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Melanocortin 4 Receptor Sequence Variations Are Seldom a Cause of Human Obesity: The Swedish Obese Subjects, the HERITAGE Family Study, and a Memphis Cohort

Human Genomics Laboratory (P.J., O.U., T.R., E.E.S., C.B.), Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808; Department of Internal Medicine and Biocenter Oulu (O.U.), University of Oulu, FIN-90220 Oulu, Finland; School of Kinesiology and Leisure Studies (A.S.L.), University of Minnesota, Minneapolis, Minnesota 55455; Division of Biostatistics and Departments of Genetics and Psychiatry (D.C.R.), Washington University School of Medicine, St. Louis, Missouri 63110; Department of Kinesiology (J.S.S.), Indiana University, Bloomington, Indiana 46405; Department of Health and Kinesiology (J.H.W.), Texas A&M University, College Station, Texas 77843-4243; Departments of Medicine (L.L., L.S.) and Diagnostic Radiology (L.L.), Sahlgrenska University Hospital, 413 45 Göteborg, Sweden; and Department of Surgery (G.S.C.), College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38112

Address all correspondence and requests for reprints to: Claude Bouchard, Ph.D., Human Genomics Laboratory, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808-4124. E-mail: bouchac{at}pbrc.edu.

Abstract

The prevalence of mutations within and in the flanking regions of the gene encoding the melanocortin 4 receptor was investigated in severely obese and normal-weight subjects from the Swedish Obese Subjects study, the Health, Risk Factors, Exercise Training, and Genetics (HERITAGE) Family study, and a Memphis cohort. A total of 433 white and 95 black subjects (94% females) were screened for mutations by direct sequencing. Three previously described missense variants and nine novel (three missense, six silent) variants were detected. None of them showed significant association with obesity or related phenotypes. In addition, two novel deletions were found in two heterozygous obese women: a -65_-64delTG mutation within the 5' noncoding region and a 171delC frameshift mutation predicted to result in a truncated nonfunctional receptor. No pathogenic mutations were found among obese blacks or nonobese controls. Furthermore, none of the null mutations found in other populations was present in this sample. In conclusion, our results do not support the prevailing notion that sequence variation in the melanocortin 4 receptor gene is a frequent cause of human obesity.

HUMAN OBESITY IS a complex trait influenced by interacting environmental and genetic factors (1). Several rodent models exist in which obesity is caused by a single mutated gene (2). One such model is the Agouti mouse, the obesity phenotype that is of particular interest because of its resemblance to human nonsyndromic obesity, i.e. maturity onset, intact fertility, moderate metabolic aberrations, and adipocyte hypertrophy (3, 4). Characterization of the Agouti phenotype at the molecular level has uncovered the impact of the melanocortin pathway on energy balance (5, 6, 7). In short, neurons within the arcuate nucleus and other hypothalamic nuclei project to the lateral hypothalamus and paraventricular nuclei in which they express -melanocyte stimulating hormone derived from posttranslational processing by enzymes, such as proconvertase-1 and -2, of the hypothalamic/pituitary prohormone proopiomelanocortin. The -melanocyte stimulating hormone stimulates the melanocortin 4 receptor (MC4R), resulting in inhibition of feeding and increase in energy expenditure (8, 9, 10). MC4R is naturally antagonized by the agouti-related protein, which is expressed in neurons distinct from those expressing proopiomelanocortin (6, 8). The melanocortin pathway is targeted by a peripheral feedback signal from adipose tissue mediated by the circulating hormone leptin (11). Disruption of this pathway, at various levels, including the MC4R, has been shown to cause obesity in rodents (7, 12, 13, 14, 15). MC4R-deficient mice exhibit an obesity phenotype similar to the Agouti obesity syndrome, albeit with larger weight gain and higher levels of insulin and glucose. Heterozygous MC4R deficiency results in an obesity phenotype intermediate to those of the homozygous mutant and the wild type (7).

Several human studies have reported associations between MC4R mutant variants and early-onset morbid obesity, particularly in females (16, 17, 18, 19, 20, 21, 22, 23, 24). Dominant as well as recessive modes of inheritance with variable expressivity or penetrance have been proposed. Based on the high frequencies of mutations found in some populations, it has been suggested that MC4R mutations constitute the most common monogenic cause of human obesity with up to 4% of all patients with morbid obesity attributable to mutations in this gene (22). To verify this hypothesis and establish whether these results could be replicated, we sequenced the MC4R gene for mutations in samples of severely obese and normal-weight blacks and whites from three different cohorts.

