF2 RNA levels by real-time RT-PCR
100
80
60
40
20
H19 promoter
H19 promoter
KvDMR1 in
KCNQ1OT1
Control 1
Control 2
BWS1
BWS2
Ind 1
Ind 7
Ind 6's family
Ind 6
Ind 8
6 kb
Ind 3
Ind 4
Ind 6
Ind 2
Control 3
Father
Mother
Tw i n
0
1
2
3
4
5
6
6 twin
7
8
9
BWS1
4.2 kb
1.8 kb
1 kb
ab
Figure 1 Methylation analysis of KCNQ1OT1 and H19 in individuals with SRS. (a) Methylation analysis of KvDMR1 in KCNQ1OT1 and the H19 promoter in
individuals with SRS (including individual 6’s twin and parents), two individuals with BWS and three control subjects. Individual BWS1 has isolated
hypermethylation of the H19 promoter; individual BWS2 has isolated demethylation of KvDMR1 in KCNQ1OT1. The upper bands (6 and 1.8 kb) are
methylated and correspond to the maternal (KvDMR1 in KCNQ1OT1) and paternal (H19) alleles, respectively. The lower bands (4.2 and 1 kb) are
unmethylated and correspond to the paternal (KvDMR1 in KCNQ1OT1) and maternal (H19) alleles, respectively. (b) Quantitative representation of MIs for
the H19 promoter in individuals with SRS (including individual 6’s twin), individual BWS1 with isolated hypermethylation of the H19 promoter and control
subjects. The gray area corresponds to the range of normal MI values.
Table 1 Clinical and molecular characteristics of individuals with SRS
Individual 1 2 3 4 5 6
a
6stwin789
Sex F F M M F F F F F M
Growth
Gestational age, weeks 38 40 34 39 40 38 38 32 34 37.5
Birth height, cm (s.d.)
b
40 (–4.4) 45 (–3.3) 39 (–3.2) 42 (–3.9) 44 (–3.8) 38 (–5.3) 48 (–1.0) 38 (–2.5) 31 (–7.6) 41.5 (–3.8)
Birth weight, g (s.d.)
b
1600 (–4.2) 2410 (–2.3) 1270 (–3.5) 2270 (–2.4) 2500 (–2.1) 1780 (–3.7) 3450 (+0.6) 1380 (–1.2) 980 (–4.7) 1970 (–1.9)
Head circumference at birth,
cm (s.d.)
b
32 (–2) 35 (–0.1) 30 (–1.0) 33 (–1.6) 32 (–2.9) 33 (–1.1) NA 27.4 (–1.6) 30.5 (–0.7) 28.5 (–4.4)
Others
Typical facial features Yes Yes Yes Yes Yes Yes No Yes Yes Yes
Body asymmetry Yes Yes No Yes Yes Yes No Yes Yes Yes
Clinodactyly or brachydactyly Yes No Yes Yes Yes Yes No No Yes No
Genital abnormalities No No No No No No No No No Yes
Cafe
´
au lait spots NA No Yes No Yes No No No No No
Feeding difficulties No Yes Yes Yes No Yes No Yes Yes Yes
Delayed speech No No No No No Yes No Yes Yes Yes
Molecular characteristics
Demethylation of the H19
promoter (MI, %)
Yes (25.1) No (55.3) Yes (29.2) Yes (32.3) No (50.1) Yes (25.2) Yes (27) No (48) Yes (8.5) No (48.4)
Demethylation of ICR1
(MI, %)
Yes (29.9) No (60.6) Yes (37.6) Yes (40.8) No (53.4) Yes (18.2) Yes (25) No (54.3) Yes (10) No (55.2)
Demethylation of CTCF site 6
(MI, %)
ND ND Yes (21) ND ND ND ND ND Yes (4.8) ND
Demethylation of IGF2 DMR2
(MI, %)
Yes (34) No (50.2) Yes (30.9) Yes (22.3) No (50) Yes (20.7) Yes (22.6) No (51) Yes (10) ND
a
Individual 6 is a monozygotic (monochorial, biamniotic) twin; her twin had no SRS phenotype.
b
The number of standard deviations from the mean is indicated in parentheses.
NA, not available; ND, not done.
