Inversed impaired osteogenic activity in children with severe obesity due to MC4R deficiency compared to LEP and LEPR deficiency

Inversed impaired osteogenic activity in children with severe obesity due to MC4R deficiency compared to LEP and LEPR deficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Inversed impaired osteogenic activity in children with severe obesity due to MC4R deficiency compared to LEP and LEPR deficiency Philippe Froguel, Qasim Janjua, Roohia Khanam, Sadia Saeed, Jaida Manzoor, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6595544/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in International Journal of Obesity → Version 1 posted 10 You are reading this latest preprint version Abstract OBJECTIVE Chronic obesity is associated with impaired bone health. However, few investigations have been conducted to assess bone physiology in early-onset obesity. In this study, we measured specific bone turnover and metabolic biomarkers in children with severe obesity with biallelic loss-of-function variants of the leptin ( LEP) , leptin receptor (LEPR) , or melanocortin 4 receptor ( MC4R) genes. METHODS Forty-one children aged 0.3–13 years with a BMI SDS ≥ 3, previously identified with pathogenic variants in LEP , LEPR , or MC4R , were recruited for the current study. Additionally, 13 age-matched children with severe obesity who tested negative for variants in known obesity-related genes were included, and another 15 unrelated age-matched children with normal body weight served as the control group. Serum osteocalcin, osteopontin, osteoprotegerin, and sclerostin levels were assessed using multi-analyte profiling. Serum leptin, insulin, and cortisol levels were determined using ELISA. RESULTS Serum levels of osteocalcin and osteopontin, specific markers of bone formation, were significantly lower in subjects with LEP and LEPR biallelic variants than in the control group. In contrast, the values of these two biomarkers in subjects with MC4R deficiency were significantly higher than those in the other groups. No differences were observed in the bone resorption markers osteoprotegerin and sclerostin. Hyperleptinemia was more pronounced in subjects with LEPR deficiency. Serum insulin concentrations were elevated in subjects with MC4R deficiency, whereas serum cortisol levels were significantly higher in subjects with LEP deficiency than in all other groups. CONCLUSION Our data demonstrate that osteogenic activity (but not resorption activity) is differentially affected in children with complete genetic disruption of the leptin signaling pathway. Children with MC4R deficiency showed higher osteogenic markers, but children with LEP and LEPR deficiencies showed the opposite. Our results support the usefulness of bone turnover biomarkers for the assessment and management of bone health in different types of obesity. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity Health sciences/Health care/Paediatrics Biological sciences/Physiology/Metabolism/Metabolic diseases/Obesity Monogenic obesity Bone metabolism biomarkers LEP LEPR MC4R INTRODUCTION The prevalence of childhood and adolescent obesity has escalated significantly in the wake of the obesity pandemic over the past three decades and is emerging as a major global public health concern. According to the World Health Organization, by 2022, 37 million children with obesity are under 5 years of age, and more than 390 million young children and adolescents (5-19 years) were classified as overweight [1]. Currently, the global increase in the prevalence of childhood obesity is anticipated to reach approximately 380 million by 2035 [2]. Childhood obesity substantially increases the risk of chronic diseases in young or middle-aged adults, such as type 2 diabetes, cardiovascular diseases, certain forms of cancer, sleep apnea, psychological distress, and other metabolic dysfunctions [3]. Notably, children with obesity are more susceptible to health-related complications than adults [4]. In this regard, we recently demonstrated that childhood obesity caused by recessive genetic defects may be associated with dramatic mortality and chronic and infectious disease, rates, largely due to lung and gut infections [5, 6]. Previous studies also suggested that obesity influences bone metabolism and modulation, potentially leading to bone abnormalities [7]. Historically, obesity has been thought to have an advantageous and positive role in sustaining bone health by averting bone loss and osteoporosis due to mechanical load [8, 9]; however, several recent studies have indicated that surplus fat mass is related to low bone mineral density and total mineral content [10, 11]. The dynamic process of bone remodeling, essential for maintaining bone strength, involves the coordinated activities of osteoblasts forming new bone and osteoclasts resorbing old bone [12]. This process is regulated by the interplay between neural, endocrine, and paracrine signals, thus emphasizing the complex relationship between body composition and skeletal integrity. Among other factors, the adipocyte-derived hormone leptin (encoded by LEP ) plays an important role in bone metabolism. The involvement of leptin signaling in bone metabolism is further strengthened by the presence of the leptin receptor (LEPR) [13] in ossifying cartilage [14], chondrocytes, and adult primary osteoblasts [15]. Previous studies have demonstrated that leptin influences the dynamics of bone remodeling thus affecting both bone formation and resorption [16-18]. Peripheral and central administration of leptin has been shown to increase osteogenesis, bone density, and mineralization in mice with Lep deficiency (ob/ob) [19-21]. The effects of leptin replacement on bone metabolism in patients with LEP deficiency have been sparse and mostly inconsistent and related to case reports or small series of patients from diverse ethnicities (or origins) [22-24]. Contrary to individuals with LEP deficiency, children with melanocortin 4 receptor (MC4R) deficiency have high bone mass linked to a decrease in bone resorption, attributed mainly to overexpression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus [25, 26]. Mc4r -/- mice also have increased hypothalamic CART and exhibit high bone mass due to the inhibition of osteoclast proliferation and function [25]. The bone phenotype of mice with Mc4r deficiency has been shown to be corrected by decreasing CART expression [26], demonstrating that increased bone mass due to Mc4r deficiency is mainly mediated by increased CART signaling. Most studies on bone metabolism in the context of obesity have utilized dual-energy X-ray absorptiometry to assess bone mass and mineral density but lack insights into the physiological processes of bone resorption and formation [27-29]. Only a few studies have been conducted on bone health in children with early onset severe obesity owing to monogenic or undetermined plausible genetic causality [30, 31]. However, the available data are conflicting and based on case reports or a small sample size [22, 32, 33]. Furthermore, a limited number of studies have addressed changes in bone metabolism related to increased fat mass using bone turnover markers in children with severe forms of obesity [34, 35]. Bone markers such as osteocalcin (OC), bone-specific alkaline phosphatase, and