Association of the BDNF Val66Met variation with obesity in women

Molecular Genetics and Metabolism 95 (2008) 110–112

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Molecular Genetics and Metabolism j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y m g m e

Brief Com­mu­ni­ca­tion

Association of the BDNF Val66Met variation with obesity in women Sigri Beckers a,1, Armand Peeters a,1, Doreen Zegers a, Ilse Mertens b, Luc Van Gaal b, Wim Van Hul a,* a b

Depart­ment of Med­i­cal Genet­ics, Uni­ver­sity of Antwerp and Antwerp Uni­ver­sity Hos­pi­tal, Uni­ver­site­itsp­lein 1, B-2610 Wil­rijk, Antwerp, Bel­gium Depart­ment of Endo­cri­nol­ogy, Dia­be­tol­ogy and Metab­o­lism, Antwerp Uni­ver­sity Hos­pi­tal, Wil­rijkstraat 10, B-2650 Ede­gem, Bel­gium

a r t i c l e

i n f o

Article history: Received 19 June 2008 Accepted 19 June 2008 Available online 29 July 2008 

a b s t r a c t Brain-derived neu­ro­tro­phic fac­tor (BDNF) has been implied in the reg­u­la­tion of food intake. In the pres­ ent study, we gen­o­typed the Val66Met poly­mor­phism in a Bel­gian cohort of 532 obese women and 197 healthy female con­trols and were, for the first time, able to show an asso­ci­a­tion of the 66Met allele with obes­ity, at least in our female cohort. © 2008 Else­vier Inc. All rights reserved.

Key­words: Brain-derived neu­ro­tro­phic fac­tor Obes­ity Asso­ci­a­tion Women Poly­mor­phism

Intro­duc­tion Brain-derived neu­ro­tro­phic fac­tor (BDNF) is a fac­tor known to be crit­i­cally involved in the dif­fer­en­ti­a­tion of the cen­tral ner­vous sys­tem. Fur­ther­more, BDNF also plays a role in obes­ity and loco­ mo­tor activ­ity as was dem­on­strated through the gen­er­a­tion of het­ ero­zy­gous and con­di­tional knock-out mice. These mice develop a matu­rity-onset obes­ity phe­no­type, accom­pa­nied by aggres­sive­ ness and abnor­mal­i­ties in loco­mo­tor activ­ity [1–3]. Recently, a muta­tion in the gene encod­ing BDNF was iden­ti­fied in a patient with obes­ity [4]. Gray and col­leagues recently described a patient with hyper­pha­gia and obes­ity that har­bours a chro­mo­somal inver­ sion of the region includ­ing the BDNF gene [5]. Also, a muta­tion in TrkB, the recep­tor for BDNF, was shown to be asso­ci­ated with an obese phe­no­type [6,7]. BDNF has also been asso­ci­ated with eat­ing dis­or­ders in sev­eral cohorts [8–10]. In the pres­ent study, we hypoth­e­sized that the Val66Met poly­ mor­phism in BDNF may be impli­cated in the path­o­gen­e­sis of obes­ ity. There­fore, we gen­o­typed a large cohort of obese women and healthy con­trol sub­jects and com­pared allele fre­quen­cies of this cod­ing var­i­ant between these groups. Mate­ri­als and meth­ods Five hun­dred and thirty-two obese women (mean BMI 37.9 ± 0.3 kg/m2) were recruited from patients con­sult­ing the out­ * Cor­re­spond­ing author. Fax: +32 3 820 25 66. E-mail addresses: si­gri.bec­[email protected] (S. Beckers), wim.van­[email protected] (W. Van Hul). 1 These authors con­trib­uted equally to this work. 1096-7192/$ - see front matter © 2008 Else­vier Inc. All rights reserved. doi:10.1016/j.ymgme.2008.06.008