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in skin fibroblasts from individuals 6 and 8 and compared them
with those of individual 6’s twin and a control subject of the same
age, respectively. IGF2 levels in individual 6 were half those in her
twin (Fig. 3c), and IGF2 levels in individual 8 were one-fifth those
in the age-matched control subject (Fig. 3d), showing that loss
of methylation at ICR1 and IGF2 DMR2 led to the downregulation
of IGF2.
One of the individuals with hypomethylation at ICR1, the
H19 promoter and IGF2 DMR2 (individual 6) was informative
for the RsaI polymorphism of the fourth exon of H19.We
analyzed parental imprinting of H19 by RT-PCR analysis of total
RNA extracted from the blood cells of the proband, her twin and
her parents. Analysis of the digested cDNA PCR products (Fig. 4)
showed that transcripts from both parental alleles were present
in blood cells from individual 6 and her phenotypically normal
twin. This demonstrates relaxation of imprinting with biallelic expres-
sion of H19.
Our data indicate that the imprinted 11p15 region is involved in
SRS. The epimutation consists of a partial loss of paternal methylation
marks at three different loci (ICR1, the H19 promoter and IGF2
DMR2) in the telomeric imprinted domain of the 11p15 region.
In contrast, the methylation status of the centromeric domain is
maintained. This loss of methylation of the paternal allele results in
the relaxation of imprinting of H19 and expression of the normally
silent paternal allele. The intragenic DMR2 is necessary for high-level
transcription of the paternal IGF2 allele, and in vitro methylation of
DMR2 increases IGF2 transcript levels
13
. We show here that demethy-
lation of ICR1 and IGF2 DMR2 is associated with a decrease in IGF2
RNA levels. Our data for SRS are comparable to those for the recently
published mouse model depleted in CpG residues in the repeats of the
H19 DMR
14
. When paternally inherited, these mutations disrupt
maintenance of H19 DMR and H19 promoter methylation, resulting
in biallelic H19 expression, decrease in Igf2 expression and fetal
growth retardation.
The reciprocal imprinting of maternally expressed H19 and pater-
nally expressed IGF2 depends on differential methylation of ICR1
upstream of H19, which acts as an insulator. CTCF binds to maternal
unmethylated ICR1 and prevents the IGF2 promoter from interacting
with enhancers downstream of H19, resulting in transcriptional
silencing of the maternal IGF2 allele
15–17
. CTCF also maintains the
unmethylated state of ICR1 in the maternal allele
18,19
.TheH19 DMR
interacts differentially with the Igf2 DMRs in mice
20
.TheH19 DMR
100
80
60
A
CTCF 1
RH RH
Control
BWS1
M
23
Probe
Leukocytes
456 7
H H25
R4
B7 B6 B5 A2 B4 B3 B2 B1 A1
R5 R6
HH
HH HH H H H H
R7 A
1,000 bp
40
Control 1 Control 2 BWS1 Ind 3 Ind 8
20
H19-IGF2 ICR1
0
1
2
3
4
5
6
6 twin
7
8
9
BWS1
100
80
60
40
20
CTCF site 6
0
1
2
3
4
5
6
6 twin
7
8
9
BWS1
C1
C2
RH
Ind 3
RH
Ind 4
RH
Ind 5
RH
Ind 8
RH
Mother
Ind 6's family
Fibroblasts
RH
Tw i n
RH
Tw i n
RH
Ind 6
RH
1.5 kb
(R4-R5)
Ind 6
RH
Father
0.7 kb
(H25-R5)
bc
de
a
Figure 2 Methylation analysis of H19-IGF2 ICR1 in individuals with SRS. (a) Structure of H19-IGF2 ICR1. A, ApaIsite;R,RsaI site; H, HpaII site. RsaI
and HpaII sites are numbered as previously described
26
. The location of the probe used for analysis of the methylation status of H19-IGF2 ICR1 is
indicated. (b) DNA methylation of H19-IGF2 ICR1 determined by Southern blotting. Leukocyte and fibroblast DNA was digested with RsaI(R),RsaIand
HpaII (H) and RsaIandMspI (M), and Southern blots were hybridized with the probe indicated in a. Individual BWS1 has isolated hypermethylation of the
H19 promoter. (c) Quantitative representation of MIs for H19-IGF2 ICR1 in individuals with SRS (including individual 6’s twin), individual BWS1 with
isolated hypermethylation of the H19 promoter and control subjects. The gray area corresponds to the range of normal MI values. (d) DNA methylation
profiles of CTCF site 6 analyzed by bisulfite genomic sequencing in two individuals with SRS, individual BWS1 with isolated hypermethylation of the H19
promoter and two control subjects. Each line corresponds to a single cloned DNA molecule template, and each circle corresponds to a CpG dinucleotide.
