DNA methylation and body mass index from birth to adolescence: meta-analyses of epigenome-wide association studies.

RESEARCH Open Access DNA me hyla ion and body mass index om bi h o adolescence: me a-analyses o epigenome-wide associa ion s udies Flo ianne O. L. Vehmeije 1,2,3† , Leanne K. Küpe s 4,5,6† , Gemma C. Sha p 4 , Lucas A. Salas 7,8,9,10 , Saman ha Len 11 , De eje D. Jima 12,13 , Gwen Tindula 14 , Sa ah Reese 15 , Cancan Qi 16,17 , Olena G uzie a 18,19 , Ch is ian Page 20,21 , Faisal I. Rezwan 22,23 , Philip E. Mel on 24,25 , Ellen Noh 26,27 , Geò gia Esca amís 10,28,29 , Pe e Rzehak 30 , Anni Heiskala 31 , Tong Gong 32 , Samuli T. Tuominen 33 , Lu Gao 34 , Jason P. Ross 35 , Anne P. S a ling 36,37 , John W. Holloway 23,38 , Paul Youse i 4 , Gunn Ma i Aas ang 39 , Law ence J. Beilin 40 , Anna Be gs öm 18,19 , Elisabe h Binde 41,42 , Leda Cha zi 43 , E a Co peleijn 6 , Da ina Czama a 41 , B enda Eskenazi 44 , Susan Ewa 45 , Na alia Fe e 46 , Vei G o e 30 , Da iusz G usz eld 47 , Si i E. Håbe g 20 , Ca h ine Hoyo 13,48 , Ka en Huen 14 , Robe Ka lsson 32 , Inge Kull 49,50 , Jean-Paul Langhend ies 51 , Johanna Lepeule 52 , Ma ia C. Magnus 4,5,20 , Rachel L. Magui e 48,53 , Pe e L. Molloy 35 , Clai e Monne eau 1,3 , T e o A. Mo i 40 , Emily Oken 54 , Ka i Räikkönen 33 , She yl Ri as-Shiman 54 , Ca los Ruiz-A enas 8,9,10 , Syl ain Sebe 31 , Vilhelmina Ullema 32 , El i a Ve duci 55 , Judi h M. Vonk 6,17 , Cheng-jian Xu 16,17,56,57 , I ana V. Yang 36,58,59 , Hongmei Zhang 60 , Weiming Zhang 61 , Wil ied Ka maus 60 , Dana Dabelea 36,37,62 , Be e ly S. Muhlhausle 63 , Ca ie V. B e on 34 , Ja i Lah i 33,64 , Ca a ina Almq is 32,65 , Ma jo-Rii a Ja elin 31,66,67,68 , Be hold Kole zko 30 , Ma ine V ijheid 8,9,10 , Tho kild I. A. Sø ensen 4,69 , Rae-Chi Huang 70 , Syed Hasan A shad 38,71 , Wenche Nys ad 72 , E ik Melén 49,50 , Ge a d H. Koppelman 16,17 , S ephanie J. London 15 , Nina Holland 14 , Ma iona Bus aman e 8,9,10 , Susan K. Mu phy 53 , Ma ie-F ance Hi e 54,73,74 , And ea Bacca elli 75† , Ca oline L. Rel on 4† , Ha old Sniede 6† , Vincen W. V. Jaddoe 1,2,3† and Janine F. Felix 1,2*† Abs ac Backg ound: DNA me hyla ion has been shown o be associa ed wi h adiposi y in adul hood. Howe e , whe he simila DNA me hyla ion pa e ns a e associa ed wi h childhood and adolescen body mass index (BMI) is la gely unknown. Mo e insigh in o his ela ionship a younge ages may ha e implica ions o u u e p e en ion o obesi y and i s ela ed ai s. (Con inued on nex page) © The Au ho (s). 2020 Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle's C ea i e Commons licence, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle's C ea i e Commons licence and you in ended use is no pe mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his licence, isi h p://c ea i ecommons.o g/licenses/by/4.0/. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed in a c edi line o he da a. * Co espondence: [email p o ec ed] † Flo ianne O.L. Vehmeije and Leanne K. Küpe s a e au ho s who con ibu ed equally o he wo k. † And ea Bacca elli, Ca oline L. Rel on, Ha old Sniede , Vincen W.V. Jaddoe, and Janine F. Felix a e au ho s join ly supe ised he wo k. 1 The Gene a ion R S udy G oup, E asmus MC, Uni e si y Medical Cen e Ro e dam, Room Na-2918, E asmus MC, PO Box 2040, 3000 CA Ro e dam, he Ne he lands 2 Depa men o Pedia ics, E asmus MC, Uni e si y Medical Cen e Ro e dam, Ro e dam, he Ne he lands Full lis o au ho in o ma ion is a ailable a he end o he a icle Vehmeije e al. Genome Medicine (2020) 12:105 h ps://doi.o g/10.1186/s13073-020-00810-w (Con inued om p e ious page) Me hods: We examined whe he DNA me hyla ion in co d blood and whole blood in childhood and adolescence was associa ed wi h BMI in he age ange om 2 o 18 yea s using bo h c oss-sec ional and longi udinal models. We pe o med me a-analyses o epigenome-wide associa ion s udies including up o 4133 child en om 23 s udies. We examined he o e lap o indings epo ed in p e ious s udies in child en and adul s wi h hose in ou analyses and calcula ed en ichmen . Resul s: DNA me hyla ion a h ee CpGs (cg05937453, cg25212453, and cg10040131), each in a di e en age ange, was associa ed wi h BMI a Bon e oni signi icance, P<1.06×10 −7 , wi h a 0.96 s anda d de ia ion sco e (SDS) (s anda d e o (SE) 0.17), 0.32 SDS (SE 0.06), and 0.32 BMI SDS (SE 0.06) highe BMI pe 10% inc ease in me hyla ion, espec i ely. DNA me hyla ion a nine addi ional CpGs in he c oss-sec ional childhood model was associa ed wi h BMI a alse disco e y a e signi icance. The s eng h o he associa ions o DNA me hyla ion a he 187 CpGs p e iously iden i ied o be associa ed wi h adul BMI, inc eased wi h ad ancing age ac oss childhood and adolescence in ou analyses. In addi ion, co ela ion coe icien s be ween e ec es ima es o hose CpGs in adul s and in child en and adolescen s also inc eased. Among he op indings o each age ange, we obse ed inc easing en ichmen o he CpGs ha we e p e iously iden i ied in adul s (bi h P en ichmen = 1; childhood P en ichmen =2.00×10 −4 ;adolescenceP en ichmen =2.10×10 −7 ). Conclusions: The e we e only minimal associa ions o DNA me hyla ion wi h childhood and adolescen BMI. Wi h he ad ancing age o he pa icipan s ac oss childhood and adolescence, we obse ed inc easing o e lap wi h al e ed DNA me hyla ion loci epo ed in associa ion wi h adul BMI. These indings may be compa ible wi h he hypo hesis ha DNA me hyla ion di e ences a e mos ly a consequence a he han a cause o obesi y. Keywo ds: Body mass index, Childhood obesi y, DNA me hyla ion, Epigene ics Backg ound An accumula ing body o e idence sugges s ha exposu es in ea ly li e a e associa ed wi h childhood BMI [1]. I is hy- po hesized ha changes in DNA me hyla ion may unde lie he associa ions o ea ly-li e exposu es wi h childhood adi- posi y [2–4]. Thus a , mos o he e idence ega ding DNA me hyla ion and adiposi y s ems om adul s udies [5–9]. The la ges epigenome-wide associa ion s udy (EWAS) in adul s iden i ied c oss-sec ional associa ions be ween DNA me hyla ion a 187 loci and BMI in o e 10, 000 pa icipan s [5]. P e ious s udies o he associa ions be ween epigenome-wide DNA me hyla ion and childhood and adolescen adiposi y we e small and inconclusi e [10– 16]. Candida e gene s udies in childhood iden i ied associa- ions o DNA me hyla ion in co d and childhood blood wi h measu es o adiposi y [17–24]. Epigenome-wide asso- cia ion s udies in child en and adolescen s, wi h sample sizes anging om 40 o 700 indi iduals, iden i ied a limi ed numbe o cy osine-phospha e-guanine si es (CpGs) associ- a ed wi h BMI [11–13,15,25]. Al hough indings o some s udies sugges ha di e ences in DNA me hyla ion may p ecede he de elopmen o adiposi y, ecen s udies in adul s, using me hods such as Mendelian andomiza ion, posi ha al e a ions in DNA me hyla ion a e p edomin- an ly he consequence o adiposi y, a he han he cause [4,5,9,26,27]. The di ec ion o any causal pa hway has no been obus ly app aised in child en. Ob aining mo e knowledge on he associa ion be ween DNA me hyla ion and adiposi y al eady in childhood may ha e implica ions o u u e p e en ion o obesi y and i s ela ed ai s. We pe o med a me a-analysis o epigenome-wide asso- cia ion s udies o BMI in up o 4133 pa icipan s om 23 s udies. We assessed associa ions o DNA me hyla ion in co d blood, in childhood and adolescence wi h BMI in child en aged 2–18 yea s. We also compa ed he e ec es- ima es and examined whe he he e was en ichmen in ou da a o CpGs p e iously iden i ied o hei associ- a ion wi h adolescen and adul adiposi y. Me hods Pa icipan s We me a-analyzed epigenome-wide associa ion s udies o co d o whole blood me hyla ion wi h childhood o adolescen body mass index (BMI). We used da a om up o 4133 pa icipan s om 23 s udies collabo a ing in he P egnancy And Childhood Epigene ics (PACE) Con- so ium, Li eCycle P ojec , and Nu iP og am P ojec (Addi ional ile 1: Table S1A-D and Addi ional ile 2: Supplemen a y Me hods) [28,29]: ALSPAC, BAMSE, CHAMACOS, CHOP S udy, CHS, DOMInO T ial, GECKO D en he coho , Gene a ion R S udy, GOYA s udy, Heal hy S a S udy, HELIX, INMA, IOW F1, IOW F2, MoBa1, MoBa2, NEST, NFBC 1986, PIAMA s udy, PREDO s udy, P ojec Vi a, Raine, and STOPPA ( ull names in Supplemen a y Me hods). Coho pa ici- pan s we e mainly o Eu opean ances y, bu he e we e also coho s wi h (pa ly) non-Eu opean e hnici ies (A - ican, Hispanic, and Abo iginals). Mos coho s a e p o- spec i e bi h coho s. We excluded mul iple bi hs, siblings (maximum one child pe amily), physician- diagnosed synd omic obesi y cases, and any ype o ma- e nal diabe es (including ges a ional diabe es). In o med consen was ob ained o all pa icipan s, and all s udies Vehmeije e al. Genome Medicine (2020) 12:105 Page 2 o 15 ecei ed app o al om hei local e hics commi ees (see Addi ional ile 2: Supplemen a y Me hods). DNA me hyla ion DNA me hyla ion was measu ed in co d blood and whole blood samples, in child en and adolescen s using he Illumina In inium® HumanMe hyla ion450 BeadChip assay (Illumina, San Diego, CA, USA) [30]. Each coho independen ly conduc ed hei p e e ed quali y con ol and no maliza ion me hod, see Addi ional ile 2: Supple- men a y Me hods o de ails. Un ans o med no malized be a alues o indi idual CpG si es we e used as expos- u e a iables. I mul iple measu emen s o DNA me hy- la ion and BMI we e a ailable wi hin an age ange, we used he oldes age wi hin ha ange o which BMI and DNA me hyla ion we e a ailable a he same ime poin . Ou lying me hyla ion be a alues we e excluded using he ollowing me hod: alues < (25 h pe cen ile −3*in e - qua ile ange (3IQR)) and alues > (75 h pe cen ile + 3IQR) we e emo ed [31]. DNA me hyla ion is exp essed as he p opo ion o alleles a which he DNA was me hyla ed a a speci ic si e and hence akes alues om ze o o one. Childhood BMI Heigh and weigh we e measu ed in each s udy using es ablished p o ocols as desc ibed in de ail in he Add- i ional ile 2: Supplemen a y Me hods. The p ima y ou - come was BMI, calcula ed as weigh /heigh 2 in kg/m 2 ,ona con inuous scale measu ed in h ee age anges: 2–5 yea s (ea ly childhood), 5–10 yea s (la e