Author's Accepted Manuscript

Genome-wide methylomic analysis of monozygotic twins discordant for adolescent depression Emma L Dempster, Chloe C Y Wong, Kathryn J Lester, Joe Burrage, Alice M Gregory, Jonathan Mill, Thalia C. Eley

www.elsevier.com/locate/bps

PII: DOI: Reference:

S0006-3223(14)00297-2 http://dx.doi.org/10.1016/j.biopsych.2014.04.013 BPS12198

To appear in:

Biological Psychiatry

Cite this article as: Emma L Dempster, Chloe C Y Wong, Kathryn J Lester, Joe Burrage, Alice M Gregory, Jonathan Mill, Thalia C. Eley, Genome-wide methylomic analysis of monozygotic twins discordant for adolescent depression, Biological Psychiatry, http: //dx.doi.org/10.1016/j.biopsych.2014.04.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.



‡’•–‡”etal.

Genomewidemethylomicanalysisofmonozygotictwinsdiscordantfor adolescentdepression EmmaLDempster1~,ChloeCYWong2,KathrynJLester2,JoeBurrage1,AliceM Gregory3,JonathanMill1,2,*,ThaliaC.Eley2,* 1,UniversityofExeterMedicalSchool,ExeterUniversity,StLuke’sCampus,Exeter,U.K 2,SGDPCentre,InstituteofPsychiatry,King’sCollegeLondon,DenmarkHill,London,U.K 3,DepartmentofPsychology,Goldsmiths,UniversityofLondon,London,UK *Theseauthorscontributedequallytothiswork.  Correspondingauthor: ƒ‡’•–‡” ‹˜‡”•‹–›‘ˆš‡–‡”‡†‹…ƒŽ…Š‘‘Ž š‡–‡”‹˜‡”•‹–› –—‡ǯ•ƒ’—• š‡–‡” ͳʹ Ǥ ‡Ǧƒ‹Žǣ‡ǤŽǤ†‡’•–‡”̷‡š‡–‡”Ǥƒ…Ǥ— Š‘‡ǣΪͶͶȋͲȌͳ͵ͻʹ͹ʹ͸Ͷʹͻ  KEYWORDS ‡’”‡••‹‘ǡ‘‘œ›‰‘–‹…™‹•ǡ’‹‰‡‡–‹…ǡ‡–Š›Žƒ–‹‘ǡ ‡‘‹…•ǡ†‘Ž‡•…‡–‡’”‡••‹‘ WordCount „•–”ƒ…–αʹͷͲ ƒ‹–‡š–αʹǡͻͺ͸ NumbersofFiguresandtables ƒ„Ž‡•αͳ ‹‰—”‡•α͵ —’’Ž‡‡–ƒ”›ƒ–‡”‹ƒŽαͳ 



ͳ

‡’•–‡”etal.



ABSTRACT Background Adolescentdepressionisacommonneuropsychiatricdisorderthatoftencontinuesinto adulthoodandisassociatedwithawiderangeofpooroutcomesincludingsuicide.Whilst therehavebeenanumberofstudiesofgeneticmarkersassociatedwithdepression,therole ofepigeneticvariationremainsrelativelyunexplored.Weselectedmonozygotic(MZ)twins fromanadolescenttwinstudydesignedtoinvestigatetheinterplayofgeneticand environmentalfactorsinthedevelopmentofemotionalandbehaviouraldifficulties. Methods EighteenpairsofMZtwinswereidentifiedinwhichonememberscoredconsistentlyhigher (groupmeanwithintheclinicallysignificantrange)onselfrateddepressionthantheother. WeassessedgenomewidepatternsofDNAmethylationintwinbuccalcellDNAusingthe Illumina450KHumanMethylationarray.Qualitycontrolanddatapreprocessingwas undertakenusingtheWateRmelonpackage.Differentiallymethylatedprobes(DMP)were identifiedusingananalysisstrategytakingintoaccountboththesignificanceandmagnitude ofDNAmethylationdifferences.ThetopdifferentiallymethylatedDMPwassuccessfully validatedbybisulfitepyrosequencingandidentifiedDMPsweretestedinpostmortembrain samplesobtainedfrommajordepressivedisorderpatients(n=14)andmatchedcontrols (n=15). Results TworeproducibledepressionassociatedDMPswereidentified,includingthetoprankedDMP thatwaslocatedwithinSTK32C,whichencodesaserine/threoninekinase,ofunknown function. Conclusions OurdataindicatethatDNAmethylationdifferencesareapparentinMZtwinsdiscordantfor adolescentdepressionandthatsomeofthediseaseassociatedvariationobservedinbuccal cellDNAismirroredinadultbraintissueobtainedfromclinicallydepressedindividuals. 





ʹ



‡’•–‡”etal.

