C L I N I C A L R E S E A R C H A R T I C L E

Novel Genetic and Biochemical Findings of DLK1

in Children with Central Precocious Puberty:
A Brazilian–Spanish Study

Luciana Montenegro,1,* José I. Labarta,2,* Maira Piovesan,1 Ana P.M. Canton,1

Raquel Corripio,3 Leandro Soriano-Guillén,4 Lourdes Travieso-Suárez,5

Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020

Álvaro Martín-Rivada,5 Vicente Barrios,5 Carlos E. Seraphim,1 Vinicius N. Brito,1
Ana Claudia Latronico,1,* and Jesús Argente5,*
1
Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/
LIM42, Hospital das Clínicas, Disciplina de Endocrinologia e Metabologia, Faculdade de Medicina da
Universidade de São Paulo, São Paulo, Brazil; 2Pediatric Endocrinology Unit, Department of Pediatrics,
Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria de Aragón, Zaragoza, Spain;
3
Pediatric Endocrinology Department, Corporació Parc Taulí Hospital Universitari, Institut d’Investigació i
Innovació Parc Taulí I3PT, Universitat Autònoma de Barcelona, Sabadell, Spain; 4Pediatric Endocrinology
Unit, Institute of Biomedical Research – Hospital Universitario Fundación Jiménez Díaz, Universidad
Autónoma de Madrid. Madrid, Spain; and 5Department of Pediatrics, Universidad Autónoma de Madrid.
Departments of Pediatrics and Pediatric Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto
de Investigación La Princesa, Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad
y Nutrición (CIBEROBN), Instituto de Salud Carlos III, IMDEA Food Institute. Madrid, Spain

ORCiD numbers: 0000-0002-8401-7613 (L. Montenegro); 0000-0001-6782-693X (A. C. Latronico);

0000-0001-5826-0276 (J. Argente).

Background:  Central precocious puberty (CPP) has been associated with loss-of-function
mutations in 2 paternally expressed genes (MKRN3 and DLK1). Rare defects in the DLk1 were
also associated with poor metabolic phenotype at adulthood.

Objective:  Our aim was to investigate genetic and biochemical aspects of DLK1 in a Spanish
cohort of children with CPP without MKRN3 mutations.

Patients:  A large cohort of children with idiopathic CPP (Spanish PUBERE Registry) was studied.
Genomic deoxyribonucleic acid was obtained from 444 individuals (168 index cases) with CPP
and their close relatives. Automatic sequencing of MKRN3 and DLK1 genes were performed.

Results:  Five rare heterozygous mutations of MKRN3 were initially excluded in girls with familial
CPP. A rare allelic deletion (c.401_404 + 8del) in the splice site junction of DLK1 was identified in a
Spanish girl with sporadic CPP. Pubertal signs started at 5.7 years. Her metabolic profile was normal.
Familial segregation analysis showed that the DLK1 deletion was de novo in the affected child.
Serum DLK1 levels were undetectable (<0.4 ng/mL), indicating that the deletion led to complete lack
of DLK1 production. Three others rare allelic variants of DLK1 were also identified (p.Asn134=; g.-
222 C>A and g.-223 G>A) in 2 girls with CPP. However, both had normal DLK1 serum levels.

Conclusion:  Loss-of-function mutations of DLK1 represent a rare cause of CPP, reinforcing a

significant role of this factor in human pubertal timing. (J Clin Endocrinol Metab 105: 1–8, 2020)

Freeform/Key Words:  precocious puberty, DLK1, genetics, mutations

ISSN Print 0021-972X  ISSN Online 1945-7197 *Drs. Montenegro, Labarta, Latronico, and Argente contributed equally to this article.
Printed in USA Abbreviations:  ACMG, American College of Medical Genetics; BMI, body mass index;
© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals. CPP, central precocious puberty; DNA, deoxyribonucleic acid; EGF, epidermal growth
permissions@oup.com factor; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH,
Received 17 March 2020. Accepted 14 July 2020. luteinizing hormone; LHRH, luteinizing hormone releasing hormone; MFE, minimum
First Published Online 17 July 2020. free energy; MRI, magnetic resonance imaging; mRNA, messenger ribonucleic acid;
Corrected and Typeset 17 August 2020. PCR, polymerase chain reaction.

doi:10.1210/clinem/dgaa461 J Clin Endocrinol Metab, October 2020, 105(10):1–8   https://academic.oup.com/jcem   1

