Group Title: BMC Medical Genetics
Title: Prevalence of pathogenetic MC4R mutations in Italian children with early Onset obesity, tall stature and familial history of obesity
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 Material Information
Title: Prevalence of pathogenetic MC4R mutations in Italian children with early Onset obesity, tall stature and familial history of obesity
Physical Description: Book
Language: English
Creator: Santoro, Nicola
Cirillo, Grazia
Xiang, Zhimin
Tanas, Rita
Greggio, Nella
Morino, Giuseppe
Iughetti, Lorenzo
Vottero,Alessandra
Salvatoni, Alessandro
Di Pietro, Mario
Balsamo, Antonio
Crino,A ntonino
Grandone, Anna
Haskell-Luevano, Carrie
Perrone, Laura
del Giudice, Emanuele
Publisher: BMC Medical Genetics
Publication Date: 2009
 Notes
Abstract: BACKGROUND:Melanocortin-4-receptor (MC4R) mutations represent the most frequent genetic cause of non-syndromic early onset obesity. Children carrying MC4R mutations seem to show a particular phenotype characterized by early onset, severe obesity and high stature. To verify whether MC4R mutations are associated with this particular phenotype in the Italian pediatric population, we decided to screen the MC4R gene in a group of obese children selected on the basis of their phenotype.METHODS:To perform this study, a multicentric approach was designed. Particularly, to be enrolled in the study subjects needed to meet the following criteria: Body mass index = 3 deviation scores according to age and sex, familiar history of obesity (at least one parent obese), obesity onset before the 10 years old, height = 2 deviation scores. The coding region of MC4R gene was screened in 240 obese children (mean age 8.3 ± 3.1, mean BMI 30.8 ± 5.4) and in 200 controls (mean age 8.1 ± 2.8; mean BMI 14.2 ± 2.5).RESULTS:Three mutations have been found in five obese children. The S127L (C380T), found in three unrelated children, had been described and functionally characterized previously. The Q307X (C919T) and the Y332H (T994C) mutations were found in two patients. Functional studies showed that only Q307X impaired protein function.CONCLUSION:The low prevalence of MC4R mutations (1.6%) in this group of obese children selected according to the obesity degree, the tall stature and the family history of obesity was similar to the prevalence observed in previous screenings performed in obese adults and in not phenotypically selected obese children.
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Research article


Prevalence of pathogenetic MC4R mutations in Italian children with
early Onset obesity, tall stature and familial history of obesity
Nicola Santoro', Grazia Cirillo1, Zhimin Xiang2, Rita Tanas3, Nella Greggio4,
Giuseppe Morino5, Lorenzo lughetti6, Alessandra Vottero7,
Alessandro Salvatoni8, Mario Di Pietro9, Antonio Balsamo'0,
Antonino Crino11, Anna Grandonel, Carrie Haskell-Luevano2,
Laura Perronel and Emanuele Miraglia del Giudice*1

Address: 'Dipartimento di Pediatria "F. Fede", Seconda Universita degli Studi di Napoli, Napoli, Italy, 2Department of Medicinal Chemistry,
University of Florida, Gainesville, FL 32610, USA, 3Azienda Ospedaliero-Universitaria Arcispedale S. Anna, Ferrara, Italy, 4Dipartimento di
Pediatria Universita di Padova, Padova, Italy, 5Ospedale Pediatrico Bambin Gesu, Roma, Servizio di dietologia clinic, Roma, Italy, 6Universita
di Modena e Reggio Emilia, Dipartimento di Pediatria, Modena, Italy, 7Universita di Parma, Clinica Pediatrica, Dipartimento dell'eta evolutiva,
Parma, Italy, 8Universita dell'Insubria, Clinica Pediatrica, Dipartimento di Scienze Cliniche e Biologiche, Italy, 9Ospedale S. Liberatore di Atri, UO
di Pediatria, Teramo, Italy, loPoliclinico S. Orsola-Malpighi, Dipartimento di Scienze pediatriche, Bologna, Italy and 11Ospedale Pediatrico
Bambin Gesu, Roma, Paediatric and Autoimmune Endocrine Diseases Unit, Roma, Italy
Email: Nicola Santoro nicolasantoro@hotmail.com; Grazia Cirillo grazia.cirillo@unina2.it; Zhimin Xiang nmanuele.miraglia@unina2.it;
Rita Tanas manuele.miraglia@unina2.it; Nella Greggio manuele.miraglia@unina2.it; Giuseppe Morino manuele.miraglia@unina2.it;
Lorenzo lughetti nicolasantoro@hotmail.com; Alessandra Vottero nicolasantoro@hotmail.com;
Alessandro Salvatoni nicolasantoro@hotmail.com; Mario Di Pietro nicolasantoro@hotmail.com;
Antonio Balsamo nicolasantoro@hotmail.com; Antonino Crino nicolasantoro@hotmail.com; Anna Grandone annagrandone@inwind.it;
Carrie Haskell-Luevano nicolasantoro@hotmail.com; Laura Perrone laura.perrone@unina2.it; Emanuele Miraglia del
Giudice* emanuele.miraglia@unina2.it
* Corresponding author