childhood), and 14–18 yea s (adolescence). I mul iple BMI and DNA me hyla ion measu emen s we e a ailable, we used he measu emen s a heoldes agewi hin heage ange o whichBMIand DNA me hyla ion we e a ailable a he same ime poin . BMI alues we e hen ans o med in o sex- and age- adjus ed s anda d de ia ion sco es (SDS) using LMSG ow h [32–34]. The In e na ional Obesi y Task Fo ce (IOTF) s anda d was used o de ine cu o s o BMI o unde - weigh , no mal weigh , o e weigh , and obesi y in child en, c ea ed wi h he B i ish 1990 g ow h e e ence and in o - ma ion o pa icipan s on BMI, sex, and age [35,36]. In sec- onda y analyses, we used a bina y ou come a iable wi h no mal-weigh child en as con ols and o e weigh o obese child en as cases. Unde weigh child en we e ex- cluded om hese seconda y analyses. I a s udy had ≤10 pa icipan s in one o he (case o con ol) g oups, his s udy was excluded om he seconda y analyses. Co a ia es Co a ia es included in all models we e ma e nal co a i- a es: ma e nal age, ma e nal educa ional le el (coho de - ini ion), ma e nal smoking s a us du ing p egnancy (any smoking e sus no smoking), ma e nal p e-p egnancy o ea ly p egnancy BMI and pa i y (mul ipa ous e sus nul- lipa ous), and ges a ional age a bi h. Fo de ails on coho -speci ic collec ion me hods, see Addi ional ile 2: Supplemen a y Me hods. We es ima ed whi e blood cell p opo ions (B cells, CD8+ T cells, CD4+ T cells, g anulo- cy es, NK cells, and monocy es) using he e e ence-based Houseman me hod wi h he Reinius e e ence in he min i package in R [37–40]. A sensi i i y analysis using he co d blood-speci ic Bakulski e e ence was pe o med in he Gene a ion R and ALSPAC s udies [41]. Ba ch e ec s we e adjus ed o using coho -speci ic me hods, see Add- i ional ile 2: Supplemen a y Me hods. Addi ional co a i- a es added in he c oss-sec ional childhood analyses we e bi h weigh and b eas eeding. The adolescen analyses we e addi ionally adjus ed o adolescen age, sex, own smoking s a us, and pube y s a us. Pube y s a us was ca ego ized in o ea ly pube y (i bo h b eas and pubic hai Tanne s ages (o compa able classi ica ion) we e 1, 2, o 3 and i gi ls we e p e-mena cheal o boys did no ha e oice change ye ) and la e pube y (i ei he b eas o pubic hai Tanne s ages (o compa able classi ica ion) we e 4 o 5 o i gi ls we e pos -mena cheal o boys had had hei oice change) [42–44]. Fu he de ails a e p o- ided in he s udy-speci ic Addi ional ile 2: Supplemen- a y Me hods. S udy-speci ic analyses Associa ions o DNA me hyla ion wi h childhood o adolescen BMI we e pe o med in indi idual s udies on pa icipan s wi h comple e da a on all co a ia es. In s udies wi h mo e han one e hnic g oup, each g oup was analyzed sepa a ely. We used obus linea eg es- sion models o he con inuous ou come o BMI-SDS and gene alized linea eg ession models o he case/ con ol analyses o o e weigh and obesi y e sus no mal weigh , acco ding o a p e-speci ied analysis plan. EWAS analyses we e conduc ed using DNA me hyla ion a h ee ime poin s: bi h, childhood and adolescence, and BMI da a collec ed a h ee ime poin s: ea ly childhood (2–5 yea s), la e childhood (5–10 yea s), and adolescence (12-18y) (Table 1). We ca ego ized he childhood pe iod in o ea ly and la e childhood o o e come any age- speci ic e ec s and he po en ial in luence o he adipos- i y ebound on he esul s [45]. Depending on da a a ail- abili y, coho s pa icipa ed in one o mo e o ou analyses: (analysis A) longi udinal associa ions o co d blood DNA me hyla ion wi h ea ly childhood BMI (2– 5 yea s; 3295 child en om 13 s udies), (analysis B) lon- gi udinal associa ions o co d blood DNA me hyla ion wi h la e childhood BMI (5–10 yea s; 4133 child en om 12 s udies), (analysis C) c oss-sec ional associa ions o childhood blood DNA me hyla ion wi h childhood BMI (2–10 yea s; 3371 child en om 11 s udies), and (ana- lysis D) c oss-sec ional associa ions o adolescen blood Vehmeije e al. Genome Medicine (2020) 12:105 Page 3 o 15 DNA me hyla ion wi h adolescen BMI (14–18 yea s; 2842 adolescen s om 7 s udies) (Table 1). Pa icipa ing s udies pe analysis a e shown in Addi ional ile 1: Table S1A-D. Co d blood analyses we e adjus ed o ma e nal age, educa ional le el, smoking s a us, p e-p egnancy o ea ly p egnancy BMI, pa i y, ges a ional age, ba ch, and es i- ma ed cell ype p opo ions. The c oss-sec ional analysis in childhood was addi ionally adjus ed o child co a i- a es bi h weigh and b eas eeding; in con as , he c oss-sec ional analysis in adolescence was adjus ed o he same co a ia es as analysis C plus adolescen sex, age, and smoking and pube y s a us. Me a-analyses A e pe o ming quali y con ol on all s udies, we com- bined esul s in a ixed-e ec s in e se a iance-weigh ed me a-analysis using METAL [46,47]. All ollow-up ana- lyses we e conduc ed in R [39]. The me a-analyses we e done independen ly by wo s udy g oups, and he esul s we e compa ed. A e exclusion o p obes ha we e measu ed in only one s udy, ha mapped o X and Y ch omosomes and p obes ha co-hyb idized o al e na e sequences (c oss- eac i e