pa­tient obes­ity clinic at the Uni­ver­sity Hos­pi­tal. Inclu­sion cri­te­ ria were obes­ity (BMI 7 30 kg/m2) and age between 18 and 55 years. Patients with dia­be­tes or impaired glu­cose tol­er­ance were excluded from this study. Other exclu­sion cri­te­ria were preg­ nancy, Cush­ing’s dis­ease and hypo- or hyper­thy­roid­ism. As our patient sam­ple was seek­ing treat­ment, it might be expected that they are enriched for genetic defects caus­ing obes­ity. How­ever, after screen­ing 95 patients of our cohort for mel­a­no­cor­tin-4 recep­tor (MC4R) muta­tions, the most com­mon cause of mono­ genic obes­ity, no muta­tions were found [11]. There­fore, we con­ clude that the fre­quency of MC4R muta­tions in our sam­ple will be less than 1%. Con­trol indi­vid­u­als (197 women; mean BMI 22.1 ± 0.1 kg/m2) were recruited among the uni­ver­sity and hos­pi­tal per­son­nel and from cou­ples seek­ing pre­na­tal coun­sel­ling (due to high mater­nal age or increased tri­ple test) at the Depart­ment of Med­i­cal Genet­ics. Cou­ples seek­ing pre­na­tal genetic coun­sel­ling because of famil­ial dis­ease his­tory were excluded. No infor­ma­tion was avail­able about the pres­ence of eat­ing dis­or­ders among the con­trol sub­jects. Clin­ i­cal char­ac­ter­is­tics of cases and con­trols are shown in Table 1. All sub­jects were Cau­ca­sian and at en­rol­ment none were involved in an ongo­ing weight man­age­ment pro­gram. The study pro­to­col was approved by the Local Eth­ics Com­mit­tee and all sub­jects gave their informed con­sent. Height was mea­sured to the near­est 0.5 cm; body weight was mea­sured with a dig­i­tal scale to the near­est 0.1 kg. BMI was cal­cu­ lated as weight (in kg) over height (in m) squared. Geno­typ­ing of the Val66Met poly­mor­phism in BDNF (rs6265) was done using a Hy­bP­robe assay designed by Tib Mol­Bi­ol (Ber­lin, Ger­many). PCR was per­formed using stan­dard con­di­tions accord­ing to man­u­fac­turer’s rec­om­men­da­tions. Anal­y­sis was done



S. Bec­kers et al. / Molecular Genetics and Metabolism 95 (2008) 110–112

Table 1 Clin­i­cal char­ac­ter­is­tics of patients and healthy con­trol sub­jects (mean ± SE) Param­e­ter

Cases

Con­trols

Age (years) BMI (kg/m2) Weight (kg) Height (m)

37.5 ± 0.4 37.9 ± 0.3 102.7 ± 0.7 1.65 ± 0.00

35.0 ± 0.6 22.1 ± 0.1 61.9 ± 0.5 1.66 ± 0.01

SE, stan­dard error.

