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Department of Medicine and Research Program in Molecular Medicine (K.V.-J., L.O., K.K., C.S.-J.), University of Helsinki, FIN-00290 Helsinki, Finland; The Hospital for Children and Adolescents (M.L.-N.) and Department of Endocrinology (C.S.-J.), Helsinki University Hospital, FIN-00290 Helsinki, Finland; and Department of Medicine (A.N.H., C.B.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Camilla Schalin-Jäntti, M.D., Ph.D., Departments of Medicine and Endocrinology, Helsinki University Hospital, Haartmaninkatu 4, P.O. Box 340, FIN-00290 Helsinki, Finland. E-mail: camilla.schalin-jantti{at}hus.fi.
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Abstract |
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40 kg/m2), were screened for melanocortin-4 receptor (MC4R) mutations. We identified a pathogenic mutation (S127L) in one child, causing severe early-onset obesity. We describe the phenotype of this particular mutation for the first time. We also identified a novel (I226T) polymorphism in the coding and two new variations (-439delGC and 1059C>T) outside the coding region of the MC4R gene. Three previously described polymorphisms (V103I, T112M, and I125L) were identified. In vitro functional studies of variants T112M, S127L, and I226T supported a pathogenic role of the S127L mutation, because signaling properties of the receptor in response to the MC4R agonists 
-MSH, ß-MSH, and 
1-MSH were impaired. The S127L mutation did not affect receptor inhibition by the antagonist agouti-related protein. Localization of the three variant receptors was similar to that of wild type. In conclusion, a pathogenic MC4R mutation was found among subjects with severe early-onset obesity but not among morbidly obese adults. Impaired function of the S127L receptor was due to reduced activation, not a defect of protein transport to the cell membrane. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-MSH and the antagonist agouti-related protein (AGRP) (3). There is also growing evidence that -MSH is not the most abundant form of MSH in the brain and that other pro-opiomelanocortin-derived peptides also could function as MC4R agonists (4, 5, 6). The critical role of MC4R in the regulation of body weight was first shown in rodents. Mice lacking both alleles of MC4R develop a severe obesity syndrome, whereas heterozygous mice lacking one MC4R allele have an intermediate obesity phenotype (7). In humans, the importance of MC4R in the regulation of body weight was highlighted in 1998, when two groups reported heterozygous frameshift mutations in the MC4R as a cause of dominant, severe early-onset obesity (8, 9). Subsequently, several groups have reported associations between MC4R mutant variants and early-onset morbid obesity (10, 11, 12, 13, 14). These mutations represent the most common monogenic defect causing human obesity so far reported, accounting for up to 4% of morbid obesity (12). However, there are also studies challenging this view (15, 16, 17). The present study was undertaken to investigate whether MC4R mutations underlie morbid obesity in Finns, a homogenous ethnic population in which MC4R has not been studied hitherto, and whether the results would differ between subjects with severe early-onset obesity compared with adult morbid obesity. The inheritance of the MC4R variants detected was investigated in the respective families. Functional properties of the mutant receptor variants were investigated in vitro using a variety of MC4R agonists and the antagonist AGRP. Subcellular localization of the mutant receptor variants was assessed by ELISA. The phenotype of one mutation carrier was carefully characterized. |
Subjects and Methods |
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The first cohort includes 56 children, who were referred to the Helsinki University Hospital of Children and Adolescents because of severe, early-onset obesity (relative weight for height greater than or equal to +70% before age 10; 29 females and 27 males; mean age at present ± SD, 13.6 ± 4.7 yr). The inclusion criteria included a height-specific relative weight greater than 60% and/or age-specific relative body mass index (BMI) greater than the 98th percentile value. Weight and length chart data from birth onward were available for all children. The patients were carefully examined with regard to weight, height, waist-hip ratio, and blood pressure. Pubertal stage was classified according to Tanner (18). Blood samples were drawn for DNA analysis and serum glucose, lipid, and hormone measurements. The second cohort consisting of 252 morbidly obese adults (BMI, 40 kg/m2; 182 females and 70 males; mean age ± SD, 48.6 ± 8.9 yr) has been previously described (19). All subjects were referred to the Department of Endocrinology, Helsinki University Central Hospital, at adult age because of morbid obesity (19). History of weight development in the adult group was assessed by a questionnaire. Five individuals reported that they were obese at 10 yr of age, but none of the subjects was diagnosed with or treated for severe childhood obesity. Blood samples from 321 healthy blood donors (161 females and 160 males) were obtained from Dr. Tom Krusius (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) and were used for determination of allele frequencies in the background population. Written informed consent was obtained from all participants or their guardians, and the study was approved by the local ethics committee.
