Heterozygous versus homozygous phenotype caused by the same MC4R mutation: novel mutation affecting a large consanguineous kindred

Obesity, affecting more than 30% of adults and children worldwide [1], is a complex trait affected by diet in conjunction with environmental and genetic factors [2]. The melanocortin-4 receptor (MC4R) is a key player in the leptin-regulated melanocortin circuit, essential for central energy regulation [3]. Predominantly expressed in the hypothalamus, the G-protein-coupled-receptor MC4R acts in signaling satiety, consequently decreasing food intake: upon binding of its endogenous ligand, the neuropeptide melanocyte stimulating hormone (α-MSH), MC4R activates adenylate cyclase, enhancing cAMP synthesis and levels, thereby generating a satiety signal. In line with the role of MC4R in satiety signaling and regulation of food intake, MC4R null mutant (MC4R ^−/−) mice develop severe obesity, while heterozygous (MC4R ^+/−) mice present a mildly obese intermediate phenotype [4].

Mutations in members of the leptin-regulated melanocortin circuit have been shown to result in human obesity. Most notably, human obesity has been associated with mutations in leptin (LEP) [5], leptin receptor (LEPR) [6], prohormone convertase 1 (PC1) [7], proopiomelanocortin (POMC) [8] and melanocortin-4 receptor (MC4R) [9–15]. In fact, heterozygous loss-of-function MC4R mutations have been shown to be the most common known genetic cause of human obesity. Initial studies by Farooqi et al. [16] estimated that about 6% of early onset morbidly obese patients harbor MC4R mutations. Consequently, other large cohort studies, mostly in Caucasian European and American populations, demonstrated lower calculated prevalence of about < 2% [17–21]. The prevalence of MC4R mutations in severely obese populations in other ethnicities, such as Asians, was found to be low or not relevant [16].

Homozygous or compound heterozygous carriers of MC4R mutations are rare [15, 16, 22, 23]. Previous studies suggest that the obesity in these rare cases develops earlier in life, and is more severe than for heterozygous carriers; notably, these homozygous individuals do not display any discernible additional unrelated phenotypes [16]. Only few families have been described to date in which multiple heterozygotes and homozygotes of the same mutation are found [16]. We now describe large consanguineous inbred kindred with individuals that are either homozygous or heterozygous carries of the same novel MC4R mutation, and review the literature of such families described to date.

MC4R mutations are the most common known genetic cause of obesity, affecting 2–3% of the population in various cohorts tested [16, 29]. To date, about 200 MC4R genetic variants have been identified, including at least 122 missense mutations, 2 in-frame deletion mutations, 7 nonsense mutations and dozens of frameshift mutations [3, 30], altogether affecting more than 30% of the receptor coding sequence. While it has been suggested that obesity due to MC4R mutations can be caused by either haplo-insufficiency or dominant negative activity exerted by the mutant receptor, co-transfection studies show that the extreme majority of mutations analyzed do not have dominant negative activity [3, 15, 31, 32]. Therefore, it is suggested that haplo-insufficiency is the main route through which these mutations exert their effect. While loss-of-function mutations in MC4R cause familial forms of obesity, two rare gain-of-function MC4R polymorphisms have been identified that are associated with protection against obesity [19]. In fact, we show that in our cohort of 120 control Bedouin whole exome sequences, 4 individuals are heterozygous for one of these variants, namely c.307G > A, p.V103I. Meta analysis of previously published data showed that this gain-of function mutation has a modest negative association with obesity [19]. It is of interest that the prevalence of the p.V103I variant in the Israeli Bedouin community seems to be higher than worldwide (ExAC frequency 0.01743). Notably, another gain-of-function c.751A > C p.I251L MC4R variant, that is more clearly negatively associated with obesity worldwide [19], was not found in our Bedouin cohort. Obviously, larger cohorts within this large inbred Bedouin community [33] should be tested to validate statistical significance of these observations.

