Novel Melanocortin-3 and -4 Receptor Functional Variants in Asian Children With Severe Obesity

Abstract

Context

Genetic variants in melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) genes are strongly associated with childhood obesity.

Objective

This study aims to identify and functionally characterize MC3R and MC4R variants in an Asian cohort of children with severe early-onset obesity.

Methods

Whole-exome sequencing was performed to screen for MC3R and MC4R coding variants in 488 Asian children with severe early-onset obesity (body mass index for age ≥97^th percentile). Functionality of the identified variants were determined via measurement of intracellular cyclic adenosine monophosphate (cAMP) concentrations and luciferase activity.

Results

Four MC3R and 2 MC4R heterozygous nonsynonymous rare variants were detected. There were 3 novel variants: MC3R c.151G > C (p.Val51Leu), MC4R c.127C > A (p.Gln43Lys), and MC4R c.272T > G (p.Met91Arg), and 3 previously reported variants: MC3R c.127G > A (p.Glu43Lys), MC3R c.97G > A (p.Ala33Thr), and MC3R c.437T > A (p.Ile146Asn). Both MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants demonstrated defective downstream cAMP signaling activity. The MC4R c.127C > A (p.Gln43Lys) variant showed reduced cAMP signaling activity at low substrate concentration but the signaling activity was restored at high substrate concentration. The MC3R c.151G > C (p.Val51Leu) variant did not show a significant reduction in cAMP signaling activity compared to wild-type (WT) MC3R. Coexpression studies of the WT and variant MC3R/MC4R showed that the heterozygous variants did not exhibit dominant negative effect.

Conclusion

Our functional assays demonstrated that MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants might predispose individuals to early-onset obesity, and further studies are needed to establish the causative effect of these variants in the pathogenesis of obesity.

childhood obesity, variants, MC3R, MC4R

The prevalence of childhood obesity has been on the rise in Singapore and has been imposing a huge burden on our healthcare infrastructure (1). Obesity is a complex, multifactorial disease involving excess body fat accumulation that increases the risk of debilitating cardiometabolic conditions that can potentially reduce quality of life and life expectancy (2). Obesity evolves from the interaction between modern “obesogenic” environment and genetic susceptibility to excessive weight gain (3). Genetic factors are especially important in the development of early-onset obesity as children have minimal exposure to extrinsic environmental factors (4).

Melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) are G protein–coupled receptors that are activated by α-melanocyte stimulating hormone (α-MSH) via stimulatory heterotrimeric G proteins leading to cyclic adenosine monophosphate (cAMP) production that regulates feeding behavior and metabolism pathways (5-10). The knockout (KO) of MC3R and MC4R genes in mice has been shown to generate an obese phenotype (11, 12). However, the obesity features differ between the MC3R KO and MC4R KO models. MC3R KO rats displayed a mild obesity phenotype characterized by hypophagia, higher fat mass, and lower lean mass (12, 13), while MC4R KO rats were shown to exhibit a severe obesity phenotype characterized by hyperphagia, hyperglycemia, and hyperinsulinemia (13).

Studies have revealed multiple mutations in the MC3R and MC4R genes that are strongly associated with early-onset obesity (14-25). MC4R mutations account for about 5% of severe obesity, which makes it the most common cause of monogenic obesity (17), whereas a fewer number of MC3R mutations have been reported to be associated with early-onset obesity (26). MC3R and MC4R mutations have been shown to impair receptor signaling through disruption of receptor expression/trafficking, alteration in substrate binding, and reduction in downstream signaling pathway related to appetite control (16, 18, 21, 22, 27-30).

Groundbreaking pharmacological research has led to the development of the small-molecule MC4R agonist setmelanotide, which has recently been approved by the US Food and Drug Administration for the treatment of obesity associated with genetic defects upstream of the MC4R pathway (31, 32). Studies have shown that setmelanotide can lead to weight loss in individuals who have MC4R mutation-driven obesity via a “chaperone effect” whereby the small-molecule MC4R agonist rescues the variant MC4R by increasing cell surface expression (33-36). Setmelanotide also activates the MC3R signaling pathway (33). The identification and functional characterization of MC3R and MC4R variants may guide the use of genetic-driven drugs, such as setmelanotide, for the treatment of obesity.

