Melanocortin 3 receptor gene and melanocortin 4 receptor gene mutations: the Asian Perspective
Associate Professor Lee Yung Seng,
Department of Paediatrics,
Yong Loo Lin School of Medicine,
NUHS Tower Block, Level 12,
1E Kent Ridge Road,
Singapore 119228, Singapore
Melanocortin 4 receptor (MC4R) deficiency resulting from disruption of one or both MC4R alleles represents the commonest monogenic form of human obesity to date. Human MC4R deficiency was reported to affect 4 and 5.8% of severely obese French and British populations respectively. However, studies elsewhere reported low incidence of MC4R mutations in their obese populations. The significance of MC4R mutations in Asian obese populations has not been adequately examined, though small studies in Japan, China, and Singapore reported few or no pathogenic mutations, suggesting a low prevalence in this part of the world. There were also few common mutations described across populations, suggesting a relative lack of founder effect. The pathogenic role of melanocortin 3 receptor gene (MC3R) mutations in human obesity is not as well described and accepted as MC4R mutations, though it is gradually gaining ground. Two common single nucleotide polymorphisms Thr6Lys and Val81Ile within the coding region were associated with higher body fat and leptin levels in obese children, supported by impaired signaling activity in vitro. There were also reports of missense mutations enriched in obese populations. While MC3R mutations are unlikely to result in an autosomal dominant form of monogenic obesity given the lack of strong co-segregation in family studies, the studies so far provided evidence that MC3R can be one of the genes which contributes to increased adiposity, and exert an effect on the human phenotype.
The pathogenesis of obesity is multifactorial, where our modern undesirable eating habits and sedentary lifestyle interact with an underlying genetic susceptibility to result in the obese phenotype. While this genetic predisposition is likely polygenic in the vast majority of obese individuals, it has been estimated from the body of data accumulated to date that about 5% may have genetic obesity, who harbour single gene defects resulting in disruption of the biological weight regulation mechanism. While the search for obesity genes has posed a major challenge, we have witnessed significant milestones in obesity gene research in the last decade, in the discovery of novel single gene defects which result in human obesity, such as leptin deficiency, leptin receptor deficiency, proopiomelanocortin (POMC) deficiency, prohormone convertase 1 deficiency (PC1), and melanocortin 4 receptor (MC4R) deficiency. These monogenic forms of human obesity resulted in deficiency of critical molecules and disruption of the leptin-melanocortin system which lead to the obese phenotype, and thus provide validation of the role of the leptin-melanocortin system in energy homeostasis, and unravel the molecular circuitry of human weight regulation.
The Leptin-Melanocortin system
Various human and murine genetic studies have shed light on the biological weight regulation mechanism, akin to pieces of a jigsaw puzzle being put together which progressively unravel this integral system. Excess food intake is stored in adipose tissue. Adipose tissue secretes leptin in response to increased fat storage, which circulates as an afferent satiety signal and activates hypothalamic neurons expressing pro-opiomelanocortin (POMC) located in the arcuate nucleus, which innervates other hypothalamic regions known to regulate feeding behaviour [2–4]. Pro-opiomelanocortin (POMC) is a polypeptide that undergoes tissue-specific post-translational processing, the products of which include the melanocortin peptides α, β, and γ-melanocyte-stimulating hormones (MSH) . One or more of the three melanocortin peptides is/are involved in the anorectic response by stimulating MC4R on neurons downstream in the paraventricular nucleus (PVN) [6–10], and melanocortin-3 receptors (MC3R) to reduce feed efficiency, which is the ability to convert food to fat stores [11–15]. The melanocortin system thus mediates the anorexigenic effects of leptin, reducing food intake and increasing energy expenditure. MC3R is also located on POMC expressing neurons in the arcuate nucleus, and may form part of a feedback loop which negatively regulates the anorexic tone of the POMC expressing neurons , where melanocortin peptides from activated POMC neurons negatively autoregulate further POMC expression through their inhibitory actions at the MC3R. Recent evidence suggests that the tyrosine kinase B receptor and the brain derived neurotrophic factor [17,18] and nesfatin  are critical mediators downstream of MC4R. Leptin also inhibits neurons co-expressing the orexigenic neuropeptide Y and agouti-related peptide in the arcuate nucleus, which will otherwise promote feeding activity .
