Search

Funder: National Institute for Health Research (NIHR), Serotonin reuptake inhibitors and receptor agonists are used to treat obesity, anxiety and depression. Here we studied the role of the serotonin 2C receptor (5-HT2CR) in weight regulation and behavior. Using exome sequencing of 2,548 people with severe obesity and 1,117 control individuals without obesity, we identified 13 rare variants in the gene encoding 5-HT2CR (HTR2C) in 19 unrelated people (3 males and 16 females). Eleven variants caused a loss of function in HEK293 cells. All people who carried variants had hyperphagia and some degree of maladaptive behavior. Knock-in male mice harboring a human loss-of-function HTR2C variant developed obesity and reduced social exploratory behavior; female mice heterozygous for the same variant showed similar deficits with reduced severity. Using the 5-HT2CR agonist lorcaserin, we found that depolarization of appetite-suppressing proopiomelanocortin neurons was impaired in knock-in mice. In conclusion, we demonstrate that 5-HT2CR is involved in the regulation of human appetite, weight and behavior. Our findings suggest that melanocortin receptor agonists might be effective in treating severe obesity in individuals carrying HTR2C variants. We suggest that HTR2C should be included in diagnostic gene panels for severe childhood-onset obesity.

Context While severe obesity due to congenital leptin deficiency is rare, studies in patients before and after treatment with leptin can provide unique insights into the role that leptin plays in metabolic and endocrine function. Objective The aim of this study was to characterize changes in peripheral metabolism in people with congenital leptin deficiency undergoing leptin replacement therapy, and to investigate the extent to which these changes are explained by reduced caloric intake. Design Ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) was used to measure 661 metabolites in 6 severely obese people with congenital leptin deficiency before, and within 1 month after, treatment with recombinant leptin. Data were analyzed using unsupervised and hypothesis-driven computational approaches and compared with data from a study of acute caloric restriction in healthy volunteers. Results Leptin replacement was associated with class-wide increased levels of fatty acids and acylcarnitines and decreased phospholipids, consistent with enhanced lipolysis and fatty acid oxidation. Primary and secondary bile acids increased after leptin treatment. Comparable changes were observed after acute caloric restriction. Branched-chain amino acids and steroid metabolites decreased after leptin, but not after acute caloric restriction. Individuals with severe obesity due to leptin deficiency and other genetic obesity syndromes shared a metabolomic signature associated with increased BMI. Conclusion Leptin replacement was associated with changes in lipolysis and substrate utilization that were consistent with negative energy balance. However, leptin’s effects on branched-chain amino acids and steroid metabolites were independent of reduced caloric intake and require further exploration.

Peer reviewed: True, Funder: Bernard Wolfe Health Neuroscience Endowment, Funder: Botnar Fondation, Funder: NIHR Cambridge Biomedical Research Center, Funder: NIHR Senior Investigator Award, Obesity, defined as an excess of adipose tissue that adversely affects health, is a major cause of morbidity and mortality. However, to date, understanding the structure and function of human adipose tissue has been limited by the inability to visualize cellular components due to the innate structure of adipocytes, which are characterized by large lipid droplets. Combining the iDISCO and the CUBIC protocols for whole tissue staining and optical clearing, we developed a protocol to enable immunostaining and clearing of human subcutaneous white adipose tissue (WAT) obtained from individuals with severe obesity. We were able to perform immunolabelling of sympathetic nerve terminals in whole WAT and subsequent optical clearing by eliminating lipids to render the opaque tissue completely transparent. We then used light sheet confocal microscopy to visualize sympathetic innervation of human WAT from obese individuals in a three-dimensional manner. We demonstrate the visualization of sympathetic nerve terminals in human WAT. This protocol can be modified to visualize other structures such as blood vessels involved in the development, maintenance and function of human adipose tissue in health and disease.

Context Genetic variants affecting the nuclear hormone receptor coactivator steroid receptor coactivator, SRC-1, have been identified in people with severe obesity and impair melanocortin signaling in cells and mice. As a result, obese patients with SRC-1 deficiency are being treated with a melanocortin 4 receptor agonist in clinical trials. Objective Here, our aim was to comprehensively describe and characterize the clinical phenotype of SRC-1 variant carriers to facilitate diagnosis and clinical management. Methods In genetic studies of 2462 people with severe obesity, we identified 23 rare heterozygous variants in SRC-1. We studied 29 adults and 18 children who were SRC-1 variant carriers and performed measurements of metabolic and endocrine function, liver imaging, and adipose tissue biopsies. Findings in adult SRC-1 variant carriers were compared to 30 age- and body mass index (BMI)-matched controls. Results The clinical spectrum of SRC-1 variant carriers included increased food intake in children, normal basal metabolic rate, multiple fractures with minimal trauma (40%), persistent diarrhea, partial thyroid hormone resistance, and menorrhagia. Compared to age-, sex-, and BMI-matched controls, adult SRC-1 variant carriers had more severe adipose tissue fibrosis (46.2% vs 7.1% respectively, P = .03) and a suggestion of increased liver fibrosis (5/13 cases vs 2/13 in controls, odds ratio = 3.4), although this was not statistically significant. Conclusion SRC-1 variant carriers exhibit hyperphagia in childhood, severe obesity, and clinical features of partial hormone resistance. The presence of adipose tissue fibrosis and hepatic fibrosis in young patients suggests that close monitoring for the early development of obesity-associated metabolic complications is warranted.

