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The Changing Brain

August 18 – 20, 2025 Irvine, California, USA

• The Evolving Brain

• The Learning Brain

• States of the Brain

• The Developing Brain

• The Dynamic Brain

• The Disordered Brain

Paola Arlotta, Harvard University

Xiangmin Xu, University of California, Irvine

Confirmed Speakers: Conference Organizers:

Ishmail Abdus-Saboor, Columbia University

Paola Arlotta, Harvard University

Carlos Brody, Princeton University

Beth Buffalo, University of Washington

Edward Chang, UCSF

Anne Churchland, UCLA

Yang Dan, University of California, Berkeley

Catherine Dulac, Harvard University

Guoping Feng, MIT

Zhigang He, Harvard University

Hailan Hu, Zheijang University, China

Josh Huang, Duke University

Sten Linnarsson, Karolinska Institutet, Sweden

Liqun Luo, Stanford University

Hongkui Zeng, Allen Institute for Brain Science

Guillermina López-Bendito, UMH-CSIC, Spain

Liqun Luo, Stanford University

Michelle Monje, Stanford University

John Ngai, National Institutes of Health

Tom Nowakowski, UCSF

Vanessa Ruta, Rockefeller University

Bernardo Sabatini, Harvard University

Nelson Spruston, Howard Hughes Medical Institute

Karel Svoboda, Allen Institute for Neural Dynamics

Li-Huei Tsai, MIT

Pierre Vanderhaeghen, Leuven Brain Institute of Technology

Hongkui Zeng, Allen Institute of Brain Science

Larry Zipursky, UCLA

Editor-in-Chief

JulioLicinio, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

PublishingManager

Ma-LiWong, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

EditorialBoard

HudaAkil, UniversityofMichigan,AnnArbor,Michigan48109,USA

MauricioArcos-Burgos, UniversidaddeAntioquia,Medellín,Colombia OleA.Andreassen, UniversityofOslo,0318Oslo,Norway

BernhardBaune, UniversityofMünster,48149Münster,Germany

StefanR.Bornstein, TUDDresdenUniversityofTechnology,01307Dresden,Germany

KristenBrennand, YaleUniversitySchoolofMedicine,NewHaven,Connecticut06511,USA

AvshalomCaspi, DukeUniversity,Durham,NorthCarolina27708,USA

MosesChao, NewYorkUniversityLangoneMedicalCenter,NewYork,NewYork10016,USA

SvenCichon, UniversityofBasel,4031Basel,Switzerland

IanDeary, UniversityofEdinburgh,Edinburgh,EH89JZ,Scotland,UK

YogeshDwivedi, UniversityofAlabamaatBirmingham,Birminagm,Alabama35294,USA

StephenFaraone, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

JaniceFullerton, NeuroscienceResearchAustralia&UniversityofNewSouthWales,Randwick,NSW2031,Australia

FredH.Gage, SalkInstituteforBiologicalStudies,LaJolla,California92037,USA

SamuelE.Gandy, IcahnSchoolofMedicineatMountSinai,NewYork,NewYork10029-5674,USA

PatriciaGaspar, INSERMParisBrainInstitute,HôpitalSalpêtrière,75013Paris,France

AnthonyA.Grace, UniversityofPittsburgh,Pittsburgh,Pennsylvania15260,USA

ToddD.Gould, UniversityofMarylandSchoolofMedicine,Baltimore,Maryland21201,USA

RaquelE.Gur, UniversityofPennsylvania,Philadelphia,Pennsylvania19104,USA

Jan-ÅkeGustafsson, UniversityofHouston,Houston,Texas77204,USA

SirJohnHardy, UniversityCollegeLondonDementiaResearchInstitute,London,WC1E6B,UK

NoboruHiroi, UniversityofTexasHealthSanAntonio,SanAntonio,Texas78229,USA.

YasminHurd, IcahnSchoolofMedicineatMountSinai,NewYork,NewYork10029,USA.

