Promoter methylation of serotonin transporter gene is associated with obesity measures: a monozygotic twin study.

0.28 MB·English

by  Zhao, J

InternationalJournalofObesity(2013)37,140--145
&2013MacmillanPublishersLimited Allrightsreserved0307-0565/13
www.nature.com/ijo
ORIGINAL ARTICLE
Promoter methylation of serotonin transporter gene is associated
with obesity measures: a monozygotic twin study
JZhao1, JGoldberg2,3 and VVaccarino4
OBJECTIVE: Epigenetic mechanisms are increasingly beingrecognized asan importantfactor forobesity. Theserotonin
transporter gene(SLC6A4) hasacritical role inregulating food intake, bodyweightand energybalance. This study
examinesthe potential association betweenSLC6A4 promoter methylation andobesity measuresina monozygotic(MZ)
twin sample.
METHODS: Westudied 84MZtwinpairsdrawn fromtheVietnamEra TwinRegistry. Obesitymeasuresinclude body mass
index (BMI), bodyweight,waist circumference (WC)and waist-hip ratio (WHR).The SLC6A4promoter methylation profile in
peripheral bloodleukocytes wasquantified by bisulfite pyrosequencing. The association between methylation variationand
obesity parameters wasexaminedby mixed-model regressionand matched pairanalysis, adjustingfor age,smoking, alcohol
consumption, physical activity and totaldailyenergy intake.Multiple testing wascontrolled using theadjustedfalse discovery
rate (q-value).
RESULTS: Mean methylation levelwas positivelycorrelated withBMI (r¼0.29; P¼0.0002), bodyweight(r¼0.31;Po0.0001)
and WC (r¼0.20; P¼0.009), butnot WHR. Intra-pair differences inmean methylation weresignificantly correlated with
intra-pair differences in BMI,body weightandWC, butnot WHR.On average, a1% increasein meanmethylation was
associated with0.33kgm(cid:2)2 increaseinBMI (95% CI:0.02--0.65; P¼0.03),1.16kgincrease inbody weight(95%CI, 0.16--2.16;
P¼0.02) and0.78cm increaseinWC (95% CI, 0.05--1.50;P¼0.03)after controlling forpotential confounders.
CONCLUSIONS: SLC6A4 promoter hypermethylationis significantly associated with anincreased prevalence ofobesity within
a MZtwinstudy.
International Journal ofObesity (2013) 37,140--145; doi:10.1038/ijo.2012.8; publishedonline 31January 2012
Keywords: DNA methylation; serotonintransporter gene; SLC6A4;monozygotic twins
INTRODUCTION central nervous system inhibit food intake and promote weight
Obesityhasreachedepidemicproportionsgloballyinallageand loss.6,7Theserotonintransporterisresponsibleforthereuptakeof
ethnic groups. It is associated with an increased risk of type 2 5-HT into presynaptic neuron and regulates the extracellular 5-HT
diabetes, cardiovascular disease and certain types of cancer, concentration, the strength of 5-HT signals and the duration of
posing a substantial public health and economic burden on postsynaptic responses to 5-HT. The serotonin transporter has a
societies.1 Despite significant effort, our understanding of the pivotalroleinserotoninturnoverandserotoninlevelinthesynaptic
molecular processes underlying obesity remains incomplete. The cleft.8Consequently,anydefectsofserotonintransportermaylead
current obesity epidemic most likely results from the complex to dysregulation of the serotonergic system and could potentially
interplaybetweengenetic,behavioralandenvironmentalfactors. contribute to energy imbalance and influence obesity. Indeed, a
Epigenetic factors act at the interface between the internal promoter gene polymorphism in the serotonin transporter gene
genetic landscape and external environmental factors, and thus hasbeenassociatedwitheatingdisorder,9obesity10,11andtype2
could be the basis for some specific features of obesity, such as diabetes,12,13thoughmixedresultsexist.14,15
discordance within monozygotic (MZ) twins, and the close The goal of this study was to investigate whether promoter
relationship with lifestyle factors. Therefore, epigenetic modifica- methylation of the serotonin transporter gene (SLC6A4) is
tions,especiallyDNAmethylation,mayhaveanimportantrolein associated with body mass index (BMI), body weight, waist
thepathogenesisof obesityand itsassociated phenotypes.2 circumference (WC) and waist-hip ratio (WHR). We tested this
Obesityandoverweightresultfromanimbalancebetweenfood hypothesis using a MZ twin sample because epigenetic mechan-
intake and energy expenditure, leading to storage of excessive isms are in part under genetic control.16 MZ twins are matched
calories asbody fat. Serotonin(5-hydroxytryptamine,5-HT) hasan on their DNA and many other known and unknown factors, the
importantroleinthecontrolofenergybalanceandbodyweightin use of MZ twins could eliminate the confounding by genotype
the central nervous system.3 Depletion of central serotonin using and sex and many other unknown or unmeasured factors, thus
selectiveneurotoxinsresultsinhyperphagiaandobesity,4,5whereas providingausefulmodelforepigeneticresearchofcomplextraits
pharmacological agents that increase serotonin activity in the such asobesity.