on a Light­cycler® 480 Real-Time PCR Sys­tem (Roche, Penz­berg, Ger­many). v2 square anal­y­sis was used to com­pare allele fre­quen­cies among obese patients and con­trols. We com­pared geno­types 2 £ 2 using Mann–Whit­ney U tests to see whether a dom­i­nant or reces­ sive model could be pro­posed. After this anal­y­sis, we com­pared geno­types under a reces­sive model (GG + GA vs. AA) by performing Mann–Whit­ney U tests to iden­tify dif­fer­ences in BMI. Logistic regres­sion was used to cal­cu­late odds ratios. In a pre­vi­ous study we ana­lyzed the FTO gene (rs1421085 and rs9939609) in our cohort and found a sig­nif­i­cant asso­ci­a­tion with com­mon obes­ity [12]. There­fore, we checked whether the BDNF rs6265 had an inde­ pen­dent effect on obes­ity in our cohort using a like­li­hood ratio test. A model includ­ing FTO SNPs rs1421085 and rs9939609 was com­pared to a model includ­ing the two FTO SNPs and the BDNF rs6265 using a like­li­hood ratio test. Sig­nif­i­cance level was set at P = 0.05. All sta­tis­ti­cal anal­y­ses were per­formed using SPSS ver­sion 12.0 (SPSS, Chi­cago, IL, USA). The HWE pro­gram from LINK­UTIL (http://www.gene­map­ping.cn/util.htm) was used to check Hardy– Wein­berg equi­lib­rium. Results First, we deter­mined that Hardy–Wein­berg equi­lib­rium was pres­ent for the Val66Met poly­mor­phism (P = 0.78). After geno­typ­ ing, the allele fre­quen­cies in obese patients and con­trols were com­ pared: the 66Met allele was pres­ent in 23.7% of obese patients and 18.3% of healthy women (P = 0.027). The alle­lic odds ratio was cal­ cu­lated and found to be 1.36 (P = 0.034). As we pre­vi­ously found two FTO SNPs (rs1421085 and rs9939609) to be sig­nif­i­cantly asso­ci­ ated to com­mon obes­ity in our study pop­u­la­tion [12], we checked whether the BDNF Val66Met poly­mor­phism had an inde­pen­dent effect on obes­ity. After com­par­ing a model with the FTO SNPs to a model also con­tain­ing the Val66Met, we found that BDNF Val66Met was inde­pen­dently asso­ci­ated to a higher risk of becom­ ing obese (adjusted OR = 1.38, P = 0.031). We then went on to study if the Met allele had a dom­i­nant or reces­sive effect on BMI in our cohort. We com­pared Val/Val to Val/Met car­ri­ers and found that these indi­vid­u­als did not dif­fer in BMI (P = 0.38). How­ever, it was seen that Val/Val car­ri­ers had a sig­nif­i­cantly dif­fer­ent BMI than Met/Met car­ri­ers (P = 0.002). A dif­fer­ence in BMI was also seen when com­par­ing Val/Met to Met/ Met car­ri­ers (P = 0.0003). There­fore, we decided to ana­lyse our data under the assump­tion that the 66Met allele has a reces­sive effect. When look­ing at BMI val­ues in obese and con­trol women together, it became clear that women homo­zy­gous for 66Met had a higher mean BMI than women homo­zy­gous or het­ero­zy­gous for 66Val (Met/Met 37.1 kg/m2 vs. Val/Val + Val/Met 33.4 kg/m2, P = 0.001). Dis­cus­sion To our knowl­edge this is the first asso­ci­a­tion study link­ing the Val66Met poly­mor­phism in BDNF to obes­ity. From our results, we can con­clude that the 66Met allele is involved in the path­o­gen­e­sis of obes­ity in women as the fre­quency of this allele is sig­nif­i­cantly higher in obese women when com­pared to a con­trol group. Fur­

