https://orcid.org/0000-0002-5647-362X
Abstract
Melanocortin receptor-4 (MC4R) gene mutations are associated with early-onset severe obesity, and the identification of potential pathological variants is crucial for the clinical management of patients with obesity.
To explore whether and how a novel heterozygous MC4R variant (MC4R-F313Sfs*29), identified in a young boy (body mass index [BMI] 38.8 kg/m2) during a mutation analysis conducted in a cohort of patients with obesity, plays a determinant pathophysiological role in the obesity development.
The genetic screening was carried out in a total of 209 unrelated patients with obesity (BMI ≥ 35 kg/m2). Structural and functional characterization of the F313Sfs*29-mutated MC4R was performed using computational approaches and in vitro, using HEK293 cells transfected with genetically encoded biosensors for cAMP and Ca2+.
The F313Sfs*29 was the only variant identified. In vitro experiments showed that HEK293 cells transfected with the mutated form of MC4R did not increase intracellular cAMP or Ca2+ levels after stimulation with a specific agonist in comparison with HEK293 cells transfected with the wild type form of MC4R (∆R/R0 = -90% ± 8%; P < 0.001). In silico modeling showed that the F313Sfs*29 mutation causes a major reorganization in the cytosolic domain of MC4R, thus reducing the affinity of the putative GalphaS binding site.
The newly discovered F313Sfs*29 variant of MC4R may be involved in the impairment of α-MSH-induced cAMP and Ca2+ signaling, blunting intracellular G protein-mediated signal transduction. This alteration might have led to the dysregulation of satiety signaling, resulting in hyperphagia and early onset of obesity.
Obesity is a chronic disease rapidly becoming more prevalent in many countries worldwide (1). The prevalence of overweight and obesity among children and adolescents aged 5 to 19 has risen dramatically in the last 2 decades and is still rising (2). Melanocortin 4 receptor (MC4R) is a G protein-coupled receptor expressed by neurons in the hypothalamic paraventricular nucleus (PVN), involved in the leptin/melanocortin pathway and the regulation of energy balance, food intake, and body weight (3). Melanocortin 4 receptor gene mutations are associated with early-onset severe obesity (4), and more than 200 variants have been described, classified into classes depending on their molecular effects (5), although not all variants are linked to the obese phenotype and, on the contrary, could protect against obesity. While most MC4R variants are loss of function, a subset causes gain of function and are associated with a significantly lower body mass index (BMI) via a signaling bias toward β-arrestin recruitment and increased activation of mitogen-activated protein kinase (MAPK) (6).
The α-Melanocyte-stimulating hormone (α-MSH) is a nonselective agonist ligand that binds MC4R and acts as a satiety signal, modulating feeding behavior and energy homeostasis (7). It has been observed that the MC4R C-terminal region is of critical importance for the correct signal transduction downstream of the receptor (8), which results in the intracellular accumulation of cAMP and subsequent protein kinase A (PKA) activation (9). An impairment in MC4R function, caused by a mutation involving a protein site that is fundamental for the correct functioning of the receptor, can lead to a defective anorectic action, resulting in hyperphagia, reduced energy expenditure, hyperinsulinemia, and a consequent early onset of obesity (10, 11). It has been shown that restoring the correct functioning of the leptin/melanocortin system by the administration of MC4R agonists can reduce the calorie intake and body weight in subjects affected by monogenic obesity (12, 13). However, not all the obese patients carrying MC4R mutations respond in the same way in terms of weight loss (14). Therefore, the identification of potential pathological variants and their functional characterization are crucial for the pathophysiological understanding and clinical management of patients with obesity.
Materials and Methods
Patients
A total of 299 unrelated Caucasian patients with BMI > 35 were enrolled in the Center for the Study and the Integrated Treatment of Obesity of the University Hospital of Padova, Italy. Declaration of Helsinki guidelines were followed and informed consent was obtained from all participants. All patients agreed to perform the diagnostic test and to the anonymous use of the test data. Patients with intellectual and developmental impairment, congenital malformations, visual impairment and/or deafness, and abnormal growth parameters who were already diagnosed with a genetic obesity disorder (ie, Alström syndrome, Bardet-Biedl syndrome, Prader-Willi syndrome) were not included. Clinical and anthropometric data were collected for each subject. Inclusion criteria for genetic testing were BMI > 35 and at least 1 of the following: the early prepubertal onset of obesity, a family history of obesity, hyperphagia, extreme obesity (BMI >50 kg/m2), repeated surgery after weight regain, or insufficient weight loss.
Mutation analysis
The coding region of human MC4R, leptin (LEP), and leptin receptor (LEPR) genes were amplified starting from genomic deoxyribonucleic acid (DNA) isolated from whole blood of each patient. Six nested primers were used to sequence the resulting polymerase chain reaction (PCR) product on both strands using the BigDye terminator chemistry and an ABI 3100 automated DNA sequencing analyzer (Applied Biosystems, Foster City, California).
DNA cloning and construction of wild type and mutant MC4R expression vectors
The PCR products of MC4R (both the wild type gene and the F313Sfs*29 mutated gene) isolated from the genomic DNA of the patient carrying the mutation, were amplified by using the following primers (forward 5’-GGATCCCACCATGGTGAACTCCACCCAC-3’ and reverse 5’-GAATTCTTAGTCATCGTCTTTGTAGTCATATCTGCTAGACAAGTCACAAAGG-3’), extracted by using PureLink Quick Gel Extraction Kit, inserted into pCR2.1 TOPO vector using a TOPO Ta-Cloning Kit and subcloned into the eukaryotic expression vector pcDNA3.1 using the pcDNA3.1 Directional TOPO Expression Kit (all from Thermo Fisher Scientific, Waltham, Massachusetts), which allowed the expression of mRNA coding for the MC4R gene. Sequences of the constructs containing the wild type form (pMC4Rwt) or the F313Sfs*29 mutated form (pMC4Rmut) were confirmed by sequencing.
Cell cultures and transfection
HEK293 cells were cultured in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS) and 1 mM sodium pyruvate (all from Thermo Fisher Scientific, Waltham, Massachusetts). For the functional fluorescence resonance energy transfer (FRET) and immunofluorescence experiments subconfluent HEK293 cells plated onto 15-mm glass coverslips were transiently co-transfected using a plasmid encoding the exchange protein directly activated by cAMP (EPAC)-based, cAMP-sensitive FRET sensor EPACSH187 (H187): (mTurquoise2Δ-Epac(CD,ΔDEP,Q270E)-tdcp173Venus) together with either pcDNA33.1 (control), pMC4Rwt or pMC4Rmut, or both, using Lipofectamine 2000 according to the manufacturer’s instructions. Assays were performed 24 to 48 hours after transfection.
