International Journal of Obesity (2003) 27, 70–74
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PAPER
Melanocortin-3-receptor gene variants
in morbid obesity
C Schalin-Jäntti1,2*, K Valli-Jaakola1, L Oksanen1, E Martelin3, K Laitinen1, T Krusius4, P Mustajoki5,
M Heikinheimo3,6 and K Kontula1
1
Department of Medicine, University of Helsinki, Helsinki, Finland; 2Department of Endocrinology, Helsinki University Hospital,
Helsinki, Finland; 3Department of Pediatrics, University of Helsinki, Helsinki, Finland; 4Finnish Red Cross Blood Transfusion
Service, Helsinki, Finland; 5Peijas Hospital, Vantaa, Finland; and 6Department of Pediatrics, Washington University in St. Louis,
MO, USA
BACKGROUND: Linkage and knock-out mice studies suggest that the melanocortin-3-receptor (MC3R) is a candidate gene for
obesity.
OBJECTIVE: To evaluate whether MC3R mutations underlie morbid obesity.
SUBJECTS AND METHODS: MC3R coding and 50-flanking regions were sequenced in 48 subjects and the detected variants
genotyped in 252 morbidly obese (BMI 40 kg/m2) Finns. Gel shifts were used to examine whether a mutation in the putative
promoter alters GATA-factor binding.
RESULTS: Three common MC3R variants were found: a 17C > A variant, changing Thr6?Lys in 16%, a 241G > A variant
changing Val81?Ile in 15%, and a 7239A > G substitution in the GATA binding site in 21% of the subjects. Four other variants
were detected in the 50 flanking region. Frequencies of the three common variants did not differ between obese and contol
subjects. Among the obese, the 17C > A and 241G > A variants were coinherited and associated with increased insulin – glucose
ratios (P < 0.05) and leptin levels (P < 0.05). GATA-4 bound efficiently to wild type oligonucleotide, but only weakly to the
oligonucleotide with the 7239A > G mutation.
CONCLUSIONS: MC3R gene variants are common and do not explain human morbid obesity. These variants associated with
subtle changes in onset of weight gain, hyperleptinemia and insulin – glucose ratios. The 7239A > G mutation abolishes binding
of GATA-4 to the MC3R promoter region.
International Journal of Obesity (2003) 27, 70 – 74. doi:10.1038=si.ijo.0802184
Keywords: melanocortin-3-receptor; MC3R gene variants; GATA-4; EMSA; leptin; insulin resistance; insulin – glucose ratio;
obesity; energy balance; brain
Introduction
The proportion of body fat is reflected by circulating leptin
and insulin levels, and long-term homeostasis of body
weight is accomplished by integration of these hormonal
signals by hypothalamic centers.1 – 3 Neuropeptides derived
from the ACTH precursor pro-opiomelanocortin (POMC)
and melanocortin receptors (MCRs) play a key role in this
complex control of appetite and body weight.1,2 MC3R and
MC4R are both highly expressed in brain areas involved in
regulation of energy balance.2 The MC4R plays an important
role in energy homeostasis, as its targeted disruption causes
*Correspondence: C Schalin-Jäntti, Departments of Medicine and
Endocrinology, University of Helsinki, BOX 340, Haartmaninkatu 4,
FIN-00290 Helsinki, Finland.
E-mail: [email protected]
Received 6 February 2002; revised 10 June 2002;
accepted 22 July 2002
hyperphagia and obesity in mice.4 MC4R mutations underlie
up to 4% of severe early-onset or adult obesity.5,6 Observed
linkage between obesity and the chromosomal region 20q13
also makes the MC3R gene a plausible candidate gene for
human obesity.7 Furthermore, disruption of MC3R in mice
results in increased fat mass, hyperleptinemia and insulin
resistance.8,9 We examined whether mutations in the MC3R
gene underlie morbid obesity and/or are associated with
changes in metabolic parameters.
Subjects and methods
A cohort of 252 morbidly obese (BMI 40 kg/m2, 182
females/70 males, age 21 – 67 y, prevalence of diabetes and
hypertension 24 and 45%, respectively) patients was collected between 1989 – 1995.10 History of weight development
MC3R gene in morbid obesity
C Schalin-Jäntti et al
was assessed by questionnaire. For mutation detection,
1400 bp of the 50-flanking region and the whole coding
region of the MC3R gene were sequenced in 48 subjects and
the variants detected genotyped in the rest of the cohort.
