OBJECTIVE-Melanocortin-4 receptor (MC4R) deficiency is the most frequent genetic cause of obesity. However, there is uncertainty regarding the degree of penetrance of this condition, and the putative impact of the environment on the development of obesity in MC4R mutation carriers is unknown.RESEARCH DESIGN AND METHODS-We determined the MC4R sequence in 2,257 obese individuals and 2,677 nonobese control subjects of European origin and established the likely functional impact of all variants detected. We then included relatives of probands carriers and studied 25 pedigrees, including 97 carriers and 94 noncarriers from three generations.RESULTS-Of the MC4R nonsynonymous mutations found in obese subjects, 68% resulted in a loss of function in vitro. They were found in 1.72% of obese versus 0.15% of nonobesed subjects (P ϭ 6.9 ϫ 10 Ϫ10 ). Among the families, abnormal eating behavior was more frequent in both MC4R-deficient children and adults than in noncarriers. Although BMI was inversely associated with educational status in noncarrier adults, no such relationship was seen in MC4R mutation carriers. We observed a generational effect, with a penetrance of 40% in MC4R-deficient adults aged Ͼ52 years, 60% in 18-to 52-year-old adults, and 79% in children. The longitudinal study of adult carriers showed an increasing age-dependent penetrance (37% at 20 years versus 60% at Ͼ40 years).CONCLUSIONS-We have established a robust estimate of age-related penetrance for MC4R deficiency and demonstrated a generational effect on penetrance, which may relate to the development of an "obesogenic" environment. It remains to be seen whether appropriate manipulation of environmental factors may contribute to preventing the development of obesity even in those strongly genetically predisposed to it.

Objective: Genetic components of energy homeostasis contributing to childhood obesity are poorly understood. Genome scans were performed to identify chromosomal regions contributing to physical activity and dietary intake traits in Hispanic children participating in the VIVA LA FAMILIA Study. Research Methods and Procedures: We report linkage findings on chromosome 18 for physical activity and dietary intake in 1030 siblings from 319 Hispanic families. Measurements entailed physical activity by accelerometry, dietary intake by two 24‐hour recalls, and genetic linkage analyses using SOLAR software. Results: Significant heritabilities were seen for physical activity and dietary intake, ranging from 0.46 to 0.69, except for vigorous activity (h2 = 0.18). Percentage time in sedentary activity mapped to markers D18S1102‐D18S64 on chromosome 18 [logarithm of the odds (LOD) score = 4.07], where melanocortin 4 receptor gene (MC4R) resides. Quantitative trait loci (QTLs) for total activity counts, percentage time in light or in moderate activity, and carbohydrate intake and percentage of energy intake from carbohydrates were detected in the same region (LOD = 2.28, 2.79, 2.2, 1.84, and 1.51, respectively). A novel loss of function mutation in MC4R (G55V) was detected in six obese relatives, but not in the rest of the cohort. Removal of these MC4R‐deficient subjects from the analysis reduced the LOD score for sedentary activity to 3.94. Discussion: Given its role in the regulation of food intake and energy expenditure, MC4R is a strong positional candidate gene for the QTL on chromosome 18 detected for physical activity and dietary intake in Hispanic children.

Mutations in the melanocortin 4 receptor (MC4R) gene are the most common known cause of monogenic human obesity. The MC4R gene was sequenced in 2000 subjects with severe early-onset obesity. We detected seven different nonsense and 19 nonsynonymous mutations in a total of 94 probands, some of which have been reported previously by others. We functionally characterized the 11 novel obesity associated missense mutations. Seven of these mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) showed impaired cell surface trafficking, reduced level of maximal binding of the radioligand [125I]NDP-MSH, and reduced ability to generate cAMP in response to ligand. Four mutant MC4Rs (G55V, G55D, S136F, and A303T) displayed cell surface expression and agonist binding similar to the wild-type receptor but showed impaired cAMP production, suggesting that these residues are likely to be critical for conformational rearrangement essential for receptor activation. Homology modeling of these mutants using a model of MC4R based on the crystal structure of the beta2-adrenoreceptor was used to provide insights into the possible structural basis for receptor dysfunction. Transmembrane (TM) domains 1, 3, 6, 7, and peripheral helix 8 appear to participate in the agonist-induced conformational rearrangement necessary for coupling of ligand binding to signaling. We conclude that G55V, G55D, S136F, and A303T mutations are likely to strengthen helix-helix interactions between TM1 and TM2, TM3 and TM6, and TM7 and helix 8, respectively, preventing relative movement of these helices during receptor activation. The combination of functional studies and structural modeling of naturally occurring pathogenic mutations in MC4R can provide valuable information regarding the molecular mechanism of MC4R activation and its dysfunction in human disease.

