Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome
J Clin Endocrinol Metab, July
Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome
Sophie Geoffron 14
Walid Abi Habib 14
Sandra Chantot-Bastaraud 13
Be´ atrice Dubern 11
Virginie Steunou 14
Salah Azzi 14
Alexandra Afenjar 19
Tiffanny Busa 18
Ana Pinheiro Canton 14 17
Christel Chalouhi 16
Marie-Noe¨ lle Dufourg 14
Blandine Esteva 14
M e´lanie Fradin 21
David Genevie` ve 15 20
Solveig Heide 13
Bertrand Isidor 8
Agn e`s Linglart 6 7 9
Fanny Morice Picard 4
Catherine Naud-Saudreau 5
Isabelle Oliver Petit 2
Nicole Philip 18
Catherine Pienkowski 2
Marl e`ne Rio 0 3
Sylvie Rossignol 10 12
Maith e´ Tauber 1 2
Thuy-Ai Vu-Hong 14
Madeleine D Harbison
Fr e´d e´ric Brioude 14
Ir e`ne Netchine 14
Eloı¨se Giabicani 14
0 INSERM UMR 1163, Paris Descartes- Sorbonne Paris Cit e ́ Universite ́ , Institut Imagine , F-75015 Paris , France
1 INSERM U1043, Centre de Physiopathologie de Toulouse Purpan , Universit e ́
2 CHU de Toulouse, Hoˆ pital des Enfants, Unit e ́ d'Endocrinologie, Ob e ́sit e ́, Maladies Osseuses, G e ́n e ́tique et Gyn e ́cologie M e ́dicale , 31059 Toulouse cedex 9 , France
3 APHP, Hoˆ pital Necker- Enfants-Malades, Service de G e ́ne ́ tique , F-75015 Paris , France
4 CHU de Bordeaux, Hoˆ pital Pellegrin-Enfants, Department of Pediatric Dermatology, National Centre for Rare Skin Disorders , 33076 Bordeaux cedex , France
5 Bretagne Sud Hospital Center , Pediatric Endocrinology and Diabetology, 56322 Lorient cedex , France
6 APHP, Bic eˆtre Paris Sud Hospital, Department of Pediatric Endocrinology and Diabetology , 94270 Le Kremlin Bic eˆtre , France
7 INSERM U1169, Bic eˆtre Paris Sud Hospital , 94270 Le Kremlin Bic eˆtre, Universit e ́ Paris-Saclay , France
8 CHU Nantes, Service de G e ́n e ́tique M e ́dicale , 44093 Nantes cedex 1 , France
9 APHP, Bic eˆtre Paris Sud Hospital, Reference Center for Rare Mineral Metabolism Disorders (Fili e`re OSCAR) and the Plateforme d'Expertise Paris Sud Maladies Rares , 94270 Le Kremlin Bic eˆtre , France
10 INSERM U1112, Laboratoire de G e ́n e ́tique M e ́dicale, Institut de G e ́n e ́tique M e ́dicale d'Alsace, Facult e ́ de M e ́decine de Strasbourg , 67091 Strasbourg cedex , France
11 Sorbonne Universit e ́, INSERM, UMRS U1166 (Eq 6) Nutriomics, Institut de Cardiom e ́tabolisme et Nutrition, APHP, Hoˆ pital Armand Trousseau, Service de Nutrition et de Gastroent e ́rologie P e ́diatriques , F-75012 Paris , France
12 Hoˆ pitaux Universitaires de Strasbourg, Service de P e ́diatrie , 67091 Strasbourg cedex , France
13 APHP, Hoˆ pital Armand Trousseau, D e ́partement de G e ́n e ́tique, UF de G e ́n e ́tique Chromosomique , F-75012 Paris , France
14 Sorbonne Universit e ́, INSERM, UMR_S 938 Centre de Recherche Saint Antoine, Assistance Publique - Hoˆ pitaux de Paris (APHP), Hoˆ pital Armand Trousseau, Explorations Fonctionnelles Endocriniennes , F-75012 Paris , France
15 INSERM U1183, Institute of Regenerative Medicine and Biotherapie, Montpellier University, CHU Montpellier , 34295 Montpellier cedex 5 , France
16 APHP, ~ Hoˆ pital Necker-Enfants-Malades, Service de P e ́diatrie G e ́n e ́rale , F-75015 Paris , France
17 Unidade de Endocrinologia Gen e ́tica, Laborato ́ rio de Endocrinologia Celular e Molecular LIM25, Disciplina de Endocrinologia, Faculdade de Medicina da Universidade de S
