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Steno Diabetes Center and Hagedorn Research Institute (L.H.L., S.M.E., O.P.), 2820 Gentofte, Denmark; Exiqon (S.M.E.), 2950 Vedbaek, Denmark; Danish Epidemiology Science Centre (L.H.L., T.I.A.S.), Institute of Preventive Medicine, Copenhagen University Hospital, 1357 Copenhagen, Denmark; Roskilde County Hospital (T.A.), University of Copenhagen, 4000 Roskilde, Denmark; Department of Molecular Pharmacology (B.S.W.), Novo Nordisk, 2760 Maaloev, Denmark; and Faculty of Health Science (O.P.), University of Aarhus, 8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: Lesli Hingstrup Larsen, Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 6, NSK 1.14, 2820 Gentofte, Denmark. E-mail: LieL{at}Steno.dk.
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Abstract |
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MSH, whereas the Ala219Val variant showed a significantly impaired melanotan II induction of cAMP, compared with the wild-type receptor. The remaining 11 mutations have previously been reported, but selected MC4R variants were further characterized in vitro in the present study. A previously identified nonsense mutation, Tyr35stop, had a relatively high allele frequency (0.6%), suggesting a possible founder effect in the Danish population. This study shows a carrier frequency of 2.5% of pathogenic mutations in the MC4R gene in a population-based study of obese men. Thus, variation in this gene is the most common known specific genetic cause of obesity among Scandinavian men. |
Introduction |
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MSH, a melanocortin produced by proconvertase-1-dependent cleavage of proopiomelanocortin. The binding of the agonist to the MC4R induces an increased synthesis of cAMP through the interaction of the Gs-G protein. Several superpotent MSH analogs have been developed, among these melanotan II (MTII) and [Nle4,D-Phe7] (NDP)-MSH. MC4R signaling is modulated by the antagonist agouti-related protein, which is also expressed in the hypothalamic arcuate nucleus (2). The MC4R is one of the downstream targets for the adipocyte signaling molecule, leptin, which among other molecules acts through the activation and inhibition of proopiomelanocortin and agouti-related protein, respectively.
The actions of MSH on the MC4R lead to a decrease in food intake, and mice with null mutations in MC4R demonstrate a very obese phenotype accompanied by hyperinsulinemia, whereas heterozygous MC4R-deficient mice present an intermediate phenotype with female mice being more obese than males (3, 4). In humans, several studies identified rare obesity-associated MC4R mutations cosegregating with obesity and defective binding or signaling properties of the variant receptors when tested in vitro (5, 6, 7). In these studies the prevalence of pathogenic mutations of the MC4R gene varies considerably, from 0.5% in some reports of adult obesity to a relatively high prevalence of 5.8% among severely obese British children. MC4R deficiency in man is characterized by hyperphagia; hyperinsulinemia; and increased fat mass, lean body mass, bone mineral density, and linear growth rate.
In the present study, we report on the variation of the coding region of MC4R in a population-based sample of 750 Danish men [mean age 19.8 ± 1.8 yr (SD)] with juvenile-onset obesity and 706 matched control subjects [mean age 20.0 ± 1.9 yr (SD)] who were examined in a case-cohort design. Moreover, selected MC4R variants were functionally characterized in vitro.
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Subjects and Methods |
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The mutation analysis was performed on genomic DNA from 1456 Danish men, who around age 20 yr [mean age: 19.9 ± 1.8 yr (SD)] were examined at the draft board and who, in addition, were examined at the Copenhagen City Heart Study Programe in 1981–1983 (8) and again in 1992–1994 after 27.4 ± 8.4 (SD) yr of follow-up (9). The cohort of men consisted of two groups: one obese group (n = 750) with juvenile-onset obesity, i.e. with body mass index (BMI) 31 kg/m2 or greater at the draft board examination (BMI 33.3 ± 2.4 kg/m2) and a control group (n = 706) involving the randomly selected one half percentage of the young men at the draft board (BMI 21.4 ± 2.1 kg/m2). The control group was recruited without selecting for or against BMI according to the principles of a case-cohort sampling strategy. Genomic DNA was obtained from blood samples, drawn at the last examination at the Copenhagen City Heart Study. The mean age at reexamination was 43.1 ± 6.1 yr and the mean BMI was 35.7 ± 5.7 kg/m2. Clinical variables were measured as reported by Schnohr et al. (10). The study was approved by the Ethical Committee of Copenhagen and was in accordance with the principles of the declaration of Helsinki II.
