https://www.cell.com/cell/pdf/S0092-8674(07)00447-3.pdf

The Hormonal Control of Food Intake

The melanocortin-4 receptor (MC4R) was cloned in 1993 by degenerate PCR; however, its function was unknown. Subsequent studies suggest that the MC4R might be involved in regulating energy homeostasis. This hypothesis was confirmed in 1997 by a series of seminal studies in mice. In 1998, human genetic studies demonstrated that mutations in the MC4R gene can cause monogenic obesity. We now know that mutations in the MC4R are the most common monogenic form of obesity, with more than 150 distinct mutations reported thus far. This review will summarize the studies on the MC4R, from its cloning and tissue distribution to its physiological roles in regulating energy homeostasis, cachexia, cardiovascular function, glucose and lipid homeostasis, reproduction and sexual function, drug abuse, pain perception, brain inflammation, and anxiety. I will then review the studies on the pharmacology of the receptor, including ligand binding and receptor activation, signaling pathways, as well as its regulation. Finally, the pathophysiology of the MC4R in obesity pathogenesis will be reviewed. Functional studies of the mutant MC4Rs and the therapeutic implications, including small molecules in correcting binding and signaling defect, and their potential as pharmacological chaperones in rescuing intracellularly retained mutants, will be highlighted.

The Melanocortin-4 Receptor: Physiology, Pharmacology, and Pathophysiology

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. The authors 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. They also examined the effects of an MC4R agonist administered for 7 days in 28 overweight or obese volunteers.

Modulation of blood pressure by central melanocortinergic pathways.

The frequency of a haplotype comprising one allele at each of two loci can be expressed as a cubic equation (the 'Hill equation'), the solution of which gives that frequency. Most haplotype and linkage disequilibrium analysis programs use iteration-based algorithms which substitute an estimate of haplotype frequency into the equation, producing a new estimate which is repeatedly fed back into the equation until the values converge to a maximum likelihood estimate (expectation-maximisation). We present a program, "CubeX", which calculates the biologically possible exact solution(s) and provides estimated haplotype frequencies, D', r2 and χ2 values for each. CubeX provides a "complete" analysis of haplotype frequencies and linkage disequilibrium for a pair of biallelic markers under situations where sampling variation and genotyping errors distort sample Hardy-Weinberg equilibrium, potentially causing more than one biologically possible solution. We also present an analysis of simulations and real data using the algebraically exact solution, which indicates that under perfect sample Hardy-Weinberg equilibrium there is only one biologically possible solution, but that under other conditions there may be more. Our analyses demonstrate that lower allele frequencies, lower sample numbers, population stratification and a possible |D'| value of 1 are particularly susceptible to distortion of sample Hardy-Weinberg equilibrium, which has significant implications for calculation of linkage disequilibrium in small sample sizes (eg HapMap) and rarer alleles (eg paucimorphisms, q < 0.05) that may have particular disease relevance and require improved approaches for meaningful evaluation.

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Cubic exact solutions for the estimation of pairwise haplotype frequencies: implications for linkage disequilibrium analyses and a web tool 'CubeX'

The global obesity epidemic is clearly driven by environmental factors; however, inherited factors can also influence human adiposity. In this Review, the authors focus on the genes implicated in monogenic obesity syndromes. These genes encode components of the leptin–melanocortin pathway, which is critical for regulation of food intake and body weight. Obesity is associated with increased morbidity and mortality from cardiovascular disease, diabetes mellitus and certain cancers. The prevalence of obesity is increasing rapidly throughout the world and is now recognized as a major global public-health concern. Although the increased prevalence of obesity is undoubtedly driven by environmental factors, the evidence that inherited factors profoundly influence human fat mass is equally compelling. Twin and adoption studies indicate that up to 70% of the interindividual variance in fat mass is determined by genetic factors. Genetic strategies can, therefore, provide a useful tool with which to dissect the complex (and often heterogeneous) molecular and physiologic mechanisms involved in the regulation of body weight. In this Review, we have focused our attention on monogenic disorders, which primarily result in severe, early-onset obesity. The study of these genetic disorders has provided a framework for our understanding of the mechanisms involved in the regulation of body weight in humans and how these mechanisms are disrupted in obesity. The genes affected in these monogenic disorders all encode ligands and receptors of the highly conserved leptin–melanocortin pathway, which is critical for the regulation of food intake and body weight.

Mutations in ligands and receptors of the leptin-melanocortin pathway that lead to obesity.

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.

Prevalence and functionality of paucimorphic and private MC4R mutations in a large, unselected European British population, scanned by meltMADGE.

384-well MADGE for high-throughput DNA-bank studies

High-resolution MADGE

Microplate-array diagonal-gel electrophoresis (MADGE) systems for high-throughput electrophoresis

The first era of DNA research, from the discovery of DNA itself by Friedrich Miescher in 1869 (Harbers 1969) to the completion of the human genome sequencing project scheduled for the year 2005 (Table 2.1), is about to end. The argument put forward in this chapter is that the existing DNA sampling and storing techniques were set up to satisfy different goals and needs than those that will be required in the forth coming era of genomics and of large-scale population genetics. The human genome diversity project aims at the definition and explanation of every polymorphism, every rare or sporadic germ-line or somatic mutation, every Mendelian, polygenic or complex disease and every inherited phenotypic trait on a global scale (Cavalli-Sforza et al. 1991; Kidd et al. 1993; Cavalli-Sforza 1998; Weiss 1998). Such projects may seem immense today but so did the various genome projects a few years ago. Research for high-throughput analytical methodologies is producing most encouraging results. There are already analytical techniques for genotyping several thousands of individuals per technician per day (Day et al. 1998) and DNA extraction is becoming a bottleneck of progress. 
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Landmarks in DNA research

Emmanuel Spanakis

Papers

Prevalence and functionality of paucimorphic and private MC4R mutations in a large, unselected European British population, scanned by meltMADGE.

384-well MADGE for high-throughput DNA-bank studies

High-resolution MADGE

Microplate-array diagonal-gel electrophoresis (MADGE) systems for high-throughput electrophoresis

Human DNA Sampling and Banking