Melanocortin 4 receptor gene (MC4R)
The human MC4R is a 332 amino acid protein encoded by a single exon localised on chromosome 18q22 [21,22]. The MC4R is a seven transmembrane G-protein coupled receptor highly expressed in hypothalamic nuclei which regulate energy homeostasis [23,24]. MC4R is modulated by the endogenous agonist α-melanocyte stimulating hormone (MSH) and antagonist agouti-related protein, and signals through activation of adenylate cyclase . Mice with inactivation of both copies of the MC4R genes produced an obesity syndrome with hyperphagia associated with pathological lack of satiety, hyperinsulinaemia with hyperglycaemia, and increased linear growth, but unlike leptin deficient mice, had normal reproductive function . Heterozygotes had an intermediate weight between the homozygotes and wild-type mice, and females were more affected than males. MC4R knockout mice continue to increase feeding on a high fat diet, but do not increase thermogenesis. Interestingly, the MC4R knockout mouse exhibits normal feeding and returns to previous weight in response to food restriction. Thus MC4R does not appear to be required for normal feeding or metabolic response to fasting. However, MC4R is required for normal response to high fat diet by maintaining satiety, and increasing thermogenesis and metabolic rate.
MC4R deficiency resulting from disruption of one or both MC4R alleles represents the commonest monogenic form of human obesity [8,10]. Obese individuals with MC4R deficiency displayed a common, nonsyndromic form of obesity and were not characterized by any peculiar phenotypic abnormalities. The subjects with MC4R mutations were obese from an early age, but with increase in both fat and lean masses, were excessively hungry from 6–8 months of age with persistent food-seeking behaviour, and become distressed if food was not provided. They had higher food intake when compared to obese controls when assessed with ad-libitum meals. There was increased growth velocity in childhood, where those with MC4R mutations were taller than matched obese controls, and the bone age exceeded the chronological age by 1–4.9 years. Pubertal onset and secondary sexual characteristics were normal. They also had significantly higher insulin levels compared to matched controls, but the majority were not diabetic. The proportions of type 2 diabetic or glucose intolerant subjects, triglyceride levels, and leptin levels were not statistically different between both mutated and nonmutated obese groups. The affected subjects did not have any developmental, intellectual or behavioural problems, and there were no dysmorphic features.
In-vitro function of mutant MC4-receptors correlated with the severity of the clinical phenotype, indicating that weight regulation is sensitive to amount of functional MC4 receptors. Subjects with inactivating (null) MC4R mutations were heavier, taller, and more hyperphagic than those with partially active MC4R mutations .
Most family studies revealed autosomal co-dominant pattern of inheritance. Homozygotes for MC4R mutations exhibited a more severe phenotype than heterozygotes, where they were heavier and taller. Transmission of the mutations in the affected families indicated variable penetrance and expressivity that is not related to the functional severity of the mutations in-vitro. Family studies of heterozygous probands demonstrated co-segregation of mutation with early onset obesity with 100% penetrance, while that of homozygous probands demonstrated early onset obesity only in 68% of heterozygous family members. There is variable age of onset of obesity as well as its severity, even for the same mutation within the same family. The reason for this variability in penetrance and expressivity is yet to be fully elucidated.
