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Genetic Screening for Melanocortin-4 Receptor Mutations in a Cohort of Italian Obese Patients: Description and Functional Characterization of a Novel Mutation

Department of Endocrinology and Metabolism (F.S., G.C., C.P., G.S., V.R., A.M., S.L., M.T., P.A., C.M., P.V., A.P.), and Dulbecco Telethon Institute at Department of Endocrinology and Metabolism (M.M., C.C.), University of Pisa, 56124 Pisa, Italy; and Endocrinology Unit, University of Pavia, Fondazione Salvatore Maugeri Istituto di Ricovero e Cura a Carattere Scientifico (L.C.), 27100 Pavia, Italy

Address all correspondence and requests for reprints to: Ferruccio Santini, M.D., Department of Endocrinology, University of Pisa, Via Paradisa, 2, 56124 Pisa, Italy. E-mail: fsantini{at}endoc.med.unipi.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Mutations in the human melanocortin-4 receptor (MC4-R) gene may account for up to 5.8% of morbid nonsyndromic obesity. We have screened 120 unrelated obese patients for variants of the MC4-R gene. Four heterozygous missense variants were detected, including two polymorphisms (Val¹⁰³Ile and Ile²⁵¹Leu) previously described in the literature. A novel heterozygous mutation (Glu³⁰⁸Lys) was detected in a 36-yr-old female patient. Compared with the wild-type receptor, cells expressing the mutated receptor showed a reduced stimulation of cAMP production and a reduction of radioactive MSH binding. No segregation of the mutation with the obese phenotype could be demonstrated. A second, potentially pathogenic mutation (Ser³⁰Phe) was detected in a 31-yr-old female patient. Functional analysis of the mutated receptor showed no change in the affinity to the natural ligand MSH nor limited ability to stimulate cAMP production. Sixty lean subjects were also screened, and no additional variants of the MC4-R gene were observed, except for two individuals with the Val¹⁰³Ile polymorphism. In conclusion, we have screened a population of Italian obese subjects for MC4-R variants, demonstrating a 1.7% prevalence of potentially pathogenic mutations. A novel heterozygous missense mutation (Glu³⁰⁸Lys) that impairs MC4-R functional activity in vitro was characterized.

	Introduction

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THE PREVALENCE OF obesity has been continuously increasing over the last decades in most countries with established market economies. This trend over time has been explained as the result of an increase in the average energy supply per capita associated with a sedentary lifestyle, which is typical of Western societies (1, 2). However, individual susceptibility to developing obesity may be extremely different within a population, and there is abundant evidence that the ability to accumulate adipose tissue differs considerably among ethnic groups. Thus, genetic factors appear to be as important as environmental factors in the pathogenesis of obesity (3). The assimilation, storage, and utilization of nutrient energy include a number of metabolic pathways that control body weight and body fat content by a set-point mechanism. This system involves a pool of genes, several of which have been recently identified on the basis of their known roles in energy homeostasis in animals combined with the finding of gene mutations that appear associated with obesity phenotypes in humans (1, 2, 3, 4, 5). Among these genes, those regulating the melanocortin system have recently attracted substantial attention, and sequence variations of these genes appear to be a common reason for genetic predisposition to obesity (6, 7). In particular, mutations in the human melanocortin-4 receptor (MC4-R) gene may account for up to 5.8% of morbid nonsyndromic obesity (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Obesity is usually transmitted as an autosomal dominant trait, most likely as a result of haploinsufficiency, but variable penetrance and expressivity of obesity in different families argue that MC4-R acts together with other genes as a component of a "thrifty genotype" (20). The precise contributions of different MC4-R gene mutations to the pathogenesis of obesity and their exact prevalences in various ethnic groups remain a matter of investigation. In this study we screened a sample of obese and normal weight Italian subjects for variants of the MC4-R gene, and we have functionally characterized two mutations of the gene.