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ther­more, we were able to dem­on­strate that this allele has a reces­ sive effect on BMI. A lim­i­ta­tion of our study is that we only stud­ied women. We chose this approach because we do not have a suf­fi­ciently large male con­trol sam­ple to per­form ade­quately pow­ered asso­ci­a­tion stud­ies. The potential role of BDNF in obes­ity was first dis­cov­ered through gen­er­a­tion of KO mice which dis­play an obese phe­no­type [1–3]. In 2003, it was shown that BDNF acts down­stream of the mel­a­no­cor­tin-4 recep­tor. The effect on food intake was reg­u­lated through acti­va­tion of its TrkB recep­tor [13]. Later, muta­tion anal­y­ ses dis­cov­ered BDNF and TrkB muta­tions in patients with obes­ity [4–7]. Our pres­ent results fur­ther strengthen the belief that BDNF has an impor­tant func­tion in the path­o­gen­e­sis of obes­ity. Pre­vi­ously, Fri­edel and col­leagues also inves­ti­gated the potential role of the Val66Met var­i­ant in obes­ity, but they did not find any sig­nif­i­cant asso­ci­a­tions when com­par­ing extremely obese chil­dren and ado­les­cents to a group of under­weight con­trols [4]. This dif­fer­ ence between stud­ies may be caused by the fact that they looked at chil­dren and ado­les­cents, while we only inves­ti­gated obese adult females. It could be pos­si­ble that a gen­der spe­cific effect is pres­ent. How­ever, as we did not study men we can­not draw con­clu­sions on this mat­ter. Fur­ther­more, our sam­ple size is larger, com­par­ing 532 patients to 197 con­trols, while Fri­edel stud­ied a group of 183 patients and 187 con­trols. Addi­tion­ally, it was also seen that the het­ero­zy­gous BDNF KO mouse dis­plays a matu­rity-onset obes­ity phe­no­type [1–3], which sug­gests that the effect of the Met allele may not be seen when anal­y­sing chil­dren. Together, these results sug­gest a role for the Val66Met poly­ mor­phism in the reg­u­la­tion of food intake. The observed asso­ci­ a­tion could also occur by chance or could be the result of high link­age dis­equi­lib­rium to the causal SNP. How­ever, when look­ing at the Hap­Map data for the CEPH pop­u­la­tion [14], we see that rs6265 is only in high LD to rs2049045, an intronic SNP. There­ fore, as Val66Met itself is a non-syn­on­y­mous cod­ing var­i­ant, it is spec­u­lated that this SNP may be the func­tional var­i­ant respon­ si­ble for these asso­ci­a­tions. Sev­eral stud­ies have already shown that the 66Met allele affects the intra­cel­lu­lar traf­fick­ing and secre­tion of proB­DNF in neu­ro­nal cells [15,16]. It has also been sug­gested that the 66Met allele might pre­vent proper cleav­age of proB­DNF to mature BDNF [17]. We hy­poth­es­ise that the 66Met allele leads to reduced cleav­ age of proB­DNF to BDNF. As proB­DNF pref­er­en­tially binds to the p75NTR recep­tor [18], instead of the TrkB recep­tor implied in food intake reg­u­la­tion, we expect that the reduced amount of mature BDNF causes less inhi­bi­tion on food intake, even­tu­ally lead­ing to obes­ity. In con­clu­sion, we have found for the first time a sig­nif­i­cant asso­ci­a­tion between the 66Met allele and obes­ity in a cohort of women. Due to the nature of the SNP, we expect Val66Met to be the func­tional var­i­ant caus­ing this asso­ci­a­tion. Fur­ther func­tional stud­ies are nec­es­sary to con­firm the exact mech­a­nism through which this SNP influ­ences food intake reg­u­la­tion. Acknowl­edg­ments This study was sup­ported by a BOF-grant from the Uni­ver­sity of Antwerp and by a grant from the FWO-Flan­ders (G.0028.05) both to L.V.G. and W.V.H. S.B. and D.Z. hold a spe­cial­iza­tion schol­ ar­ship from the “Insti­tute for the Pro­mo­tion of Inno­va­tion through Sci­ence and Tech­nol­ogy in Flan­ders (IWT-Vla­and­er­en)”. Ref­er­ences [1] S.G. Ker­nie, D.J. Lie­bl, L.F. Pa­ra­da, BDNF reg­u­lates eat­ing behav­ior and loco­mo­ tor activ­ity in mice, EMBO J. 19 (2000) 1290–1300.