Mutation analysis of the of the coding region of the MC4R gene
PCR amplification was carried out in a reaction volume of 35 µl containing 100 ng genomic DNA, 1x PCR buffer, 0.2 µmol/liter of each primer, 0.2 mmol/liter deoxynucleotide triphosphate, 1.5 mmol/liter MgCl2, and 0.35 U Amplitaq-polymerase (Applied Biosystems, Foster City, CA). The cycle program was an initial denaturation at 95 C for 5 min followed by 35 cycles of denaturation at 95 C for 30 sec, annealing at 55 C for 30 sec, and elongation at 72 C for 30 sec with a final elongation step at 72 C for 10 min using a GeneAmp PCR System 9700 (Applied Biosystems). Primers were selected from the published GenBank sequence of MC4R (accession no. S77415) for PCR amplification of two segments: segment AB, primer A forward 5'-ATCAATTCAGGGGGACACTG-3' and primer B reverse 5'-TCCACTGCAATTGAAAGCAG-3' and segment DE, primer D forward 5'-TGTAGCTCCTTGCTTGCATC-3' and primer E reverse 5'-TGCATGTTCCTATATTGCGTG-3'. Denaturing HPLC was performed on a WAVE DNA fragment analysis system (Transgenomic, Crewe, UK). To enhance heteroduplex formation, the PCR product was denaturated at 95 C for 5 min and allowed to renaturate at room temperature for 30 min. Samples were automatically loaded onto the DNA separation column (Transgenomic) and eluted with a linear gradient of acetonitrile (J.T. Baker, Phillipsburg, NJ) in a 0.1 M triethylamine acetate buffer (pH 7), at a constant flow rate of 0.9 ml/min. The start and end points were adjusted according to segment size. Analysis time per amplified segment was 6.3 min including column regeneration and equilibration. Samples were analyzed at the melting temperatures determined to cover the entire amplified segment using the WAVE Maker (version 4.0, Transgenomic). Eluted segments were detected by an UV-C detector (Transgenomic). Samples displaying variation in the dHPLC melting profile were reamplified using the same primers with 5' modifications for –21 M13 uniprimer or –29 M13-rev primer, purified using Microcon centrifugal filter devices YM-100 (Millipore, Bedford, MA), and directly sequenced using ABI Prism dye primer cycle sequencing kit with Amplitaq DNA polymerase FS on ABI Prism 377 (Applied Biosystems).
In vitro analysis of mutated receptors
Receptor constructs. Identified variants were introduced into MC4R cDNA containing PC1-DNA3.1 vector using Quickchange site-directed mutagenesis (Stratagene, La Jolla, CA) according to the manufacturer’s instructions (mutation primers can be requested from the corresponding author). Plasmid DNA was generated using the plasmid maxikit (Qiagen, Santa Clarita, CA) and sequenced using –29 M13 and –21 uni-M13 primers (MWG Biotech, Ebersberg, Germany).