The prevalence of pathogenic MC4R mutations reported in various obese populations varied widely, ranging from 0.5 to 5.8% [8,10,25–31]. Human MC4R deficiency was reported to affect 4% and 5.8% of severely obese French and British populations respectively [8,10]. However, studies elsewhere reported low incidence of MC4R mutations in their respective obese populations [25–33]. One possible explanation is differences between study cohorts in terms of ethnicity, age of onset, and severity of obesity. A British study of about 500 subjects with early onset severe obesity reported the highest prevalence of 5.8% , while a French study of adults with severe obesity where a third were obese since childhood, yielded a prevalence of 4% . However, a German cohort of extremely obese children found only 1.9% , and an Italian study of 208 children with severe early onset obesity found only one patient with a pathogenic MC4R mutation (<0.5%) . The significance of MC4R mutations in Asian obese populations has not been adequately examined. The prevalence and spectrum of MC4R mutations in Asia has not been well studied. A small Japanese study of 50 obese adults revealed no pathogenic mutations , while two studies involving Chinese adult and paediatric subjects identified a few mutations of uncertain significance in less than 1.5% of the cohort [30,31], including Cys40Arg, Val166Ile, Arg310Lys, and Cys277Stop. A Singapore study of 227 Asian (Chinese, Malay and Indian) children and adolescents with early onset severe obesity similar to those of the British and French studies [8,10] found only three mutations in three subjects (1.3%) – a missense mutation Tyr157Ser (c.470A>C) in homozygosity; a heterozygous mutation with T deletion at nucleotide position 976 (c.976delT); a heterozygous mutation with 4 bp deletion of nucleotides 631–634 (c.631–634delCTCT) . Taken together with the results of other studies, it is unlikely that the higher prevalence reported in some cohort studies were due to selection of study subjects with early onset severe obesity, but rather that MC4R mutations are truly more prevalent in northern European obese populations [8,10].
Though there were a few common mutations described across populations, such as the c.631–634delCTCT mutation in a Malay subject in the Singapore study, which was previously reported in English and German Caucasian subjects [34,35], a significant number of novel and ‘exclusive’ or ‘private’ mutations were reported in different obese populations from various parts of the world, and these mutations were not frequent even within these populations [8,10,25–31,34]. One possible reason for this relative lack of founder effect is that the pathogenicity of MC4R mutations in obesity may be a fairly recent event in human evolution.
Our family studies in Singapore demonstrated generational phenotypic variability, where the children and younger subjects were more severely affected compared to the adults/parents. At least two large studies have shown incomplete penetrance and variable expressivity in their cohort of MC4R mutation carriers [10,36]. Heterozygous mc4r+/– mice also display a broad variability in phenotype with an adult weight ranging from that of wild-type to that of homozygous mc4r–/– mice . This is indeed plausible, considering that the environment, behaviour, and other modifier genes can contribute to accumulation of adiposity to varying extent. The current environment may be more ‘obesogenic’ compared to that of the previous generation, and exposure to disease modifying factors such as the environment at critical stages of childhood may determine the eventual phenotype. Another possibility is amelioration of phenotype as the young mutation carriers become adults. Young British subjects with MC4R mutations were described to have early onset hyperphagic obesity, higher lean body mass, increased linear growth with higher height SDS scores, hyperinsulinaemia and higher bone mineral density compared to matched obese controls, and amelioration of hyperphagia and degree of hyperinsulinaemia observed in adulthood . Recently a large European study of probands and relatives also observed generational effect with increasing penetrance in MC4R deficient individuals at younger ages – 40% in adults more than 52 years old, 60% in 18–52 year-olds, and 79% in children . However, longitudinal study of the adult carriers showed an increasing age-dependent penetrance (37% at 20 years vs. 60% at >40 years). This again may be related to the temporal development of an increasingly ‘obesogenic’ environment over the decades which succeeding generations are exposed to, as discussed previously.
The MC4R has two common variants Val103Ile and Ile25Leu which were thought to be common polymorphisms, though an in-vitro study revealed these two variants could be possible gain of function mutations . A large European study (n = 16797) of nine cohorts and a UK study (n = 8304) of three cohorts as well as meta-analysis  reported that these polymorphisms are protective against obesity. The prevalence of the 103Ile variant in the UK cohorts ranged 2–4%, and it was estimated that the carriers had 18% less risk of obesity. A meta-analysis of six East Asian studies (n = 3526) revealed that the prevalence of the 103Ile variant is higher at 5.8–7.2% in this East Asian populations, and the 103Ile carriers had 31% less risk of obesity . Thus not only is the prevalence of MC4R variants Val103Ile different between populations, the extent of biological protective effect also differs. While these results augmented the critical role of the melanocortin system in human weight regulation, they also demonstrated the different impact of the protective alleles in different populations and its contribution to population risk reduction for obesity.