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[2] M. Rios, G. Fan, C. Fek­e­te, J. Kelly, B. Bates, R. Ku­ehn, R.M. Le­chan, R. Jae­nisch, Con­di­tional dele­tion of brain-derived neu­ro­tro­phic fac­tor in the post­na­tal brain leads to obes­ity and hyper­ac­tiv­ity, Mol. Endo­cri­nol. 15 (2001) 1748– 1757. [3] W.E. Lyons, L.A. Mamo­un­as, G.A. Ri­ca­urte, V. Cop­pola, S.W. Reid, S.H. Bora, C. Wih­ler, V.E. Ko­li­at­sos, L. Tes­sar­ol­lo, Brain-derived neu­ro­tro­phic fac­tordefi­cient mice develop aggres­sive­ness and hyper­pha­gia in con­junc­tion with brain sero­to­ner­gic abnor­mal­i­ties, Proc. Natl. Acad. Sci. USA 96 (1999) 15239– 15244. [4] S. Fri­edel, F.F. Hor­ro, A.K. Werm­ter, F. Gel­ler, A. Demp­fle, K. Reich­wald, J. Smidt, G. Bron­ner, K. Kon­rad, B. Her­pertz-Dahl­mann, A. Warn­ke, U. Hemm­ in­ger, M. Lin­der, H. Kie­fl, H.P. Golds­ch­midt, W. Sieg­fried, H. Rems­ch­midt, A. Hin­ney, J. Hebe­brand, Muta­tion screen of the brain derived neu­ro­tro­phic fac­tor gene (BDNF): iden­ti­fi­ca­tion of sev­eral genetic vari­ants and asso­ci­a­ tion stud­ies in patients with obes­ity eat­ing dis­or­ders and atten­tion-def­i­ cit/hyper­ac­tiv­ity dis­or­der, Am. J. Med. Genet. B Neu­ro­psy­chi­atr. Genet. 132 (2005) 96–99. [5] J. Gray, G.S. Yeo, J.J. Cox, J. Mor­ton, A.L. Ad­lam, J.M. Ke­ogh, J.A. Ya­nov­ski, A. El Gha­rbawy, J.C. Han, Y.C. Tung, J.R. Hod­ges, F.L. Ray­mond, S. O’Rah­il­ly, I.S. Far­ooqi, Hyper­pha­gia severe obes­ity impaired cog­ni­tive func­tion and hyper­ac­ tiv­ity asso­ci­ated with func­tional loss of one copy of the brain-derived neu­ro­ tro­phic fac­tor (BDNF) gene, Dia­be­tes 55 (2006) 3366–3371. [6] J. Gray, G. Yeo, C. Hung, J. Ke­ogh, P. Clay­ton, K. Baner­jee, A. McAu­lay, S. O’Rah­ il­ly, I.S. Far­ooqi, Func­tional char­ac­ter­iza­tion of human NTRK2 muta­tions iden­ ti­fied in patients with severe early-onset obes­ity, Int. J. Obes. (Lond) 31 (2007) 359–364. [7] G.S. Yeo, C.C. Con­nie Hung, J. Roch­ford, J. Ke­ogh, J. Gray, S. Siv­ar­a­ma­krish­nan, S. O’Rah­il­ly, I.S. Far­ooqi, A de novo muta­tion affect­ing human TrkB asso­ci­ated with severe obes­ity and devel­op­men­tal delay, Nat. Neu­ro­sci. 7 (2004) 1187– 1189. [8] M. Ri­bas­es, M. Grata­cos, L. Ar­men­gol, R. de Cid, A. Badia, L. Jime­nez, R. So­lan­o, J. Val­lejo, F. Fer­nan­dez, X. Es­tiv­ill, Met66 in the brain-derived neu­ro­tro­phic fac­ tor (BDNF) pre­cur­sor is asso­ci­ated with anorexia nerv­osa restric­tive type, Mol. Psy­chi­a­try 8 (2003) 745–751. [9] M. Ri­bas­es, M. Grata­cos, F. Fer­nan­dez-Aran­da, L. Bel­lod­i, C. Boni, M. An­der­luh, M.C. Cav­al­lin­i, E. Cel­lin­i, D. Di Bella, S. Erz­ego­ves­i, C. Fo­u­lon, M. Gab­rovsek, P. Gor­wood, J. Hebe­brand, A. Hin­ney, J. Hol­li­day, X. Hu, A. Kar­wautz, A. Kip­man,

[10]

[11]

[12]

[13]

[14] [15]

[16]

[17]

[18]