p obes), we included 429,959 p obes o analysis A, 429,959 p obes o analysis B, 429, 957 p obes o analysis C, and 428,967 p obes o ana- lysis D [48,49]. In he esul iles o he main me a- analyses, we lagged p obes ha map o DNA con aining a single nucleo ide polymo phism (SNP), o epe i i e sequence elemen s, o o DNA ha bo ing an INDEL (Addi ional ile 3: Table S2A-D) [48,49]. We co ec ed o mul iple es ing using bo h he Bon e oni co ec- ion, which gi es a signi icance h eshold o P< 1.16 × 10 −7 (0.05/429,959), and he less s ingen alse disco e y a e (FDR) h eshold using he me hod by Benjamini and Hochbe g [50]. EWAS esul s we e summa ized as mean (and s anda d e o ) di e ences in BMI-SDS pe 10% inc ease in me hyla ion o each CpG. We c ea ed olcano plo s o isualize magni ude and di ec ion o e - ec ( educed o inc eased me hyla ion) along wi h he le el o s a is ical signi icance. We calcula ed he I 2 s a - is ic o explo e he e ogenei y ac oss s udies. The I 2 es i- ma es he p opo ion o a ia ion in he me a-analysis esul s o each CpG si e ha is due o be ween-s udy di e ences a he han andom/sampling a ia ion. He - e ogenei y was de ined as an I 2 alue o > 50 and shown g aphically in o es plo s. We pe o med lea e-one-ou analyses, in which we e an he main me a-analysis e- pea edly wi h one o he 23 s udies emo ed each ime, o explo e i any s udy in luenced indi idual indings. We enhanced he anno a ion p o ided by Illumina using he UCSC Genome B owse . All o he anno a ions use he human Feb ua y 2009 (GRCh37/hg19) assembly. We upda ed he gene names manually in all esul iles using HUGO gene nomencla u e, and in case hey we e no ound he e, we used he NCBI gene websi e on No em- be 5, 2019 [51–53]. To explo e he associa ions o he ex eme uppe alues o he BMI dis ibu ion, we pe o med case/con- ol analyses (o e weigh and obesi y e sus no mal weigh ). Unde weigh child en we e excluded om hese analyses, leading o sample sizes o N= 491 cases and 2540 con ols (analysis A), N= 707 cases and 3217 con- ols (analysis B), N= 644 cases and 2567 con ols (ana- lysis C), and N= 507 cases and 2188 con ols (analysis D) (Addi ional ile 4: Table S3A-D). To examine whe he any o he Bon e oni-signi ican o FDR-signi ican CpGs in ou analyses we e close o Table 1 O e iew o main analyses, seconda y analyses, and sensi i i y analyses Analysis Main analyses Seconda y analyses: bina y model (N), cases = o e weigh and obesi y, con ols = no mal weigh Sensi i i y analyses DNA me hyla ion in he blood BMI SD sco es NEu opeans only (N) Wi hou s udies > 30% o e weigh and obesi y (N) Co d blood analyses A Bi h (co d blood) Ea ly childhood (2–5 yea s) 3295 Cases = 491 Con ols = 2540 2902 2989 B Bi h (co d blood) La e childhood (5–10 yea s) 4133 Cases = 707 Con ols = 3217 3657 3489 C oss-sec ional analyses C Childhood (whole blood) Childhood (2–10 yea s) 3371 Cases = 644 Con ols = 2567 3026 3171 D Adolescence (whole blood) Adolescence (12–18 yea s) 2842 Cases = 507 Con ols = 2188 NA NA Analyses A and B we e adjus ed o ma e nal age, educa ional le el, smoking s a us, p e-p egnancy o ea ly p egnancy BMI, pa i y, ges a ional age a bi h, ba ch, and es ima ed cell ype p opo ions Analyses C was adjus ed o ma e nal age, educa ional le el, smoking s a us, p e-p egnancy o ea ly p egnancy BMI, pa i y, ges a ional age a bi h, ba ch, es ima ed cell ype p opo ions, bi h weigh , and b eas eeding Analyses D was adjus ed o ma e nal age, educa ional le el, smoking s a us, p e-p egnancy o ea ly p egnancy BMI, pa i y, ges a ional age a bi h, ba ch, es ima ed cell ype p opo ions, bi h weigh , b eas eeding, adolescen sex, age smoking and pube y s a us Vehmeije e al. Genome Medicine (2020) 12:105 Page 4 o 15 BMI SNPs, we assessed i hese CpGs we e loca ed wi hin a 4-Mb window (± 2 Mb) su ounding he 15 gen- e ic loci associa ed wi h childhood body mass index [2, 54]. Fo he FDR-signi ican CpGs ha we e lagged be- cause hey we e po en ially in luenced by a SNP, we isually inspec ed densi y plo s in he Gene a ion R S udy o see whe he hese de ia ed om unimodali y (Addi ional ile 5: Supplemen a y In o ma ion, Fig. S6). To explo e DNA me hyla ion pa e ns in he egions a ound he signi ican CpGs, we assessed he associa- ions o all CpGs loca ed wi hin a 10-kb window (± 5 kb) su ounding hese CpGs wi h BMI in he ele an models (Addi ional ile 6: Table S4). Sensi i i y analyses To explo e whe he e hnic he e ogenei y may ha e a - ec ed ou esul s, we epea ed he me a-analyses includ- ing s udies wi h pa icipan s o Eu opean ances y only (N= 2902 (excluding h ee s udies o analysis A), N= 3657 (excluding h ee s udies o analysis B), N=3026 (excluding wo s udies o analysis C)), he la ges e hnic subg oup (Addi ional ile 7: Table S5A-C). E hnici y was de ined using sel - epo ed ques ionnai es unless speci- ied o he wise in he s udy-speci ic Supplemen a y Me hods (Addi ional ile 2). We pe o med addi ional analyses excluding s udies wi h a high pe cen age (> 30% (pe cen age calcula ed a e exclusion o unde weigh child en)) o child en wi h o e weigh and obesi y o ex- plo e whe he any