18 Assistance Publique des Hoˆ pitaux de Marseille, Hoˆ pital Timone Enfants, Centre de R e ́f e ́rence Anomalies du D e ́veloppement et Syndromes Malformatifs Provence Alpes Coˆ te d'Azur, D e ́partement de Ge ́ n e ́tique M e ́dicale et G e ́nomique Fonctionnelle, Aix Marseille Universit e ́ , 13284 Marseille cedex 7 , France
19 Sorbonne Universit e ́, APHP, Hoˆ pital Armand Trousseau, D e ́partement de G e ́n e ́tique Clinique et Centre de R e ́f e ́rence Anomalies du D e ́veloppement et Syndromes Malformatifs et D e ́ficiences Intellectuelles de Causes Rares , F-75012 Paris , France
20 H oˆpital Arnaud de Villeneuve, Unit e ́ de G e ́n e ́tique Clinique, D e ́partement de G e ́n e ́tique M e ́dicale, Maladies Rares et M e ́decine Personnalis e ́e , 34090 Montpellier , France
21 Centre Hospitalier Universitaire (CHU) Hoˆ pital Sud, Service de G e ́n e ́tique Clinique, Centre de R e ́f e ́rence Maladies Rares Centre Lab e ́llis e ́ 'Anomalies du D e ́veloppement'-Ouest , 35203 Rennes cedex 2 , France
*These authors are co-first authors.
Abbreviations: aGNRH, gonadotrophin-releasing hormone analog; BMI, body mass index;
GH, growth hormone; IG-DMR, intergenic differentially methylated region; LOM, loss of
methylation; MLMD, multilocus methylation defect; NH-CSS, Netchine-Harbison clinical
scoring system; SDS, standard deviation score; SGA, small for gestational age; SRS,
SilverRussell syndrome; TS, Temple syndrome.
Paul-Sabatier, 31330 Toulouse, France; 23Centre de R e´f e´rence du Syndrome de Prader Willi, 31059 Toulouse
cedex 9, France; 24CHU Dijon, Hoˆ pital d’Enfants, Centre de G e´n e´tique et Centre de R e´f e´rence “Anomalies
du D e´veloppement et Syndromes Malformatifs,” 21079 Dijon cedex, France; 25CHU Grenoble-Alpes,
Hoˆ pital Couple-Enfants, Centre de G e´n e´tique, Centre de R e´f e´rence “Anomalies du D e´veloppement et
Syndromes Malformatifs,” 38700 La Tronche, France; 26Icahn School of Medicine at Mount Sinai,
Department of Pediatrics, New York, New York 10029; and 27The MAGIC Foundation, Russell-Silver
Syndrome/Small for Gestational Age Research & Education Fund, Warrenville, Illinois 60555
Context: Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and
Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders
with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and
Objective: To describe the clinical overlap between SRS and TS and extensively study the molecular
aspects of TS.
Patients: We retrospectively collected data on 28 patients with disruption of the 14q32.2 imprinted
region, identified in our center, and performed extensive molecular analysis.
Results: Seventeen (60.7%) patients showed loss of methylation of the MEG3/DLK1 intergenic
differentially methylated region by epimutation. Eight (28.6%) patients had maternal uniparental
disomy of chromosome 14 and three (10.7%) had a paternal deletion in 14q32.2. Most patients
(72.7%) had a Netchine-Harbison SRS clinical scoring system $4/6, and consistent with a clinical
diagnosis of SRS. The mean age at puberty onset was 7.2 years in girls and 9.6 years in boys; 37.5%
had premature pubarche. The body mass index of all patients increased before pubarche and/or the
onset of puberty. Multilocus analysis identified multiple methylation defects in 58.8% of patients.
We identified four potentially damaging genetic variants in genes encoding proteins involved in the
establishment or maintenance of DNA methylation.