Filled circles indicate methylated cytosines; open circles, unmethylated cytosines. (e) Quantitative representation of MIs for CTCF site 6 in two individuals
with SRS, individual BWS1 with isolated hypermethylation of the H19 promoter and two control subjects (C1 and C2).
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interacts with Igf2 DMR1 on the maternal allele and with Igf2 DMR2
on the paternal allele, partitioning maternal and paternal chromatin to
distinct loops.
The loss of paternal methylation marks in individuals with SRS may
have resulted from a deficient acquisition of methylation during sper-
matogenesis or from a lack of maintenance of methylation after
fertilization. Depletion of CpG residues in the H19 DMR in mice
does not affect acquisition of methylation in sperm but does affect the
maintenance of methylation after fertilization and before implanta-
tion
14
. The five individuals with SRS that carry the epimutation had
only a partial loss of methylation, and four individuals had body asym-
metry. These data suggest that the loss of methylation occurred after
fertilization and resulted in a mosaic distribution of the epimutation.
The epimutation described in individuals with SRS is the exact
opposite of one of the molecular defects responsible for BWS: B10%
of individuals with BWS have hypermethylation of the H19 promo-
ter
5
. H19 hypermethylation is associated with loss of expression of
H19, hypermethylation of IGF2 DMR2 and biallelic expression
of IGF2 (ref. 21). The most common epimutation in individuals
with BWS involves the centromeric 11p15 subdomain and consists of
a loss of methylation of the maternal KCNQ1OT1 allele. The paternal
inheritance of a null KCNQ1OT1 allele results in fetal growth
retardation by 20–25% (ref. 2) but does not affect expression of H19
or IGF2. None of the individuals with SRS that we report here showed
a gain of methylation of the paternal KCNQ1OT1 allele.
One of the five individuals with the epimutation was a monozygotic
twin, and her twin had no clinical features of SRS. Both twins had a
loss of methylation of the telomeric 11p15 domain in their leukocyte
DNA and biallelic expression of H19 in their blood cells. In skin
fibroblasts, only the affected twin showed abnormal methylation. This
observation is consistent with results obtained from BWS-discordant
monozygotic twins. Monozygotic twins are over-represented among
individuals with BWS
22
, and BWS is almost always discordant in pairs
of identical twins. The affected and unaffected twins always have the
same epigenetic defect in leukocyte DNA (loss of methylation of
KCNQ1OT1 in the 11p15 centromeric domain)
5,22
. In skin fibroblasts,
only the affected twins have altered methylation of KCNQ1OT1
(ref. 22), suggesting that the presence of the epigenetic defect in
blood cells of both twins results from shared fetal circulation.
The precise mechanism responsible for the loss of paternal methyla-
tion of the 11p15 telomeric domain in some individuals with SRS
remains to be elucidated. Sparago et al.
23
recently showed that two of
seven individuals with BWS with H19 hypermethylation had a
maternally transmitted microdeletion in ICR1. Although there was
no growth retardation phenotype in subjects with a paternally trans-
mitted microdeletion of ICR1 (ref. 23), we ruled out the possibility of
a deletion of ICR1 in the individuals with SRS reported here. Further
studies will be required to determine the exact incidence of this
epigenetic defect of the 11p15 region in individuals with intrauterine
growth retardation and, particularly, with SRS phenotypes.
