Transcript Slide 1

Tutorial: two case studies in
genetics of obesity
Prof. Karine Clément
Inserm U872 team Nutriomique
Université Paris 6/Cordelier Research Centre
Endocrinology and Nutrition Dept, Pitié-Salpêtrière
Hospital, Paris
Two case studies in genetics of
obesity
Contents
Slides
1. Gene discovery
3-20
2. Implication of a candidate gene in obesity
21-50
Further reading
51
Abbreviations used
52-53
BMI Curves of 3 sisters in a French family
B
A
C
52
66
70
50
64
68
62
66
60
64
58
62
56
60
54
58
48
46
44
42
56
52
40
54
50
52
38
48
50
46
36
48
44
34
46
42
32
30
44
40
42
38
40
36
38
34
36
28
26
34
32
bmi
24
22
bmi
+1 ds
30
-1 ds
28
+1 ds
+2 ds
- 2 ds
20
+3 ds
-3 ds
18
26
-1 ds
+2 ds
28
+2 ds
+3 ds
22
N
+1 ds
30
- 2 ds
24
bmi
32
-1 ds
- 2 ds
26
+3 ds
-3 ds
24
-3 ds
11416
22
S
20
16
20
18
14
18
16
16
14
12
14
12
10
12
10
8
10
8
6
8
6
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
6
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0
1
2
3
4
5
6
7
Severe obesity form occurring very early in life
Can we gather arguments for a genetic origin ?
8
9
10
11
12
13
14
15
16
17
18
Clinical observation
of affected members in the French family
Clinical Phenotype
Biological phenotype

BMI : 52, 65, 71kg/m2

Hypogonadotropic hypogonadism

Early onset of weight gain

Blunted growth hormone

Short stature

Abnormal eating behavior

Central hypothyroidism

Compulsive behavior

Normal hypothalamic pituitary

Normal RMR

No pubertal development (17y)