Sequence analysis of the MC4R gene
The MC4R gene was amplified by PCR using six primer pairs, covering the entire coding region and 1139 and 251 bp of 5'- and 3'-flanking regions, respectively. Four of the primer pairs used have been described previously (20). In addition, the following two pairs of primers were used: PPF, 5'-TCAGTATCCTAGCCAAAAAGCA-3', PPR, 5'-TCCTTGAAGCTGCCATGC-3'; and PDF, 5'-TTTTCCTGAGTTCAGTGTAAGCA-3', PDR 5'-CCCTAGAGGCCATTCTGTGA-3'. The amplified fragments were analyzed by denaturing HPLC with WAVE nucleic acid fragment analysis system HSM 3500A (Transgenomic, Omaha, NE) and/or direct sequencing by ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). Genotyping of the -493delGC, V103I, T112M, S127L, I226T, I251L, and 1059C>T variants was performed using restriction enzymes Hin6I, BpiI, HpyCH4IV, MlyI, BoxI, Kpn2I, and BstNI, respectively.
Site-directed mutagenesis
A human (h)MC4R cloned in pcDNA3 (Invitrogen, Carlsbad, CA) and a hemagglutinin (HA)-tagged hMC4R green fluorescent protein (GFP) fusion protein (HA-hMC4R-GFP) encoded in pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA) were kindly provided by Dr. Jeffrey S. Flier (Division of Endocrinology, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA) (21). The mutations were incorporated into the hMC4R and HA-hMC4R-GFP constructs by QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).
Cell culture and transfection
The 293T cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM, high glucose, supplemented with 10% (vol/vol) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 C in a humidified atmosphere containing 5% CO2. All transfections were performed with Lipofectamine reagent (Invitrogen) in OPTI-MEM I reduced serum medium (Invitrogen). Receptor signaling was studied by transiently transfecting 293T cells with wild-type (wt) or mutant MC4R and a cAMP-responsive TRH luciferase reporter (TRH-Luc) (22). A ß-galactosidase (ß-gal) expression vector under control of a CMV promoter (ß-gal-CMV) was included in all transfections as an internal control. After transfection and overnight incubation, cells were stimulated with increasing amounts of the MC4R agonists -MSH (Sigma-Aldrich, St. Louis, MO), ß-MSH, or 1-MSH (Phoenix Pharmaceuticals, Belmont, CA) and incubated for 5 h at 37 C and 5% CO2. To evaluate the effect of the MC4R antagonist AGRP (83–132) (Phoenix Pharmaceuticals) on the mutant receptors, cells were pretreated with increasing concentrations of AGRP for 30 min and incubated for 5 h in a constant 100 nM -MSH concentration. The luciferase assay and measurement with an EG&G Berthold LB 9501 luminometer (Berthold Technologies, Bad Wilbad, Germany) was performed as described previously (23). All values were normalized for transfection efficiency by determination of ß-gal activity with the Galacto-Light kit (Applied Biosystems).
Fluorescence microscopy
The 293T cells were plated on poly-D-lysine (Sigma-Aldrich)-coated Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) and transfected with HA-hMC4R-GFP or the pEGFP-N1 vector. Cells were washed once with cold PBS (Invitrogen), fixed with 3% paraformaldehyde in PBS, and mounted with Vectashield mounting medium with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Cells were visualized on an Axioskop 2 fluorescence microscope, and pictures were taken with an AxioCam digital camera (Carl Zeiss, Thornwood, NY).