METHODS
Subjects. Nine individuals with SRS were included in this study on the basis of
the following criteria: (i) intrauterine growth retardation; (ii) postnatal growth
retardation; (iii) presence of at least three of the following facial characteristics:
triangular face, micrognathia, bossed forehead, craniofacial disproportion in
early infancy, relative macrocephaly, downward slanting corners of the mouth
and irregular spacing of teeth; and (iv) presence of at least two of the following
100
A
Ind 1
AA AAH AH AH AH A AH A AH A AH
80
60
40
20
IGF2 DMR2
0
1
2
3
4
5
6
6 twin
7
8
9
BWS1
C1
C2
C3
120
100
80
60
Relative expression
40
20
0
120
100
80
60
Relative expression
40
20
0
Ind 6 Twin Ind 8 Control
Ind 2 Ind 3 Ind 4 Ind 5 Cont 1 Cont 2
AAH
Ind 8
AAH
Mother
AAH
1.1 kb
Father
0.9 kb
AAH
Tw i n
AAH
Ind 6
Ind 6's family
a
bcd
Figure 3 Methylation and expression analyses of IGF2 in individuals with SRS. (a) Methylation analysis of IGF2 DMR2 in the ninth exon of IGF2 in
individuals with SRS (including individual 6’s twin and parents) and two control subjects. Blood leukocyte DNA was digested with AvaII (A) and with AvaII
and HpaII (AH). (b) Quantitative representation of MIs for IGF2 DMR2 in individuals with SRS (including individual 6’s twin), individual BWS1 with isolated
hypermethylation of the H19 promoter and three control subjects (C1–C3). (c,d) Determination of IGF2 RNA levels. IGF2 expression was analyzed in skin
fibroblasts from individual 6 and her twin (c) and from individual 8 and an age-matched control subject (d) by quantitative real-time RT-PCR using primers
specific for IGF2 and 18S RNA.
110 bp
Mother
Tw i n
Ind 6
Genomic DNA
Father
Control
80 bp
Mother
Tw i n
Ind 6
cDNA
Father
Control
Figure 4 Analysis of parental expression of H19 in blood cells from
individual 6, her twin, her parents and a control subject. Genomic DNA and
cDNA PCR products were digested with RsaI.
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features: body asymmetry, clinodactyly or brachydactyly of the fifth digits,
genital abnormalities, cafe
´
au lait spots, feeding difficulties and delayed speech
(Tab le 1 and Supplementary Table 1 online). One of the nine individuals
(individual 6) was a monozygotic twin whose twin had no clinical features of
SRS (Tab l e 1 ). None of the individuals was born as a result of assisted
reproductive technology. All the individuals had normal karyotypes, growth
hormone secretion, thyroid function and biparental inheritance of chromosome
7. We obtained informed consent from all affected individuals or their parents
in accordance with national ethics rules and the ethical board of the Trousseau
hospital, University of Paris 6, France.
Extraction of nucleic acids. We extracted DNA from leukocytes and fibroblasts
as previously described
5
. We extracted total RNA from blood cells and
fibroblasts using RNAble (Eurobio) in accordance with the protocol supplied
by the manufacturer.
Methylation analysis of the 11p15 chromosomal region. We assessed
methylation of KvDMR1 in KCNQ1OT1,theH19 promoter, IGF2 DMR2
and H19-IGF2 ICR1 by Southern-blot analysis. We assessed the methylation
status of KvDMR1 in KCNQ1OT1,theH19 promoter and IGF2 DMR2 as
previously described
5,24
. We digested genomic DNA with BamHI and then
with the methylation-sensitive enzyme NotIforKvDMR1inKCNQ1OT1; with
PstI and the methylation-sensitive enzyme SmaI for the H19 promoter; and
with AvaII and the methylation-sensitive enzyme HpaII for IGF2 DMR2. We
subjected the digested samples to electrophoresis in 0.7% or 1.2% agarose
gels, blotted them onto Hybond XL membranes and hybridized them with the
HLHAY79 KCNQ1OT1 probe (corresponding to EST 68627; ATCC), the H19
cDNA probe
25
or the IGF2 cDNA probe
24
. For analysis of the methylation of
H19-IGF2 ICR1, we digested genomic DNA with RsaI and the methylation-
sensitive enzyme HpaII and subjected it to electrophoresis in 1.3% agarose gels.
We obtained the probe used for hybridization (Fig. 2a) by PCR using primers
(Supplementary Table 2 online) that amplify a 879-bp fragment (nucleotides
5,401–6,279; ref. 26). We assessed MIs by densitometry of autoradiographs
using a Storm PhosphorImager (Molecular Dynamics, Inc.).
Bisulfite sequencing of CTCF site 6. We treated 1 mg of genomic DNA with
sodium bisulfite as previously described
27
. We used previously reported primers
for the analysis of CTCF site 6 (ref. 28). For sequencing individual clones, we
subcloned the PCR products into a TOPO TA Cloning vector (Invitrogen) in
accordance with the manufacturers instructions.