	Subjects and Methods

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Subjects

One hundred and twenty unrelated patients with body mass index (BMI) of 35 or more who turned to the obesity center of our department for diagnosis and cure of their weight problem were enrolled after informed consent was obtained. Clinical, hematological, and instrumental examinations of each patient were performed following the Italian guidelines for obesity, and each patient was treated according to appropriate protocols for his/her condition. Self-reported age of onset of weight excess and family history of obesity were collected for each subject. Comorbidities and drugs taken were also recorded. Sixty-five subjects (19 men and 46 women) had developed severe obesity before the age of 11 yr, 23 (five men and eight women) subjects during adolescence (before the age of 18 yr), and 32 (eight men and 24 women) subjects during adulthood. Anthropometric measures (body weight and height, and waist and hip circumferences) were determined after an overnight fast. Body weight was measured to the nearest kilogram, and body height and circumferences were determined to the nearest centimeter. Subject characteristics are shown in Table 1.

Direct nucleotide sequencing of the MC4-R gene

Two primers, MC4-R forward (5'-AATAACTGAGACGACTCCCTGAC-3') and MC4-R reverse (5'-CAGAAGTACAATATTCAGGTAGGG-3'), were used in a PCR reaction to amplify the MC4-R gene from genomic DNA isolated from whole blood. PCR was performed using Taq polymerase (Invitrogen, San Diego, CA) and was carried out under standard conditions, with 35 cycles of 95 C for 30 sec, 57 C for 30 sec, and 72 C for 50 sec.

PCR for the sequencing was performed with an annealing temperature of 57 C, using the following primers: MC4-RF1 (5'-TGAGACGACTCCCTGACCCAG-3'), MC4-RF2 (5'-CATCACCCTATTAAACAGTACAG-3'), MC4-RF3 (5'-AGGCTTCACATTAAGAGGATTG-3'), MC4-RR1 (5'-TACAATATTCAGGTAGGGTAAGA-3'), MC4-RR2 (5'-TTGGCGGATGGCACCAGTGC-3'), and MC4-RR3 (5'-CACTGTGAAACTCTGTGCATC-3'). Sequencing was carried out using a CEQ 2000 XL sequencer (Beckman Coulter, Fullerton, CA).

Cell cultures

COS-7 cells were grown on 10-cm petri dishes in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2% penicillin/streptomycin (Invitrogen), 1% fungizone (Invitrogen), and 1% sodium pyruvate (Invitrogen) and were incubated at 37 C in humidified air containing 5% CO₂. Cells were harvested using a trypsin-EDTA mixture 24 h before transfection and were seeded (150,000 cells/dish) in 3-cm dishes. Cells at 70–80% confluence were used for transfections.

Studies of mutant receptor function

Wild-type (normal) and mutant MC4-Rs were cloned using primer MC4-R forward (see above) and MC4-R RevClon (5'-CACTCGAGCAGAAGTACAATATTCAGGTACGGTAAG-3'). MC4-R forward flanks an endogenous EcoRI site, whereas MC4-R RevClon contains the XhoI restriction site, thus facilitating cloning of the PCR product into the mammalian expression vector pcDNA3 (Invitrogen). After sequencing of the different constructs to verify the absence of cloning artifacts, the wild-type and mutant MC4-R genes were transiently transfected into COS-7 using the diethylaminoethyl-dextran method, followed by a 2-min 10% dimethylsulfoxide shock (21). Forty-eight hours after transfection, cells were deprived of medium, and various concentrations of [Nle⁴,D-Phe⁷]MSH (Calbiochem, San Diego, CA) were added. As activation of MC4-R increases intracellular cAMP, this latter was measured by RIA using a commercial anti-cAMP antibody (Research Products International Corp., Mount Prospect, IL) and in-house radioiodinated cAMP succinyl-methyl-esther (Sigma-Aldrich Corp., St. Louis, MO) (22). Cells were incubated for 1 h with MSH and 0.5 mmol/liter isobutylmethylxanthine (Sigma-Aldrich Corp.) at 37 C in a final volume of 1 ml isotonic medium. The medium was then discarded, and 1 ml 0.1 N HCl was added to each dish. After 20 min at room temperature, the HCl solution was collected, and wells were washed with 1 ml H₂O. Cell extracts were dried in a vacuum concentrator and suspended in 0.2 ml H₂O. Ten microliters of this solution were incubated in triplicate in the presence of 0.19 ml 0.05 mol/liter citrate buffer, 0.1 ml 1:350 diluted cAMP antibody, and approximately 12,000 cpm [¹²⁵I]cAMP in 0.1 ml citrate buffer. After incubation at 4 C for 16–18 h, 1 ml 0.1% dextran T70/charcoal-activated BSA was added, and the mixture was centrifuged at 1,000 x g for 30 min to separate bound from free radioactivity. cAMP was expressed as picomoles per dish, using a nine-point standard curve.