Cell culture and transient transfection. Human embryonic kidney (HEK)293 cells were routinely maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY) and penicillin/streptomycin (90 units ml–1 and 90 µg ml–1, respectively). Cells were seeded in T75 flasks, transfected with 8.5 µg receptor DNA and 0.5 µg ß-galactosidase construct using the FuGEne6 transfection reagent (Roche, Stockholm, Sweden), harvested 24–48 h after transfection, and applied directly to the activity experiments or plasma membrane preparations. ß-Galactosidase activity was measured as a control for transfection efficiency according to the manufacturer’s instruction (Promega, Madison, WI).
cAMP activity assay. HEK293 cells transiently expressing the wild-type or mutated MC4R were harvested using Versene (Life Technologies) and resuspended in assay buffer (Flashplate, NEN Life Science Products, Boston, MA) to a cell density of 2.0 x 106 cells/ml. MTII was diluted in dH2O or dH2O with 0.02% Tween20. Cells (50 µl) and MTII (50 µl) were mixed gently for 5 min in 96-well Flashplates (NEN Life Science Products) and incubated for 25 min at room temperature. The amount of cAMP produced was measured by displacement of radiolabeled cAMP bound to anti-cAMP antibodies in the Flashplates in accordance with the manufacturer’s instructions.
Preparation of plasma membranes. Before preparation of plasma membranes, cells were aliqouted for ß-galactosidase assay. The remaining cells were harvested, resuspended in 5 ml cold buffer A [10 mM Tris-HCL (pH 7.4) and 1 mM EDTA], and subjected to homogenization by a 10-sec burst using a polytron homogenizer and subsequent centrifugation at 15 min at 10,000 rpm (11,951 x g) and 4 C. The resulting pellet was resuspended in 5 ml cold buffer A and centrifuged as above.
The final pellet was resuspended in 3 ml buffer A. The amount of protein in each preparation was measured by Coomassie blue staining and measured at 520 nm on a spectrophotometer. Aliquots were frozen at –80 C.
Binding assay.
Freshly thawed membrane preparations and NDP-MSH were diluted in assay buffer (50 mM HEPES-NaOH, 5 mM NaCl2, 5 mM EGTA, 0.005% Tween 20). Membranes, cold NDP-MSH, and 125I-NDP-MSH tracer (Bachem, Torrance, CA) were mixed in 96-well 0.65-µm filter microtiter plates (Millipore) and incubated for 2 h at 25 C. Next, bound and unbound peptide/tracer was flushed away using a vacuum manifold (Millipore), and plates were washed twice in 100 µl/well cold 1 mM NaOH and left at 30 C to dry. Filters were exercised using the Millipore punch system and the amount of bound radioligand was determined using a Packard 
-counter (Packard, Ramsey, MI).
Data analysis and statistics
Fisher’s exact test was applied to test for the significance of differences in allele frequencies. A t test for comparison of means for independent samples with equal variance in SPSS (version 11.0, SPSS Inc., Chicago, IL) was used in genotype-phenotype association studies. All genotype groups were in Hardy-Weinberg equilibrium. The activity and binding data were studied applying nonlinear regression analyses, i.e. using Prism (version 4.00, GraphPad Software, Inc., San Diego, CA).
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Results |
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MSH binding or impaired cAMP response (Table 1
and Figs. 1 and 2). Three known variants, Ile103Val, Ile251Leu, and Thr112Met, had the same allele frequencies in the obese and control groups (Table 1). Because these variants in preceding studies were demonstrated to possess normal receptor function, they were not further examined in the present study. Moreover, we identified two nucleotide substitutions, 117G>A and 714T>G; each of them was found in only one obese subject (Table 1). Because these mutations merely predict synonymous substitutions, they were not considered to have any functional impact on the MC4R protein and were thus not further characterized. The Ala175Thr variant was found in one obese subject (Table 1) and has already been reported to induce partial inactivity of the MC4R (7). The codon 175 variant was thus categorized as partially active and was not characterized in this study. The previously identified missense mutation, Arg165Gln, had an allele frequency of 0.4% in the obese group and was not found in the control group (Table 1). This mutation was significantly associated with juvenile-onset obesity (P = 0.02); in addition, the encoded mutated codon 165Gln MC4R protein did not show any binding of NDP-MSH (Fig. 1).