SiegfriedKasper, CenterforBrainResearch,MedicalUniversityofVienna,1090Vienna,Austria

KennethS.Kendler, VirginiaCommonwealthUniversity,Richmond,Virginia23298,USA

LorenzoLeggio, NationalInstitutesofHealth,Baltimore,Maryland21224,USA

ChunyuLiu, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

Xin-YunLu, MedicalCollegeofGeorgiaatAugustaUniversity,Augusta,Georgia30912,USA

RobertMalenka, StanfordUniversity,Stanford,California94305,USA

NickMartin, QIMRBerghoferMedicalResearchInstitute,Brisbane,Queensland4029,Australia

AndrewMcIntosh, UniversityofEdinburgh,Edinburgh,EH105HF,Scotland,UK

MariaOquendo, UniversityofPennsylvania,Philadelphia,Pennsylvania19104,USA

SirMichaelOwen, CardiffUniversity,Cardiff,CF244HQ,Wales,UK

AarnoPalotie, InstituteforMolecularMedicine,UniversityofHelsinki,00014Helsinki,Finland

CarlosN.Pato,RutgersUniversity,Piscataway,NewJersey08854,USA

MichelePato, RutgersUniversity,Piscataway,NewJersey08854,USA

MaryL.Phillips, UniversityofPittsburghSchoolofMedicine,Pittsburgh,Pennsylvania15213,USA

RobertPlomin, InstituteofPsychiatryPsychologyandNeuroscienceatKing’sCollege,London,SE58AF,UK

MaurizioPopoli, UniversitàdegliStudidiMilano,20133Milan,MI,Italy

JamesPotash, JohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland21287,USA

JohnRubenstein, UniversityofCalifornia,SanFrancisco,California94158,USA

CarloSala, L’IstitutodiNeuroscienzedelCNR,UniversiyofMilan–Bicocca,20854VedanoalLambro,MB,Italy

AlanF.Schatzberg, StanfordUniversity,Stanford,California94305,USA

JairSoares, UniversityofTexasHealthScienceCenter,McGovernSchoolofMedicine,Houston,Texas77054,USA.

ThomasC.Südhof, StanfordUniversity,Stanford,California94305,USA

KristiinaTammimies, KarolinskaInstitutet,17177Stockholm,Sweden

GiuseppeTesta, UniversitàdegliStudidiMilano,HumanTechnopole,20157Milan,MI,Italy

GustavoTurecki, McGillUniversity,Montréal,QuébecH4H1R3,Canada

MonicaUddin, UniversityofSouthFlorida,Tampa,Florida33612,USA

MyrnaWeissman, ColumbiaUniversity,NewYorkStatePsychiatricInstitute,NewYork,NewYork10032,USA

XiangminXu, UniversityofCalifornia,Irvine,California92697,USA

TakeoYoshikawa, RIKENBrainScienceInstitute,Saitama,351-0198,Japan

MoneZaidi, IcahnSchoolofMedicineatMountSinai,NewYork,NewYork10029,USA

GenomicPsychiatry ispublishedbyGenomicPress.

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GenomicPsychiatry

Volume1 Number2 March2025

EDITORIAL

Fromvulnerabilitytoprotection:ThedualnatureofADNPvariants

INNOVATORS&IDEAS:RISINGSTAR

Pierre-EricLutz:Theroleofepigenomicplasticityintheemergenceandprotractedcourseofpsychiatricdisorders

Pierre-EricLutz

INNOVATORS&IDEAS:RESEARCHLEADERS

KerryJ.Ressler:Exploringthetranslationofamygdalafunctionatthecellularandgenomiclevelstounderstandstress,fear,andtrauma disorders,suchaspost-traumaticstressdisorder(PTSD)

KerryJ.Ressler

CathyBarr:Geneticsandneurobiologyofchildhoodpsychiatricandcognitivedisorders

CathyBarr

YogeshDwivedi:Pre-clinicalandtranslationalresearchfocusingongeneregulationthroughepigeneticandepitranscriptomicmechanisms andtheirimplicationsinmooddisordersandsuicidalbehavior

VIEWPOINT

Theassociationbetweentraceamine-associatedreceptor1(TAAR1)geneticmutationsandneuropsychiatricdisorders

BrittoShajan,TarunBastiampillai,andPramodC.Nair

CirculatinglongnoncodingRNA:Newfrontiersinbiomarkerresearch formooddisorders

BhaskarRoy,AnujKumarVerma…YogeshDwivedi

Rolesofalternativepolyadenylationinpsychiatricdisorderrisk MichellePaff,StevenF.Grieco…XiangminXu

