Copy number variation (CNV) analysis and mutation analysis of the 6q14.1-6q16.3 genes SIM1 and MRAP2 in Prader Willi like patients.

BACKGROUND: Prader-Willi syndrome (PWS), caused by a paternal defect on 15q11.2-q13, is the most common form of syndromic obesity. However, patients clinically diagnosed with PWS do not always show this defect on chromosome 15q and are therefore molecularly categorized as Prader Willi like (PWL). Deletions at 6q14.1-q16.3 encompassing MRAP2 and SIM1 were reported in some individuals with a PWL phenotype. In addition, a few mutations in SIM1 and MRAP2 were also previously identified in cohorts of obese individuals. Therefore, we decided to perform copy number variation analysis of the 6q14.1-6q16.3 region followed by mutation analysis of SIM1 and MRAP2 in a PWL cohort. METHODS: A genome-wide microarray analysis was performed in a group of 109 PWL patients. Next, we screened 94 PWL patients for mutations in SIM1 and MRAP2 using high-resolution melting curve analysis and Sanger sequencing. Additionally, 363 obese children and adolescents were screened for mutations in MRAP2. RESULTS: No gene harboring deletions were identified at the 6q14.1-q16.3 region in the 109 PWL patients. SIM1 mutation analysis resulted in the identification of one very rare nonsynonymous variant p.P352S (rs3734354). Another rare nonsynonymous variant, p.A40S, was detected in the MRAP2 gene. No variants were identified in the 363 obese individuals. CONCLUSIONS: In contrast to literature reports, no gene harboring deletions were identified in the SIM1 and MRAP2 regions in our PWL cohort. Secondly, taking into account their very low minor allele frequencies in public sequencing databases and the results of in silico prediction programs, further functional analysis of p.P352S found in SIM1 and p.A40S found in MRAP2 is useful. This would provide further support for a possible role of SIM1 and MRAP2 in the pathogenesis of the PWL phenotype albeit in a limited number of patients.

Nucleotide variation of sFRP5 gene is not associated with obesity in children and adolescents.

Because sFRP5 was shown to be an important extracellular modulator of the Wnt pathway, regulating adipogenesis, we wanted to investigate the role of sFRP5 variants in human, monogenic obesity by performing mutation analysis. We screened the complete sFRP5 coding region in 622 obese children and adolescents and 503 lean control individuals by high-resolution melting curve analysis and direct sequencing. We found a total of 15 sequence variants in sFRP5, 10 of which resulted in a non-synonymous amino acid change. Five of these variants were, to our knowledge, not previously reported. For one of the variants (c.-3G>A), we identified a trend towards association between the variant frequency and the obese phenotype. We argue that, when looking at conservation and location inside known protein domains, several of the identified variants (D103N, A113V, K212N and H317L), may affect sFRP5 protein function. In addition, we found c.-3G>A, residing in the Kozak sequence, with a lower frequency in cases compared to controls. However, functional studies investigating the effect of sFRP5 variants on protein function are necessary to determine the true role of sFRP5 genetic variation in human, monogenic obesity.

Genetic and structural variation in the SH2B1 gene in the Belgian population.

OBJECTIVE: Animal studies, genome-wide association and genomic structural variation studies have identified the SH2B1 gene as a candidate gene for obesity. Therefore, we have designed an extensive mutation and copy number variation (CNV) analysis investigating the prevalence of genetic and structural variations in SH2B1 in the Belgian population. DESIGN AND METHODS: In the first part of this study, we performed a mutation screen for variants in the SH2B1 coding region in 581 obese children and adolescents and 433 healthy, lean individuals with high-resolution melting curve analysis followed by direct sequencing. In the second part of this study, Multiplex Amplicon Quantification (MAQ) analysis was used to identify CNVs in the distal SH2B1-containing chr.16p11.2 region in 421 obese children and adolescents with no developmental delay or behavioral phenotype. RESULTS: Mutation analysis resulted in the identification of fifteen rare non-synonymous heterozygous variants. Several of these were found both in lean and obese subjects, suggesting that these are neutral polymorphisms. However, six private, heterozygous, non-synonymous variations were present in obese children only. Furthermore, we also identified six missense variants solely in lean individuals. CNV analysis could not identify carriers of the distal 16p11.2 deletion in our population. CONCLUSION: Our mutation analysis has demonstrated that variation in the SH2B1 gene is frequent in both lean and obese groups, with distinctive variations being present on either side of the weight spectrum. Although the equal variation frequency does not immediately support disease causality, it cannot be excluded that some variations are weight-increasing or -decreasing. Further functional testing of the variants will be necessary to fully understand the impact of these variants on SH2B1. We were not able to detect carriers of the distal 16p11.2 deletion in our study population. As we excluded patients with developmental or behavioral problems, we suggest that in addition to obesity, the distal deletion might predispose for these traits. Further characterization of the phenotype is therefore necessary to clearly identify the phenotype of the distal 16p11.2 microdeletion syndrome.