1Department ofBiostatisticsandEpidemiology,CollegeofPublicHealth,UniversityofOklahomaHealthSciencesCenter, Oklahoma City,OK,USA;2SeattleEpidemiologic
Research&InformationCenter,VeteransAffairsOfficeofResearch&Development,Seattle,WA,USA;3DepartmentofEpidemiology,UniversityofWashington,Seattle,WA,USA
and4DepartmentofEpidemiology,EmoryUniversitySchoolofPublicHealth,andDepartmentofMedicine,DivisionofCardiology,EmoryUniversitySchoolofMedicine,Atlanta,
GA,USA.Correspondence:DrJZhao,DepartmentofBiostatisticsandEpidemiology,CollegeofPublicHealth,UniversityofOklahomaHealthSciencesCenter,OklahomaCity,
OK73104,USA.
E-mail:[email protected]
Received2November2011;revised15December2011;accepted3January2012;publishedonline31January2012
Methylationofserotonintransportergeneassociatedwithobesity
JZhaoetal
141
MATERIALSANDMETHODS Table1. TargetedregionsofSLC6A4bypyrosequencing
Study population
GenomicDNAtarget 50-CGCCGCCAAAGAGCTCTTGAAGAATTTTT
TwinsincludedinthisstudyweredrawnfromtheVietnamEraTwinRegistry,
sequence GCG-30
oneofthelargesttwinregistriesintheUnitedStates.17Alltwinsweremale 50-TCACTTTGAGGCGAATAAACTTAATGCTT
veteranswhowerebornbetween1946and1956.Atotalof307twinpairs CCC-30
(whowereraisedinthesamehousehold)wererecruitedbytheEmoryTwin
Studies(ETS),whichincludedtwocompanionstudiestoinvestigatetherole Bisulfiteconverted 50-YGTYGTTAAAGAGTTTTTGAAGAATTTTT
targetsequence GYGTTA-30
of psychological, behavioral and biological risk factors for subclinical
50-TTTTGAGGYGAATAAATTTAATGTTTTTT-30
cardiovasculardiseaseintwins.TheEmoryTwinStudiesincludemale--male
twinpairs,including187MZpairsand120dizygoticpairs,withaninclusion Pyrosequencingassay 50-GTCTGTCGCTAGAGTTGAGATTAGTCGTAT
of two samples of twin pairs discordant for major depression or (dispensationorder) TGATGTCGAT-30
posttraumaticstressdisorder(PTSD).TheEmoryTwinStudiesprotocolhas
PCRproductlength(bp) 205
been described elsewhere.18 This research was approved by the Emory
InstitutionalReviewBoardandalltwinssignedaninformedconsent.
The current analysis included 84 MZ twin pairs from the Emory Twin CpG sites, we designed pyrosequencing assays and tested for PCR
Studies. These twin pairs were selected based on the availability of DNA preferential amplification and quantitative pyrosequencing analysis. The
samplesandphenotypedataforbothmembersofatwinpair.Alltwinswere target sequences (genomic DNA and bisulfite converted DNA) from the
examinedinpairsattheEmoryUniversityGeneralClinicalResearchCenter pyrosequencingassaysarelistedinTable1.Table2presentsthegenomic
between2002and2010,wheretheirmedicalhistorywasupdated.Alltwins locationofthetargetedCpGsites(withrespecttotranscriptionalstartsite)
wereCaucasians.ZygosityinformationwasdeterminedbyDNAanalysis. bypyrosequencing.
The bisulfite conversion was performed with 500ng genomic DNA
Measurement ofobesityparameters isolatedfromperipheralbloodleukocytesusingtheEZDNAmethylation
kit(ZymoResearch,Inc.,Irvine,CA,USA).ThePCRreactionwasperformed
Body weight (kilograms) and height (centimeters) were measured when
participantsworelightclothesandnoshoesbytrainedresearchstaff.BMI with0.2mMofeachprimerwithoneofthePCRprimersbeingbiotinylated
inordertopurifythefinalPCRproductusingsepharosebeads.ThePCR
wascalculatedbydividingweightinkilogramsbythesquareofheightin
productwasboundtostreptavidinsepharoseHP(AmershamBiosciences,
meters.Normalbodyweight,overweightorobesitywasdefinedas18.5o
BMI p24.9kgm(cid:2)2, 25.0 o BMI p29.9kgm(cid:2)2 or BMI X30.0kgm(cid:2)2, Uppsala, Sweden), and the sepharose beads containing the immobilized
PCR product were purified, washed and denatured using 0.2M NaOH
respectively.WCwasmeasuredatthelevelmidwaybetweenthelowestrib
solution and rewashed using the pyrosequencing Vacuum Prep Tool
andtheuppermostiliaccrestwiththesubjectsstanding.Hipcircumference
(QiagenPyrosequencing,Valencia,CA,USA)asrecommendedbythemanu-
wasmeasuredatthelevelofwidestcircumferenceovergreatertrochanters
with the legs close together. WHR was calculated as WC divided by hip facturer.Then0.5mMpyrosequencingprimerwasannealedtothepurified
single-stranded PCR product. A total of 10ml of the PCR products were
circumference. All anthropometric measurements were performed twice
sequencedbypyrosequencingPSQ96HSSystem(QiagenPyrosequencing)
andthemeanvalueofthetwomeasurementswasusedforanalysis.
