Prevalence of pathogenetic MC4R mutations in Italian children with early Onset obesity, tall stature and familial history of obesity

Prevalence of pathogenetic MC4R mutations in Italian children with early Onset obesity, tall stature and familial history of obesity
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  BioMed   Central Page 1 of 9 (page number not for citation purposes) BMC Medical Genetics Open Access Research article Prevalence of pathogenetic MC4R mutations in Italian children with early Onset obesity, tall stature and familial history of obesity NicolaSantoro 1 , GraziaCirillo 1 , ZhiminXiang  2 , RitaTanas 3 , NellaGreggio 4 , GiuseppeMorino 5 , LorenzoIughetti 6 , AlessandraVottero 7 ,  AlessandroSalvatoni 8 , MarioDi Pietro 9 , AntonioBalsamo 10 ,  AntoninoCrinò 11 , AnnaGrandone 1 , CarrieHaskell-Luevano 2 , LauraPerrone 1  and EmanueleMiragliadel Giudice* 1  Address: 1 Dipartimento di Pediatria "F. Fede", Seconda Università degli Studi di Napoli, Napoli, Italy, 2 Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, USA, 3  Azienda Ospedaliero-Universitaria Arcispedale S. Anna, Ferrara, Italy, 4 Dipartimento di Pediatria – Università di Padova, Padova, Italy, 5 Ospedale Pediatrico Bambin Gesù, Roma, Servizio di dietologia clinica, Roma, Italy, 6 Università di Modena e Reggio Emilia, Dipartimento di Pediatria, Modena, Italy, 7 Università di Parma, Clinica Pediatrica, Dipartimento dell'età evolutiva, Parma, Italy, 8 Università dell'Insubria, Clinica Pediatrica, Dipartimento di Scienze Cliniche e Biologiche, Italy, 9 Ospedale S. Liberatore di Atri, UO di Pediatria, Teramo, Italy, 10 Policlinico S. Orsola-Malpighi, Dipartimento di Scienze pediatriche, Bologna, Italy and 11 Ospedale Pediatrico Bambin Gesù, Roma, Paediatric and Autoimmune Endocrine Diseases Unit, Roma, Italy Email:;;;;;;;;; MarioDi;; AntoninoCrinò;;;; EmanueleMiragliadel Giudice* * Corresponding author Abstract Background: Melanocortin-4-receptor (MC4R) mutations represent the most frequent geneticcause of non-syndromic early onset obesity. Children carrying MC4R mutations seem to show aparticular phenotype characterized by early onset, severe obesity and high stature. To verifywhether  MC4R mutations are associated with this particular phenotype in the Italian pediatricpopulation, we decided to screen the MC4R gene in a group of obese children selected on the basisof their phenotype. Methods: To perform this study, a multicentric approach was designed. Particularly, to be enrolledin the study subjects needed to meet the following criteria: Body mass index ≥  3 deviation scoresaccording to age and sex, familiar history of obesity (at least one parent obese), obesity onsetbefore the 10 years old, height ≥  2 deviation scores. The coding region of MC4R gene was screenedin 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 inthree unrelated children, had been described and functionally characterized previously. The Q307X Published: 12 March 2009 BMC Medical Genetics  2009, 10 :25doi:10.1186/1471-2350-10-25Received: 9 October 2008Accepted: 12 March 2009This article is available from:© 2009 Santoro et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  BMC Medical Genetics   2009, 10 :25 2 of 9 (page number not for citation purposes) (C919T) and the Y332H (T994C) mutations were found in two patients. Functional studies showedthat only Q307X impaired protein function. Conclusion: The low prevalence of  MC4R mutations (1.6%) in this group of obese childrenselected according to the obesity degree, the tall stature and the family history of obesity wassimilar to the prevalence observed in previous screenings performed in obese adults and in notphenotypically selected obese children. Background Prevalence of obesity has dramatically increased in chil-dren and adolescents in the past 25 years [1] and studiesconcerning 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 cascadeof leptin [3]. In the hypothalamus, the POMC derivedpeptides α -MSH and β -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 throughthis pathway is highly susceptible to quantitative varia-tions in  MC4-R expression or function. These variationsmay be consequence of a reduced ligand binding or of areduced receptor expression. Molecular screeningsallowed 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 inhumans. Clinical characteristics of subjects carrying muta-tions on  MC4R have been carefully described [10]. 