Published: 12 March 2009 Received: 9 October 2008
BMC Medical Genetics 2009, 10:25 doi:10.1186/1471-2350-10-25 Accepted: 12 March 2009
This article is available from: http://www.biomedcentral.com/1471-2350/10/25
2009 Santoro et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Melanocortin-4-receptor (MC4R) mutations represent the most frequent genetic
cause of non-syndromic early onset obesity. Children carrying MC4R mutations seem to show a
particular phenotype characterized by early onset, severe obesity and high stature. To verify
whether MC4R mutations are associated with this particular phenotype in the Italian pediatric
population, we decided to screen the MC4R gene in a group of obese children selected on the basis
of their phenotype.
Methods: To perform this study, a multicentric approach was designed. Particularly, to be enrolled
in the study subjects needed to meet the following criteria: Body mass index > 3 deviation scores
according to age and sex, familiar history of obesity (at least one parent obese), obesity onset
before the 10 years old, height > 2 deviation scores. The coding region of MC4R gene was screened
in 240 obese children (mean age 8.3 3.1, mean BMI 30.8 5.4) and in 200 controls (mean age 8.1
+ 2.8; mean BMI 14.2 2.5).
Results: Three mutations have been found in five obese children. The S127L (C380T), found in
three unrelated children, had been described and functionally characterized previously. The Q307X



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(C919T) and the Y332H (T994C) mutations were found in two patients. Functional studies showed
that only Q307X impaired protein function.
Conclusion: The low prevalence of MC4R mutations (1.6%) in this group of obese children
selected according to the obesity degree, the tall stature and the family history of obesity was
similar to the prevalence observed in previous screenings performed in obese adults and in not
phenotypically selected obese children.


Background
Prevalence of obesity has dramatically increased in chil-
dren and adolescents in the past 25 years [1] and studies
concerning molecular basis of obesity have been encour-
aged.

Melanocortinergic system represents the most interesting
known system involved in the central regulation of body
weight. Blockade of the melanocortin signalling pathway
leads to hyperphagia, reduced energy expenditure and,
ultimately, obesity [2]. Proopiomelanocortin (POMC)
represents a key step in the anorexigenic signaling cascade
of leptin [3]. In the hypothalamus, the POMC derived
peptides a-MSH and P3-MSH bind the melanocortin-4-
receptor (MC4R), a seven transmembrane receptor [4,5]
causing a reduction of appetite and an increase of energy
expenditure. Regulation of energy homoeostasis through
this pathway is highly susceptible to quantitative varia-
tions in MC4-R expression or function. These variations
may be consequence of a reduced ligand binding or of a
reduced receptor expression. Molecular screenings
allowed to identify mutations on POMC and MC4R asso-
ciated with early onset obesity [6-8].

MC4R mutations represent the most frequent cause of
non syndromic early onset obesity, with prevalence rang-
ing from 0.5% to 5% [9,10]. Several mutations on MC4R
have been found and functionally characterized in
humans. Clinical characteristics of subjects carrying muta-
tions on MC4R have been carefully described [101. Their
particular features are early onset, severe, obesity, acceler-
ated height velocity (height is usually more than 2 DS),
advanced bone age and hyperinsulinemia.

Surprisingly, recent studies on large cohorts of obese
adults and children described mutations in the obese
patients, but also in the normal weight individuals [11-
13], and failed to establish that the early onset of obesity
[12] as well as the tall stature [13] of the obese children
are specific clinical characteristics of functionally relevant
heterozygous MC4R mutation carriers.

In a previous study we observed a prevalence of MC4R
mutations among Italian obese children and adolescent
of 0.5%, lower than that previously showed by other
authors in obese children from different geographic areas


[9]. The low prevalence that we found was attributed to
the selection criteria and to the method used for molecu-
lar screening [14]. To understand whether the previous
data were biased by not strict selection criteria in patients
recruitment, we have screened MC4R in a group of Italian
obese children selected, with a multicentric approach, on
the basis of their phenotype (i.e.; familiar history of obes-
ity, BMI > 3 deviation scores, obesity onset before the 10
years old, height> 2 deviation scores).

Methods
Recruitment criteria
The Childhood Obesity Study Group of the Italian Society
of Pediatric Endocrinology and Diabetes (SIEDP) was
involved in the study and the following nine childhood
obesity services from nine different cities participated: Fer-
rara, Varese, Bologna, Modena, Parma and Padova
(Northern Italy), Roma and Atri, (Central Italy) and
Napoli (Southern Italy).