RNA analysis. We carried out genotyping of the RsaI polymorphism of H19 by
PCR using genomic DNA and primers H19RSA1F and H19RSA1R (Supple-
mentary Table 2 online) and analyzed samples with heterozygous genotypes
for allelic expression by RT-PCR as previously described
29
. We amplified
leukocyte genomic DNA and cDNA samples by PCR using the H19RSA1F
and gP
32
-labeled H19RSA1R primer pair. We used DNase-treated RNA PCR
products as a control for evaluating DNA contamination of the RNA samples.
We digested products with RsaI and subjected them to 10% SDS-PAGE.
We assessed expression of IGF2 by Sybr Green I real-time RT-PCR amplifi-
cation (PE Applied Biosystems)
30
. Reactions were run on an Mx 3000P
Sequence Detector. We used 200 nM of each target IGF2 gene primer
(Supplementary Table 2 online) and 50 nM of each reference 18S primer in
each reaction. We collected and analyzed data with Mx 3000P Sequence
Detector software (Stratagene).
Accession codes. GenBank: H19, AF125183.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank A. Munnich for providing skin fibroblasts from a control subject and
J.-P. Siffroi for culture fibroblasts from individuals with SRS. This work was
supported by Pharmacia-Pfizer and Pierre Chatelain, INSERM U515, Universite
´
Pierre et Marie Curie and Assistance Publique Ho
ˆ
pitaux de Paris. S.R. was a
recipient of NovoNordisk France.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 14 March; accepted 6 July 2005
Published online at http://www.nature.com/naturegenetics/
1. DeChiara, T.M., Efstratiadis, A. & Robertson, E.J. A growth-deficiency phenotype in
heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting.
Nature 345, 78–80 (1990).
2. Fitzpatrick, G.V., Soloway, P.D. & Higgins, M.J. Regional loss of imprinting and growth
deficiency in mice with a targeted deletion of KvDMR1. Nat. Genet. 32, 426–431
(2002).
3. Leighton, P., Ingram, R., Eggenswiler, J., Efstratiadis, A. & Tilghman, S. Disruption of
imprinting caused by deletion of the H19 gene region in mice. Nature 375,3439
(1995).
4. Sun, F., Dean, W., Kelsey, G., Allen, N. & Reik, W. Transactivation of IGF2 in a mouse
model of Beckwith-Wiedemann syndrome. Nature 389, 809–815 (1997).
5. Gaston, V. et al. Analysis of the methylation status of the KCNQ1OT and H19 genes in
leukocyte DNA for the diagnosis and prognosis of Beckwith-Wiedemann syndrome. Eur.
J. Hum. Genet. 9, 409–418 (2001).
6. Price, S.M., Stanhope, R., Garrett, C., Preece, M.A. & Trembath, R.C. The spectrum of
Silver-Russell syndrome: a clinical and molecular genetic study and new diagnostic
criteria. J. Med. Genet. 36, 837–842 (1999).
7. Hitchins, M.P., Stanier, P., Preece, M.A. & Moore, G.E. Silver-Russell syndrome: a
dissection of the genetic aetiology and candidate chromosomal regions. J. Med. Genet.
38, 810–819 (2001).
8. Hannula, K., Kere, J., Pirinen, S., Holmberg, C. & Lipsanen-Nyman, M. Do patients
with maternal uniparental disomy for chromosome 7 have a distinct mild Silver-Russell
phenotype? J. Med. Genet. 38, 273–278 (2001).
9. Fisher, A.M. et al. Duplications of chromosome 11p15 of maternal origin result in a
phenotype that includes growth retardation. Hum. Genet. 111, 290–296 (2002).
10. Eggermann, T. et al. Is maternal duplication of 11p15 associated with Silver-Russell
syndrome? J. Med. Genet. 42, e26 (2005).
11. Obermann, C. et al. Searching for genomic variants in IGF2 and CDKN1C in Silver-
Russell syndrome patients. Mol. Genet. Metab. 82, 246–250 (2004).
12. Meyer, E., Wollmann, H.A. & Eggermann, T. Analysis of genomic variants in the
KCNQ1OT1 transcript in Silver-Russell syndrome patients. Mol. Genet. Metab. 84,
376–377 (2005).
13. Murrell, A. et al. An intragenic methylated region in the imprinted Igf2 gene augments
transcription. EMBO Rep. 2, 1101–1106 (2001).