INTRODUCTION ‡’”‡••‹‘‹•ƒ…‘‘…Š”‘‹…’•›…Š‹ƒ–”‹…†‹•‘”†‡”…Šƒ”ƒ…–‡”‹•‡†„›‡’‹•‘†‡•‘ˆ•‡˜‡”‡ Ž‘™‘‘†Ǥ‡’”‡••‹‘”ƒ••‡…‘†‹‰Ž‘„ƒŽ†‹•‡ƒ•‡„—”†‡ȋͳȌ„—–†‡•’‹–‡‡š–‡•‹˜‡ ”‡•‡ƒ”…Š‹–•‡–‹‘Ž‘‰›‹•‘–™‡ŽŽ—†‡”•–‘‘†ǤŠ‡Š‡”‹–ƒ„‹Ž‹–›‘ˆ†‡’”‡••‹‘‹•‡•–‹ƒ–‡†–‘ „‡„‡–™‡‡͵ͳΨƒ†ͶʹΨȋʹȌǡƒŽ–Š‘—‰Š‰‡‘‡Ǧ™‹†‡ƒ••‘…‹ƒ–‹‘•–—†‹‡•Šƒ˜‡„‡‡ —ƒ„Ž‡–‘‹†‡–‹ˆ›”‡’”‘†—…‹„Ž‡ƒ••‘…‹ƒ–‹‘•‹‰ƒŽ•(3).Š‹Ž‡•‘‡‘ˆ–Š‹•Žƒ…‘ˆ ”‡’Ž‹…ƒ–‹‘‹‰‡‡–‹…•–—†‹‡•…ƒ„‡‡š’Žƒ‹‡†„›–Š‡Š‡–‡”‘‰‡‡‘—•ƒ–—”‡‘ˆ †‡’”‡••‹‘ǡ‹…”‡ƒ•‡†—†‡”•–ƒ†‹‰ƒ„‘—––Š‡ˆ—…–‹‘ƒŽ…‘’Ž‡š‹–›‘ˆ–Š‡‰‡‘‡Šƒ• Ž‡†–‘‰”‘™‹‰”‡…‘‰‹–‹‘ƒ„‘—––Š‡Ž‹‡Ž›”‘Ž‡‘ˆ‘Ȃ•‡“—‡…‡Ǧ„ƒ•‡†˜ƒ”‹ƒ–‹‘‹ †‹•‡ƒ•‡‡–‹‘Ž‘‰›ȋͶǡͷȌǤ  ‡•’‹–‡–Š‡ƒ’’ƒ”‡–Ž›Š‹‰ŠŠ‡”‹–ƒ„‹Ž‹–›‘ˆ†‡’”‡••‹‘ǡ–Š‡”‡‹•…‘•‹†‡”ƒ„Ž‡†‹•…‘”†ƒ…‡ ™‹–Š‹‘‘œ›‰‘–‹…ȋȌ–™‹’ƒ‹”•ȋ͸Ȍǡ‹’Ž‹…ƒ–‹‰ƒ”‘Ž‡ˆ‘”‘Ǧ‰‡‡–‹…ǡ’”‡•—ƒ„Ž› ‡˜‹”‘‡–ƒŽǡˆƒ…–‘”•Ǥ‡‡…Šƒ‹•„›™Š‹…Š–Š‡‡˜‹”‘‡–…ƒ‹’ƒ…–‘ ‹†‹˜‹†—ƒŽ†‡˜‡Ž‘’‡–‹•˜‹ƒ‡’‹‰‡‡–‹……Šƒ‰‡•–‘‰‡‡‡š’”‡••‹‘Ǥ’‹‰‡‡–‹… ‡…Šƒ‹••‡†‹ƒ–‡†‡˜‡Ž‘’‡–ƒŽ…Šƒ‰‡•‹‰‡‡ˆ—…–‹‘‹†‡’‡†‡–‘ˆ •‡“—‡…‡˜ƒ”‹ƒ–‹‘ǡ’”‹…‹’ƒŽŽ›–Š”‘—‰ŠƒŽ–‡”ƒ–‹‘•‹‡–Š›Žƒ–‹‘ƒ†…Š”‘ƒ–‹ •–”—…–—”‡ȋ͹ȌǤ’‹‰‡‡–‹…’”‘…‡••‡•Šƒ˜‡„‡‡•Š‘™–‘—†‡”Ž‹‡—…Š‘ˆ–Š‡’Žƒ•–‹…‹–›‹ –Š‡ƒ–—”‡…‡–”ƒŽ‡”˜‘—••›•–‡ǡ’Žƒ›‹‰ƒ‡›”‘Ž‡‹Ž‡ƒ”‹‰ƒ†‡‘”›ǡƒ† ‡ƒ„Ž‹‰–Š‡„”ƒ‹–‘”‡•’‘†–‘‡˜‹”‘‡–ƒŽ•–‹—Ž‹ȋͺȌǤŠ‡”‡‹•ƒ……——Žƒ–‹‰ ‡˜‹†‡…‡ˆ”‘ƒ‹ƒŽ‘†‡Ž•–Šƒ–ƒ›‘ˆ–Š‡‡ƒ”Ž›ǦŽ‹ˆ‡‡˜‹”‘‡–ƒŽ•–”‡••‘”• ƒ••‘…‹ƒ–‡†™‹–Šƒ‹…”‡ƒ•‡†”‹•‘ˆ†‡’”‡••‹‘…ƒ’”‡…‹’‹–ƒ–‡‡’‹‰‡‡–‹……Šƒ‰‡•‹–Š‡ „”ƒ‹ȋ•‡‡ȋͻȌȌǡƒŽ–Š‘—‰Š‘•–”‡•‡ƒ”…ŠŠƒ•ˆ‘…—••‡†‘ƒ•‹‰Ž‡‰‡‡‘”ˆ—…–‹‘ƒŽ ’ƒ–Š™ƒ›ȋͳͲǦͳ͵ȌǤ‡”‰‹‰‡˜‹†‡…‡‹’Ž‹…ƒ–‡•ƒ”‘Ž‡ˆ‘”‡’‹‰‡‡–‹…‘†‹ˆ‹…ƒ–‹‘•ƒ…”‘•• –Š‡‰‡‘‡‹•‡˜‡”ƒŽŠ—ƒ‡—”‘’•›…Š‹ƒ–”‹…†‹•‘”†‡”•ǡ‹…Ž—†‹‰†‡’”‡••‹‘ȋͶȌǤ Ž–Š‘—‰Šˆ‡™‡’‹”‹…ƒŽ•–—†‹‡•Šƒ˜‡•›•–‡ƒ–‹…ƒŽŽ›‡šƒ‹‡†–Š‡”‘Ž‡‘ˆƒŽ–‡”‡† ‡’‹‰‡‡–‹…’”‘…‡••‡•‹†‡’”‡••‹‘ǡ”‡…‡–ƒƒŽ›•‡•’”‘˜‹†‡‡˜‹†‡…‡ˆ‘”ƒŽ–‡”‡† ‡–Š›Žƒ–‹‘ƒ†Š‹•–‘‡‘†‹ˆ‹…ƒ–‹‘•‹–Š‡†‹•‘”†‡”ȋͳͶǦͳ͹ȌǤ †‘Ž‡•…‡…‡‹•ƒ—‹“—‡’‡”‹‘†…Šƒ”ƒ…–‡”‹œ‡†„›„‘–Š’Š›•‹…ƒŽƒ†‡‘–‹‘ƒŽ †‡˜‡Ž‘’‡–ƒ†‡š’‘•—”‡–‘‘˜‡Ž‡˜‹”‘‡–ƒŽ•–”‡••‘”•ȋͳͺȌǤ –‹•ƒ’‡”‹‘†‘ˆƒ…–‹˜‡ „”ƒ‹ƒ–—”ƒ–‹‘ǡ…Šƒ”ƒ…–‡”‹œ‡†„›’”‘…‡••‡••—…Šƒ••›ƒ’–‹…’”—‹‰ȋͳͻȌǡƒ†‹•–Š‡ †‡˜‡Ž‘’‡–ƒŽ•–ƒ‰‡™Š‡–Š‡ˆ‹”•–•‹‰•‘ˆƒ›’•›…Š‹ƒ–”‹…†‹•‘”†‡”•ˆ‹”•–„‡…‘‡