Immunofluorescence
Untransfected HEK293 cells and HEK293 cells transfected with H187 together with either the empty vector pcDNA3.1, or pMC4Rwt or pMC4Rmut, were washed twice with PBS and fixed with a 4% paraformaldehyde solution for 15 minutes at 37°C. Cells were then washed twice with phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Unspecific binding was reduced by blocking with 2% fetal bovine serum for 30 minutes. Cells were incubated overnight at 4°C with polyclonal rabbit anti-MC4R antibody (#24233, dilution 1:500; AbCam, Cambridge, MA, USA) and monoclonal mouse β-arrestin (#13140, dilution 1:200; Santa Cruz, Biotechnology, Dallas, TX, USA). After incubation, cells were extensively washed with PBS. Goat anti-rabbit IgG Alexa Fluor 647 (dilution 1:1000; Thermo Fisher Scientific, Waltham, Massachusetts) and goat anti-mouse IgG Alexa Fluor 488 (dilution 1:1000) were used as secondary antibodies for 1 hour. Unbound antibodies were removed by washing 3 times with PBS. Coverslips were then incubated with Hoechst 33342 (dilution 1:6000; Sigma-Aldrich, St. Louis, Missouri) for 1 minute to counterstain the nuclei, washed in PBS, and mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific, Waltham, Massachusetts). Images were collected with a DMI6000B microscope and analyzed by using LAS software (all from Leica Microsystem, Wezlar, Germany). Hoechst staining was visualized using a 340- to 380-nm excitation filter, a 400 dichroic mirror, and an LP 425 emission filter. Yellow fluorescent protein (YFP) fluorescence and Alexa488-conjugated antibody were visualized using the 488-nm argon laser line as excitation and a 505 to 550 band pass filter in emission. Alexa647-conjugated antibody was excited with the 543-nm HeNe laser line, and emission was collected through a 585-nm long pass filter. Mean fluorescence intensity was quantified using ImageJ software, as previously described (15).
Fluorescence resonance energy transfer imaging
The day before transfection, HEK293 cells were plated onto glass coverslips; 24 to 48 hours after transfection, cells were mounted onto an open perfusion chamber RC-25F (Warner Instruments, Holliston, MA, USA). Cells were perfused with Ringer’s modified buffer (NaCl 125 mM; KCL 5 mM; Na3PO4 1 mM; MgSO4 1 mM; Hepes 20 mM; glucose 5.5 mM; CaCl2 1 mM; pH adjusted to 7.4 using 1 M NaOH) using a homemade gravity-fed perfusion system with a velocity of 1 mL/minute. Experiments were performed on an Olympus IX71 inverted microscope equipped with a beam-splitter (OptoSplit II, Teledyne Photometrics, Tucson, AZ, USA) and a charge-coupled device (CCD) camera (CoolSNAP HQ2). A light-emitting diode source excited the cyan fluorescent protein (mTurquoise for H187) at 430 nm; the emission fluorescence was collected for both donor and acceptor fluorophores at 480 nm and 545 nm, respectively, every 5 to 10 seconds. Automatic image collection and preliminary analysis were performed using MetaFluor software (Molecular Devices, LLC. San Jose, CA, USA). Raw data (16) were transferred to Excel (Microsoft, Redmond, Washington, USA) for background subtraction and generation of the ratios; graphs were generated by Origin software (OriginLab, Corporation Northampton, MA, USA). Fluorescence resonance energy transfer responses were expressed as percentage of the maximum (∆R/∆Rmax, where ∆R = RαMSH-R0; and ∆Rmax= RF/I-R0. R0 is the ratio of intensity at time = 0 seconds; RαMSH is the ratio at time = t seconds; and RF/I is the ratio achieved by Forskolin and IBMX, a treatment that saturates the sensor’s signal).
In silico modeling
The MC4R-wt and the F313Sfs*29 variant were initially modelled in silico using MODELLER (17) on the crystal structure of MC4R in complex with SHU9119 (PDB ID: 6W25) (18). After 50 ns of relaxation with Gromacs 2016.1 (19), the resulting structures were investigated with University of California, San Francisco (UCSF) Chimera (20). The prediction of the docking between MC4R and GalphaS was performed using HADDOCK (21), using the binding region information provided by PetiMap (22). The MC4R-F313Sfs*29-GalphaS and MC4R-wt-GalphaS complex were analyzed using Protein Interfaces, Surfaces and Assemblies (PISA) (23).
[Ca2+]cyt measurements
HEK293 cells were grown on 13-mm poly-L-lysine-coated round glass coverslips at 50% confluence and cotransfected with an aequorin-based probe (cytAeq) (24) together with the indicated plasmid (the mock vector pcDNA3.1 was used as a control). Two days after transfection, cells were incubated with 5 μM of coelenterazine for 1 to 2 hours in Krebs-Ringer modified buffer (KRB: 125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4) at 37°C supplemented with 1 mM CaCl2, and then transferred to the perfusion chamber. All aequorin measurements were carried out in KRB. Agonists and other drugs were added to the same medium, as specified in the text. The experiments were terminated by lysing cells with 100 μM digitonin in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca2+] values by an algorithm based on the Ca2+ response curve of aequorin at physiological conditions of pH, [Mg2+], and ionic strength, as previously described (25). All the results are expressed as mean ± standard deviation (SD) and are representative of at least 3 independent transfections.
Translocation of β-arrestin
β-arrestin translocation was evaluated using the bioluminescent procedure described in (26). Briefly, HEK293 cells were grown on 13-mm poly-L-lysine-coated round glass coverslips at 50% confluence and cotransfected with a β-arrestin-aequorin chimera (a kind gift of Paolo Pinton) together with the indicated plasmid. Two days after transfection, cells were incubated with 5 μM of coelenterazine for 1hour in KRB supplemented with 100 µM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). After incubation, cells were stimulated for 10 minutes by the addition of 100 nM NDP-MSH and then transferred to the perfusion chamber. After 1 minute, cells were stimulated with KRB supplemented with 1 mM CaCl2. The experiments were terminated by lysing cells with 100 μM digitonin in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca2+] values, as specified above. All the results are expressed as mean ± SD and are representative of at least 2 independent transfections.