14. Engel, N., West, A.G., Felsenfeld, G. & Bartolomei, M.S. Antagonism between DNA
hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG
mutations. Nat. Genet. 36, 883–888 (2004).
15. Bell, A.C. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls
imprinted expression of the Igf2 gene. Nature 405, 482–485 (2000).
16. Hark, A.T. et al. CTCF mediates methylation-sensitive enhancer-blocking activity at the
H19/Igf2 locus. Nature 405, 486–489 (2000).
17. Kanduri, C. et al. The 5¢ flank of mouse H19 in an unusual chromatin conformation
unidirectionally blocks enhancer-promoter communication. Curr. Biol. 10, 449–457
(2000).
18. Schoenherr, C.J., Levorse, J.M. & Tilghman, S.M. CTCF maintains differential methy-
lation at the Igf2/H19 locus. Nat. Genet. 33, 66–69 (2003).
19. Fedoriw, A.M., Stein, P., Svoboda, P., Schultz, R.M. & Bartolomei, M.S. Transgenic
RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303,
238–240 (2004).
20. Murrell, A., Heeson, S. & Reik, W. Interaction between differentially methylated
regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin
loops. Nat. Genet. 36, 889–893 (2004).
21. Reik, W. et al. Imprinting mutations in the Beckwith-Wiedemann syndrome suggested
by altered imprinting pattern in the IGF2–H19 domain. Hum. Mol. Genet. 4,
2379–2385 (1995).
22. Weksberg, R. et al. Discordant KCNQ1OT1 imprinting in sets of monozygotic twins
discordant for Beckwith-Wiedemann syndrome. Hum. Mol. Genet. 11, 1317–1325
(2002).
23. Sparago, A. et al. Microdeletions in the human H19 DMR result in loss of IGF2
imprinting and Beckwith-Wiedemann syndrome. Nat. Genet. 36, 958–960 (2004).
24. Schneid, H. et al. Parental allele specific methylation of the human insulin-like growth
factor II gene and Beckwith-Wiedemann syndrome. J. Med. Genet. 30, 353–362
(1993).
25. Brannan, C., Dees, E., Ingram, R. & Tilghman, S. The product of the H19 gene may
function as an RNA. Mol. Cell. Biol. 10, 28–36 (1990).
26. Frevel, M.A., Sowerby, S.J., Petersen, G.B. & Reeve, A.E. Methylation sequencing
analysis refines the region of H19 epimutation in Wilms tumor. J. Biol. Chem. 274,
29331–29340 (1999).
27. Grunau, C., Clark, S. & Rosenthal, A. Bisulfite genomic sequencing: systematic
investigation of critical experimental parameters. Nucleic Acids Res. 29, E65–5
(2001).
28. Dupont, J.M., Tost, J., Jammes, H. & Gut, I.G. De novo quantitative bisulfite
sequencing using the pyrosequencing technology. Anal. Biochem. 333, 119–127
(2004).
29. Ulaner, G.A. et al. CTCF binding at the insulin-like growth factor-II (IGF2)/H19
imprinting control region is insufficient to regulate IGF2/H19 expression in human
tissues. Endocrinology 144, 4420–4426 (2003).
30. Corpechot, C. et al. Hypoxia-induced VEGF and collagen I expressions are associated
with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology 35,1010
1021 (2002).