Absence of diabetes

Abnormal social behavior

Normal TG & CT

No mental retardation
secretion
adrenal axis
Severe obesity form occurring very early in life, with severe
anomalies of food intake behavior and endocrine dysfunction
Examination of the family pedigree
5
1
2
4
3
4
5
6
3
4
3
7
8
9
10
11
12
2
3
13
14
15
4
16
17
18
51 ans
27,7
19
47 ans
33
1
20
21
22
23
24
8 ans
25
12 ans
16,2
26
13 ans
68,9
27
16 ans
29,75
28
17 ans
33,69
29
19 ans
66
30
19 ans
52,61
31
23 ans
25,4
32
24 ans
27,3
Summary
• Severe obesity form (early onset)
• Severe anomalies of food intake
• Endocrine anomalies (can also be mental retardation,
sensorial deficiency…)
• Consanguineous family
• The origin is probably genetic
• But which gene is involved?
Leptin levels in the HD family
3 obese sisters
800
leptin ng/ml
3 normal
weight
600
400
200
0
0
20
40
BMI kg/m2
60
80
100
Leptin levels in the HD family
Sibs years
height
weight
Leptin (ng/ml)
51y
47y
180cm
90kg
155cm
80kg
BMI :27,78
BMI :33,30
145
362
12
13
16
17ans
19ans
19ans
23ans
24ans
151cm
37kg
153cm
149kg
171cm
87kg
175cm
103,2kg
154cm
165kg
159cm
133kg
173cm
76kg
160cm
70kg
BMI :16,23
BMI :68,90
BMI :29,75
BMI :33,69
BMI :66
BMI :52,61
BMI :25,4
BMI :27,3
212
88
600
526
5.6
Clément, Nature, 1998
670
240
294
Hypothesis
•
•
•
•
•
•
•
Severe obesity form (early onset)
Severe anomalies of food intake (compulsive behavior)
Endocrine anomalies (growth and reproductive functions)
(can also be mental retardation, sensorial deficiency…)
Consanguineous family
The origin is probably genetic
Since leptin levels are higher than would have been
expected based on body fat mass…
…could it be a mutation in the leptin receptor gene ?
LEPR is a member of the cytokine receptor family
Ob-R (leptin receptor)
Gp130
IL-4-R
GH-R
IL-12-R
Mutation screening of Ob-R
Strategy ?
Study design
T+ M P T+
Exon 14
Exon 15
T
C
C
A
T
A
A
C
A
T
Exon16
Ob-R
SSCP polyacrylamide gel
Clément, Nature, 1998
T
C
T
A
T
A
A
C
A
T
......CACCAAAG
gtattgtac
Exon16
a
Consequences of the ObRhd mutation
Ž
Exon 15
Exon 16
gt
Exon 17
Ž
Exon 15
Exon 16
at
Exon 17
Exon17
Exon15
AAT AGT TTC ACT CAA GAA TGA AAA
Asn Ser Phe Thr Gln Glu
826 827 828 829 830 831
Clément, Nature, 1998
*
RT-PCR of Ob-R exon15⇒17 in the HD family
M
300bp
200bp
Clément, Nature, 1998
Leptin levels in the HD family
Genotype
Leptin (ng/ml)
8ans
MN
51ans
47ans
180cm
90kg
155cm
80kg
BMI :27,78
BMI :33,30
MN
145
MN
362
12ans
13ans
16ans
17ans
19ans
19ans
23ans
24ans
151cm
37kg
153cm
149kg
171cm
87kg
175cm
103,2kg
154cm
165kg
159cm
133kg
173cm
76kg
160cm
70kg
BMI :16,23
BMI :68,90
BMI :29,75
BMI :33,69
BMI :66
BMI :52,61
BMI :25,4
BMI :27,3
MN
212
NN
MM
600
MM
526
NN
5.6
Clément, Nature, 1998
MM
670
88
MN
240
MN
294
LEPR: a member of the cytokine receptor family
Mutated LEPR
LEPR Isoforms
X X X X
OBRa OBRc
OBRb
Cytokines
830 AAs
OBRe
OBRd
Gp130 IL-4-R
GH-R
IL-12-R
Following questions
• Are the high leptin levels in LEPR mutation
subjects related to the interruption of the leptinbrain loop?
• Can we demonstrate that?
Human monogenic obesities : models for biology
Ob-Ra
Ob-Rc
Ob-Rd
Mutation
Human LeptinR *
mutation
X
Ob-Rb
XX
Leptin receptor
White adipocytes