ELISA
ELISA was performed as a quantitative analysis of receptors on the cell surface. The 293T cells were grown on poly-D-lysine-coated 24-well plates and transfected with HA-hMC4R-GFP or the pEGFP-N1 vector. ELISA was performed as previously described by Shinyama et al. (21), and the absorbance of the samples was measured at 450 nm.
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Results |
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Among severely obese children, we identified two single-nucleotide substitutions, predicted to result in S127L and V103I amino acid changes, a novel -439delGC deletion in the putative promoter region of the MC4R gene, and a novel 1059C>T change in the 3' untranslated region. Among adult obese subjects, we detected a novel heterozygous missense mutation I226T and the previously described V103I, T112M, and I251L polymorphisms (Table 1
). In addition, four common polymorphisms (-1042C>T, -1005C>T, -896C>T, and -719G>A) were detected in the 5' flanking region of the MC4R gene. All mutations and polymorphisms were found in heterozygous form. The proband of the S127L mutation was also a carrier of the V103I polymorphism. Blood samples were collected from available T112M, S127L, and I226T family members, and the transmission of these mutations was tested within the families (Fig. 1A).
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The ability of MC4R to generate cAMP in response to increasing concentrations of -MSH was studied by cotransfecting 293T cells with wt or mutant MC4R and a cAMP-responsive TRH-luciferase promoter construct. This assay revealed impaired response of the S127L mutant receptor to -MSH (Fig. 2A). The mutant receptors were also tested for ß-MSH and 1-MSH. The activity of S127L mutant receptor was impaired for all tested peptides, whereas the other mutants gave responses similar to the wt receptor (Fig. 2, B and C). No difference was seen between the different mutants or the wt receptor when tested for inhibition by AGRP (Fig. 2, D and E). Before evaluation of mutant receptor expression and subcellular localization, we confirmed that intracellular signaling in response to -MSH in 293T cells transfected with HA-hMC4R-GFP was similar to signaling in cells transfected with hMC4R (data not shown).
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Cellular localization of the mutant and wt MC4-receptors was examined by fluorescence microscopy and ELISA. All mutants and the wt receptor could be detected on the cell membrane by fluorescence microscopy (Fig. 3A). Quantification with ELISA was performed to demonstrate that same amounts of mutant and wt receptors were transported to the cell membrane (Fig. 3B).
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The S127L mutation was found in a 14.8-yr-old boy with severe early-onset obesity. His abnormal weight gain began in the second year of life (Fig. 1B
). On physical examination, pubertal stage was G4P3, and he presented with acanthosis nigricans in the neck and axillae. His weight was 111 kg; height, 174 cm (height SD score, +0.8); BMI, 36.7 kg/m2; waist circumference, 123 cm; hip, 121 cm; and waist-hip ratio, 1.02. Blood pressure was 126/72 mm Hg. On laboratory testing, his fasting blood glucose was 4.7 mmol/liter; insulin, 33 mU/liter; leptin, 21 µg/liter; TSH, 1.28 mU/liter; and free T4, 14 pmol/liter. Total cholesterol was 4.2 mmol/liter; high-density lipoprotein cholesterol, 1.31 mmol/liter; low-density lipoprotein cholesterol, 2.31 mmol/liter; and triglycerides, 1.13 mmol/liter. An oral glucose tolerance test (OGTT) performed 6 months later, revealed normal glucose tolerance but marked hyperinsulinemia. Glucose levels (sampling at 0, 30, 60, and 120 min) were 4.9, 6.5, 6.1, and 6.5 mmol/liter, and the corresponding insulin values were 47, 213, 139, and 181 mU/liter, respectively. Testosterone, FSH, and LH levels were 7.0 nmol/liter (normal range for corresponding pubertal stage, 2–13 nmol/liter), 1.2 IU/liter (1–7 IU/liter), and 0.8 IU/liter (1–9 IU/liter), respectively. All available relatives of the S127L proband were genotyped with restriction enzyme analysis. Only the proband’s moderately obese father was proved to be positive for the S127L mutation, which could not be found in the grandparents (Fig. 1A). Nonpaternity in the family was excluded by microsatellite analysis.