Resistin polymorphisms show associations with obesity, but not with bone parameters in men: results from the Odense Androgen Study.

Resistin is an obesity-related adipokine which has also been implicated in bone metabolism. Therefore, we designed a study to investigate the possible role of resistin gene variation in both obesity and bone mineral density. We included 1,155 individuals from the Odense Androgen Study (663 young subjects and 492 older subjects), a population-based, prospective, observational study on the inter-relationship between endocrine status, body composition, muscle function, and bone metabolism in men, in an association study with resistin (RETN) polymorphisms. Three RETN variants (rs1862513, rs3745367 and rs3745369) were genotyped with TaqMan Pre-Designed Genotyping assays. Linear regression was performed to investigate the possible association of these variants with several obesity- and bone-related parameters. After genotyping 1,155 Danish men, 663 young subjects and 492 older subjects, we found that rs3745367 was associated with several obesity-related measures in both the young and elderly cohort. Rs3745369 was only associated with obesity-phenotypes in the elderly cohort. When studying the combined cohorts, we could confirm the associations of rs3745367 with several obesity-related parameters. We were unable to identify any association between RETN polymorphisms and bone-related measurements. Together, these results illustrate resistin's role in the development of obesity. Rs3745367 gives the most consistent results in the current study and these should be confirmed in other populations. Research into its possible functional effect might also be required. A role for RETN variants in determining bone mineral density seems unlikely from our results.

No important role for genetic variation in the Chibby gene in monogenic and complex obesity.

Chibby (CBY) has been identified as a potent proadipogenic factor required for adipocyte differentiation. It has been shown that CBY inhibits the canonical Wnt pathway, and therefore promotes the development of new fat cells. Our objective therefore is to investigate the contribution of rare and common genetic variation in CBY to the development of human obesity. A mutation analysis was performed on a total of 566 obese patients and 432 lean individuals. To investigate the involvement of CBY in complex obesity, we performed a genetic association analysis of the entire CBY gene region on 1,011 obese individuals and 523 control samples. Four rare, novel variants were identified in either obese patients or lean control subjects, among which two non-synonymous variations and one frameshift mutation. In addition, four previously reported CBY variants were found. In the association analysis, logistic and linear regression showed no association between common genetic variation in CBY and obesity parameters. Several novel variations were found, but no definite role in the pathogenesis of obesity could be confirmed. Results from the association analysis suggest that common variation in CBY is not a cause for obesity in the Belgian population.

Monogenic and complex forms of obesity: insights from genetics reveal the leptin-melanocortin signaling pathway as a common player.

The role of genetic factors involved in the etiology of human obesity is beyond question. The identification of the hypothalamic leptin-melanocortin signaling pathway as a critical regulator in energy homeostasis and food intake has been essential for genetic research. In this review, we discuss the involvement of established and novel genes from this pathway in the pathogenesis of obesity. Their roles in monogenic and complex forms of obesity are illustrated by discussing the results of mutation analysis, candidate gene and genome-wide association studies, as well as copy number analysis. While we can conclude from these outcomes that the leptin-melanocortin pathway is of immense importance, there is still a lot of heritability that currently cannot be explained. Future studies implementing genome-wide association studies, genome-wide copy number variant analysis, and whole exome and whole-genome sequencing might aid in finding new variation in the conventional pathways and might reveal new biological pathways implicated in the pathogenesis of obesity.

Identification of mutations in the NUCB2/nesfatin gene in children with severe obesity.