associa ions ound may be d i en by mo e ex eme alues o BMI (included N= 2989 (exclud- ing wo s udies o analysis A), N= 3489 (excluding ou s udies o analysis B), N= 3171 (excluding one s udy o analysis C) (Addi ional ile 8: Table S6A-C). We also pe o med a hi d, conse a i e, sensi i i y analysis in all age g oups, excluding coho s o non-Eu opeans, s udies wi h a high pe cen age (> 30%) o child en wi h o e - weigh o obesi y, and s udies in which he sample was selec ed on o en iched o any pa icula exposu e o ou come (Addi ional ile 9: Table S7A-D). Compa ison wi h p e ious indings We explo ed whe he CpG si es associa ed wi h child- hood, adolescen , o adul BMI in p e ious s udies we e associa ed wi h BMI in ou da a. Fo p e ious candida e gene s udies and smalle EWASs (N< 1000), we pe - o med a look-up o he hi s, using a Bon e oni-adjus ed P alue cu o pe s udy, so o each s udy, he cu o was 0.05/(NCpGs om ha s udy) (Addi ional ile 10:Table S8) [7,15,17,18,20,21,23,24,55]. I he speci ic CpGs om a s udy we e no a ailable in ou da ase , we looked up all CpGs anno a ed o he ele an genes [17,24]. To es ablish whe he he CpG si es associa ed wi h BMI in p e iously epo ed la ge EWASs (N≥1000) we e o e - ep esen ed among ou CpGs wi h he smalles P alues, we examined he absolu e o e lap o he op CpGs om li e a u e wi h he op CpGs in ou analyses [5,6,9,25, 26,56–59]. The la e we e de ined using wo cu o s: a s ingen cu o o P alue < 1 × 10 −5 and a mo e lenien one o P alue < 0.05. (Addi ional ile 11: Table S9). We used a hype geome ic es o calcula e en ichmen wi h he phype unc ion in he R S a s package in R. We examined he 187 CpGs iden i ied in he la ges adul s udy (N= 10,261) o da e in mo e de ail in ou e- sul s [5]. We es ed whe he he en iched CpGs signi i- can ly o e lapped be ween ou analyses using chi-squa e es s. We used Pea son’s co ela ion coe icien s o examine he co ela ions be ween he e ec es ima es o hese 187 CpGs in adul s and hose in ou analyses [5]. Using Fishe ’s exac es , we calcula ed whe he he co - ela ion coe icien s a he a ious ages we e signi ican ly di e en om each o he . Func ional analyses We explo ed he po en ial unc ional in e p e a ion o he mos signi ican ly associa ed CpGs (P alue < 1 × 10 −4 ) in all models using Gene On ology (GO) and Kyo o Encyclopedia o Genes and Genomes (KEGG) en- ichmen analyses. We used he missMe hyl package, which enabled us o co ec o he numbe o p obes pe gene on he 450K a ay, based on he May 5, 2020, e sion o he GO and he Oc obe 23, 2019, e sion o he KEGG sou ce da abases [60]. To il e ou he la ge, gene al pa hways, we se he numbe o genes o each gene se be ween 5 and 2000, espec i ely. We epo nominal P alues < 0.05 and FDR o en ichmen (Add- i ional ile 12, Table S10). Resul s Pa icipan s We included 2842 o 4133 pa icipan s om 23 independ- en coho s om he P egnancy And Childhood Epigene - ics (PACE) Conso ium [28]. We assessed associa ions o DNA me hyla ion in co d blood wi h BMI in ea ly child- hood (2–5yea s)(N= 3295, analysis A), DNA me hyla ion in co d blood wi h BMI in la e childhood (5–10 yea s) (N= 4133, analysis B), DNA me hyla ion in childhood wi h BMI in childhood (2–10 yea s) (N= 3371, analysis C), and DNA me hyla ion in adolescence wi h BMI in adolescence (12–18 yea s) (N= 2842, analysis D). De ails o pa icipan s and s udies used in he di e en analyses a e p esen ed in Table 1, Addi ional ile 1: Table S1A-D and Addi ional ile 2: Supplemen a y Me hods. Me a-analyses The main, seconda y, and sensi i i y analyses a e ou - lined in Table 1. Genomic in la ion ac o s (lambdas) o he main me a-analyses anged be ween 0.97 and 1.27 (Addi ional ile 5: Supplemen a y in o ma ion, Fig. 1a-d). Vehmeije e al. Genome Medicine (2020) 12:105 Page 5 o 15 Genomic in la ion ac o s (lambdas) o all coho -speci ic analyses a e shown in Addi ional ile 1: Table S1A-D. The main esul s a e shown in Table 2and Fig. 1. We did no iden i y associa ions a genome-wide signi icance o DNA me hyla ion in co d blood wi h BMI in ea ly childhood (analysis A, Fig. 1a, and Addi ional ile 3: Table S2A). DNA me hyla ion a one CpG, cg05937453 (SFRP5), in co d blood was signi ican ly associa ed wi h la e- childhood BMI (analysis B, Fig. 1b, and Addi ional ile 3: Table S2B). Fo each 10% inc ease in DNA me hyla ion a cg05937453 in co d blood, la e-childhood BMI inc eased 0.96 SD (s anda d e o (SE) 0.17). Co d blood DNA me hyla ion a his CpG was nominally signi ican ly asso- cia ed wi h BMI in ea ly-childhood (P alue = 0.004), bu DNA me hyla ion in childhood and adolescence was no associa ed wi h BMI in he c oss-sec ional analyses (Add- i ional ile 13: Table S11). In he c oss-sec ional analysis (analysis C), childhood DNA me hyla ion a cg25212453 (SLC43A2) was associa ed wi h childhood BMI a e Bon e oni co ec ion. A 10% in- c ease in DNA me hyla ion a cg25212453 was associa ed wi h a 0.32 SD (SE 0.06) inc ease in childhood BMI (Fig. 1c and Addi ional ile 3:TableS2C).DNAme hyla iona his CpG a bi h and in adolescence was no associa ed wi h BMI (Addi ional ile 13: Table