͵



‡’•–‡”etal.

ƒ‹ˆ‡•–ȋʹͲȌǤŠ‡’”‡˜ƒŽ‡…‡‘ˆ†‡’”‡••‹‘†”ƒƒ–‹…ƒŽŽ›‹…”‡ƒ•‡•†—”‹‰ƒ†‘Ž‡•…‡…‡ǡ ’ƒ”–‹…—Žƒ”Ž›‹ˆ‡ƒŽ‡•ȋʹͳȌǤ…‡‡•–ƒ„Ž‹•Š‡†ǡƒ†‘Ž‡•…‡–†‡’”‡••‹‘…‘‘Ž› …‘–‹—‡•‹–‘ƒ†—Ž–Š‘‘†ƒ†‹•ƒ••‘…‹ƒ–‡†™‹–Šƒ™‹†‡”ƒ‰‡‘ˆ’‘‘”‘—–…‘‡•‹…Ž—†‹‰ •—‹…‹†‡ȋʹʹǡʹ͵ȌǤ ‘‹†‡–‹ˆ›‡’‹‰‡‡–‹…˜ƒ”‹ƒ–‹‘ƒ••‘…‹ƒ–‡†™‹–Šƒ†‘Ž‡•…‡–†‡’”‡••‹‘™‡‡šƒ‹‡† ‰‡‘‡Ǧ™‹†‡’ƒ––‡”•‘ˆ‡–Š›Žƒ–‹‘‹„—……ƒŽ…‡ŽŽ†‡”‹˜‡†ˆ”‘‘‘œ›‰‘–‹… ȋȌ–™‹Ǧ’ƒ‹”•†‹•…‘”†ƒ–ˆ‘”ƒ†‘Ž‡•…‡–†‡’”‡••‹‘•›’–‘•ƒ••‡••‡†ƒ–—Ž–‹’Ž‡ –‹‡’‘‹–•ǤŠ‡—•‡‘ˆ†‹•‡ƒ•‡Ǧ†‹•…‘”†ƒ––™‹•”‡’”‡•‡–•ƒ’‘™‡”ˆ—Ž•–”ƒ–‡‰›‹ ‡’‹‰‡‡–‹…‡’‹†‡‹‘Ž‘‰›„‡…ƒ—•‡‹†‡–‹…ƒŽ–™‹•ƒ”‡ƒ–…Š‡†ˆ‘”‰‡‘–›’‡ǡƒ‰‡ǡ•‡šǡ ƒ–‡”ƒŽ‡˜‹”‘‡–ǡ’‘’—Žƒ–‹‘…‘Š‘”–‡ˆˆ‡…–•ƒ†‡š’‘•—”‡–‘ƒ›•Šƒ”‡† ‡˜‹”‘‡–ƒŽˆƒ…–‘”•ȋʹͶǡʹͷȌǤ‡–Š›Žƒ–‹‘†‹ˆˆ‡”‡…‡•Šƒ˜‡„‡‡ƒ••‘…‹ƒ–‡†™‹–Š –™‹†‹•…‘”†ƒ…‡ˆ‘”•‡˜‡”ƒŽ…‘’Ž‡š’Š‡‘–›’‹…–”ƒ‹–•ǡ‹…Ž—†‹‰’•›…Š‘•‹•ȋʹ͸Ȍǡ ƒ—–‹•ȋʹ͹Ȍƒ†›’‡ͳ†‹ƒ„‡–‡•ȋʹͺȌǤ –Š‡…‘–‡š–‘ˆ†‡’”‡••‹‘ǡ–Š‡—•‡‘ˆƒ†‘Ž‡•…‡– –™‹•Šƒ•–Š‡ƒ†˜ƒ–ƒ‰‡–Šƒ–‡–Š›Žƒ–‹‘‹•Ž‡••Ž‹‡Ž›–‘„‡‹ˆŽ—‡…‡†„›‘–Š‡” ‡˜‹”‘‡–ƒŽˆƒ…–‘”•ǡŽ‹ˆ‡Ǧ•–›Ž‡…Š‘‹…‡•ǡƒ†‡†‹…ƒ–‹‘‡š’‘•—”‡ǡ™Š‹…Š…ƒ…‘ˆ‘—† •–—†‹‡•‹‘Ž†‡”…Ž‹‹…ƒŽ…‘Š‘”–•Ǥ  MaterialsandMethods Sampledescription Š‹••–—†›™ƒ•‡•–‡†™‹–Š‹–Š‡ ‡‡•‹•ͳʹǦͳͻ•–—†›ȋ ͳʹͳͻȌǡƒƒ†‘Ž‡•…‡––™‹ƒ† •‹„Ž‹‰•–—†›ȋαͳ͵ͲͲ’ƒ‹”•Ȍ†‡•‹‰‡†–‘‹˜‡•–‹‰ƒ–‡–Š‡†‡˜‡Ž‘’‡–‘ˆ‡‘–‹‘ƒŽƒ† „‡Šƒ˜‹‘—”ƒŽ†‹ˆˆ‹…—Ž–‹‡•ˆ‘…—••‹‰‘–Š‡‹–‡”’Žƒ›‘ˆ‰‡‡•ƒ†‡˜‹”‘‡–ȋͳͺȌǤŠ‡ –™‹•™‡”‡”‡…”—‹–‡†˜‹ƒ–Š‡ˆˆ‹…‡‘ˆƒ–‹‘ƒŽ–ƒ–‹•–‹…•ǡƒ†ˆ‘—”™ƒ˜‡•‘ˆ†ƒ–ƒŠƒ˜‡ „‡‡…‘ŽŽ‡…–‡†–‘†ƒ–‡Ǥ–‡ƒ…Š™ƒ˜‡’ƒ”–‹…‹’ƒ–•…‘’Ž‡–‡†–Š‡•Š‘”–‘‘†ƒ† ‡‡Ž‹‰• —‡•–‹‘ƒ‹”‡ȋ ȌȋʹͻȌǡƒ•‡ŽˆǦ”‡’‘”–‡ƒ•—”‡‘ˆ†‡’”‡••‹‘•›’–‘•Ǥ…‘”‡•‘–Š‹• ‡ƒ•—”‡”ƒ‰‡ˆ”‘œ‡”‘–‘ʹ͸ǡ™‹–Šƒ‡ƒ‘ˆ͹ǤŠ‡‡ƒ†‹ˆˆ‡”‡…‡•…‘”‡™‹–Š‹–Š‡