Conclusions: Most patients with 14q32.2 disruption fulfill the criteria for a clinical diagnosis of SRS.
These clinical data suggest similar management of patients with TS and SRS, with special attention
to their young age at the onset of puberty and early increase of body mass index. (J Clin Endocrinol
Metab 103: 2436–2446, 2018)
Irisk of perinatal morbidity and mortality and metabolic
mpaired fetal growth is associated with an increased
problems later in life, according to the
Developmental Origins of Health and Disease theory (
regions are known to play an important role in fetal
). Paternally expressed genes are mostly
involved in growth promotion, whereas maternally
expressed genes repress it. Most imprinted regions are
methylated on the maternal allele. In humans, only two
regions are methylated in the male germ line (
11p15 H19/IGF2 intergenic differentially methylated region
(IG-DMR) and the 14q32.2 MEG3/DLK1:IG-DMR,
involved in Silver-Russell syndrome (SRS)/Beckwith-Wiedemann
syndrome, and Temple syndrome (TS)/Kagami-Ogata
SRS is characterized by fetal and postnatal growth
retardation and feeding difficulties (
resulting in the loss of methylation (LOM) of H19/IGF2:
IG-DMR on the paternal allele, is identified in 50% of
SRS cases (
). In this region, the imprinting center
H19/IGF2:IG-DMR is methylated on the paternal allele,
resulting in IGF2 expression. When unmethylated, as on the
maternal allele, it allows H19 expression, a long noncoding
RNA (Fig. 1). The key role of IGF2 in prenatal growth is
well-established. Maternal uniparental disomy for
chromosome 7 [upd(
)mat] is seen in ;5% to 10% of patients
with SRS (
). However, for 35% to 40% of patients with
SRS, the molecular etiology remains unknown.
TS, first clinically and molecularly described in 1991,
associates fetal and postnatal growth retardation,
hypotonia, obesity, and early puberty (
). TS is caused
by disruption of the 14q32.2 imprinted region, where
MEG3/DLK1:IG-DMR is methylated on the paternal
allele. MEG3/DLK1:IG-DMR methylation results in
DLK1, RTL1, and DIO3 expression, whereas long
noncoding RNAs (MEG3 and MEG8), microRNAs, and
small nucleolar RNAs are expressed when it is
unmethylated (as on the maternal allele) (Fig. 1). In a
metaanalysis of 51 patients with TS, the molecular anomalies
identified consisted mostly of maternal uniparental
disomy of chromosome 14 [upd(
epimutation of MEG3/DLK1:IG-DMR on the paternal
allele (11.8%), and paternal deletion of the MEG3/DLK1
domain (9.8%) (
). A cohort of 32 patients with
Clinical assessment and definitions
The Netchine-Harbison clinical scoring
system (NH-CSS), recently adopted as the
clinical definition of SRS by the first
international consensus on this syndrome
), was applied to each of the 28 patients.
This scoring system defines a suspicion of
SRS if at least four of the six following
criteria are met: (
) being born small for
gestational age (SGA) [birth weight and/or birth
length #22 standard deviation score (SDS)
for gestational age], (
) postnatal growth
failure (height at 24 6 1 months #22 SDS or
height #22 SDS from midparental target
) relative macrocephaly at birth
(head circumference at birth $1.5 SDS
above birth weight and/or length SDS),
) protruding forehead (forehead
projecting beyond the facial plane on a side
view as a toddler), (
) body asymmetry
[leg length discrepancy $0.5 cm or arm asymmetry or leg
length discrepancy ,0.5 cm with at least two other
asymmetrical body parts (one nonface)], and (
) low body mass
index (BMI) (BMI #22 SDS at 24 months) and/or feeding
difficulties defined by the use of a feeding tube and/or
cyproheptadine for appetite stimulation. See the Supplemental
Materials and Methods for auxologic methods.
Premature pubarche was defined by the appearance of pubic or
axillary hair occurring before eight years in girls and nine years in
). Precocious puberty was defined by breast development
(thelarche) before age 8 years in girls and testicular enlargement
before age 9 years in boys (
). Exaggerated adrenarche was
defined by high levels of serum dehydroepiandrosterone sulfate for
age (after other diseases were excluded) (
Methylation studies at both 11p15 H19/IGF2:IG-DMR and
MEG3/DLK1:IG-DMR loci are described in the Supplemental
Data. All patients had hypomethylation at
MEG3/DLK1:IGDMR. We distinguished three different mechanisms: upd(
deletion, and LOM by epimutation.