Results
A novel MC4R mutation identified in a young patient with obesity
Out of 209 obese patients screened for mutations, 58 (27.8%) had early onset obesity and 151 (72.2%) had at least 1 of the other criteria of which only 1 was necessary for inclusion. Anthropometric and clinical characteristics of the patients and most frequent comorbidities are summarized in Table 1. A young male patient had 1 novel mutation (Fig. 1A), suggesting a low prevalence of MC4R variants in the adult obese population in northern Italy. This variant is a deletion in position 938 (c.del938T) that causes a change in the amino acid sequence in position 313 of a phenylalanine replaced by a serine (F>S) and a consequent frameshift, leading to an aberrant extended protein of 342 amino acids (Fig. 1D) instead of 332 of the wild type form (Fig. 1C). After the identification of the novel mutation in the patient, the DNA of his parents was also tested for MC4R mutations and the same variant was identified in the mother (Fig. 1B), who is affected by morbid obesity (BMI 40.5 kg/m2), had menarche at 10 years, biliary stones, and primary hypothyroidism treated with levothyroxine. The mutation was not present in the DNA of the father, who is normal weight (BMI 23.4 kg/m2), had a regular pubertal development, and did not report any chronic illness (Fig. 1C). The MC4R F313Sfs*29 mutation has not been described in the SNP database (dbSNP) (27) nor in 6503 controls (13 006 chromosomes) included in the Exome Variant Server database of the NHLBI Exome Sequencing Project (28). In addition, none of the 209 obese patients tested carried any mutation in MC4R gene and no mutations in LEP or LEPR genes were detected in our selected population.
Anthropometric and clinical characteristics of patients with obesity
| Early Onset (n = 58) | Other Criteria (n = 151) | P-value | |
|---|---|---|---|
| BMI: Median (IQR) | 47.1 (41.7–54.1) | 42.2 (38.6–48.7) | 0.004* |
| Gender: M/F | 24/34 | 60/91 | 0.885 |
| Age: Median (IQR) | 43.5 (18.2–52.7) | 49 (35.2–56) | 0.136 |
| Comorbidities: n (%) | |||
| Metabolic | |||
| Type 2 Diabetes | 32 (54.5) | 57 (37.9) | 0.022* |
| Insulin Resistance | 8 (13.6) | 21 (13.7) | >0.999 |
| NAFLD | 8 (13.6) | 18 (12) | 0.713 |
| Dyslipidemia | 3 (4.5) | 16 (10.3) | 0.288 |
| Hypercholesterolemia | 5 (9) | 13 (8.6) | 0.997 |
| Hyperinsulinemia | 5 (9) | 10 (6.9) | 0.616 |
| Hyperleptinemia | 3 (4.5) | 6 (3.9) | 0.710 |
| Metabolic Syndrome | 3 (4.5) | 5 (3.4) | 0.687 |
| Endocrine | |||
| Hypothyroidism | 11 (18.1) | 26 (17.2) | 0.767 |
| Hyperthyroidism | 11 (18.1) | 13 (8.6) | 0.035* |
| PCOS | 12 (20.6) | 11 (7.3) | 0.005* |
| Hyperandrogenism | 5 (9) | 11 (7.3) | 0.745 |
| Hypotestosteronemia | 3 (4.5) | 8 (5.1) | >0.999 |
| Hyperprogesteronism | 3 (4.5) | 5 (3.4) | 0.687 |
| Hypogonadotropic hypogonadism | 3 (4.5) | 4 (2.6) | 0.399 |
| Cardiovascular | |||
| Hypertension | 29 (50) | 81 (53) | 0.636 |
| Heart disease | 8 (13.6) | 13 (8.6) | 0.264 |
| Respiratory | |||
| OSAS | 18 (31.8) | 36 (24.1) | 0.287 |
| Asthma | 5 (9) | 5 (3.4) | 0.107 |
| Respiratory insufficiency | 3 (4.5) | 5 (3.4) | 0. 687 |
| None | 3 (4.5) | 10 (6.9) | 0.697 |
| Early Onset (n = 58) | Other Criteria (n = 151) | P-value | |
|---|---|---|---|
| BMI: Median (IQR) | 47.1 (41.7–54.1) | 42.2 (38.6–48.7) | 0.004* |
| Gender: M/F | 24/34 | 60/91 | 0.885 |
| Age: Median (IQR) | 43.5 (18.2–52.7) | 49 (35.2–56) | 0.136 |
| Comorbidities: n (%) | |||
| Metabolic | |||
| Type 2 Diabetes | 32 (54.5) | 57 (37.9) | 0.022* |
| Insulin Resistance | 8 (13.6) | 21 (13.7) | >0.999 |
| NAFLD | 8 (13.6) | 18 (12) | 0.713 |
| Dyslipidemia | 3 (4.5) | 16 (10.3) | 0.288 |
| Hypercholesterolemia | 5 (9) | 13 (8.6) | 0.997 |
| Hyperinsulinemia | 5 (9) | 10 (6.9) | 0.616 |
| Hyperleptinemia | 3 (4.5) | 6 (3.9) | 0.710 |
| Metabolic Syndrome | 3 (4.5) | 5 (3.4) | 0.687 |
| Endocrine | |||
| Hypothyroidism | 11 (18.1) | 26 (17.2) | 0.767 |
| Hyperthyroidism | 11 (18.1) | 13 (8.6) | 0.035* |
| PCOS | 12 (20.6) | 11 (7.3) | 0.005* |
| Hyperandrogenism | 5 (9) | 11 (7.3) | 0.745 |
| Hypotestosteronemia | 3 (4.5) | 8 (5.1) | >0.999 |
| Hyperprogesteronism | 3 (4.5) | 5 (3.4) | 0.687 |
| Hypogonadotropic hypogonadism | 3 (4.5) | 4 (2.6) | 0.399 |
| Cardiovascular | |||
| Hypertension | 29 (50) | 81 (53) | 0.636 |
| Heart disease | 8 (13.6) | 13 (8.6) | 0.264 |
| Respiratory | |||
| OSAS | 18 (31.8) | 36 (24.1) | 0.287 |
| Asthma | 5 (9) | 5 (3.4) | 0.107 |
| Respiratory insufficiency | 3 (4.5) | 5 (3.4) | 0. 687 |
| None | 3 (4.5) | 10 (6.9) | 0.697 |
Comparisons of dichotomous variables were performed using chi-square or Fisher’s exact test if the expected frequency in a cell was less than 5. Continuous variables were analyzed with Student’s t-test or the Mann-Whitney U Test if variables were not normally distributed. P-value below 0.05 (*) was considered significant.