*Clément, Vaisse et al, Nature 1998
• Lack of leptin signal consequences?
• Follow hypothalamic dysfunction
• Measurements of leptin fractions (free and
bound, leptin displacement)
• Gene expression in peripheral tissue
Gene expression of leptin in patients with LEPR
loss of function
Ob mRNA (atoml/ug)
70
60
Controls
50
Affected
girls
40
30
20
10
0
35
40
45
50
55
Percent body fat
60
65
70
Clément et al, IJO 2002
Gel filtration chromatography of circulating leptin
in the LEPR deficient family
Wild type
Homozygous
Heterozygous
I-125
leptindisplacement
displacement
I125 Leptin
1600
25
2
Ho mozyg ou s
Ho mozyg ou s
Hétérozyg ou s
Wild typ e
20
1200
c t i o
n
1400
ra
1
/
F
1000
800
L
ep
t i n
cp
n
m
g
15
600
10
400
5
200
Fra ctio n Num ber
60
58
56
54
52
50
48
46
44
42
9
4
40
6
4
38
3
4
36
0
4
34
7
3
32
4
3
30
1
3
28
8
2
26
5
2
24
2
2
22
9
1
20
6
1
Lahlou, Clement, Diabetes, 2000
18
3
1
Fractio n Nu mber
16
0
7
4
1
1
0
0
Two case studies in genetics of
obesity
2: Implication of a candidate
gene in human obesity
Leptin and melanocortin mutations
Food intake
Energy expenditure
Mice
Humans
Ob
Db
5*
POMC-/-
3*
Rare syndromes
Adapted from D Cummings, 2003
6*
fat
1*
Mc4r (-/-)
?
Phenotype
Obese phenotype
Mouse models: obesity with no other
dysfunction
Weight
ob/ob
db/db
MC4R-/MC4R+/Wt
Age
Clement 2002
Case situation
• MC4R: very good candidate gene
– Melanocortin pathway (mutations in upstream genes lead to
severe forms of obesity)
– MC4R KO leads to severe obesity in mice
– Not located in linked loci
• Is MC4R implicated in obesity development ?
First Questions
- Which human cases (isolated vs. syndromes)
- How many subjects we should screen (monogenic vs.
polygenic)
- Which techniques (1000 bp)
Is MC4R gene an obesity gene ?
• Case-control association study
– Frequency in large populations and controls
• Family analysis (co-segregation mutation family)
• Phenotypes of subjects carrying homozygous and
heterozygous mutations
• Functional studies of mutated receptors
Is MCR an obesity gene ?
• Frequency in large populations and controls
• Family analysis (co-segregation mutation family)
• Phenotypes of subjects carrying homozygous and
heterozygous mutation
• Functional studies
Association studies
Adapted from Redden et al, 2007
Identifying Disease Genes
Family Based Linkage
Studies
Members of a family Affected by the Same
disease share Identical disease genes
Population Based
Association Studies
Distribution of disease alleles is Different
between Cases and Controls
MC4R is an obesity gene ?
(frequency)
A
25
S
N
L
H
R
A
S
Y35stop
35
D37V
G
G
P48S
40 C
Y
L
S
15
R N
W L
H L
stop
10
H M
T
S
T
T11S
T11A
G
10
H M
R
G R
5
H
T
S
H T
S
V
N
5
N V
hMC4R
M
hMC4R
M
S
D
V50M
• In the French population : 774
subjects, 21 (2.7 %) mutations
K
G
Y
T37M
G
D
T
D
I102SG
T
I102T
40 C
S
42
Y
G98R105
L 42N
A
C271Y
C271R
M200V
Q
S
I125K
FT
D A Q
T
D
D
S
S
S
D S
S
A
S
A
P
N
V253I
V
C
Q
NP
InsGT
N274S
PQ Y C
Y
C
V
C
V
F
C
V I
I S
Y
120
267
S
F Y
G252S
C P
195
T
M 281
F
VT
I I
S
I
stop
I
Y
H S
120
267 F Y IH L
L C
I N
Q E
185
195
D
281
S
M
V
F L
N
I
I I
N F
C TI
L IF F L H S
L
N
260
F
M
L
E
V
L
H
V
I
T
Q
105
F
P
185
D
I L
NI
P S
D
F L
N
Y
I
L
F
L
I
201
N97D
S295P
S
I
T
F