In this study, we aimed to identify and functionally characterize MC3R and MC4R variants in an Asian cohort of children with severe early-onset obesity.

Materials and Methods

Study Participants

A total of 571 children with obesity, and of Chinese and Malay ethnicity, were recruited from hospital (National University Hospital) and community settings (Singapore Health Promotion Board) under the OBesity in Singaporean Children (OBiSC) study. The recruitment criteria were 1) onset of obesity at younger than 10 years, 2) body mass index (BMI) for age greater than or equal to the 97th percentile, and 3) no syndromic causes of obesity. Family members of children with obesity were also recruited if available. Family history of obesity was obtained during the study visit interview. Standard anthropometric parameters such as weight, height, and body fat percentage were measured, and pubertal assessment was conducted. Blood samples were collected in a fasted state. The study was performed in accordance with the Declaration of Helsinki, and ethics approval was obtained from Domain Specific Review Board of National Healthcare Group, Singapore (reference No. 2015/00314). Written informed consent was obtained from all study participants. The study is registered under clinicaltrials.gov (NCT02418377).

Whole-Exome Sequencing

Genomic DNA was extracted from peripheral blood leukocytes. Sequencing libraries were constructed from 1 μg of DNA using the Ultra II DNA Library Prep Kit for Illumina (NEBNext). Exonic regions were captured genomewide by hybridization with SeqCap EZ exome v3.0 probes (Nimblegen). Libraries were assessed using a Bioanalyzer High Sensitivity DNA chip (Agilent) sequenced on a HiSeq 4000 (Illumina) platform. To maximize sequencing accuracy, each sample was sequenced to an average of 40× coverage, using 2 × 150 bp read lengths.

Paired-ended sequencing reads were aligned with the reference sequence MC3R: NM_019888.3, MC4R: NM_005912.3 using Burrows Wheeler Aligner. Variant quality score recalibration was performed in the raw variant call set and single-nucleotide variations (SNVs) with low confidence were excluded based on the following parameters: quality score normalized by depth less than 2, mapping quality less than 40, strand bias greater than 60, mapping quality bias less than 12.5, and bias in read position of alternative allele versus reference allele less than −8. High-quality common variants with a minor allele frequency greater than 5% and SNV call rate greater than 95% were used to perform sample quality control (QC) procedures in our study data set. Samples with depth of coverage less than 10, call rate less than 95%, extreme heterozygosity levels, or with first-degree relation/outliers from reported ethnicity were excluded.

Selection of Melanocortin 3 Receptor and Melanocortin 4 Receptor Genetic Variants

Each variant that passed the QC was annotated using ANNOVAR (ver20140915) (37) for determination of reference sequence gene symbol and the frequency of reference populations from the Exome Aggregation Consortium database (37). Novel (not found in Genome Aggregation Database, gnomAD) or rare nonsynonymous variants with an allele frequency of less than 0.1% in Asian population, from exonic regions of obesity susceptibility genes MC3R and MC4R, were identified among our QC data set of Chinese and Malay childhood-obesity cases, which were compared against controls consisting of 1762 Chinese and Malay normal-weight (BMI 18.5-23) Singaporean adults selected from other cohorts. Sanger sequencing was then performed to reconfirm the identity of these variants. Novel variants were submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).

Conservational Analysis and Prediction of Functional Effect of Melanocortin 3 Receptor and Melanocortin 4 Receptor Variants

Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used to align and compare the degree of conservation of sequence region surrounding the MC3R or MC4R variants among different species. Bioinformatics tools such as Sorting Intolerant From Tolerant (SIFT) (https://sift.bii.a-star.edu.sg/) and Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) were used to predict the possible impact of the MC3R or MC4R variants on protein function. The classification of variant function (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) was based on the American College of Medical Genetics and Genomics criteria (38).