Melanocortin 3 receptor gene (MC3R)
The Melanocyte Stimulating Hormone (MSH) is the principal agonist of the neuronal melanocortin receptors, MC3R and MC4R, both of which are critical for weight regulation in rodents [7,11,12]. MC3R is a seven-transmembrane G-protein coupled receptor  expressed in hypothalamic nuclei known to regulate energy homeostasis. It exhibits a more restricted distribution than MC4R in the central nervous system , and has a dominant role in inhibition of energy storage [11,12]. Mc3r−/− mice homozygous for knockout mutations of MC3R gene had increased body fat [11,12] not caused by increased food intake but by increased feed efficiency. The Mc3r−/− mice were hypophagic with hyperleptinemia compared to wildtype littermates . These mice were unusually susceptible to high fat diet-induced obesity, and were relatively inactive, which partly explained the obesity. The mice showed no perturbations in metabolic rate, thyroid hormone levels, respiratory exchange ratio or body temperature. Male mice developed mild hyperinsulinaemia. Mice lacking both MC3R and MC4R have exacerbated obesity, which supports the notion that both are important and nonredundant . MC4R mutations causing human obesity are well described [8,10], but the search for human MC3R mutations has been unsuccessful [13,44–47]. Hence its pathogenic role in human obesity was less well accepted, though recent reports of rare loss-of-function mutations [14,48] continue to lend support to the critical role of MC3R.
The human MC3R has two common nonsynonymous single nucleotide polymorphisms (SNP) Thr6Lys and Val81Ile in near complete linkage disequilibrium, and at least two studies of obese children from different populations have independently shown the association of these SNPs with childhood adiposity, supported by reduced variant MC3R activity in-vitro compared to wild type MC3R [13,14]. The US study of Afro-American and Caucasian obese children showed that those homozygous for the variants had higher BMI SDS score, body fat mass and percentage body fat , and the Singapore study of Asian children similarly found those with the variants have higher percentage ideal weight for height, percentage body fat and leptin levels, supported by an additive effect, where the subjects who were heterozygous had intermediate phenotype . A recent report of three cohorts of normal and overweight children 6–19 years old (n = 302) found that the carriers of the variants had greater energy intake when given ad libitum meals, and did not demonstrate altered resting energy expenditure (indirect calorimetry), total energy expenditure (measured by doubly labeled water), or physical activity level . Interestingly, case control studies had failed to demonstrate that these variants were enriched in the obese populations [14,44]. A possible explanation is that the two MC3R variants only exert an effect on the phenotype in the obese state. A subanalysis of the data from US and Singapore  cohorts showed that the frequency of the variants differ greatly between ethnic groups (Table 1). The variants are more prevalent in Afro-American obese children than White American obese children, while the prevalence among Chinese and Malay obese children is intermediate. Thus the impact and contribution of these MC3R variants in predisposing to increased adiposity will vary significantly from population to population.
Table 1. The prevalence of MC3R variants Thr6Lys/Val81Ile in US  and Singapore  cohorts
To date, there are at least 11 rare missense and nonsense MC3R mutations associated with human obesity [14,48,49], and like MC4R mutations there is a relative lack of founder effect. Common obesity is a polygenic trait resulting from interaction of multiple genetic loci with the environment. Sequence variants in a large set of genes implicated in energy regulation could predispose an individual to excessive weight gain in a given environment. While MC3R mutations do not result in autosomal co-dominant form of human obesity like MC4R deficiency, there is increasing evidence that MC3R variants and mutations may contribute to human obesity as predisposing factors at the very least. The wide variation in the adiposity of the individuals with common and rare variants may be due to other modifying genetic and environmental factors. We believe that further studies will continue to unravel the phenotype and support the role of MC3R in human weight regulation and pathogenesis of obesity.
Conflicts of interest
The author declares that there is no conflict of interest in the preparation of this manuscript.