ͳʹͳͻ–™‹Ȁ•‹„Ž‹‰’ƒ‹”•ƒ–‡ƒ…Š™ƒ˜‡”ƒ‰‡†„‡–™‡‡ͶǤͲ͸ƒ†ͷǤͳ͵™‹–Š•–ƒ†ƒ”† †‡˜‹ƒ–‹‘•‘ˆ„‡–™‡‡͵Ǥͻͻƒ†ͶǤͺͲǤ‡†‡•‹‰ƒ–‡†ƒ†‹ˆˆ‡”‡…‡‘ˆ͸ȋ‹Ǥ‡Ǥƒ’’”‘š‹ƒ–‡Ž› ‘‡ƒ†ƒŠƒŽˆ–‹‡•–Š‡•–ƒ†ƒ”††‡˜‹ƒ–‹‘‘ˆ–Š‡†‹ˆˆ‡”‡…‡Ȍƒ•”‡’”‡•‡–‹‰•—ˆˆ‹…‹‡– †‹•…‘”†ƒ…›–‘„‡‘ˆ‹–‡”‡•–ˆ‘”–Š‡…—””‡–•–—†›ƒ†‹†‡–‹ˆ‹‡†ͳͺ–™‹’ƒ‹”•ȋͳ͵ ˆ‡ƒŽ‡’ƒ‹”•ǡͷƒŽ‡’ƒ‹”•ǡƒŽŽ™Š‹–‡ƒ—…ƒ•‹ƒȌ™‹–Š…‘•‹•–‡–†‹•…‘”†ƒ…›‘atleast 

Ͷ



‡’•–‡”etal.