N
F
A
L
I
260 P F C W
L
G F
I
V
T L
FMM T
127
V E
V
290YL
T P IS V
D I 179 V SI L
201
V F
F
A
L
L
V
I 291
I I I 127 S 131
G
T A
EE 100 T I C
C WV F V
290 L I
C M
253
F51L
S C I V 131
V ST
97 N V GF SV
A
MM L
179 C
L T
S
291
L
V
I
A
G
L
E
N
V
55
174T
G C M
L A A 253
100
I251L
S
S
97 N G LS
W
I S
A C
V F
L T
LG I
211M L S A
I V
L S
NI
55 S G V
C IA
S A L A175T
L S
W 174
I S
D 298
V SI
L I
211 L HS V Y
T II 249
I VV
S137
C
L P
I IS
C I S A168
MS L
M
TT L
I
I
E L
V Y
P D 298
S
I 168
90 D L
L VL S 137
H
L
L
I
C
249
L
L
I
G
F
I
M
M
T
63 I N
I
S
Y
E A
V
L
L
90 D
P299H
216
GI A
L
63 I AN V
K RI
A M
Y I
A
I S L 145 S
V G
L F
G KA
I V
L
A V A
L
K RV 163
A M L R216
K242
M
V S
A
I301T
VA I
145
A
M N
V 163
242
A V
L R
I I C
S LR D
A N
750insGAA
312
G A 312
A VF
R D V
A I
I C Y
I
Y
V95I
Q
T
L
F
G
70
F
A
H
I316T
R
F
T
Q
80 Y
T
L
F I
70
N K
H
T F
80 Y151
I R I
N K
T
A
151 I
T305 R
M M
I
305 R
P M
S58C
235 235 A
I
K
I317T
II
L
F
P M
K
L
L250W
F
F
G
I
F
G
S
N
I CTCT 211 K
S
S
N
Y
I
S
Y
I
K
N
R
N
R
L H
L S127L
H
A L Q A YL H
H
K
A244E
R305Q
T
K
Q Y
T
319
319
Q
Q
R
C
R
C
N62S
K
K
G
G
E C
E C E
I I
P
E
P
I69M
P78L
A A
I170V
V LV L
R165W
308
308
Y
Y
stop I137T
F
R165Q
T150I
A154Y
P
G231S
Y
R
Y SR
S
D C
SL S
L
L
P
L
L
G G
D
C
L
• More than 3000 subjects
screened since 1998
– 2-3% of the population
T5T
G
S
D
H
S
G
Y
A18C
A18H
N
15
L
L
K
35
N W
E
30
S30F
R
20
S S
L R Y
S
L
G
20
S S
25
S H
S
E
30
Y
G
G
–
–
–
–
–
Dominant transmission
Early onset obesity (74% vs 57%)
Variable expression in the families
« common » obesity forms
Heterogeneity of functional
consequences
Vaisse Nature genet 1998, JCI 2000
Lubrano Berthelier, 2003
MC4R mutation in human obesity
Study country
n
Age
BMI
Carriers
% mutants MC4R
France
769
≥18
≥35 kg/m2
20
2.6
USA
165
18-48
≥40 kg/m2
6
2.6
Switzerland
469
≥18
≥35 kg/m2
5
1.07
Spain
159
≥18
≥30 kg/m2
1
0.63
Denmark
750
20
≥31 kg/m2
19
2.5
France
63
≤18
≥99thp*
4
6.35
UK
500
≤18
≥99thp
29
5.8
France
172
≤18
≥95thp
3
1.74
Germany
808
≤18
≥95thp
15
1.86
France
516
≤18
≥85thp
8
1.74
110
2.52%
TOTAL
4371
* p = percentile
Lubrano-Berthelier et al 2004
Dubern et al 2002
Lubrano-Berthelier et al 2003
Lubrano-Berthelier et al 2004
Extracellular
20
25
A
N
S
H
L
R
Y
S
S
R
N W
15
L
H
L
10
S
T
H
M
30
5
G
S
C
E
H
S
H
L
A
F*
L
R
NH2
M
T
S N
V
S
G
H
K
G
35
Y
L
S
S
V*
D
Y
G
P
G
M
C
40
T
Y
S
T
D
D
A
Q
S
F
S
Q E 42
V
S
Y
120
P
N
K
T
T
R
I
D
S
S
A
V
I
I
195
C
C P
267
F
Y
C
V
Q
I
185
Y
I
S
L
I
F
N
H S
M
281
H
I
L C
I N
L N
L
V
260
N F
N D
I
L F
L
105 T L
T
I
V
F F
M
Y
P
P S
V
L
G
I
F
290
P
I
A
I L
201
L
F
L
I I
E
R
W
127 S D
179 V S
T
T
V
C
T
M
V
I
V F
E 100
M
V
S*
I
C
C
C
H
A L
T
131
N G S
A A
D
F V
P
55 G L
N
W 174
M L
253 V
S
S S
S
S
G
L
I
97
V
A
I
C
I
S
L
I
C
S V
I
L I
S
L S
T
I
L S
A
211
I
249
T
Y
L V
V
D 298
L
I S
L
V
I
R
P
C
M
E
137
K
L
T
N
D
M H
Q
I
S
168 I
216
G
63 I
T
90
L L
L F
G A
R V
V A
Y I
L
K
S
V
A
S
K
242
M
I
A
M
V 163
A I
L
N
145 -S
L R
A
V
A
312
V
S
C
A
T
A I
I
D
Q G
F
T
Y R
70
K
H
I R
F
T I
N
L
N
F
Y
235
Q W
80
T
T
I
M
M
A
151
E
I
305 R L F I
P
K
M
I
F
S R K C
G
S
T
K
N
Y
N L H
C
I
Q K E C
H
A
T
320
R
L Q Y
Y
E
L
W
G
I
A V L P
D
P
308
L
P
S
G
S
G
L
L
C
D
Y R S S L
S
F
L
Intracellular
Dubern B, Lubrano C, Clement K
COOH