Nesfatin-1 is the N-terminal fragment of nucleobindin-2 (NUCB2) that was identified as a novel satiety molecule in rodents. The protein is reported to exert anorexigenic effects and appears to play an important role in hypothalamic pathways regulating energy homeostasis and food intake. In this study, we hypothesized that mutations in the nesfatin encoding gene NUCB2 might cause obesity in humans. Therefore, we screened the entire coding region of the NUCB2 gene for mutations in a population of 471 obese children and adolescents. Mutation analysis of NUCB2 identified a total of seven sequence variants of which four were previously reported as polymorphisms. The remaining three variants included ex9+6G>C, L125H and K178X and were found in 3 unrelated individuals in the obese population only (0.6%). Biochemical experiments including ELISA and western blot were performed on plasma samples of the obese patient carrying the nonsense mutation K178X. However, neither NUCB2/nesfatin-1 immunoreactive plasma levels of the patient, nor expression of full length NUCB2 differed significantly from matched obese control individuals. In conclusion, we have identified the first genetic variants in the NUCB2 gene in obese individuals, although further functional characterization will be essential to verify disease causality of the mutations.

Association between polymorphisms of the Nesfatin gene, NUCB2, and obesity in men.

Nesfatin-1, which originates from its precursor protein nucleobindin-2 (NUCB2), is a novel appetite-regulating molecule that might be associated with the melanocortin signalling pathway in the hypothalamus. The secreted protein appears to play an important role in metabolic control through its anorexigenic and anti-hyperglycemic effects. Therefore, we hypothesized that polymorphisms in the NUCB2 gene might influence the susceptibility for the development of obesity. In this study, we investigated the association of NUCB2 polymorphisms with the development of obesity in an extensive Caucasian population comprising 1049 obese subjects and 315 normal weight control individuals. We selected 8 tagSNPs, which after additional analysis of 6 multi-marker tests, cover most information on common genetic variation in the selected region. We found association with obesity for 3 SNPs (rs1330, rs214101 and rs757081) and 3 multi-marker tests, only when analyzing the male population separately. We subsequently performed linear regression analysis, again in the male population only, and found that several SNPs were associated with BMI, weight and fat free mass. These data indicate that polymorphisms in the NUCB2 gene could play an important role in the protection against the development of obesity in male subjects and might have an influence on energy homeostasis. Nevertheless, further research including replication of our results and elucidation of the molecular mechanism remains necessary.

Association study of MC4R with complex obesity and replication of the rs17782313 association signal.

Recently, genome-wide association studies have discovered several single nucleotide polymorphisms (SNPs) involved in the etiology of complex obesity. A variant downstream from the melanocortin-4 receptor gene (MC4R), a gene known to be involved in monogenic obesity, was reported to be highly associated with BMI. In the present study, we performed a replication study with the previously reported SNP rs17782313. We also included 3 tagSNPs (rs8087522, rs11872992, and rs1943226) for the MC4R gene region in our study to understand the role of this gene in complex obesity. We genotyped all 4 SNPs in a population of 1049 obese cases (mean BMI=38.2±6.2) and 312 healthy lean individuals (mean BMI 22.0±1.7). We could confirm that rs17782313 is highly associated with complex obesity in our population (odds ratio=1.42, 95% CI 1.14-1.77, P=0.002). Furthermore, we found this SNP to be associated with BMI (B=0.92, 95% CI 0.19-1.65, P=0.01) and body weight (B=2.44, 95% CI 0.28-4.60, P=0.03). In addition, we could also detect an association between rs11872992 and complex obesity (odds ratio=0.74, 95% CI 0.57-0.98, P=0.03). Through conditional analysis, we demonstrate that this effect is independent from the rs17782313 association signal. No associations with obesity could be found for rs8087522 and rs1943226. In conclusion, we could replicate the previously reported association between rs17782313 and complex obesity. Furthermore, our data do not support the hypothesis that a SNP in MC4R causes the rs17782313 association signal.

The role of the leptin-melanocortin signalling pathway in the control of food intake.

Obesity is one of the most important health problems today. Obesity is mostly caused by a complex interaction between environmental and genetic factors. However, several monogenic forms of obesity also exist. The mutations causing these forms of obesity were all found in genes involved in the leptin-melanocortin pathway: leptin, leptin receptor, proopiomelanocortin, prohormone convertase 1, and melanocortin-4 receptor. Recently, several novel players with a role in this pathway have been identified and have increased our knowledge on the regulation of food intake. These include the melanocortin-3 receptor, BDNF, SIM1, and nesfatin-1. In this review, we will discuss the most important players involved in this pathway. We will focus on genetic studies concerning mouse models involving these genes and reported human variation in these genes. We intend to provide an extensive overview of all currently known proteins with a significant role in this pathway. Together, these data demonstrate the importance of this pathway in the regulation of food intake and the pathogenesis of obesity.

