In e na ional Jou nal o
Molecula Sciences
A icle
Lep in Recep o Gene Va ian s11804091 Is
Associa ed wi h BMI and Insulin Resis ance
in Spanish Female Obese Child en: A
Case-Con ol S udy
Josune Olza 1,2,3 ID , Azaha a I. Rupé ez 1, Me cedes Gil-Campos 2,4, Rosau a Leis 2,5,
Ramón Cañe e 2,4, Ra ael Tojo 5,Ángel Gil 1,2,3 ID and Concepción M. Aguile a 1,2,3,*ID
1
Depa men o Biochemis y and Molecula Biology II, Facul y o Pha macy, Ins i u e o Nu i ion and Food
Technology, Uni e si y o G anada, A . Del Conocimien o s/n., 18016 G anada, Spain; jolza@ug .es (J.O.);
azaha a upe ez@ug .es (A.I.R.); agil@ug .es (Á.G.)
2CIBER Fisiopa ología de la Obesidad y la Nu ición (CIBEROBN), Ins i u o de Salud Ca los III,
28029 Mad id, Spain; [email p o ec ed] (M.G.C.); ma ia [email p o ec ed] (R.L.);
[email p o ec ed] (R.C.)
3Ins i u o de In es igación Biosani a ia ibs.GRANADA, 18012 G anada, Spain
4Paedia ic Resea ch and Me abolism Uni , Reina So ía Uni e si y Hospi al, Maimonides Ins i u e o
Biomedical Resea ch (IMIBIC), A . Menendez Pidal s/n., 14010 Có doba, Spain
5Uni o In es iga ion in Nu i ion, G ow h and Human De elopmen o Galicia, Paedia ic Depa men ,
Clinic Uni e si y Hospi al o San iago, Uni e si y o San iago de Compos ela, T a esia de Choupana,
15706 Galicia, Spain; [email p o ec ed]
*Co espondence: caguile @ug .es; Tel.: +34-958-242335
Recei ed: 11 July 2017; Accep ed: 28 July 2017; Published: 3 Augus 2017
Abs ac :
Lep in is an endoc ine ho mone ha has a c i ical ole in body weigh homoeos asis and
media es i s e ec s ia he lep in ecep o (LEPR). Common polymo phisms in he genes coding
lep in ecep o s ha e been associa ed wi h me abolic abno mali ies. We assessed he associa ion o
28 LEPR polymo phisms wi h body mass index (BMI) and hei ela ionship wi h obesi y- ela ed
pheno ypes, in lamma ion and ca dio ascula disease isk bioma ke s. A mul icen e case-con ol
s udy was conduc ed in 522 child en (286 wi h obesi y and 236 wi h no mal-BMI). All an h opome ic,
me abolic ac o s and bioma ke s we e highe in child en wi h obesi y excep apolipop o ein
(Apo)-AI, choles e ol, high-densi y lipop o ein choles e ol (HDL-c), and adiponec in, which we e
lowe in he obesi y g oup; and glucose, low-densi y lipop o ein choles e ol (LDL-c), and ma ix
me allop o einase-9 ha did no di e be ween g oups. We iden i ied he associa ions be ween
s11208659, s11804091, s10157275, s9436303 and s1627238, and BMI in he whole popula ion,
as well as he associa ion o s11804091, s10157275, and s1327118 wi h BMI in he emale g oup,
al hough only he s11804091 emained associa ed a e Bon e oni co ec ion (p= 0.038). This single
nucleo ide polymo phisms (SNP) was also associa ed wi h insulin (p= 0.004), homeos asis model
assessmen o insulin esis ance (HOMA-IR) (p= 0.006), quan i a i e insulin sensi i i y check index
(QUICKI) (p= 0.005) and adiponec in (p= 0.046) a e adjus ing o age, Tanne s age and BMI. Ou
esul s show a sex-speci ic associa ion be ween he s11804091 and obesi y sugges ing an in luence o
his SNP on insulin esis ance.
Keywo ds: LEPR gene; gene ic polymo phism; obesi y; child; insulin esis ance
1. In oduc ion
Paedia ic obesi y is a complex condi ion o igina ed om bo h en i onmen al and gene ic
ac o s [
1
]. I has become a wo ldwide public heal h conce n as i has d ama ically inc eased o e
In . J. Mol. Sci. 2017,18, 1690; doi:10.3390/ijms18081690 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2017,18, 1690 2 o 14
he pas h ee decades [
2
,
3
]. Gene ic associa ion s udies ha e p o ided insigh s in o he gene ics o
ea ly-onse obesi y, iden i ying s ongly associa ed genes wi h la ge pheno ypic e ec s such as he
lep in (LEP) o lep in ecep o (LEPR) genes [
4
]. Lep in is an endoc ine ho mone mainly p oduced by
he adipose issue, which has a c i ical ole in body weigh homeos asis [
5
]; i s ci cula ing le els a e
co ela ed wi h he amoun o body a and e lec he nu i ional s a us [
6
]. Lep in media es i s e ec s
ia he lep in ecep o , which is a membe o he Class I cy okine ecep o amily and consis s o an
ex acellula ligand-binding ansmemb ane domain and a cy oplasmic signalling domain [
7
]. Once in
he bloods eam, lep in eaches he b ain by c ossing he blood–b ain ba ie (BBB), and binds o i s
ecep o in he hypo halamus a cua e nucleus; he e, he ac i a ion o he ecep o inhibi s he ac ion
o o exigenic pep ides and s imula es ano ec ic neu opep ides, ensuing in he con ol o appe i e and
ood in ake [
8
]. Lep in also pa icipa es in he egula ion o glucose homeos asis and insulin sensi i i y,
since hese wo ho mones exe opposi e e ec s and egula e each o he in such a way ha lep in
inhibi s insulin, and insulin s imula es lep in syn hesis and sec e ion [9].