osteopontin (OPN) [36-38] have been used to assess bone formation, whereas sclerostin (SOST) and osteoprotegerin (OPG) are considered biomarkers for bone resorption [39, 40]. Furthermore, there is a paucity of information regarding the dynamics of bone turnover in children with monogenic forms of obesity [4, 41]. Errors in bone metabolism in children at an early age can have long-term consequences for bone health, potentially increasing the risk of fractures and osteoporosis later in life [7]. Here, we assessed bone health using bone turnover markers in an ethnically homogenous cohort of children from consanguineous families from Pakistan carrying pathogenic biallelic variants in LEP , LEPR , or MC4R, drawn from one of the world’s largest cohorts of children with severe obesity [5]. Additionally, we have included age-matched children with severe obesity who were negative for variants in these three genes or other known genetic variants associated with obesity and a group of children with normal body weight as controls. All study participants shared a common ethnicity and similar socio-economic and environmental conditions. MATERIALS AND METHODS Participants This study included 54 children from the Severe Obesity in Pakistani Population (SOPP) cohort belonging to consanguineous families with a BMI standard deviation score (SDS) ≥ 3 and a history of hyperphagia. Among these, 41 children were previously identified to have homozygous loss-of-function variants in the LEP , LEPR , or MC4R genes (LEP: NM_000230.3; LEPR: NM_002303.6; MC4R: NM_005912.3) [5]. In addition, 13 age-matched children with severe obesity who tested negative for variants in these three genes or other known monogenic causes of severe obesity based on whole-exome sequencing were included. Fifteen age-matched children with normal body weight from the same population served as the control group. The study protocol was approved by the institutional ethics committee. Patients and/or their parents were interviewed regarding their family and medical history. Written informed consent to participate in this study was obtained from all patients/parents. Physical examination and anthropomorphic measurements were performed. A 4-5 ml blood sample was obtained between 1000 and 1200 h, in each case, for serum extraction and subsequent biochemical estimations. The study was performed in strict accordance with the principles of the Declaration of Helsinki. Biochemical determinations Simultaneous quantification of the bone turnover markers OC, OPN, OPG, and SOST was carried out in serum samples by multi-analyte profiling using EMD Millipore’s MILLIPLEX MAP Human Bone Magnetic Bead Kit (Cat. # HBNMAG-51K) (Billerica, MA, USA). Luminex analyzer MAGPIX (Austin, Texas, USA), was used for reading the plate. Serum levels of leptin, insulin, and cortisol were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (leptin, Labor Diagnostika Nord GmbH, Nordhorn, Germany; insulin and cortisol, Monobind Inc., Lake Forest, CA, USA) and an automated EIA analyzer (Bio-Rad Laboratories, Hercules, CA, USA). All assays were performed in duplicate according to the manufacturer’s instructions. The inter- and intra-assay variations were < 11% in all cases. Statistical analysis The significance of the differences between the groups was analyzed using Scheffe's multiple comparison test. Statistical significance was set at P < 0.05. All calculations were performed using Statistical Package for the Social Sciences version 20 (SPSS, Inc., Chicago, IL, USA). RESULTS Physical characteristics The physical characteristics of children with monogenic obesity caused by LEP , LEPR , or MC4R biallelic variants, age-matched individuals with severe obesity with unknown genetic etiology, and those with normal body weight are summarized in Table 1. Although all affected individuals were characterized by early onset severe obesity, the majority of children with LEP and LEPR deficiencies presented with hyperphagia at a much earlier stage than children with MC4R deficiency [5] (Table 1). Notably, there was an increase in linear growth in children with MC4R deficiency but not with LEP or LEPR deficiency, compared to age-matched (4.3 to 12 years) children with normal body weight (124.9±4.4 kg vs 113.0±3.1 kg). Bone turnover biomarkers Mean serum OC concentrations in individuals with MC4R deficiency were >3-fold higher compared to children with LEP or LEPR deficiency and were also significantly raised over the individuals with severe obesity negative for these variants and children with normal body weight (185.2±25.3 vs 70.4±7.2 and 107.7±10.8 ng/ml, respectively). Remarkably, the levels of this biomarker of bone formation were significantly lower in children with LEP and LEPR variants than in children with normal body weight (Table 2). The mean serum OC levels in children with severe obesity with unknown genetic etiology were intermediate to the control values and in those with LEP or LEPR deficiency. The mean serum levels of OPN were comparable in individuals with MC4R deficiency and children with normal body weight and were significantly higher than those in the LEP and LEPR mutant groups (Table 2). The mean serum OPN levels observed in children with severe obesity with unknown genetic etiology were comparable to those in children with LEP or LEPR deficiency. No statistically significant difference was evident in the levels of the bone resorption biomarkers SOST and OPG in children with severe obesity compared to children with normal body weight. Endocrine profile As expected, serum leptin levels were undetectable in children with LEP variants. Hyperleptinemia was evident in the remaining three groups of children with obesity and was most severe in children with LEPR deficiency (Table 2). The mean leptin levels in individuals with MC4R deficiency were comparable to those of children with severe obesity with unknown genetic etiology. Serum insulin levels were significantly elevated in children with MC4R deficiency compared to individuals with pathogenic biallelic variants in LEP or LEPR genes, and children with severe obesity with unknown genetic etiology. The mean insulin levels in children with LEP and LEPR variants tended to be higher than the control values; however, this difference was not statistically significant (Table 2). Similarly, mean serum insulin concentrations in age-matched individuals with severe obesity of unknown genetic etiology though discernably higher than the LEP - and LEPR -deficient, were lower compared to those of children with MC4R deficiency (32.0±8.7 vs 48.5±10.2 ng/ml) but the difference was not statistically significant. Serum cortisol concentrations were significantly higher in children with LEP deficiency than in the other groups. No significant differences in serum cortisol levels were discernible in the other three groups of children with obesity and were comparable to those of the children with normal body weight (Table 2). DISCUSSION Our findings demonstrate that in children with biallelic MC4R variants, there is a 2-3-fold increase in the bone formation biomarkers, OC, and OPN, compared to an osteogenic deficit observed in the other children with obesity. This contrasts with earlier studies that showed no significant changes in serum OC levels in children with only heterozygous MC4R variants [26]. This suggests that partial maintenance of MC4R integrity is sufficient to maintain normal osteogenesis. However, our results corroborate previous findings that used dual-energy X-ray absorptiometry scans to demonstrate increased bone mass in MC4R deficiency [31, 33]. Mice with Mc4r deficiency have also been shown to exhibit increased bone formation [26]. Additionally, increased body height has been reported in adolescents and adults with MC4R deficiency [5, 42]. Although the precise molecular events involved in increased bone formation as a result of MC4R dysfunction are yet to be elucidated, the main contributing factor to this increased bone mass is ascribed to CART overexpression. In turn, CART inhibits the osteoclastogenic factor receptor activator of nuclear factor kappa B ligand (RANKL), thereby decreasing bone resorption [43, 44]. In contrast, no conclusive data are available on bone resorption markers in early-onset severe obesity. A decrease in the levels of the bone resorption markers carboxy-terminal telopeptides of type-I collagen [26] and deoxypyridinoline [25] in individuals with pathogenic variants in MC4R has previously been reported. In our study, the levels of serum SOST, a biomarker for bone resorption [45], tended to be lower in the MC4R group than in the other groups. However, this trend needs to be confirmed in a larger cohort. This suggests that MC4R complete invalidation has a stronger effect on osteogenesis than on bone resorption. In contrast, in children with leptin signaling deficiency due to loss-of-function biallelic variants in LEP or LEPR, reduced osteogenic activity was evidenced by significantly decreased serum levels of OC and OPN compared to the reference values in age-matched children with normal body weight. Importantly, in our group of children with severe consanguineous obesity of unknown genetic etiology, the profile of bone metabolic biomarkers was comparable to that of children with leptin signaling deficiency. A deficit in bone formation in children with elevated leptin levels could partly be due to ‘leptin resistance’ or ‘cellular leptin resistance’ [46]. The possible mechanisms underlying leptin resistance associated with hyperleptinemia in rodents and humans have been extensively reviewed [46]. In individuals with general or monogenic forms of obesity, hyperleptinemia resulting from excessive fat mass can lead to hypothalamic and cellular leptin resistance, thereby disrupting the negative feedback of leptin on hypothalamic neurons (e.g., POMC, AgRP). MC4R deficiency has, therefore, been termed as the ‘purest’ form of leptin resistance [47]. Leptin also increases the expression of CART, which suppresses RANKL, thereby inhibiting bone resorption and favoring bone formation [25]. These factors disrupt the delicate balance of bone remodeling by promoting osteoclastogenesis and inhibiting osteoblast activity, ultimately favoring bone resorption over bone formation. Such processes also contribute to a paradoxical state in which, despite the higher bone mineral density often observed in individuals with obesity, their bones exhibit reduced quality and structural integrity, resulting in an increased risk of fractures [48-50]. The reduced osteogenic activity observed in cases of severe obesity could also be partly due to the absence of leptin's regulatory effects on other hormones and factors, such as cytokines, which are crucial for normal bone growth and maintenance [17]. Our analysis did not reveal significant changes in the bone resorption biomarkers SOST and OPG in children with LEP or LEPR deficiency compared to those of children with normal weight (Table 2). According to a recent consensus, leptin has a stabilizing effect on bone metabolism and influences the simultaneous processes of bone formation and resorption. By binding to its receptors in the ventromedial nucleus of the hypothalamus, leptin increases adrenergic tone, which inhibits CART and activates RANKL, thereby promoting osteoclastogenesis. As leptin receptors have been identified in osteoblasts, leptin may also affect peripheral bone formation by the direct activation of these cells [25]. As anticipated, in our study participants, serum leptin levels were undetectable (<0.5 ng/ml) in LEP mutants and markedly elevated in children with LEPR and MC4R deficiencies. In addition, hyperleptinemia has previously been reported in patients with LEPR and MC4R deficiencies [51] and has been attributed to a lack of responsiveness to leptin signaling [52]. The mean serum insulin values were remarkably higher (P<0.05) in children with MC4R deficiency than those in the other groups. Consistent with our observations, elevated insulin levels, and hyperleptinemia have been reported in children with MC4R deficiency [31, 53]. We also observed significantly increased cortisol levels in children with LEP deficiency compared to the other three groups of patients with obesity, possibly due to a higher chronic stress level in children with LEP deficiency. These findings are consistent with those of a previous study reporting hypercortisolemia in children with LEP deficiency [51]. CONCLUSION Our results, based on the levels of bone metabolism biomarkers, indicated a significant increase in osteogenic activity in children with total MC4R deficiency. Conversely, pronounced remission of bone formation was observed in children with leptin-signaling deficiency, supporting the osteogenic role of leptin. A similar tendency of bone remission was noted in children with severe obesity negative for known genetic causes, suggesting impaired leptin signaling/resistance. Our data support the view that severe obesity, particularly in children, does not necessarily lead to abnormal bone metabolism but is related to the etiology of the affliction. Based on these data, we believe that it is desirable to use specific bone turnover markers in addition to imaging techniques for a comprehensive evaluation of bone health in children with the onset of severe obesity at an early age and to make informed decisions about possible options for its management. Recombinant leptin has been used to treat children with leptin deficiency for more than two decades and has been reported to have a beneficial effect on bone health [54]. More recently, setmelanotide, an MC4R agonist that bypasses leptin resistance, has been approved for the treatment of monogenic obesity due to LEPR -, POMC -, or PCSK1 -deficient states [55-57]. However, the effect of this agonist on bone health in these forms of monogenic obesity remains to be elucidated on a long-term basis. The advent of new drugs based on GLP and GIP receptor agonists [58, 59] for the treatment of common obesity necessitates a comprehensive evaluation of bone health and metabolism. In initial studies, GLP-1 receptor agonists and their analogs have recently been investigated in preclinical trials to improve bone fragility disorders [59]. Declarations ACKNOWLEDGMENTS We thank the patients and their families for their participation in this study. We thank Frédéric Allegaert, Timothée Beke, and Stefan Gaget for their technical assistance. This work was supported by funding from the Medical Research Council (MRC) MR/S026193/1 (PF) and the Pakistan Academy of Sciences (MA). Further support was provided by the National Center for Precision Diabetic Medicine, PreciDIAB, which is jointly supported by the French National Agency for Research (ANR-18-IBHU-0001), European Union (FEDER), Hauts-de-France Regional Council, and European Metropolis of Lille (MEL). AUTHOR CONTRIBUTIONS MA and PF conceptualized the study. QMJ, RK, and MA wrote the first draft of the manuscript. QMJ, RK, SS, and MA contributed to the amendment of the first draft. RK, QMJ, AH, and MA collected the samples and performed the biochemical analysis. SS, AB, and PF analyzed the genetic data. QMJ, RK, and SH performed the statistical analysis. JM, RK, QMJ, and MA identified and recruited families with obesity. 