following the manufacturer’s instructions (Qiagen Pyrosequencing). The
methylation status of each CpG site was analyzed individually as an
Other measurements artificial T/C SNP using QCpG software (Qiagen Pyrosequencing). The
Allmeasurementswereperformedinthemorningafteranovernightfast, methylationlevelateachCpGsiteforeachsamplewascalculatedasthe
and both members of a pair were tested at the same time. A medical percentage of the methylated alleles over the sum of methylated and
historyandaphysicalexamwereobtainedfromalltwins.Physicalactivity unmethylated alleles. The mean methylation level was calculated using
wasassessedbymeansofamodifiedversionoftheBaeckeQuestionnaire methylation levels of all measured CpG sites within the targetedregion.
of Habitual Physical Activity used in the Atherosclerosis Risk in Commu- Each pyrosequencing assay was done on duplicate samples, and each
nities Study,19 a 16-question instrument documenting level of physical pyrosequencingassaywasperformedaminimumoftwotimes.Forquality
activityatwork,duringsportsandnon-sportsactivities.Thetotalphysical control,eachexperimentincludednon-CpGcytosinesasinternalcontrols
activityscorewasusedintheanalysis.Dietaryintakewasassessedusing to verify efficient sodium bisulfite DNA conversion. We also included
the Willett self-administered semi-quantitative food frequency question- unmethylatedandmethylatedDNAsascontrolsineachrun.Inaddition,
naire,20 which collected dietary data for the 12-months before testing. we performed PCR bias testing using pyrosequencing by mixing the
Cigarette smoking was classified into current smoker (any number of unmethylatedDNAcontrolandinvitromethylatedDNAatdifferentratios
cigarettes) versus never or past smoker. Pack-years of smoking were (0, 20, 40 up to 100%) followed by bisulfite modification, PCR and
calculatedasthenumberofpacksofcigarettessmokedperdaytimesthe pyrosequencing analysis. The percent methylation obtained from the
number of years smoked. Information on alcohol consumption was mixing study showed high correlation with expected methylation
collected by askingabout the number of alcoholic drinks (beer, wine or percentages(r2X0.97),indicatinghigh-qualitymethylationdata.
liquor) consumed in a typical week. The total amount of alcohol
consumption(ingrams)perweekwasestimatedbasedonthefollowing Statistical analyses
algorithms:4ozofwinecontains10.8g,12ozofbeercontains13.2gand
BecausemethylationdataatsomeCpGsitesarenotnormallydistributed,we
1.5ozofliquorcontains15.1gofethanol.
performednonparametricstatisticalanalysis.Toadjustformultipletesting,
weusedtheBenjamini--Hochbergfalsediscoveryrateprocedure21tocorrect
DNAmethylation analysesby quantitativebisulfite for the number of CpG sites evaluated. An false discovery rate-adjusted
pyrosequencing P-value(q-value)thresholdof0.05wasusedtodeterminesignificance.The
Promotermethylationleveloftheserotonintransportergene(SLC6A4)in PROCMULTTESTprocedureinSAS9.2wasusedtocalculatetheq-value.The
peripheral blood leukocytes was determined using quantitative bisulfite following statistical analyses were performed to examine the association
pyrosequencing by the EpigenDx Inc. (Worcester, MA, USA). Briefly, we betweenSLC6A4promotermethylationlevelandobesity:
assayed20CpGdinucleotidesinthepromoterregionoftheSLC6A4from- Regression analyses by treating twins as individuals: We examined
209to-67bpsfromthetranscriptionalstartsite,basedonEnsemblGene whetherDNAmethylationvariationwasassociatedwithobesitymeasures
IDENSG00000108576andtheTranscriptIDENST00000394821.TheSLC6A4 (BMI,bodyweight,WCandWHR),adjustingforage,smokingandalcohol
assay was designed to capture the CpG sites in the promoter region consumption (g per week). These analyses were done using mixed
immediatelyupstreamofthetranscriptionalstartsite.Theassayistargeted modeling,inwhichtwinpairwasincludedasrandomeffecttoaccountfor
to the antisense sequence of SLC6A4 gene. To sequence these selected thewithintwinpaircorrelations.
