No evidence for pathogenic variants or maternal effect
of ZFP57 as the cause of Beckwith–Wiedemann
Susanne E Boonen*,1,2, Johanne MD Hahnemann1, Deborah Mackay3,4, Niels Tommerup2,
Karen Brøndum-Nielsen1,5, Zeynep Tu ¨mer1,5and Karen Grønskov1,5
Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome, which, in 50–60% of sporadic cases, is caused by
hypomethylation of KCNQ1OT1 differentially methylated region (DMR) at chromosome 11p15.5. The underlying defect of this
hypomethylation is largely unknown. Recently, recessive mutations of the ZFP57 gene were reported in patients with transient
neonatal diabetes mellitus type 1, showing hypomethylation at multiple imprinted loci, including KCNQ1OT1 DMR in some. The
aim of our study was to determine whether ZFP57 alterations were a genetic cause of the hypomethylation at KCNQ1OT1 DMR
in patients with BWS. We sequenced ZFP57 in 27 BWS probands and in 23 available mothers to test for a maternal effect.
We identified three novel, presumably benign sequence variants in ZFP57; thus, we found no evidence for ZFP57 alterations
as a major cause in sporadic BWS cases.
European Journal of Human Genetics (2012) 20, 119–121; doi:10.1038/ejhg.2011.140; published online 24 August 2011
Keywords: Beckwith–Wiedemann syndrome; imprinting; hypomethylation; ZFP57; maternal effect genes; germline alterations
Beckwith–Wiedemann syndrome (BWS, OMIM 130650) is an
imprinting syndrome with an incidence of 1:13700.1The most
prominent clinical features are pre- and postnatal overgrowth
(497th centile), macroglossia and abdominal wall defects.1–3The
underlying pathology of BWS includes genetic and epigenetic altera-
tions on chromosome 11p15.5, resulting in dysregulation of growth
regulatory genes.1–3Hypomethylation of the differentially methylated
region (DMR) of imprinting control region 2/KCNQ1OT1 DMR on
the maternal allele occurs in 50–60% of BWS cases and is mostly
due to an apparently pure epigenetic modification.1,2
Recently, mechanisms influencing imprinting disorders in trans
have been identified. Hypomethylation of multiple imprinted
loci (HIL) is described in different imprinting syndromes including
transient neonatal diabetes mellitus type 1 (TNDM1; MIM 601410),4
BWS,5–7Silver–Russell syndrome (SRS; MIM 180860)/growth restric-
tion,8,9and in a single patient with the clinical phenotype of BWS and
Prader–Willi syndrome.10Recessive ZFP57 mutations were identified
in more than half of the TNDM1 cases displaying HIL.11ZFP57 is
localized at chromosome 6p22.1 and encodes the KRAB zinc finger
protein ZFP57, containing seven zinc finger domains (ZF1–ZF7).11In
mice, when the zygotic function of Zfp57 is lost (Zfp57?/?), offsprings
show partial hypomethylation at multiple maternally and paternally
imprinted loci, but when both maternal and zygotic zfp57 function are
lost (Zfp57?/?offspring of Zfp57?/?mothers), the offsprings show
complete loss of methylation. This indicates that Zfp57 is a maternal-
zygotic effect gene required for maintenance of DNA methylation
imprints.12,13The maternal effect of a gene is the genetic phenomenon
in which a phenotype in the progeny is caused by a genetic alteration
in the maternal genome rather than an alteration of its own; this
has been observed both in humans7,14and in mice.12,13Mutations in
NLRP7 were described in females with familial recurrent biparental
complete hydatidiform moles.14These females had normal methyla-
tion, but failed to establish methylation imprints in their oocytes.