Subjects and Methods

Subject characteristics are shown in Table 1. The obese and control subjects were defined as having body mass index (BMI) of at least 30 kg/m² or BMI less than 30 kg/m², respectively. Approximately three-quarters (n = 196) of all obese individuals had a current or reported maximum BMI of 40.0 kg/m² or more, with 77 patients above 45 and 22 above 50 kg/m².

To investigate the role of MC4R sequence variation in obesity across ethnic groups, black females from the Health, Risk Factors, Exercise Training, and Genetics (HERITAGE) Family Study and a Memphis cohort were included.

The HERITAGE Family Study is designed to study the genetics of the variability in response of cardiovascular disease and type 2 diabetes mellitus risk factors to endurance training in sedentary North American individuals. Details of the HERITAGE study have been published elsewhere (27). For the present study, 18 obese and 48 nonobese black women from this cohort were analyzed. The sample included seven pairs of relatives: four mother-daughter pairs (all nonobese controls) and three obesity-discordant sibling pairs (mean BMI difference 16.4 kg/m²). All obese subjects were unrelated. This cohort was ascertained based on sedentary lifestyles rather than obesity.

The Memphis cohort consists of extremely obese patients who underwent bariatric surgery in a surgical obesity clinic. From this cohort, 29 obese black women were included in the present study.

For all study groups, data collected before any interventions were used. This study was conducted in accordance with the guidelines proposed in The Declaration of Helsinki and was formally approved by the appropriate institutional review boards. Informed consent was obtained from all subjects.

Obesity-related phenotypes

Measurements were performed after an overnight fast. Body mass was measured on calibrated balances or electronic scales to the nearest 0.1 kg. Body height was measured to the nearest centimeter. BMI was calculated as body mass (kilograms) divided by body height (meters) squared. The subjects among the Swedish siblingships underwent more extensive phenotyping: fat-free mass (FFM) was calculated from total body potassium, as derived from whole-body ⁴⁰K counting, and fat mass (FM) was obtained by subtracting FFM from measured body mass. Abdominal visceral fat (AVF) was estimated from a computed tomography scan at the level of the iliac crest. Intraabdominal adipose tissue area was determined as previously described (28). Resting metabolic rate (RMR) was obtained from indirect calorimetry measured in the supine position for 30 min in a ventilated hood system after a 15-min rest. RMR was expressed as kilocalories per 24 h.

Statistical analyses

Descriptive statistics were expressed as means and SD. Differences in genotype frequencies (obese vs. controls, by race) were analyzed using Fisher’s exact test, and P values smaller than 0.05 were considered significant. FM was adjusted for the effects of age and sex. AVF was log transformed to obtain approximate normality and was adjusted for sex, age, and FM. RMR was adjusted for sex, age, age², age³, body height, FM and FFM. Genotype associations with FM, AVF, and RMR were assessed using general linear models, and genotype effect size was defined as the type III sum of squares to the corrected-total sum of squares ratio. Measures of nucleotide diversity were calculated as described (29). The SAS statistical software package (Windows version 8.1, SAS Institute Inc., Cary, NC) was used for all statistical analyses.

Sequencing analysis of the MC4R gene

MC4R is a 332 amino acid seven-transmembrane G protein-coupled receptor, translated from a single exon. The entire 999-bp coding region was sequenced along with 393 and 280 bp of the 5' and 3' flanking regions, respectively. Genomic DNA was isolated from either whole blood or permanent lymphoblastoid cell lines. Coding and noncoding regions of the gene were amplified by PCR using four primer pairs, thus generating four overlapping fragments, in which the fragment generated by primer pair 1 covered the 5' untranslated region (UTR), 2 and 3 the coding region, and 4 the 3' UTR. Oligonucleotide sequences modified from reference (30) were: 1 forward (f) 5'-AGCTTCCGAGAGGCAGCCGA-3'; 1 reverse (r) 5'-CGGTTCCAGAGGTGCAGAGAAG-3'; 2(f) 5'-ATCAATTCAGGGGGACACTG-3'; 2(r) 5'-GACAGCACTACTATCTGAGT-3'; 3(f) 5'-ATGCTCTCCAGTACCATAAC-3'; 3(r) 5'-TGCAGAAGTACAATATTCAGG-3'; 4(f) 5'-CACTCCGGAGTCAAGAACTG-3'; 4(r) 5'-AAGCTTTTAATAAGGACTTTTCT-3'.