two‡ƒ•—”‡‡–‘……ƒ•‹‘•ǤŠ‡‡ƒ •…‘”‡ˆ‘”ƒ…”‘••‡ƒ…Š‘ˆ–Š‡†‡’”‡••‡†–™‹ ’ƒ‹”‡„‡”•™ƒ•ͳʹǤ͵ͺȋ‘”‡–Šƒ‘‡•–ƒ†ƒ”††‡˜‹ƒ–‹‘ƒ„‘˜‡–Š‡…Ž‹‹…ƒŽ–Š”‡•Š‘Ž† ‘ˆͺȋʹͻȌȌƒ†–Š‡…‘Ǧ–™‹‰”‘—’‡ƒ™ƒ•͸ǤͶʹǤ™‹•†‹†‘–•‹‰‹ˆ‹…ƒ–Ž›†‹ˆˆ‡”‘ —„‡”‘”•‡˜‡”‹–›‘ˆ•‡ŽˆǦ”‡’‘”–‡†Ž‹ˆ‡‡˜‡–•ƒ–ƒ›‘ˆ–Š‡ƒ••‡••‡––‹‡’‘‹–•Ǥ• –Š‹•™ƒ•ƒ’‘’—Žƒ–‹‘Ǧ„ƒ•‡†•ƒ’Ž‡™ƒ•‘„–ƒ‹‡†ˆ”‘„—……ƒŽ…‡ŽŽ•ȋ‡ƒƒ‰‡αͳ͸Ǥͺ ›‡ƒ”•ȋαʹǤͶȌȌƒ†‹•‘Žƒ–‡†—•‹‰ƒ•–ƒ†ƒ”†’”‘–‘…‘Žȋ͵ͲȌǤ  Methylomicprofiling ™ƒ•…‘˜‡”–‡†™‹–Š•‘†‹—„‹•—Žˆ‹–‡‹†—’Ž‹…ƒ–‡—•‹‰–Š‡Ǧͻ͸‡–Š›Žƒ–‹‘ ‹–ȋ›‘”‡•‡ƒ”…ŠǡǡȌƒ†’”‘ˆ‹Ž‡†—•‹‰–Š‡ ˆ‹‹——ƒ‡–Š›Žƒ–‹‘ͶͷͲ ‡ƒ†Š‹’ȋͶͷͲȌȋ ŽŽ—‹ƒ …ǤȌƒ•’”‡˜‹‘—•Ž›†‡•…”‹„‡†ȋ͵ͲȌ.ƒ…Š–™‹’ƒ‹”™ƒ• ’”‘…‡••‡†–‘‰‡–Š‡”‘–Š‡•ƒ‡ƒ””ƒ›–‘‡‰ƒ–‡„ƒ–…Š‡ˆˆ‡…–•Ǥƒ™†ƒ–ƒ™ƒ•’”‡Ǧ ’”‘…‡••‡†—•‹‰ ‡‘‡–—†‹‘ȋ ŽŽ—‹ƒ …ȌǤ–”‹‰‡–“—ƒŽ‹–›…‘–”‘Ž…Š‡…•ǡ“—ƒ–‹Ž‡ ‘”ƒŽ‹œƒ–‹‘ƒ†•‡’ƒ”ƒ–‡„ƒ…‰”‘—†ƒ†Œ—•–‡–‘ˆ‡–Š›Žƒ–‡†ƒ†—‡–Š›Žƒ–‡† ‹–‡•‹–‹‡•‘ˆ›’‡ ƒ† ’”‘„‡•™‡”‡‹’Ž‡‡–‡†—•‹‰–Š‡wateRmelon’ƒ…ƒ‰‡‹ ȋ͵ͲȌǤŽ›•ƒ’Ž‡•™‹–ŠδͷΨ‘ˆ•‹–‡•™‹–Šƒ†‡–‡…–‹‘pǦ˜ƒŽ—‡εͲǤͲͷ™‡”‡‹…Ž—†‡†ǡƒ† ’”‘„‡•™‹–ŠεͷΨ‘ˆ•ƒ’Ž‡•™‹–Šƒ†‡–‡…–‹‘pǦ˜ƒŽ—‡εͲǤͲͷ‘”ƒ„‡ƒ†…‘—–Ž‡••–Šƒ͵‹ ͷΨ‘ˆ•ƒ’Ž‡•™‡”‡”‡‘˜‡†ˆ”‘–Š‡ƒƒŽ›•‹•Ǥ –‘–ƒŽǡͶ͵ͻǡͺͶ͸’”‘„‡•™‡”‡‹…Ž—†‡†‹ –Š‡ˆ‹ƒŽƒƒŽ›•‹•ƒ†ƒŽŽ•ƒ’Ž‡•’ƒ••‡†‘—”•–”‹‰‡–“—ƒŽ‹–›…‘–”‘Žˆ‹Ž–‡”Ǥ‘Ž›‘”’Š‹… …‘–”‘Ž’”‘„‡•Ž‘…ƒ–‡†‘–Š‡ƒ””ƒ›™‡”‡—•‡†–‘…‘ˆ‹”–Šƒ–ƒŽŽ–™‹’ƒ‹”•™‡”‡ ‘‘œ›‰‘—•Ǥ‹–Š–Š‡ƒ‹‘ˆ‹†‡–‹ˆ›‹‰”‡ƒŽǡ„‹‘Ž‘‰‹…ƒŽŽ›”‡Ž‡˜ƒ–™‹–Š‹Ǧ–™‹ƒ† „‡–™‡‡Ǧ‰”‘—’‡–Š›Žƒ–‹‘†‹ˆˆ‡”‡…‡•ǡ™‡—•‡†ƒƒƒŽ›–‹…ƒ’’”‘ƒ…Š–Šƒ– ‹…‘”’‘”ƒ–‡•„‘–Š–Š‡•‹‰‹ˆ‹…ƒ…‡ȋ–Šƒ–‹•ǡ’ƒ‹”‡†tǦ–‡•–•–ƒ–‹•–‹…Ȍƒ†ƒ‰‹–—†‡ȋ–Šƒ–‹•ǡ ƒ„•‘Ž—–‡†‡Ž–ƒǦȾȋȟȾȌȌ‘ˆƒ›‘„•‡”˜‡††‹ˆˆ‡”‡…‡•–‘’”‘†—…‡ƒ”ƒ‡†Ž‹•–‘ˆ †‹ˆˆ‡”‡–‹ƒŽŽ›‡–Š›Žƒ–‡†’”‘„‡•ȋ•Ȍȋʹ͸ǡʹ͹ȌǤ „”‹‡ˆǡ’”‘„‡•™‡”‡”ƒ‡†•‡’ƒ”ƒ–‡Ž› „›’ƒ‹”‡†–Ǧ–‡•–’Ǧ˜ƒŽ—‡ƒ†ȟȾȌƒ†–Š‡”ƒ•™‡”‡•—‡†ȋ•‡‡Supplementary FigureS1ˆ‘”‘”‡†‡–ƒ‹Ž‘ˆƒƒŽ›•‹••–”ƒ–‡‰›ȌǤFǦ–‡•–™ƒ•—•‡†–‘‡šƒ‹‡™Š‡–Š‡” –Š‡˜ƒ”‹ƒ…‡•„‡–™‡‡ƒˆˆ‡…–‡†ƒ†—ƒˆˆ‡…–‡†‰”‘—’•ˆ‘”‡ƒ…Š‹†‹˜‹†—ƒŽ’”‘„‡†‹ˆˆ‡”‡†Ǥ ‘–‡•––Š‡‘˜‡”ƒŽŽ‡ƒ˜ƒ”‹ƒ…‡ƒ…”‘••ƒŽŽ’”‘„‡•„‡–™‡‡–Š‡–™‘‰”‘—’•–Š‡˜ƒ”‹ƒ…‡ ™ƒ•…ƒŽ…—Žƒ–‡†ˆ‘”‡ƒ…Š’”‘„‡‹‡ƒ…Š‰”‘—’•‡’ƒ”ƒ–‡Ž›ƒ†–Š‡”‡•—Ž–‹‰†‹•–”‹„—–‹‘• ™‡”‡–Š‡…‘’ƒ”‡†—•‹‰–Š‡‹Ž…‘š‘•‹‰‡†Ǧ”ƒ–‡•–Ǥ‡‰‹‘Ž‡˜‡ŽƒƒŽ›•‹•™ƒ•



ͷ



‡’•–‡”etal.