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Discussion |
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In line with the study by Jacobson et al. (17), no pathogenic MC4R mutations were found in the present large cohort of morbidly obese adult patients. In contrast, among subjects with severe early-onset obesity from the same geographical area, the prevalence of pathogenic MC4R mutations in the present study was 1.8% (95% confidence intervals, 0–5.0%). As the child cohort was fairly small (n = 56), this figure might change as more children are investigated and should be regarded as an approximate. The figure is, however, close to the frequency of 2.9%, which represents an estimate from all available MC4R studies up to October 2002 (24). Furthermore, Hirschhorn and Altshuler (24) argued that even this figure may be too high, because some of the missense changes identified in obese subjects do not impair function in vitro and therefore may not represent functional MC4R mutations. In line with this, the frequency of pathogenic MC4R mutations in a recent large screening study of 808 extremely obese German children and adolescents was 1.9% (95% confidence intervals, 1.0–3.0%) (25). Somewhat surprisingly, Jacobson et al. (17) found no MC4R mutations in a subgroup of adults with childhood onset of obesity. Because this grouping was defined based on self-reported estimates of age at onset of weight problems (17), it is likely that the subjects had a much less severe phenotype in childhood compared with children with a verified diagnosis of severe, early-onset obesity. This might provide one explanation why Jacobson et al. (17) found no pathogenic MC4R mutations.
It is clear that environmental factors may have a large impact on the development of morbid obesity in adult age, probably diminishing the likelihood of finding dominant mutations in these subjects. Interestingly, the obesity phenotype associated with MC4R mutations becomes less prominent with age (26). It is therefore possible that adult subjects with MC4R mutations are more likely to be found among the large group of moderately, rather than morbidly obese adults.
The two novel changes in the noncoding region of the MC4R gene (-439delGC and 1059C>T) were both found in single individuals of the cohort of obese children. The 1059C>T is located 60 bp after the translation stop codon and 108 bp before the polyadenylation signal. Further studies on these variants are needed to evaluate their possible significance.
Three of the variants (T112M, S127L, and I226T) were selected for functional studies, based on the facts that the T112M and S127L mutations were in our material present in obese individuals only, and no functional information was available for the I226T variant. Because there is growing evidence that -MSH may not be the most important MC4R agonist in the brain (4, 5, 6), we took a novel approach and studied the signaling properties of the mutant receptors, in addition to stimulation with -MSH, also with ß-MSH and 1-MSH. These studies confirmed the pathogenic role of the S127L mutation, showing decreased responses to all the agonists tested. The data indicate that obesity in S127L carriers may, in addition to decreased signaling in response to -MSH, also be due to decreased signaling in response to ß-MSH. In contrast, 1-MSH is a poor ligand for MC4R in vitro, and it seems unlikely that the observed decreased response to very high doses of 1-MSH plays a significant role in the pathogenesis of obesity in S127L carriers. In line with this, -MSH peptides are believed to signal mainly through the MC3R and/or a hitherto-uncharacterized -MSH receptor (27). We also tested the hypothesis that mutant MC4R receptors could respond differently, in a mutation-dependent manner to these peptides. The results do not, however, support such a hypothesis. In contrast to the S127L, the responses of the previously described T112M and the novel I226T variants did not differ from that of wt receptor. The variant receptors were also tested for inhibition by AGRP. It can be concluded that the mutations do not affect AGRP-mediated inhibition of the MC4-receptor in vitro. The cell membrane localization of the three variants, T112M, S127L, and I226T, did not differ from that of wt, confirming that the impaired S127L function is the result of a signaling defect and not a defect of transport to the cell membrane.