The initial PCR amplification was carried out in a volume of 60 µl containing 250 ng DNA, 200 µM of each deoxynucleotide triphosphate (Amersham Pharmacia Biotech Inc., Piscataway, NJ), 0.3 µM of each primer, and 1.25 U Taq polymerase with 6 µl of the manufacturer’s PCR buffer 10x (QIAGEN, Valencia, CA). PCR was performed in a thermocycler (Eppendorf Mastercycler Gradient, New York, NY) at 95 C for 3 min, 55 C for 60 sec and 72 C for 2 min, followed by 40 cycles at 95 C for 30 sec, 55 C for 30 sec, and 72 C for 75 sec terminated by one cycle at 72 C for 10 min. Bidirectional sequencing of the PCR products was performed using Big Dye terminator chemistry (Applied Biosystems, Foster City, CA) as recommended by the manufacturer. The primers used in the initial PCR were also used for sequencing. The purified PCR products were directly sequenced using an ABI3700 automated sequencer (Applied Biosystems) as previously described (31). Sequences were assembled and examined using the Sequencer software (Gene Codes, Ann Arbor, MI). Sequences suggesting heterozygous deletions were verified after subcloning the PCR products into the pPCR-Script Amp SK+ cloning vector (Stratagene Cloning Systems, La Jolla, CA) with subsequent transformation into Epicurian Coli XL10-Gold ultracompetent cells, in concordance with the manufacturer’s protocol. Ten representative clones were then sequenced as outlined above.

Results

A total of 12 nucleotide substitutions was detected among the 528 subjects of the study. The Val103Ile, Thr112Met, and Ile251Leu polymorphisms have been reported earlier by other groups (18, 19, 21, 22, 30, 32) and have been shown to be equivalent to the wild type in terms of pharmacological properties (18, 22, 33) as well as being equally common among obese and nonobese people. All other variants reported here were previously unknown. Among them, two were silent, three were missense, and four were in noncoding regions. The genotype frequencies in obese subjects and normal-weight people are shown in Table 2. Except for the -178A>C variant, all were in the heterozygous form. There were four cases of compound heterozygosity: -184G>A + Thr102Ile (black normal-weight), -178A>C + Ile251Leu (white obese), Val103Ile + Thr112Met (white obese), and Val103Ile + 1123G>A (white obese).

There was a tendency for the -178A>C variant, after pooling blacks and whites, to cluster in the uppermost parts of the BMI distribution: 10 of the 77 subjects with BMI of 45 or more and 5 of the 22 subjects with BMI of 50 or more were carriers. Unadjusted P values for frequency differences between normal-weight controls and subjects with BMIs of either 45 or more or 50 or more were 0.09 and 0.02, respectively. One of the two -178A>C homozygotes in the black cohort was a normal-weight control. Taken together and considering its 5' UTR location, we consider the -178A>C association a chance finding for the moment.

By and large, there were no significant associations with obesity for any of the missense variants found in the whole sample, neither when the analyses were done separately by race (Table 2) nor after pooling the data.

In addition to base substitutions, we detected two novel deletions in the heterozygous state. A -65_-64delTG mutation was found in one morbidly obese white female, 40 yr of age. Self-reported onset of weight problems was late (34 yr), and there were no comorbidities or metabolic aberrations except hyperinsulinemia. The reported BMIs of her mother and father was 29.3 and 26.2 kg/m², respectively. Because this mutation is located in the 5' UTR, any effect on gene function would be at the transcription or expression level, which cannot be predicted and will require further study. A heterozygous 171delC mutation in codon 57 was found in an obese white female, 41 yr of age. This deletion is predicted to cause a frameshift, yielding aberrant sequence of six consecutive amino acids, culminating in a premature stop codon at position 64 (Fig. 1). This truncation is within the first transmembrane domain and is presumably incompatible with a functional protein because domains closer to the C terminus are essential for receptor signaling and cell surface localization (33). The BMI of the carrier was 40.0 kg/m², body height above average (1.73 m), and blood pressure was 155/100 mm Hg. In addition, she exhibited hyperinsulinemia and hypertriglyceridemia. Notably, self-reported age of onset of weight problems, 15 yr, was not as early as has been reported in other null allele heterozygotes. There was no history of obesity in her parents (neither of which were genotyped, however).