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action. This study set out to identify novel gene variants that may contribute to human obesity, by combining human exosome sequencing analyses with systematic functional screening in Drosophila. This identifies a number of novel obesity-associated genes which control adiposity in flies, and uncovers a potential role for the Hippo signaling pathway in obesity.

Objective People who are severely obese due to melanocortin-4 receptor (MC4R) deficiency experience hyperphagia and impaired fullness after a meal (satiety). Meal-induced satiety is influenced by hormones, such as peptide-YY (PYY), which are released by enteroendocrine cells upon nutrient delivery to the small intestine. Design We investigated whether gastric emptying and PYY levels are altered in MC4R deficiency. Methods Gastric emptying was measured with a gastric scintigraphy protocol using technetium-99m (99 Tcm )-Tin Colloid for 3.5 h in individuals with loss of function MC4R variants and a control group of similar age and weight. In a separate study, we measured plasma PYY levels before and at multiple time points after three standardised meals given to individuals with MC4R deficiency and controls. Fasting PYY (basal secretion) and postprandial PYY levels were measured and the area under the curve and inter-meal peak were calculated. Results We found that gastric emptying time was significantly delayed and percentage meal retention increased in individuals with MC4R deficiency compared to obese controls. In addition, fasting and mean PYY secretion throughout the day were decreased in MC4R deficiency, whereas postprandial PYY secretion was unaltered. Conclusion Delayed gastric emptying and reduced basal PYY secretion may contribute to impaired satiety in people with obesity due to MC4R deficiency.

Background Frontotemporal dementia (FTD) is associated with complex changes in eating behavior and metabolism, which potentially affect disease pathogenesis and survival. It is currently not known if body composition changes and changes in fat deposition also exist in FTD, the relationship of these changes in eating behavior and appetite, and whether these changes are centrally mediated. Methods Body composition was measured in 28 people with behavioral‐variant frontotemporal dementia (bvFTD), 16 with Alzheimer’s disease (AD), and 19 healthy controls, using dual energy x‐ray absorptiometry. Changes in body composition were correlated to brain grey matter atrophy using voxel‐based morphometry on high‐resolution magnetic resonance imaging. Results Behavioral‐variant FTD was characterized by changes in body composition, with increased total fat mass, visceral adipose tissue area (VAT area), and android: gynoid ratio compared to control and AD participants (all P values

Summary The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy., Graphical abstract, Highlights • Obesity-associated MC4R mutations that do not reduce cAMP disrupt other processes • MC4R mutations impact receptor homodimerization, endocytosis, and trafficking • Obesity-protecting mutations increase plasma membrane MC4Rs and enhance signaling • Multiple mechanisms regulate melanocortin tone to a physiologically relevant level, Using mutations in the human Melanocortin-4 Receptor (MC4R), Brouwers et al. identify receptor trafficking and endocytosis, coupling to Gαs/β-arrestins, and homodimerization as mechanisms involved in the regulation of body weight that may be targeted for weight loss therapy.

Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders.

Summary Obesity is genetically heterogeneous with monogenic and complex polygenic forms. Using exome and targeted sequencing in 2,737 severely obese cases and 6,704 controls, we identified three genes (PHIP, DGKI, and ZMYM4) with an excess burden of very rare predicted deleterious variants in cases. In cells, we found that nuclear PHIP (pleckstrin homology domain interacting protein) directly enhances transcription of pro-opiomelanocortin (POMC), a neuropeptide that suppresses appetite. Obesity-associated PHIP variants repressed POMC transcription. Our demonstration that PHIP is involved in human energy homeostasis through transcriptional regulation of central melanocortin signaling has potential diagnostic and therapeutic implications for patients with obesity and developmental delay. Additionally, we found an excess burden of predicted deleterious variants involving genes nearest to loci from obesity genome-wide association studies. Genes and gene sets influencing obesity with variable penetrance provide compelling evidence for a continuum of causality in the genetic architecture of obesity, and explain some of its missing heritability., Graphical Abstract, Highlights • Three genes (PHIP, DGKI, and ZMYM4) are linked to severe childhood obesity • Wild-type PHIP enhances POMC transcription, but variants in PHIP repress POMC • Rare variants in BMI-associated loci from GWAS are enriched in severe obesity • Genetic architecture of severe childhood obesity reveals a continuum of causality, Childhood obesity can be caused by penetrant mutations in a number of genes controlling appetite and body weight. Marenne et al. identify three genes with mutations with variable penetrance in a continuum of causality in childhood obesity, and demonstrate that variants in PHIP repress POMC transcription.

Objective Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. Methods In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. Results We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. Conclusions OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis., Highlights • A mouse Otp mutation alters hypothalamic neuropeptide expression leading to increased food intake, obesity and anxiety. • In severe early onset obesity, we found a heterozygous LOF variant in a patient with attention deficit disorder features. • These studies show for the first time that mutations in the Otp/OTP gene cause obesity.

Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss., Neurons expressing pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here the authors show that Steroid Receptor Coactivator-1 (SRC-1) regulates the function of Pomc expressing neurons, and that rare heterozygous variants found in obese individuals lead to loss of SRC-1 function.

Obesity is genetically heterogeneous with monogenic and complex polygenic forms. Using exome sequencing followed by targeted sequencing in 2,737 severely obese cases and 6,704 controls, we identified three genes (PHIP, DGKI, ZMYM4) with an excess burden in cases of variants (MAF

The variation in weight within a shared environment is largely attributable to genetic factors. Whilst many genes/loci confer susceptibility to obesity, little is known about the genetic architecture of healthy thinness. Here, we characterise the heritability of thinness which we found was comparable to that of severe obesity (h2 = 28.07 vs 32.33% respectively), although with incomplete genetic overlap (r = -0.49, 95% CI [-0.17, -0.82], p = 0.003). In a genome-wide association analysis of thinness (n = 1,471) vs severe obesity (n = 1,456), we identified 10 loci previously associated with obesity, and demonstrate enrichment for established BMI-associated loci (pbinomial = 3.05x10-5). Simulation analyses showed that different association results between the extremes were likely in agreement with additive effects across the BMI distribution, suggesting different effects on thinness and obesity could be due to their different degrees of extremeness. In further analyses, we detected a novel obesity and BMI-associated locus at PKHD1 (rs2784243, obese vs. thin p = 5.99x10-6, obese vs. controls p = 2.13x10-6 pBMI = 2.3x10-13), associations at loci recently discovered with much larger sample sizes (e.g. FAM150B and PRDM6-CEP120), and novel variants driving associations at previously established signals (e.g. rs205262 at the SNRPC/C6orf106 locus and rs112446794 at the PRDM6-CEP120 locus). Our ability to replicate loci found with much larger sample sizes demonstrates the value of clinical extremes and suggest that characterisation of the genetics of thinness may provide a more nuanced understanding of the genetic architecture of body weight regulation and may inform the identification of potential anti-obesity targets., Author summary Obesity-associated disorders are amongst the leading causes of morbidity and mortality worldwide. Most genome-wide association studies (GWAS) have focused on body mass index (BMI = weight in Kg divided by height squared (m2)) and obesity, but to date no genetic association study testing thin and healthy individuals has been performed. In this study, we recruited a first of its kind cohort of 1,471 clinically ascertained thin and healthy individuals and contrasted the genetic architecture of the trait with that of severe early onset obesity. We show that thinness, like obesity, is a heritable trait with a polygenic component. In a GWAS of persistent healthy thinness vs. severe obesity with a total sample size of 2,927, we are able to find evidence of association in loci that have only been recently discovered using large cohorts with >40,000 individuals. We also find a novel BMI-associated locus at PKHD1 in UK Biobank highlighted by our association study. This work illustrates the value and increased power brought upon by using clinically ascertained extremes to study complex traits and provides a valuable resource on which to study resistance to obesity in an increasingly obesogenic environment.

STUDY OBJECTIVES: The rise in obesity has been paralleled by a decline in sleep duration in epidemiological studies. However, the potential mechanisms linking energy balance and the sleep/wake cycle are not well understood. We aimed to examine the effects of manipulating energy balance on the sleep/wake cycle. METHODS: Twelve healthy normal weight men were housed in a clinical research facility and studied at three time points: baseline, after energy balance was disrupted by 2 days of caloric restriction to 10% of energy requirements, and after energy balance was restored by 2 days of ad libitum/free feeding. Sleep architecture, duration of sleep stages, and sleep-associated respiratory parameters were measured by polysomnography. RESULTS: Two days of caloric restriction significantly increased the duration of deep (stage 4) sleep (16.8% to 21.7% of total sleep time; P = 0.03); an effect which was entirely reversed upon free feeding (P = 0.01). Although the apnea-hypopnea index stayed within the reference range (< 5 events per hour), it decreased significantly from caloric restriction to free feeding (P = 0.03). Caloric restriction was associated with a marked fall in leptin (P < 0.001) and insulin levels (P = 0.002). The fall in orexin levels from baseline to caloric restriction correlated positively with duration of stage 4 sleep (Spearman rho = 0.83, P = 0.01) and negatively with the number of awakenings in caloric restriction (Spearman rho = -0.79, P = 0.01). CONCLUSIONS: We demonstrate that changes in energy homeostasis directly and reversibly impact on the sleep/wake cycle. These findings provide a mechanistic framework for investigating the association between sleep duration and obesity risk.

Summary Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin’s effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species., Graphical Abstract, Highlights • Leptin is the link between obesity and increased blood pressure • Leptin acts through the dorsomedial hypothalamus to increase blood pressure • Blockade of leptin signaling reduces blood pressure in obese mice • Humans with defects in leptin signaling are protected from obesity hypertension, Leptin is found to be the link between obesity and increased blood pressure. Blocking leptin action reduces blood pressure in obese mice with clinical studies in humans, suggesting that defects in leptin signaling may protect against hypertension associated with obesity.

SummaryKinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.PaperFlick

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.

Objective Meals high in protein induce greater intermeal satiety than meals high in fat and carbohydrates. We studied the gut hormone response and subsequent food intake after breakfasts high in protein, carbohydrate or high in fat controlled for volume, calories and appearance. Design and Methods Eight healthy volunteers participated in this randomized three-way crossover study. Study breakfasts were calculated to provide 20% of daily energy requirements and provided either 60% of energy from protein, fat or carbohydrate. Blood was drawn half-hourly for 4 h; energy intake at a subsequent ad libitum meal was measured. Results Total ghrelin decreased after food intake equally with the three breakfasts. PYY levels were highest after the high protein breakfast (P = 0.005). Indeed, PYY at 240 min was highest after the high protein breakfast compared to the high fat breakfast and to the high carbohydrate breakfast (P = 0.011 and P = 0.012, respectively). GLP-1 levels were highest after the high protein breakfast (P = 0.041) at 120 min and remained higher throughout the study. These differences in gut hormones did not translate into differences in food intake (1023 ± 390 kcal after high protein, 1016 ± 388 kcal after high fat and 1158 ± 433 kcal after high carbohydrate). Conclusion We conclude that a high protein meal increases circulating concentrations of the gut hormones PYY and GLP-1, but when meals are matched for volume, appearance and caloric value, these gut hormone changes do not translate into a reduction in ad libitum food intake.

Genetic studies in patients with severe early-onset obesity have provided insights into the molecular and physiological pathways that regulate body weight in humans. We report a 19-year-old male with hyperphagia and severe obesity, mild learning difficulties and hypogonadism, in whom diagnostic tests for Prader-Willi syndrome (PWS) had been negative. We carried out detailed clinical and metabolic phenotyping of this patient and investigated the genetic basis of this obesity syndrome using Agilent 185 k array comparative genomic hybridization (aCGH) and Affymetrix 6.0 genotyping arrays. The identified deletion was validated using multiplex ligation-dependent probe amplification and long-range PCR, followed by breakpoint sequencing which enabled precise localization of the deletion. We identified a approximately 187 kb microdeletion at chromosome 15q11-13 that encompasses non-coding small nucleolar RNAs (including HBII-85 snoRNAs) which were not expressed in peripheral lymphocytes from the patient. Characterization of the clinical phenotype revealed increased ad libitum food intake, normal basal metabolic rate when adjusted for fat-free mass, partial hypogonadotropic hypogonadism and growth failure. We have identified a novel deletion on chromosome 15q11-13 in an individual with hyperphagia, obesity, hypogonadism and other features associated with PWS, which is normally caused by deficiency of several paternally expressed imprinted transcripts within chromosome 15q11-13, a region that includes multiple protein-coding genes as well as several non-coding snoRNAs. These findings provide direct evidence for the role of a particular family of non-coding RNAs, the HBII-85 snoRNA cluster, in human energy homeostasis, growth and reproduction.

The adipocyte-derived hormone leptin is crucial for energy homeostasis in mammals; mice1 and humans2,3 without it suffer from a voracious appetite and extreme obesity. The effect on energy balance of variations in plasma leptin above a minimal threshold is uncertain, however, particularly in humans. Here we examine a group of individuals who are genetically partially deficient in leptin, and show that differences in circulating leptin levels within the range found in normal human populations can directly influence the laying down of fat tissue (adiposity).

Roux-en-Y gastric bypass (RYGB) is one of the most effective long-term therapies for the treatment of severe obesity. Recent evidence indicates that RYGB effects weight loss through multiple physiological mechanisms, including changes in energy expenditure, food intake, food preference, and reward pathways.Because central melanocortin signaling plays an important role in the regulation of energy homeostasis, we investigated whether genetic disruption of the melanocortin-4 receptor (MC4R) in rodents and humans affects weight loss after RYGB.Here we report that MC4R(-/-) mice lost substantially less weight after surgery than wild-type animals, indicating that MC4R signaling is necessary for the weight loss effects of RYGB in this model. Mice heterozygous for MC4R remain fully responsive to gastric bypass. To determine whether mutations affect surgically induced weight loss in humans, we sequenced the MC4R gene in 972 patients undergoing RYGB. Patients heterozygous for MC4R mutations exhibited the same magnitude and distribution of postoperative weight loss as patients without such mutations, suggesting that although two normal copies of the MC4R gene are necessary for normal weight regulation, a single normal copy of the MC4R gene is sufficient to mediate the weight loss effects of RYGB.MC4R is the first gene identified that is required for the sustained effects of bariatric surgery. The need for MC4R signaling for the weight loss effects of RYGB in mice underscores the physiological mechanisms of action of this procedure and demonstrates that RYGB both influences and is dependent on the normal pathways that regulate energy balance.