2  Montenegro et al   DLK1 Deficiency in Central Precocious Puberty
G
enomic imprinting has been demonstrated to play a
relevant role in regulation of pubertal timing in hu-
mans. Several loss-of-function mutations of 2 maternally
imprinted genes, MKRN3 and Delta-like 1 homolog
(DLK1), were described in children with familial cen-
tral precocious puberty (CPP) (1-4). Interestingly, a po-
tential interaction between 2 imprinted regions where
these CPP genes are located (chromosomes 15q11.2 and
14q32.2) was determined in vitro, suggesting a unique
epigenetic regulation involving these loci (5).
Notably, loss-of-function mutations of MKRN3
are very well-established causes of familial PCC (first
description in 2013; 800 studied patients) (1, 3). The
frequency of these mutations is particularly high in
Occidental countries (up to 46% in European studies)
and to date, more than 30 different inactivating muta-
tions have been described in girls and boys with familial
CPP from different ethnic backgrounds (3). In contrast,
DLK1 defects in children with CPP are recent and ap-
parently uncommon, with scarce information on geno-
type–phenotype correlations.
DLK1, also known as preadipocyte factor 1 (Pref-1)
and fetal antigen (FA1), is an epidermal growth factor-
like (EGF-like) membrane-bound protein. It contains 6
tandem EGF-like repeats, a juxtamembrane region with
a TACE (ADAM17)-mediated cleavage site, a trans-
membrane domain, and a short intracellular tail (6, 7).
DLK1 is part of the Notch signaling pathway that con-
trols many developmental processes. It is well known
that DLK1 prevents the differentiation of preadipocytes
into mature adipocytes (8-10) and Dlk1 knockout mice
present obesity, growth retardation, and skeletal malfor-
mations (9). Conversely, mice expressing a Dlk1 trans-
gene in adipose tissue are lean and show decreased fat
pad weight (11). In humans, common allelic variants of
the DLK1 gene have been associated with severe obesity
(Trio families study) in children and early menarche
(GWAS studies) in European women (12).
In 2017, Dauber et al. (2). described a complex de-
fect of DLK1 (∼14-kb deletion and 269-bp duplication)
in 5 members of a multigenerational Brazilian family
with nonsyndromic CPP by using linkage analysis and
whole-genomic sequencing. This deletion included the
5′ untranslated region and the first exon of DLK1. Only
family members who inherited the defect from their
father were shown to have precocious puberty, con-
sistent with the known pattern of imprinting of DLK1.
This very rare genomic defect was the first descrip-
tion of severe isolated deficiency of DLK1 in humans.
More recently, 3 novel frameshift mutations located at
the extracellular domain of DLK1 (p.Gly199Alafs*11,
p.Val271Cysfs*14, and p.Pro160Leufs*50) have been
J Clin Endocrinol Metab, October 2020, 105(10):1–8
described in 5 women with familial CPP (4 Brazilian
women and 1 English woman) (13). Metabolic abnor-
malities, such as overweight/obesity, early-onset glucose
intolerance/type 2 diabetes mellitus, and hyperlipidemia,
were more prevalent in the women with DLK1 muta-
tions than in the idiopathic CPP group. Interestingly,
2 sisters who carried the p.Gly199Alafs*11 mutation
also exhibited polycystic ovary syndrome and infertility
(13). The high prevalence of metabolic alterations in
adult women who experienced precocious menarche or
Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020
CPP due to DLK1 defects suggests that DLK1 repre-
sents a new link between reproduction and metabolism.
A soluble form of DLK1 with a molecular weight of
50  kDa can be generated through TACE (ADAM17)-
mediated cleavage of its extracellular domain. Serum
DLK1 concentrations are undetectable in women with
CPP caused by deleterious defects of DLK1, suggesting
that this accessible biochemical measurement could be
a potential screening assay for the diagnosis of familial
CPP due to a rare deficiency of DLK1.
In the present study, comprehensive investigation
of genetic and biochemical aspects of DLK1 was per-
formed in a large cohort of Spanish children with CPP
(Spanish PUBERE Registry) without MKRN3 muta-
tions. This study represents a collaborative initiative be-
tween Brazilian and Spanish universities for continuous
elucidation of the genetic basis of CPP in children.
Novel DLK1 findings were demonstrated in the Spanish
cohort that had been previously diagnosed with idio-
pathic CPP, reinforcing a significant role of this factor in
human pubertal timing.
Patients and Methods
Spanish cohort
Spanish children with idiopathic CPP evaluated in 55 cen-
ters throughout the country were studied. This cohort consti-
tutes the Spanish PUBERE Registry that emerged to address
different issues related to CPP, such as epidemiological and
clinical information (14). The PUBERE Registry is supported
by the Spanish Society for Pediatric Endocrinology (Sociedad
Española de Endocrinología Pediátrica).
Genomic deoxyribonucleic acid (DNA) was obtained from
444 individuals, including 168 cases with CPP (index cases)
and their relatives. Idiopathic CPP was defined as girls diag-
nosed with progressive thelarche before the age of 8  years
and boys with testicular volume greater than 4  mL (Prader
orchidometer) before 9 years of age, with the diagnosis con-
firmed with luteinizing hormone releasing hormone (LHRH)
testing; luteinizing hormone (LH) peak after LHRH stimula-
tion (100 µg/m2) greater than 7 IU/L; bone age minus chrono-
logical age equals more than 1 year.
The CPP group was mainly composed of female patients
(161 girls and 7 boys), with a mean chronological age at pres-