’‡”ˆ‘”‡†—•‹‰–Š‡‹‘…‘†—…–‡”’ƒ…ƒ‰‡ ŽŽ—‹ƒ‡–Š›Žƒ–‹‘ƒŽ›œ‡”ȋ Ȍȋ͵ͳȌǤ

‡‡•™‡”‡ƒ••‹‰‡†–‘’”‘„‡•—•‹‰–Š‡ ’ƒ…ƒ‰‡ȋ͵ʹȌ ȋŠ––’ǣȀȀ„‡Œ‡”ƒ‘Ǥ•–ƒˆ‘”†Ǥ‡†—Ȁ‰”‡ƒ–Ȁ’—„Ž‹…ȀŠ–ŽȌ–ƒ‹‰‹–‘ƒ……‘—––Š‡ˆ—…–‹‘ƒŽ •‹‰‹ˆ‹…ƒ…‡‘ˆcisǦ”‡‰—Žƒ–‘”›”‡‰‹‘•Ǥƒ–Š™ƒ›ƒ†‡–™‘”ƒƒŽ›•‹•™ƒ•’‡”ˆ‘”‡† —•‹‰ ‰‡—‹–›ƒ–Š™ƒ›ƒŽ›•‹•ȋ Ȍȋ ‰‡—‹–››•–‡•ǡ‡†™‘‘†‹–›ǡǡǢ ™™™Ǥ‹‰‡—‹–›Ǥ…‘ȌǤ”‹…Š‡–ˆ‘”•’‡…‹ˆ‹…’ƒ–Š™ƒ›•ƒ†„‹‘Ž‘‰‹…ƒŽˆ—…–‹‘•™ƒ• †‡–‡”‹‡†”‡Žƒ–‹˜‡–‘–Š‡’”‘’”‹‡–ƒ”›†ƒ–ƒ„ƒ•‡ǡ—•‹‰ƒ”‹‰Š–Ǧ–ƒ‹Ž‡† ‹•Š‡”ǯ•–‡•–ƒ–ƒ •‹‰‹ˆ‹…ƒ…‡Ž‡˜‡ŽofpδͲǤͲͷǤ  DMPvalidationusingbisulfitepyrosequencing ‡’‡”ˆ‘”‡†‹†‡’‡†‡–˜‡”‹ˆ‹…ƒ–‹‘ƒƒŽ›•‡•‘–Š‡Š‹‰Š‡•–”ƒ‡††‡’”‡••‹‘Ǧ ƒ••‘…‹ƒ–‡†’”‘„‡ȋ…‰Ͳ͹ͲͺͲͲͳͻǡ͵ʹȌ‘‹ƒ–‡†ˆ”‘–Š‡‰‡‘‡Ǧ™‹†‡‹…”‘ƒ””ƒ› ƒƒŽ›•‹•—•‹‰„‹•—Žˆ‹–‡’›”‘•‡“—‡…‹‰ǤŠ‡ƒ••ƒ›•’ƒ‡†—Ž–‹’Ž‡’ •‹–‡•ǡ‹…Ž—†‹‰ –Š‡•’‡…‹ˆ‹…’ ‹–‡””‘‰ƒ–‡†‘–Š‡ ŽŽ—‹ƒͶͷͲƒ””ƒ›Ǥ”‹‡ˆŽ›ǡͷͲͲ‰ˆ”‘‡ƒ…Š ‹†‹˜‹†—ƒŽ™ƒ•‹†‡’‡†‡–Ž›–”‡ƒ–‡†™‹–Š•‘†‹—„‹•—Žˆ‹–‡‹†—’Ž‹…ƒ–‡—•‹‰–Š‡ͻ͸Ǧ ‡–Š›Žƒ–‹‘‹–ƒ•†‡•…”‹„‡†ƒ„‘˜‡Ǥ‹•—Žˆ‹–‡Ǧƒ’Ž‹ˆ‹…ƒ–‹‘™ƒ•’‡”ˆ‘”‡†‹ †—’Ž‹…ƒ–‡ƒ†“—ƒ–‹–ƒ–‹˜‡‡–Š›Žƒ–‹‘ƒƒŽ›•‹•™ƒ•…‘†—…–‡†—•‹‰–Š‡›”‘ƒ” ʹͶ’›”‘•‡“—‡…‡”ȋ‹ƒ‰‡ǡƒŽ‡…‹ƒǡǡȌȋ•‡‡SupplementaryTableS2ˆ‘”ƒ••ƒ› †‡–ƒ‹Ž•ȌǤ  Replicationinpostmortembraintissue ‘•–Ǧ‘”–‡…‡”‡„‡ŽŽ—•ƒ’Ž‡•™‡”‡‘„–ƒ‹‡†ˆ”‘–Š‡–ƒŽ‡›‡†‹…ƒŽ‡•‡ƒ”…Š •–‹–—–‡ȋ‡—”‘’ƒ–Š‘Ž‘‰›‘ŽŽ‡…–‹‘Ȍȋ͵͵Ȍˆ”‘‹†‹˜‹†—ƒŽ•™‹–Šƒ†‹ƒ‰‘•‹•‘ˆƒŒ‘” †‡’”‡••‹˜‡†‹•‘”†‡”ȋȌȋαͳͶȌƒ†—ƒˆˆ‡…–‡†…‘–”‘Ž•ƒ’Ž‡•™‹–Š‘†‹ƒ‰‘•‡† ’•›…Š‹ƒ–”‹…†‹•‘”†‡”ȋαͳͷȌǤ‡–ƒ‹Ž‡††‡‘‰”ƒ’Š‹…‹ˆ‘”ƒ–‹‘ˆ‘”–Š‡•‡•ƒ’Ž‡•‹•‰‹˜‡ ‹SupplementaryTableS3Ǥ™ƒ•‹•‘Žƒ–‡†ˆ”‘̱͹Ͳ‰‘ˆ–‹••—‡—•‹‰ƒ•–ƒ†ƒ”† ’Š‡‘ŽǦ…ŠŽ‘”‘ˆ‘”‡š–”ƒ…–‹‘’”‘–‘…‘Žǡ•‘†‹—„‹•—Žˆ‹–‡–”‡ƒ–‡†‹†—’Ž‹…ƒ–‡ǡƒ†’”‘ˆ‹Ž‡† —•‹‰–Š‡ ˆ‹‹——ƒ‡–Š›Žƒ–‹‘ͶͷͲ‡ƒ†Š‹’ƒ•†‡•…”‹„‡†ƒ„‘˜‡Ǥƒ•‡•ƒ† …‘–”‘Ž•™‡”‡”ƒ†‘‹œ‡†ƒ†”—ƒ•ƒ•‹‰Ž‡„ƒ–…Š–‘‹‹‹œ‡ƒ›–‡…Š‹…ƒŽ …‘ˆ‘—†‹‰Ǥ‡–Š›Žƒ–‹‘†ƒ–ƒˆ‘”•’‡…‹ˆ‹…’”‘„‡•‘‹ƒ–‡†ˆ”‘‘—”†‹•…‘”†ƒ– –™‹ƒƒŽ›•‹•™‡”‡ƒ••‡••‡†ˆ‘”ƒƒ••‘…‹ƒ–‹‘™‹–ŠƒŒ‘”†‡’”‡••‹‘Ǥ 