&2013MacmillanPublishersLimited InternationalJournalofObesity(2013) 140--145
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Table2. SLC6A4promoterCpGsitesexaminedbypyrosequencing Table3. Demographic,clinicalandlaboratorycharacteristics
ofthetwins
Targeted Distancerelative Genomicposition
CpGsites toTSS(bp) (GCRh37/hg19) Characteristic Mean±s.d.or%
1 --69 Chr17:28562784 Age(years) 55.1±2.8
2 --72 Chr17:28562787 Type2diabetes(%) 11.4
3 --99 Chr17:28562814 Hypertension(%) 36.5
4 --112 Chr17:28562827 Currentsmoking(pack-year) 22.9±24.1
5 --133 Chr17:28562848 Alcoholconsumption(gperweek) 12.5±36.3
6 --135 Chr17:28562850 Overweight(%) 43.6
7 --139 Chr17:28562854 Obesity(%) 40.6
8 --141 Chr17:28562856 Bodyweight(kg) 90.4±15.9
9 --147 Chr17:28562862 Bodyheight(cm) 175.4±6.5
10 --149 Chr17:28562864 Bodymassindex(kgm(cid:2)2) 29.4±4.8
11 --155 Chr17:28562870 Waistcircumference(cm) 99.3±12.5
12 --170 Chr17:28562885 Hipscircumference(cm) 104.4±9.3
13 --174 Chr17:28562889 Waist-to-hipratio 0.95±0.06
14 --188 Chr17:28562903 Systolicbloodpressure(mmHg) 129.3±17.5
15 --190 Chr17:28562905 Diastolicbloodpressure(mmHg) 81.4±11.6
16 --195 Chr17:28562910 High-densitylipoprotein(mgdl(cid:2)1) 37.6±10.7
17 --200 Chr17:28562915 Low-densitylipoprotein(mgdl(cid:2)1) 123.3±36.8
18 --207 Chr17:28562922 Totaltriglyceride(mgdl(cid:2)1) 177.5±102.5
19 --209 Chr17:28562924 MeanSLC6A4methylationlevel(%) 11.0±4.2
20 --213 Chr17:28562928
Abbreviation:Chr,chromosome.
11.0%, with the highest and lowest being 16.7% and 3.0%,
respectively. Mean methylation levels in normal, overweight and
Matchedpairanalysesbyconsideringtwinsasmembersofatwinpair:We
obese twins were 10.1%, 11.1% and 11.4%, respectively. Mean
firstcalculatedintra-pairdifferenceinmethylationlevelwithinatwinpair,
methylation level was positively correlated with BMI (r¼0.29;
defined as either the absolute difference or the actual difference in
P¼0.0002), body weight (r¼0.31; Po0.0001) and WC (r¼0.20;
methylation level between two members of a twin pair. The intra-pair
P¼0.009), but not body height (r¼0.05; P¼0.48) and
differences in obese measures and other continuous variables were
WHR (r¼0.06; P¼0.41). DNA methylation levels of the two
similarly calculated. Spearman’s rank correlations between intra-pair
members within a pair were highly correlated at all studied
difference in each of the obese measures, separately, and intra-pair
CpG sites (all P’s o0.0001). The mean DNA methylation level of
differenceinDNAmethylationlevelateachCpGsitewerethencalculated.
the two twins within a pair was also significantly correlated
In addition, we conducted robust regression by regressing the intra-pair
(r¼0.41,P¼0.0001).
difference in obesity parameter (dependent variable) on the intra-pair
Mixed model regression analyses by treating twins as indivi-
difference in DNA methylation level (independent variable) at each CpG
duals demonstrate that methylation levels at most of the
site,adjustingforintra-pairdifferencesinsmoking(pack-year)andalcohol
examined CpG sites were significantly associated with BMI, body
consumption(gperweek)betweentwomembersofatwinpair.
weight and WC, but not body height and WHR, after adjusting
Sensitivityanalyses:Asdescribedbefore,ourstudyincludedarandom
for age, smoking and alcohol consumption. Mean methylation
sampleoftwinswithdepressionorPTSD.Toexaminewhetherdepression
level of the examined CpG sites across the promoter region
(asmeasuredbyBDIscores)orPTSDpotentiallyconfoundstheresults,we
of SLC6A4 was also significantly associated with these three
conducted sensitivity analyses by further adjusting for depressive
obesity parameters (Table 4). On average, a 1% increase in DNA
symptomsorPTSD(n¼35,including9pairsand17singletons).Moreover, methylationwasassociatedwith0.33kgm(cid:2)2increaseinBMI(95%
we performed statistical analyses to examine the potential impact of
CI, 0.02--0.65; P¼0.03), 1.16kg increase in body weight (95%
physicalactivityand dieton ourresultsby furtheradjustingfor physical
CI, 0.16--2.16; P¼0.02) and 0.78cm increase in WC (95% CI,
activity level and total daily energy intake (Kcal per day) in the above
0.05--1.50;P¼0.03).
statisticalmodels.