Association of the BDNF Val66Met variation with obesity in women.

Brain-derived neurotrophic factor (BDNF) has been implied in the regulation of food intake. In the present study, we genotyped the Val66Met polymorphism in a Belgian cohort of 532 obese women and 197 healthy female controls and were, for the first time, able to show an association of the 66Met allele with obesity, at least in our female cohort.

CNV analysis and mutation screening indicate an important role for the NPY4R gene in human obesity.

OBJECTIVE: Genome-wide copy number variation (CNV) analyses have associated the 10q11.22 CNV with obesity. As the NPY4R gene is the most interesting candidate gene in this region, it was hypothesized that both genetic and structural variation in NPY4R might be implicated in the pathogenesis of obesity. METHODS: In the first part of this study, 326 children and adolescents with obesity and 298 healthy lean individuals were screened for CNV in the NPY4R-containing chr.10q11.22 region. In the second part of this study, a mutation screen for variants in the NPY4R coding region was performed in 356 children and adolescents with obesity and 337 healthy lean adults. RESULTS: Our CNV analysis demonstrated a significantly higher frequency of NPY4R containing 10q11.22 CNV loss in the patient population (P = 0.0003), while CNV gain in this region was more prevalent in the control population (P = 0.031). Mutation analysis resulted in the identification of 15 rare non-synonymous heterozygous variants. For two variants that could only be identified in the patient population, receptor dysfunction and thus a pathogenic effect were demonstrated. CONCLUSIONS: In conclusion, these data support an essential role for genetic and structural variation within the NPY4R gene in the pathogenesis of obesity.

Association study of PNPLA2 gene with histological parameters of NAFLD in an obese population.

INTRODUCTION: The prevalence of non-alcoholic fatty liver disease (NAFLD) and the closely associated metabolic syndrome is high and is related to risk factors such as obesity and type 2 diabetes. A genetic basis for NAFLD has been suggested, but only few causal genes have been identified. The most significant association reported to date is the robust association of the PNPLA3 I148M variant with susceptibility to NAFLD. We therefore hypothesized that the PNPLA2 gene might also be involved in NAFLD pathogenesis, because of its close sequence similarity with PNPLA3 and its possible involvement in ectopic fat accumulation. METHODS: In this study, we investigated the association of PNPLA2 polymorphisms with the development of non-alcoholic fatty liver disease in a prospectively recruited Belgian obese population comprising 633 individuals with varying degrees of fatty liver disease. We selected 3 PNPLA2 SNPs for genotyping, including 2 tagSNPs that cover most information on common genetic variation in the selected region. RESULTS: After performing linear regression analysis, we found that 2 of the analyzed PNPLA2 SNPs were associated with anthropometric and metabolic parameters. In our subcohort of patients that underwent liver biopsy (n=372/633 or 58.7%), we assessed the influence of the PNPLA2 variants on the severity of histologically determined liver damage, but we did not find convincing evidence for association. CONCLUSION: Although we found evidence for moderate association between PNPLA2 tagSNPs and anthropometric and metabolic parameters in our cohort, no evidence for association between polymorphisms in the PNPLA2 gene and the presence and severity of NAFLD was identified.

Screening for rare variants in the PNPLA3 gene in obese liver biopsy patients.

BACKGROUND: Previous research has clearly implicated the PNPLA3 gene in the etiology of nonalcoholic fatty liver disease as a polymorphism in the gene was found to be robustly associated to the disease. However, data on the involvement of rare PNPLA3 variants in the development of nonalcoholic fatty liver disease (NAFLD) is currently limited. Therefore, we performed an extensive mutation analysis study on a cohort of obese liver biopsy patients to determine PNPLA3 variation and its correlation with fatty liver disease. METHODS: We screened the entire coding region of the PNPLA3 gene in DNA samples of 393 obese liver biopsy patients with varying degrees of fatty liver disease. Mutation analysis was performed by high-resolution melting curve analysis in combination with direct sequencing. RESULTS: We identified several common polymorphisms as well as one rare synonymous variant (c.867G>A rs139896256), one rare intronic variant (c.979+13C>T) and 3 nonsynonymous coding variants (p.A76T, p.A104V and p.T200M) in the PNPLA3 gene. In silico analysis indicated that the p.A104V variant will probably have no functional effect, whereas for the p.A76T and p.T200M variant a possible pathogenic effect is suggested. CONCLUSION: Overall, we showed that novel variants in PNPLA3 are very rare in our liver biopsy cohort, thereby indicating that their impact on the etiology of NAFLD is probably limited. Nevertheless, for the three rare coding variants that were identified in patients with advanced liver disease, further functional characterization will be essential to verify their potential disease causality.