Abbreviations: BMI, body mass index; F, female; IQR, interquartile range; M, male; NALFD, nonalcoholic fatty liver disease; OSAS, obstructive sleep apnea syndrome; PCOS, polycystic ovary syndrome;
Anthropometric and clinical characteristics of patients with obesity
| Early Onset (n = 58) | Other Criteria (n = 151) | P-value | |
|---|---|---|---|
| BMI: Median (IQR) | 47.1 (41.7–54.1) | 42.2 (38.6–48.7) | 0.004* |
| Gender: M/F | 24/34 | 60/91 | 0.885 |
| Age: Median (IQR) | 43.5 (18.2–52.7) | 49 (35.2–56) | 0.136 |
| Comorbidities: n (%) | |||
| Metabolic | |||
| Type 2 Diabetes | 32 (54.5) | 57 (37.9) | 0.022* |
| Insulin Resistance | 8 (13.6) | 21 (13.7) | >0.999 |
| NAFLD | 8 (13.6) | 18 (12) | 0.713 |
| Dyslipidemia | 3 (4.5) | 16 (10.3) | 0.288 |
| Hypercholesterolemia | 5 (9) | 13 (8.6) | 0.997 |
| Hyperinsulinemia | 5 (9) | 10 (6.9) | 0.616 |
| Hyperleptinemia | 3 (4.5) | 6 (3.9) | 0.710 |
| Metabolic Syndrome | 3 (4.5) | 5 (3.4) | 0.687 |
| Endocrine | |||
| Hypothyroidism | 11 (18.1) | 26 (17.2) | 0.767 |
| Hyperthyroidism | 11 (18.1) | 13 (8.6) | 0.035* |
| PCOS | 12 (20.6) | 11 (7.3) | 0.005* |
| Hyperandrogenism | 5 (9) | 11 (7.3) | 0.745 |
| Hypotestosteronemia | 3 (4.5) | 8 (5.1) | >0.999 |
| Hyperprogesteronism | 3 (4.5) | 5 (3.4) | 0.687 |
| Hypogonadotropic hypogonadism | 3 (4.5) | 4 (2.6) | 0.399 |
| Cardiovascular | |||
| Hypertension | 29 (50) | 81 (53) | 0.636 |
| Heart disease | 8 (13.6) | 13 (8.6) | 0.264 |
| Respiratory | |||
| OSAS | 18 (31.8) | 36 (24.1) | 0.287 |
| Asthma | 5 (9) | 5 (3.4) | 0.107 |
| Respiratory insufficiency | 3 (4.5) | 5 (3.4) | 0. 687 |
| None | 3 (4.5) | 10 (6.9) | 0.697 |
| Early Onset (n = 58) | Other Criteria (n = 151) | P-value | |
|---|---|---|---|
| BMI: Median (IQR) | 47.1 (41.7–54.1) | 42.2 (38.6–48.7) | 0.004* |
| Gender: M/F | 24/34 | 60/91 | 0.885 |
| Age: Median (IQR) | 43.5 (18.2–52.7) | 49 (35.2–56) | 0.136 |
| Comorbidities: n (%) | |||
| Metabolic | |||
| Type 2 Diabetes | 32 (54.5) | 57 (37.9) | 0.022* |
| Insulin Resistance | 8 (13.6) | 21 (13.7) | >0.999 |
| NAFLD | 8 (13.6) | 18 (12) | 0.713 |
| Dyslipidemia | 3 (4.5) | 16 (10.3) | 0.288 |
| Hypercholesterolemia | 5 (9) | 13 (8.6) | 0.997 |
| Hyperinsulinemia | 5 (9) | 10 (6.9) | 0.616 |
| Hyperleptinemia | 3 (4.5) | 6 (3.9) | 0.710 |
| Metabolic Syndrome | 3 (4.5) | 5 (3.4) | 0.687 |
| Endocrine | |||
| Hypothyroidism | 11 (18.1) | 26 (17.2) | 0.767 |
| Hyperthyroidism | 11 (18.1) | 13 (8.6) | 0.035* |
| PCOS | 12 (20.6) | 11 (7.3) | 0.005* |
| Hyperandrogenism | 5 (9) | 11 (7.3) | 0.745 |
| Hypotestosteronemia | 3 (4.5) | 8 (5.1) | >0.999 |
| Hyperprogesteronism | 3 (4.5) | 5 (3.4) | 0.687 |
| Hypogonadotropic hypogonadism | 3 (4.5) | 4 (2.6) | 0.399 |
| Cardiovascular | |||
| Hypertension | 29 (50) | 81 (53) | 0.636 |
| Heart disease | 8 (13.6) | 13 (8.6) | 0.264 |
| Respiratory | |||
| OSAS | 18 (31.8) | 36 (24.1) | 0.287 |
| Asthma | 5 (9) | 5 (3.4) | 0.107 |
| Respiratory insufficiency | 3 (4.5) | 5 (3.4) | 0. 687 |
| None | 3 (4.5) | 10 (6.9) | 0.697 |
Comparisons of dichotomous variables were performed using chi-square or Fisher’s exact test if the expected frequency in a cell was less than 5. Continuous variables were analyzed with Student’s t-test or the Mann-Whitney U Test if variables were not normally distributed. P-value below 0.05 (*) was considered significant.
Abbreviations: BMI, body mass index; F, female; IQR, interquartile range; M, male; NALFD, nonalcoholic fatty liver disease; OSAS, obstructive sleep apnea syndrome; PCOS, polycystic ovary syndrome;
Direct sequencing of the coding region of MC4R revealed a frameshift mutation (A) chromatograms of the DNA sequences of the wild type and mutated (del938T) MC4R gene, isolated from peripheral blood DNA of the proband. B: Pedigree of the family carrying the F313Sfs*29 mutation and body mass index (BMI) of each subject. C–D: The deletion of a T in position 938 causes a substitution of a phenylalanine replaced by a serine in position 313 (red bubble) and an aberrant extended tail (red letters) in C-terminal region (C-term) of the receptor. Abbreviations: DNA, deoxyribonucleic acid; ECL, extracellular loop; ICL, intracellular loop; T, timine; MC4R, Melanocortin 4 receptor. Schematic adaptation from GPCRDB Tools (48).