Is MCR an obesity gene ?
• Frequency in large populations and controls
• Family analysis (co-segregation mutation family)
• Phenotypes of subjects carrying homozygous and
heterozygous mutation
• Functional studies
Family with severe MC4R mutation
a.
I
2
1
†73y
>35kg/m2
78y
37kg/m2
Z=2.95
II
III
2
1
58y
51kg/m2
Z=6.48
59y
28kg/m2
Z=0.73
3
35y
57kg/m2
Z=10.14
36y
23kg/m2
Z=-0.55
34y
50kg/m2
Z=8.54
IV
11y
30kg/m2
Z=6.5
100bp
200bp
Vaisse C, Clément, Nature Genet 1998
24y
33kg/m2
Z=4.58
2
1
b.
Clement Brest 2002
4
3
2
1
50y
28kg/m2
Z=0.87
5y
14kg/m2
Z=-1.5
Mutations in MC4R (examples)
Age
P, T
IMC
Mutation Val50Met
Mutation Ser58Cys
Dubern et al, J Pediatr 2001
Dubern et al, J Pediatr 2001
39 ans
100 kg, 173 cm
33 kg/m²
M
M
Age
Poids
Taille
Z-score de l’IMC
6 ans
38.1 kg
126 cm
5.4 DS
Dubern, J Pediatr 2001
NM
35 ans
70 kg, 162 cm
26,7 kg/m²
NM
NM
13 ans
70 kg
165 cm
2.6 DS
11 ans
58 kg
155 cm
2.9 DS
Age
P, T
IMC
39 ans
110 kg, 189 cm
30,8 kg/m²
Age
Poids
Taille
Z-score de l’IMC
35 ans
85 kg, 165 cm
31.2 kg/m²
M
NM
M
NM
M
16 ans
75 kg
180 cm
1.1 DS
14 ans
69 kg
183 cm
0.9 DS
12.5 ans
92 kg
167 cm
4.2 DS
NM
9 ans
40 kg
145 cm
1.7 DS
Is MCR an obesity gene ?
• Frequency in large populations and controls
• Family analysis (co-segregation mutation family)
• Phenotypes of subjects carrying homozygous and
heterozygous mutation
• Functional studies
Complete loss of function leads to a severe phenotype
25
S
N
A
L
H
R
Y
20
S
S
R
N
W
15
L
H
S
E
30
L
S
T
10
H M
G
R
5
H
T
S
N
V
M
S
L
G
K
G
35
Y
S
D
G
D
T
G
S
L
63
V
I
70
A
L
M
I
80 Y F
P M
S
K
N
L
L
V
A
V
C
K
N
S
V
A
V
A
I
D
120
V
N
D
I
3 y, Weight 27.4 kg
BMI 27 kg/m2
Impulsivity
Familial Obesity
Consanguinity
N
I
E 100
S
G
N
90
I
I
S
L
E
N
I
97
V
I
N
L
L
T
T
L
G
55
E
V
F
V
105
T
V
S
P
F
T
S
E 42
Q
L
S
T
Y
F
Q
D
C
40
A
•
•
•
•
•
L
S
• Homozygous deletion of MC4R gene
(delAG 346-347)
F
H
Dubern, J Pediatr, 2007
MC4R carriers and food intake behavior ?
• Branson et al « Binge eating as a major phenotype of
Melanocortin receptor gene mutations » NEJM 2003
• Not confirmed (Farooqi et al. 2003; Gotoda 2003; Herpertz et al.
2003; Hebebrand et al. 2004, Lubrano-Berthelier, JCEM 2006)
Phenotypes of MCAR carriers
Children
NM
M
n = 59
n=4
Age (y)
11.6 ± 0.4
11.9 ± 1.5
BMI Z-score (SD)
4.6 ± 0.1
4.5 ± 0.5
Obesity early onset (y)
2.6 ± 0.3
2.5 ± 1.0
Adiposity rebound (y)
2.5 ± 0.2
3.3 ± 0.7
Fat mass (%)
43.8 ± 0.9
40.6 ± 4.7
Insulin (µUml)
20.2 ± 1.9
17.7 ± 7.2
Food Intake, JCI 2000
control
n = 254
mut n = 201
mut +
n=8
age (y)
52 +/- 7
42 +/- 11
39 +/-12
BMI (kg/m²)
21 +/- 2
51.5 +/- 7.5
52.5 +/- 8.0
2023 +/- 551
2393 +/- 900
2800 +/- 763
38.4 +/- 5
39 +/- 7.4
40.5 +/- 9.0
kcal/24h
Lipids (%)
MC4R carriers and height growth
Increased Bone density
Decreased bone resorption
(Eleftriou Nature 2005
Ahn & Dubern Endocrinology 2006)
Farooqi, NEJM, 2003
Evidence that MC4R is an “obesity gene”
• Biological candidate
• Invalidation in mouse
models leads to obesity
(KO)
• Population based
association studies
• “Co-segregation” of
genotype and phenotype
in families
• Loss of function of variant
receptors
Review in Govaerts Peptide 2005
European populations screened
(>5000 obese screened, >8000 controls)