Sexdifferencesinalcoholandtobaccousedisordersamongindividualswithpanicdisorder:across-sectionalanalysisfromthegenomic psychiatrycohort

MichaelJ.Chung,PenelopeGeorgakopoulos…MicheleT.Pato

Protectiveinheritedmutationsinactivity-dependentneuroprotectiveprotein(ADNP):thegood,thebad,andtheugly IllanaGozes,ShulaShazman…JosephLevine

CoverArt:Anartisticvisualizationcapturingthemolecularmechanismsofactivity-dependentneuroprotectiveprotein(ADNP)functioninbraindevelopment.Acentral DNAdoublehelix,renderedinluminescentblue,representstheADNPgene,whilethesphericalstructureswithradiatingpointsillustratethe14-3-3proteinbinding sitescriticalforADNPprotein’snuclear-cytoplasmicshuttling.Thecrystallinetextureofthesemolecularstructureshighlightstheircomplex interactions,whilethesoft orangebokeheffectsinthebackgroundsuggestthebroaderneuronalenvironmentwheretheseinteractionsoccur.Thisvisualizationreflectsthepaper’sfindingsabout protectiveinheritedmutationsinADNPandtheirimpactonneurodevelopment.Formoreinformation,pleasereferto“Protectiveinheritedmutationsinactivity-dependent neuroprotectiveprotein(ADNP):thegood,thebad,andtheugly”byIllanaGozesetal.onpages49-55inthisissue. Coverdesigncreatedthroughextensiveanditerativehuman-AIcollaborationusingClaudeandGrokAIassistants.ThefinalcoverislicensedunderCreativeCommons Attribution-NonCommercial-NoDerivatives4.0InternationalLicense(CCBY-NC-ND4.0).Thiscovermaybereproducedwithoutpermissionunderthe termsofthislicense, providedappropriatecreditisgiventoGenomicPress,andthecontentisnotmodifiedorusedforcommercialpurposes.

Thisissueisnowavailableat https://genomicpress.kglmeridian.com/view/journals/genpsych/genpsych-overview.xml

Genomic Press

GenomicPsychiatry

EDITORIAL

Fromvulnerabilitytoprotection:ThedualnatureofADNPvariants

©TheAuthor(s),2025.ThisarticleisunderexclusiveandpermanentlicensetoGenomicPress

GenomicPsychiatry March2025;1(2):1–3;doi: https://doi.org/10.61373/gp025d.0017

Inthissecondissueof GenomicPsychiatry (1),wehighlightareportby IllanaGozesandherteamfromTelAvivasourcoverarticle.InanindepthGenomicPressinterviewpublishedin BrainMedicine (2),Gozes providedinsightsintoherbodyofworkonactivity-dependentneuroprotectiveprotein(ADNP)(3).HerresearchdemonstratedADNP’sessentialroleinbrainformation,neurodevelopment,generegulation,and proteininteractions.SheidentifiedADNP’sinvolvementinautophagy, schizophrenia,andautismthroughcriticalbindingwithSHANK3and actin.Inthereportpublishedinthisissue,herteampresentsneworiginaldatathatchallengestheconventionalnarrativesurroundinggenetic variation(4).TheauthorsidentifiedanovelvariantofADNP.Whatsets thisapartisthemolecularinsightitprovidesandthephilosophicalramificationsitbearsaboutthenatureofgeneticdeterminism.Moreover, wecanseehereinonepersonwhatmaybehappeningatthepopulationlevel:beneficialvariantsmakedeleteriousmolecularevolutionless deleterious.

ADNPwasfirstidentifiedinGozes’labovertwodecadesago,and itsroleascriticalforcerebraldevelopmentisnowwellestablished(5). Pathogenicvariantsinthissinglegenehavefrequentlybeenassociated withclinicalphenotypesacrossneurodevelopmental,neuropsychiatric, andneurodegenerativespectra.Atthesevereendofthisclinicalarc liesHelsmoortel-VanDerAasyndrome—colloquiallyreferredtoasADNP syndrome—adisordermarkedbydenovoloss-of-functionpathogenic variantsinADNPandcharacterizedbyprofounddevelopmentalimpairments.