Lep in esis ance de ines a s a e o obesi y whe e hype lep inemia o diminished esponsi eness
o his ho mone is obse ed. The lack o esponse o lep in due o he de elopmen o esis ance may
dis u b cen al and pe iphe al ac ions o his ho mone. Al hough some p oposals ha e been made
such as ailu e o lep in c ossing he BBB, inhibi ion o he lep in signalling cascade, o a dec ease in he
exp ession o lep in ecep o s, o da e he mechanisms unde lying lep in esis ance emain unclea [
10
].
In bo h he LEP and LEPR genes, homozygous mu a ions ha e been desc ibed ha de i e in
ex eme obesi y [
11
–
13
]. In addi ion, a a ie y o single nucleo ide polymo phisms (SNPs) in di e en
loci ha e been associa ed wi h ci cula ing lep in le els and obesi y [
14
–
16
]. In he LEPR gene, di e en
SNPs con e ing inc eased suscep ibili y o common o ms o obesi y ha e been iden i ied in adul s [
16
],
amilies [
16
–
23
] and child en [
24
–
30
] wi h di e en e hnic backg ounds. In addi ion, o he a ian s
such as Gln223A g ( s1137101) in his gene ha e been associa ed wi h ype 2 diabe es [
31
]. Howe e ,
al hough he common o ms o obesi y a e a majo public heal h p oblem and he impai men o he
ac ion o lep in and i s ecep o a e implica ed in he onse o obesi y, ew s udies ha e in es iga ed
he con ibu ion o LEPR gene ic a ian s o he suscep ibili y o childhood obesi y also ocusing on
hei associa ion wi h ci cula ing bioma ke s.
Wi h all his in mind, he p esen s udy was unde aken wi h he aim o elucida ing he possible
associa ion ega ding LEPR a ian s in he se ing o de elopmen o obesi y in child en. Fo his, we
examined he associa ion o 28 LEPR polymo phisms wi h body mass index (BMI) and analysed hei
ela ionship wi h bioma ke s o insulin esis ance, in lamma ion and ca dio ascula disease (CVD)
isk in Spanish child en.
2. Resul s
2.1. Gene al Cha ac e is ics o he Popula ion
Table 1shows he an h opome ic, clinical and me abolic cha ac e is ics o he s udied g oups,
as p e iously published [
32
]. As expec ed, weigh , heigh , BMI, BMI z-Sco e (z-BMI) and wais
ci cum e ence (WC) we e signi ican ly highe in child en wi h obesi y compa ed o child en wi h
no mal-BMI. Sys olic and dias olic blood p essu e (BP), as well as plasma iacylglyce ols (TAG),
apolipop o ein (Apo)-B, insulin, and homeos a ic model assessmen o insulin esis ance (HOMA-IR),
we e highe in child en wi h obesi y, whe eas he quan i a i e insulin sensi i i y check index
(QUICKI), plasma o al choles e ol, high-densi y lipop o ein-choles e ol (HDL-c) and Apo-AI we e
lowe in his g oup when compa ed wi h no mal-weigh child en. Fas ing plasma glucose and
low-densi y lipop o ein-choles e ol (LDL-c) concen a ions showed no di e ences be ween g oups.
The concen a ions o alanine ansaminase and
γ
-glu amyl anspep idase we e highe in he obesi y
g oup, while ha o aspa a e ansaminase was lowe .
Plasma lep in concen a ion was signi ican ly highe in subjec s wi h obesi y han in he
no mal-BMI subjec s, whe eas adiponec in was lowe . In lamma ion and mos o he CVD isk
In . J. Mol. Sci. 2017,18, 1690 3 o 14
bioma ke s di e ed be ween g oups. C- eac i e p o ein (CRP), in e leukin (IL) 6, IL-8, and umou
nec osis ac o alpha (TNF-
α
) we e highe in he obesi y g oup compa ed wi h he no mal-BMI g oup.
Likewise, plasma soluble in acellula adhesion molecule-1 (sICAM-1), soluble endo helial selec in
(sE-selec in), myelope oxidase (MPO) and ac i e and o al plasminogen ac i a o inhibi o (PAI-1) we e
highe in child en wi h obesi y, whe eas ma ix me allop o einase-9 (MMP-9) showed no di e ences
be ween g oups.