͸



‡’•–‡”etal.

 Results SitespecificDNAdifferencesarewidespreadinMZdiscordantdepressiontwins •‹‰ƒƒƒŽ›•‹•‡–Š‘††‡•‹‰‡†–‘‹†‡–‹ˆ›–Š‡Žƒ”‰‡•–ƒ†‘•–•‹‰‹ˆ‹…ƒ–†‹ˆˆ‡”‡…‡• ‹‡–Š›Žƒ–‹‘ƒ–‹†‹˜‹†—ƒŽ’ •‹–‡•ǡ™‡‹†‡–‹ˆ‹‡†—Ž–‹’Ž‡’ •‹–‡•ƒ…”‘••–Š‡ ‰‡‘‡‡šŠ‹„‹–‹‰•‹‰‹ˆ‹…ƒ–†‡’”‡••‹‘Ǧƒ••‘…‹ƒ–‡††‹ˆˆ‡”‡–‹ƒŽ‡–Š›Žƒ–‹‘ǤŠ‡ –‡–‘’Ǧ”ƒ‡††‹ˆˆ‡”‡–‹ƒŽŽ›‡–Š›Žƒ–‡†’‘•‹–‹‘•ȋ•Ȍƒ”‡•Š‘™‹ƒ„Ž‡ͳƒ† Figure1ǡ™‹–Š–Š‡ͳͲͲ–‘’Ǧ”ƒ‡†•Ž‹•–‡†‹SupplementaryTable1ǤŠ‡–‘’ ”ƒ‡†ȋ…‰Ͳ͹ͲͺͲͲͳͻǡȟȾαΪͲǤͲ͹ǡpαͲǤͲͲͲͳȌ‹•Ž‘…ƒ–‡†‹–Š‡ͺ–Š‹–”‘‘ˆ͵ʹ‘ …Š”‘‘•‘‡ͳͲ“͵͸Ǥ͵ǡ‡…‘†‹‰ƒ•‡”‹‡Ȁ–Š”‡‘‹‡‹ƒ•‡‘ˆ—‘™ˆ—…–‹‘–Šƒ–‹• Š‹‰ŠŽ›‡š’”‡••‡†‹–Š‡„”ƒ‹Ǥ‡’”‡••‹‘Ǧƒ••‘…‹ƒ–‡†Š›’‡”‡–Š›Žƒ–‹‘ƒ––Š‹•™ƒ• •—……‡••ˆ—ŽŽ›˜ƒŽ‹†ƒ–‡†„›„‹•—Žˆ‹–‡Ǧ’›”‘•‡“—‡…‹‰ȋpαͲǤͲʹȌȋFigure2ȌǤ‡–Š›Žƒ–‹‘ ƒ––Š‡–Š”‡‡ƒ††‹–‹‘ƒŽ’ •‹–‡•ƒ••‡••‡†‹‘—”„‹•—Žˆ‹–‡Ǧ’›”‘•‡“—‡…‹‰ƒ••ƒ›™ƒ• •–”‘‰Ž›…‘””‡Žƒ–‡†™‹–Š…‰Ͳ͹ͲͺͲͲͳͻȋ”ʹεͲǤͻͺȌ™‹–Š‡ƒ‡–Š›Žƒ–‹‘ƒ…”‘••–Š‡ ƒ’Ž‹…‘„‡‹‰•‹‰‹ˆ‹…ƒ–Ž›ƒ••‘…‹ƒ–‡†™‹–Š†‡’”‡••‹‘ȋpαͲǤͲͳȌǤ —„‡”‘ˆ–Š‡–‘’Ǧ”ƒ‡†•ƒ”‡ƒ••‘…‹ƒ–‡†™‹–Š‰‡‡•–Šƒ–Šƒ˜‡„‡‡’”‡˜‹‘—•Ž› ‹’Ž‹…ƒ–‡†‹†‡’”‡••‹‘ƒ†‘–Š‡””‡Žƒ–‡†‡—”‘’•›…Š‹ƒ–”‹…†‹•‘”†‡”•ȋ‹…Ž—†‹‰Ͷ ȋ͵ͶȌǡͳȋ͵ͷȌǡͳȋ͵͸Ȍǡͷȋ͵͹Ȍǡ͵ͳȋ͵ͺȌƒ† ͵ȋ͵ͻȌǤ‡–™‘”ƒƒŽ›•‹• ‘ˆ–Š‡ͳ͹ͷ‰‡‡•ƒ••‘…‹ƒ–‡†™‹–Š–Š‡ͳͲͲ–‘’Ǧ”ƒ‡†’”‘„‡•—•‹‰ Š‹‰ŠŽ‹‰Š–‡† •‹‰‹ˆ‹…ƒ–‡”‹…Š‡–‘ˆ’ƒ–Š™ƒ›•‹˜‘Ž˜‡†‹‡”˜‘—••›•–‡†‡˜‡Ž‘’‡–ƒ† ˆ—…–‹‘ǡ‡—”‘Ž‘‰‹…ƒŽ†‹•‡ƒ•‡ƒ†’•›…Š‘Ž‘‰‹…ƒŽ†‹•‘”†‡”•ȋSupplementaryTablesS4 6ȌǤ‡”˜‘—••›•–‡†‡˜‡Ž‘’‡–ƒ†ˆ—…–‹‘™ƒ•–Š‡–‘’Ǧ”ƒ‡†„‹‘Ž‘‰‹…ƒŽˆ—…–‹‘ ȋpα͸ǤͳͷǦͲͷȌǡ‹…Ž—†‹‰ƒ—„‡”‘ˆ‰‡‡•”ƒ‡†Š‹‰ŠŽ›‹‘—”’”‘„‡Ǧ™‹•‡ƒƒŽ›•‹•ȋ‡Ǥ‰ǤǢ ͶǡͳǡͳͲǡ ͷȌǤ‡‰‹‘Ž‡˜‡ŽƒƒŽ›•‹•—•‹‰ ‹†‡–‹ˆ‹‡†•‡˜‡”ƒŽ †‡’”‡••‹‘Ǧƒ••‘…‹ƒ–‡††‹ˆˆ‡”‡–‹ƒŽŽ›‡–Š›Žƒ–‡†”‡‰‹‘•ȋ•ȌȋSupplementaryTable S3ȌǤŠ‡–‘’Ǧ”ƒ‡†™ƒ•ƒ͵ͲͲ„’”‡‰‹‘‹–Š‡ͳ•–‹–”‘‘ˆ͵ǡ‡…‘’ƒ••‹‰ Ͷ’”‘„‡•Š›’‡”‡–Š›Žƒ–‡†‹ƒˆˆ‡…–‡†–™‹•ȋȟȾαǦͲǤͲʹͶǢ’αͲǤͲͲͲͷȌȋSupplementary FigureS2ȌǤ ‘—”‰‡‡•Š‹‰ŠŽ‹‰Š–‡†ˆ”‘–Š‡ ”‡‰‹‘ƒŽƒƒŽ›•‹•ƒŽ•‘ˆ‡ƒ–—”‡†‹–Š‡–‘’ ͳͲͲ”ƒ‡†‹†‹˜‹†—ƒŽ’”‘„‡•ˆ”‘–Š‡ƒ‹ƒƒŽ›•‹•ǣ͵ʹȋ–Š‡‘˜‡”ƒŽŽ–‘’”ƒ‡† ’”‘„‡Ȍǡ͸ȋ”ƒ‡†ͳ͸Ȍǡʹ͵ȋ”ƒ‡†͸ͳȌƒ†ʹ ȋ”ƒ‡†͹ʹȌǤ‘‡‘ˆ–Š‡ –‘’Ǧ”ƒ‡†’”‘„‡••Š‘™‡†•‹‰‹ˆ‹…ƒ–†‹ˆˆ‡”‡…‡•„‡–™‡‡ƒŽ‡ƒ†ˆ‡ƒŽ‡•ƒ’Ž‡•Ǥ