Single nucleotide polymorphism microarray analysis
We analyzed the DNA samples using Illumina CytoSNP-12
arrays (Illumina, San Diego, CA) to distinguish between MEG3/
DLK1:IG-DMR epimutation, upd(
)mat, and large copy
number variations. See the Supplemental Materials and Methods
IG-DMR and exome variant sequencing
Library preparation, exome capture, sequencing, and data
analysis were performed by IntegraGen SA (Evry, France). The
sequencing methods and bioinformatics analysis are detailed in
the Supplemental Materials and Methods.
The characteristics of the population are described as
percentages for qualitative variables or as SDS and mean (range) for
14q32.2 anomalies has recently been reported and, again,
most had upd(
)mat (71.9%), whereas only 18.8% had
). Clinical overlap between SRS and TS
has been previously highlighted in reports of patients
presenting with a clinical diagnosis of SRS with no 11p15
disruption or upd(
)mat, but for whom chromosome
14q32.2 anomalies were identified (
). Thus, these
syndromes overlap in terms of phenotype and may be
caused by anomalies of imprinted regions sharing
similar molecular organization, both methylated in the male
We identified 28 patients with chromosome 14q32.2
disruption in our molecular diagnostic laboratory. The
purpose of this study was to clinically and
molecularly characterize these patients to determine the clinical
overlap with patients with SRS. Furthermore, we sought
to identify the mechanism involved in the onset of
Patients and Methods
The study population consisted of 28 patients with
chromosome 14q32.2 disruption. The molecular diagnosis of 25
patients was performed in our laboratory and three upd(
mat were identified without methylation analysis in other
diagnostic laboratories. All patients were either followed in
our clinic or were referred by other clinical centers for
molecular analysis. A clinical file, including extensive clinical
data, growth charts, a detailed phenotypic description, and
pictures was completed for all patients. Each patient had been
examined by a geneticist and/or a pediatric endocrinologist.
Written informed consent for participation was received either
from the patients themselves or their parents, in accordance
with French national ethics rules for patients recruited in
France (Assistance Publique – Hoˆ pitaux de Paris authorization
no. 681) and with the institutional review board I00000204 of
the Mount Sinai School of Medicine, New York, for patients
recruited in the United States.
continuous variables. For subgroup comparisons, we used the
Wilcoxon Mann-Whitney test and the Fischer test.
Our cohort was composed of 28 patients (17 girls). Three
patients (10.7%) were conceived with the aid of medically
assisted procreation: two in vitro fecundations and one
intrauterine insemination. The median maternal age was 29.1
(19.8–41.5) years and paternal age was 30.7 (25.8-44.8) years.
Classical molecular anomalies found in SRS [i.e., 11p15
epimutation and upd(
)mat] were ruled out for all but three
patients with upd(
)mat not identified in our laboratory.
All patients presented with chromosome 14q32.2
hypomethylation at the MEG3/DLK1:IG-DMR, which was
secondary to upd(
)mat in eight (28.6%) patients or to a
paternal deletion of DLK1/MEG3 region in three (10.7%),
whereas 17 (60.7%) had MEG3/DLK1:IG-DMR LOM
caused by epimutation on the paternal allele. This was
ascertained after ruling out a upd(
)mat or deletion of the
DLK1/MEG3 region by single nucleotide polymorphism
The median age at the end of the study was 7.5
(1.3 to 21.6) years. Birth parameters, postnatal growth,
dysmorphic anomalies, psychomotor development, and
associated malformations are summarized in Table 1.
Dysmorphic features such as protruding forehead,
prominent heel, tented appearance of the mouth, and acromicria
are presented in Supplemental Fig. 1.