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 obeseadults and children described mutations in the obesepatients, 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 tothe selection criteria and to the method used for molecu-lar screening [14]. To understand whether the previousdata were biased by not strict selection criteria in patientsrecruitment, we have screened MC4R in a group of Italianobese children selected, with a multicentric approach, onthe 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) wasinvolved in the study and the following nine childhoodobesity services from nine different cities participated: Fer-rara, Varese, Bologna, Modena, Parma and Padova(Northern Italy), Roma and Atri, (Central Italy) andNapoli (Southern Italy).Recruitment started on January 2005 and stopped on Jan-uary 2006. To be enrolled in the study children needed tomeet 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 theDepartment 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, thesubject being undressed, height was measured by aHarpenden Stadiometer and BMI was calculated. Stand-ard deviations scores for BMI was calculated by using theLMS method [15]. The LMS method fits growth standardsto all forms of anthropometry by making the simpleassumption of a skew normal distribution. Standard devi-ation of height (SD-height) and pubertal stage were eval-uated according to Tanner [16]. Waist circumference wasmeasured with an anelasticated tape, the subject being instanding position; the tape is applied horizontally mid- way between the lowest rib margin and the iliac crest. Toassess the age of obesity onset during early childhood, the  BMC Medical Genetics   2009, 10 :25 3 of 9 (page number not for citation purposes) records of the patients were reviewed. In these records theanthropometric measurements made in the pediatrician'ssurgeries within the ambit of children health balancesmade annually are reported [17]. The control group  A control group composed by 200 age and sex matchedlean children was recruited as previously shown [18].Briefly, lean children who were age and sex matched andbelonged 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 theSecond 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 bloodcells. Amplification of the  MC4R coding region was performedusing 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, α  -MSH, 4-norleucine-7- D -phenylalanine (NDP)-MSH, ACTH(1–24),  β  -MSH, γ  2 -MSH, were purchased from commercial sources (Bachem, Terrance, CA, USA). The melanocortin tetrapeptideJRH887–9 (Ac-His- D -Phe-Arg-Trp-NH 2 ) was synthesizedas previously reported [19]. hMC4R in vitro receptor mutagenesis  The human wild-type (WT) N-terminal Flag-taggedhMC4R cDNA mutagenesis was performed as describedpreviously [19]. Amino acid modifications of the hMC4R  were introduced using a complementary set of primerscontaining the nucleotide mutation(s) resulting in thedesired residue change. The construction of hMC4R con-taining the desired mutant has been described previously [19]. Complete Flag-hMC4R sequences were confirmedfree 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 cAMPresponse element (CRE)/  β  -galactosidase reporter gene aspreviously described [19]. Briefly, forty-eight hours post-transfection, the cells were stimulated with the peptide for  α  -MSH, NDP-MSH, and ACTH(1–24) and the peptide for  γ  2 -MSH,  β  -MSH, and JRH887–9 or forskolin control inassay medium for 6 h. Subsequently, substrate buffer [60mM sodium phosphate, 1 mM MgCl 2 , 10 mM KCl, 5 mM  β  -mercaptoethanol, 2 mg/mL of o -nitrophenyl-  β  -D-galactopyranoside (ONPG)] was added to the cell lysateplates, and were incubated at 37°C. The sample absorb-ance was measured and data points were normalized bothto 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 describedabove. The peptide NDP-MSH was used to competitively displace the 125 I-radiolabeled peptides NDP-MSH. Dose-response curves (10 -6 to 10 -12 M) and IC 50  values 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. Eachexperiment was performed using duplicate data pointsand 