More recently, a homozygous NLRP2 mutation was found in a female;
both her sons had BWS with hypomethylation of the KCNQ1OT1
DMR, and furthermore, one of them also had hypomethylation of
PEG1 DMR.7In this study, we present the mutation analysis of ZFP57
in sporadic BWS patients and their mothers to test two hypotheses:
(1) mutations in ZFP57 lead to isolated hypomethylation of
KCNQ1OT1 DMR and thereby lead to BWS; (2) mutations in
ZFP57 in a mother may lead to BWS in her children.
MATERIAL AND METHODS
Twenty-seven sporadic BWS probands (14 females and 13 males), 23 from
Scandinavia, three from other parts of E`urope and one of unknown origin,
were referred for diagnostic molecular genetic testing for BWS. Patients with
hypomethylation at KCNQ1OT1 DMR were selected for the study. This was
tested either by Southern blot analysis using a KCNQ1OT1 probe and a
methylation sensitive restriction enzyme, or by methylation-specific MLPA
the Netherlands). The mean methylation indices were for Southern blot
analysis 0.12 (based on nine patients; minimum 0.05 and maximum 0.24;
median 0.07) and for MS-MLPA 0.09 (based on four patients; minimum 0.01
Received 25 January 2011; revised 29 June 2011; accepted 30 June 2011; published online 24 August 2011
1Center for Applied Human Molecular Genetics, The Kennedy Center, Glostrup, Denmark;2Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular
and Molecular Medicine, Panum Institute, University of Copenhagen, Copenhagen N, Denmark;3Wessex Genetics Service, Southampton University Hospital Trusts, Southampton
SO16 5Y, UK;4Salisbury Hospital NHS Foundation Trust, Salisbury, UK;5Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
*Correspondence: Dr SE Boonen, Center for Applied Human Molecular Genetics, The Kennedy Center, Gl. Landevej 7, 2600 Glostrup, Denmark. Tel: +45 43 26 01 68;
Fax: + 45 43 43 11 30; E-mail: email@example.com
European Journal of Human Genetics (2012) 20, 119–121
& 2012 Macmillan Publishers Limited All rights reserved 1018-4813/12
and maximum 0.14; median 0.10). Ten patients were further tested for HIL by
MS-PCR as described previously.4Three of the 27 probands were from
discordant twin pairs (one from a pair of female monozygotic (MZ) twins,
one from a pair of female twins with unknown zygosity, and one from a pair of
male twins of unknown zygosity). Twenty-three mothers of BWS probands
were available for investigation, and all had normal methylation at KCNQ1OT1
DMR tested as described above. To our knowledge, none of the probands were
from consanguineous families. We have no information about whether the
probands were conceived by assisted reproductive technology. The control
group consisted of 50 normal Danish individuals.
procedures. The five coding exons of ZFP57 and 20bp of the flanking intronic
sequences were amplified in eight fragments by PCR as previously described.11
PCR primer sequenced are listed in Supplementary Table 1 and PCR conditions
are available upon request. All ZFP57 alterations are described according
to den Dunnen and Antonarakis,15with GenBank accession number:
DNA was extracted from peripheral blood by standard
In silico analysis
The possible impact of the novel amino acid substitutions was evaluated
using the SIFT database, (http://sift.jcvi.org/) and the PolyPhen-2 database,
By sequencing ZFP57 in 27 sporadic BWS patients with hypomethyla-
tion at KCNQ1OT1 DMR and in 23 mothers with normal methyla-
tion, we identified three novel sequence variations. All the variations
were located in exon 6. Ten out of the 27 patients were investigated
for HIL, but no signs of HIL were observed (data not shown).
In the affected twin from the MZ female twins (from Scandinavia),
(p.Ser168Phe) affecting a non-conserved amino acid in ZF1. The
variation was also found in the non-affected twin and in the father,
but not in the mother. The SIFT database predicts the variation to be
tolerated with a score of 0.30 (normal 40.05), but the PolyPhen-2
database predicts it to be probably damaging with a score of 0.971
(sensitivity: 0.56; specificity: 0.91). The variant was not detected in 50
controls or present in the 1000 genomes database (http://browser.