At the time of inclusion, clinical characteristics of the patient were obesity (BMI 38.8 kg/m2; >97th percentile), hyperinsulinism, celiac sprue, Perthes disease, and iron-deficiency anemia. He was born at term by cesarean section, with a birth weight of 3550 g. There were no complications in the perinatal or neonatal periods. The patient had early-onset obesity: rapid progressive weight gain and short stature (-2 SD) were reported in early childhood and hyperphagia was described beginning at 1 year of age. Weight, height, and BMI growth curves are described in Fig. 2A and the values of weight, height, BMI, BMI z-score, and percentile measured throughout the patient follow-up are displayed in Fig. 2B. The patient had growth hormone deficiency (diagnosed at 8 years old by performing provocative growth hormone (GH) testing: GH peak after arginine administration was 2.8 ng/ml and after clonidine administration was 1.4 ng/ml) treated with recombinant GH starting at age 9; low serum Insulin-like growth factor 1 (IGF-1) levels: 116 ng/ml (-0.4 SD) at age 8 and 642 ng/ml (+1.36 SD) at age 16 undergoing GH treatment; hypogonadotropic hypogonadism (diagnosed at age 14 by performing a GnRH test) treated with hormone replacement therapy (intramuscular [IM] testosterone 100 mg monthly starting at age 14) and a mild learning disability.
Anthropometric characteristics of the proband weight (red dots), height (blue dots), and body mass index (BMI, orange dots) of the proband carrying the F313Sfs*29 mutation are plotted on the growth charts of the Center for Disease Control (CDC)/ National Center for Health Statistics (A). BMI, z-score and percentile measured throughout the patient follow-up (from 8–17 years) are displayed in panel B.
Expression of wild type MC4R and F313Sfs*29-mutated MC4R in HEK293 cells
HEK293 cells were transfected with the wild type and the F313Sfs*29-mutated form of MC4R. The expression of MC4R was evaluated by immunofluorescence staining with a specific anti-MC4R antibody (Fig. 3). As controls, untransfected HEK293 cells or HEK293 cells transfected only with empty vector pcDNATM3.1 and plasmid H187 were used. The 2 control conditions did not show any MC4R-positive staining (Fig. 3A and 3B), proving the absence of endogenous expression of the receptor in HEK293 cells. Only cells transfected with H187 showed a positive signal at 505 to 550 nm emission, due to the presence of a yellow fluorescent protein (YFP) in the construct (Fig. 3B–3D). In fact, the FRET-based H187 sensor consists of the cAMP effector Epac1 fused to a cyan fluorescent protein (CFP) donor (mTurquoise2) and an optimized YFP acceptor (a tandem of the circular permutated version of Venus, cp173Venus). These results confirmed the correct expression of the cAMP sensor in all the cells transfected with H187 plasmid. Both the wild type and F313Sfs*29-mutated MC4R were properly expressed on the surface of the cells transfected with the wild type and the F313Sfs*29-mutated plasmids (Fig. 3C and 3D), without any significant difference in terms of distribution, expressed as the mean of fluorescence intensity divided by the area of each cell analyzed (Fig. 3E). No significant differences between wild type and F313Sfs*29-mutated MC4R expression in terms of fluorescence intensity or distribution were present after staining the same cells in the absence of a permeabilization treatment, confirming that the mutation does not affect the cellular localization of MC4R (data not shown).
Cellular expression of wild type MC4R and F313Sfs*29-mutated MC4R in HEK293 cells. Untransfected HEK293 cells were used as control (A). HEK293 cells were co-transfected with Epac-based FRET sensor (H187) together with either the empty vector pcDNA™3.1 (B), or pMC4Rwt (C) or pMC4Rmut (D) plasmids, the last 2 coding for the wild type and the mutated form of MC4R, respectively. Differential interference contrast (DIC) microscopy and fluorescence microscopy was used to assess the expression of these plasmids in the cells 48 hours after transfection. Immunofluorescence staining of nuclear-specific dye Hoechst (blue), YFP (yellow) and anti-MC4R (pink) are shown in HEK293 cells in the same viewing field. Scale bar: 20µm. Mean fluorescence intensity was measured in 50 different cells positively stained for anti-MC4R in each condition; images were background corrected by subtracting mean pixel values of a cell-free region of interest (results are expressed as mean +/- SEM, where the signal was detectable) (E). Abbreviations: FRET, fluorescence resonance energy transfer; MC4R, Melanocortin 4 Receptor; SEM, standard error of the mean; YFP, yellow fluorescent protein.
The F313Sfs*29 substitution abolishes the MC4R-dependent cAMP production in response to NDP-MSH
In order to assess the impact of the F313Sfs*29 substitution on the function of MC4R, we compared its ability to produce cAMP to that of the wild type protein. We directly measured cAMP production in response to (Nle4, D-Phe7)-alpha-MSH (NDP-MSH), a potent analog of the endogenous α-MSH, using the FRET-based cAMP sensitive sensor EPACSH187 (29, 30). As shown in Fig. 4, HEK293 cells expressing H187 did not respond to NDP-MSH (Fig. 4A), while, as expected, cells expressing the sensor and MC4RWT responded to the stimulus with a fast and significant increase in intracellular cAMP levels (Fig. 4B). On the contrary, when cells expressing the mutant MC4R-F313Sfs*29 were challenged with NDP-MSH we were unable to detect any FRET changes (Fig. 4C), strongly suggesting that the mutation significantly affects the ability of the receptor to respond to extracellular signals. These previous experiments tested the function of MC4R-F313Sfs*29 in homozygosis (since HEK293 cells do not express the wild type receptor); therefore, we next set to investigate cAMP signaling in heterozygosis, which is the condition observed in our patient. In order to simulate heterozygosis in HEK293 cells, we overexpressed both MC4R wild type and MC4R F313Sfs*29 together with the FRET sensor H187. As shown in Fig. 5A–5D, cells expressing both MC4R receptor versions (wild type and mutant) responded after NDP-MSH stimulus indicated that the MC4R F313Sfs*29 mutation does not act as dominant negative; however, the production of cAMP, especially at the basal level was significantly decreased when compared with that produced by the expression of MC4R wild type. Indeed, it is worth noting that the basal ratio of H187, which is an indicator of basal cAMP levels, was low in nontransfected HEK293 cells (0.33 ± 0.086) and in cells expressing the F313Sfs*29 mutant (0.32 ± 0.14), but was high in the cells expressing MC4R wild type (0.71 ± 0.26). Interestingly, when the 2 receptors were co-expressed, the basal ratio of H187 assumed an intermediate value between that of the mutant and wild type (0.49 ± 0.15) (Fig. 5E). While it is important to point out that the simultaneous exogenous expression of 2 receptors will have some element of variability, these experiments clearly suggest that in heterozygosis conditions, such as those of our patient, the levels of cAMP produced in response to endogenous MC4R ligands will be significantly lower than those produced in nonpathological wild type conditions.