Table 1. An h opome ic, clinical, and biochemical pa ame e s o he s udied child en.
No mal-BMI Obese p
n236 286
An h opome y
Sex (M/F) 133/103 146/140 0.252
Age (y) 9.72 ±0.16 9.43 ±0.15 0.188
Tanne S age (P epube /Pube )
Male 113/20 122/24
Female 83/20 108/32
Weigh (kg) 32.9 ±0.7 55.9 ±1.0 <0.001
Heigh (m) 1.37 ±0.01 1.41 ±0.01 0.002
BMI (kg/m2)17.14 ±0.13 27.59 ±0.24 <0.001
BMI z-Sco e −0.17 ±0.04 3.50 ±0.08 <0.001
Wais ci cum e ence (cm)
60.3 ±0.5 84.0 ±0.9 <0.001
Clinical and Me abolic Bioma ke s
Sys olic BP (mm Hg) 98 ±1 111 ±1 <0.001
Dias olic BP (mm Hg) 60 ±1 69 ±1 <0.001
Glucose (mg/dL) 84 ±1 85 ±1 0.816
Insulin (mU/L) 5.89 ±0.23 11.53 ±0.52 <0.001
HOMA-IR 1.26 ±0.05 2.45 ±0.12 <0.001
QUICKI 0.383 ±0.003 0.347 ±0.002 <0.001
T iacylglyce ols (mg/dL)
55 ±1 75 ±2 <0.001
Apo-AI (mg/dL) 149 ±2 132 ±2 <0.001
Apo-B (mg/dL) 67 ±1 71 ±1 0.006
Choles e ol (mg/dL) 171 ±2 165 ±2 0.024
HDL-c (mg/dL) 64 ±1 51 ±1 <0.001
LDL-c (mg/dL) 94 ±2 97 ±2 0.136
AST (U/L) 23.70 ±0.48 21.23 ±0.40 <0.001
ALT (U/L) 16.80 ±0.57 20.88 ±0.51 <0.001
GGT (U/L) 8.44 ±0.27 10.90 ±0.30 <0.001
Adiponec in (mg/L) 28.23 ±0.77 22.53 ±0.66 <0.001
Resis in (µg/L) 9.67 ±0.34 11.77 ±0.35 <0.001
Lep in (µg/L) 4.30 ±0.26 23.15 ±0.87 <0.001
In lamma ion Bioma ke s
C- eac i e p o ein
(mg/L) 0.97 ±0.23 3.44 ±0.25 <0.001
In e leukin 6 (ng/L) 4.55 ±0.54 7.03 ±0.76 0.008
In e leukin 8 (ng/L) 1.57 ±0.11 2.17 ±0.15 0.002
TNF-α(ng/L) 3.04 ±0.11 4.00 ±0.13 <0.001
Ca dio ascula Disease Risk Bioma ke s
MMP-9 (µg/L) 79.72 ±3.17 87.98 ±3.92 0.714
MPO (µg/L) 13.18 ±1.18 21.70 ±1.73 <0.001
sE selec in (µg/L) 22.91 ±0.77 31.36 ±1.06 <0.001
sICAM-1 (mg/L) 0.153 ±0.004 0.174 ±0.005 <0.001
Ac i e PAI-1 (µg/L) 5.07 ±0.26 11.92 ±0.58 <0.001
To al PAI-1 (µg/L) 18.82 ±0.85 27.11 ±1.12 <0.001
Mean
±
s anda d e o o he mean (SEM). M: male; F: emale; y: yea ; BMI: body mass index; BP: blood p essu e;
HOMA-IR: homeos asis model assessmen o insulin esis ance; QUICKI: quan i a i e insulin sensi i i y check
index; Apo: apolipop o ein; HDL-c: high-densi y lipop o ein choles e ol; LDL-c: low-densi y lipop o ein choles e ol;
ALT: alanine ansaminase; AST: aspa a e ansaminase; GGT: gamma-glu amyl anspep idase; TNF-
α
: umou
nec osis ac o alpha; MMP-9: me allop o einase-9; MPO: myelope oxidase; sICAM-1: soluble in acellula adhesion
molecule-1, PAI-1: plasminogen ac i a o inhibi o .
In . J. Mol. Sci. 2017,18, 1690 4 o 14
2.2. Associa ion o LEPR SNPs wi h Obesi y
Among he 28 analysed SNPs, only he s11208659, s11804091, s10157275, s9436303, and
s1627238 we e signi ican ly associa ed wi h obesi y in child en, a e age, sex and Tanne s age
adjus men unde an addi i e model (Table 2). Howe e , none o he SNPs emained signi ican ly
associa ed a e Bon e oni co ec ion. When we pe o med he analysis sepa a ely by sex, we obse ed
ha s11804091 and s10157275, and addi ionally, s1327118, we e associa ed wi h BMI only in he
emale g oup, al hough only s11804091 emained s a is ically signi ican a e Bon e oni co ec ion
(OR = 2.73 o allelic e ec , 95% CI: 1.47–5.08, p= 0.038). No associa ion was obse ed be ween hese
SNPs and obesi y in he male g oup; only s11208659 showed a nega i e associa ion wi h obesi y
in males ha was los a e Bon e oni co ec ion (Table 3). Howe e , he p e iously desc ibed in
adul s SNPs, s1137101, s1137100, and s8179183, we e no associa ed wi h BMI in ou popula ion.