Recruitment started on January 2005 and stopped on Jan-
uary 2006. To be enrolled in the study children needed to
meet the following criteria: a BMI > 3 SD (severe obesity)
according to age and sex, familiar history of obesity (at
least one parent should be obese or ex obese), obesity
onset before the 10 years old, height > 2 deviation scores.
All patients data and blood samples were sent to the
Department of Pediatrics of the Second University of
Naples. The ethical committee of the Second University of
Study of Naples approved the study. Informed consent
was obtained by parents.

Clinical data
Body weight was measured by a balance beam scale, the
subject being undressed, height was measured by a
Harpenden Stadiometer and BMI was calculated. Stand-
ard deviations scores for BMI was calculated by using the
LMS method [15]. The LMS method fits growth standards
to all forms of anthropometry by making the simple
assumption of a skew normal distribution. Standard devi-
ation of height (SD-height) and pubertal stage were eval-
uated according to Tanner [16]. Waist circumference was
measured with an anelasticated tape, the subject being in
standing position; the tape is applied horizontally mid-
way between the lowest rib margin and the iliac crest. To
assess the age of obesity onset during early childhood, the

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records of the patients were reviewed. In these records the
anthropometric measurements made in the pediatrician's
surgeries within the ambit of children health balances
made annually are reported [17].

The control group
A control group composed by 200 age and sex matched
lean children was recruited as previously shown [18].
Briefly, lean children who were age and sex matched and
belonged to the region of Napoli were recruited as con-
trols (mean age, 10.7 2.2 yr; mean BMI z-score, 0.5
0.4). They consulted the Department of Pediatrics of the
Second University of Naples for presumed diseases and
were found to be normal. Informed consent from parents
was obtained before entry in the study.

Genotyping
Melanocortin- 4-receptor (MC4R)
Genomic DNA was collected from nucleated white blood
cells.

Amplification of the MC4R coding region was performed
using five primer pairs and the condition previously
described [9]. PCR products were analysed by an auto-
matic sequencer (ABI PRISM 310, Perkin Elmer, USA).

Functional Study
Materials
Peptides used in this study, a-MSH, 4-norleucine-7-D-
phenylalanine (NDP)-MSH, ACTH(1-24), fi-MSH, 7-2
MSH, were purchased from commercial sources (Bachem,
Terrance, CA, USA). The melanocortin tetrapeptide
JRH887-9 (Ac-His-D-Phe-Arg-Trp-NH2) was synthesized
as previously reported [19].

hMC4R in vitro receptor mutagenesis
The human wild-type (WT) N-terminal Flag-tagged
hMC4R cDNA mutagenesis was performed as described
previously [19]. Amino acid modifications of the hMC4R
were introduced using a complementary set of primers
containing the nucleotide mutation(s) resulting in the
desired residue change. The construction of hMC4R con-
taining the desired mutant has been described previously
[19]. Complete Flag-hMC4R sequences were confirmed
free of PCR nucleotide base errors by DNA sequencing
(University of Florida sequencing core facilities).

cAMP-based functional bioassay
Human embryonic kidney-293 cells stably expressing WT
and mutant receptors were transfected with cAMP
response element (CRE)//3-galactosidase reporter gene as
previously described [19]. Briefly, forty-eight hours post-
transfection, the cells were stimulated with the peptide for
a-MSH, NDP-MSH, and ACTH(1-24) and the peptide for
72-MSH, fi-MSH, and JRH887-9 or forskolin control in
assay medium for 6 h. Subsequently, substrate buffer [60


mM sodium phosphate, 1 mM MgCl2, 10 mM KC1, 5 mM
gq-mercaptoethanol, 2 mg/mL of o-nitrophenyl-) -D-
galactopyranoside (ONPG)] was added to the cell lysate
plates, and were incubated at 37C. The sample absorb-
ance was measured and data points were normalized both
to the relative protein content and non-receptor-depend-
ent forskolin values. Assays were performed using dupli-
cate data points and repeated in at least four independent
experiments. Means and standard errors (SE) are reported.

Receptor-binding studies
Human embryonic kidney-293 cells stably expressing the
WT and mutant receptors were maintained as described
above. The peptide NDP-MSH was used to competitively
displace the 125I-radiolabeled peptides NDP-MSH. Dose-
response curves (10-6 to 10-12 M) and IC50values were gen-
erated and analyzed by non-linear least-squares analysis
[19]. The percentage total specific binding was deter-
mined based upon the non-specific values obtained using
10-6 M NDP-MSH for the radiolabeled peptide. Each
experiment was performed using duplicate data points
and repeated in at least two independent experiments.