MC4R-dependent cAMP production after NDP-MSH stimulation in HEK293 cells. MC4R-deficient HEK293 cells were transfected with the cAMP FRET sensor H187 in combination with a control pcDNA3.1 plasmid (A), or pMC4Rwt (B) or pMC4Rmut (C) plasmids, the last 2 coding for the wild type and the mutated form of MC4R, respectively. As summarized in (D), NDP-MSH challenge had no effect on the cells transfected with the control plasmid (HEK). On the contrary, cells expressing the MC4R wild type responded to NDP-MSH with a significant increase in cAMP production (HEK MC4Rwt), while cells expressing mutated MC4R F313Sfs*29 (HEK MC4Rmut) behaved similar to the empty vector control cells, with no response to the challenge. All cells responded to the addition of the adenylyl cyclase activator forskolin (FSK) together with the PDE inhibitor 3-Isobutyl-1-methylxanthine (IBMX) used as control treatment. Each experiment was repeated at least 5 times, FRET traces are an average of 42 cells for control, 12 cells for MC4R wild type, and 27 cells for MC4R F313Sfs*29 (bar graphs are mean +/- SEM, ***< 0.001). Abbreviations: FRET, fluorescence resonance energy transfer; HEK, Human embryonic kidney; MC4R, Melanocortin 4 Receptor; NDP-MSH, (Nle4,D-Phe7)-alpha-MSH; PDE, phosphodiesterase; SEM, standard error of the mean.
FRET-based cAMP measurements in HEK293 cells expressing MC4R variants. HEK293 cells expressing H187 and challenged with NDP-MSH did not induce cAMP production (A). Overexpression of MC4R wild type resulted in the production of cAMP in response to NDP-MSH (B) while the overexpression of MC4R-F313Sfs*29-mutant version showed no response (C). In cells transfected with equal amounts of both mutant and wild type receptors, we observed cAMP production, suggesting that the mutant does not act in a dominant manner (D). Bar graphs of the basal H187 FRET ratio in all conditions mirror the basal cAMP levels. No difference was observed between nontransfected cells and cells expressing the mutant MC4R. Overexpression of wild type MC4R resulted in high basal cAMP levels, while co-expression of wild type and mutant resulted in intermediate cAMP rises (E). Abbreviations: FRET, fluorescence resonance energy transfer; MC4R, Melanocortin 4 Receptor; NDP-MSH, (Nle4,D-Phe7)-alpha-MSH.
The F313Sfs*29 mutation’s impacts on the structure of the cytosolic domain of the MC4 receptor
The F313Sfs*29 is a frameshift mutation that leads to a change in amino acids located in the cytoplasmic C-terminal tail of the receptor. We thus investigated the structural impact of this variant using an in silico approach. Homology modeling of the mutant MC4R revealed a significant derangement of the cytoplasmic region. In particular, the comparison between the mutant and wild type structures shows a partial unfolding of the C-terminal helix H8 (Fig. 6A), which generates a global reorganization of the cytosolic domain containing the site for G protein binding. Accordingly, molecular docking revealed a decrease in binding affinity between the GalphaS subunit and mutant MC4R (Fig. 6B and 6C).
In silico modeling of the MC4R-F313Sfs*29 variant. The top and the side view of the MC4R-F313Sfs*29 variant (red) and MC4R-wt (transparent yellow) show different folding of the H8 domains induced by the mutated amino acids (A, orange). The top view of the MC4R-F313Sfs*29 variant (B) and MC4R-wt (C) show the disappearance of the cytoplasmic binding pocket (green). Abbreviations: H8, putative 8th helix; MC4R, Melanocortin 4 Receptor; wt, wild type.
The F313Sfs*29 mutation blunts global G protein–dependent signaling
We next wondered whether mutant MC4R could activate other unwanted non-Gαs signaling cascades and/or impair the recruitment of β-arrestin. First, in order to look for putative activation of Gαq-dependent pathways, we monitored cytoplasmic Ca2+ concentration ([Ca2+]cyt) using a genetically encoded Ca2+ indicator based on the Ca2+-sensitive photoprotein aequorin (25). As shown in Fig. 7A, mock transfected HEK293 cells showed no response to NDP-MSH, thus further supporting the lack of endogenous MC4R. Importantly, these cells showed a transient increase of [Ca2+]cyt in response to ATP stimulation (Fig. 7C), since they have endogenous expression of Gαq-coupled P2Y1/2 receptors (31). Surprisingly, HEK293 cells expressing wild type MC4R showed a clear rise in [Ca2+]cyt in response to NDP-MSH (Fig. 7B), maybe indicating pleiotropic signaling in response to receptor activation. In any case, mutant MC4R showed no response to the selective MC4R agonist, further supporting the idea of F313Sfs*29 as a loss-of-function mutation.
![MC4R-dependent [Ca2+]cyt measurements, MC4R after NDP-MSH, and ATP stimulation in HEK293 cells. MC4R-deficient HEK293 cells were transfected with cytosolic aequorin and probed together with a control pcDNA3.1 (HEK), pMC4Rwt (MC4R wt) or pMC4Rmut (MC4R mut) plasmids, the last 2 coding for the wild type and the mutated form of MC4R, respectively. A: Representative traces of [Ca2+] measurements in cells stimulated with NDP-MSH and ATP. B: Average of [Ca2+] peaks triggered by NDP-MSH, a specific agonist for exogenous MC4R. C: Average of [Ca2+] peaks triggered by ATP, a nonspecific agonist for endogenous IP3-coupled P2Y receptors. Bar graphs are mean +/- SD, *< 0.05, n ≥ 4. Abbreviations: ATP, Adenosine triphosphate; HEK, Human embryonic kidney; MC4R, Melanocortin 4 Receptor; NDP-MSH, (Nle4,D-Phe7)-alpha-MSH; SD, standard deviation of the mean; wt, wild type.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/106/3/10.1210_clinem_dgaa885/3/m_dgaa885_fig7.jpeg?Expires=1747866277&Signature=I9tgyWZPtHss1H3I1tpM6YLfZP5fWPBlXYa3rNBf05UDUF0hWMTAhEm1meuh-tcYeuS2ZUPaY2hcesHsGmAkkuuJXU5K-gfSewQMK550~5gT89tGJOPteWgXLW56g8rAeOQ2j7uxIk3qR-43RE4aTmJut5cZ9rVpOfzKKOWgOwP~SyblQilBqqHzSFDSWKIwmCR~Zod363-8qsybnjqBYZmifbqz46YzGlQtPgOMD1QmJ2gBKSY2TeLfHvqZSlVUDa-Eza-3wCiO0galHM5AXLoUMge~n1kFMroGHhV9lOdT5k-w-V38889LOJRdzrpzV5s39mvW9etSDBqgLr2j4Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MC4R-dependent [Ca2+]cyt measurements, MC4R after NDP-MSH, and ATP stimulation in HEK293 cells. MC4R-deficient HEK293 cells were transfected with cytosolic aequorin and probed together with a control pcDNA3.1 (HEK), pMC4Rwt (MC4R wt) or pMC4Rmut (MC4R mut) plasmids, the last 2 coding for the wild type and the mutated form of MC4R, respectively. A: Representative traces of [Ca2+] measurements in cells stimulated with NDP-MSH and ATP. B: Average of [Ca2+] peaks triggered by NDP-MSH, a specific agonist for exogenous MC4R. C: Average of [Ca2+] peaks triggered by ATP, a nonspecific agonist for endogenous IP3-coupled P2Y receptors. Bar graphs are mean +/- SD, *< 0.05, n ≥ 4. Abbreviations: ATP, Adenosine triphosphate; HEK, Human embryonic kidney; MC4R, Melanocortin 4 Receptor; NDP-MSH, (Nle4,D-Phe7)-alpha-MSH; SD, standard deviation of the mean; wt, wild type.