Yetaglimmerofbiologicalresilienceemergesamidthatbleak landscapeofpathology.Gozesandcolleagueshavenowidentified aninheritedADNPvariation—ADNP_Glu931Glyfs12—that,paradoxically, appearstoconferprotectionratherthandeficit(see Figure1 foraconceptualrepresentation).Thisvariantwasdiscoveredinamother(VB) whoseadaptivefunctioning,measuredviatherigorousVinelandAdaptiveBehaviorScales,surpassedpopulationaverages.Herson(HB),who inheritedthissame(protective)variantalongsidea denovo pathogenic ADNPvariant(p.Arg730Thrfs∗ 5),demonstratedaclinicalphenotypefar milderthanwouldbeexpectedinthecontextofadual-mutantADNP profile.Thatfindingalonewouldbenoteworthy;themechanismsbehinditelevateittotherealmofscientificprovocation.Ofnote,Chen etal.predictedthatinevolutionaryterms,beneficialmutationspartiallynegativelyoffsetdeleteriousmutations.Theiroutstandingpaper isentitled“FromDrifttoDraft:HowMuchDoBeneficialMutationsActuallyContributetoPredictionsofOhta’sSlightlyDeleteriousModelof MolecularEvolution?”(6).Essentially,Chenetal.concludethatthedeleteriousmodelofmolecularevolutionisindeedultimatelydeleterious. However,itwouldhavebeenevenmoredeleterioushaditnotbeenfor thecompensatingeffectsofbeneficialvariants–whichisexactlythe casewithVB’sson.Itishighlythought-provokingthatGozes’sfindings illustrateinasinglecasewhatmaybehappeningmuchmorebroadly population-wise.

Usingadvancedstructuralmodelingandmolecularanalyses,they showthattheprotectivevariantcreatesanewbindingmotiffor14-3-3

Received:25March2025.Accepted:28March2025. Publishedonline:22April2025.

Figure1. Aconceptualillustrationofabrainwithglowingneuronsformingaprotectivenetwork.ThisisinspiredbyGozesetal.’sfindings,where theADNP_Glu931Glyfs∗ 12mutationenhancesneuroprotectionandresilience againstpathogenicvariantsinneurodevelopmentaldisorders(4).ImagegeneratedbyGrok(xAI,2025)withactiveauthorinput.

proteins—afamilyofcentralchaperonesofintracellulartrafficandsignaling.Morenotably,thisvariantseemstoenhancetheinteractionbetweenADNPandNAPVSIPQ(NAP),anendogenouslyoccurringneuroprotectivepeptidewithintheADNPsequencethathasbeendevelopedas davunetideandstudiedinclinicaltrials.Essentially,thevariantdoesnot just“escapeharm”—ittunestheprotein-proteinandinternalproteininteractionnetworksinwaysthatincreasefunctionalresilience.

Suchfindingshavecausedmetorethinkmyassumptions.Frameshift mutationshavelongbeenconceptualizedasdetrimentalanddisruptiveevents;however,inrarecases,theymaycreatenewfunctionalmotifsthatenhance,ratherthanimpair,proteinfunction.Thisisfarmore thanjustatechnicaldistinction.Itredefineshowweconceptualizegeneticarchitecture—notasabinaryofhealthversusdisease,butasa complexcontinuumwherecertaindisruptionsyieldunanticipatedadvantages.Wecanalmostseethetheoryofevolutioninactionhere: somerare,randomvariantsofferadvantagesandmaybeselectedover generations.

Theimplicationsareimmense.Protectivemutations—raregenetic chanceeventsthatgivetheircarriersabiologicallegup—havehistoricallygottenlessplaythanpathogenicones.In2010H.AllenOrr statedthat“thepopulationgeneticstudyofadvantageousmutationshas laggedbehindthatofdeleteriousandneutralmutations”(7).Yetthey mightprovidethekeytotherapeuticmimicry.Soifnature,viaevolutionaryhappenstance,makesvariantsthatbufferagainstdisease,then

pharmacologicalapproachesthatmimictheseconfigurationsmightbe bothpossibleandprofoundlyeffective.Saneetal.haverecentlyshown that“shiftsinmutationspectramayevolveunderselectionandcandirectlyaltertheoutcomeofadaptiveevolutionbyfacilitatingaccessto beneficialmutations”(8).