FACS analysis of wild-type and mutant Flag-tagged hMC4 receptors
Fluorescence-activated cell sorting (FACS) analysis of N-
terminally Flag-tagged WT and mutant hMC4R was per-
formed as described previously [19]. For cell surface and
intracellular detection of the Flag-hMC4Rs an allophyco-
cyanin (APC)-conjugated anti-Flag monoclonal antibody
(Prozyme, San Leandro, CA, USA) was used. To detect the
total (surface and intracellular) receptor expression cells
were subsequently permeabilized with saponin buffer and
stained with the APC-conjugated anti-Flag monoclonal
antibody. Unlabeled cells were used to set the background
fluorescence staining for these analyses. BD Biosciences
(San Jose, CA, USA) FACS Calibur flow cytometers were
used to collect both stained cell percentages (surface and
total) and mean fluorescence data were measured from a
minimum of 10 000 collected events per sample. Experi-
ments were repeated three independent times and the
mean is reported.

Results
We enrolled 240 obese children and adolescents (135
girls) with a mean age 8.3 3.1 years, a mean age at onset
of obesity of 2.8 2.0 years, the mean SD-height was 2.3
0.2, mean BMI 30.8 + 5.4 and z-score BMI 4.2 + 0.9.

Molecular screening of the MC4R showed the substitution
of a serine with a leucine at the codon 127 (S127L) conse-
quent to the substitution of a cytosine in position 380
with a thymidine, a substitution of the glutamine with a
stop codon at the codon 307 (Q307X) caused by the sub-
stitution of a cytosine with a thymidine in position 919
(C919T) and the substitution of a tyrosine with a histidine
at the codon 332 (Y332H) caused by the substitution of a

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thymidine with a cytosine in position 994 (T994C). The
S127L mutation was found in three non-consanguineous
individuals from three different centers (Modena, Bolo-
gna, Napoli), the two mutations, Y332H and Q307X were
found in a child from Napoli and in a child from Ferrara,
respectively (table 1). Four out of five families were inves-
tigated and mutations co-segregated with obesity in 9 out
of 10 individuals harboring a variant (figures 1 and 2).

None lean subject showed the above described mutations.
Four obese subjects (3 from Napoli and one from
Modena) and three lean controls (males) showed a com-
mon, polymorphism (I 103V) previously described as neg-
atively associated with obesity [10].


I






II Age:52
BMI: 32





III


When the ability of the Q307X and Y332H mutated
MC4Rs to generate cAMP in response to increasing con-
centrations of a-MSH was tested, the Q307X MC4R did
not evocate any response, while the response evocated by
the agonist binding the Y332H MC4R (6.29 1.93) was
similar to that evocated by binding the wild type receptor
(6.85 3.23) (figure 3)

To evaluate if some ligand may pharmacologically rescue
the agonist response of the mutated Q307X MC4R, the
endogenous melanocortin agonists ACTH14, P3-MSH, y2-
MSH, and the synthetic agonist JRH887-9 were tested.
None was able to rescue the functional activity of the
mutated protein (figure 3).


Age: 10 Age:8


Z-score BMI: 3.2


Z-score BMI: 3.1


C i


Age: 47

BMI: 36


Age: 10

Z-score BMI: 3.6


Age: 47

BMI: 26


Age: 2

Z-score BMI: 0.9


Age: 45

BMI: 38


Age: 8
Age: 8


Z-score BMI: 4


Figure I
Family trees of the probands carrying the S 127L variant. This variant was found in three unrelated subjects from three
different obesity Services. A show the family tree of subject I (see Table I). The figure shows that all the carriers but the sub-
ject I 2 (grandmother) were obese. B and C show the family trees of patients 2 and 3, respectively (see Table I). The half black
square indicates males heterozygotes for the mutation, the half black circle indicates the females heterozygotes for the muta-
tion.



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B I


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A0 1


Age: 39

BMI:41


4


Age: 37

BMI:33


Age: 4
ore BMI:


Age (years)


Figure 2
A. The essential family tree (father, mother and daughter) of the proband carrying the Q307X mutation is
shown. B and C show the BMI chart for the subject II I (proband) and for the subject I I (the father), respectively. Both carry
the mutation, but, while subject II I shows an early onset of obesity, the subject I I became obese after he was 20 years old.
The half black square indicates males heterozygotes for the mutation, the half black circle indicates the females heterozygotes
for the mutation.


Binding data obtained using the radiolabelled NDP-MSH
showed that, in the case of the Q307X mutation, the
endogenous agonist was unable to stimulate the receptor
(figure 4).