NATURE G ENETI CS VOLUME 37
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NUMBER 9
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SEPTEMBER 2005 1007
LETTERS
© 2005 Nature Publishing Group http://www.nature.com/naturegenetics
    • "Specific imprinted regions are affected by methylation changes in SRS SRS is one of several disorders associated with imprinted regions (Ishida and Moore 2013). Approximately, 50 % of SRS cases display hypomethylation at the H19 ICR1, while a further 5–10 % of SRS patients exhibit mUPD7 (Gicquel et al. 2005; Preece et al. 1997). We determined the prevalence of SRS-specific differences in methylation at imprinted loci in a genome-wide, systematic way. "
    [Show abstract] [Hide abstract] ABSTRACT: Silver–Russell syndrome (SRS) is a clinically heterogeneous disorder characterised by severe in utero growth restriction and poor postnatal growth, body asymmetry, irregular craniofacial features and several additional minor malformations. The aetiology of SRS is complex and current evidence strongly implicates imprinted genes. Approximately, half of all patients exhibit DNA hypomethylation at the H19/IGF2 imprinted domain, and around 10 % have maternal uniparental disomy of chromosome 7. We measured DNA methylation in 18 SRS patients at >485,000 CpG sites using DNA methylation microarrays. Using a novel bioinformatics methodology specifically designed to identify subsets of patients with a shared epimutation, we analysed methylation changes genome-wide as well as at known imprinted regions to identify SRS-associated epimutations. Our analysis identifies epimutations at the previously characterised domains of H19/IGF2 and at imprinted regions on chromosome 7, providing proof of principle that our methodology can detect DNA methylation changes at imprinted loci. In addition, we discovered two novel epimutations associated with SRS and located at imprinted loci previously linked to relevant mouse and human phenotypes. We identify RB1 as an additional imprinted locus associated with SRS, with a region near the RB1 differentially methylated region hypermethylated in 13/18 (~70 %) patients. We also report 6/18 (~33 %) patients were hypermethylated at a CpG island near the ANKRD11 gene. We do not observe consistent co-occurrence of epimutations at multiple imprinted loci in single SRS individuals. SRS is clinically heterogeneous and the absence of multiple imprinted loci epimutations reflects the heterogeneity at the molecular level. Further stratification of SRS patients by molecular phenotypes might aid the identification of disease causes.
    Full-text · Article · Jan 2015
    • "Loss of methylation at the H19/IGF2 ICR results in short body length and low birth weight, both in rodent models (DeChiara et al., 1990) as well as in humans, such as patients with Silver-Russell syndrome, a developmental disorder characterized by intrauterine and postnatal growth retardation (Gicquel et al., 2005). This has also been observed in humans who were periconceptually exposed to famine (Heijmans et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Diabetes mellitus represents a group of complex metabolic diseases that result in impaired glucose homeostasis, which includes destruction of β-cells or the failure of these insulin-secreting cells to compensate for increased metabolic demand. Despite a strong interest in characterizing the transcriptome of the different human islet cell types to understand the molecular basis of diabetes, very little attention has been paid to the role of long non-coding RNAs (lncRNAs) and their contribution to this disease. Here we summarize the growing evidence for the potential role of these lncRNAs in β-cell function and dysregulation in diabetes, with a focus on imprinted genomic loci.
    Full-text · Article · Jul 2014
    • "The IGF2/H19 ICR contains several differentially methylated regions (DMRs), which are all predominantly methylated on the paternally inherited allele: the IGF2 DMR0 (located between exons 2 and 3 of IGF2), the IGF2 DMR2 (between exons 8 and 9) and the H19 DMR, located 4 kb upstream of the transcription start of H19[15], which are all methylated to a level of 40-50% [10,16,17]. The methylation status of the IGF2 DMR0 is more likely to be indicative of changes in IGF2 transcription from the active allele given it has been suggested to possess promoter activity [18]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background There is a substantial genetic component for birthweight variation, and although there are known associations between fetal genotype and birthweight, the role of common epigenetic variation in influencing the risk for small for gestational age (SGA) is unknown. The two imprinting control regions (ICRs) located on chromosome 11p15.5, involved in the overgrowth disorder Beckwith-Wiedemann syndrome (BWS) and the growth restriction disorder Silver-Russell syndrome (SRS), are prime epigenetic candidates for regulating fetal growth. We investigated whether common variation in copy number in the BWS/SRS 11p15 region or altered methylation levels at IGF2/H19 ICR or KCNQ10T1 ICR was associated with SGA. Methods We used a methylation-specific multiplex-ligation-dependent probe amplification assay to analyse copy number variation in the 11p15 region and methylation of IGF2/H19 and KCNQ10T1 ICRs in blood samples from 153 children (including 80 SGA), as well as bisulfite pyrosequencing to measure methylation at IGF2 differentially methylated region (DMR)0 and H19 DMR. Results No copy number variants were detected in the analyzed cohort. Children born SGA had 2.7% lower methylation at the IGF2 DMR0. No methylation differences were detected at the H19 or KCNQ10T1 DMRs. Conclusions We confirm that a small hypomethylation of the IGF2 DMR0 is detected in peripheral blood leucocytes of children born SGA at term. Copy number variation within the 11p15 BWS/SRS region is not an important cause of non-syndromic SGA at term.
    Full-text · Article · Jun 2014
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