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). Analysis of the binding properties of the Gly181Asp and Leu325Phe showed that neither of these mutated receptors did bind NDP-MSH (Fig. 1). The Ser36Tyr, Ile102Thr, and Ala219Val MC4R variants bound NDP-MSH with the same affinity as the wild-type MC4R (Fig. 1), but they showed various degrees of MTII cAMP activation, compared with the wild-type receptor (Fig. 2). The activities of the Ser36Tyr, Ile102Thr, and Ala219Val variants were also assayed using MSH. The mean EC50 with MSH for the wild-type, Ser36Tyr, Ile102Thr, and Ala219Val receptors were 1.7 x10–7, 2.6 x 10–7, 2.4 x 10–7, and 5.4 x 10–7 (n = 2), respectively, and the maximal activity obtained with MSH showed the same effect of these mutant receptors, compared with the wild-type receptor as the results for MTII (data not shown). The Ser36Tyr and Ile102Thr mutations showed nonsignificant impairments of cAMP-induced activity (86 and 81%, respectively, relative to wild-type) in response to MTII, whereas the Ala219Val variant showed a significantly reduced activity, compared with the wild-type receptor (34%) (Fig. 2). The haplotype, consisting of the nonsense mutation Tyr35stop and the nucleotide substitution 110A>T, was found in nine obese subjects (Table 1). The allele frequency of the haplotype was 0.6% in the obese group, and no carriers of this haplotype were found in the control group (Table 1). Thus, the haplotype was significantly associated with juvenile-onset obesity (P = 0.004). Moreover, when tested for binding of NDP-MSH, the haplotype showed no binding of the NDP-MSH ligand (Fig. 1).
Thus, in total we identified four MC4R variants that completely abolished the binding of NDP-MSH to the receptor, and these mutations are considered loss-of-function mutations (Fig. 1). One variant, the Ala219Val, showed a significant reduction in cAMP signal properties of the receptor and is, together with the Ala175Thr receptor variant, categorized as partially active (Fig. 2).
When the variants showing no binding of NDP-MSH or significantly impaired cAMP activity in vitro, compared with the wild-type receptor, are summed, defective MC4R mutations in the present study are highly associated with obesity among Danish men (P < 0.0001). Phenotypic characteristics (BMI and height at baseline; BMI, height, waist to hip ratio, and sagittal diameter at follow-up; and average BMI gain per year from baseline to follow-up) for carriers of the inactive or partially active mutations were compared with the remaining group of men with obesity. No consistent differences in the examined phenotypes could, however, be demonstrated (data not shown). Similarly, we were unable to show any differences in anthropometrics between obese subjects carrying nonsense, compared with missense, mutations (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We identified nine carriers of a Tyr35stop mutation and six carriers of an Arg165Gln variant, both of which did not show any binding of NDP-MSH (Fig. 1). In previous studies these mutations have shown cosegregation with obesity, and the encoded receptors display impaired in vitro function (11, 12, 14). In the present protocol, we did not have access to family materials. However, we show that the Arg165Gln variant failed to bind NDP-MSH, whereas a recent study by Farooqi et al. (7) found that it only partially inhibited the receptor. Because abolishment of binding to the Arg165Gln mutant receptor is observed, it is assumed that this receptor will not be able to transduce any signal giving rise to any cAMP activity. Thus, the discrepancy between these results could be explained by the difference in the applied methods. We measured the direct binding of a ligand, whereas Farooqi et al. (7) measured the produced amount of intracellular cAMP indirectly through a reporter assay using a cAMP response element. Further support for an absolute abolishment of the binding ability by this variant comes from a study by Nijenhuis et al. (26), who found the Arg165Gln variant to be completely retained in intracellular vesicles and no MC4R protein transportation to the cell membrane.
The Tyr35stop variant encodes a truncated receptor, and in this study no binding of this mutated receptor was seen, which is consistent with a previous study (15). The Tyr35stop variant was in complete linkage disequilibrium with an 110A
T nucleotide substitution, and consequently it constituted a haplotype in the Danish population. The same MC4R haplotype has been reported in the German population (6, 11, 12). The high prevalence of this variant in the Danish population may indicate the existence of an ancestral founder of the Tyr35stop/110AT haplotype possibly shared with the German population. For obvious reasons, we cannot, however, in our study exclude a family relationship between these haplotype carriers.