In a proband from Europe, we found two silent sequence variations,
c.723C4T (p.¼) and c.1026T4C (p.¼). The c.723C4T is located in
ZF4 and the c.1026T4C is located between ZF4 and ZF5. Both
variations were inherited from the unaffected father and thus were in
cis. Both variations were absent in the mother. The variants were not
detected in 50 controls or in the 1000 genome database.
In a proband from Scandinavia, we identified a sequence variation
c.499C4T (p.Arg167Cys) affecting a non-conserved amino acid in
ZF1. The variation was inherited from the mother. This variation
was present in seven out of 50 controls and was reported in the SNP
database (http://ncbi.nlm.nih.gov/snp) (rs61730330). The SIFT data-
base predicts the variation to affect protein function with a score
of 0.02 (normal 4 0.05) and the PolyPhen-2 database predicts it to
be probably damaging with a score of 0.914 (sensitivity: 0.67;
The etiology of the hypomethylation of KCNQ1OT1 DMR observed in
BWS patients is unknown. Recently, mutations in ZFP57 were found
to cause HIL in some TNDM1 patients.11As KCNQ1OT1 DMR
is among the loci observed to be hypomethylated in TNDM HIL
patients, and further, as some BWS patients were also shown to have
HIL, we investigated whether sporadic BWS could be caused by
mutations in ZFP57.
We identified three novel sequence variations in ZFP57. Two of the
variations are silent mutations, and furthermore, not located at the
splice sites. The causative effect of the third variation is questionable as
suggested by in silico analysis. We also identified a known SNP.
None of the probands or mothers was homozygous or compound
heterozygous for variations in ZFP57.
It could be argued that ZFP57 mutations would only be found in
patients with HIL, but notably the methylation profile for the different
imprinted loci varies considerably in patients with ZFP57 mutations.11
Other studies reported 11–25% of BWS patients5,6and 14% of SRS
patients8,9to have HIL. In our BWS cohort, none of the 10 investi-
gated patients had HIL, but the cohort is too small to deduce
conclusions. In SRS patients, mutational analysis of ZFP57 similarly
has shown no pathogenic alterations.16In contrast to BWS, however,
the hypomethylation in SRS affects a paternally imprinted locus
(H19 DMR), albeit cases of both SRS and BWS have been reported
with hypomethylation at both maternally as well as paternally methy-
Other trans-acting factors and mechanisms involved in the estab-
lishment and maintenance of methylation in DNA imprints could be
responsible for the methylation and demethylation at KCNQ1OT1,
maybe in cooperation with ZFP57, including DNA methyltransferases,
methyl-binding proteins, imprinting genes network and/or RNA
interference. Furthermore, alterations in cis are known to cause
aberrant methylation, for example, in SRS and growth retardation
where deletions and translocations of the H19/IGF2 enhancer region
can cause hypomethylation of the IGF2P0 promoter element.19
Mice studies have shown that Zfp57 is a maternal effect gene, and it
has been hypothesized that a gradual loss of DNA methylation takes
place in the Zfp57?/? progeny of Zfp57?/? mothers and account for
the spatial and temporal discrepancy of the molecular defect and the
phenotypic effect;13however, we do not find ZFP57 to be a major
maternal effect gene in sporadic BWS, neither do our data support the
hypothesis that alterations in ZFP57 are a major cause of KCNQ1OT1
DMR hypomethylation in sporadic BWS cases.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Wilhelm Johannsen Centre for Functional Genome Research is established
by the Danish National Research Foundation. This work was supported by
a grant from the Danish Agency for Science, Technology and Innovation,
the University of Copenhagen. Furthermore, financial support was granted
by Director Jacob Madsen and wife Olga Madsens Foundation and
King Christian X fond.
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Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
ZFP57 variations in Beckwith–Wiedemann syndrome
SE Boonen et al
European Journal of Human Genetics