Results for the association between SLC6A4 methylation and
depressive symptoms using absolute and actual intra-pair
differences are very close. Recognizing that twin 1 and twin 2 in
RESULTS a pair were allocated randomly, here we choose to present the
Table 3 presents the demographic characteristics of the twins results by absolute difference. Intra-pair differences in DNA
included in this analysis. The age of the twins ranged from methylation level were significantly correlated with intra-pair
48 to 61 years with a mean of 55. About 41% and 44% of the differencesinBMIandbodyweight(CpGsites12,13,15,16and
twin participants are obese and overweight, respectively. Except 20;all rX0.24, allP’s p0.04) aswell as WC (CpG sites 1, 3, 7, 10
for body height, all other obese measures (BMI, body weight, and 12; all r X0.22, all P’s p0.05). However, there was no
WC and WHR) are highly correlated (all P’s o0.0001). Twins correlationbetweenintra-pairdifferenceinDNAmethylationand
included in the current analysis were not different from those intra-pairdifference inbody heightandWHR.
notincludedintermsofobesemeasures(e.g.,BMI,bodyweight, Results for regression analyses using intra-pair differences are
WC and WHR) and other covariates (e.g., pack-year, alcohol shown in Table 5. It shows that intra-pair differences in DNA
consumption, physical activity level, total energy intake and BDI methylation level were significantly associated with intra-pair
scores). differencesinBMIandbodyweight(atotalofnineCpGsitesoutof
Among the 20 examined CpG sites, methylation levels at 19 the twenty studiedCpGsites)aswellasWC(atotalofeightCpG
siteswerehighlycorrelated(allP’so0.0001).Methylationlevelof sites out of the twenty examined CpG sites), after adjusting for
one CpG site (position 3) was significantly correlated (r¼0.72; smoking and alcohol consumption. On average, a 1% increase in
Po0.0001) with that of another site (position 4), but not others. thedifferenceinmeanDNAmethylationlevelwasassociatedwith
Mean methylation level across the 20 promoter CpG sites was 0.12kgm(cid:2)2increase in the difference in BMI (95% CI: 0.01--0.22;
InternationalJournalofObesity(2013) 140--145 &2013MacmillanPublishersLimited
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Table4. AssociationbetweenSLC6A4promotermethylationandobesitymeasuresbymixedmodel
CpGsite BMI(kgm(cid:2)2)(95%CI;P) BW(kg)(95%CI;P) WC(cm)(95%CI;P)
Position
1 0.43((cid:2)0.08to0.94;0.10) 1.53((cid:2)0.02to3.08;0.05) 1.01((cid:2)0.01to2.03;0.05)
2 0.12((cid:2)0.39to0.62;0.65) 0.49((cid:2)1.28to2.27;0.59) 0.27((cid:2)0.85to1.38;0.64)
3 0.06((cid:2)0.08to0.19;0.39) 0.08((cid:2)0.37to0.53;0.72) 0.01((cid:2)0.27to0.27;0.97)
4 0.05((cid:2)0.02to0.13;0.16) 0.08((cid:2)0.18to0.34;0.56) 0.06((cid:2)0.11to0.22;0.49)
5 0.29(0.05to0.54;0.01)a 1.03(0.28to1.78;0.007)a 0.69(0.12to0.25;0.01)a
6 0.37(0.07to0.67;0.01)a 1.27(0.31to2.23;0.007)a 0.86(0.17to1.55;0.01)a
7 0.26(0.03to0.55;0.01)a 0.88(0.01to1.34;0.03)a 0.62(0.01to1.28;0.03)
8 0.41(0.03to0.79;0.03)a 1.39(0.18to2.60;0.02)a 0.94(0.05to1.83;0.03)a
9 0.17((cid:2)0.03to0.37;0.09) 0.62((cid:2)0.02to1.25;0.05) 0.43((cid:2)0.03to0.89;0.06)
10 0.31(0.02to0.39;0.03)a 1.06(0.06to2.06;0.03)a 0.70(0.01to1.40;0.04)a