Construction of Wild-type and Variant Melanocortin 3 Receptor and Melanocortin 4 Receptor Plasmids

The pBuDCE4.1 dual-expression vector (Thermo Fisher), which consists of 2 multiple cloning sites (MCS) under 2 constitutive promoters respectively, was used to create the MC3R or MC4R plasmids. The sequence for Renilla luciferase (internal control) was first cloned into one of the MCS of the dual-expression vectors using HindIII and BamHI restriction sites, and the wild-type (WT) MC3R or MC4R sequence was then cloned into the other MCS of the same dual-expression vector using the XhoI and BglII restriction sites to generate the MC3R or MC4R WT plasmids. The MC3R or MC4R variants were introduced into the respective MC3R or MC4R WT plasmids via site-directed mutagenesis (GenScript) to generate variant MC3R or MC4R plasmids. The sequences of all plasmids were verified by Sanger sequencing.

Measurement of Cyclic Adenosine Monophosphate Levels

Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's Modified Eagle's Medium (Cytiva) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (Cytiva). Cells were incubated at 37 °C with 5% CO₂. The cells were grown to 90% confluent on a 6-well plate, and each well was transfected with 2 μg of either MC3R or MC4R plasmid (WT or variant plasmid) using Lipofectamine 2000 (Invitrogen). The transfected cells were incubated at 37 °C with 5% CO₂ overnight for 24 hours. Thereafter, the cells were stimulated with α-MSH of differing concentrations ranging from 100 pmol/L to 10 µmol/L for 16 hours. Cells were lysed with 0.1 M HCl and assayed for cAMP levels using a cAMP competitive enzyme-linked immunosorbent assay kit (Abcam catalog No. ab65355, RRID: AB_2909598), in which absorbance was measured at OD450 nm by the Synergy 2 Microplate Reader (Biotek Instruments). The assay was performed in technical duplicates and each experiment was performed in biological triplicates. Levels of cAMP were expressed as fold increase over basal reading of nonstimulated cells.

Measurement of Dual Luciferase Activity

HEK293 cells were grown to 90% confluent on a 24-well plate, and each well was cotransfected with 0.5 μg of MC3R or MC4R plasmid (WT or variant plasmid) and 0.5 μg of pGL4.29 cAMP response element (CRE) firefly luciferase reporter vector (Promega) using Lipofectamine 2000 (Invitrogen). On binding of α-MSH to MC3R or MC4R, cAMP will be produced and the cAMP will bind to CRE to promote the expression of firefly luciferase. Concurrently, HEK293 cells were cotransfected with 0.25 μg of variant plasmids, 0.25 μg of WT plasmids and 0.5 μg of CRE reporter vector to investigate for a possible dominant-negative effect of variant MC3R or MC4R. Transfected cells were incubated overnight for 24 hours and then stimulated with α-MSH of differing concentrations ranging from 100 pmol/L to 1 µmol/L for 16 hours. The activity of variant MC3R or MC4R was determined by a dual-luciferase reporter assay (Promega) according to the manufacturer's instructions, and luciferase activity was measured using the Glomax Luminometer (Promega). The assay was performed in technical duplicates and each experiment was performed in biological triplicates. Luciferase activity was normalized against Renilla activity (internal control for transection efficiency) and expressed as fold over basal reading of nonstimulated cells.

Statistical Analysis

A 3-parameter sigmoidal dose-response curve was fitted to normalized cAMP and luciferase values from all replicates using GraphPad Prism 8 for Windows (version 8.4.3 GraphPad Software). Extra sum-of-squares F-test was used to compare the differences in EC₅₀ and E_max.

Relative E_max was determined based on cAMP levels and luciferase activity measured at 10⁻⁵M and 10⁻⁶M α-MSH, respectively. For variant MC3R or MC4R that did not mount a response with α-MSH stimulation, no fit curve was generated, and t test (SPSS, version 27.0, IBM) was used to compare differences in E_max.