Addi ionally, haplo ype analyses showed ha none o he signi ican ly associa ed SNPs we e in linkage
disequilib ium (LD) wi h he h ee men ioned abo e, ei he conside ing he whole popula ion o when
analyses we e pe o med sepa a ely by sex (Figu es S1–S3).
Since ou popula ion came om wo di e en ci ies o Spain, we pe o med a me a-analysis o
a oid popula ion s a i ica ion biases o a geno yping ba ch e ec . The esul s o his analysis (p alues
o QCoch ane: ( s11208659, Q= 0.997; s11804091, Q= 0.228; s10157275, Q= 0.304; s9436303,
Q= 0.166; and s1627238, Q= 0.887) indica es li le de ec able he e ogenei y o he wo conside ed
popula ions o he s udy.
To in es iga e he po en ial unc ional ole o hese a ian s, di e en web-based ools designed
o in silico p edic ion o SNP unc ion we e que ied using he SNP IDs, including he FuncPRED ool
o he Na ional Ins i u es o Heal h [
33
], RegulomeDB [
34
], Mi SNP [
35
], and RegSNP [
36
]. The sea ch
showed no de e minan e ec o he signi ican ly obesi y associa ed SNPs on he binding o known
ansc ip ion ac o s o mic oRNAs. Mo eo e , o he a ian s ound o be in LD wi h he associa ed
SNPs, including one missense polymo phism, we e no p edic ed o in luence he unc ion o he
p o ein o he binding o ansc ip ion ac o s o mic oRNAs o he LEPR gene.
2.3. Associa ion o SNP s11804091 wi h Obesi y-Rela ed T ai s
Table 4shows he associa ion o s11804091 in he emale popula ion wi h an h opome ic, clinical,
in lamma ion and CVD isk ma ke s adjus ed by age and Tanne s age. This SNP was signi ican ly
posi i ely associa ed wi h weigh , z-BMI, sys olic BP, insulin, HOMA-IR, Apo-AI, lep in, and TNF-
α
;
and signi ican ly nega i ely associa ed wi h heigh , QUICKI and adiponec in. This nega i e associa ion
wi h heigh could be due o he lowe mean age o he GG geno ype g oup (7.9 yea s). A e an
addi ional adjus men o BMI, insulin, HOMA-IR, QUICKI and adiponec in emained signi ican ly
associa ed ((
β
= 0.09 mU/L; 95% CI: 0.03, 0.15; p= 0.004), (
β
= 0.09; 95% CI: 0.03, 0.15; p= 0.006),
(
β
=
−
0.019 mg/L; 95% CI:
−
0.028,
−
0.009; p= 0.005) and (
β
=
−
3.09 mg/L; 95% CI:
−
6.11,
−
0.07;
p= 0.046), espec i ely).
In . J. Mol. Sci. 2017,18, 1690 5 o 14
Table 2. Geno ypic dis ibu ions o he LEPR analysed polymo phisms and i s associa ion wi h obesi y in child en.
Polymo phism Func ion Allele
1/Allele 2
Case Con ol Mino
Allele
Mino Allele
OR (95% CI) ppa
11 12 22 11 12 22 Case Con ol
s11208659 In on T/C 240 44 2 173 61 2 C 0.084 0.138 0.54 (0.35–0.81) 0.003 0.076
s11804091 In on A/G 193 76 8 181 46 2 G 0.166 0.109 1.64 (1.13–2.39) 0.010 0.251
s10157275 In on C/T 195 83 8 184 46 6 T 0.173 0.123 1.53 (1.08–2.18) 0.017 0.444
s9436303 In on A/G 162 100 24 152 73 11 G 0.259 0.201 1.36 (1.02–1.81) 0.036 0.926
s1627238 In on C/T 186 87 13 167 60 5 T 0.198 0.151 1.40 (1.01–1.94) 0.046 1
s17412175 In on T/A 82 146 58 57 120 58 A 0.458 0.502 0.82 (0.64–1.06) 0.133 1
s9436739 In on T/A 231 53 2 178 56 2 A 0.100 0.127 0.74 (0.50–1.10) 0.135 1
s1137101
Gln223A g
A/G 85 135 65 76 117 41 G 0.465 0.425 1.16 (0.91–1.48) 0.243 1