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Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med. 1999; 341:879–84. Qamar, S, Mallik, R, Makaronidis, J. Setmelanotide: a melanocortin-4 receptor agonist for the treatment of severe obesity due to hypothalamic dysfunction. US Endocrinol. 2024; 20:1–10. Argente, J, Verge, CF, Okorie, U, Fennoy, I, Kelsey, MM, Cokkinias, C, et al. Setmelanotide in patients aged 2–5 years with rare MC4R pathway-associated obesity (venture): a 1 year, open-label, multicenter, phase 3 trial. Lancet Diabetes Endocrinol. 2025; 13:29–37. Collet, TH, Dubern, B, Mokrosinski, J, Connors, H, Keogh, JM, Mendes de Oliveira, E, et al. Evaluation of a melanocortin-4 receptor (MC4R) agonist (setmelanotide) in MC4R deficiency. Mol Metab. 2017; 6:1321–9. Wu, Z, Deng, W, Ye, Y, Xu, J, Han, D, Zheng, Y, et al. Liraglutide, a glucagon-like peptide-1 receptor agonist, inhibits bone loss in an animal model of osteoporosis with or without diabetes. Front Endocrinol (Lausanne). 2024; 15:1378291. Bouvard, B, Mabilleau, G. Gut hormones and bone homeostasis: potential therapeutic implications. Nat Rev Endocrinol. 2024; 20:553–64. Tables Tables are available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose Supplementary Files Table1.xlsx Table 1 Table2.xlsx Table 2 Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2026 Read the published version in International Journal of Obesity → Version 1 posted Editorial decision: revise 20 Jun, 2025 Review # 2 received at journal 12 Jun, 2025 Review # 1 received at journal 11 Jun, 2025 Reviewer # 2 agreed at journal 30 May, 2025 Reviewer # 1 agreed at journal 27 May, 2025 Reviewers invited by journal 15 May, 2025 Submission checks completed at journal 14 May, 2025 First submitted to journal 13 May, 2025 Unknown event 06 May, 2025 Editor assigned by journal 05 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Oman","correspondingAuthor":false,"prefix":"","firstName":"Qasim","middleName":"","lastName":"Janjua","suffix":""},{"id":457307964,"identity":"31cc42cb-6758-46f2-8c65-e15e64cb4b04","order_by":2,"name":"Roohia Khanam","email":"","orcid":"https://orcid.org/0000-0001-6826-7471","institution":"Forman Christian College (A Chartered University)","correspondingAuthor":false,"prefix":"","firstName":"Roohia","middleName":"","lastName":"Khanam","suffix":""},{"id":457307965,"identity":"c9aabf2e-7a98-48f2-aa87-90c660af7299","order_by":3,"name":"Sadia Saeed","email":"","orcid":"https://orcid.org/0000-0003-3144-7772","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Sadia","middleName":"","lastName":"Saeed","suffix":""},{"id":457307966,"identity":"10b9bd26-a26d-49c8-9b23-ecc1e6aa1e18","order_by":4,"name":"Jaida 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07:09:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":496664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6595544/v1/035c5282-6b2e-48b9-9d9e-dc6ee8197f34.pdf"},{"id":83106124,"identity":"08992477-81b9-40d8-a8b5-24cb47601cf0","added_by":"auto","created_at":"2025-05-20 06:17:01","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":12028,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6595544/v1/84a8b7c268495cd6daec2a1b.xlsx"},{"id":83106764,"identity":"3dac854c-da58-4a98-bdf5-03a2c74236db","added_by":"auto","created_at":"2025-05-20 06:25:01","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11920,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6595544/v1/4c8cd44d978b78097b1c781b.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"\u003cp\u003eInversed impaired osteogenic activity in children with severe obesity due to \u003cem\u003eMC4R\u003c/em\u003e deficiency compared to \u003cem\u003eLEP \u003c/em\u003eand \u003cem\u003eLEPR\u003c/em\u003e deficiency\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe prevalence of childhood and adolescent obesity has escalated significantly in the wake of the obesity pandemic over the past three decades and is emerging as a major global public health concern. According to the World Health Organization, by 2022, 37 million children with obesity are under 5 years of age, and more than 390 million young children and adolescents (5-19 years) were classified as overweight\u0026nbsp;[1]. Currently, the global increase in the prevalence of childhood obesity is anticipated to reach approximately 380 million by 2035 [2]. Childhood obesity substantially increases the risk of chronic diseases in young or middle-aged adults, such as type 2 diabetes, cardiovascular diseases, certain forms of cancer, sleep apnea, psychological distress, and other metabolic dysfunctions [3]. Notably, children with obesity are more susceptible to health-related complications than adults [4]. In this regard, we recently demonstrated that childhood obesity caused by recessive genetic defects may be associated with dramatic mortality and chronic and infectious disease, rates, largely due to lung and gut infections [5, 6].\u003c/p\u003e\n\u003cp\u003ePrevious studies also suggested that obesity influences bone metabolism and modulation, potentially leading to bone abnormalities [7]. Historically,\u0026nbsp;obesity has been thought to have an advantageous and positive role in sustaining bone health by averting bone loss and osteoporosis due to mechanical load [8, 9]; however, several recent studies have indicated that surplus fat mass is related to low bone mineral density and total mineral content [10, 11]. The dynamic process of bone remodeling, essential for maintaining bone strength, involves the coordinated activities of osteoblasts forming new bone and osteoclasts resorbing old bone [12]. This process is regulated by the interplay between neural, endocrine, and paracrine signals, thus emphasizing the complex relationship between body composition and skeletal integrity.\u003c/p\u003e\n\u003cp\u003eAmong other factors, the adipocyte-derived hormone leptin (encoded by \u003cem\u003eLEP\u003c/em\u003e) plays an important role in bone metabolism. The involvement of leptin signaling in bone metabolism is further strengthened by the presence of the leptin receptor (LEPR) [13] in ossifying cartilage [14], chondrocytes, and adult primary osteoblasts [15]. Previous studies have demonstrated that leptin influences the dynamics of bone remodeling thus affecting both bone formation and resorption [16-18]. Peripheral and central administration of leptin has been shown to increase osteogenesis, bone density, and mineralization in mice with \u003cem\u003eLep\u003c/em\u003e deficiency (ob/ob) [19-21]. The effects of leptin replacement on bone metabolism in patients with \u003cem\u003eLEP\u003c/em\u003e deficiency have been sparse and mostly inconsistent and related to case reports or small series of patients from diverse ethnicities (or origins) [22-24].