Results

Identification of Novel Melanocortin 3 Receptor and Melanocortin 4 Receptor Variants in Asian Children With Early-Onset Obesity

After sample QC, 211 patients of Malay ethnicity (43.2%) and 277 cases of Chinese ethnicity (56.8%) were available for downstream analysis. These patients were 67.6% male (32.4% female), and had a mean age of 12.7 ± 3.54 years, BMI of 133 ± 20% of 95th percentile, body fat percentage of 47.9 ± 9.93% and waist-to-hip ratio of 0.95 ± 0.07. Six heterozygous rare nonsynonymous variants (4 in MC3R and 2 in MC4R) were identified in 6 unrelated children with severe obesity. These 6 cases did not possess pathogenic variants in other candidate obesity-susceptibility genes. All identified variants were visually inspected using the integrated genomics viewer. The Variant Quality Score Log-odds (VQSLOD) for these variants ranged from 6.37 to 11.36, indicating high sequence quality, and there were good sequencing depths ranging from 12 to 156 in the samples identified to contain these variants. The 6 heterozygous rare nonsynonymous variants were reconfirmed by Sanger sequencing (Supplementary Fig. S1) (39).

We identified 1 novel MC3R variant, NM_019888.3 (MC3R): c.151G > C (p.Val51Leu), and 2 novel MC4R variants, NM_005912.3 (MC4R): c.127C > A (p.Gln43Lys) and NM_005912.3 (MC4R): c.272T > G (p.Met91Arg) in our cohort of Asian children with early-onset obesity. We also identified 3 other MC3R variants that have previously been reported: NM_019888.3 (MC3R): c.127G > A (p.Glu43Lys) is a variant of uncertain significance; NM_019888.3 (MC3R): c.97G > A (p.Ala33Thr) is a likely benign variant; and NM_019888.3 (MC3R): c.437T > A (p.Ile146Asn) is a likely pathogenic variant (21, 25, 40, 41). Characteristics and functional prediction/classification of each variant is represented in Table 1. Human sequences surrounding the MC3R and MC4R variants are highly conserved in other species (Supplementary Fig. S2) (39). The location of each variant on MC3R or MC4R proteins are shown in Supplementary Fig. S3 (39).

Table 1.

Characteristics and functional prediction/classification of MC3R and MC4R variants

SNV	Location	Genotype	Frequency in obese cohort	Frequency in control cohort	SIFT prediction	Polyphen-2 prediction	Allele frequency in Asians	Variant classification	Accession No.
MC3R (NM_019888.3)
c.97G > A (p.Ala33Thr)	Extracellular N-terminus	Heterozygous	1	0	Tolerated	Benign	0.00008	Likely benign	rs770261378
c.127G > A (p.Glu43Lys)	First transmembrane helix	Heterozygous	2	0	Tolerated	Possibly damaging	<0.00001	Uncertain significance	rs375750359
c.151G > C (p.Val51Leu)a	First transmembrane helix	Heterozygous	1	0	Tolerated	Benign	—	Uncertain significance	SCV002540643
c.437T > A (p.Ile146Asn)	Second intracellular loop	Heterozygous	1	0	Damaging	Probably damaging	0.00018	Likely pathogenic	rs74315393
MC4R (NM_005912.3)
c.127C > A (p.Gln43Lys)a	Extracellular N-terminus	Heterozygous	1	0	Tolerated	Possibly damaging	—	Uncertain significance	SCV002540644
c.272T > G (p.Met91Arg)a	Second transmembrane helix	Heterozygous	1	0	Damaging	Probably damaging	—	Likely pathogenic	SCV002540645

Characteristics of each variant including location, genotype, frequency, novelty, and predicted functionality are described. Frequency refers to the number of individuals within each cohort that are found to possess the respective variant. None of the variants were detected in 1762 normal-weight adult controls. Bioinformatics tools such as Sorting Intolerant From Tolerant (SIFT) and Polyphen-2 were used to predict functional significance of these variants. The allele frequency of each variant in Asians were extracted from the Genome Aggregation Database (gnomAD). The pathogenicity of each variant was classified based on the American College of Medical Genetics and Genomics criteria.