TheGozesreportpaystributeasmuchtostructuralbiologyasto translationalimagination.Theteam’scomputationalmodelsprovideavisualandmechanisticbridgefromsequencevariationtophenotypicoutcome,illustratinghowastructure-functionanalysisconvertsgenotype intoactionableinsight.Identifyingenhancedinteractionswith14-3-3 andSH3domainsfurthersolidifiesthatspecificvariantsdonotexistin isolation;theyparticipateinproteincrosstalkthatmayrewireentiresignalingpathways.

Thiscase,elegantinitsanomaly,alsoillustratesthestaggering complexityofneurodevelopmentalsyndromes.Withinthesamegene— ADNP—mutationalheterogeneityproduceswildlydivergentclinicaltrajectories.Itispreciselythisvariancethatunderscorestheurgencyofprecisionmedicine.Thepatientisnotthevariantbutthesumofvariants, modifiergenes,andenvironment.Onlybymappingthisintricateconstellationcanwehopetointervenewithprecision.

Thetherapeuticechoofthisstudyismostpalpableinitsrekindlingof interestindavunetide.Longinvestigatedasaneuroprotectiveagentin murinemodelsandhumantrials,itsinteractionwiththepathwaymodulatedbytheprotectivevariantbreathesnewrelevanceintoitspharmacologictrajectory.Couldtheefficacyofdavunetideinbroaderclinicalpopulationsbesharpenedingeneticallydefinedsubgroups?Thedata suggestso.

Interestingly,cerebrospinalfluidbiomarkersofsynapticdysfunction, including14-3-3arealteredinParkinson’sdiseaseandrelatedneurodegenerativetauopathies(9),withtauopathiesbeingtargetedinpreviousdavunetideclinicaltrials.Furtherspeakingofgeneticdifferences, onethatisglaringandoftenignoredissexdifferences.Importantly,in collaborationwiththeToyo-Okagroup,theGozesgroupshowedthat thecytoplasmiclocalizationofADNPthrough14-3-3promotessexdependentneuronalmorphogenesis,corticalconnectivity,andcalcium signaling(10).Inthisrespect,theGozesgroupfurtherdiscoveredunexpectedsexdifferencesinthepureneurodegenerativetauopathyprogressivesupranuclearpalsy(PSP),revealingfasterdeteriorationinwomen. Sexstratificationofaplacebo-controlledPhase2/3studyclinicaltrial resultsshowedthatdavunetideoffersstatisticallysignificantneuroprotectivebenefitsinfemalesubjectsinoneoftheco-primaryendpointsofthestudy,theSchwabandEnglandActivitiesofDailyLiving (SEADL)scale.Analysisofthesecondco-primaryendpoint,PSPRatingScale(PSPRS),revealedthatwhiledavunetidehadtrendingbeneficialeffectsinthefemalesubjectpopulation,themalesubjectpopulationshowedastatisticallysignificantdeteriorationcomparedtoplacebo, stronglyindicatingasex-basedeffectofdavunetide(11).Assuch,ExoNavisTherapeuticsLtd.Isdevelopingdavunetideforwomensufferingfrom PSP(12).

Furthermore,theGozesgrouprecentlyshowedthatADNPisessentialforsex-dependenthippocampalneurogenesis,throughmaleunfoldedproteinresponseandfemalemitochondrialgeneregulation,with davunetide’sprotection(13).Assuch,davunetideisfurtherbeingdevelopedforADNPsyndrome(ExoNavis).

Fromafutureboldstrategicvantage,thenextstepsareclear.Adeeper structuralinterrogationofADNPvariants—pathogenicandprotective— isneeded.Simultaneously,theidentificationofsmallmoleculesthatenhance14-3-3orSH3domaininteractionsopensfertilegroundfordrug development.Thesearenotmarginalpursuits;theyrepresentaconceptualpivotfromreactivemedicinetowardanticipatorydesign.