11 0.24(0.01to0.47;0.03)a 0.85(0.13to1.56;0.02)a 0.54(0.01to1.07;0.04)a
12 0.26(0.02to0.45;0.03)a 0.88(0.05to1.35;0.02)a 0.64(0.03to1.29;0.03)a
13 0.19(0.01to0.38;0.04)a 0.68(0.06to1.29;0.03)a 0.45(0.01to0.91;0.02)a
14 0.19(0.01to0.36;0.03)a 0.65(0.09to1.19;0.02)a 0.44(0.03to0.85;0.02)a
15 0.31(0.02to0.60;0.03)a 1.10(0.17to2.03;0.02)a 0.73(0.06to1.39;0.03)a
16 0.22(0.02to0.44;0.02)a 0.81(0.11to1.50;0.02)a 0.53(0.03to1.03;0.03)a
17 0.25((cid:2)0.04to0.54;0.08) 0.94(0.03to1.85;0.04)a 0.65((cid:2)0.01to1.30;0.05)
18 0.26(0.03to0.50;0.03)a 0.91(0.01to1.61;0.03)a 0.63((cid:2)0.02to1.27;0.06)
19 0.41(0.05to0.77;0.02)a 0.15(0.36to2.62;0.01)a 0.97(0.11to1.82;0.02)a
20 0.20(0.02to0.38;0.03)a 0.73(0.14to1.31;0.01)a 0.46(0.04to0.88;0.03)a
Mean 0.33(0.02to0.65;0.03)a 1.16(0.16to2.16;0.02)a 0.78(0.05to1.50;0.03)a
Abbreviations:BMI,bodymassindex;BW,bodyweight;WC,waistcircumference.AllP-valuesadjustedforage,smokingandalcoholconsumption.Those
highlightedinboldindicatethattheassociationsremainedstatisticallysignificantafterfurtheradjustingforphysicalactivity,totaldailyenergyintakeand
depressivesymptoms.aP-valuesremainedsignificantaftercorrectionformultipletestingbyadjusted-FDR(q-value)atthe0.05level.
Table5. Changeinthedifferenceinobesemeasuresper1%increaseinthedifferenceinSLC6A4methylationlevelbetweentwomembersofa
twinpairbyrobustregression
CpGsite DifferenceinBMI(95%CI;P) DifferenceinBW(95%CI;P) DifferenceinWC(95%CI;P)
Position
1 0.16((cid:2)0.02to0.26;0.11) 0.63((cid:2)0.37to1.62;0.22) 0.39((cid:2)0.54to1.32;0.41)
2 0.26((cid:2)0.15to0.67;0.21) 1.01((cid:2)0.14to2.16;0.08) 0.46((cid:2)0.62to1.53;0.41)
3 0.04((cid:2)0.22to0.31;0.75) 0.14((cid:2)0.65to0.93;0.72) 0.37((cid:2)0.34to1.07;0.31)
4 0.03((cid:2)0.11to0.05;0.49) 0.04((cid:2)0.28to0.19;0.72) 0.08((cid:2)0.13to0.29;0.45)
5 0.16(0.05to0.28;0.007)a 0.57(0.24to0.91;0.0009)a 0.37(0.04to0.69;0.02)a
6 0.13((cid:2)0.03to0.28;0.11) 0.41((cid:2)0.05to0.88;0.08) 0.39((cid:2)0.02to0.81;0.06)
7 0.06((cid:2)0.04to0.16;0.26) 0.22((cid:2)0.08to0.53;0.16) 0.27((cid:2)0.02to0.55;0.07)
8 0.16(0.004to0.27;0.05) 0.65(0.10to1.20;0.02)a 0.56(0.04to1.08;0.03)a
9 0.06((cid:2)0.01to0.12;0.11) 0.21((cid:2)0.01to0.43;0.06) 0.20(0.002to0.34;0.05)
10 0.08((cid:2)0.08to0.25;0.32) 0.30((cid:2)0.22to0.82;0.26) 0.23((cid:2)0.23to0.70;0.33)
11 0.07((cid:2)0.03to0.16;0.16) 0.26((cid:2)0.03to0.56;0.07) 0.20((cid:2)0.06to0.46;0.14)
12 0.12(0.01to0.25;0.05) 0.44(0.06to0.82;0.02)a 0.31(0.04to0.63;0.02)a
13 0.09(0.006to0.16;0.03)a 0.31(0.05to0.54;0.02)a 0.20(0.005to0.41;0.05)
14 0.08(0.008to0.15;0.03)a 0.26(0.04to0.48;0.02)a 0.21(0.004to0.42;0.03)a
15 0.14(0.002to0.27;0.04) 0.43(0.002to0.88;0.05) 0.28(0.004to0.71;0.05)
16 0.12(0.01to0.23;0.03)a 0.45(0.10to0.79;0.01)a 0.20((cid:2)0.13to0.52;0.24)
17 0.11((cid:2)0.02to0.26;0.08) 0.39((cid:2)0.002to0.79;0.05) 0.22((cid:2)0.14to0.59;0.24)
18 0.08((cid:2)0.04to0.22;0.19) 0.28((cid:2)0.10to0.68;0.15) 0.26((cid:2)0.08to0.60;0.14)
19 0.20(0.04to0.36;0.01)a 0.63(0.15to1.11;0.009)a 0.60(0.12to1.08;0.01)a
20 0.07(0.01to0.14;0.05) 0.27(0.01to0.53;0.04) 0.20(0.01to0.42;0.05)
Mean 0.12(0.01to0.22;0.03)a 0.41(0.01to0.81;0.03)a 0.35(0.002to0.48;0.04)
Abbreviations:BMI,bodymassindex;BW,bodyweight;WC,waistcircumference.AllP-valuesadjustedfordifferencesinpack-yearandalcoholconsumption.