Eight of the 23 patients, for whom data were
available, were treated with recombinant growth hormone
(GH) therapy from a mean age of 4.7 (1.1 to 11.3) years
according to the SGA indication and posology (
Among patients for whom all items of the NH-CSS
were available, 72.7% (16/22) had a score $4/6, and
consistent with a clinical diagnosis of SRS. One item was
missing in six patients, of whom two had an NH-CSS
score of 4/5 (compatible with a diagnosis of SRS), one
had a score of 3/5 and three had scores of 2/5, which does
not fulfill the criteria for a clinical diagnosis of SRS
(Supplemental Fig. 2). Among the six patients who did
not fulfill the NH-CSS criteria, five (83.37%) had an
epimutation and one a deletion (case 26).
Puberty and pubarche
We collected data on puberty and pubarche for all
patients but one (one girl for whom data were not available).
At the end of the study, 11 patients had gone into puberty,
eight girls and three boys; the oldest among the other
nonpubertal patients was an 8.7-year-old girl. Of these 11
patients, six (54.5%) had precocious puberty, including five
(62.5%) girls and one (33.3%) boy. Four (66.7%) had
epimutations and two (33.3%) had upd(
Puberty occurred early for the other three girls, before age
9 years, and was rapidly progressive, with menarche
,1 year after breast development for two of them. Puberty
also started early for the other two boys, at 10.0 and 10.2
years (Table 2). Six (54.5%) patients were treated with
gonadotrophin-releasing hormone analogs (aGnRHs) to
suppress puberty at a mean age of 7.9 (5.0 to 10.3) years. Six
(54.5%) patients had exaggerated adrenarche; four among
them were treated with cyproterone acetate.
Epiphyseal fusion occurred early in four patients, at 12.7
(11.2 to 13.8) years in girls (n = 3) and 13.8 years in one boy,
without aGnRH treatment. The mean final height of the girls
(n = 4) was 143.5 (141.0 to 145.0) cm, corresponding to 23.6
(24.0 to 23.3) SDS, according to Sempe´ (
), with a mean
pubertal growth spurt of 12.8 (10.0 to 17.0) cm without
aGnRH and 25.5 cm for the girl who was treated. One boy
had a final height of 150.0 cm (23.9 SDS), with a pubertal
growth spurt of 19.3 cm; the second had a final height of
169 cm (20.8 SDS), far from his target height (+2.7 SDS).
None of these patients received recombinant GH treatment.
All clinical data concerning puberty and pubarche of
these 11 patients are summarized in Supplemental Table 1.
The age of adiposity rebound was precocious for
93.8% (15/16 for whom data were available) of the
patients, with a mean at 2.1 (1.0 to 6.5) years. Twelve
patients (75.0%) had adiposity rebound by the age of 2 years.
Among patients with precocious adiposity rebound, only
one needed enteral feeding. For this patient, nutrition intake
is on the decrease but she experienced complete anorexia.
For all other patients, BMI had spontaneously grown
precociously. The BMI of all patients for whom puberty had
started increased markedly (.1 SDS) before the onset of
pubarche and/or central puberty.
Extensive molecular analysis
Methylation analysis of 18 imprinted loci
We studied the methylation levels at 18 imprinted
loci, using TaqMan allele-specific methylated
multiplex real-time quantitative polymerase chain reaction,
as previously described (
), and methylation specific
multiplex ligation-dependent probe amplification (for
chromosomes 6, 7, and 14) for 23 patients (Fig. 2). We
found that 58.8% of patients with epimutation of the
MEG3/DLK1:IG-DMR had methylation defects within
at least one of the other studied loci. The upd(
group, as well as one patient with a paternal deletion (case
25), had normal methylation levels at all other studied
loci, as expected for patients with a cytogenetic defect of
the 14q32.2 region. We did not perform these analysis for
two patients with deletions and the three with upd(
not identified in our laboratory.
Screening for regulatory cis-element defects
We searched for large deletions or duplications of the
14q32.2 region, using Illumina CytoSNP-12 arrays and
multiplex ligation-dependent probe amplification to
identify a potential cis-element defect within the MEG3/DLK1:
IG-DMR and the MEG3:TSS-DMR that could lead to
epimutations. No genetic defects were identified. We then
looked for point mutations or small insertions/deletions by
sequencing the entire MEG3/DLK1:IG-DMR and MEG3:
TSS-DMR. We identified a homozygous variation (C.A)
within the IG-DMR at NC_000014.8:g.101274313C.A
in one patient. All other copy number variations that we
found were already reported in public single nucleotide
polymorphism databases, making it highly unlikely that
they disturb any regulatory cis-elements of either DMR.