Thereis,too,apersonalresonance.Myownworkontheleptin pathway—initiallyanarrowinvestigationintoanexceedinglyrareobesitysyndrome—ultimatelyrevealedprinciplescentraltometabolism,behavior,andendocrineregulation(14, 15).Inasimilarfashion,thissingle protectiveADNPvariantmayilluminatepathwaysfundamentaltocognition,synapticintegrity,andneuroprotection.Theoutlierisoftenthe oracle.

Thisstudy,initsfusionofmolecularrigorandclinicalnuance,reminds usthatgeneticdiagnosesarenotdeterministicverdicts.Theyaredynamic startingpoints.Whatmattersisthecontext—theprecisenatureofthe variant,thebackgroundinwhichitoccurs,andthedownstreamnetworks itengagesordisrupts.TheADNP_Glu931Glyfs∗ 12variantisnotaglitch. Itis,ratherastonishingly,aprotectivesignaturewritteninthelanguage oferror.

Atitscore,thisworkreaffirmstheeleganceofmolecularneuroscience—thebeautyofwatchingmolecularshiftsrippleintohuman behaviorandtheaudacityofattemptingtounderstandthemindbyinterrogatingthemolecule.Intheconfluenceofcomputationalmodeling,geneticanalysis,andclinicalobservation,weareremindedofwhat genomicpsychiatryatitsbestcanachieve.

Asweadvanceintoanerawherewenolongeraskwhatagenedoes ingeneralbutratherwhatitdoesinaparticularpatient,theGozespaper emergesasacasestudyofscientificcreativityandbiologicalhumility.It suggeststhatnotallgeneticmistakesareerrors.Someareinnovations— quietrevolutionsetchedintothegenome,awaitingdiscovery.

JulioLicinio1 1 Editor-in-Chief, GenomicPsychiatry

,GenomicPress,NewYork,NY10036,USA e-mail: julio.licinio@genomicpress.com

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12.ExoNavisTherapeutics.ADNPSyndrome.Phase.32025[Availablefrom: https:// exonavis.com/adnp-clinical-trial-phase-3/

13.ShapiraG,KarmonG,Hacohen-KleimanG,GanaiemM,ShazmanS,TheotokisP,etal. ADNPisessentialforsex-dependenthippocampalneurogenesis,throughmaleunfoldedproteinresponseandfemalemitochondrialgeneregulation.MolPsychiatry. 2024.DOI: 10.1038/s41380-024-02879-w.PMID:39715923

14.LicinioJ,CaglayanS,OzataM,YildizBO,deMirandaPB,O’KirwanF,etal.Phenotypic effectsofleptinreplacementonmorbidobesity,diabetesmellitus,hypogonadism,and behaviorinleptin-deficientadults.ProcNatAcadSciUSA.2004;101(13):4531–36. DOI: 10.1073/pnas.0308767101.384781.PMID:15070752;PMCID: PMC384781

15.Paz-FilhoG,WongML,LicinioJ.Tenyearsofleptinreplacementtherapy.ObesRev. 2011;12(5):e315–23.DOI: 10.1111/j.1467-789X.2010.00840.x.PMID:21410864

withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

Modifiedversionsoftheworkcannotbedistributed.(4)Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulatedinthelicense.Public domainmaterialsorthosecoveredbystatutoryexceptionsareexemptfromthese terms.Thislicensedoesnotcoverallpotentialrights,suchaspublicityorprivacy rights,whichmayrestrictmaterialuse.Third-partycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunlessotherwisestated.Ifuseexceeds thelicensescopeorstatutoryregulation,permissionmustbeobtainedfromthe copyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/ licenses/by-nc-nd/4.0/.Thelicenseisprovidedwithoutwarranties.