No cosegregation of the T112M or I226T variants can be seen with the obesity phenotype (Fig. 1A). In line with this, the activity of the T112M variant did not differ significantly from that of wt receptor in two recent studies (28, 29). In contrast, in the S127L family, a segregation pattern compatible with a dominant inheritance of this mutation can be seen, because both mutation carriers are obese and the other family members are lean (Fig. 1A).
The S127L mutation was found in a 14.8-yr-old boy, in whom abnormal weight gain began in infancy (Fig. 1B) and whose current BMI is 36.7 kg/m2. In line with previous phenotypic data from other MC4R mutation carriers (26), the patient had severe fasting hyperinsulinemia. His fasting insulin values were 33 and 47 IU/ml, respectively, as measured at baseline and 6 months later during an OGTT. In comparison, the mean (±SD) fasting insulin value for 11 age-matched (13.5 ± 0.9 yr; BMI, 35.1 ± 3.9 kg/m2) boys from the same cohort was only 19.5 ± 5.0 IU/ml. Furthermore, the patient demonstrated marked hyperinsulinemia during an OGTT, whereas glucose tolerance remained normal. He also presented with acanthosis nigricans, which together with hyperinsulinemia points toward marked insulin resistance (30, 31). In contrast to previous data from children with other MC4R mutations (11), his blood pressure has been normal for age. Furthermore, his height is normal (SD score, +0.8) and close to the final height expected from his parents. At the age of 12.8 yr, he was moderately advanced in skeletal development with a bone age of 14.0 yr. Consequently, he was tall for his age, as obese children in general are (32, 33). The father, who carries the same mutation, suffered from severe obesity in childhood, whereas this phenotype became less apparent in adult age.
While this work was in progress, Lubrano-Berthelier et al. reported that the majority of childhood obesity-associated heterozygous MC4R mutations lead to intracellular retention of the mutated receptor (34). However, in line with our data, the S127L mutation was characterized by decreased activation of the receptor by -MSH, but not by impaired cell surface expression (34). The S127L mutation was also detected in compound heterozygous form in two subjects with severe, early-onset obesity in the very recent study by Hinney et al. (25). In contrast to the present study and the study by Lubrano-Berthelier et al. (34), Hinney et al. (25) concluded that the S127L mutation led to constitutively active receptor function, because they found a slight 2-fold increase in basal S127L activity compared with the wt receptor. Hinney et al. used a 1-h cAMP assay as opposed to the 5-h transcriptional assay used in the present study, which might provide one explanation for the differing results. However, importantly, in the study by Hinney et al. (25), there was an 80-fold increase in EC50 of the S127L mutant receptor compared with that of the wt receptor (4003 ± 615 vs. 51 ± 18 nM -MSH). This major shift to the right of the dose-response curve is compatible with a severely impaired function of the S127L receptor and in line with the data of the present study and that by Lubrano-Berthelier et al. (34). The S127L mutation has thus been detected in four subjects with severe early-onset obesity worldwide, with the present study providing the first clinical characteristics of the mutation carrier.
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Acknowledgments |
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Footnotes |
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Abbreviations: AGRP, Agouti-related protein; BMI, body mass index; ß-gal, ß-galactosidase; GFP, green fluorescent protein; h, human; HA, hemagglutinin; MC4R, melanocortin-4 receptor; OGTT, oral glucose tolerance test; wt, wild type.
Received July 8, 2003.
Accepted November 10, 2003.
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References |
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-, ß- and 2-melanocyte-stimulating hormones on hypothalamic neuronal activation and feeding in the fasted rat. Neuroscience 108:437–445[CrossRef][Medline]
-melanocyte-stimulating hormone genes in severely obese children. J Pediatr 139:204–209[CrossRef][Medline]
-MSH peptides in the pituitary: effects, target cells, and receptors. Ann NY Acad Sci 994:123–132[Medline]
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