UK (Farooqi &Yeo NEJM 03, Hum Mol Genet 03,
JCI 00, Nature Genet 98)


Finland (Valli-Jaakola, JCEM 04)
France (Lubrano, Dubern, Clément Vaisse,
Diabetes 04, JCEM 04, Hum Mol Genet 03, J Ped
00, JCI 00, Nature genet 98, JCEM 06)

Germany (Hinney & Hebebrand, Am J Hum
Genet 04, JCEM 03, Mol Psy02, Am J Hum Gent
99, Biebermann H, JCEM 06)

Italy (Miraglia Del Giudice, JIO 02, Buono 05)
Spain (Marti IJO 03)
Switzerland (Branson, NEJM 03)
Denmark (Larsen, JCEM 2005)

And others…



Is MCR an obesity gene ?
• Frequency in large populations and controls
• Family analysis (co-segregation mutation family)
• Phenotypes of subjects carrying homozygous and
heterozygous mutation
• Functional studies
Cloning of MC4R mutants
1. PCR amplification of MC4R gene from genomic DNA of patients carrying mutations;
2. Preparation of Insert and vector:
1) Insert
- Digestion of purified PCR products with EcoRI / BspEI;
2) Vector
- Digestion of wild-type plasmid (Pc) with EcoRI / BspEI;
- Running on 1.5% agarose gel, cutting vector band and making gel extraction and
purification with QIAGEN kit;
3. Ligation of vector (wt) and inserts (mutations):
- Ratio of vector to insert = 1 / 5;
4. Transformation:
- DH5 competent cells;
5. Miniculture;
6. MiniPrep to obtain the new plasmids with mutations (QIAGEN Kit);
7. Sequencing of the new plasmids.
Assay of cAMP activity stimulated by -MSH
1. Cell culture:
Human embryonic kidney (HEK 293) cell line;
2. Transfection of HEK293 cells with MC4R plasmid:
- Lipofectamine with Plus reagent;
- Transfection with Flag-GFP chimerical wild-type MC4R:
Assay of cAMP activity stimulated by -MSH
3. cAMP activity stimulated by -MSH in MC4R-transfected cells:
- Enzymeimmunoassay (EIA) with cAMP assay kit;
- Transfection with wild-type or mutant MC4R (Pc):
100
cAMP (% of untreatment)
2500
80
% B/B0
60
40
2000
1500
1000
500
20
no
n0
no
nno 1
n10
0
Pc
-0
Pc
-1
Pc
-1
00
M
10
M
11
M
110
0
M
20
-0
M
20
-1
M
20
-1
00
0
0
10
100
1000
10000
cAMP (fm ol/w ell)
Standard curve of cAMP concentrations
alpha-MSH (nM)
cAMP activity stimulated by -MSH
(non: non-transfected; Pc: MC4R wt; M1 & M20: mutants No. 1 & No. 20)
aMSH responses
Lubrano-Berthelier et al, Hum Mol Genet, 2002
125
W T 1 .0 4 ± 0 .1 6 n M
V 5 0 M 1 .6 4 ± 0 .6 4 n M
100
75
S 5 8 C 1 5 .8 8 ± 7 .1 6 n M
I1 0 2 S
50
25
0
-1 3
-1 2
-11
-1 0
-9
 -M S H lo g M
-8
-7
-6
Membrane expression
Lubrano-Berthelier et al, Hum Mol Genet, 2002
N Flag
MC4R
GFP
C
Wild-type
receptor
V
V
X
Ile102Ser
Functional effects of obesity associated
Melanocortin-4 Receptor Mutations
Base Change
A-307-G
A-751-C
47-48insG
750 ins GATT
A-31-G
C-52-T
C-449-T
A-508-G
C-493-T
T-749-A
T-902-C
Effect on amino acid
sequence
Va l103 Ile
Ile251 Leu
16 +12 amino-acids
Frames hift at 6thTM
Thr11Ser
Arg18Cys
Thr150Ile
Ile170Va l
Arg165Trp
Leu250G ln
Ile301 Thr
Vaisse C, UCSF, 2002
NIe = normal
Membrane
expression
Nle
Nle
0
0
Nle
Nle
Nle
Nle