Resistance to thyroid hormone (RTH), a dominantly inherited disorder usually associated with mutations in thyroid hormone receptor beta (THRB), is characterized by elevated levels of circulating thyroid hormones (including thyroxine), failure of feedback suppression of thyrotropin, and variable tissue refractoriness to thyroid hormone action. Raised energy expenditure and hyperphagia are recognized features of hyperthyroidism, but the effects of comparable hyperthyroxinemia in RTH patients are unknown. Here, we show that resting energy expenditure (REE) was substantially increased in adults and children with THRB mutations. Energy intake in RTH subjects was increased by 40%, with marked hyperphagia particularly evident in children. Rates of muscle TCA cycle flux were increased by 75% in adults with RTH, whereas rates of ATP synthesis were unchanged, as determined by 13C/31P magnetic resonance spectroscopy. Mitochondrial coupling index between ATP synthesis and mitochondrial rates of oxidation (as estimated by the ratio of ATP synthesis to TCA cycle flux) was significantly decreased in RTH patients. These data demonstrate that basal mitochondrial substrate oxidation is increased and energy production in the form of ATP synthesis is decreased in the muscle of RTH patients and that resting oxidative phosphorylation is uncoupled in this disorder. Furthermore, these observations suggest that mitochondrial uncoupling in skeletal muscle is a major contributor to increased REE in patients with RTH, due to tissue selective retention of thyroid hormone receptor alpha sensitivity to elevated thyroid hormone levels.

Weight gain and weight loss are associated with changes in blood pressure through unknown mechanisms. Central melanocortinergic signaling is implicated in the control of energy balance and blood pressure in rodents, but there is no information regarding such an association with blood pressure in humans.We assessed blood pressure, heart rate, and urinary catecholamines in overweight or obese subjects with a loss-of-function mutation in MC4R, the gene encoding the melanocortin 4 receptor, and in equally overweight control subjects. We also examined the effects of an MC4R agonist administered for 7 days in 28 overweight or obese volunteers.The prevalence of hypertension was markedly lower in the MC4R-deficient subjects than in the control subjects (24% vs. 53%, P=0.009). After the exclusion of subjects taking antihypertensive medications, blood-pressure levels were significantly lower in MC4R-deficient subjects than in control subjects, with mean (+/-SE) systolic blood pressures of 123+/-14 mm Hg and 131+/-12 mm Hg, respectively (P=0.02), and mean diastolic blood pressures of 73+/-10 mm Hg and 79+/-7 mm Hg, respectively (P=0.03). As compared with control subjects, MC4R-deficient subjects had a lower increase in heart rate on waking (P=0.007), a lower heart rate during euglycemic hyperinsulinemia (P0.001), and lower 24-hour urinary norepinephrine excretion (P=0.04). The maximum tolerated daily dose of 1.0 mg of the MC4R agonist led to significant increases of 9.3+/-1.9 mm Hg in systolic blood pressure and of 6.6+/-1.1 mm Hg in diastolic blood pressure (P0.001 for both comparisons) at 24 hours, as compared with placebo. Differences in blood pressure were not explained by changes in insulin levels; there were no significant adverse events.Results of our genetic and pharmacologic studies implicate melanocortinergic signaling in the control of human blood pressure through an insulin-independent mechanism.

Context: Mutations in the proopiomelanocortin (POMC) gene that impair the synthesis or structure of POMC-derived peptides predispose to human obesity. Objective: Our objective was to identify and characterize novel mutations in the POMC gene found in patients with early-onset obesity. Design and Patients: The POMC gene was screened in 500 patients with severe early-onset obesity. The biosynthesis, processing, sorting, and secretion of wild-type POMC and two newly identified POMC mutants was studied using metabolic labeling, Western blotting, and immunoassay analysis of lysates and conditioned media of transiently transfected β-TC3 cells. Results: Two novel heterozygous missense mutations in POMC (C28F and L37F) were identified in unrelated probands with early-onset obesity and their overweight or obese family members. Both mutations lie in a region of the N terminus of POMC that has been suggested to be involved in its sorting to the regulated secretory pathway. Metabolic labeling studies indicate that whereas the mutations do not reduce intracellular levels of POMC, both mutations (C28F>L37F) impair the ability of POMC to be processed to generate bioactive products. Studies of the secretion of POMC products suggest, particularly with C28F, that the impaired propeptide processing of these mutations results, at least in part, from a mistargeting of mutant POMC to the constitutive rather than the regulated secretory pathway. Conclusion: These mutations in patients with early-onset obesity represent a novel molecular mechanism of human POMC deficiency whereby naturally occurring mutations in its N-terminal sequence impair the ability of POMC to enter the trafficking pathway in which serial propeptide processing normally occurs.

Context: Congenital leptin deficiency is a very rare cause of severe early-onset obesity. We recently characterized a mutation in the leptin gene (p.D100Y), which was associated with detectable leptin levels and bioinactivity of the hormone. Case Description: We now describe two siblings, a 9-year-old girl and a 6-year-old boy with severe early-onset obesity and hyperphagia, both homozygous for a c.309C>A substitution in the leptin gene leading to a p.N103K amino acid exchange in the protein and detectable circulating levels of leptin. In vitro experiments in a heterologous cell system demonstrated that the mutated protein was biologically inactive. Treatment with sc recombinant human leptin led to rapid improvement of eating behavior and weight loss. Conclusions: Sequencing of the leptin gene may need to be considered in hyperphagic, severely obese children with detectable levels of circulating leptin.