View larger version (23K): [in this window] [in a new window]   Figure 1. Chromatogram showing plasmid sequence and predicted amino acid sequences of subcloned alleles from a 171delC heterozygote. Mutation site is indicated by a vertical box.

Discussion

It has been proposed that MC4R mutations are a major cause of monogenic human obesity, accounting for up to 4% of all patients with morbid obesity (22). We tested this hypothesis by mutation screening of severely obese and normal-weight individuals from three cohorts. Whereas sequence variation was not uncommon, none of the previously reported null alleles was present in this relatively large sample of 528 individuals. Moreover, no significant associations were found between any of the detected missense variants and obesity. It could be argued that the sample size of the black obese group was too small to allow rejection of a common-allele hypothesis. However, the binomial probability of finding zero carriers in 47 unrelated individuals, assuming a prevalence of 4%, is less than 0.15. This corresponds to a risk of type II error comparable with that of studies in which a power of 80–90% is deemed sufficient. The corresponding probability for the white obese group (considering only unrelated individuals) was 0.0002. Therefore, we conclude that MC4R mutations are not as frequent a cause of obesity as has been proposed.

An interesting observation was the high frequency of variant sites at the MC4R locus. We compared raw and normalized variant frequencies and average heterozygosity in MC4R coding and noncoding regions to those from a recent report based on mutation screening of 106 genes in a sample of 114 chromosomes (36) (Table 3). The most striking difference was the more than 3-fold higher frequency () of nonsynonymous substitution sites in MC4R, notwithstanding missense variants reported by other groups. The observed lower average heterozygosity per base pair () in our sample is due in part to the difference in sample sizes. Although the two estimates of genetic variation, () and , are expected to be of similar magnitude under the assumption that all mutations are neutral, it has been shown that the correlation between them decreases with increasing sample size (37). Conversely, the normalized difference between and (Tajima’s D statistic) has been implemented as a means of testing whether the assumption of mutation neutrality is valid. In the present study, D values for noncoding, synonymous, and nonsynonymous variants were -1.17, -1.09, and -1.51, respectively. Based on the ß distribution of D (mean, 0; variance, 1), none of these values is significantly smaller than zero [95% confidence interval -1.715 to 2.150 (37)]. Thus, the hypothesis of mutation neutrality was not rejected, although there was a nonsignificant trend (P < 0.1) for nonsynonymous variants toward significant negative divergence, which could suggest the presence of selection against deleterious mutations. Consistently, noncoding and synonymous variants are likely to be selectively neutral, as suggested by the nonsignificant divergence of their D values.

Acknowledgments

We thank Dr. Cynthia K. Buffington for providing the data from the Memphis cohort.

Footnotes

The SOS study was supported by Swedish Medical Research Council Grant 05239 (to L.S.). The HERITAGE Family Study is supported by National Heart, Lung, and Blood Institute through Grants HL-45670 (to C.B.), HL-47323 (to A.S.L.), HL-47317 (to D.C.R.), HL-47327 (to J.S.S.) and HL-47321 (to J.H.W.). This work was partially supported by the Henry L. Taylor Endowed Professorship in Exercise Science and Health Enhancement (to A.S.L.) and the George A. Bray Chair in Nutrition (to C.B.).

Abbreviations: AVF, Abdominal visceral fat; BMI, body mass index; FFM, fat-free mass; FM, fat mass; HERITAGE, Health, Risk Factors, Exercise Training, and Genetics Family study; MC4R, melanocortin 4 receptor; RMR, resting metabolic rate; SOS, Swedish Obese Subjects study; UTR, untranslated region.

Received April 10, 2002.

Accepted July 9, 2002.

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