\u003c/p\u003e\n\u003cp\u003eContrary to individuals with \u003cem\u003eLEP\u003c/em\u003e deficiency, children with melanocortin 4 receptor (MC4R) deficiency have high bone mass linked to a decrease in bone resorption,\u0026nbsp;attributed mainly to overexpression of\u0026nbsp;the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus [25, 26]. \u003cem\u003eMc4r\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice also have increased hypothalamic CART and exhibit high bone mass due to the inhibition of osteoclast proliferation and function [25]. The bone phenotype of mice with \u003cem\u003eMc4r\u003c/em\u003e deficiency has been shown to be corrected by decreasing CART expression [26], demonstrating that increased bone mass due to \u003cem\u003eMc4r\u003c/em\u003e deficiency is mainly mediated by increased CART signaling.\u003c/p\u003e\n\u003cp\u003eMost studies on bone metabolism in the context of obesity have utilized dual-energy X-ray absorptiometry to assess bone mass and mineral density but lack insights into the physiological processes of bone resorption and formation [27-29]. Only a few studies have been conducted on bone health in children with early onset severe obesity owing to monogenic or undetermined plausible genetic causality [30, 31]. However, the available data are conflicting and based on case reports or a small sample size [22, 32, 33]. Furthermore, a limited number of studies have addressed changes in bone metabolism related to increased fat mass using bone turnover markers in children with severe forms of obesity [34, 35]. Bone markers such as osteocalcin (OC), bone-specific alkaline phosphatase, and osteopontin (OPN) [36-38] have been used to assess bone formation, whereas sclerostin (SOST) and osteoprotegerin (OPG) are considered biomarkers for bone resorption [39, 40].\u003c/p\u003e\n\u003cp\u003eFurthermore, there is a paucity of information regarding the dynamics of bone turnover in children with monogenic forms of obesity [4, 41]. Errors in bone metabolism in children at an early age can have long-term consequences for bone health, potentially increasing the risk of fractures and osteoporosis later in life [7]. Here, we assessed bone health using bone turnover markers in an ethnically homogenous cohort of children from consanguineous families from Pakistan carrying pathogenic biallelic variants in \u003cem\u003eLEP\u003c/em\u003e, \u003cem\u003eLEPR\u003c/em\u003e, or \u003cem\u003eMC4R,\u003c/em\u003e drawn from one of the world\u0026rsquo;s largest cohorts of children with severe obesity [5]. Additionally, we have included age-matched children with severe obesity who were negative for variants in these three genes or other known genetic variants associated with obesity and a group of children with normal body weight as controls. All study participants shared a common ethnicity and similar socio-economic and environmental conditions.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eParticipants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included 54 children from the Severe Obesity in Pakistani Population (SOPP) cohort belonging to consanguineous families with a BMI standard deviation score (SDS) \u0026ge; 3 and a history of hyperphagia. Among these, 41 children were previously identified to have homozygous loss-of-function variants in the\u003cem\u003e\u0026nbsp;LEP\u003c/em\u003e, \u003cem\u003eLEPR\u003c/em\u003e, or \u003cem\u003eMC4R\u003c/em\u003e genes (LEP: NM_000230.3; LEPR: NM_002303.6; MC4R: NM_005912.3) [5]. In addition, 13 age-matched children with severe obesity who tested negative for variants in these three genes or other known monogenic causes of severe obesity based on whole-exome sequencing were included. Fifteen age-matched children with normal body weight from the same population served as the control group. The study protocol was approved by the institutional ethics committee. Patients and/or their parents were interviewed regarding their family and medical history. Written informed consent to participate in this study was obtained from all patients/parents. Physical examination and anthropomorphic measurements were performed. A 4-5 ml blood sample was obtained between 1000 and 1200 h, in each case, for serum extraction and subsequent biochemical estimations. The study was performed in strict accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical determinations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimultaneous quantification of the bone turnover markers OC, OPN, OPG, and SOST was carried out in serum samples by multi-analyte profiling using EMD Millipore\u0026rsquo;s MILLIPLEX MAP Human Bone Magnetic Bead Kit (Cat. # HBNMAG-51K) (Billerica, MA, USA). Luminex analyzer MAGPIX (Austin, Texas, USA), was used for reading the plate. Serum levels of leptin, insulin, and cortisol were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (leptin, Labor Diagnostika Nord GmbH, Nordhorn, Germany; insulin and cortisol, Monobind Inc., Lake Forest, CA, USA) and an automated EIA analyzer (Bio-Rad Laboratories, Hercules, CA, USA). All assays were performed in duplicate according to the manufacturer\u0026rsquo;s instructions. The inter- and intra-assay variations were \u0026lt; 11% in all cases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe significance of the differences between the groups was analyzed using Scheffe\u0026apos;s multiple comparison test. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. All calculations were performed using Statistical Package for the Social Sciences version 20 (SPSS, Inc., Chicago, IL, USA).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePhysical characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physical characteristics of children with monogenic obesity caused by \u003cem\u003eLEP\u003c/em\u003e, \u003cem\u003eLEPR\u003c/em\u003e, or \u003cem\u003eMC4R\u003c/em\u003e biallelic variants, age-matched individuals with severe obesity with unknown genetic etiology, and those with normal body weight are summarized in Table 1. Although all affected individuals were characterized by early onset severe obesity, the majority of children with \u003cem\u003eLEP\u003c/em\u003e and \u003cem\u003eLEPR\u0026nbsp;\u003c/em\u003edeficiencies presented with hyperphagia at a much earlier stage than children with \u003cem\u003eMC4R\u003c/em\u003e deficiency [5] (Table 1). Notably, there was an increase in linear growth in children with \u003cem\u003eMC4R\u003c/em\u003e deficiency but not with \u003cem\u003eLEP\u003c/em\u003e or \u003cem\u003eLEPR\u003c/em\u003e deficiency, compared to age-matched (4.3 to 12 years) children with normal body weight (124.9\u0026plusmn;4.4 kg vs 113.0\u0026plusmn;3.1 kg).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone turnover biomarkers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean serum OC concentrations in individuals with \u003cem\u003eMC4R\u003c/em\u003e deficiency were \u0026gt;3-fold higher compared to children with \u003cem\u003eLEP\u0026nbsp;\u003c/em\u003eor \u003cem\u003eLEPR\u003c/em\u003e deficiency and were also significantly raised over the individuals with severe obesity negative for these variants and children with normal body weight (185.2\u0026plusmn;25.3 vs 70.4\u0026plusmn;7.2 and 107.7\u0026plusmn;10.8 ng/ml, respectively). Remarkably, the levels of this biomarker of bone formation were significantly lower in children with \u003cem\u003eLEP\u003c/em\u003e and \u003cem\u003eLEPR\u0026nbsp;\u003c/em\u003evariants than in children with normal body weight (Table 2). The mean serum OC levels in children with severe obesity with unknown genetic etiology were intermediate to the control values and in those with \u003cem\u003eLEP\u003c/em\u003e or \u003cem\u003eLEPR\u003c/em\u003e deficiency.