Ligand binding assay

Ligand binding assays were carried out on intact COS-7 cells, transiently transfected with wild-type or mutant MC4-R construct as described above. Cells were washed with binding buffer (1 mg/ml BSA in Ca²⁺/Mg²⁺ PBS) 48 h after transfection, and were incubated at room temperature for 4 h with a binding buffer containing 100,000 cpm of ¹²⁵I-labeled [Lys¹¹,Nle⁴,D-Phe⁷]MSH (Amersham Biosciences Italia, Milan, Italy; specific activity, 2000 Ci/mmol)/dish. Specific binding was determined using a concentration of 10^-6 mol/liter unlabeled MSH as competitor. Four hours after incubation, the binding medium was removed, and 0.1 N NaOH was added. Radioactivity was counted with a -counter.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Gene screening of MC4-R

A total of four heterozygous missense variants were detected among the 120 obese subjects of the study, including two polymorphisms (Val¹⁰³Ile and Ile²⁵¹Leu) previously described in literature. A novel heterozygous missense mutation (Glu³⁰⁸Lys) was detected in a 36-yr-old female patient, resulting in an amino acid substitution at the C-terminal cytoplasmic tail of the receptor. The proband reported a regular body weight at birth (3600 g) and a gradual excessive increase in her body mass starting from early infancy. She weighed 96 kg at the age of 13 yr, and at the time of this study weighed 112 kg (BMI, 44.9 kg/m²), with a waist/hip ratio of 0.85. Serum leptin was 43.5 ng/ml, appropriate for her BMI. Figure 1 shows the pedigree, BMIs, and genotypes of the patient’s family members. Her mother, one sister, and her son harbored the same mutation, although they all had a BMI below 25 kg/m² at the time of this study and had no history of obesity. The proband’s daughter showed no mutations of the MC4-R gene, although she had a BMI in the uppermost percentile range for her age (31 kg/m²).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Pedigree, clinical characteristics, and genotypes of members of the family with MC4-R Glu308Lys mutation. The proband is indicated by an arrow. Age, BMI, and MC4-R genotype of the family members, if available, are shown below each symbol. •, Subject with severe early-onset obesity; M, mutant allele (Glu308Lys); N, normal allele; X, deceased; /, not tested.

No additional variants of the MC4-R gene were observed, except for two individuals with the Val¹⁰³Ile polymorphism, among the 60 control subjects.

Functional characterization of Glu³⁰⁸Lys and Ser³⁰Phe mutations in MC4-R

To explore the functional properties of mutated MC4-Rs, vectors expressing the wild-type MC4-R or mutated variant types (Glu³⁰⁸Lys or Ser³⁰Phe) were transiently transfected in COS-7 cells. Cells were then exposed to increasing concentrations of MSH, and cAMP production was measured by RIA. As shown in Fig. 2, cells expressing the wild-type receptor exhibited a sigmoidal dose response to MSH. Compared with those with the wild-type receptor, cells expressing the Glu³⁰⁸Lys receptor showed a reduced stimulation of cAMP production at various concentrations of MSH. Conversely, the Ser³⁰Phe mutation did not affect MSH-induced cAMP production, compared with the wild-type receptor (data not shown). Treatment with forskolin, a direct activator of adenylate cyclase, was performed in parallel experiments, producing equal cAMP production in wild-type and mutant-transfected cells.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Dose-response curve of MSH-stimulated cAMP production in COS-7 cells transiently transfected with vectors expressing the wild-type or Glu308Lys mutated MC4-R genes. Results are expressed as the mean of triplicate determinations ± SE. Closely agreeing results were obtained in two additional experiments.

View larger version (10K): [in this window] [in a new window]   FIG. 3. Binding of radiolabeled MSH to COS-7 cells transiently transfected with vectors expressing the wild-type (WT) or mutant (Glu308Lys or Ser30Phe) MC4Rs. Cells were exposed to tracer amounts of 125I-labeled [Lys11,Nle4,D-Phe7]MSH, in the absence and presence of 10-6 mol/liter unlabeled MSH to determine specific binding, and the ability of cells to bind the radioligand was measured. Data are expressed as counts per minute per dish. Results are expressed as the mean of triplicate determinations ± SD. Closely agreeing results were obtained in two additional experiments. *, P < 0.005 vs. WT receptor, by t test.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

One of the two mutations we found, Glu³⁰⁸Lys, was a novel variant. Functional and pedigree studies of this mutation produced interesting and apparently contrasting results. On the one hand, the mutation clearly affects the biological properties of MC4-R by reducing its ability to bind MSH and to stimulate adenylate cyclase activity. On the other hand, no segregation of the mutation with obese phenotype could be demonstrated, in that three of four family members harboring the mutation were actually of normal weight.