The neurotrophin brain-derived neurotrophic factor (BDNF) inhibits food intake, and rodent models of BDNF disruption all exhibit increased food intake and obesity, as well as hyperactivity. We report an 8-year-old girl with hyperphagia and severe obesity, impaired cognitive function, and hyperactivity who harbored a de novo chromosomal inversion, 46,XX,inv(11)(p13p15.3), a region encompassing the BDNF gene. We have identified the proximal inversion breakpoint that lies 850 kb telomeric of the 5′ end of the BDNF gene. The patient’s genomic DNA was heterozygous for a common coding polymorphism in BDNF, but monoallelic expression was seen in peripheral lymphocytes. Serum concentration of BDNF protein was reduced compared with age- and BMI-matched subjects. Haploinsufficiency for BDNF was associated with increased ad libitum food intake, severe early-onset obesity, hyperactivity, and cognitive impairment. These findings provide direct evidence for the role of the neurotrophin BDNF in human energy homeostasis, as well as in cognitive function, memory, and behavior.

Congenital deficiency of proopiomelanocortin (POMC) results in a syndrome of hypoadrenalism, severe obesity, and altered skin and hair pigmentation. The concept that subtle variation in POMC expression and/or function might contribute to common obesity is suggested by studies reporting linkage of obesity-related traits to a locus on chromosome 2p22 encompassing the POMC gene. We identified a novel homozygous frameshift (C6906del) mutation in POMC in a child of Turkish origin with severe obesity and hypoadrenalism. This mutation would be predicted to lead to the loss of all POMC-derived peptides. The availability of a large extended pedigree provided the opportunity to address whether loss of one copy of the POMC gene was sufficient to alter obesity risk. Twelve relatives were heterozygous for the mutation and 7 were wild type. Of the heterozygotes, 11 of 12 heterozygotes were obese or overweight compared with only 1 of 7 of the wild-type relatives. The mean BMI SD score was 1.7 ± 0.5 in heterozygotes and 0.4 ± 0.4 in the wild-type relatives. Parametric linkage analysis of the trait “overweight” provided statistically significant evidence of linkage with this locus, with a maximum “location score” (comparable with multipoint logarithm of odds scores) of 3.191. We conclude that loss of one copy of the POMC gene predisposes to obesity in humans. Thus, genetic variants having relatively subtle effects on POMC expression and function could influence susceptibility to obesity.

The gene encoding the melatonin-related receptor (GPR50) is highly expressed within hypothalamic nuclei concerned with the control of body weight and metabolism. We screened GPR50 for mutations in an obese cohort and identified an insertion of four amino acid residues (TTGH) at position 501, two common coding polymorphisms (T528A and V602I), and one noncoding polymorphism (C-16X2GPR50T). Single-nucleotide polymorphisms were then typed in 500 English Caucasian subjects, and associations were sought to intermediate obesity phenotypes. Although no association was seen with body mass index, carriers of two copies of the mutant allele at C-16X2GPR50T, Ins501Del, and A1582G had significantly higher fasting circulating triglyceride levels (P < 0.05). In a separate set of 585 subjects, the associations were replicated, with statistically significant effects of similar magnitude and direction. The association of C-16X2GPR50T with fasting triglycerides was highly significant (P < 0.001). In addition, a significant association between C-16X2GPR50T and circulating HDL levels was observed in the combined population, with C-16X2GPR50T carriers having significantly lower circulating HDL-cholesterol levels (1.39 mM) than wild-type subjects (1.47 mM) (P < 0.01). These findings suggest a previously unexpected role for this orphan receptor in the regulation of lipid metabolism that warrants further investigation.

The melanocortin-4 receptor (MC4R) plays a critical role in the control of energy balance. Of its two pro-opiomelanocortin (POMC)-derived ligands, alpha- and beta-MSH, the majority of attention has focused on alpha-MSH, partly reflecting the absence of beta-MSH in rodents. We screened the POMC gene in 538 patients with severe, early-onset obesity and identified five unrelated probands who were heterozygous for a rare missense variant in the region encoding beta-MSH, Tyr221Cys. This frequency was significantly increased (p < 0.001) compared to the general UK Caucasian population and the variant cosegregated with obesity/overweight in affected family members. Compared to wild-type beta-MSH, the variant peptide was impaired in its ability to bind to and activate signaling from the MC4R. Obese children carrying the Tyr221Cys variant were hyperphagic and showed increased linear growth, both of which are features of MC4R deficiency. These studies support a role for beta-MSH in the control of human energy homeostasis.