Screening for regulatory trans-acting factors
We carried out extensive mutation screening by whole
exome-sequencing to look for trans-acting factor defects
that could be involved in the establishment, maintenance,
or reading of DNA methylation marks and/or related to
imprinting disorders for all patients with epimutations.
We first looked for heterozygous or homozygous
missense/nonsense mutations or frameshifts insertions/
deletions in a common defective gene or different genes
from the same family. Several genes were mutated in
at least four patients, but their function or cellular
localization made them very unlikely to be involved in the
hypomethylation of the MEG3/DLK1:IG-DMR. We
then examined genes encoding proteins known to
be involved in the establishment/maintenance of DNA
methylation marks related to imprinting, along with
some of their binding proteins. The variations found
within genes that could be involved in the hypomethylation
defect are presented in Table 3. We validated these
variations by Sanger sequencing and determined their
Here we report clinical and molecular data on a large
cohort of 28 patients with 14q32.2 imprinted region
disruptions. The underlying mechanism for most of the
patients (60.7%) is epigenetic, with a hypomethylation at
aBicornurate uterus (n = 1), renal agenesis (n = 1), bilateral cryptorchidism (n = 2), nephrocalcinosis (n = 1).
bMultiple agenesis (n = 3), crowded teeth (n = 3), delayed tooth eruption (n = 2).
cAneurysm of the interatrial septum (n = 2), interatrial communication (n = 1).
the paternal MEG3/DLK1:IG-DMR, whereas it has been
reported to be between 11.8% and 18.8% in recent
publications, consisting mostly of upd(
We have described as many 14q32.2 epimutations as
those already published (n = 17; Table 4).
Epimutations at 14q32.2 have been rarely reported,
and the mechanisms responsible for their occurrence
are unknown. We therefore extensively studied the
molecular pattern of the 17 patients with epimutations. We
first looked for genetic disruption of MEG3/DLK1:
Characteristics of Pubarche and Puberty for 11 Patients >9 Years of Age
Age at pubarche onset, y
Bone age advancement during puberty
Age at thelarche or testicle enlargement, y
Age at menarche, ya
Delay between thelarche and menarche, ya
At pubarche onset (SDS)
At central puberty onset (SDS)
aWithout GnRH analogs.
1.9 (20.9 to 6.1)
Girls (n = 8)
1.8 (20.9 to 6.1)
Boys (n = 3)
IG-DMR; however, we found no relevant sequence
anomaly within this DMR. A large proportion of these
patients (58.8%) had multilocus methylation defects
(MLMDs). MLMDs have already been reported in patients
with SRS with epimutations of H19/IGF2:IG-DMR and
Beckwith-Wiedemann syndrome (another imprinted disorder
leading to overgrowth secondary to abnormal methylation of
the 11p15 region), although in much lower proportions, at
10% and 25%, respectively (
). The high proportion of our
patients with MLMDs prompted us to look for genetic
anomalies in trans-acting factors involved in the regulation of
specific parental methylation at imprinted loci. However, we
did not find any commonly disrupted genes in these patients,
and only four patients had variations within genes known to
be involved in the establishment or maintenance of
methylation. Two of these patients, for whom we identified two
paternally inherited variants in the KAP1 (rare variant) and
UHRF1BP1 (unreported variation) genes, showed no
evidence of MLMD, making it unlikely that these candidate
genes are involved in the hypomethylation process of MEG3/
DLK1:IG-DMR. Another patient (case 13), with MLMD
affecting six loci aside from 14q32.2, inherited an unreported
maternal variation in the ARID4A gene, which has been
shown to be involved in the maintenance of methylation at the
Snrpn locus in mice. Finally, the fourth patient inherited a rare
compound heterozygous variant of the SETDB1 gene, which
is also a key player in the methylation process of imprinted
genes. This patient showed hypomethylation only at 14q32.2
and no evidence of MLMD. These variations, as well as the
;60% of MLMDs identified among these patients, and the
absence of cis-regulatory element defects within either
MEG3/DLK1:IG-DMR or MEG3:TSS-DMRs, strongly
)mat: maternal uniparental disomy of chromosome 14. In our cohort, short stature was defined as length ,22 SDS at 24 mo; no definition for
other cohorts. References 5–14 can be found in the Supplemental Data.
suggest that these imprinting disturbances may be secondary
to the dysfunction of one or several trans-acting factors.