Abbreviation: SNV, single-nucleotide variation.

^aNovel variants identified in this study.

Clinical Characteristics and Family Pedigrees of Probands

MC3R c.97G > A (p.Ala33Thr)

This heterozygous variant was detected in a 17-year-old Chinese girl who developed obesity at age 3 years. She had a BMI of 105.6% of 95th percentile and body fat percentage of 41.2%. She had type 2 diabetes mellitus and was on metformin treatment. Her 52-year-old father was overweight, while her 45-year-old mother and 14-year-old brother were not overweight. However, DNA of family members were not available for genotyping (Fig. 1).

Figure 1.

Family pedigrees of probands with MC3R or MC4R variants. Age (in years), race, onset of obesity (in years), genotype, body mass index (BMI), percentage (%) of 95th percentile BMI, and body fat percentage (%) are listed. Heterozygotes are half shaded. Arrows represent probands with early-onset obesity. A, Family of proband with MC3R c.97G > A (p.Ala33Thr) variant. B, Family of proband with MC3R c.127G > A (p.Glu43Lys) variant. C, Family of proband with both MC3R c.127G > A (p.Glu43Lys) and c.151G > C (p.Val51Leu) variants. D, Family of proband with MC3R c.437T > A (p.Ile146Asn) variant. E, Family of proband with MC4R c.127C > A (p.Gln43Lys) variant. F, Family of proband with MC4R c.272T > G (p.Met91Arg) variant.

MC3R c.127G > A (p.Glu43Lys)

This heterozygous variant was identified in a 7-year-old Malay boy who developed obesity at age 3 years. He had a BMI of 188.4% of 95th percentile and body fat percentage of 66.9% with no existing medical conditions. His 43-year-old mother was overweight, but the DNA of the mother was not available for genotyping (see Fig. 1).

MC3R c.151G > C (p.Val51Leu) and c.127G > A (p.Glu43Lys)

This novel heterozygous variant, MC3R c.151 G > C, was discovered in a 9-year-old Malay boy who had been obese since infancy. This boy was also found to have the MC3R c.127G > A (p.Glu43Lys) variant. He had a BMI of 160.6% of 95th percentile and body fat percentage of 66.2% with no existing medical conditions. His 41-year-old mother was found to have the heterozygous MC3R c.151G > C (p.Val51Leu) variant but not the MC3R c.127G > A (p.Glu43Lys) variant. His mother had been obese since age 7. She had a BMI of 36.3 kg/m² and body fat percentage of 50.6%. His 14-year-old brother was overweight, while his 49-year-old father and 11-year-old sister were not overweight. However, DNA samples of the father and siblings were not available for genotyping (see Fig. 1).

MC3R c.437T > A (p.Ile146Asn)

This heterozygous variant was detected in a 14-year-old Malay girl who had been obese since age 3. She had a BMI of 170.0% of 95th percentile and body fat percentage of 68.3% with no existing medical conditions. Her 53-year-old mother who was obese with a BMI of 29.30 kg/m² and body fat percentage of 40.4% did not possess the variant. Her father and 20-year-old sister were overweight, while her 19-year-old sister was not overweight. However, DNA samples of the father and siblings were not available for genotyping (see Fig. 1).

MC4R c.127C > A (p.Gln43Lys)

This novel heterozygous variant was discovered in a 14-year-old Malay girl who had been obese since age 1. She had a BMI of 149.8% of 95th percentile and body fat percentage of 57.3% with no existing medical conditions. Both her 41-year-old father and 39-year-old mother were obese, while her older sister and younger brother were not overweight. However, DNA of family members were not available for genotyping (see Fig. 1).

MC4R c.272T > G (p.Met91Arg)

This novel heterozygous variant was discovered in an 11-year-old Chinese boy who had been obese since age 5. He had a BMI of 107.4% of 95th percentile and body fat percentage of 50.6% with no existing medical conditions. His 49-year-old father was overweight, while his younger brother and sister were not overweight. However, DNA samples of family members were not available for genotyping (see Fig. 1).