entation of 6.87 years ± 0.84 in girls and 6.24 years ± 0.53 in
doi:10.1210/clinem/dgaa461
boys; the mean bone age at diagnosis was 9.55 years ± 1.85 in
girls and 9.35 years ± 0.65 in boys. Familial CPP cases were
diagnosed around 20% of the Spanish cohort. No magnetic
resonance imaging (MRI) lesions were identified in this group
of CPP patients.
Sanger sequencing
DNA was collected from the index cases, their parents,
and first- or second-degree family members when available.
Genomic DNA was extracted from peripheral blood lympho-
cytes according to standard protocols (15). The MKRN3
sequencing was first performed and followed by DLK1 ana-
lysis in negative MKRN3 cases. The entire coding regions of
MKRN3 (1 exon—GenBank accession number NM_005664)
and DLK1 (5 exons—GenBank accession number
NM_003836) were amplified by polymerase chain reaction
(PCR) followed by purification and automatic sequencing of
the products by using the Sanger sequencing method. DNA
sequences obtained were compared to the human GenBank
MKRN3 and DLK1 sequence using Sequencher sequence
alignment software. Familial segregation analysis of poten-
tial pathogenic variants was performed by Sanger sequencing.
PCR primers and conditions are available upon request.
DLK1 serum measurements
Serum DLK1 levels were measured in the affected family
members and pubertal controls by using a soluble DLK1
enzyme-linked immunosorbent assay from Immuno-Biological
Laboratories, Inc. (IBL-America, Minneapolis, MN). The
intra- and interassay variations were 5.6% and 8.1%, respect-
ively. The sensitivity limit was 0.34 ng/mL.
Microsatellite analysis
The biological paternity was confirmed by the AmpFlSTR®
Identifiler® PCR Amplification Kit, a short tandem repeat
multiplex assay that amplifies 15 tetranucleotide repeat loci
and the Amelogenin gender-determining marker using manu-
facture instruction (Life Technologies, Carlsbad, CA).
In silico analysis
In silico analysis of splice regions was performed by
NNSplice (16), Human Splice Finder (17), FSplice (Softberry,
Mount Kisco, NY) and NetGene2 (18). The promoter region
of DLK1 was studied using RNAfold WebServer (http://rna.
tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). The
5′ untranslated region messenger ribonucleic acid (mRNA)
of DLK1 was submitted with default parameters to predict
the potential secondary structure for the wildtype and the
DLK1 g.-223 G>A and g.-222 C>A mutant mRNA. Potential
minimum free energy (MFE) structures, centroid structures,
and positional entropies were obtained (19). The American
College Medical Genetics and Genomics classification and the
pathogenicity sites prediction was performed by Varsome (20).
Results
MKRN3 analysis
Loss-of-function mutations of MKRN3 are frequent
genetic causes of familial CPP with paternal inheritance
https://academic.oup.com/jcem  3
and they were first excluded. Five rare variants were iden-
tified, including 4 missense mutations and 1 frameshift
mutation, in girls with CPP. Two variants have previously
been associated with CPP phenotype (Table 1) (21).
DLK1 analysis
A heterozygous deletion (c.401_404 + 8del) affecting
exon 4 and intron 4-5 of DLK1 was identified in a
girl with sporadic CPP (Patient 1). The deletion com-
prised the splice site junction. Both of her parents had
Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020
a wildtype sequence of exon 4 of DLK1 (Fig. 1A). The
biological paternity was confirmed using microsatellite
analysis in this case.
A rare heterozygous synonymous allelic variant was
identified in a girl (Patient 2) with sporadic CPP (Fig. 1B).
The variant c.402C>T p.Asn134= (rs150016759) has
a minor allele frequency less than 0.01 in the normal
population and 0.0008825 in European population
(according to gnomAD data). In silico analysis showed
that this synonymous variant was located in a potential
donor 5′ splice region, characterized by a conserved se-
quence GU at the 5′ end of the intron 4-5. Although the
alteration does not modify the amino acid sequence, this
nucleotide change could disrupt RNA splicing resulting
in the loss of exons or the inclusion of introns, leading
to an altered protein-coding sequence. Segregation ana-
lysis showed that it was inherited from her father and
was absent in her mother.
Two consecutive rare allelic variants (g.-222 C>A
and g.-223 G>A) in the DLK1 promoter region were
identified in an adopted girl (Patient 3)  with CPP
(Fig.  1C). According to the Encyclopedia of DNA
Elements (ENCODE consortium), both variants af-
fected regions that are DNA sequences for poten-
tial binding of 4 transcription factors—Retinoic Acid
Receptor Alpha (RARA), E2F Transcription Factor 1
(E2F1), Eomesodermin (EOMES), and Retinoic Acid
Receptor Gamma (RARG). Therefore, both variants
could impair gene transcription, increasing or reducing
its expression. The allele frequencies of the g.-222 C>A
(rs1442192598) and g.-223 G>A (rs1186489952) vari-
ants were 0.00002449 and 0.00002222, respectively.
These variants were not identified in European or Latin
American populations. As this patient was adopted,
her biological parents were not available for genetic
analysis.
In silico analysis was performed using the software
RNA Fold, which predicts the secondary structures of
single-stranded RNA. The software calculates the energy
necessary to form the secondary structure of the RNA
based on MFE algorithms. The MFE of a RNA molecule
can be affected by 3 properties of nucleotides in the se-
quence: their number, composition, and arrangement.
4  Montenegro et al   DLK1 Deficiency in Central Precocious Puberty J Clin Endocrinol Metab, October 2020, 105(10):1–8