However, the potential involvement of the identified genetic
variants in the LOM mechanism will require further
investigation and/or identification of variants of these genes in
Molecular analysis allowed us to distinguish among
three types of anomalies in the 14q32.2 region:
)mat, and deletions. Identification of
deletions in these patients is important because of the risk
of recurrence if inherited from the father. Aside from the
need for genetic counseling, the identification of deletions
would also modify the therapeutic strategy. Indeed, some
reports have highlighted an increased risk of thyroid
cancer [papillar carcinoma (
)] in patients with a large
deletion in the 14q32.2 region and/or mental disability in
those with a YY1 deletion, a gene within the same 14q32
). Among the three patients with deletions in this
study, two had an already identified recurrent deletion,
leading to cognitive delay and thyroid cancer
(Supplemental Figure 3). This cancer risk must be considered and
carefully evaluated for patients who are candidates for
recombinant GH treatment because of short stature.
We compared the clinical data from all published cases
with our cohort (Table 4). We noticed the relatively high
frequencies of NH-CSS items among the previously
published patients (being born SGA, relative
macrocephaly, feeding difficulties, short stature, and protruding
forehead). Asymmetry was rare, as in our patients. In
patients with SRS, asymmetry is also the less frequent sign
when looking at the overall SRS group (57.3%) (
in our study, neonatal hypotonia, acromicria, obesity,
and early puberty were frequent signs among patients
with 14q32.2 anomalies. In our cohort, we found no
substantial differences between the clinical features
associated with epimutations and those associated with upd
The overlap between TS and SRS phenotypes has
already been established in reported cases (
of the patients in our cohort with a 14q32.2 disruption
meet the criteria for a clinical diagnosis of SRS, as do
other recently reported patients (
). Features classically
reported in TS, such as neonatal hypotonia, acromicria,
clinodactyly precocious puberty, obesity, and
psychomotor delay, can also be identified in patients with SRS
with 11p15 epimutations or upd(
)mat. Indeed, although
certain classic characteristics are shared across the
different molecular causes of SRS, others may be more
common to one molecular cause than another (
7, 9, 11
As such, patients with SRS can have a large spectrum of
symptoms that are not included in the NH-CSS. The
NHCSS was developed to provide easy, rapid, and sensitive
diagnosis of SRS but is not meant to be exhaustive.
As recently published in the first SRS international
), SRS is a clinical diagnosis, and
molecular testing is useful for confirmation and stratification of
the diagnosis. In our cohort, almost 3/4 of patients with
14q32.2 disruption fulfill the clinical criteria of the
NHCSS ($4/6) and therefore may be considered as falling
within the SRS phenotype. As also proposed in the SRS
consensus for CDKN1C and IGF2 mutation, 14q32.2
disruption may be an alternative molecular diagnosis
In this retrospective analysis, we focused on pubarche and
puberty in the oldest patients. All but three patients of the
cohort were born SGA and puberty was precocious or
early for all patients. SGA patients are known to develop
early and rapidly progressive puberty (
). These data
are concordant with our clinical experience (work under
submission) with patients with SRS with 11p15 disruption or
upd(7)mat, who frequently present with aggressive early
adrenarche and puberty. GnRH analogs have been shown to
be beneficial for patients born SGA of small stature at
pubertal onset (with or without a clinical diagnosis of SRS),
together with recombinant GH treatment (
therapy is now considered to be a possible personalized
treatment of patients with SRS (9). Adding aGnRH to
recombinant GH treatment may also be considered for
patients with 14q32.2 anomalies. Although the role of DLK1
in the regulation of puberty is still unknown, a recent familial
report suggests a link between DLK1 anomalies and pubertal
All of our patients started pubarche or puberty after a
substantial rise in BMI. DLK1 expression has been
implicated in the homeostasis of fat metabolism in studies in
mice and humans, showing a role in preadipocyte
differentiation, regulation of hypothalamic satiety, circulating
leptin concentrations, peripheral adipose tissue activity,
and muscle mass development (
). As in patients with
SRS, the BMI of patients with 14q32.2 anomalies should
be carefully followed to avoid an excessive and
uncontrollable increase during infancy (
). The SRS
international consensus recommends maintaining a ratio of
weight/expected weight for height at 75% to 85% and BMI
at 12 to 14 kg/m2 until the age of 4 years in those with SRS
(9). Likely, it is also very important for patients with 14q32
disruption to try to limit their weight gain in their early life
to avoid the onset of obesity and metabolic complications
usually observed in this group of patients.