s6673591 In on A/G 83 129 74 55 121 60 G 0.484 0.511 0.89 (0.70–1.13) 0.325 1
s17412723 In on A/G 71 155 60 58 114 62 G 0.481 0.509 0.88 (0.68–1.14) 0.329 1
s6697315 In on T/C 126 125 35 92 113 30 C 0.341 0.368 0.88 (0.68–1.14) 0.329 1
s6704167 In on A/T 92 138 56 68 114 52 T 0.437 0.466 0.88 (0.69–1.13) 0.330 1
s8179183
Lys656Asn
G/C 195 82 9 153 72 10 C 0.175 0.196 0.87 (0.63–1.19) 0.379 1
s1327118 PRO G/C 73 143 59 62 121 42 C 0.475 0.456 1.10 (0.85–1.43) 0.472 1
s1137100
Lys109A g
A/G 155 112 19 133 89 14 G 0.262 0.248 1.09 (0.82–1.45) 0.552 1
s3806318 PRO A/G 158 104 24 116 101 16 G 0.266 0.285 0.92 (0.70–1.21) 0.562 1
s970468 In on T/G 123 135 28 96 113 26 G 0.334 0.351 0.93 (0.71–1.22) 0.588 1
s3790429 In on A/T 187 93 5 161 64 8 T 0.181 0.172 1.09 (0.78–1.51) 0.630 1
s9436740 In on A/T 143 115 24 119 89 26 T 0.289 0.301 0.95 (0.73–1.24) 0.704 1
s1475397 In on C/T 149 118 19 122 95 19 T 0.273 0.282 0.95 (0.72–1.25) 0.712 1
s11585329 In on T/G 215 64 7 171 61 4 T 0.136 0.146 0.94 (0.66–1.33) 0.718 1
s4655802 In on A/G 92 133 55 74 111 41 G 0.434 0.427 1.03 (0.80–1.32) 0.828 1
s6678033 In on G/A 107 136 43 91 111 34 A 0.388 0.379 1.03 (0.80–1.33) 0.829 1
s6672331 In on G/C 273 13 0 224 12 0 C 0.023 0.025 0.94 (0.42–2.12) 0.886 1
s1137099
Th 85Ala
A/ 286 0 0 236 0 0 0 0 - - -
s13306526
Ile503Val
A/ 286 0 0 236 0 0 0 0 - - -
CI: con idence in e al; OR: odds a io; PRO: p omo e . OR adjus ed o age, sex and Tanne s age unde he addi i e model.
a
p alues a e Bon e oni co ec ion. The bold is he s a is ic
signi icance o he ows whe e pis lowe han 0.05.
In . J. Mol. Sci. 2017,18, 1690 6 o 14
Table 3. Geno ypic dis ibu ions o he signi ican LEPR polymo phisms and i s associa ion wi h obesi y by sex in child en.
Polymo phism Allele
1/Allele 2
Case Con ol Mino
Allele
Mino Allele OR (95% CI) ppa
11 12 22 11 12 22 Case Con ol
Females
s11208659 T/C 113 26 1 72 3 1 C 0.100 0.155 0.56 (0.31–1.00) 0.050 1
s11804091 A/G 87 42 4 82 19 0 G 0.188 0.094 2.73 (1.47–5.08) 0.001 0.038
s10157275 C/T 99 37 4 84 17 2 T 0.161 0.102 1.77 (1.01–3.12) 0.045 1
s9436303 A/G 77 52 11 68 30 5 G 0.264 0.194 1.49 (0.95–2.32) 0.080 1
s1627238 C/T 93 41 6 75 23 2 T 0.189 0.135 1.59 (0.95–2.32) 0.079 1
s1327118 G/C 32 73 30 29 54 12 C 0.493 0.411 1.53 (1.01–2.32) 0.048 1
Males
s11208659 T/C 127 18 1 101 31 1 C 0.068 0.124 0.50 (0.27–0.92) 0.026 0.681
s11804091 A/G 106 34 4 99 27 2 G 0.146 0.121 1.23 (0.76–2.02) 0.837 1
s10157275 C/T 96 46 4 100 29 4 T 0.185 0.139 1.40 (0.89–2.21) 0.147 1
s9436303 A/G 85 48 13 84 43 6 G 0.253 0.207 1.28 (0.87–1.87) 0.218 1
s1627238 C/T 93 46 7 92 37 3 T 0.206 0.163 1.33 (0.86–2.04) 0.202 1
s1327118 G/C 41 70 29 33 67 30 C 0.457 0.489 0.88(0.63–1.24) 0.465 1
CI: con idence in e al; OR: odds a io. OR adjus ed o age and Tanne s age unde he addi i e model.
a
p alues a e Bon e oni co ec ion. The bold is he s a is ic signi icance o he
ows whe e pis lowe han 0.05.
Table 4. Associa ion o s11804091 wi h an h opome ic, clinical, in lamma ion and CVD isk bioma ke s in gi ls.