The lower the thermodynamic energy of the structure,

advancement
the more stable it generally is. The secondary structure
is directly associated to the RNA function. The results
Bone age

suggested a potential change in the MFE necessary for

the formation of the second RNA structure and centroid

1.7
1.1

2.6

2.1

0.5
secondary structure.

Clinical features of girls with rare DLK1 variants

Chronological age of

Patient  1. The girl with a heterozygous deletion

puberty onset

(c.401_404 + 8del) affecting exon 4 and intron 4-5

Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020

of DLK1 showed progressive CPP. Pubertal signs
appeared at 5.7  years of age. The first clinical visit
took place at 6.3 years of age and the physical exam-
6.8

6.8

6.5

6.4

7.3

ination showed thelarche III, pubarche III, height

125.5 cm (1.6 SDS [standard deviation score]), weight
Female

Female

Female

Female

Female

30.7  kg (2.1 SDS), body mass index (BMI) 19.5  kg/

Abbreviations: ACMG, American College of Medical Genetics; CPP, central precocious puberty; MAF minor allele frequency; NA, not available
Sex

m2 (1.5 SDS), bone age 10.5 years with target height

of 146.6  cm. Personal antecedents were uneventful.
Likely pathogenic

Likely pathogenic

Biochemical analysis confirmed early activation of

classification
Table 1.   Rare allelic variants of MKRN3 identified in patients with CPP from a Spanish cohort

the central gonadotrophic axis. Basal and peak LH

Pathogenic

Pathogenic

Pathogenic

levels after LHRH stimulation were 1.7  mIU/mL

Highest minor allelic frequency observes including 1000 genome Phase 3, NHLBI Exome Sequencing Project and gnomAD.
ACMG

and 32.77  mIU/mL, respectively, and basal and peak

follicle-stimulating hormone (FSH) after LHRH stimu-
lation were 6.32 mIU/mL and 19.89 mIU/mL, respect-
ively. MRI of the hypothalamic–pituitary region was
description in

normal and pelvic ultrasound demonstrated increased

ovarian volume with follicles consistent with CPP.
(21)

(1)
Previous

The patient was treated with gonadotropin-releasing

CPP

hormone (GnRH) analogues during 3.8 years (6.3 to

NA

NA

NA

10.1 years) with no side effects. Menarche occurred at

11.2 years and at 15.9 years her height was 153.5 cm
segregation

(–1.4 SDS), weight 41.6  kg, BMI 17.6  kg/m2 (–1.2

SDS), and she has regular menses without hirsutism,
Familial

Father

Father

Father

Father

obesity, or hyperandrogenism.
NA

Patient 2.  The heterozygous synonymous allelic variant

(c.402C>T p.Asn134=) was identified in a girl with
Allelic frequencya

sporadic CPP and in her father. Pubertal signs appeared

MAF < 0.001

MAF < 0.001

at 7  years of age. The first clinical visit took place at

Phenotype

MAF < 0.01

MAF < 0.01

MAF < 0.01

7.25 years of age and the physical examination showed

thelarche II, pubarche II, height 131.2  cm (2.2 SDS),
weight 31 kg (1.8 SDS), BMI 18 kg/m2 (0.65 SDS), and
bone age 8 years and 10 months. Personal antecedents
p.Ala162Glyfs

p.Met297Arg

were uneventful. Biochemical analysis confirmed early

p.Phe417Leu

p.Arg242Trp

p.Tyr418His

activation of the central gonadotrophic axis. Basal

Protein

and peak LH levels after LHRH stimulation were

<0.07 mIU/mL and 7.7 mIU/mL, respectively, and basal
and peak FSH after LHRH stimulation were 2.1 mIU/
mL and 19 mIU/mL, respectively. MRI of the hypothal-
(rs1277371835)