The fact that different effects of mutations in the MC4R has been demonstrated, among these a recessive mutation (14), a constitutively active mutation (5), and some functional mutations with reduced penetrance (14), points to the existence of various functional mechanisms for the different variants of the MC4R. Our finding of a relatively high prevalence of the truncated Tyr35stop/110AT haplotype among obese subjects may suggest that a common impact of mutations in the MC4R could be due to haploinsufficiency. This suggestion is supported by in vitro coexpression studies of two truncated MC4Rs, which do not show any evidence of dominant-negative effects (27). In contrast, a recent study identified a loss-of-function MC4R variant, which appears to have an additional dominant-negative impact based on receptor dimerization (28). This may prove to be relevant in modulating the effect of some functional MC4R variants. Intriguingly, investigations of subjects with hemizygous chromosomal deletions of parts of chromosome 18q including the MC4R locus did not show that subjects hemizygous for the MC4R were more obese, compared with subjects carrying both alleles (29). However, a more in-depth analysis of the healthy alleles or mutations at other loci is necessary to fully address these inconsistencies.
The rare variant, Gly181Asp, has been identified previously, and our in vitro study of the variant is in agreement with the functional implications reported (6, 7). The Ile102Thr variant showed an activity of 81% of the wild-type activity in the present study and was thus assumed to have wild-type activity, an interpretation that is in line with earlier reports in which no cosegregation with obesity was shown (16, 30).
Two polymorphisms, Val103Ile and Leu251Ile, were found in both the obese group and the control group with similar allele frequencies (P = 0.63 and P = 0.70, respectively). Again, this is consistent with previous studies (5, 7, 12, 14, 27, 31). The two substitutions are conservative and have in in vitro studies no implications on MC4R binding or signaling (31). However, a Swedish study by Rosmond et al. (32) demonstrated that heterozygous carriers have lower waist to hip ratio than wild-type subjects. In the present study, we were unable to show that the codon 103 variant is associated with indices of obesity (Table 1), and prior investigations of the functional impact on the expressed protein of this polymorphism in vitro did not show any difference (27). Yet we cannot at the present exclude that the codon 103 variant may have a slight impact on metabolism without influencing binding or activity of the MC4R in vitro by affecting genome-associated functions before transcription, or it might be in linkage disequilibrium with some as-yet-unknown variants in the MC4R locus.
One already known amino acid variant, Thr112Met, was in the present study found in one obese and one control subject. This mutation has been previously reported with a similar carrier frequency in obese and nonobese subjects and does not change the in vitro function of the encoded receptor protein (12, 31).
We failed to show anthropometric differences between obese carriers of the MC4R mutations and the remainder part of the obese group. That the carriers were not taller than the rest of the obese men and did not have an altered pattern for changes in BMI during 27 yr of follow-up might suggest that altered phenotypic features of MC4R mutation carriers are to be identified in childhood, a suggestion made by Farooqi et al. (7), who observed an age-related decrease in hyperphagia and hyperinsulinemia of obese children with MC4R mutations.
This study includes only men, and as has been shown in both rodent and human studies, females or women have a tendency to gain more weight or have a higher BMI than males or men with identical deletions or mutations (3, 11).
In conclusion, among 750 Danish men with juvenile-onset obesity, the carrier frequency of pathogenic mutations in the MC4R gene was 2.5%, providing strong confirmation that variations in this gene are the most common genetic cause of obesity identified to date.
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Acknowledgments |
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Footnotes |
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First Published Online October 14, 2004
Abbreviations: BMI, Body mass index; HEK, human embryonic kidney; MC4R, melanocortin 4 receptor; MTII, melanotan II; NDP, [Nle4,D-Phe7].
Received March 12, 2004.
Accepted October 1, 2004.
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-melanocyte-stimulating hormone genes in severely obese children. J Pediatr 139:204–209[CrossRef][Medline]
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