In families with MC4R-associated obesity, obesity tends to have an autosomal dominant mode of transmission, but the penetrance of the disease can be incomplete and the clinical expression variable (moderate to severe obesity), underscoring the role of the environment and other possible modulating genetic factors [34, 35]. As heterozygous MC4R mutation carriers are obese, yet present with partial penetrance of the mutations, O’Rahilly and colleagues concluded that the mode of inheritance in MC4R deficiency is codominance with modulation of expressivity and penetrance of the phenotype [36]. It has been suggested that the varying onset and severity of obesity in heterozygous MC4R mutation carriers are related to the severity of the functional effects of the mutations. In fact, with many human MC4R mutations identified, several research groups (Tao et al. [3], MacKenzie [29], Vaisse et al. [14], Farooqi et al. [15]) classified the MC4R mutations based on possible functional consequences: mutations that cause intracellular-retaining of the receptor, defective expression, defective binding, defects in both basal and ligand-induced signaling, etc. However, as only a minority of the variants underwent in-depth functional analysis, validity of such classifications in the context of clinical phenotypic association awaits further studies.

Homozygous or compound heterozygous carriers of MC4R mutations are rare [15, 22, 23]. Only few families have been described to date in which multiple heterozygotes and homozygotes of the same mutation are found. We now identified a novel MC4R truncation mutation, putatively abolishing all 7 transmembrane domains of the molecule (Fig. 1c). Previous studies reported phenotypic variation in consequences of heterozygous MC4R deletion mutations [19, 20]. As the cohort we studied is small, while the average BMI in heterozygous individuals was higher than in wild type family members (29.22 versus 24.24.46, respectively), this difference was not statistically significant, neither in adults nor in children.

Unique to our study, we delineated the mutation-related phenotype in large consanguineous kindred with 4 homozygous and 7 heterozygous individuals, as well as 5 wild type family members. This unique kindred, of few identified thus far, allows insights as to phenotypic effects in heterozygotes versus homozygotes of the same mutation. As evident in Table 2, although the cohort is too small to establish statistical significance, the data clearly point to early-onset obesity in individuals homozygous for the MC4R mutation: of the children (ages 1–7 years) within the kindred, the two homozygotes were morbidly obese (BMI 37 and 37; Z scores 3.12, 3.08) as compared to the two wild type individuals (BMI 18 and 17) and the heterozygous individual (BMI 17) that were within normal BMI values (85th BMI-per age percentile). In fact, in spite of the fact that a single kindred is described, the clear morbid obesity (average BMI 44.83) in homozygous individuals is statistically significant compared to the overweight (average BMI 29.22) in heterozygous individuals and the higher end of normal weight (average BMI 24.46) seen in wild type family members. This is in line with previous reports, showing a dramatically more consistent and severe obesity phenotype in homozygotes for MC4R mutations than in heterozygotes [16]. Furthermore, the obesity in the homozygotes is of early onset, with BMI Z scores ~ 3 in individuals ages 5–10 years. While previous studies of MC4R heterozygotes have shown age-dependent differences in expressivity and a stronger effect in females [16, 18–21, 34], the cohort in the present study is too small to reach conclusions in this regard. Interestingly, although all affected individuals share the same mutation and reside in the same environment (practically the same household), there is variability in phenotypic expression, in the heterozygous individuals in particular, suggesting possible effects of modifier genes. In fact, future studies of such kindreds might be conducive to elucidation of such modifiers.

Previous reports of homozygotes vs heterozygotes of the same MC4R mutations did not systematically describe related blood biochemistry values. Fasting blood triglycerides, cholesterol, LDL, HDL, glucose and HbA1C levels were measured for most individuals in our studied kindred. As seen in Table 2, only the eldest of the 4 homozygous individuals (III:5, Fig. 1a, age 30–35 years) had extremely high levels of cholesterol, triglycerides, LDL, glucose and HbA1C, while the other 3 (ages 5–10 and 25–30 years) had normal values. Similarly, of the 7 heterozygotes, high levels of triglycerides, glucose and HbA1C were found only in the eldest individual (age range 55–60 years vs age ranges 5–10, 20–25, 25–30, 25–30, 25–30, 30–35).

Through studies of a large inbred kindred we demonstrate a novel MC4R mutation, practically eliminating all functional domains of the encoded protein. We show that the phenotype in homozygotes for the mutation is significantly more severe than that of heterozygous carriers of the same mutation. While obesity, as delineated through BMI measurements, is evident in homozygotes (yet not necessarily in heterozygotes) at early ages, the metabolic consequences of MC4R-related obesity appear in both heterozygotes and homozygotes only at later ages.