Peptide-YY (PYY) is secreted from endocrine L-cells of the gastrointestinal tract in response to caloric ingestion and may mediate postprandial satiety through the hypothalamic neuropeptide Y2 receptor (Y2R). We examined whether variants in the genes encoding PYY and Y2R might be associated with obesity-related phenotypes in humans. Among 101 subjects with severe early-onset obesity and a history of hyperphagia, we found two rare sequence variants—L73P and IVS2 + 32delG—in PYY and three rare missense mutations—L40F, F87I, and A172T—in Y2R. Although none of these were found in 100 normal-weight white control subjects, L73P in PYY and F87I and A172T in Y2R did not segregate with obesity in family studies, and family data were unavailable for IVS2 + 32delG in PYY and L40F in Y2R. Two common single nucleotide polymorphisms (SNPs), R72T and IVS3 + 68C>T, in PYY were in tight linkage disequilibrium but showed no association with BMI in a large white population. In the Y2R, two SNPs, 585T>C and 936T>C, were found and were in tight linkage disequilibrium. Men, homozygous for the rarer variant, had significantly lower BMI (P = 0.017), waist-to-hip ratio (P = 0.013), and, surprisingly, higher nonesterified fatty acid levels (P = 0.01). In conclusion, mutations in PYY and Y2R are not commonly found in humans with severe early-onset obesity. The relationship between common variants in Y2R and obesity-related traits deserves further exploration in other populations.

Loss of function mutations in the small heterodimer partner (SHP) gene have been reported to cause obesity and increased birth weight. We examined the relation between genetic variation in SHP and birth weight, adiposity, and insulin levels in three independent populations. The coding regions and 562 bases of the SHP promoter were screened for mutations in 329 subjects with severe early-onset obesity. Two novel missense mutations, R34G and R36C, were identified; these were not found in control subjects and did not cosegregate with obesity in family studies. Two common polymorphisms, G171A and −195CTGAdel, were found in 12 and 16% of subjects, respectively. Within the obese cohort, G171A and −195CTGAdel carriers had higher and lower birth weights, respectively, than wild-type subjects, the rare homozygotes for G171A being particularly large at birth. In a U.K. population-based cohort of 1,079 children, the 171A allele was associated with higher BMI (P < 0.05) and waist circumference (P = 0.001). Children carrying the G171A variant had higher 30-min insulin responses to a glucose load (P = 0.03). In conclusion, although mutations in SHP are not a common cause of severe human obesity, genetic variation in the SHP locus may influence birth weight and have effects on BMI, possibly through effects on insulin secretion.

Prolactin-releasing peptide (PrRP) and its G-protein-coupled receptor, GPR10, have been implicated in the central control of appetite and blood pressure. To determine whether mutations in these genes might contribute to morbid obesity, we screened both genes in 94 subjects with severe early-onset obesity. Four rare silent variants in PrRP and eight polymorphisms in GPR10 were found, two of which (V283I and P305L) altered amino acid sequence but were also found in U.K. Caucasian control subjects. Cells expressing the P305L variant receptor generated less intracellular calcium in response to PrRP than cells expressing the wild-type receptor. To examine whether genetic variation of the GPR10 locus might be associated with phenotypes relevant to obesity and/or blood pressure, the most common noncoding (G-62A) and coding (C914T [P305L]) polymorphisms were typed in 1,084 U.K. Caucasians. While no association was found with BMI, carriers of the P305L allelic variant had significantly lower systolic (123.95 vs. 128.55 mmHg, P < 0.05) and diastolic (74.90 vs. 78.20 mmHg, P < 0.01) blood pressure than wild-type subjects. In conclusion, we have conducted the first genetic study of GPR10 and its ligand PrRP in relation to metabolic phenotypes and have identified an association between GPR10 polymorphisms and diastolic and systolic blood pressure. The alteration in signaling properties of the receptor produced by P305L may provide a functional basis for this association.

Melanocortin 4 receptor (MC4R) deficiency is the commonest monogenic form of obesity. However, the clinical spectrum and mode of inheritance have not been defined, pathophysiological mechanisms leading to obesity are poorly understood, and there is little information regarding genotype-phenotype correlations.We determined the nucleotide sequence of the MC4R gene in 500 probands with severe childhood obesity. Family studies were undertaken to examine cosegregation of identified mutations with obesity. Subjects with MC4R deficiency underwent metabolic and endocrine evaluation; the results were correlated with the signaling properties of mutant receptors.Twenty-nine probands (5.8 percent) had mutations in MC4R; 23 were heterozygous, and 6 were homozygous. Mutation carriers had severe obesity, increased lean mass, increased linear growth, hyperphagia, and severe hyperinsulinemia; homozygotes were more severely affected than heterozygotes. Subjects with mutations retaining residual signaling capacity had a less severe phenotype.Mutations in MC4R result in a distinct obesity syndrome that is inherited in a codominant manner. Mutations leading to complete loss of function are associated with a more severe phenotype. The correlation between the signaling properties of these mutant receptors and energy intake emphasizes the key role of this receptor in the control of eating behavior in humans.