As intracellular retention of mutated MC4Rs is a common
obesity-causing defect, to allow for the rapid evaluation of
Table I: Clinical features of obese children with MC4R mutations


cell surface expression of Q307X relative to total expres-
sion of the receptor in individual transiently transfected
cells, a method based on immunostaining and fluores-
cence detection by flow cytometry has been used. As com-
pared to the wild type expression, total Q307X expression
was 88%, surface expression was 31% and, therefore,
intracellular retention was about 57% (figure 5).


Mutation (sex)

Age (years)
Age at obesity onset (years)
z-score BMI
Height-sds


S127L (M) S127L (F) S127L (F) Q307X (F)


Y332H (F)

II
3
4.0
2.2


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* a-MSH
A NDP-MSH
v ACTH(1-24)
0 y2-MSH
* P-MSH
DE JRH887-9


WT hMC4R


-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
Log Agonist Concentration (M)


Q307Stop hMC4R


-12 -11 -f0 -9 -8 -7 -6 -5
Log Agonist Concentration (M)


1.25


0.75/
0.50
0.25
Y332H hMC4R
0.00
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
Log Agonist Concentration (M)


Figure 3
Functional Agonist Data. Illustration of the activity of a-MSH, ACTH 1-24, P-MSH, y2-MSH, and synthetic agonist JRH887-9
at the wild type MC4R, Q307X MC4R and Y332H MC4R. Agonists did not evocate any response by binding the Q307X
MC4R. When the agonists were binding the Y332H MC4R the evocated response was similar to that observed for the wild
type.


Given that the Y332H variant did not affect the function
of MC4R, this latter mutation was not included in the cal-
culation of the prevalence of pathogenetic relevant MC4R
gene mutations in our population, that can be conse-
quently considered 1.6% (4 out of 240 patients).

Discussion
MC4R mutations functional analysis
The S127L variant has been functionally studied previ-
ously [20], while the Q307X has been described but not
functionally characterized [21]. The S127L impairs the
signaling in response to a-, P- and y-MSH [20], but not the
MC4R expression on cell surface [14] and is, therefore,
highly likely to be causative, rather than incidental.

Of the two mutations, Q307X and Y332H, only the
Q307X showed a functional relevance. At our best knowl-




| 75. WT hMC4R
0 Q307Stop hMC4R
"50. o Y332H hMC4R


S25

0
-12 -11 -10 -9 -8 -7 -6 -5
Log NDP-MSH Concentration

Figure 4
Binding Data. Competitive displacement binding affinity
studies of the polymorphic hMC4Rs examined in this study.
1125NDP-MSH was used to competitively displace non-labeled
NDP-MSH in a dose-response fashion. The endogenous ago-
nists were unable to stimulate the Q307X MC4R at up to I
iM concentrations.


edge, at date, this is the most C-terminal nonsense muta-
tion on MC4R. This mutation produces a protein lacking
the last 26 amino acids. Among these, five are residues at
100% conserved during evolution in vertebrates [22]. The
C-terminal tail has previously been identified as a region
important for targeting to the plasma membrane. Accord-
ingly, we have demonstrated some intracellular retention


200

o 150u

100-
X 100

S75

r' 50

. a 25,
^ 2


|DTotal Cell Receptor Expression
MCell Surface Receptor Expression


Figure 5
FACS Data. Fluorescence activated cell sorting analysis
(FACS) of the MC4R Q307X mutation expressed in HEK-
293. The total cell receptor expression levels were deter-
mined using permeabilized cells measuring both cell surface
and intracellular protein expression. The cell surface expres-
sion levels were determined using non-permeabilized cells.
Cell expression levels are presented relative to the wild type
human-MC4R control. Total Q307X expression was 88%,
surface expression was 3 1% and intracellular retention was
about 57%.



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i 1.25
1.00,
I 0.75,


3 0.25,
0.00i


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for the Q307X protein. This finding is consistent with
those of Ho and MacKenzie [23] who demonstrated that
an artificial receptor lacking amino acids 306-319 within
the C-terminal tail was unable to traffic to the plasma
membrane [23]. Rather surprisingly given its location in
the C-terminal tail of the receptor, the Q307X mutant
showed also a decreased binding affinity for agonist. This
unexpected finding has been yet showed in another MC4R
mutation affecting the tail of the receptor (I316S) [24]. It
is conceivable that in both cases the mutations may
induce a sufficient alteration of the receptor tertiary struc-
ture of the ectodomain, or that they may affect interaction
with intracellular kinases or other enzymes which might
post-translationally modify the receptor and modulate
receptor-ligand interaction [24]. Concluding, the
impaired signaling of the Q307X mutation is due to a
combination of defects, i.e.; both reduced cell surface
expression and decreased affinity of the receptor for a-
MSH.