Bioma ke s AA AG GG β(95% CI) p p a
n169 61 4
An h opome y
Heigh (m) 1.37 ±0.01 1.41 ±0.02 1.35 ±0.05 −0.014 (−0.025, −0.004) 0.010 –
Weigh (kg) 43.1 ±1.4 49.9 ±2.5 50.5 ±7.9 6.1 (2.6, 9.6) 0.001 –
BMI (kg/m2)22.44 ±0.47 24.41 ±0.78 27.33 ±2.15 2.20 (0.70, 3.70) 0.004 –
BMI z-Sco e 1.57 ±0.15 2.08 ±0.23 3.51 ±0.45 0.70 (0.22, 1.18) 0.004 –
Wais ci cum e ence (cm) 71.56 ±1.31 76.07 ±2.14 80.75 ±2.39 5.46 (2.62, 9.59) 0.055 0.667
In . J. Mol. Sci. 2017,18, 1690 7 o 14
Table 4. Con .
Bioma ke s AA AG GG β(95% CI) p p a
Clinical and Me abolic Bioma ke s
Sys olic BP (mm Hg) 104 ±1 108 ±2 120 ±3 5.56 (1.97, 9.14) 0.003 0.092
Dias olic BP (mm Hg) 65 ±1 66 ±1 73 ±6 2.49 (−0.51, 5.49) 0.105 0.569
Glucose (mg/dL) 84 ±1 85 ±1 79 ±2−0.08 (−1.94, 1.78) 0.934 0.989
Insulin (mU/L) 9.11 ±0.60 12.64 ±1.15 10.95 ±3.25 0.14 (0.07, 0.21) 0.0001 0.004
HOMA-IR 1.91 ±0.13 2.70 ±0.28 2.17 ±0.69 0.14 (0.07, 0.22) 0.0002 0.006
QUICKI 0.367 ±0.003 0.342 ±0.004 0.349 ±0.014 −0.019 (−0.028, −0.009) 0.0001 0.005
T iacylglyce ols (mg/dL) 69 ±3 70 ±4 130 ±32 8.50 (−0.17, 17.17) 0.056 0.376
Apo-AI (mg/dL) 139 ±2 131 ±3 122 ±9−8.19 (−15.01, −1.37) 0.019 0.129
Choles e ol (mg/dL) 168 ±2 169 ±4 180 ±14 2.01 (−5.11, 0.55) 0.604 0.334
HDL-c (mg/dL) 55 ±2 52 ±2 62 ±17 −1.83 (−5.68, 2.01) 0.350 0.649
Adiponec in (mg/L) 26.67 ±0.95 20.96 ±1.48 23.33 ±2.30 −4.77 (−7.94, −1.60) 0.004 0.046
Lep in (µg/L) 13.66 ±1.03 19.01 ±2.13 19.81 ±3.69 5.10 (0.04, 1.06) 0.006 0.314
In lamma ion Bioma ke s
C- eac i e p o ein (mg/L) 2.29 ±0.41 3.42 ±0.52 1.65 ±0.61 0.84 (−0.45, 2.13) 0.202 0.633
IL-6 (ng/L) 6.03 ±0.89 5.38 ±0.99 18.16 ±9.07 0.92 (−1.91, 3.77) 0.522 0.737
IL-8 (ng/L) 1.80 ±0.13 1.79 ±0.25 3.63±1.51 0.21 (−0.26, 0.69) 0.381 0.637
TNF-α(ng/L) 3.26 ±0.15 3.84 ±0.31 4.02 ±0.94 0.55 (0.04, 1.06) 0.035 0.090
Ca dio ascula Disease Risk Bioma ke s
MMP-9 (µg/L) 84.18 ±4.44 78.82 ±6.59 75.67 ±13.56 −5.36 (−19.92, 9.11) 0.494 0.489
MPO (µg/L) 17.27 ±1.47 21.59 ±4.28 22.05 ±7.14 3.47 (−2.63, 9.58) 0.266 0.572
sE-Selec in (µg/L) 26.78 ±1.22 29.67 ±2.44 21.99 ±5.74 2.54 (−1.89, 6.97) 0.263 0.518
sICAM-1 (mg/L) 0.164 ±0.005 0.160 ±0.009 0.218 ±0.047 0.002 (−0.016, 0.021) 0.766 0.889
Ac i e PAI-1 (µg/L) 9.51 ±0.69 9.63 ±1.12 15.55 ±6.81 0.89 (−1.47, 3.26) 0.461 0.441
To al PAI-1 (µg/L) 23.75 ±1.29 23.56 ±2.54 28.67 ±9.17 0.62 (−3.98, 5.23) 0.713 0.707
CI: Con idence in e al; BMI: body mass index; BP: blood p essu e; HOMA-IR: homeos asis model assessmen o insulin esis ance; QUICKI: quan i a i e insulin sensi i i y check
index; HDL-c: high-densi y lipop o ein choles e ol; IL: in e leukin; TNF-
α
: umou nec osis ac o alpha; MMP-9: ma ix me allop o einase-9; MPO: myelope oxidase; sICAM-1: soluble
in acellula adhesion molecule-1; sE-selec in: soluble endo helial selec in; PAI-1: plasminogen ac i a o inhibi o .