R. Ko­mel, B. Nac­mias, H. Rems­ch­midt, V. Ric­ca, S. Sor­bi, G. Wag­ner, J. Trea­sure, D.A. Col­lier, X. Es­tiv­ill, Asso­ci­a­tion of BDNF with anorexia bulimia and age of onset of weight loss in six Euro­pean pop­u­la­tions, Hum. Mol. Genet. 13 (2004) 1205–1212. M. Ri­bas­es, M. Grata­cos, F. Fer­nan­dez-Aran­da, L. Bel­lod­i, C. Boni, M. An­der­luh, M. Cri­sti­na Cav­al­lin­i, E. Cel­lin­i, D. Di Bella, S. Erz­ego­ves­i, C. Fo­u­lon, M. Gab­ rovsek, P. Gor­wood, J. Hebe­brand, A. Hin­ney, J. Hol­li­day, X. Hu, A. Kar­wautz, A. Kip­man, R. Ko­mel, B. Nac­mias, H. Rems­ch­midt, V. Ric­ca, S. Sor­bi, M. To­mor­i, G. Wag­ner, J. Trea­sure, D.A. Col­lier, X. Es­tiv­ill, Asso­ci­a­tion of BDNF with restrict­ing anorexia nerv­osa and min­i­mum body mass index: a fam­ily-based asso­ci­a­tion study of eight Euro­pean pop­u­la­tions, Eur. J. Hum. Genet. 13 (2005) 428–434. S. Bec­kers, I. Mer­tens, A. Pe­et­ers, L. Van Gaal, W. Van Hul, Screen­ing for mel­a­no­ cor­tin-4 recep­tor muta­tions in a cohort of Bel­gian mor­bidly obese adults and chil­dren, Int. J. Obes. (Lond) 30 (2006) 221–225. A. Pe­et­ers, S. Bec­kers, A. Ver­rij­ken, P. Roe­vens, P. Pe­et­ers, L. Van Gaal, W. Van Hul, Vari­ants in the FTO gene are asso­ci­ated with com­mon obes­ity in the Bel­ gian pop­u­la­tion, Mol. Genet. Metab. 93 (2008) 481–484. B. Xu, E.H. Goul­ding, K. Zang, D. Ce­poi, R.D. Cone, K.R. Jones, L.H. Te­cott, L.F. Re­ic­hardt, Brain-derived neu­ro­tro­phic fac­tor reg­u­lates energy bal­ance down­ stream of mel­a­no­cor­tin-4 recep­tor, Nat. Neu­ro­sci. 6 (2003) 736–742. Inter­na­tional Hap­Map Pro­ject. www.hap­map.org Accessed on 12 June 2008. M.F. Egan, M. Koj­ima, J.H. Cal­li­cott, T.E. Gold­berg, B.S. Kola­cha­na, A. Ber­to­li­no, E. Za­it­sev, B. Gold, D. Gold­man, M. Dean, B. Lu, D.R. Wein­ber­ger, The BDNF val66met poly­mor­phism affects activ­ity-depen­dent secre­tion of BDNF and human mem­ory and hip­po­cam­pal func­tion, Cell 112 (2003) 257–269. Z.Y. Chen, P.D. Patel, G. Sant, C.X. Meng, K.K. Teng, B.L. Hemp­stead, F.S. Lee, Var­ i­ant brain-derived neu­ro­tro­phic fac­tor (BDNF) (Met66) alters the intra­cel­lu­lar traf­fick­ing and activ­ity-depen­dent secre­tion of wild-type BDNF in neu­ro­se­cre­ tory cells and cor­ti­cal neu­rons, J. Neu­ro­sci. 24 (2004) 4401–4411. G. Oro­szi, L. Lapt­eva, E. Davis, C.H. Yar­boro, T. We­ick­ert, T. Roe­buck-Spencer, J. Blei­berg, D. Ro­sen­stein, M. Pao, P.E. Lip­sky, D. Gold­man, R.H. Lip­sky, G.G. Il­lei, The Met66 allele of the func­tional Val66Met poly­mor­phism in the brainderived neu­ro­tro­phic fac­tor gene con­fers pro­tec­tion against neu­ro­cog­ni­tive dys­func­tion in sys­temic lupus ery­th­e­mat­o­sus, Ann. Rheum. Dis. 65 (2006) 1330–1335. R. Lee, P. Ker­man­i, K.K. Teng, B.L. Hemp­stead, Reg­u­la­tion of cell sur­vival by secreted proneu­ro­tro­phins, Sci­ence 294 (2001) 1945–1948.