Nle

Basal
Activity
Nle
Nle
0
0







EC50 for MSH
stimulation
Nle
Nle
Nle
Nle



Nle/

C Vaisse, NAASO, 2001
Functional consequences of MC4R mutations
Data from the French population
AGRP
N
Membrane expression
MSH
-
Intracellular retention
=> 56% of MC4R mutations
+
C
X
Prot Gs
AC
AMPc
?
Food Intake
Energy
homeostasis
Lubrano-Berthelier et al, HMG 2003
Srinivasan et Lubrano-Berthelier et al JCI 2004
Lubrano-Berthelier et al, JCEM 2006
Receptor activity
Deficit of MSH response
80% of MC4R mutation
Decreased basal activity
=> 76% of MC4R mutations
Genotype-phenotype Relationships
Intracellular retention associated with early onset
obesity
Evidence that MC4R is an “obesity gene”
Further reading: three useful reviews
1: O'Rahilly S.
Human obesity and insulin resistance: lessons from experiments of nature.
Biochem Soc Trans. 2007 Feb;35(Pt 1):33-6. Review.
PMID: 17212584 [PubMed - indexed for MEDLINE]
2: Mutch DM, Clement K.
Unraveling the genetics of human obesity.
PLoS Genet. 2006 Dec 29;2(12):e188. Review.
PMID: 17196040 [PubMed - indexed for MEDLINE]
3: Farooqi IS.
Monogenic human obesity syndromes.
Prog Brain Res. 2006;153:119-25. Review.
Abbreviations
used I
AC
Acetylcholine
ACTH
Adrenocorticotropic hormone
AGRP
Agouti related peptide
ARC
Arculate nucleus
BBS
Bardet-Biedl syndrome
BDNF
Brain-derived neurotrophic factor
CEPH
Centre d’Etudes du Polymorphisme Humain
CLIP
corticotropin-like intermediate lobe peptide or ACTH18-39
CPE
Carboxypeptidase E
CPH
Carboxypeptidase H
CTX
Collagen fragment peptide AHDGGR
Dpd
Deoxypyridinoline (bone resorption marker)
EcoR1, BspE1
Restriction enzymes
GFP
Green fluorescent protein
GHR
Ghrelin receptor
GLP
Glucagon-like peptide
Gs, Gi, Go
Guanine binding proteins (s = stimulating, I = inhibiting)
IBD
Identical by descent
IFT
Intraflagellar transport
IL
Interleukin
IR
Insulin receptor
Abbreviations
used II
LepR
Leptin receptor
LOD
Logarithmic odds
LPH
Lipotropic pituitary hormone
M
Mutant
MC4R
Melanocortin4 receptor
MSH (α-, etc)
Melanocyte stimulating hormone
N
Normal
NIe
Normal
NPY
Neuropeptide Y
PC (1, etc)
Prohormone convertase 1
POMC
Pro-opiomelanocortin
PPARG
Peroxisome proliferative activated receptor, gamma
PVN
Paraventricular nucleus
PYY
Pancreatic Peptide YY3-36
RMR
Resting metabolic rate
SIM1
Drosophila single-minded gene
SNP
Single nucleotide polymorphism
SSCP
Single strand conformation polymorphism
TDT
Transmission disequilibrium test
Z-score
Number of standard deviations from an age/sex adjusted mean