Blood samples from 312 healthy blood donors (153 females
and 159 males) from the Finnish Red Cross Blood Transfusion
Service served as controls for estimation of allele frequencies
in the background population. Leptin concentrations were
determined by radioimmunoassay (Linco Research Inc.,
St Charles, MO, USA) with intra- and interassay CV’s of less
than 5%. Glucose, insulin and lipid levels were measured as
previously described.10
DNA sequencing and genotyping
After PCR, the MC3R gene was sequenced using primers pairs:
50-TGACCAGAGCAGACTACTTTCA-30 and 50-TGAGCAAAGACAACAGCCACT-30; 50-TCTTCTTCCACCCCAGACTC-30
and 50-GCAAGACAGGGGATGTGTTA-30; 50-CCCTTGGCCAATATGAAAAA-30 and 50-CCAGATACGTCTTTTGGATGC-30;
50-TCTCTACCCTCCCCATCCTT-30 and 50-GGGCATTGGACA50-CATCGTCAGTCTGCTGGAAA-30
and
CACTTACC-30;
0
5 -GAGCATCATGGCGAAGAAC-30; and 50-CTCGGAGAGCAAAATGGTCA-30 and 50-TCACGTGGATGGAAAGTCAA-30.
Sequencing was carried out using the ABI Prism 377 DNA
sequencer. Genotyping of the 7239A > G, 17C > A and
241G > A variants were performed using restriction
enzymes AlwI, HpyCH4IV and BseDI, respectively, and gel
electrophoresis.
Electrophoretic mobility shift assay (EMSA)
Nuclear proteins were prepared11 from a mouse Sertoli tumor
cell MSC-1, Jurkat T-cell lymphoma and NIH 3T3 fibroblast
lines, expressing GATA-4 and GATA-6 (MSC-1), GATA-3
(Jurkat) and no known GATA proteins (NIH-3T3). Probes
were prepared by annealing oligonucleotides 50-TTTCTATGTAAACAAGATAAAAACTGCTCCTCCT-30 (corresponding to
nucleotides 7253 to 7220) and 50-AGGAGGAGCAGTTTTTATCTTGTTTACATAGAAA-30 (MC3R), 50-TTTCTATGTAAACAGGATAAAAACTGCTCCTCCT-30 and 50-AGGAGGAGCAGTTTTTATCCTGTTTACATAGAAA-30 (MCR3mut, 7239A >
G mutation underlined), and 50-CCCATAAAGATAGGGA-30
and 50-TCCCTATCTTTATGGG-30 (corresponding to nucleotides 7182 to 7163 of the steroidogenic factor-1 (SF-1)
gene). One of the oligonucleotides of each probe was
50-end-labelled with [g-32P]ATP before annealing. Binding
reactions were carried out as described.13 The antibodies
were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Statistical analyses
Mann – Whitney rank-sum test was used to analyse differences between group means and Fisher’s exact test for
frequency distributions.
71
Results
MC3R gene variants
Two common missense mutations were found in the MC3R
coding region: nucleotide 17C > A changing amino acid
6 from threonine to lysine and 241G > A changing
amino acid 81 from valine to isoleucine. Five additional
variants, 7939G > C, 7911G > A, 7803T > C, 7373G > T
and 7239A > G were detected in the putative promoter
region. The 7239A > G variant was localized in a consensus
GATA transcription factor binding site13 (aaacaaGATAaaaact), comprising nucleotides 7244 to 7228 of the
MC3R gene (common allelic form in bold and underlined).
The genotype frequencies for the 17C > A, 241G > A and
7239A > G variants did not differ between morbidly obese
and the control population (Table 1A).
The 7239A > G variant and GATA binding
In EMSAs, nuclear extracts abundantly containing GATA-4/
GATA-6 yielded a strong retarded band A with the wild type
probe (Figure 1, lane 1), whereas assays using the MC3Rmut
oligonucleotide revealed only a weak protein – DNA complex (Figure 1, lanes 9 and 10). In an excess of unlabelled
Table 1A
MC3R genotypes in morbidly obese and control subjects
Variant
Morbidly obese n (%)
Control subjects n (%)
7239A > G
AA
AG
GG
198 (79)
51 (20)
3 (1)
238 (76)
67 (21)
7 (2)
Total
252 (100)
312 (100)*
17C > A (Thr6?Lys)
CC
CA
AA
204 (84)
37 (15)
3 (1)
217 (80)
46 (17)
8 (3)
Total
244 (100)
271 (100)
241G > A (Val81?Ile)
GG
GA
AA
206 (85)
35 (14)
3 (1)
186 (83)
35 (16)
4 (2)
Total
244 (100)
225 (100)*
Table 1B Relationship between coding region MC3R gene variants and
metabolic parameters in morbidly obese subjects
17C
Variant
n
Insulin – glucose ratio
Leptin (ng/ml)
2
BMI (kg/m )
2
BMI at 20 y (kg/m )
241G
CC
AC/AA
GG
AG/AA
204
3.1 0.2
38.5 1.5
42.7 0.5
26.9 0.4
40
4.1 0.4*
46.4 3.6*
43.3 1.1
29.3 1.4
206
3.1 0.2
38.5 1.5
42.7 0.5
26.9 0.4
38
4.0 0.5**
47.0 3.7**
43.2 0.5
29.0 1.4
*P < 0.05 vs CC; **P < 0.05 vs GG. Values are means s.e.m.