The functional loss of both alleles of the human pro-opiomelanocortin (POMC) gene leads to a very rare syndrome of hypoadrenalism, red hair and early-onset obesity. In order to examine whether more subtle genetic variants in POMC might contribute to early-onset obesity, the coding region of the gene was sequenced in 262 Caucasian subjects with a history of severe obesity from childhood. Two children were found to be heterozygous for a missense mutation, R236G, which disrupts the dibasic cleavage site between beta melanocyte-stimulating hormone (beta-MSH) and beta-endorphin. Beta-TC3 cells transfected with the mutant POMC cDNA produced a mutant beta-MSH/beta-endorphin fusion protein. This fusion protein bound to the human melanocortin-4 receptor (hMC4R) with an affinity similar to its natural ligands, but had a markedly reduced ability to activate the receptor. This variant co-segregated with early-onset obesity over three generations in one family and was absent in 412 normal weight UK Caucasian controls. Combining the results in UK Caucasians with a new case-control study in French subjects and three previously published reports, mutations disrupting this processing site were present in 0.88% of subjects with early-onset obesity and 0.22% of normal-weight controls. These results suggest that the R236G mutation may confer an inherited susceptibility to obesity through the production of an aberrant fusion protein that has the capacity to interfere with central melanocortin signalling.

Context: Mutations in the melanocortin-4 receptor (MC4R) represent the commonest genetic form of obesity and are associated with hyperphagia. Objective: The aim of this study was to investigate whether melanocortin signaling modulates anticipatory food reward by studying the brain activation response to food cues in individuals with MC4R mutations. Design/Setting/Participants/Main Outcome Measure: We used functional magnetic resonance imaging to measure blood oxygen level-dependent responses to images of highly palatable, appetizing foods, bland foods, and non-food objects in eight obese individuals with MC4R mutations, 10 equally obese controls, and eight lean controls with normal MC4R genotypes. Based on previous evidence, we performed a region-of-interest analysis centered on the caudate/putamen (dorsal striatum) and ventral striatum. Results: Compared to non-foods, appetizing foods were associated with activation in the dorsal and ventral striatum in lean controls and in MC4R-deficient individuals. Surprisingly, we observed reduced activation of the dorsal and ventral striatum in obese controls relative to MC4R-deficient patients and lean controls. There were no group differences for the contrast of disgusting foods with bland foods or non-foods, suggesting that the effects observed in response to appetizing foods were not related to arousal. Conclusion: We identified differences in the striatal response to food cues between two groups of obese individuals, those with and those without MC4R mutations. These findings are consistent with a role for central melanocortinergic circuits in the neural response to visual food cues.

OBJECTIVE: To investigate whether genetic variation at the UCP3 locus contributes to human obesity. SUBJECTS: Ninety-one obese children (BMI>4 standard deviations from age related mean) and 419 Caucasian adults from the Isle of Ely Study. DESIGN: Single strand conformation polymorphism (SSCP) analysis was used to scan the coding region of the UCP3 gene in 91 severely obese children. A common polymorphism identified in this gene (c-55t) has been shown to associate with lower UCP3 mRNA expression. Polymerase chain reaction-based forced restriction digestion was used to detect this allele in Caucasian adults. Multiple regression analysis was used to determine associations between the c-55t genotype and anthropometric, energetic and biochemical indices relevant to obesity. MEASUREMENTS: For the obese children, SSCP analysis and sequencing of variants were carried out. For the Isle of Ely Study, c-55t genotype and anthropometric (body mass index, waist–hip ratio, percentage body fat), energetic (dietary fat intake, physical activity index, adjusted metabolic rate, maximum oxygen consumption) and biochemical indices (pre- and post-glucose challenge plasma triglycerides, non-esterified fatty acids, insulin and glucose) were determined. RESULTS: A previously reported missense mutation (V102I) was detected in a single obese Afro-Carribean child. Twenty-one percent of the genes examined in the Isle of Ely study carried the c-55t promoter variant. Age-adjusted body mass index (BMI) was significantly (P=0.0037) lower in carriers of this variant. CONCLUSION: Mutations in the coding sequence of UCP3 are unlikely to be a common monogenic cause of severe human obesity. In a Caucasian population the UCP3 c-55t polymorphism is negatively associated with BMI. International Journal of Obesity (2001) 25, 472–477

Over 20 severely obese subjects in 11 independent kindreds have been reported to have pathogenic heterozygous mutations in the gene encoding the melanocortin 4 receptor (MC4R), making this the most common known monogenic cause of human obesity. To date, the detailed clinical phenotype of this dominantly inherited disorder has not been defined, and no homozygous subjects have been described. We determined the nucleotide sequence of the entire coding region of the MC4R gene in 243 subjects with severe, early-onset obesity. A novel two-base pair GT insertion in codon 279 was found in two unrelated subjects, and four novel missense mutations, N62S, R165Q, V253I, C271Y, and one mutation (T112M) reported previously were found in five subjects. N62S was found in homozygous form in five children with severe obesity from a consanguineous pedigree. All four heterozygous carriers were nonobese. Several features of the phenotype, e.g. hyperphagia, tendency toward tall stature, hyperinsulinemia, and preserved reproductive function, closely resemble those reported previously in Mc4r knock-out mice. In addition, a marked increase in bone mineral density was seen in all affected subjects. In transient transfection assays, the N62S mutant receptor showed a responsiveness to alphaMSH that was intermediate between the wild-type receptor and mutant receptors carrying nonsense and missense mutations associated with dominantly inherited obesity. Thus MC4R mutations result in a syndrome of hyperphagic obesity in humans that can present with either dominant or recessive patterns of inheritance.