\u003c/p\u003e\n\u003cp\u003eThe mean serum levels of OPN were comparable in individuals with \u003cem\u003eMC4R\u003c/em\u003e deficiency and children with normal body weight and were significantly higher than those in\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe\u003cem\u003e\u0026nbsp;LEP\u003c/em\u003e and \u003cem\u003eLEPR\u003c/em\u003e mutant groups (Table 2). The mean serum OPN levels observed in children with severe obesity with unknown genetic etiology were comparable to those in children with \u003cem\u003eLEP\u003c/em\u003e or \u003cem\u003eLEPR\u0026nbsp;\u003c/em\u003edeficiency. No statistically significant difference was evident in the levels of the bone resorption biomarkers SOST and OPG in children with severe obesity compared to children with normal body weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndocrine profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs expected, serum leptin levels were undetectable in children with \u003cem\u003eLEP\u003c/em\u003e variants. Hyperleptinemia was evident in the remaining three groups of children with obesity and was most severe in children with \u003cem\u003eLEPR\u003c/em\u003e deficiency (Table 2). The mean leptin levels in individuals with \u003cem\u003eMC4R\u003c/em\u003e deficiency were comparable to those of children with severe obesity with unknown genetic etiology.\u003c/p\u003e\n\u003cp\u003eSerum insulin levels were significantly elevated in children with \u003cem\u003eMC4R\u003c/em\u003e deficiency compared to individuals with pathogenic biallelic variants in \u003cem\u003eLEP\u0026nbsp;\u003c/em\u003eor \u003cem\u003eLEPR\u003c/em\u003e genes, and children with severe obesity with unknown genetic etiology. The mean insulin levels in children with \u003cem\u003eLEP\u003c/em\u003e and \u003cem\u003eLEPR\u003c/em\u003e variants tended to be higher than the control values; however, this difference was not statistically significant (Table 2).\u003c/p\u003e\n\u003cp\u003eSimilarly, mean serum insulin concentrations in age-matched individuals with severe obesity of unknown genetic etiology though discernably higher than the \u003cem\u003eLEP\u003c/em\u003e- and \u003cem\u003eLEPR\u003c/em\u003e-deficient, were lower compared to those of children with \u003cem\u003eMC4R\u003c/em\u003e deficiency (32.0\u0026plusmn;8.7 \u003cem\u003evs\u0026nbsp;\u003c/em\u003e48.5\u0026plusmn;10.2 ng/ml) but the difference was not statistically significant. Serum cortisol concentrations were significantly higher in children with \u003cem\u003eLEP\u003c/em\u003e deficiency than in the other groups. No significant differences in serum cortisol levels were discernible in the other three groups of children with obesity and were comparable to those of the children with normal body weight (Table 2).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur findings demonstrate that in children with biallelic \u003cem\u003eMC4R\u003c/em\u003e variants, there is a 2-3-fold increase in the bone formation biomarkers, OC, and OPN, compared to an osteogenic deficit observed in the other children with obesity. This contrasts with earlier studies that showed no significant changes in serum OC levels in children with only heterozygous \u003cem\u003eMC4R\u0026nbsp;\u003c/em\u003evariants [26]. This suggests that partial maintenance of MC4R integrity is sufficient to maintain normal osteogenesis. However, our results corroborate previous findings that used dual-energy X-ray absorptiometry scans to demonstrate increased bone mass in \u003cem\u003eMC4R\u003c/em\u003e deficiency [31, 33]. Mice with \u003cem\u003eMc4r\u003c/em\u003e deficiency have also been shown to exhibit increased bone formation [26]. Additionally, increased body height has been reported in adolescents and adults with \u003cem\u003eMC4R\u003c/em\u003e deficiency [5, 42]. Although the precise molecular events involved in increased bone formation as a result of MC4R dysfunction are yet to be elucidated, the main contributing factor to this increased bone mass is ascribed to CART overexpression. In turn, CART inhibits the osteoclastogenic factor receptor activator of nuclear factor kappa B ligand (RANKL), thereby decreasing bone resorption [43, 44].\u003c/p\u003e\n\u003cp\u003eIn contrast, no conclusive data are available on bone resorption markers in early-onset severe obesity. A decrease in the levels of the bone resorption markers carboxy-terminal telopeptides of type-I collagen [26] and deoxypyridinoline [25] in individuals with pathogenic variants in \u003cem\u003eMC4R\u0026nbsp;\u003c/em\u003ehas previously been reported.\u0026nbsp;In our study, the levels of serum SOST, a biomarker for bone resorption [45], tended to be lower in the \u003cem\u003eMC4R\u003c/em\u003e group than in the other groups. However, this trend needs to be confirmed in a larger cohort. This suggests that \u003cem\u003eMC4R\u003c/em\u003e complete invalidation has a stronger effect on osteogenesis than on bone resorption. In contrast, in children with leptin signaling deficiency due to loss-of-function biallelic variants in \u003cem\u003eLEP\u0026nbsp;\u003c/em\u003eor \u003cem\u003eLEPR,\u0026nbsp;\u003c/em\u003ereduced osteogenic activity was evidenced by significantly decreased serum levels of OC and OPN compared to the reference values in age-matched children with normal body weight. Importantly, in our group of children with severe consanguineous obesity of unknown genetic etiology, the profile of bone metabolic biomarkers was comparable to that of children with leptin signaling deficiency. A deficit in bone formation in children with elevated leptin levels could partly be due to \u0026lsquo;leptin resistance\u0026rsquo; or \u0026lsquo;cellular leptin resistance\u0026rsquo; [46]. The possible mechanisms underlying leptin resistance associated with hyperleptinemia in rodents and humans have been extensively reviewed [46].