͹



‡’•–‡”etal.

  SeveraltoprankedDMPsarealsoassociatedwithMDDinthecerebellum ™‘‘ˆ–Š‡–‡–‘’Ǧ”ƒ‡†•‹†‡–‹ˆ‹‡†‹„—……ƒŽ…‡ŽŽˆ”‘‘—”†‹•…‘”†ƒ––™‹• ™‡”‡ƒŽ•‘ˆ‘—†–‘„‡•‹‰‹ˆ‹…ƒ–Ž›ƒ••‘…‹ƒ–‡†™‹–Š‹’‘•–Ǧ‘”–‡…‡”‡„‡ŽŽ— •ƒ’Ž‡•ǤŠ‹•‹…Ž—†‡†‘—”–‘’Ǧ”ƒ‡†’”‘„‡ǡ…‰Ͳ͹ͲͺͲͲͳͻǡ™Š‹…Š™ƒ•ˆ‘—†–‘„‡ •‹‰‹ˆ‹…ƒ–Ž›Š›’‡”‡–Š›Žƒ–‡†‹…‡”‡„‡ŽŽ—•ƒ’Ž‡•…‘’ƒ”‡†–‘ƒ–…Š‡†…‘–”‘Ž •ƒ’Ž‡•ȋƒ˜‡”ƒ‰‡„‡–ƒ†‹ˆˆ‡”‡…‡αͲǤͲʹǡpαͲǤͲ͵ȌȋFigure2CȌǤ”‘„‡…‰ͲͻͲͻͲ͵͹͸ǡ ƒ’’‹‰–‘–Š‡ͳ•–‹–”‘‘ˆ͹ƒ†”ƒ‡†ͺ–Š‹‘—”†‹•…‘”†ƒ––™‹ƒƒŽ›•‹•ǡƒŽ•‘ ‡šŠ‹„‹–‡†•‹‰‹ˆ‹…ƒ–†‹ˆˆ‡”‡…‡•‹–Š‡’‘•–‘”–‡„”ƒ‹••ƒ’Ž‡•…‘•‹•–‡––‘–Š‘•‡ ‘„•‡”˜‡†‹„—……ƒŽ…‡ŽŽ•ˆ”‘–Š‡†‹•…‘”†ƒ––™‹’ƒ‹”•ȋƒ˜‡”ƒ‰‡„‡–ƒ†‹ˆˆ‡”‡…‡α ͲǤͲ͵ǡpαͲǤͲ͵ȌȋFigure3ȌǤ  ThereisincreasedvariabilityofDNAmethylationindepressedtwins •‡š’‡…–‡†ǡ‘†‹ˆˆ‡”‡…‡•‹‘˜‡”ƒŽŽ‡ƒ‰‡‘‡Ǧ™‹†‡‡–Š›Žƒ–‹‘™ƒ••‡‡ „‡–™‡‡†‡’”‡••‡†ƒ†—ƒˆˆ‡…–‡†–™‹•ȋpαͲǤͺͶȌǤ‘™‡˜‡”ǡ–Š‡†‹ˆˆ‡”‡…‡‹˜ƒ”‹ƒ…‡ ™ƒ••‹‰‹ˆ‹…ƒ–(p