Onset of Puberty and Stature Among Probands With Melanocortin 3 Receptor or Melanocortin 4 Receptor Variants

MC3R rare variants were found to be associated with delayed onset of puberty and reduced linear growth (42), while MC4R is reported to be involved in regulation of growth (43). Hence, we examined the onset of puberty and stature in our probands with MC3R and MC4R variants. There was no delayed onset of puberty among the female probands while all male probands are at prepubertal stage likely due to young age. We did not observe short stature among the probands (Supplementary Table S1) (39).

Signaling Activities of Variant Melanocortin 3 Receptor or Melanocortin 4 Receptor

The activities of variant MC3R or MC4R were assessed by the intracellular level of cAMP, which is a downstream messenger of the MC3R and MC4R signaling pathways. MC3R c.97G > A (p.Ala33Thr) and MC3R c.151G > C (p.Val51Leu) variants demonstrated similar signaling activities compared to MC3R WT with no significant differences in EC₅₀ and E_max (Fig. 2A and 2C). In contrast, MC3R c.127G > A (p.Glu43Lys) and MC3R c.437T > A (p.Ile146Asn) variants resulted in a complete loss of receptor function with negligible response to increasing α-MSH concentrations (Figs. 2B and 2D). The presence of both MC3R c.127G > A (p.Glu43Lys) and MC3R c.151G > C (p.Val51Leu) variants also resulted in a complete loss of receptor function (Fig. 2E).

Figure 2.

Signaling activities of variant MC3R or MC4R. Cyclic adenosine monophosphate (cAMP) results were normalized against basal nonstimulated readings. Each data point represents the mean and SE of at least 3 independent experiments performed in technical duplicates. A, MC3R c.97G > A (p.Ala33Thr) variant; B, MC3R c.127G > A (p.Glu43Lys) variant; C, MC3R c.151G > C (p.Val51Leu) variant; D, MC3R c.437T > A (p.Ile146Asn) variant; E, MC3R c.[127G > A;151G > C] (p.[Glu43Lys;Val51Leu]) variant; F, MC4R c.127C > A (p.Gln43Lys) variant; G, MC4R c.272T > G (p.Met91Arg) variant. ^MC4R c.127C > A (p.Gln43Lys) included stimulation with 50 µM α-melanocyte stimulating hormone (α-MSH). Three-parameter sigmoidal dose-response curve was fitted to normalized cAMP values. EC₅₀ values are presented in µM. E_max values are a measure of normalized ratios against basal readings. Data are represented as mean ± SE of at least 3 independent experiments performed in technical duplicates. The asterisk indicates a statistically significant (P < .05) difference in EC₅₀ or E_max values when the variant MC3R or MC4R was compared against wild-type MC3R or MC4R.

The MC4R c.127C > A (p.Gln43Lys) variant demonstrated a modest reduction in receptor signaling activity at lower concentrations of α-MSH compared to WT but the function of the variant MC4R was restored to that of WT at higher concentrations of α-MSH (Fig. 2F). In contrast, MC4R c.272T > G (p.Met91Arg) resulted in a complete loss of receptor function with negligible response to increasing α-MSH concentrations (Fig. 2G).

Figure 3.

Dominant negative effect of variant MC3R or MC4R. Luciferase levels were normalized against the internal control, Renilla levels, to account for variation in transfection efficiency. Each data point represents the mean and SE of at least 3 independent experiments performed in technical duplicates. A, MC3R c.97G > A (p.Ala33Thr) variant; B, MC3R c.127G > A (p.Glu43Lys) variant; C, MC3R c.151G > C (p.Val51Leu) variant; D, MC3R c.437T > A (p.Ile146Asn) variant; E, MC3R c.[127G > A;151G >C] (p.[Glu43Lys;Val51Leu]) variant; F, MC4R c.127C > A (p.Gln43Lys) variant; G, MC4R c.272T > G (p.Met91Arg) variant. Three-parameter sigmoidal dose-response curve was fitted to normalized luciferase values. EC₅₀ values are presented in nM. E_max values are a measure of normalized ratios against basal readings. Data are represented as mean ± SE of at least 3 independent experiments performed in duplicates. The asterisk indicates a statistically significant (P < .05) difference in EC₅₀ or E_max values when comparing among the wild-type, heterozygous variant, and homozygous variant MC3R or MC4R.