Thosehighlightedinboldindicatethattheassociationsremainedstatisticallysignificantafterfurtheradjustingforphysicalactivity,totaldailyenergy
intakeanddepressivesymptoms.aP-valuesremainedsignificantaftercorrectionformultipletestingbyadjusted-FDR(q-value)atthe0.05level.
P¼0.03),0.41kgincreaseinthedifferenceinbodyweight(95%CI, DISCUSSION
0.01--0.81; P¼0.03) and 0.35cm increase in the difference in WC We demonstrate that promoter methylation of the serotonin
(95% CI, 0.002--0.48; P¼0.04). However, intra-pair differences in transportergeneinperipheralbloodleukocytesweresignificantly
DNA methylation level were not associated with intra-pair associatedwithobesityparameters.Specifically,SLC6A4promoter
differences in body height and WHR. Further adjustments for hypermethylationwassignificantlyassociatedwithanincreasein
physical activity, total energy intake and depressive symptoms/ obesitymeasures(e.g.,BMI,bodyweightorWC).Theserotonergic
PTSD slightly attenuated the associations but results remained system is involved in the complex behavioral, psychological and
statisticallysignificant. physiological processes in maintaining energy balance.22,23
&2013MacmillanPublishersLimited InternationalJournalofObesity(2013) 140--145
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JZhaoetal
144
Asufficientamount ofserotonin inthecentral nervous systemis and WC are useful and reliable measures for total body fat or
necessaryforthenormalfunctioningofthesebiologicalactivities. abdominal fat, they provide no detailed information on body fat
Animal studies have shown that the loss of brain serotonin was distributionorbodycomposition,36whichmayfunctiondifferently
associated with overeating and increased body weight,5 and in leading to obesity or its related disorders.37 For example,
serotonin transporter deficiency increases abdominal fat.24 More- subjects with excess visceral fat are reported to be at higher risk
over,serotoninagonistsorserotoninreuptakeinhibitorsdecreases for diabetes, hypertension and insulin resistance than those with
food intake and induces weight loss in rodents.25 Studies in an excess in subcutaneous fat.34 It is therefore necessary to
humans have also demonstrated that abnormal hypothalamic conduct more precise measurements using imaging methods,
serotonergic neurotransmission is associated with eating dis- such as magnetic resonance imaging or computed tomographic
turbances.6,26 In this context, our finding about the association scanning, to distinguish visceral and subcutaneous adipose
betweenhigherDNAmethylationlevelintheserotonintranspor- tissues38 in future studies. Fifth, our sample included twins with
ter gene promoter region and an increased risk for obesity is in oversampling of either major depression or PTSD, which may
agreementwithprevious literatures. influence the risk of overweight or obesity.39,40 However, the
DNAmethylationusuallyinhibitsorsilencesgeneexpression.27 observed association between aberrant DNA methylation and
Hypermethylation in the serotonin transporter gene promoter obese measures is unlikely to be confounded by these two
regioncould lead to reduced orsilenced gene expression, which psychiatric conditions because the observed associations per-
inturnleadstolow-serotoninuptakeandadeficiencyofserotonin sistedafterfurtheradjustmentsfordepressivesymptomsorPTSD.
activity. Indeed, evidence of serotonin deficiency has been Sixth, though we were able to control many of the potential
reported in obese individuals.28 Alternatively, given the critical confounders,wecannotentirelyexcludethepossibilityofresidual
role of serotonergic system in stress responses, it is also possible confoundingbyunknownorunmeasuredriskfactors.Finally,our
that hypermethylation-induced serotonin deficiency could result twin sample was derived from a middle-aged sample of male
in abnormal responses to stress, a mechanism known to be military veterans; therefore, the generalizability to females and
involvedinthe pathogenesisof obesity.29 otheryoungerorolderpopulationsisnotknown.Lastly,because
DNA methylation is site specific. We therefore presented the our study was cross-sectional, we are unable to determine
associationofmethylationvariationateachindividualCpGsiteas whether the observed epigenomic signatures are epiphenomena
wellastheirmeanwithobesitymeasures.AsshowninTable4,it orpartofthecausalpathwaysleadingtoanincreasedprevalence
appears that there exist three epigenetic domains ((cid:2)133 to ofobesity.