S11). DNA me hyla ion in childhood a nine addi ional CpGs in o nea o he genes was associa ed wi h childhood BMI using FDR P alue < 0.05 (Fig. 1c and Addi ional ile 3:TableS2C).DNAme hy- la ion in adolescence a cg10040131 (SFXN5) was associ- a ed wi h adolescen BMI a e Bon e oni co ec ion (analysis D, Fig. 1d and Addi ional ile 3: Table 2d). A 10% inc ease in DNA me hyla ion a cg10040131 was associa ed wi h a 0.32 SD (SE 0.06) highe BMI in adolescence. DNA me hyla ion a his CpG in childhood was nominally signi ican ly associa ed wi h childhood BMI (P alue = 0.0002). The associa ion o DNA me hyla ion a his CpG in co d blood and BMI in childhood was no sig- ni ican (Addi ional ile 13: Table S11). Associa ions o DNA me hyla ion wi h BMI did no show a p e e en ial di ec ion o e ec in any o he analyses ( ol- cano plo s, Addi ional ile 5: Supplemen a y In o ma ion, Fig. S2A-D). We obse ed e y li le e idence o he e ogen- ei y be ween s udies among he Bon e oni-signi ican ly associa ed CpG si es, wi h all I 2 ≤50 (Addi ional ile 3: Table 2a-d and o es plo s, Addi ional ile 5: Supplemen- a y In o ma ion, Fig. S3A, B and L). We ound e idence o Fig. 1 Manha an plo s o he me a-analyses o DNA me hyla ion and childhood o adolescen BMI. Manha an plo s showing he me a-analysis esul s o associa ions o DNA me hyla ion in co d blood wi h ea ly childhood BMI (a) and la e childhood BMI (b), o DNA me hyla ion in whole blood in childhood wi h childhood BMI (c), and o DNA me hyla ion in whole blood in adolescence wi h adolescen BMI (d). The g ay line shows he Bon e oni-co ec ed signi icance h eshold o mul iple es ing (P< 1.06 × 10 −7 ). The o ange line shows he FDR-co ec ed signi icance h eshold o mul iple es ing Vehmeije e al. Genome Medicine (2020) 12:105 Page 6 o 15 be ween-s udy he e ogenei y (I 2 > 50) o 3 o he 9 FDR- signi ican ly associa ed CpG si es (Addi ional ile 3:Table2c and o es plo s, Addi ional ile 5: Supplemen a y In o ma- ion, Fig. S3C-K). The esul s o he wel e Bon e oni o FDR-signi ican ly associa ed CpGs we e s able a e omi - ing one s udy a a ime (lea e-one-ou analyses, Add- i ional ile 5: Supplemen a y In o ma ion, Fig. S4A-L). When BMI was dicho omized in o no mal and o e - weigh /obesi y, only one CpG in he c oss-sec ional model in childhood, cg06991974 (PRDM16-DT), showed e idence o associa ion. In he c oss-sec ional model du ing childhood, which included 644 child en wi h o e weigh /obesi y and 2567 no mal-weigh child en, DNA me hyla ion a cg06991974 was associa ed wi h an inc eased isk o o e weigh /obesi y in childhood (odds a io (OR) 3.10, 95% con idence in e al (CI) 2.08, 4.63) (Addi ional ile 4: Table S3A-D). None o he h ee indi idual Bon e oni-signi ican CpGs in he h ee di e en age anges no he 9 FDR-signi ican CpGs was wi hin a 4-Mb window su ounding he 15 known gene ic loci associa ed wi h childhood body mass index [54]. Fou o he 12 FDR signi ican CpGs con ained a single-nucleo ide polymo phism (SNP) [48,49]. We ound no indica ion o non-unimodal dis ibu ion o any o hese CpGs sugges ing ha me hyla ion measu e- men s a hese si es we e no ma kedly a ec ed by SNPs (Addi ional ile 5: Supplemen a y In o ma ion, Fig. S6). Two o he h ee Bon e oni-signi ican CpGs (cg05937453 and cg25212453) had o he nea by CpGs wi hin a 10-kb window (± 5 kb) measu ed on he 450K a ay (Add- i ional ile 6: Table S4). Cg05937453 (model B) was su - ounded by 24 o he CpGs, o which one was nominally signi ican ly associa ed wi h BMI (P alue < 0.05). Bo h we e loca ed in he TSS200 egion o SFRP5 wi h e ec es ima es in he same di ec ion. Cg25212453 (model C) was su - ounded by 13 o he CpGs, o which h ee we e nominally signi ican (P alues < 0.05). All we e loca ed in he gene body o SLC43A2 wi h e ec es ima es in he same di ec ion. Re- sul s o Bon e oni- and FDR-signi ican CpGs a e shown in Addi ional ile 6:TableS4. Sensi i i y analyses Findings we e consis en wi h he main esul s when e- s ic ed o up o 3657 pa icipan s o Eu opean e hnici y (Pea son co ela ion coe icien s o he e ec es ima es ac oss all CpG si es we e 0.86–0.97 and we e 0.99 ac oss op CpG si es (P alue < 1 × 10 −4 ) o all models (Add- i ional ile 7: Table S5A-C)). Simila ly, when he s udies wi h a high pe cen age (> 30%) o child en wi h o e - weigh o obesi y we e excluded, he esul s we e also consis en wi h he main analyses (Pea son co ela ion coe icien s o he e ec es ima es ac oss all CpG si es we e 0.89–0.98 and we e 0.99 ac oss op CpG si es (P alue < 1 × 10 −4 ) o all models (Addi ional ile 8: Table S6A-C)). Las ly, when he s udies o non-Eu opeans pa - icipan s, a high pe cen age o child en wi h o e weigh o obesi y and s udies in which he sample was selec ed on o en iched o any pa icula exposu e o ou come Table 2 CpG si es a which DNA me hyla ion was associa ed wi h child o adolescen BMI CpG CHR Loca ion Coe SE P alue FDR P alue Nea es gene Analysis B = associa ion o co d blood DNA me hyla ion wi h la e childhood BMI (5–10 yea s) cg05937453 10 99531765 0.96288 0.16871 1.15 × 10 −8 0.0049 SFRP5 Analysis C = c oss-sec ional associa ion o