(rs1470111765)
c.475_476insC

(rs147605349)
(rs745560329)

(rs763195944)

amic–pituitary region was normal and pelvic ultrasound

c.1249T>C

c.1252T>C

c.890T>C
c.724C>T

demonstrated increased ovarian volume. The patient

Variant

cDNA

was treated with GnRH analogues during 2.8  years

(7.4-10 years) with no side effects. Menarche occurred
a
doi:10.1210/clinem/dgaa461 https://academic.oup.com/jcem  5

Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020

Figure 1.  Sequencing data of DLK1 gene showing 3 rare allelic variants. (A) DLK1 deletion c.401_404 + 8del identified in patient 1 (index case): I,
index; II, father; III, mother; IV, sister; all of them were wildtype. (B) Patient 2 with the synonymous DLK1 c.402C>T p.Asn134= (rs150016759): I,
index case; II, father; III, mother wildtype. (C) Patient 3 with DLK1 promoter region alteration g.-222 C>A and g.-223 G>A. I, control; II, index case.

at 11 years and at 13 years her height was 163.5 cm (1.1

SDS), growth velocity <2 cm during the last year), BMI
22 kg/m2 (0.9 SDS) and she has regular menses without
hirsutism, obesity, or hyperandrogenism.

Patient  3. Two consecutive rare allelic variants (g.-

222 C>A and g.-223 G>A) in the DLK1 promoter
region were identified in an adopted girl with CPP.
Hence, genetic and biochemical information from her
parents was not available. Pubertal signs appeared at
6 years and 6 months of age. The first clinical visit took
place at 7.1 years of age and the physical examination
showed thelarche II, pubarche II, height 126.6 cm (1.19
SDS), BMI 16.6  kg/m2 (0.06 SDS), bone age 9  years
and 6  months. Personal antecedents were uneventful.
Biochemical analysis confirmed early activation of the
central gonadotrophic axis. Basal and peak LH levels
after LHRH stimulation were <0.37  mIU/mL and
7.86 mIU/mL, respectively, and basal and peak FSH after
LHRH stimulation were 2.45  mIU/mL and 6.95  mIU/
mL, respectively. Pelvic ultrasound demonstrated in-
creased ovarian volume. The patient was treated with
GnRH analogues during 3 years (7.6-10.6 years) with
no side effects. Menarche occurred at 11  years and
11  months, and at 12  years and 7  months her height
was 158.3 cm (0.7 SDS), BMI 27.42 kg/m2 (2.46 SDS),
and she has regular menses with obesity and physical
signs of hyperandrogenism.
Circulating DLK1 concentrations
To investigate the effect of the novel variants on
DLK1 production, serum DLK1 levels were measured.
DLK-1 serum levels were normalized in 62 patients
from a group of 250 healthy girls and boys throughout
development. The control group included prepubertal
(Tanner I; n = 31) and pubertal patients at the start of
puberty (Tanner II; n = 31). We have shown in the pu-
bertal group only Tanner II (Fig. 2).
Figure 2.  DLK1 serum levels in controls, obese patients, 1 patient
with CPP due to DLK1 deletion and 2 patients with CPP and variants
in DLK1. *P < .001 with control group.
Patient 1 who carried the c.401_404 + 8del deletion
had undetectable DLK1 levels (<0.4 ng/mL), supporting
that the deletion led to a complete lack of DLK1 pro-
duction in this individual. The serum DLK1 levels of
her father, mother, and sister were within normal levels
(ranged 6.36-8.98  ng/mL). Patient 2 with c.402C>T
p.Asn134= (rs150016759) had normal levels of DLK1
(6.11  ng/mL), as did her father (5.05  ng/mL). Patient
3 with 2 consecutive rare allelic variants (g.-222 C>A
and g.-223 G>A) in the DLK1 promoter region also had
normal levels of DLK1 (5.55  ng/mL). Because the pa-
tient was adopted, DLK1 levels in the biological parents
were not available.
Discussion
Loss-of-function mutations of DLK1 represent the most
recently recognized genetic cause of familial CPP with
paternal inheritance. Here, novel findings of DLK1,
including a pathogenic deletion leading to a DLK1 defi-
ciency, were identified in Spanish girls with progressive
and nonsyndromic CPP. Interestingly, none of them had
6  Montenegro et al   DLK1 Deficiency in Central Precocious Puberty J Clin Endocrinol Metab, October 2020, 105(10):1–8