International Journal of Obesity
MC3R gene in morbid obesity
C Schalin-Jäntti et al
72
Figure 1 GATA-4 binds to wild type MC3R oligonucleotide (MC3R). Nuclear extracts (NE) from MSC-1, NIH-3T3, and Jurkat cells were incubated with
32
P-labelled oligonucleotide probes. When MSC-1 extract (10 mg) and labelled MC3R were used, two major bands A and B were formed (lane 1). The
binding of proteins was competed with unlabelled MC3R in 15- and 100-fold molar excess (lanes 2 and 3), or mutated MC3R (MC3Rmut, lane 4) or SF-1
(lane 5) oligonucleotides in 100-fold molar excess. In supershift experiments, antibodies against GATA-4 (lane 6), GATA-3 (lane 7), and GATA-6 (lane 8)
were used. Labelled MC3Rmut was used as a probe with either 10 mg (lane 9) or 20 mg (lane 10) MSC-1 nuclear extract. NIH-3T3 extract (10 mg, lane 11),
without any known GATA proteins, and Jurkat cell extract (10 mg, lane 12 and 13), containing abundant GATA-3 protein, were incubated with labelled
MC3R. Supershift experiment with GATA-3 antibodies is shown on lane 13.
MC3R or SF-1 oligonucleotide, band A was clearly attenuated (Figure 1, lanes 2, 3 and 5) indicating specific binding
and suggesting that, of the various GATA proteins, GATA-4
is responsible for formation of band A. Unlabelled
International Journal of Obesity
MC3Rmut did not change the formation of band A
(Figure 1, lane 4). In supershift experiments, GATA-4 antibody abolished band A indicating that GATA-4 binds to the
MC3R probe, whereas neither GATA-3 nor GATA-6 caused
MC3R gene in morbid obesity
C Schalin-Jäntti et al
73
any supershift (Figure 1, lanes 6, 7 and 8). The mutation
7239A > G did not affect the binding of protein(s) forming
band B (Figure 1, lanes 2 – 4).
MC3R variants and metabolic parameters
The 17C > A and 241G > A gene variants were coinherited in
all but 3 cases, indicating an almost complete linkage disequilibrium. Individuals with the combined genotype of
17CC/241GG had lower insulin – glucose ratios and lower
leptin levels and tended to weigh less at 20 y than those with
the combined variant genotypes (Table 1B). A carrier status
for the 7239A > G variant was associated with a tendency
towards lower maximal BMI values when compared to wild
type homozygotes (45.7 0.7 vs 47.7 0.5 kg/m2; P ¼ 0.06).
Discussion
The GATA-binding proteins are zinc finger transcription
factors regulating gene expression, differentiation and cell
proliferation.14 We demonstrate that of the different GATA
proteins 3, 4 and 6, GATA-4 was responsible for binding to
the MC3R promoter GATA site. GATA-4 is expressed in
pituitary and hypothalamic cells, gonadal and adrenal
cells15,16 and MC3R in the brain cortex, thalamus, hippocampus, pituitary and hypothalamus.17 – 19 The expression
patterns for these two genes thus overlap, suggesting that
GATA-4 is a regulator of the MC3R in vivo. The 7239A > G
variant was associated with a tendency towards lower maximal BMI (45.7 0.7 vs 47.7 0.5 kg/m2; P ¼ 0.06) and was
also an independent predictor of maximal BMI in the obese
(stepwise forward regression analysis; R-squared increment ¼
0.019; P ¼ 0.03), indicating that it may exert a modulating
effect on development of maximal weight.
Three detected MC3R gene variants were common in the
obese as well as in control subjects and therefore do not
explain morbid obesity. The Thr6?Lys mutation is located
in the extracellular N-terminus and the Val81?Ile in the first
transmembrane part of MC3R. Within the morbidly obese,
the rare amino acid variants were associated with higher
leptin levels and insulin – glucose ratios compared to subjects
homozygous for the common variants. They also reported a
tendency towards higher BMI at 20 y. Hani et al found
increased insulin – glucose ratios in healthy subjects with
the variant MC3R alleles compared to healthy subjects
with the wild type alleles.20 Collectively, these data indicate
that the MC3R gene variants may induce subtle changes in
obesity-related parameters and that these changes are not
restricted to morbidly obese subjects.
Acknowledgements
We thank Ms Tuula Soppela-Loponen, Ms Sirpa Stick and
Ms Merja Soininen for excellent technical assistance. This
study was supported by the Yrjö Jahnsson Foundation (CS-J,
KV-J), the Stockmann Foundation (CS-J), the Finnish
Foundation for Cardiovascular Research (LO, KK), the
Maud Kuistila Foundation (LO), the Finnish Cultural Foundation (LO), the Helsinki Biomedical Graduate School, University of Helsinki (EM), the Sigrid Juselius Foundation (MH,
KK) and the Research Funds from the University Central
Hospital in Helsinki (CS-J, MH, KK)
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