We finally looked for an impairment in β-arrestin activation by looking at its recruitment at the plasma membrane after NDP-MSH stimulation. We first performed an immunolocalization in cells transfected with control and MC4R expressing plasmids, either wild type or mutated. Figure 8A shows no clear recruitment of β-arrestin to the plasma membrane in any of the conditions tested, maybe indicating that this pathway is nonfunctional in our cellular model. To further test this hypothesis, we also performed a bioluminescent procedure to evaluate protein translocation to the plasma membrane (26). This assay is based on the large [Ca2+] difference between bulk cytosolic and the subplasma membrane rim. Activation of β-arrestin pathway triggers the translocation of β-arrestin-aequorin chimera to the plasma membrane that can thus sense the local high [Ca2+] microdomain. Consequently, higher [Ca2+] peak values indicate higher translocation. In line with our immunofluorescence data, we could not detect any MC4R-dependent translocation of β-arrestin (Fig. 8B). This is likely due to the lack of some essential signaling element in our experimental model.
![MC4R-dependent translocation of β-arrestin after NDP-MSH stimulation in HEK293 cells. A: Untransfected HEK293 cells were used as control. HEK293 cells were transfected with pMC4Rwt or pMC4Rmut plasmids and treated with NDP-MSH. Differential interference contrast (DIC) microscopy, immunofluorescence staining of nuclear-specific dye Hoechst (blue), anti-β-arrestin (green), and anti-MC4R (pink) are shown in HEK293 cells in the same viewing field. Scale bar: 10µm. B: MC4R-deficient HEK293 cells were transfected with a bioluminescent probe for β-arrestin translocation (β-arrestin-aequorin), together with a control pcDNA3.1 (HEK), pMC4Rwt (MC4R wt) or pMC4Rmut (MC4R mut) plasmids, the last 2 coding for the wild type and the mutated form of MC4R, respectively. Peak values are proportional to β-arrestin translocation to the plasma membrane. Cells were treated for 10 minutes with 100 nM NDP-MSH in Ca2+-free KRB buffer, and then stimulated with KRB buffer containing 1 mM CaCl2. Bar graph shows average [Ca2+] peak values. Bar graphs show mean +/- SD, n ≥ 3. Abbreviations: HEK, Human embryonic kidney; KRB, Krebs Ringer Buffer; MC4R, Melanocortin 4 Receptor; NDP-MSH, (Nle4,D-Phe7)-alpha-MSH; SD, standard deviation of the mean; wt, wild type.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/106/3/10.1210_clinem_dgaa885/3/m_dgaa885_fig8.jpeg?Expires=1747866277&Signature=uulysoQYpu-Pxj8qszy69RExet28GQ0sQIzqq6iYHH3qRKWkRuo-NVT-7O-ijDpgDpHKFU-KbE4BmR-7mvEARUFUKntO3BMhPbS9LeC9ecnccDm4kq3~jNmBIkOePul~Q7w7WKAaBZvnDeQzcsrdtJtZG6RSaqxS3oidJl4BLIRNsf6Gkl7gUjoKBscNWBmxdqlv3qv3w8Hhi9lUFDJVWx6gPitBmTg47BGJza5YYfGc3fa66zUG7JCSEvmk1z6E9coKXTiIVSMYcW~GjlGBv~nrXfHc0EXPdTJJYIwcguGl-YDepc18OGRtaxIrpSjl0TLGogGngZ2UV-KqkLiG2g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MC4R-dependent translocation of β-arrestin after NDP-MSH stimulation in HEK293 cells. A: Untransfected HEK293 cells were used as control. HEK293 cells were transfected with pMC4Rwt or pMC4Rmut plasmids and treated with NDP-MSH. Differential interference contrast (DIC) microscopy, immunofluorescence staining of nuclear-specific dye Hoechst (blue), anti-β-arrestin (green), and anti-MC4R (pink) are shown in HEK293 cells in the same viewing field. Scale bar: 10µm. B: MC4R-deficient HEK293 cells were transfected with a bioluminescent probe for β-arrestin translocation (β-arrestin-aequorin), together with a control pcDNA3.1 (HEK), pMC4Rwt (MC4R wt) or pMC4Rmut (MC4R mut) plasmids, the last 2 coding for the wild type and the mutated form of MC4R, respectively. Peak values are proportional to β-arrestin translocation to the plasma membrane. Cells were treated for 10 minutes with 100 nM NDP-MSH in Ca2+-free KRB buffer, and then stimulated with KRB buffer containing 1 mM CaCl2. Bar graph shows average [Ca2+] peak values. Bar graphs show mean +/- SD, n ≥ 3. Abbreviations: HEK, Human embryonic kidney; KRB, Krebs Ringer Buffer; MC4R, Melanocortin 4 Receptor; NDP-MSH, (Nle4,D-Phe7)-alpha-MSH; SD, standard deviation of the mean; wt, wild type.