history of premature sexual development on their pa-
ternal side, as we had expected for a condition associated
with maternal imprinting gene defects. Indeed, patient 1
was a de novo heterozygous deletion (c.401_404 + 8del)
affecting exon 4 and intron 4-5 of DLK1 gene. Her
biological parents had no similar DLK1 abnormality,
indicating that she is a true sporadic case of CPP asso-
ciated with a DLK1 defect. The functional impact of
this deletion was clearly demonstrated by undetectable
serum DLK1 levels.
mainly as a cleaved protein and is expressed in den-
drites of neurons in multiple nuclei (arcuate, paraven-
tricular, supraoptic, suprachiasmatic, dorsomedial, and
lateral hypothalamic), suggesting a relevant neuroendo-
crine regulation for this soluble DLK1 in postnatal
development.
The long-term evaluation of the first described
women with familial precocious menarche (<9  years)
or CPP due to DLK1 variants showed multiple meta-
bolic alterations, such as overweight/obesity, type 2 dia-

Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020

DLK1 exists in both transmembrane and shed forms.
Soluble DLK1 arises by proteolytic extracellular do-
main clipping through the ADAM 17/TACE enzyme.
There are 2 processing sites in the extracellular domain,
1 located near the fourth EGF repeat and the other in
the region proximal to the transmembrane domain, re-
sulting in the larger (50 kDa) and smaller (25 kDa) sol-
uble forms (7, 22). Among the 6 pathogenic variants of
DLK1 described in patients with CPP so far, 4 of them
(the new deletion described here and 3 other frame-
shift pathogenic allelic variant—p.Gly199Alafs*11,
p.Val271Cysfs*14, and p.Pro160Leufs*50) are located
in the extracellular domain (Fig. 3) (13). More specific-
ally, only 2 consecutive exons (4 and 5) that encode the
extracellular (EFG-4 to 6) and transmembrane domain
were affected in these distinct families with CPP, sug-
gesting that this particular region could be a hot spot
for pathogenic allelic variants. These particular DLK1
defects were associated with undetectable DLK1 serum
levels. Notably, Villanueva et  al. (23) reported that
Dlk1 expression in the mouse hypothalamus occurred
betes mellitus, and hyperlipemia, in agreement with the
known physiological metabolic functions of DLK1 (an
inhibitor of adipogenesis and fat accumulation). Patient
1 who harbored a pathogenic DLK1 defect, started
pubertal development at ~5  years with no additional
medical problems and she had a satisfactory response
to the classical GnRH analog treatment, suggesting a
typical CPP at initial diagnosis. No weight excess, meta-
bolic alterations, or syndromic features were noticed
in this affected girl with the DLK1 pathogenic defect.
However, she is a teenage girl (15 years) and close clin-
ical follow-up is recommended.
Serum DLK1 levels were measured in healthy Spanish
boys and girls throughout development to establish its
normal values. Furthermore, DLK1 serum levels were
studied in prepubertal and pubertal children with severe
obesity. No differences were found between controls
and obese children (Fig.  2). Patient 2 harbored a rare
polymorphism c.402C>T p.Asn134= (rs150016759)
that was inherited from her father. Besides being a rare
variant and being suggested as a potential loss of splice