GenomicPsychiatry

INNOVATORS&IDEAS:RISINGSTAR

Pierre-EricLutz:Theroleofepigenomicplasticityintheemergenceandprotracted courseofpsychiatricdisorders

©TheAuthor(s),2024.ThisarticleisunderexclusiveandpermanentlicensetoGenomicPress

GenomicPsychiatry March2025;1(2):4–6;doi: https://doi.org/10.61373/gp024k.0037

Keywords: epigenomics,DNAmethylation,addiction,depression, neuro-epigenetics

Pierre-ÉricLutz,apermanentresearcheratFrance’sCNRS(Centre NationaldelaRechercheScientifique)andStrasbourg’sInstituteof CellularandIntegrativeNeuroscience,investigatesepigenetic mechanismsunderlyingbraindisorderssuchasaddictionand depression.Hislabemploysgeneticengineeringinmice, high-throughputmultiomicsinmousemodelsandhumancohorts,and bioinformatics.WearethrilledthatinthisGenomicPressInterview DrLutzsharesinsightsintohislifeandcareerwithourreaders.

Part1:Pierre-ÉricLutz–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience?

IwasbornandraisedinFrance,inafamilyinwhichbeingamedicaldoctor haslongbeenthedefaultoption.Asateenager,Iwasprimarilyinterested inplayingvideogames,readingbooks,andlisteningtomusic.Attheend ofhighschool,IonlyhadavagueideaofwhatIwantedtodowithmy life.Surprisinglyenough,Iendedupenteringmedicalschool.Ittookme awhiletorealizethatmymaininterestwasunderstandingdisease’sbiologicalmechanisms.Therefore,Ioptedtotakeundergraduatecoursesin molecularbiologyandbiostatisticsinadditiontomedicaltraining.Then, duringmyresidencyingeneralmedicine,Icompletedinternshipsinthe psychiatryandneurologydepartments.Thisgavemetheopportunityto workinafacilitydedicatedtothemanagementofpatientswithsevere addictions,whowerenotablyreceivingopioidsubstitutiontherapy.Duringtheseinternships,Iwasdeeplymovedbythepainfullifehistoriesof peopleaffectedbychronicpsychiatricdisorders.ThatiswhenIdecided Iwantedtounderstandhowbiological,socioeconomic,andculturalfactorsinteracttotriggersuchprofoundmaladaptivebehaviors.Inparallel tofinishingmyresidency,Icompletedmybasicsciencetrainingwitha Master’sinneuroscience.Then,IengagedinaPhD,duringwhichIworked inthelaboratoryofProfessorBrigitteKiefferattheInstituteofGenetic andMolecularandCellularBiology(IGBMC)andtheUniversityofStrasbourg,France.There,Igotfamiliarwithgeneticmanipulationsandbehavioralparadigmsinthemouse,whichweusedtounderstandbetterhow opioidreceptorsmediatetheunmatchedanalgesicpropertiesofopioids (drugssuchasmorphineorfentanyl)butalsorepresententrypointsto thedevelopmentofsubstanceuseandanxiodepressivedisorders.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourcurrentrole.Whatdefiningmomentschanneledyoutowardthis opportunity?

AfterobtainingmyMDandPhDdegreesin2012,Ihadtwosignificant objectives.First,becauseIhaddevelopedastronginterestintheemergingfieldofneuro-epigenetics,IwaslookingforalabwhereIcouldget traininginhigh-throughputsequencingapproachesandrelatedbioinformaticanalyses.Second,Iwantedtoworkinanenvironmentwitharobustclinicalperspectiveorientedtowardsthebiologicalunderstanding

Received:27May2024.Accepted:7June2024. Publishedonline:14June2024.

Pierre-ÉricLutz,MD-PhD,CentreNationaldelaRechercheScientifique,France.

ofbehavioralderegulation.ThelaboratoryofProfessorGustavoTurecki attheDouglasResearchCenter&McGillUniversityinMontréal,Canada wastheidealplacetoachievethesetwogoals.Ispentthreeyearsthere, from2013to2015,thankstofundingfromtheFyssenFoundation,the CanadianInstitutesofHealthResearch(CIHR),andtheAmericanFoundationforSuicidePrevention(AFSP).Myprojectsfocusedontheroleof epigeneticplasticityinthelong-termconsequencesofchildhoodmaltreatment,oneofthestrongestriskfactorsforpsychopathology.The originalityofourapproachwasthatweconductedmolecularanalyses ofhumanpostmortembrains,availablefromtheDouglasBellCanada BrainBank.Thisbankrepresentsauniqueresourceasitcollectsand providesaccesstopostmortemtissuesand,importantly,alsoperforms

Figure1.