When looking at the age at diagnosis, we identified two
groups: one with early diagnosis (n = 17 before 3.2 years)
and one with late diagnosis (n = 11 .8.5 years). The first
group was typically diagnosed because of being born SGA
and failure to thrive, whereas the second was diagnosed
later because of precocious puberty and obesity. Because of
the late diagnosis in these latter patients, which correspond
to the classical presentation of TS described until now,
none benefited from early care including active weight
and pubertal management or recombinant GH therapy.
Among a series of published cases with 14q32.2
disruption, only half were treated with an aGnRH during
their precocious puberty and only about a fourth were
treated with concomitant or isolated recombinant GH
). Using NH-CSS to screen these patients
allows a more precocious diagnosis and the possibility of
proposing an earlier specific management of their
metabolic, growth, and pubertal issues. Further studies will
be necessary to evaluate if patients with 14q32 disruption
will benefit from this earlier management as do patients
We provide clinical and molecular data to support that, as
raised in the SRS international consensus, 14q32.2
disruption may be considered as an alternative molecular
diagnosis of SRS. These patients should be managed with a
close follow-up for early onset of obesity, pubarche,
precocious puberty, and short stature. 14q32.2 disruption
should be investigated in case of suspected SRS without
11p15 LOM or upd(
)mat, especially in the presence of
more specific characteristics of TS such as neonatal
hypotonia, acromicria, hyperphagia, and/or early obesity. In
patients with MEG3/DLK1:IG-DMR hypomethylation,
an additional molecular analysis must be carried out to
identify any paternal deletion within the 14q32.2 region
because of the different prognosis and management of
these patients. Elucidation of the mechanisms that control
epimutation should be a research priority because of the
high prevalence of MLMD in these patients.
We thank the patients, their families and physicians, the
“Association Française des Familles ayant un enfant atteint du
Syndrome Silver-Russell ou ne´ Petit pour l’ Aˆge Gestationnel
(AFIF/PAG),” and The MAGIC Foundation for their support.
We also thank Mrs. Laurence Perin, Mrs. Nathalie Thibaut,
Mrs. Cristina Das Neves, Mrs. Marilyne le Jule, and Mrs.
Evelyne Tagodoe, the diagnostic and research technicians of
the Pediatric Endocrinology Department, Trousseau Pediatric
Hospital, and Dr. Boris Keren for his help in molecular analysis.
Financial Support: This work was supported by the
Institut National de la Sant e´ et de la Recherche Me´dicale
(INSERM) and funding from the Universit e´ Pierre et Marie
Curie (UPMC-Paris VI). W.A.H. was supported by the People
Programme Marie Curie Actions (MCA) of the European
Union’s Seventh Framework Programme FP7/ITN Ingenium
2007–2013 under REA grant agreement no. 290123 and by the
Soci e´te´ Française d’Endocrinologie et Diabe´tologie Pe´diatrique.
A.P.C. was supported by CNPq under grant no.
233262/20148. F.B. was supported by a Novo Nordisk “Growth Hormone,
Growth and Metabolism” grant. F.B. and I.N. are members
of the European Congenital Imprinting Disorders Network
European Cooperation in Science and Technology (BM1208).
Correspondence and Reprint Requests: Ire`ne Netchine,
MD, PhD, Explorations Fonctionnelles Endocriniennes,
H oˆpital Armand Trousseau, 26 avenue du Dr Arnold Netter,
75571 Paris cedex 12, France. E-mail: .
Disclosure Summary: The authors have nothing to
Geoffron et al
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