Identification of unknown mutations has remained laborious, expensive, and only viable for studies of selected cases. Population-based "reference ranges" of rarer sequence diversity are not available. However, the research and diagnostic interpretation of sequence variants depends on such information. Additionally, this is the only way to determine prevalence of severe, moderate, and silent mutations and is also relevant to the development of screening programs. We previously described a system, meltMADGE, suitable for mutation scanning at the population level. Here we describe its application to a population-based study of MC4R (melanocortin 4 receptor) mutations, which are associated with obesity. We developed nine assays representing MC4R and examined a population sample of 1,100 subjects. Two "paucimorphisms" were identified (c.307G>A/p.Val103Ile in 27 subjects and c.-178A>C in 22 subjects). Neither exhibited any anthropometric effects, whereas there would have been >90% power to detect a body mass index (BMI) effect of 0.5 kg/m(2) at P=0.01. Two "private" variants were also identified. c.335C>T/p.Thr112Met has been previously described and appears to be silent. A novel variant, c.260C>A/p.Ala87Asp, was observed in a subject with a BMI of 31.5 kg/m(2) (i.e., clinically obese) but not on direct assay of a further 3,525 subjects. This mutation was predicted to be deleterious and analysis using a cyclic AMP (cAMP) responsive luciferase reporter assay showed substantial loss of function of the mutant receptor. This population-based mutation scan of MC4R suggests that there is no severe MC4R mutation with high prevalence in the United Kingdom, but that obesity-causing MC4R mutation at 1 in 1,100 might represent one of the commonest autosomal dominant disorders in man.

We previously reported a family in which a heterozygous missense mutation in Akt2 led to a dominantly inherited syndrome of insulin-resistant diabetes and partial lipodystrophy. To determine whether genetic variation in AKT2 plays a broader role in human metabolic disease, we sequenced the entire coding region and splice junctions of AKT2 in 94 unrelated patients with severe insulin resistance, 35 of whom had partial lipodystrophy. Two rare missense mutations (R208K and R467W) were identified in single individuals. However, insulin-stimulated kinase activities of these variants were indistinguishable from wild type. In two large case-control studies (total number of participants 2,200), 0 of 11 common single nucleotide polymorphism (SNPs) in AKT2 showed significant association with type 2 diabetes. In a quantitative trait study of 1,721 extensively phenotyped individuals from the U.K., no association was found with any relevant intermediate metabolic trait. In summary, although heterozygous loss-offunction mutations in AKT2 can cause a syndrome of severe insulin resistance and lipodystrophy in humans, such mutations are uncommon causes of these syndromes. Furthermore, genetic variation in and around the AKT2 locus is unlikely to contribute significantly to the risk of type 2 diabetes or related intermediate metabolic traits in U.K. populations.

Sex-specific elimination of cells by apoptosis plays a role in sex determination in Caenorhabditis elegans. Recently, a mammalian pro-apoptotic protein named F1A␣ has been identified. F1A␣ shares extensive homology throughout the entire protein with the C. elegans protein, FEM-1, which is essential for achieving all aspects of the male phenotype in the nematode. In this report, the role of FEM-1 in apoptosis was investigated. Overexpression of FEM-1 induces caspase-dependent apoptosis in mammalian cells. FEM-1 is cleaved in vitro by the C. elegans caspase, CED-3, generating an N-terminal cleavage product that corresponds to the minimal effector domain for apoptosis. Furthermore, CED-4 associates with FEM-1 in vitro and in vivo in mammalian cells and potentiates FEM-1-mediated apoptosis. Similarly, Apaf-1, the mammalian homologue of CED-4 was found to associate with F1A␣. These data suggest that FEM-1 and F1A␣ may mediate apoptosis by communicating directly with the core machinery of apoptosis.

ObjectiveRegulation of energy balance depends on pro-opiomelanocortin (POMC)-derived peptides and melanocortin-4 receptor (MC4R). Alpha-melanocyte stimulating hormone (α-MSH) is the predicted natural POMC-derived peptide that regulates energy balance. Desacetyl-α-MSH, the precursor for α-MSH, is present in brain and blood. Desacetyl-α-MSH is considered to be unimportant for regulating energy balance despite being more potent (compared with α-MSH) at activating the appetite-regulating MC4R in vitro. Thus, the physiological role for desacetyl-α-MSH is still unclear.MethodsWe created a novel mouse model to determine whether desacetyl-α-MSH plays a role in regulating energy balance. We engineered a knock in targeted QKQR mutation in the POMC protein cleavage site that blocks the production of both desacetyl-α-MSH and α-MSH from adrenocorticotropin (ACTH1-39).ResultsThe mutant ACTH1-39 (ACTHQKQR) functions similar to native ACTH1-39 (ACTHKKRR) at the melanocortin 2 receptor (MC2R) in vivo and MC4R in vitro. Male and female homozygous mutant ACTH1-39 (Pomctm1/tm1) mice develop the characteristic melanocortin obesity phenotype. Replacement of either desacetyl-α-MSH or α-MSH over 14 days into Pomctm1/tm1 mouse brain significantly reverses excess body weight and fat mass gained compared to wild type (WT) (Pomcwt/wt) mice. Here, we identify both desacetyl-α-MSH and α-MSH peptides as regulators of energy balance and highlight a previously unappreciated physiological role for desacetyl-α-MSH.ConclusionsBased on these data we propose that there is potential to exploit the naturally occurring POMC-derived peptides to treat obesity but this relies on first understanding the specific function(s) for desacetyl-α-MSH and α-MSH.

Our data suggest that genotyping studies to investigate clopidogrel response should include CYP2C19*2 and *3 but not *17 polymorphisms in Chinese, and CYP2C19*2 and *17 polymorphisms but not *3 in Indians. All three polymorphisms should preferably be genotyped in Malays.