Figure 3.  Schematic representation of the human DLK1 gene (A) and human DLK1 protein (B) respectively. (A) Human DLK1 gene (transcript
ENST00000341267.9). Blue boxes indicate the coding sequence of the 5 exons of the gene in human. Open boxes indicate the 5′ and 3′
untranslated region of the gene, respectively. The localization of the allelic variant identified in patient with familial CPP are indicated by arrows.
In red are the pathogenic allelic variants already described (2, 13) and the 1 described in this work. (B) Human DLK1 protein structure (P80370).
The purple lozenge indicates the signal peptide; green boxes the 6 EGF-like repeats. Orange ellipse indicates the extracellular TACE (ADAM17)
proteolytic cleavage domain. The numbers represent the amino acid positions of the indicated domains.
doi:10.1210/clinem/dgaa461
site by in silico analysis, the DLK1 serum levels in this
patient and in her parents were at normal range. Similar
data were noticed in Patient 3, who carried 2 poten-
tial regulatory variants into the 5′ untranslated region
of DLK1 gene.
Human DLK1 is encoded by a paternally ex-
pressed gene located on the long arm of chromosome
14 (14q32.2), within a locus associated with Temple
syndrome, an imprinting disorder mainly charac-
terized during childhood by pre- and/or postnatal
growth failure, hypotonia, and small hands and feet
(24). Notably, 80% to 90% of patients with Temple
syndrome have been described with early puberty or
CPP (24), which seems to be much more common
than the frequency of early puberty in imprinting
disorders affecting other imprinted loci. Moreover,
patients with Temple syndrome may develop over-
weight/obesity during childhood, and hyperlipidemia
and type 2 diabetes as young adults. Interestingly, Abi
Habib et al. (25) identified barely detectable levels of
serum DLK1 in patients with Temple syndrome with
epimutations or paternal deletions at chromosome
14q32.2.
The prevalence of DLK1 pathogenic variants appears
to be significantly lower than MKRN3 defects causing
familial CPP. Among the genetic analysis of a total of
168 Spanish girls with idiopathic CPP (including fa-
milial and sporadic cases), only 1 girl presented a patho-
genic DLK1 defect. Interestingly, this Spanish cohort
was screened for MKRN3 mutations and 3.5% had po-
tentially pathogenic variants in MKRN3.
Here, we have described the first de novo DLK1 mu-
tation associated with a true case of sporadic CPP in
a girl with progressive and nonsyndromic premature
sexual development phenotype. In conclusion, DLK1
defects associated with familial and sporadic CPP are
very rare and can be associated with potential metabolic
consequences at adulthood. Furthermore, our results
indicate that the genomic deletion in DLK1 led to the
lack of serum DLK1, while the other 2 variants found in
DLK1 did not. In order to establish a genotype–pheno-
type correlation of these 2 rare variants in patients with
CPP, it remains to be determined whether their protein
product is biologically active.
Acknowledgments
Financial Support: This work was supported by grant
2008.1.1677.5.4 (to A.C.L) from the Universidade de São
Paulo (USP); grant 13/03236-5 (to A.C.L), and 2017/23892-5
(to L.R.M) from Fundação de Amparo à Pesquisa do Estado
de São Paulo (Fapesp); grant 403525/2016-0 (to A.C.L) from
https://academic.oup.com/jcem  7
the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq); Fondo de Investigación del Instituto de
Salud Carlos III (fondos FEDER); grant PI019/0166 (to J.A)
and CIBER de fisiopatología y nutrición (CIBEROBN) (to
J.A.), Madrid, Spain.
Additional Information
Correspondence and Reprint Requests: Ana Claudia Latronico,
MD, PhD, Unidade de Endocrinologia do Desenvolvimento,
Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020
Laboratório de Hormônios e Genética Molecular/LIM42,
Hospital das Clínicas, Disciplina de Endocrinologia e
Metabologia, Faculdade de Medicina da Universidade de
São Paulo, São Paulo, Brazil. E-mail: anaclusp@gmail.com;
or and Jesús Argente, Department of Pediatrics, Universidad
Autónoma de Madrid. Departments of Pediatrics and
Pediatric Endocrinology, Hospital Infantil Universitario
Niño Jesús, Instituto de Investigación La Princesa, Centro
de Investigación Biomédica en Red de Fisiopatología de la
Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos
III, IMDEA Food Institute, Madrid, Spain. E-mail: jesus.
argente@fundacionendo.org.
Disclosure Summary: The authors have nothing to disclose.
Data Availability: All data generated or analyzed during
this study are included in this published article or in the data
repositories listed in References.
References
1. Abreu AP, Dauber A, Macedo DB, et al. Central precocious pu-