Investigation of common and rare genetic variation in the BAMBI genomic region in light of human obesity.

The aim of this study was to confirm the previously identified link between BAMBI and human obesity by means of a genetic and functional analysis. We performed both a mutation analysis, using high-resolution melting curve analysis, and a genetic association study, including 8 common tagSNPs in the BAMBI gene region. Three of the identified genetic variants (R151W, H201R, and C229R) were evaluated for their Wnt signaling enhancing capacity in a Wnt luciferase reporter assay. Mutation screening of the BAMBI coding region and exon-intron boundaries on our population of 677 obese children and adolescents and 529 lean control subjects resulted in the identification of 18 variants, 10 of which were not previously reported and 12 of which were exclusively found in obese individuals. The difference in variant frequency, not taking into account common polymorphisms, between obese (3.1 %) and lean (0.9 %) subjects was statistically significant (p = 0.004). Our Wnt luciferase assay, using WT and mutant BAMBI constructs, showed a significantly reduced activity for all of the investigated variants. Logistic and linear regression analysis on our Caucasian population of 1022 obese individuals and 606 lean controls, did not identify associations with obesity parameters (p values >0.05). We found several rare genetic variations, which represent the first naturally occurring missense variants of BAMBI in obese patients. Three variants (R151W, H201R, and C229R) were shown to reduce Wnt signaling enhancing capacity of BAMBI and we believe this result should encourage further study of this gene in other obese populations. In addition, we did not find evidence for the involvement of BAMBI common variation in human obesity in our population.

Genetic association study of WNT10B polymorphisms with BMD and adiposity parameters in Danish and Belgian males.

Because of the importance of the Wnt pathway in the development and maintenance of both adipose and bone tissue, we wanted to evaluate the involvement of WNT10B, a Wnt pathway activator, in adipogenesis and osteoblastogenesis in humans. Genetic association between WNT10B polymorphisms and adiposity parameters as well as bone mineral density (BMD) measurements was analysed in two independent populations. The first is a population of 1,228 Danish men (702 aged 20-29 years; 532 aged 60-74 years) from the Odense Androgen Study (OAS), which was designed as a cross-sectional, population-based study. The second population, called SIBLOS, includes 922 Belgian men (34 ± 5 years old) and contains siblings selected from over 500 families. Four tagSNPs (rs833840, rs833841, rs10875902 and rs4018511) that capture variation of ten SNPs (MAF > 5 %) in a 15.2 kb region spanning the WNT10B gene and its flanking regions were genotyped. Although no association with body mass index was found, we found all tagSNPs to be associated with BMD parameters (BMD whole body, total hip and femoral neck) and height in the OAS population. The association of rs10875902 was most prominent (nominal p = 0.012) and confirmed a previously shown negative effect on BMD. No significant associations were observed in the SIBLOS population. In the present study, no association between WNT10B polymorphisms and adiposity parameters was found. However, our results clearly illustrate a role for WNT10B variants in determining human BMD. The effect of WNT10B polymorphisms on height should be evaluated in additional populations.

Prevalence of rare MC3R variants in obese cases and lean controls.

The role of mutations in the melanocortin-3 receptor (MC3R) gene, which is implicated in the regulation of energy homeostasis, is still under debate. Animal studies have clearly proven that, together with the melanocortin-4 receptor (MC4R), the MC3R is a critical receptor for melanocortin peptides within the leptin-melanocortin signaling cascade. However, as several mutations have been found in lean individuals and not all mutations seem to cause receptor dysfunction, results from mutation screens in obese humans remain controversial. In the present study, we screened for rare variants in the MC3R gene of obese children and lean controls to assess the prevalence of MC3R mutations in the Belgian population. We screened 249 severely overweight and obese children and adolescents and 239 lean adults for mutations in the coding region of MC3R. Mutation screening was performed by high resolution melting curve analysis and direct sequencing. We identified four non-synonymous coding variations in the obese population, all of which had been reported previously. In addition, we also found four novel rare MC3R variants in the lean control population, suggesting that not all MC3R mutations are disease-causing. Overall, the total prevalence of rare MC3R variants was 1 % in Belgian obese children and adolescents compared to 1.02 % in lean controls. Ultimately, cosegregation studies combined with comprehensive functional analysis is required to determine the potential pathogenic role of rare MC3R variants in causing human obesity.