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 andintracellular detection of the Flag-hMC4Rs an allophyco-cyanin (APC)-conjugated anti-Flag monoclonal antibody (Prozyme, San Leandro, CA, USA) was used. To detect thetotal (surface and intracellular) receptor expression cells were subsequently permeabilized with saponin buffer andstained with the APC-conjugated anti-Flag monoclonalantibody. Unlabeled cells were used to set the backgroundfluorescence staining for these analyses. BD Biosciences(San Jose, CA, USA) FACS Calibur flow cytometers wereused to collect both stained cell percentages (surface andtotal) and mean fluorescence data were measured from aminimum of 10 000 collected events per sample. Experi-ments were repeated three independent times and themean is reported. Results  We enrolled 240 obese children and adolescents (135girls) 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 substitutionof 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 astop 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 histidineat the codon 332 (Y332H) caused by the substitution of a  BMC Medical Genetics   2009, 10 :25 4 of 9 (page number not for citation purposes) thymidine with a cytosine in position 994 (T994C). TheS127L mutation was found in three non-consanguineousindividuals from three different centers (Modena, Bolo-gna, Napoli), the two mutations, Y332H and Q307X werefound 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 fromModena) and three lean controls (males) showed a com-mon, polymorphism (I103V) previously described as neg-atively associated with obesity [10]. When the ability of the Q307X and Y332H mutatedMC4Rs to generate cAMP in response to increasing con-centrations of α -MSH was tested, the Q307X  MC4R didnot evocate any response, while the response evocated by the agonist binding the Y332H  MC4R (6.29 ± 1.93) wassimilar to that evocated by binding the wild type receptor (6.85 ± 3.23) (figure 3) To evaluate if some ligand may pharmacologically rescuethe agonist response of the mutated Q307X MC4R, theendogenous melanocortin agonists ACTH 1–24 , β -MSH, γ 2-MSH, and the synthetic agonist JRH887–9 were tested.None was able to rescue the functional activity of themutated protein (figure 3). Family trees of the probands carrying the S127L variant Figure 1Family trees of the probands carrying the S127L variant . This variant was found in three unrelated subjects from three different obesity Services. A show the family tree of subject 1 (see Table 1). 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 1). The half black square indicates males heterozygotes for the mutation, the half black circle indicates the females heterozygotes for the muta-tion. 12 Age:8Z-score BMI: 3.1Age:10Z-score BMI: 3.2Age:45BMI: 34Age:43BMI: 28 1234 Age:52BMI: 32Age:50BMI: 23Age:83BMI: 25 IIIIII A 212Age: 2Z-score BMI: 0.9Age: 47BMI: 36Age: 43BMI: 291Age:10Z-score BMI: 3.6 III B 1Age: 8Z-score BMI: 412Age: 45 BMI: 38Age: 47BMI: 26 C  BMC Medical Genetics   2009, 10 :25 5 of 9 (page number not for citation purposes) Binding data obtained using the radiolabelled NDP-MSHshowed that, in the case of the Q307X mutation, theendogenous agonist was unable to stimulate the receptor (figure 4). As intracellular retention of mutated MC4Rs is a commonobesity-causing defect, to allow for the rapid evaluation of cell surface expression of Q307X relative to total expres-sion of the receptor in individual transiently transfectedcells, 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). A. The essential family tree (father, mother and daughter) of the proband carrying the Q307X mutation is shown Figure 2A. 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 1 (proband) and for the subject I 1 (the father), respectively. Both carry the mutation, but, while subject II 1 shows an early onset of obesity, the subject I 1 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. Boys: 2 to 20 yearsGirls: 2 to 20 yearsBC Age:37BMI:33Age: 4Z-score BMI: 5.1Age: 39BMI:41 III 121 A Table 1: Clinical features of obese children with MC4R mutations Mutation (sex)S127L (M)S127L (F)S127L (F)Q307X (F)Y332H (F)Age (years) 81010311 Age at obesity onset (years) 24713 z-score BMI Height-sds
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