β
Coe icien s ep esen he change in absolu e ai s alues o each
addi ional isk allele. Gene al linea o logis ic models we e used o examine associa ions, padjus ed by age and Tanne s age, paadjus ed by age, Tanne s age, and BMI.
In . J. Mol. Sci. 2017,18, 1690 8 o 14
3. Discussion
The main inding o ou s udy was he sex-speci ic associa ion be ween s11804091 and obesi y and
insulin esis ance in gi ls. We show he associa ion be ween his SNP and an h opome ic, clinical and
me abolic obesi y- ela ed ma ke s. Mo eo e , a e adjus ing o BMI, his SNP emained associa ed
posi i ely wi h insulin and HOMA-IR, and nega i ely wi h QUICKI and adiponec in. Ou esul s
sugges ha he polymo phism s11804091, o a lagged a ian in LD wi h i , migh ha e an e ec on
lep in ac ion wi h an impac on insulin signalling which does no depend en i ely on adiposi y.
Lep in and insulin a e wo body ene gy senso s ha ac in he hypo halamus h ough hei
espec i e ecep o s egula ing se e al pe iphe al unc ions. Bo h p omo e changes in he exp ession
o hypo halamic neu opep ides o egula e ene gy balance and glucose me abolism [
37
]. Two sepa a e
s udies ha e shown ha he ein oduc ion o he lep in ecep o s in he hypo halamus o LEPR null
mice educes obesi y in di e en deg ees and h ough di e en ac ions [
38
,
39
]. When LEPR we e
ein oduced in he p o-opiomelanoco in (POMC) neu ones, which usually exp ess LEPR, and o he
hypo halamic egions whe e LEPR exp ession has been associa ed wi h he egula ion o ood in ake,
he animals showed a disc e e educ ion in body weigh and adiposi y due o an inc ease in ene gy
expendi u e and also an imp o emen in he glucose and lipid me abolisms [
38
]. In e es ingly, only
male mice showed dec eased body weigh and adiposi y in ha s udy, sugges ing sex-dependan
changes in LEPR ene gy balance egula o y me abolism. Simila esul s we e obse ed in ano he
s udy in which LEPR was o e exp essed in all POMC neu ones. In his s udy, mice educed hei body
weigh by changing bo h ood in ake (dec eased) and ene gy expendi u e (inc eased); hese animals
also showed lowe plasma insulin and glucose le els [
39
]. In bo h s udies, he imp o emen in glucose
le els and insulin sensi i i y was independen o body weigh , sugges ing ha lep in signalling
in POMC neu ones has a ole in egula ing glucose homoeos asis and ha his egula o y ole is
no in luenced by adiposi y [
37
]. In ac , i has been desc ibed ha lep in pa icipa es in egula ing
glucose homeos asis and insulin sensi i i y by signalling pa hways, which include: Janus kinase (JAK),
phospha idylinosi ol 3’-kinase (PI3K) and ex acellula signal- egula ed kinase (ERK) [
40
]. The e o e,
he associa ion ound in his s udy be ween s11804091 and obesi y in emales may be ela ed o a
lowe exp ession o LEPR, which could de i e in insulin esis ance h ough mechanisms such as hose
explained abo e; howe e , exp ession analyses should be pe o med o con i m his.
Lep in ecep o plays an essen ial ole in he physiological e ec s o lep in. Al hough some s udies
ha e desc ibed e y high ci cula ing le els o lep in in ca ie s o LEPR mu a ions, o he s ha e no [
15
].
In he p esen s udy, we did no obse e associa ions be ween LEPR a ian s and ci cula ing lep in
le els, which sugges s ha SNPs in LEPR a e no impo an egula o s o ci cula ing lep in le els.
As p e iously men ioned, he obse ed ela ionship be ween he SNP s11804091 could be due o
an unknown unc ional a ian lagged by i . Indeed, he unc ional associa ion o an in onic SNP such
as s11804091 wi h a disease may a ise om di e en po en ial mechanisms such as al e ed miRNA
binding si es, o changes in TF-binding si es, ei he in he egion o he cha ac e ized a ian o in
ha o a second SNP lagged by he i s , o o a lagged missense a ian ha could ha e an impac
on he p o ein sequence and i s unc ionali y. The ac ha we did no obse e a high LD be ween
his SNP and he o he analysed a ian s indica es ha he unc ional SNP could be a any posi ion
in he genome sequence, no necessa ily nea o he candida e gene. Simila ly, we could no de ine a
unc ional ole o s11804091 a ec ing miRNA o TF binding si es, since he sea ch o he a ailable
da abases did no e ie e signi ican indings.