MC4R mutations prevalence analysis
Mutations on MC4R gene represent the most frequent
cause of monogenic non-syndromic obesity, with preva-
lence, among obese children, reaching in some cases the
5% [10]. In a previous report we observed a prevalence of
pathogenetic MC4R gene mutations in a population of
Italian obese children and adolescents of about 0.5%.
Selection criteria, particularly the lower mean BMI of the
population investigated compared to the BMI of the other
groups of patients studied, have been claimed to explain
this finding [24].

The present is the first study which aims to find mutated
children specifically selecting them on the basis of the par-
ticular phenotypic characteristics usually recognized in
the patients carrying MC4R variations (i.e.; at least one
parent obese or ex obese, obesity onset before 10 years of
age, BMI more than 3 DS and height more than 2 DS)
[11]. Anyway, we have to recognize that studies trying to
find a phenotype-genotype correlation were not conclu-
sive. In fact, studies showing mutations in the obese as
well as in the normal weight individuals [11-13] failed to
establish that the early onset of obesity [12] and the tall
stature [13] of the obese children are specific clinical char-
acteristics of functionally relevant MC4R mutations. On
the other hand, studies confuting the association between
the MC4R mutations and early onset obesity describe
populations of obese adults without objective data on
whether the obese adults had been obese as children or
not [12,13]. Prevalence of MC4R mutations in this cohort
of phenotypically selected children (1.6%) appears simi-
lar to the 1.8% reported in a previous paper investigating
a large cohort, not phenotypically selected, of European
obese children [13].


Prevalence of MC4R mutations observed in the present
study is also similar to that showed in Italian obese adults,
where it ranges from 1.8% [25] to 2.5% [26]. This obser-
vation is consistent with a recent report of Lubrano-Bethe-
lier et al. showing that obese adults carriers of functionally
relevant MC4R mutations do not specifically present with
a history of early onset obesity [121 and with the report of
Stutzman et al showing a prevalence of MC4R mutations
of 1.6% among obese adults [13].

Reduced penetrance and variable expressivity of obesity
has been found to be associated with MC4R mutations. In
fact, whereas some found a 100% penetrance of early
onset obesity in heterozygous proband, others have
described obligate carriers who were not obese also
among children [13]. The variable penetrance and expres-
sivity of obesity in heterozygous individuals argues that
the MC4R acts in concert with a number of other genes to
regulate energy storage under presumed conditions of a
sedentary lifestyle and high-fat diet [27].

It is important to note that pathogenic MC4R mutations
are "private", with individual mutations having a very low
frequency in any population [28]. We observed the same
mutation in three different non-consanguineous subjects
from three different parts of Italy. Moreover this mutation
has not been observed in the three previous screening on
Italian population [9,24,25]. This observation is not sur-
prisingly considering that the S127L variant has been
described by other groups [10,24].

We did not compare our cohort of specifically selected
obese children with a group of equivalently obese chil-
dren without increased stature. This limitation does not
allow us to firmly conclude that there are no phenotypic
characteristics of MC4R mutations other than obesity per
se.

MC4R V1031 polymorphism
The previously described polymorphism A307G resulting
in the substitution of Val with Ile at codon 103 within the
MC4R TM2 was detected at the heterozygous state in 4
obese subjects (1.6%) and in 3 lean controls (1.5%). Pre-
vious studies provided the evidence for a negative associ-
ation of the 1103 allele with obesity [29-31]. Herein we
did not observe a statistically significant difference in the
prevalence of this variant between lean and obese sub-
jects, but as noted by Geller et al. none of the individual
studies included in previous meta-analysis rendered a p
value < .05 [30]. Thus, although this observation cannot
be considered conclusive, our observation may be used
for further meta-analysis.






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Conclusion
In conclusion, we have reported the first MC4R molecular
screening performed in a population of obese children
stringently selected on the basis of their phenotype. The
prevalence of MC4R mutations (1.6%) in this group of
obese children selected according to the obesity degree,
the tall stature and the family history of obesity was simi-
lar to the prevalence observed in previous screenings per-
formed in obese adults and in not phenotypically selected
obese children.


Competing interests
The authors declare that they have no competing interests.


Authors' contributions
NS analytic framework and writing the manuscript, EmdG
had the primary responsibility for protocol development
and writing the manuscript. GC carried out the molecular
screening. AG participated in the development of the pro-
tocol, patients' recruitment and in the preliminary data
analysis. CH-L carried out the laboratory work concerning
the functional analysis. ZX was responsible for the labora-
tory work concerning the functional analysis. LP super-
vised the design and execution of the study. RT, NG, GM,
LI, AV, AS, MDP, AB and AC were responsible for selecting
and evaluating the patients in the respective centers. All
authors read and approved the manuscript.


Acknowledgements
This work has been supported in part by PRIN grant 2004 (LP) and by NIH
grants RO I DK063974 and RO I DK57080 (CHL)

The authors are indebted with Bridget Pierpont for the help in copyediting
the manuscript.