Discussion
In this study, we report the identification and the functional characterization of a new mutation of MC4R gene, found in a young patient with obesity and inherited from his similarly obese mother. The mutation was identified during a mutation analysis conducted in a cohort of 209 patients with obesity, of whom none tested positive for mutations in the MC4R, LEP, or LEPR gene. The frequency of MC4R mutations found in our cohort may seem in contrast with other studies (32, 33); however, the prevalence of MC4R mutations is dependent on the characteristics of the tested cohort with obesity, ranging from 0.5% to 5.8% (34), and the prevalence of MC4R mutations is lower in the Mediterranean population compared with the other ethnic groups (35). In particular, the prevalence of MC4R mutations in a group of selected Italian obese children with early onset obesity was 1.6% (36), and in our cohort 47.1% of the patients had early onset obesity; if we recalculate the prevalence by only including patients with early onset obesity, a prevalence of 1.72% is reached, which is more comparable with the prevalence reported in the literature.
The previously unreported F313Sfs*29 mutation that we identified was localized in the C-terminal cytoplasmic region that has been shown to be crucial for correct downstream signal transduction. In particular, while the MC4R N-terminal extracytoplasmic domain is of critical importance for binding with ligands, the C-terminal region of the receptor contains a putative 8th helix (H8) that was shown to regulate its trafficking, function, and mechanistic link to human obesity (8). The mutation in position 313 causes a single amino acid substitution of a phenylalanine with a serine in the H8 domain of MC4R, inducing a frameshift that changes the sequence of the amino acid residues downstream. Our cAMP determinations showed that in the presence of NDP-MSH, a potent agonist for MC receptors typically 10 to 100 times more effective than α-MSH, the F313Sfs*29-mutated form of MC4R failed to successfully transduce the intracellular signal in terms of cAMP production, despite the correct expression of the receptor in the transfected HEK293 cells that we tested, suggesting that the mutant is coupled with neither Gαs nor Gαi cascades. On the contrary, the wild type MC4R receptor responded to NDP-MSH with a significant cAMP elevation, indicative of efficient Gαs coupling. Although this represents the best recognized signal transduction pathway associated with this receptor, activation of the MC4R has also been shown to mobilize intracellular Ca2+ through the Gαq/PLC/IP3 axis, at least in some cellular models (37–39). This opens the possibility that mutations in MC4R could favor the coupling of the receptor to alternative signaling cascades. Our data support the idea of pleiotropic G protein-coupled receptor (GPCR) signaling associated with MC4R (40), given that specific stimulation of wild type receptor effectively triggers an increase of intracellular [Ca2+]. However, this signal could either directly depend on Gαq/PLC/IP3 activation or be secondary to cAMP elevation, at least in principle. The slow kinetic of [Ca2+] rise elicited by NDP-MSH, as opposed to the rapid increase triggered by the well-known IP3-coupled stimulus ATP (see Fig. 7A), would argue for an indirect cAMP-mediated event, but the elucidation of the exact mechanism is beyond the scope of this manuscript. Most importantly, it is clear that the newly discovered F313Sfs*29 mutation also blunts MC4R-dependent Ca2+ mobilization, thus ruling out the possibility of a potential activation of alternative signaling events. Unfortunately, our experimental system prevented the analysis of signaling pathways activated by G protein–coupled receptor kinases, since we couldn’t detect any translocation of β-arrestin to the plasma membrane, as revealed by 2 different experimental approaches. Most likely, this is due to the fact that our cellular model lacks some element to fully complement exogenous expression of MC4R.
It has been observed that some frameshift mutations of MC4R associated with human obesity lead to intracellular retention of the receptor, suggesting that the obese phenotype may be more likely an effect of haplo-insufficiency rather than a dysfunction of the receptor (41). We can rule out this possibility, as we demonstrated a similar expression of the F313Sfs*29-mutated MC4R on the surface of cells, in comparison with wild type MC4R by using immunofluorescence staining with a specific antibody directed to the extracellular N-terminal portion (aa 21–33) and also by staining the same cells in the absence of a permeabilizing agent (data not shown), as already done in previous studies (42, 43).
Moreover, in silico modeling showed that this variant causes a conformational change in the cytoplasmic region of the receptor that deprives it of the binding pocket, likely leading to the inability to bind the GalphaS subunit and, consequently, to transduce the signal downstream. This result further reinforces the hypothesis that the F313Sfs*29 mutation induces a dysfunction of MC4R, rather than its intracellular retention or its dimerization. In fact, the F at position 313 is a conserved amino acid residue among most of the MCR family receptors and finds itself at the proximity of the H8 region that has been suggested to also have a role in the dimerization of the receptor (44). However, transmembrane helix (TMH) 3, TMH 4, and intracellular loop 2 (ICL2) are primarily involved in the interface between the receptor protomers (45). The F313Sfs*29 variant induces a substantial reorganization of the H8 domain but, on the contrary, all the modifications of other transmembrane helices of the predicted model can be explained by their rigid rearrangement, which does not affect their internal organization. Therefore, while the mutation strongly affects the GalphaS binding site of MC4R, it does not remodel the domains involved in the multimerization. Taking together our structural and functional analyses, we can reasonably conclude that the F313Sfs*29 variant encodes for a loss-of-function mutant characterized by disrupted coupling to downstream G proteins.
This deficit in the paraventricular nucleus neurons of the young patient and his mother may have led to an impaired regulation of satiety signals and feeding behavior, resulting in the development of obesity. Mutations in the MC4R gene have received particular attention because they could be considered a target for newly developed drugs such as Setmelanotide, a selective MC4R agonist (46). However, not all the mutations in MC4R affect the patients in the same manner, and the administration of MC4R agonists is able to rescue the signaling only in a subset of impaired mutants. This novel MC4R gene mutation is clearly associated with the early appearance of obesity, hyperphagia, and several endocrine and metabolic abnormalities. We cannot prove the causative role of this mutation as a determinant of hypogonadism and GH deficiency, and the hyperinsulinism could be seen as a consequence of the increased body weight rather than a direct effect of the mutation on glucose metabolism and insulin secretion. In contrast to what has been reported in other cases, a short stature (-2 DS) and GH deficiency were present. In fact, it has been previously described that MC4R variants could be associated with accelerated linear growth, leading to increased adult final height (47). Despite MC4R mutation seeming to be the most common form of monogenic obesity, different variants and different phenotypes were described. The availability of a specific therapy could offer us the possibility to better define the correlations between this new variant and the endocrine and metabolic alterations found in our proband. If a tailored treatment with a MC4R agonist could be able to restore a normal hypothalamic-pituitary function, this would allow us to demonstrate a cause-effect relationship.
Acknowledgments
Financial Support: This work was supported by the Italian Ministry of Education, University and Research (grant PRIN 2017L8Z2EM COFIN to R.V.).
Additional Information
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 repository listed in References.
References