There are a number of factors that could explain these unexpected findings. 1) The in vitro environment may not reflect the in vivo situation. The Glu³⁰⁸Lys mutation is located at the cytoplasmic tail of the receptor, which is necessary for proper trafficking of the protein to the cell surface (11). Thus, the dysfunction might be revealed under conditions of maximal expression of the receptor, such as in vitro, while having a minor impact in vivo. 2) The mutation might equally affect the activities of MSH and agouti-related protein, the natural antagonist of the receptor, without altering the overall neuronal response. 3) The MC4-R gene acts in concert with other genes to regulate energy storage; therefore, additional abnormalities, beside defective melanocortin signaling, may be required for obesity to develop. 4) A protective gene against obesity could be active in the family members who are lean despite the presence of the mutation.

Evidence of a correlation between the in vitro function of mutant MC4-Rs and the severity of the clinical phenotype has been reported (18). On the other hand, a reduced penetrance of obesity associated with MC4-R mutations has been observed in heterozygous carriers of inactivating mutations (14, 18), and an obesity-associated mutation leading to constitutive activation of MC4-R has also been described (14). All of these findings suggest that obesity associated with MC4-R mutations may not be solely attributed to haploinsufficiency at this locus, and that differences in the genetic background could be crucial in determining the penetrance of the mutated allele.

The second mutation we found, Ser³⁰Phe, has been previously detected in an obese male subject who additionally harbored a missense mutation (Gly²⁵²Ser) in the sixth transmembrane domain (12). In our patient, the Ser³⁰Phe mutation was isolated and appeared to be a de novo mutation, as both parents were homozygous for the wild-type receptor. Alternatively, the father of this obese patient may not be the biological father, especially as this mutation has been previously described in a different carrier. Functional analysis of the mutated MC4-R showed no change in the affinity to the natural ligand, MSH, nor a limitation in the ability to stimulate cAMP production. These data argue against a role of the Ser³⁰Phe mutation in the pathogenesis of human obesity. However, as in the previous case, the in vitro conditions may not reflect the real biological properties of the receptor, and lean subjects harboring the mutation should be detected before classifying the Ser³⁰Phe variant as a polymorphism.

In conclusion, we have screened a large population of Italian subjects with severe obesity for MC4-R variants and have found a prevalence of potentially pathogenic mutations of approximately 2%. A novel missense mutation at the cytoplasmic tail of the receptor, which impairs its functional activity, was characterized. This mutation was associated with a low penetrance of obesity in heterozygous family members.

	Acknowledgments

 
We gratefully acknowledge the expertise of Mrs. Jennifer Cummings in the preparation of the manuscript.

	Footnotes

 
This work was supported by the following grants: Ministero dell’Istruzione, dell’Università e della Ricerca Scientifica, Programmi di Ricerca Cofinanziati 2002: Obesity: Phenotype Characterization and Relationship with Pathogenesis; Ministero della Salute, Programma Speciale di Sperimentazione Spprovato nell’Anno 2000: Strategia Multidisciplinare per la Prevenzione e la Cura dell’Obesità e dei Disturbi del Comportamento Alimentare; Ministero dell’Istruzione, dell’Università e della Ricerca Scientifica, Centro di Eccellenza AmbiSEN: Effects of Envinromental Chemical Agents on the Endocrine and Nervous System; Telethon Foundation Contract Grant TCP99016 (to M.M.); a Telethon Fellowship (to C.C.), and an Assistant Telethon Scientist Award (to M.M.).

Abbreviations: BMI, Body mass index; MC4-R, melanocortin-4 receptor.

Received July 8, 2003.

Accepted November 10, 2003.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Santini, F. Articles by Pinchera, A. Search for Related Content PubMed PubMed Citation Articles by Santini, F. Articles by Pinchera, A.