\u003c/p\u003e\n\u003cp\u003eIn individuals with general or monogenic forms of obesity, hyperleptinemia resulting from excessive fat mass can lead to hypothalamic and cellular leptin resistance, thereby disrupting the negative feedback of leptin on hypothalamic neurons (e.g., POMC, AgRP). \u0026nbsp;\u003cem\u003eMC4R\u003c/em\u003e deficiency has, therefore, been termed as the \u0026lsquo;purest\u0026rsquo; form of leptin resistance [47]. Leptin also increases the expression of CART, which suppresses RANKL, thereby inhibiting bone resorption and favoring bone formation [25]. These factors disrupt the delicate balance of bone remodeling by promoting osteoclastogenesis and inhibiting osteoblast activity, ultimately favoring bone resorption over bone formation. Such processes also contribute to a paradoxical state in which, despite the higher bone mineral density often observed in individuals with obesity, their bones exhibit reduced quality and structural integrity, resulting in an increased risk of fractures [48-50]. The reduced osteogenic activity observed in cases of severe obesity could also be partly due to the absence of leptin\u0026apos;s regulatory effects on other hormones and factors, such as cytokines, which are crucial for normal bone growth and maintenance [17]. Our analysis did not reveal significant changes in the bone resorption biomarkers SOST and OPG in children with \u003cem\u003eLEP\u003c/em\u003e or \u003cem\u003eLEPR\u003c/em\u003e deficiency compared to those of children with normal weight (Table 2). According to a recent consensus, leptin has a stabilizing effect on bone metabolism and influences the simultaneous processes of bone formation and resorption. By binding to its receptors in the ventromedial nucleus of the hypothalamus, leptin increases adrenergic tone, which inhibits CART and activates RANKL, thereby promoting osteoclastogenesis. As leptin receptors have been identified in osteoblasts, leptin may also affect peripheral bone formation by the direct activation of these cells [25].\u003c/p\u003e\n\u003cp\u003eAs anticipated, in our study participants, serum leptin levels were undetectable (\u0026lt;0.5 ng/ml) in \u003cem\u003eLEP\u0026nbsp;\u003c/em\u003emutants and markedly elevated in children with\u003cem\u003e\u0026nbsp;LEPR\u003c/em\u003e and \u003cem\u003eMC4R\u003c/em\u003e deficiencies. In addition, hyperleptinemia has previously been reported in patients with \u003cem\u003eLEPR\u003c/em\u003e and \u003cem\u003eMC4R\u003c/em\u003e deficiencies [51] and has been attributed to a lack of responsiveness to leptin signaling [52]. The mean serum insulin values were remarkably higher (P\u0026lt;0.05) in children with \u003cem\u003eMC4R\u003c/em\u003e deficiency than those in the other groups. Consistent with our observations, elevated insulin levels, and hyperleptinemia have been reported in children with \u003cem\u003eMC4R\u003c/em\u003e deficiency [31, 53]. We also observed significantly increased cortisol levels in children with \u003cem\u003eLEP\u003c/em\u003e deficiency compared to the other three groups of patients with obesity, possibly due to a higher chronic stress level in children with \u003cem\u003eLEP\u003c/em\u003e deficiency. These findings are consistent with those of a previous study reporting hypercortisolemia in children with \u003cem\u003eLEP\u003c/em\u003e deficiency [51].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur results, based on the levels of bone metabolism biomarkers, indicated a significant increase in osteogenic activity in children with total \u003cem\u003eMC4R\u003c/em\u003e deficiency. Conversely, pronounced\u0026nbsp;remission\u0026nbsp;of bone formation was observed in children with leptin-signaling deficiency, supporting the osteogenic role of leptin. A similar tendency of bone remission was noted in children with severe obesity negative for known genetic causes, suggesting impaired leptin signaling/resistance. Our data support the view that severe obesity, particularly in children, does not necessarily lead to abnormal bone metabolism but is related to the etiology of the affliction.\u003c/p\u003e\n\u003cp\u003eBased on these data, we believe that it is desirable to use specific bone turnover markers in addition to imaging techniques for a comprehensive evaluation of bone health in children with the onset of severe obesity at an early age and to make informed decisions about possible options for its management. Recombinant leptin has been used to treat children with leptin deficiency for more than two decades and has been reported to have a beneficial effect on bone health [54]. More recently, setmelanotide, an MC4R agonist that bypasses leptin resistance, has been approved for the treatment of monogenic obesity due to \u003cem\u003eLEPR\u003c/em\u003e-, \u003cem\u003ePOMC\u003c/em\u003e-, or \u003cem\u003ePCSK1\u003c/em\u003e-deficient states [55-57]. However, the effect of this agonist on bone health in these forms of monogenic obesity remains to be elucidated on a long-term basis. The advent of new drugs based on GLP and GIP receptor agonists [58, 59] for the treatment of common obesity necessitates a comprehensive evaluation of bone health and metabolism. In initial studies, GLP-1 receptor agonists and their analogs have recently been investigated in preclinical trials to improve bone fragility disorders [59].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patients and their families for their participation in this study. We thank Fr\u0026eacute;d\u0026eacute;ric Allegaert, Timoth\u0026eacute;e Beke, and Stefan Gaget for their technical assistance. This work was supported by funding from the Medical Research Council (MRC) MR/S026193/1 (PF) and the Pakistan Academy of Sciences (MA). Further support was provided by the National Center for Precision Diabetic Medicine, PreciDIAB, which is jointly supported by the French National Agency for Research (ANR-18-IBHU-0001), European Union (FEDER), Hauts-de-France Regional Council, and European Metropolis of Lille (MEL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMA and PF conceptualized the study. QMJ, RK, and MA wrote the first draft of the manuscript. QMJ, RK, SS, and MA contributed to the amendment of the first draft. RK, QMJ, AH, and MA collected the samples and performed the biochemical analysis. SS, AB, and PF analyzed the genetic data. QMJ, RK, and SH performed the statistical analysis. JM, RK, QMJ, and MA identified and recruited families with obesity. MA and PF are the guarantors of this work and, as such, had full access to all the data in the study and were responsible for the integrity of the data and the accuracy of the data analysis. All the authors contributed to the revision process and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. Obesity and overweight 2024. [cited 2025 Nov 4]. https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight\u003c/li\u003e\n\u003cli\u003eWorld Obesity Fedration. World obesity atlas 2023. [cited 2025 Nov 4]. https://data.worldobesity.org/publications/?cat=19\u003c/li\u003e\n\u003cli\u003eWestbury, S, Oyebode, O, van Rens, T, Barber, TM. Obesity stigma: causes, consequences, and potential solutions. Curr Obes Rep. 2023; 12:10\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eMust, A, Strauss, RS. Risks and consequences of childhood and adolescent obesity. 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Genetic variants in \u003cem\u003eLEP\u003c/em\u003e, \u003cem\u003eLEPR\u003c/em\u003e, and \u003cem\u003eMC4R\u003c/em\u003e explain 30% of severe obesity in children from a consanguineous population. Obesity. 2015; 23:1687\u0026ndash;95. \u003c/li\u003e\n\u003cli\u003eCrowley, VEF. Overview of human obesity and central mechanisms regulating energy homeostasis. Ann Clin Biochem. 2008; 45:245\u0026ndash;55. \u003c/li\u003e\n\u003cli\u003eMartinelli, CE, Keogh, JM, Greenfield, JR, Henning, E, Van Der Klaauw, AA, Blackwood, A, et al. Obesity due to melanocortin 4 receptor (MC4R) deficiency is associated with increased linear growth and final height, fasting hyperinsulinemia, and incompletely suppressed growth hormone secretion. J Clin Endocrinol Metab. 2011; 96:181\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eFarooqi IS, Jebb SA, Langmack G, Lawrence E, Cheetham CH, PA. Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med. 1999; 341:879\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eQamar, S, Mallik, R, Makaronidis, J. Setmelanotide: a melanocortin-4 receptor agonist for the treatment of severe obesity due to hypothalamic dysfunction. US Endocrinol. 2024; 20:1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eArgente, J, Verge, CF, Okorie, U, Fennoy, I, Kelsey, MM, Cokkinias, C, et al. Setmelanotide in patients aged 2\u0026ndash;5 years with rare MC4R pathway-associated obesity (venture): a 1 year, open-label, multicenter, phase 3 trial. Lancet Diabetes Endocrinol. 2025; 13:29\u0026ndash;37. \u003c/li\u003e\n\u003cli\u003eCollet, TH, Dubern, B, Mokrosinski, J, Connors, H, Keogh, JM, Mendes de Oliveira, E, et al. Evaluation of a melanocortin-4 receptor (MC4R) agonist (setmelanotide) in MC4R deficiency. Mol Metab. 2017; 6:1321\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eWu, Z, Deng, W, Ye, Y, Xu, J, Han, D, Zheng, Y, et al. Liraglutide, a glucagon-like peptide-1 receptor agonist, inhibits bone loss in an animal model of osteoporosis with or without diabetes. Front Endocrinol (Lausanne). 2024; 15:1378291. \u003c/li\u003e\n\u003cli\u003eBouvard, B, Mabilleau, G. Gut hormones and bone homeostasis: potential therapeutic implications. Nat Rev Endocrinol. 2024; 20:553\u0026ndash;64.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-obesity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijo","sideBox":"Learn more about [International Journal of Obesity](http://www.nature.com/ijo/)","snPcode":"41366","submissionUrl":"https://mts-ijo.nature.com/cgi-bin/main.plex","title":"International Journal of Obesity","twitterHandle":"@intjobesity","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monogenic obesity, Bone metabolism biomarkers, LEP, LEPR, MC4R","lastPublishedDoi":"10.21203/rs.3.rs-6595544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6595544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eOBJECTIVE\u003c/b\u003e\u003c/p\u003e \u003cp\u003eChronic obesity is associated with impaired bone health. However, few investigations have been conducted to assess bone physiology in early-onset obesity. In this study, we measured specific bone turnover and metabolic biomarkers in children with severe obesity with biallelic loss-of-function variants of the leptin (\u003cem\u003eLEP)\u003c/em\u003e, leptin receptor \u003cem\u003e(LEPR)\u003c/em\u003e, or melanocortin 4 receptor (\u003cem\u003eMC4R)\u003c/em\u003e genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMETHODS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eForty-one children aged 0.3\u0026ndash;13 years with a BMI SDS\u0026thinsp;\u0026ge;\u0026thinsp;3, previously identified with pathogenic variants in \u003cem\u003eLEP\u003c/em\u003e, \u003cem\u003eLEPR\u003c/em\u003e, or \u003cem\u003eMC4R\u003c/em\u003e, were recruited for the current study. Additionally, 13 age-matched children with severe obesity who tested negative for variants in known obesity-related genes were included, and another 15 unrelated age-matched children with normal body weight served as the control group. Serum osteocalcin, osteopontin, osteoprotegerin, and sclerostin levels were assessed using multi-analyte profiling. Serum leptin, insulin, and cortisol levels were determined using ELISA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRESULTS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSerum levels of osteocalcin and osteopontin, specific markers of bone formation, were significantly lower in subjects with \u003cem\u003eLEP\u003c/em\u003e and \u003cem\u003eLEPR\u003c/em\u003e biallelic variants than in the control group. In contrast, the values of these two biomarkers in subjects with \u003cem\u003eMC4R\u003c/em\u003e deficiency were significantly higher than those in the other groups. No differences were observed in the bone resorption markers osteoprotegerin and sclerostin. Hyperleptinemia was more pronounced in subjects with \u003cem\u003eLEPR\u003c/em\u003e deficiency. Serum insulin concentrations were elevated in subjects with \u003cem\u003eMC4R\u003c/em\u003e deficiency, whereas serum cortisol levels were significantly higher in subjects with \u003cem\u003eLEP\u003c/em\u003e deficiency than in all other groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCONCLUSION\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur data demonstrate that osteogenic activity (but not resorption activity) is differentially affected in children with complete genetic disruption of the leptin signaling pathway. Children with \u003cem\u003eMC4R\u003c/em\u003e deficiency showed higher osteogenic markers, but children with \u003cem\u003eLEP\u003c/em\u003e and \u003cem\u003eLEPR\u003c/em\u003e deficiencies showed the opposite. Our results support the usefulness of bone turnover biomarkers for the assessment and management of bone health in different types of obesity.\u003c/p\u003e","manuscriptTitle":"Inversed impaired osteogenic activity in children with severe obesity due to MC4R deficiency compared to LEP and LEPR deficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-20 06:16:56","doi":"10.21203/rs.3.rs-6595544/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-06-20T13:35:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-12T18:30:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-12T00:07:51+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-30T08:01:52+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-27T21:41:04+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-15T22:55:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-14T10:42:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Obesity","date":"2025-05-13T18:14:51+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-05-06T11:59:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-05T15:03:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-obesity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijo","sideBox":"Learn more about [International Journal of Obesity](http://www.nature.com/ijo/)","snPcode":"41366","submissionUrl":"https://mts-ijo.nature.com/cgi-bin/main.plex","title":"International Journal of Obesity","twitterHandle":"@intjobesity","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"57142639-5895-4b0e-894f-f228be01cdf9","owner":[],"postedDate":"May 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48725831,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity"},{"id":48725832,"name":"Health sciences/Health care/Paediatrics"},{"id":48725833,"name":"Biological sciences/Physiology/Metabolism/Metabolic diseases/Obesity"}],"tags":[],"updatedAt":"2026-03-14T07:08:31+00:00","versionOfRecord":{"articleIdentity":"rs-6595544","link":"https://doi.org/10.1038/s41366-026-02047-w","journal":{"identity":"international-journal-of-obesity","isVorOnly":false,"title":"International Journal of Obesity"},"publishedOn":"2026-03-13 04:00:00","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-05-20 06:16:56","video":"","vorDoi":"10.1038/s41366-026-02047-w","vorDoiUrl":"https://doi.org/10.1038/s41366-026-02047-w","workflowStages":[]},"version":"v1","identity":"rs-6595544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6595544","identity":"rs-6595544","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}