whole blood DNA me hyla ion wi h childhood BMI (2–10 yea s) cg25212453 17 1509953 0.31925 0.05978 9.27 × 10 −8 0.02075 SLC43A2 cg03500056 16 8814507 0.30577 0.05767 1.15 × 10 −7 0.02075 ABAT cg05281708 3 44690673 0.65856 0.12614 1.78 × 10 −7 0.02075 ZNF35 cg15125798 5 122621645 0.49705 0.09548 1.93 × 10 −7 0.02075 – cg04456029 12 113496126 0.27587 0.05358 2.63 × 10 −7 0.0226 DTX1 cg03431111 11 62621406 0.19261 0.03791 3.77 × 10 −7 0.0270 SNORD30;SNORD22;SNORD29; SNORD31;SNHG1 cg26889953 15 22915992 0.31743 0.06391 6.81 × 10 −7 0.0304 CYFIP1 cg19743522 12 113495566 0.33854 0.0682 6.92 × 10 −7 0.0304 DTX1 cg25877069 8 95003236 −0.45126 0.09092 6.94 × 10 −7 0.0304 – cg13931559 20 33146515 −0.84718 0.17082 7.07 × 10 −7 0.0304 MAP1LC3A Analysis D = c oss-sec ional associa ion o whole blood DNA me hyla ion wi h adolescen BMI (12–18 yea s) cg10040131 2 73178866 0.32434 0.0566 1.00 × 10 −8 0.0043 SFXN5 Coe icien s (Coe ) and s anda d e o s (SE) a e p esen ed pe 10% inc ease in he me hyla ion le el Analyses B was adjus ed o ma e nal age, educa ional le el, smoking s a us, p e-p egnancy o ea ly p egnancy BMI, pa i y, ges a ional age, ba ch, and es ima ed cell ype p opo ions. Analysis C was addi ionally adjus ed o child co a ia es bi h weigh and b eas eeding, whe eas analysis D was adjus ed o he same co a ia es as analysis C plus adolescen sex, age, smoking, and pube y s a us Vehmeije e al. Genome Medicine (2020) 12:105 Page 7 o 15 we e all excluded, esul s emained s ongly co ela ed o hose om he main models. Pea son co ela ion coe icien s o he e ec es ima es ac oss all CpG si es we e 0.64–0.97 and 0.95–0.99 ac oss op CpG si es (P alue < 1 × 10–4) o all models (Addi ional ile 9: Table S7A-D). Compa ison wi h p e ious indings Mos CpGs iden i ied o be associa ed wi h BMI in p e- ious candida e gene s udies o smalle EWASs (N< 1000) did no eplica e in ou esul s (Addi ional ile 10: Table S8) [7,15,17,18,20,21,23,24,55]. When com- pa ing he genome-wide signi ican indings om he la ges BMI EWASs (N> 1000) in adul s o ou mos sig- ni ican indings ac oss he ou age anges, we ound an inc easing o e lap wi h age (Table 3and Add- i ional ile 11: Table S9) [5,6,9,25,26,56–59]. We used wo cu o s o selec he mos signi ican indings in ou esul s: a P alue < 1 × 10 −5 , o iden i y “sugges i e” ind- ings, and a less s ingen , nominal P alue < 0.05. The numbe o CpGs ha me hese c i e ia a e p o ided in Table 3. Fi s , we examined he absolu e numbe o o e lapping CpGs be ween he s udies in adul s and ou indings wi h a P alue < 1 × 10 −5 and calcula ed en ich- men . Wi h ad ancing age ac oss childhood and adoles- cence, we obse ed inc easing en ichmen o he 187 CpGs p e iously epo ed o be associa ed wi h adul BMI in he la ges s udy o da e (N= 10,261) [5]. Fo he wo co d blood models, he e was no o e lap wi h he adul indings (P en ichmen = 1), o he c oss-sec ional model in childhood 2/187 adul hi s o e lapped, (P en ichmen = 0.0002), and o he c oss-sec ional model in adolescence 3/187 o e lapped (P en ichmen = 2.10 × 10 −7 )(Table3and Addi ional ile 11:Table S9). Using he less s ingen cu o (P alue < 0.05), his end was e en clea e . The o e lap be ween he 187 CpGs om he adul EWAS and he CpGs in ou da a wi h a P alue < 0.05 was 8/187 CpGs (P en ichmen = 0.77, analysis A) o he associa ion o co d blood DNA me hyla ion and ea ly childhood BMI and 11/187 CpGs (P en ichmen = 0.30, analysis B) o he as- socia ion o co d blood DNA me hyla ion and la e child- hood BMI. Fo he c oss-sec ional model in childhood, he o e lap was 61/187 CpGs (P en ichmen = 1.97 × 10 −20 ,ana- lysis C), and in adolescence, he o e lap was 77/187 CpGs (P en ichmen = 1.68 × 10 −44 , analysis D) (Table 3and Add- i ional ile 11: Table S9). Twen y-se en CpGs we e among he en iched CpGs in bo h he childhood and he adoles- cen model. This o e lap was no signi ican (P= 0.88). Co ela ion coe icien s be ween he e ec es ima es o he 187 hi s and he e ec es ima es o hose CpGs in he ou models inc eased wi h age (analysis A = −0.186 (P= 0.01), analysis B = −0.013 (P= 0.86), analysis C = 0.604 (P= 5.31 × 10 −20 ), and analysis D = 0.816 (P= 7.89 × 10 −46 ). The di e ence in co ela ion coe icien s was signi ican o all compa isons (P’s o compa ison be ween co ela ion coe icien s < 0.01) excep o he compa ison be ween models A and B (P= 0.09). E ec sizes o he associa ions o hese 187 adul BMI CpGs in ou analyses inc eased wi h ad ancing age o chil- d en in ou analyses (Addi ional ile 5: Supplemen a y In- o ma ion, Fig. S5). We ound simila ends o en ichmen o CpGs om o he EWASs in adul s and ad- olescen s (Table 3)[6,9,25,26,56–59]. O hose indings om adul s udies ha had a nominal P alue (< 0.05) in ou models, 17–35%we e epo edbymo e hanone adul s udy. Mos o hese we e ound in wo o h ee s udies, bu ou , cg06500161, cg19750657, cg12593793, and cg18181703, we e epo ed in six o se en p e ious analyses. Func ional analyses A unc ional en ichmen analysis using genes linked o he CpGs wi h P alues < 1 × 10 −4 in each o he models showed no unc ional en ichmen o Gene On ology (GO) e ms o Kyo o Encyclopedia o Genes and Genomes (KEGG) e ms (FDR < 0.05) (Addi ional ile 12: Table S10). Discussion In his la ge me a-analysis o EWASs o childhood and adolescen BMI, we ound li le e idence o an associa ion be ween DNA me hyla ion and childhood o adolescen BMI. DNA me hyla ion a h ee di e en CpGs, each one in a di e en age ange, was associa ed wi h BMI in ea ly li e. Wi h he ad ancing age o child en in ou analyses, we obse ed inc easing en ichmen o CpGs p e iously iden i ied o hei ela ion wi h adolescen o adul adi- posi y. In addi ion, o he 187 CpGs iden i ied in he la - ges p e ious s udy o adul BMI, we ound inc easing e ec sizes and inc easing co ela ions be ween he adul e ec sizes and hose in ou analyses, wi h age. In e p e a ion o main indings Childhood obesi y is a majo public heal h p oblem and associa ed wi h sho - and long- e m mo bidi y and mo - ali y [61]. Al hough he e is some e idence om p e ious s udies ha DNA me hyla ion may media e associa ions o p egnancy- ela ed exposu es wi h o sp ing adiposi y, only ew speci ic CpG si es ha e been iden i ied [4,27]. Thus a , mos o he e idence o associa ions o DNA me hyla ion wi h adiposi y s ems om adul s udies. In his s udy, we ound li le e idence o an associa ion be ween DNA me hyla ion and childhood o adolescen BMI. DNA me hyla ion a h ee CpGs (cg05937453, cg25212453, and cg10040131), each in a di e en age ange, was associa ed wi h BMI a Bon e oni signi i- cance, P< 1.06 × 10 −7 . Howe e , we did obse e inc eas- ing en ichmen and inc easing poin es ima es o CpGs Vehmeije e al. Genome Medicine (2020) 12:105 Page 8 o 15 Table 3 Absolu e numbe o o e lapping CpGs and P alues o he en ichmen o signi ican CpGs om p e ious EWASs (N> 1000) in ou da a P e ious s udy (Nsi es associa ed wi h BMI) Signi icance le el Analysis A: associa ion o co d blood DNA me hyla ion wi h ea ly childhood BMI (2–5 yea s) Analysis B: associa ion o co d blood DNA me hyla ion wi h la e childhood BMI (5–10 yea s) Analysis C: c oss- sec ional analysis o whole blood DNA me hyla ion wi h childhood BMI (2–10 yea s) Analysis D: c oss-sec ional analysis o whole blood DNA me hyla ion wi h adolescen BMI (12–18 yea s) 1×10 −5 N=7 N=8 N=51 N=26 0.05 N= 22,687 N= 20,645 N= 37,074 N= 25,292 Ali e al. [56] (3 CpGs) 1 × 10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0.05 0 P en ichmen =1 0 P en ichmen =1 1/3 P en ichmen = 0.24 0 P en ichmen =1 Aslibekyan e al. [6](8 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0.05 0 P en ichmen =1 0 P en ichmen =1 1/8 P en ichmen = 0.51 2/8 P en ichmen = 0.08 Campanella e al. [57] (26 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 1/26 P en ichmen = 0.002 0.05 3/26 P en ichmen = 0.16 1/26 P en ichmen = 0.72 6/26 P en ichmen = 0.02 11/26 P en ichmen = 1.006 × 10 −7 Geu s e al. [58] (310 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 2/310 P en ichmen = 0.0006 2/310 P en ichmen = 0.0002 0.05 12/310 P en ichmen = 0.90 13/310 P en ichmen = 0.73 103/310 P en ichmen = 3.92 × 10 −34 125/310 P en ichmen = 6.63 × 10 −70 Mendelson e al. [9] (83 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 2/83 P en ichmen = 4.67 × 10 −5 3/83 P en ichmen = 1.81 × 10 −8 0.05 4/83 P en ichmen = 0.64 8/83 P en ichmen = 0.045 28/83 P en ichmen = 1.36 × 10 −10 45/83 P en ichmen = 3.02 × 10 −33 Sayols-Baixe as e al. [59] (96 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0.05 8/96 P en ichmen = 0.13 9/96 P en ichmen = 0.04 24/96 P en ichmen = 1.53 × 10 −6 30/96 P en ichmen = 1.85 × 10 −14 Sun e al. [26]black pa icipan s (36 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 1/36 P en ichmen = 0.002 0.05 3/36 P en ichmen = 0.30 6/36 P en ichmen = 0.007 13/36 P en ichmen = 4.98 × 10 −6 22/36 P en ichmen = 1.50 × 10 −18 Sun e al. [26]whi e pa icipan s (349 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 0.05 12/349 P en ichmen = 0.959 22/349 P en ichmen = 0.12 86/349 P en ichmen = 4.13 × 10 −19 116/349 P en ichmen = 1.75 × 10 −54 Wahl e al. [5] (187 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 2/187 P en ichmen = 0.0002 3/187 P en ichmen = 2.10 × 10 −7 0.05 8/187 P en ichmen = 0.77 11/187 P en ichmen = 0.29 61/187 P en ichmen = 1.97 × 10 −20 77/187 P en ichmen = 1.68 × 10 −44 Wang e al. [25] (54 CpGs) 1×10 −5 0 P en ichmen =1 0 P en ichmen =1 0 P en ichmen =1 1/54 P en ichmen = 0.003 0.05 2/54 P en ichmen = 0.79 4/54 P en ichmen = 0.26 23/54 P en ichmen = 2.49 × 10 −11 33/54 P en ichmen = 3.98 × 10 −27 NCpGs in ≥2 adul s udies 0.05 9/52 (17.3%) 23/75 (30.7%) 98/347 (28.2%) 163/465 (35.1%) Two cu o s we e used o selec he signi ican indings in ou esul s: a P alue < 1 × 10 −5 , o iden i y “sugges i e” indings, and a less s ingen , nominal P alue < 0.05, o iden i y any ends. We used a hype geome ic es o calcula e en ichmen wi h he phype unc ion in he R S a s package in R. Resul s in bold a e nominally signi ican . O hose indings om adul s udies ha had a nominal P alue (< 0.05) in ou models, 17–35% we e epo ed by mo e han one adul s udy Vehmeije e al. Genome Medicine (2020) 12:105 Page 9 o 15