Among he s udied SNP in he p esen wo k, we demons a ed he associa ion o i e a ian s
( s11208659, s11804091, s10157275, s9436303 and s1627238) wi h obesi y in Spanish child en and
adolescen s, om which only he a ian s11208659 had been p e iously associa ed wi h se e e
ea ly-onse obesi y in Eu opean child en [
13
], and he es o he associa ions a e desc ibed o he i s
ime. We ound no associa ion be ween o he p e iously desc ibed unc ional LEPR SNPs and obesi y
such as s1137101 (Gln223A g), which has been associa ed wi h lowe and highe obesi y isk in Spanish
adul s [
16
] and gi ls [
29
], espec i ely, bu no in Tu kish [
24
], Polish [
25
,
30
], Mexican Mes izo [
26
]
In . J. Mol. Sci. 2017,18, 1690 9 o 14
o Eu opean [
27
] child en and adolescen s, as well as wi h ype 2 diabe es [
31
]. Ano he a ian , he
s1137100 (Lys109A g), has also been associa ed wi h obesi y in Eu opean [
27
] and Indian [
28
], bu
no in Mexican Mes izo [
26
], Spanish [
29
] o Danish [
41
] child en. Finally, he SNP s8179183 has been
associa ed wi h obesi y in Mexican Mes izo [
26
] bu no in Spanish [
29
], Polish [
30
] o Eu opean [
27
]
child en. The esul s o hese s udies a e inconclusi e and con o e sial and may be due o he di e en
gene ic backg ound o he popula ions, as well as o he small sample sizes used.
Ou s udy has se e al s eng hs and limi a ions, which should be men ioned. The main s eng hs
a e he high quan i y o analysed bioma ke s and he s ic SNPs’ selec ion me hod. The limi a ions
include a ela i ely small sample size o a gene ic associa ion s udy, which equi es u he alida ion
in independen and la ge popula ions; and he ac ha in o ma ion on ood in ake and ene gy
expendi u e, whose e ec s a e egula ed by lep in, was no a ailable.
In conclusion, we demons a e o he i s ime he gende -speci ic associa ion be ween s11804091
and obesi y in Spanish gi ls. Ou indings show ha his polymo phism is also associa ed wi h insulin
esis ance independen ly o obesi y in gi ls, sugges ing ha i migh ha e a po en ial e ec o lag
a unc ional polymo phism ha has an e ec on he ac ion o lep in on insulin me abolism. I will
be aluable o eplica e hese indings in la ge popula ions o alida e he esul s ob ained in he
p esen s udy.
4. Ma e ials and Me hods
4.1. S udy Design
In he p esen case-con ol mul icen e s udy, 522 child en we e ec ui ed, 286 classi ied as obese
(146 boys and 140 gi ls) and 236 as no mal weigh (133 boys and 103 gi ls) acco ding o BMI, using
he sex- and age-speci ic cu -o poin s published by Cole e al. [
42
]. The child en, aged 6–15 yea s,
we e ec ui ed in wo Spanish ci ies (Co doba and San iago de Compos ela) a p ima y ca e cen es
and schools. Inclusion c i e ia we e Eu opean-Caucasian he i age and absence o congeni al me abolic
diseases. Exclusion c i e ia we e non-Eu opean Caucasian he i age, he p esence o congeni al
me abolic diseases (e.g., diabe es o hype lipidaemia), unde nu i ion, and he use o any medica ion
o con ol BP and glucose o lipid me abolism. The e we e no siblings included in he s udy. A e
he ini ial assessmen , pa en s o child en ha ul illed he inclusion c i e ia we e in i ed o ake he
child en o he paedia ic uni o he pa icipa ing hospi als o a clinical examina ion. The s udy aims
and p ocedu es we e ully explained o pa en s o gua dians p io o w i en consen been aken, and
he child en ga e hei assen .
This s udy was complian wi h he Decla a ion o Helsinki 1975, e ised in 2008, and ollowed he
ecommenda ions o he Good Clinical P ac ice o he CEE (Documen 111/3976/88 July 1990), and
he legally en o ced Spanish egula ion, which egula es he clinical in es iga ion o human beings
(RD 223/04 abou clinical ials). The E hics Commi ee o he Reina So ía Uni e si y Hospi al o
Co doba, he E hics Commi ee on Human Resea ch o he Uni e si y o G anada and he Bioe hics
Commi ee o he Uni e si y o San iago de Compos ela app o ed he s udy (P ojec iden i ica ion
codes P06-CTS-2203 (04/05/2007) and PI 051968 (25/12/2005).
4.2. An h opome ic and Biochemical Measu emen s
The an h opome ic measu emen s we e aken wi h he child en ba e oo ed and in hei
unde wea . A s anda d beam balance was used o de e mine body weigh (kg), a p ecision s adiome e
was used o measu e heigh (cm) and o WC, wi h he child s anding, an inelas ic ape was applied
ho izon ally midway be ween he lowes ib ma gin and he iliac c es a he end o a gen le exhala ion.
BMI was calcula ed and he z-BMI was ob ained based on he Spanish e e ences [
43
]. BP was measu ed
using a me cu y sphygmomanome e wi h an app op ia e cu o he size o he child’s uppe igh
a m and ollowing in e na ional ecommenda ions [
44
]. Blood samples we e aken a e an o e nigh
as and clinical biochemical analyses we e pe o med a he labo a o ies o he pa icipa ing hospi al