References
1. Ogden CL, Flegal KM, Carrol MD, Jonhson CL: Prevalence and
trends in overweight among US children and adolescents,
1999-2000. JAMA 2002, 288:1728-32.
2. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q,
Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith
FJ, Campfield LA, Burn P, Lee F: Targeted distruption of the
melanocortin-4 receptor results in obesity in mice. Cell 1997,
88:131-141.
3. Takahashi H, Teranishi Y, Nakanishi S, Numa S: Isolation and struc-
tural organization of the human corticotropin-p-lipotropin
precursor gene. FEBS Lett 1981, 135:97-10 1.
4. Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Watson SJ, Del Valle
J, Yamada T: Molecular cloning, expression, and gene localiz-
zation of a fourth melanocortin receptor. j Biol Chem 1993,
268:15174-15179.
5. Swartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P,
Baskin DG: Leptin increases hypotalamic pro-opiomelanocor-
tin mRNA expression in the rostral arcuate nucleus. Diabetes
1997, 46:2119-2123.
6. Krude H, Biebermann H, Luck W, Rudiger H, Brabrant G, Gruters A:
Severe early onset obesity, adrenal insufficiency and red hair
pigmentation caused by POMC mutations in humans. Nat
Genet 1998, 19:155-157.
7. Hinney A, Becker I, Heibult 0, Nottebom K, Schmidt A, Ziegler A,
Mayer H, Siegfried W, Blum WF, Remschmidt H, Hebebrand J: Sys-
tematic mutation screening of the proopiomelanocortin
gene: identification of several genetic variants including
three different insertions, one nonsense and two missense


point mutations in probands of different weight extremes. j
Clin Endocrinol Metab 1998, 83:3737-41.
8. Miraglia del Giudice E, Cirillo G, Santoro N, D'Urso L, Carbone MT,
Di Toro R, Perrone L: Molecular screening of the proopi-
omelanocortin (POMC) gene in Italian obese children:
report of three new mutations. Int j Obes Relat Metab Disord
2001, 25:61-5.
9. Miraglia del Giudice E, Cirillo G, Nigro V, Santoro N, D'Urso L, Rai-
mondo P, Cozzolino D, Scafato D, Perrone L: Low frequency of
melanocortin-4 receptor (MC4R) mutations in a Mediterra-
nean population with early-onset obesity. IntJ Obes Relat Metab
Disord 2002, 26:647-51.
10. Farooqi IS, Keogh JM, Yeo GSH, Lank EJ, Cheetham T, O'Rahilly S:
Clinical spectrum of obesity and mutations in the melano-
cortin 4 receptor gene. N Englj Med 2003, 348:1085-94.
I I. Hinney A, Bettecken T, Tarnow P, Brumm H, Reichwald K, Lichtner
P, Scherag A, Nguyen TT, Schlumberger P, Rief W, Vollmert C, Illig T,
Wichmann HE, Schafer H, Platzer M, Biebermann H, Meitinger T,
Hebebrand J: Prevalence, spectrum, and functional character-
ization of melanocortin-4 receptor gene mutations in a rep-
resentative population-based sample and obese adults from
Germany. j Clin Endocrinol Metab 2006, 9:1761-9.
12. Lubrano-Berthelier C, Dubern B, Lacorte JM, et al.: Melanocortin 4
receptor mutations in a large cohort of severely obese
adults: prevalence, functional classification, genotype-phe-
notype relationship, and lack of association with binge eat-
ing. J Clin Endocrinol Metab 2006, 91:1811-8.
13. Stutzmann F, Tan K, Vatin V, Dina C, Jouret B, Tichet J, Balkau B,
Potoczna N, Horber F, O'Rahilly S, Farooqi IS, Froguel P, Meyre D:
Prevalence of melanocortin-4 receptor deficiency in Europe-
ans and their age-dependent penetrance in multigenera-
tional pedigrees. Diabetes 2008, 57:2511-8.
14. Lubrano-Berthelier C, Dubern B, Lacorte JM, Picard F, Shapiro A,
Zhang S, Bertrais S, Hercberg S, Basdevant A, Clement K, Vaisse C:
Intracellular retention is a common characteristic of child-
hood obesity-associated MC4R mutations. Hum Mol Genet
2003, 15:145-53.
15. Luciano A, Bressan F, Zoppi G: Body mass index reference
curves for children aged 3-19 years from Verona, Italy. Eurj
Clin Nutr 1997, 51:6-10.
16. Tanner JM, Whitehouse RH: Clinical longitudinal standards for
height, weight, height velocity, weight velocity, and stages of
puberty. Arch Dis Child 1976, 51:170-9.
17. Miraglia del Giudice E, Santoro N, Cirillo G, Raimondo P, Grandone
A, D'Aniello A, Di Nardo M, Perrone L: Molecular screening of
the ghrelin gene in Italian obese children: the Leu72Met var-
iant is associated with an earlier onset of obesity. Intj Obes
Relat Metab Disord 2004, 28:447-50.
18. Santoro N, Miraglia del Giudice E, Cirillo G, Raimondo P, Corsi I,
Amato A, Grandone A, Perrone L: An insertional polymorphism
of the Proopiomelanocortin (POMC) gene is associated with
fasting insulin levels in childhood obesity. j Clin End Metab 2004,
89:4846-9.
19. Proneth B, Xiang Z, Pogozheva ID, Litherland SA, Gorbatyuk OS,
Shaw AM, Millard WJ, Mosberg HI, Haskell-Luevano C: Molecular
Mechanism of the Constitutive Activation of the L250Q
Human Melanocortin-4 Receptor Polymorphism. Chemical
Biology & Drug Design 2006, 67:215-229.
20. Valli-Jaakola K, Lipsanen-Nyman M, Oksanen L, Hollenberg AN, Kon-
tula K, Bjorbaek C, Schalin-Jantti C: Identification and character-
ization of melanocortin-4 receptor gene mutations in
morbidly obese finnish children and adults. j Clin Endocrinol
Metab 2004, 89:940-5.
21. Lubrano-Berthelier C, Cavazos M, Le Stunff C, Haas K, Shapiro A,
Zhang S, Bougneres P, Vaisse C: The human MC4R promoter:
characterization and role in obesity. Diabetes 2003,
52:2996-3000.
22. Staubert C, Tarnow P, Brumm H, Pitra C, Gudermann T, Gruters A,
Schoneberg T, Biebermann H, Rompler H: Evolutionary aspects in
evaluating mutations in the melanocortin 4 receptor. Endo-
crinology 2007, 148:4642-8.
23. Ho G, MacKenzie RG: Functional characterization of muta-
tions in melanocortin-4 receptor associated with human
obesity. j Biol Chem 1999, 274:35816-22.
24. Lubrano-Berthelier C, Durand E, Dubern B, Shapiro A, Dazin P, Weill
J, Ferron C, Froguel P, Vaisse C: Intracellular retention is a com-