(cid:2)141bp, (cid:2)149 to (cid:2)195bp and (cid:2)207 to (cid:2)213bp), each of which Nonetheless, this is the first study to demonstrate the
exhibitsincreasedmethylationvariationthatmightbeassociated association of aberrant DNA methylation in the promoter region
with obesity. These differentially methylated regions are also oftheserotonintransporter genewithobesityinawell-matched
observedin cancers.30 MZtwinsample.Previousstudieshaveshownthatinterindividual
BMI is a convenient surrogate measure of total fat mass.31 epigeneticprocessesareundergeneticcontrol.41,42Therefore,the
It offers a straightforward, inexpensive and useful measure for study of the causal impact of epigenotype on disease outcome
overweight and obesity and is typically used as an indicator requires careful matching on or control for genetic background
for general obesity. Here we found that aberrant promoter andotherpotentialconfoundingvariables.MZtwinpairsprovide
methylation of the serotonin transporter gene was significantly a natural experiment to investigate the effects of epigenetics on
associated with BMI and body weight. However, SLC6A4 disease outcome, because MZ twins match exactly on genetic
methylation variation was not associated with body height, background, age and sex, thus eliminating the effects of these
suggesting that the observed association between SLC6A4 importantconfounders.43,44Inaddition,identicaltwinsingeneral
methylation and BMI is primarily driven by body weight instead share raising environment, providing further control for con-
ofbodyheight. foundingbyearlylifeexperience,whichhasalong-lastingimpact
WCandWHRarecommonlyusedanthropometricmeasuresfor ontheepigenetic plasticity ofhuman genome.45
adiposity. Although WC has been consistently correlated with In summary, this study provides the first evidence that
the proportion of abdominal fat,32 the relationship between promoter hypermethylation of the serotonin transporter gene is
WHRandabdominaladipositywasmixedinpreviousstudies.33--35 significantly associated with an increased prevalence of obesity.
In this analysis, we identified significant correlation between Because epigenetic modifications are possibly reversible, our
SLC6A4 promoter methylation variation and WC, but no correla- results, if confirmed, would suggest the potential for lifestyle or
tion was observed between DNA methylation and WHR. This therapeutic interventions for obesity, overweight and a wide
could be because of either a relatively large waist or a small hip rangeofrelated disorders.
girth, or vice versa, which leads to an uninterpretable or
misleadingWHRinrelationtotheamountofaperson’sabdominal
fat. Our findings are in line with previous studies, indicating that CONFLICTOFINTEREST
WHR is less useful in reflecting the accumulation of abdominal Theauthorsdeclarenoconflictofinterest.
adiposity. WC may be a better indicator for abdominal adiposity
thanWHR.
Our study has a few limitations. First, because of practical ACKNOWLEDGEMENTS
difficultiesinobtainingtissuesfromlivingindividuals,methylation
ThisstudywassupportedbyGrant0730100NfromtheAmericanHeartAssociation,
levelsweretestedinperipheralbloodleukocytes,butnotdirectly GrantsR21HL092363,K01AG034259,K24HL077506,R01HL68630andR01AG026255
from the primary affected organs in obesity, for example, fromthe National InstitutesofHealth. The United States Departmentof Veterans
adipocytes. Therefore, our results may not provide a direct index Affairshasprovidedfinancialsupportforthedevelopmentandmaintenanceofthe
ofDNAmethylationinthesystemofadiposemetabolism.Second, VietnamEraTwin(VET)Registry.Numerousorganizationshaveprovidedinvaluable
ourepigeneticdatawerecollectedfromDNAderivedfromwhole assistanceintheconductofthisstudy,including:DepartmentofDefense;National
blood leukocytes, which is a mixture of many cell types; as such, Personnel Records Center, National Archives and Records Administration; the
Internal Revenue Service; National Institutes of Health; National Opinion Research
we were unable to assess blood cell-specific differences in
Center;NationalResearchCouncil,NationalAcademyofSciencesandtheInstitute
methylation changes. Third, DNA methylation influences disease
forSurveyResearch,TempleUniversity.Mostimportantly,wegratefullyacknowledge
riskthroughregulatinggeneexpression,whichwasunabletobe
the continuedcooperation and participation of the members of the VET Registry
evaluated in this study because of the lack of gene expression and their families. Without their contribution this research would not have been
datafromfreshleukocytesoradiposetissues.Fourth,thoughBMI possible.
InternationalJournalofObesity(2013) 140--145 &2013MacmillanPublishersLimited
Methylationofserotonintransportergeneassociatedwithobesity
JZhaoetal
145
AUTHORCONTRIBUTIONS
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to discussion and reviewed/edited the manuscript. VV designed the study, 137--145.
contributedtodiscussionandreviewed/editedthemanuscript. 25 TallettAJ,BlundellJE,RodgersRJ.Sibutramine-inducedanorexia:potent,dose-
dependentandbehaviourally-selectiveprofileinmalerats.BehavBrainRes2009;
198:359--365.
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