Dominant Negative Effect of Variant Melanocortin 3 Receptor or Melanocortin 4 Receptor

The downstream signaling activities of the variant MC3R or MC4R were also assessed by cAMP-driven luciferase activity. The luciferase activities of the variant MC3R or MC4R were similar to that demonstrated in the cAMP assay studies. There was no significant difference in luciferase activities of MC3R c.97G > A (p.Ala33Thr) and MC3R c.151G > C (p.Val51Leu) compared to MC3R WT (Fig. 3A and 3C). MC3R c.127G > A (p.Glu43Lys), MC3R c.437T > A (p.Ile146Asn), and MC3R c.[127G > A;151G > C] (p.[Glu43Lys;Val51Leu]) showed significantly reduced luciferase activity compared to MC3R WT (Fig. 3B, 3D, and 3E). MC4R c.127C > A (p.Gln43Lys) demonstrated an impaired luciferase activity at lower α-MSH concentrations but had a similar E_max compared to MC4R WT at higher α-MSH concentrations (Fig. 3F). MC4R c.272T > G (p.Met91Arg) demonstrated minimal luciferase activity despite increasing concentrations of α-MSH (Fig. 3G).

In addition, the cAMP-driven luciferase activity was used to investigate the possible dominant-negative effect of the heterozygous MC3R or MC4R variants on the receptor function. The presence of both WT and variant alleles resulted in luciferase activity that is higher than the variant allele but lower than the WT allele. These variants did not exhibit a dominant-negative effect (see Fig. 3).

Discussion

We identified 4 MC3R and 2 MC4R heterozygous variants in a cohort of Asian children with severe obesity. The 6 variants were found in 6 unrelated children with severe obesity, and the variants were absent in 1762 normal-weight adult controls. Of the 6 variants, there were 3 novel variants: MC3R c.151G > C (p.Val51Leu), MC4R c.127C > G (p.Gln43Lys), and MC4R c.272T > G (p.Met91Arg), and 3 variants that have previously been reported: MC3R c.127G > A (p.Glu43Lys) of uncertain significance, c.97G > A (p.Ala33Thr) of likely benign function, and MC3R c.437T > A (p.Ile146Asn) of likely pathogenic function (21, 25).

A 9-year-old Malay boy was found to have both MC3R c.151G > C (p.Val51Leu) and MC3R c.127G > A (p.Glu43Lys) variants. Although the novel MC3R c.151G > C (p.Val51Leu) variant did not affect downstream receptor signaling activity, the previously reported MC3R c.127G > A (p.Glu43Lys) variant of uncertain significance was found to reduce downstream receptor signaling activity. Coexpression of both variants resulted in decreased receptor signaling activity similar to that of the MC3R c.127G > A (p.Glu43Lys) variant alone. This suggests that the boy's obese phenotype was likely attributed to the MC3R c.127G > A (p.Glu43Lys) variant. The boy likely inherited the MC3R c.151G > C (p.Val51Leu) variant from his mother who was obese and had the MC3R c.151G > C (p.Val51Leu) variant but not the MC3R c.127G > A (p.Glu43Lys) variant. His mother's obese phenotype would not have been contributed by the MC3R c.151G > C (p.Val51Leu) variant.

The MC3R c.127G > A (p.Glu43Lys) variant is located on a highly conserved residue in the first transmembrane helix that is critical for ligand binding, which supports its possible pathogenic function (44). Our analysis also showed that the site of the MC3R variant is conserved among all the 8 tested species (human, chimpanzee, monkey, pig, dog, mouse, chicken, and zebrafish). In silico functional prediction programs such as SIFT predicted that the MC3R c.127G > A (p.Glu43Lys) variant would be tolerated but our in vitro experiments demonstrated pathogenic function with significant reduction in MC3R signaling activity. This highlights the importance of in vitro experiments in functionally characterizing novel variants (45).