berty caused by mutations in the imprinted gene MKRN3. N
Engl J Med. 2013;368(26):2467-2475.
2. Dauber A, Cunha-Silva M, Macedo DB, et al. Paternally inherited
DLK1 deletion associated with familial central precocious pu-
berty. J Clin Endocrinol Metab. 2017;102(5):1557-1567.
3. Valadares LP, Meireles CG, De Toledo IP, et al. Mutations in cen-
tral precocious puberty: a systematic review and meta-analysis. J
Endocr Soc. 2019;3(5):979–995.
4. Macedo DB, Kaiser UB. DLK1, notch signaling and the timing of
puberty. Semin Reprod Med. 2019;37(4):174-181.
5. Stelzer Y, Sagi I, Yanuka O, Eiges R, Benvenisty N. The noncoding
RNA IPW regulates the imprinted DLK1-DIO3 locus in an in-
duced pluripotent stem cell model of Prader-Willi syndrome. Nat
Genet. 2014;46(6):551-557.
6. Mei  B, Zhao  L, Chen  L, Sul  HS. Only the large soluble form
of preadipocyte factor-1 (Pref-1), but not the small soluble and
membrane forms, inhibits adipocyte differentiation: role of alter-
native splicing. Biochem J. 2002;364(Pt 1):137-144.
7. Smas  CM, Chen  L, Sul  HS. Cleavage of membrane-associated
pref-1 generates a soluble inhibitor of adipocyte differentiation.
Mol Cell Biol. 1997;17(2):977-988.
8. Smas CM, Sul HS. Pref-1, a protein containing EGF-like repeats,
inhibits adipocyte differentiation. Cell. 1993;73(4):725-734.
9. Moon  YS, Smas  CM, Lee  K, et  al. Mice lacking paternally ex-
pressed Pref-1/Dlk1 display growth retardation and accelerated
adiposity. Mol Cell Biol. 2002;22(15):5585-5592.
10. Smas CM, Chen L, Zhao L, Latasa MJ, Sul HS. Transcriptional
repression of pref-1 by glucocorticoids promotes 3T3-L1 adipo-
cyte differentiation. J Biol Chem. 1999;274(18):12632-12641.
11. Lee  K, Villena  JA, Moon  YS, et  al. Inhibition of adipogenesis
and development of glucose intolerance by soluble
8  Montenegro et al   DLK1 Deficiency in Central Precocious Puberty
preadipocyte factor-1 (Pref-1). J Clin Invest. 2003;111(4):
453-461.
12. Day  FR, Thompson  DJ, Helgason  H, et  al.; LifeLines Cohort
Study; InterAct Consortium; kConFab/AOCS Investigators;
Endometrial Cancer Association Consortium; Ovarian Cancer
Association Consortium; PRACTICAL consortium. Genomic
analyses identify hundreds of variants associated with age at me-
narche and support a role for puberty timing in cancer risk. Nat
Genet. 2017;49(6):834-841.
13. Gomes LG, Cunha-Silva M, Crespo RP, et al. DLK1 is a novel link
between reproduction and metabolism. J Clin Endocrinol Metab.
2019;104(6):2112-2120.
14. Carrascosa Lezcano A, Fernández García JM, Fernández Ramos C,
et al.; Grupo Colaborador Español. [Spanish cross-sectional growth
study 2008. Part II. Height, weight and body mass index values
from birth to adulthood]. An Pediatr (Barc). 2008;68(6):552-569.
15. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure
for extracting DNA from human nucleated cells. Nucleic Acids
Res. 1988;16(3):1215.
16. Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site
detection in Genie. J Comput Biol. 1997;4(3):311-323.
17. Desmet  FO, Hamroun  D, Lalande  M, Collod-Béroud  G,
Claustres  M, Béroud  C. Human Splicing Finder: an online bio-
informatics tool to predict splicing signals. Nucleic Acids Res.
2009;37(9):e67.
J Clin Endocrinol Metab, October 2020, 105(10):1–8
18. Brunak S, Engelbrecht J, Knudsen S. Prediction of human mRNA
donor and acceptor sites from the DNA sequence. J Mol Biol.
1991;220(1):49-65.
19. Gruber  AR, Lorenz  R, Bernhart  SH, Neuböck  R, Hofacker  IL.
The Vienna RNA websuite. Nucleic Acids Res. 2008;36(Web
Server issue):W70-W74.
20. Kopanos  C, Tsiolkas  V, Kouris  A, et  al. VarSome: the
human genomic variant search engine. Bioinformatics.
2019;35(11):1978-1980.
21. Macedo  DB, Abreu  AP, Reis  AC, et  al. Central precocious pu-
berty that appears to be sporadic caused by paternally inherited
mutations in the imprinted gene makorin ring finger 3. J Clin
Endocrinol Metab. 2014;99(6):E1097-E1103.
Downloaded from https://academic.oup.com/jcem/article/105/10/dgaa461/5872717 by guest on 04 September 2020
22. Wang  Y, Sul  HS. Ectodomain shedding of preadipocyte factor
1 (Pref-1) by tumor necrosis factor alpha converting enzyme
(TACE) and inhibition of adipocyte differentiation. Mol Cell Biol.
2006;26(14):5421-5435.
23. Villanueva C, Jacquier S, de Roux N. DLK1 is a somato-dendritic
protein expressed in hypothalamic arginine-vasopressin and oxy-
tocin neurons. PLoS One. 2012;7(4):e36134.
24. Kagami M, Nagasaki K, Kosaki R, et al. Temple syndrome: com-
prehensive molecular and clinical findings in 32 Japanese pa-
tients. Genet Med. 2017;19(12):1356-1366.
25. Abi Habib W, Brioude F, Azzi S, et al. Transcriptional profiling at
the. Sci Adv. 2019;5(2):eaau9425.