Mutation analysis of WNT10B in obese children, adolescents and adults.

Wingless-type MMTV integration site family, member 10B (WNT10B) is an activator of the Wnt pathway. The Wnt pathway is known to play an important role in maintenance and differentiation of stem cells and has been implicated in the origination of obesity. To evaluate the role of genetic variation in WNT10B in obesity further, we performed a mutation analysis on Belgian obese patients and control subjects. A mutation analysis of WNT10B by means of high-resolution melting curve analysis and direct sequencing was performed on 546 obese children and adolescents (mean Z-score of 2.6 ± 0.6 and 2.5 ± 0.4 respectively), 86 morbidly obese adults (mean BMI of 48.0 ± 0.4 kg/m(2)) and 447 lean, healthy controls (mean BMI of 22.1 ± 1.7 kg/m(2)). A total of five novel non-synonymous variants were identified. R228Q was the only coding, non-synonymous variant that was exclusively found in patients, but the variant did not co-segregate with obesity in the three investigated siblings. The remaining four variants were either found both in cases and in control samples (G181D) or only in control samples (A108P, S187R and P315S). The frequency of non-synonymous variants in lean individuals (0.9 %) was higher than in obese individuals (0.3 %) and familial co-segregation of the most promising variant in patients could not be demonstrated. Therefore, we conclude that variations in WNT10B do not contribute to human monogenic obesity in our population.

Replication of the SH2B1 rs7498665 association with obesity in a Belgian study population.

OBJECTIVE: SH2B1 has been identified as an interesting candidate gene for complex obesity through genome-wide association studies. Therefore, we set out to replicate the reported association with rs7498665 in our Belgian study population and to extend our study with an additional tagSNP for the SH2B1 gene region. METHODS: We genotyped both rs7498665 and rs7201929 in a population of 1,045 obese adults and 317 healthy lean individuals. Statistical analyses were performed to evaluate the role of these polymorphisms in the development of obesity. RESULTS: We found that the rs7498665 minor allele increases obesity risk by 26% (OR(age-sex adj) = 1.26, 95% CI 1.04-1.52, nominal p = 0.016). Logistic regression showed that the rs7201929 minor allele decreases obesity risk by 24% in the population investigated (OR(age-sex adj) = 0.76, 95% CI 0.61-0.94, nominal p = 0.011). Conditional analyses showed that both associations represent the same association signal (rs7498665 OR(adjusted for rs7201929) = 1.17, 95% CI 0.95-1.45, nominal P = 0.14; rs7201929 OR(adjusted for rs7498665) = 0.82, 95% CI 0.65-1.04, nominal p = 0.10). CONCLUSION: With the current study we were able to replicate and confirm that the SH2B1 gene locus is significantly associated with complex obesity in a Caucasian population.

Identification of three novel genetic variants in the melanocortin-3 receptor of obese children.

The melanocortin-3 receptor (MC3R), a G-protein-coupled receptor expressed in the hypothalamus, is a key component of the leptin-melanocortin pathway that regulates energy homeostasis. It is suggested that an MC3R defect leads to an increased feed efficiency, by which nutrients are partitioned preferentially into fat. In this study, we hypothesized that early-onset obesity could be induced by mutations in MC3R. To investigate this hypothesis, we screened the entire coding region of the MC3R gene for mutations in obese subjects. A total of 404 overweight and obese children and adolescents, 86 severely obese adults (BMI ≥40 kg/m²), and 150 normal-weight control adults were included. Besides three synonymous coding variations in the MC3R gene (S69S, L95L, I226I), we were able to identify three novel heterozygous, nonsynonymous, coding mutations (N128S, V211I, L299V) in three unrelated obese children. None of these mutations were found in any of the control subjects. Functional studies assessing localization and signaling properties of the mutant receptors provided proof for impaired function of the L299V mutated receptor, whereas no conclusive evidence for functional impairment of the N128S and V211I mutated receptors could be established. First, these results provide supporting evidence for a role of the MC3R gene in the pathogenesis of obesity in a small subset of patients. Second, they show that caution is called for the interpretation of newly discovered mutations in MC3R.