MC3R c.97G > A (p.Ala33Thr) resides in the N-terminus, which has been shown to have a role in cell surface expression of the receptor (21). Lee et al (25) first discovered the MC3R c.97G > A (p.Ala33Thr) variant and reported that there was a significant, modest reduction in receptor signaling activity with normal cell surface expression and no dominant-negative effect of the variant. Subsequently, Yang and Tao (21) showed that this variant resulted in a nonsignificant slight reduction in signaling activity with normal ligand binding and total cell surface expression. In the present study, we revalidated the function of the MC3R c.97G > A (p.Ala33Thr) variant and found that it caused a slight reduction in receptor signaling activity compared to the WT MC3R receptor, albeit the difference was not statistically significant. We also showed that the MC3R c.97G > A (p.Ala33Thr) variant did not exert a dominant-negative effect. The lack of a significant difference as reported by Yang and Tao (21) may be attributed to the variation between biological triplicates.

MC3R c.437T > A (p.Ile146Asn), which is in the second intracellular loop that is vital for G protein–coupled receptor activation, is a pathogenic variant that leads to complete loss of protein function (25, 40). While Lee et al and Tao et al report an absence of dominant-negative effect (21, 25, 40), Rached et al (41) demonstrated a possible dominant-negative effect. We validated previous results showing that the MC3R c.437T > A (p.Ile146Asn) variant led to a loss of receptor function and did not exert a dominant-negative effect (25, 40).

A recent study by Lam et al (42) reported that rare and common MC3R variants were associated with short stature and delayed puberty. However, our children with rare MC3R variants did not have short stature or delayed puberty. The lack of significant associations between rare MC3R variants and short stature or delayed puberty in our study may be attributed to the boys who possessed the rare MC3R variants being young and at the prepubertal stage, and our small sample size. A longitudinal study of children with and without obesity will be required to examine the association between MC3R variants and childhood growth/puberty.

The novel MC4R c.272T > G (p.Met91Arg) variant resulted in a complete loss of receptor function with no downstream signaling activity. This variant resides in the second transmembrane helix, which has previously been shown to be critical for ligand binding, and conformational change in the structure of the second transmembrane helix may significantly reduce ligand binding affinity and decimate cAMP production (46).

The MC4R c.127C > A (p.Gln43Lys) variant is located on the N-terminus of the MC4R protein, which has been shown to be vital for constitutive receptor signaling activity (47). We showed that the MC4R c.127C > A (p.Gln43Lys) variant had significantly higher EC₅₀ compared to WT, but similar E_max compared to WT. The MC4R c.127C > A (p.Gln43Lys) variant may cause a decrease in binding affinity between ligand and receptor, and the binding affinity is restored with higher ligand concentration.

Our study is limited by the lack of complete family genotype data, hence we were unable to examine the variant segregation with the obese phenotype.

In conclusion, our functional assays demonstrated that the MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants might predispose individuals to early-onset obesity, and further studies are needed to establish the causative effect of these variants in the pathogenesis of obesity.

Funding

This work was supported by National Medical Research Council (NMRC), Singapore (NMRC/CIRG/1407/2014).

Disclosures

The authors declare no conflict of interest.

Data Availability

Original data generated and analyzed during this study are included in this published article or in the data repositories listed in “References.”

Abbreviations

 
• α-MSH

  α-melanocyte stimulating hormone

 
• BMI

  body mass index

 
• cAMP

  cyclic adenosine monophosphate

 
• CRE

  cAMP response element

 
• KO

  knockout

 
• MC3R

  melanocortin 3 receptor

 
• MC4R

  melanocortin 4 receptor

 
• MCS

  multiple cloning sites

 
• QC

  quality control

 
• SIFT

  Sorting Intolerant From Tolerant

 
• SNV

  single-nucleotide variation

 
• WT

  wild-type

Author notes

Siong Gim Ong and Roghayeh Dehghan are joint first authors of this work.