Page 8 of 9
(page number not for citation purposes)


BMC Medical Genetics 2009, 10:25








http://www.biomedcentral.com/1471-2350/10/25


mon characteristic of childhood obesity-associated MC4R
mutations. Hum Mol Genet 2003, 12:145-53.
25. Santini F, Maffei M, Ceccarini G, Pelosini C, Scartabelli G, Rossellini V,
Chiellini C, Marsili A, Lisi S, Tonacchera M, Agretti P, Chiovano L,
Mammoli C, Vitti P, Pinchera A: Genetic screening for melano-
cortin-4-receptor mutations in a cohort of Italian obese
patients: description and functional characterization of a
novel mutation. J Clin Endocrinol Metab 2004, 89:904-908.
26. Buono P, Pasanisi F, Nardelli C, leno L, Capone S, Liguori R, Finelli C,
Oriani G, Contaldo F, Sacchetti L: Six novel mutations in the
Proopiomelanocortin and melanocortin receptor 4 gene in
severely obese adults living in southern Italy. Clinical Chemistry
2005, 51:1358-64.
27. Cone RD: Haploinsufficiency of the melanocortin-4 receptor:
part of a thrifty genotype? j Clin Invest 2000, 106:185-7.
28. Kublaoui BM, Zinn AR: MC4R mutations weight before
screening! j Clin EndocrinolMetab 2006, 91:1671-72.
29. Heid IM, Vollmert C, Hinney A, Doring A, Geller F, Lowel H, Wich-
mann HE, Illig T, Hebebrand J, Kronenberg F, KORA Group: Associ-
ation of the 1031 MC4R allele with decreased body mass in
7937 participants of two population based surveys. j Med
Genet 2005, 42:21-8.
30. Geller F, Reichwald K, Dempfle A, Illig T, Vollmert C, Herpertz S,
Siffert W, Platzer M, Hess C, Gudermann T, Biebermann H, Wich-
mann HE, Schafer H, Hinney A, Hebebrand J: Melanocortin-4
receptor gene variant 1103 is negatively associated with
obesity. Am J Hum Genet 2004, 74:572-81.
31. Young EH, Wareham NJ, Farooqi S, Hinney A, Hebebrand J, Scherag
A, O'rahilly S, Barroso I, Sandhu MS: The V 1031 polymorphism of
the MC4R gene and obesity: population based studies and
meta-analysis of 29 563 individuals. Intj Obes 2007, 3 1:1437-41.

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