Figure2. TheoldcitycentreofStrasbourg,France,knownas“PetiteFrance,”appearseerilyquietanddesertedduringtheCOVID-19pandemic.Thepicturesque half-timberedhousesandemptycobblestonestreetsalongthecanalarebathedinasoft,ethereallight,highlightingthehistoricarchitectureintheabsence oftheusualbustlingcrowdsandvehicletraffic.ThisstrikingimagetakenbyPierre-ÉricLutzcapturesasurrealmomentintime,showcasingthebeauty and tranquilityoftheiconicneighbourhoodunderunprecedentedcircumstances.

psychologicalautopsiestoretrospectivelyassessthedemographic,clinical,anddevelopmentalhistoriesofthedonors.Ourresultsnotablyprovidedevidencesuggestingthatchildhoodmaltreatmentmayepigeneticallyreprogramcriticalbrainphysiologicalsystems(opioidreceptors) andcelltypes(oligodendrocytes)inthehumanbrain.Followingthatpostdoctoralexperience,IreturnedtoFrancein2016tojointhelaboratoryof DrsIpekYalcinandMichelBarrotattheInstituteofCellularandIntegrativeNeuroscienceinStrasbourg(figure2).Duringthistransitionperiod anduntil2018,IcontinuedtoworkontheprojectthatIhadinitiatedin Montréal,whilealsoparticipatingintheprojectsofmyhostlab.In2019, IwasrecruitedasapermanentresearcherbytheFrenchNationalCenter forScientificResearch(CNRS),andIhavesinceinitiatedmyownlineof investigation.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteresearchorprofessionalfocusarea. Ihavealwaysbeeninterestedintryingtounderstandhowthebiological processesinthebrainmayunderlieourbehaviorsandhelpexplainwhy peoplefeelsadorengageindangerousorseeminglyirrationalbehaviors. Atthesametime,duringmymedicalstudies,Idevelopedastrongtaste forgenomicsandmolecularbiology.Untilrecently,bridgingthegapbetweenthesetwoepistemologicallydistantlevelsofanalyseswasdifficult. Thishaschangedwiththeadventofneuro-epigenetics.Thisyoungdisciplineseekstodecipherhowcomplexbehavioraltraitsmaybemediated bychangesintheconformationandfunctionalpropertiesofthegenome. Myinterestinthattypeofresearchemergedduringmypost-docyears, asIusedtositnexttobioinformaticiansworkingattheircomputers.They wouldshowmesequencesofDNAordatafromlibrariesIhadgenerated fromhumanbrains,andwewouldtrytoidentifygenomicsiteswhere, forexample,changesinDNAmethylation,aprimaryepigeneticsubstrate, maycontributetothedysregulationofgenesandphysiologicalprocesses involvedinemotionalregulation.Inmylab,wenowfollowthatframework.Wetrytoleveragethepowerofthelaboratorymouse,whichallows ustomimicmaladaptivebehaviorsincontrolledexperimentalsettings, withtheinvestigationofhumancohorts,usingeasilyaccessibleperipheraltissues(suchasblood),orpostmortembrains(thankstoacontinuous collaborationwithMontréal).

Whatimpactdoyouhopetoachieveinyourfieldbyfocusingon specificresearchtopics?

Ihopemyworkwillrepresentatinycontribution,amongthoseofmany others,toabetterrecognitionofbiologicaldeterminantsofmental health.Theobjectiveisthatthisunderstandingmayhelpdecreasethe stigmathatisstillstronglyassociatedwithpsychiatricdisease.

Pleasetellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience?

Mylabiscurrentlyworkingontwomainfronts.First,werefinetheway wemodeladdiction.Todoso,wearecurrentlydevelopingaparadigmin whichmicelearntoexpressavoluntarybehaviorthattriggersthedelivery,directlyintothebrain,ofaflashoflightthat,thankstooptogenetictools,hasreinforcingeffects.Withtherepetitionofthisbehavior progressivelyemergecompulsive-likeresponsesthatincluderesistance topunishment,aberrantmotivationtogetthereinforcingstimulus,or persistentresponsewhenitisnolongeravailable.Becausethismodel canbeimplementedrelativelyquicklyinthemouse,itopensthedoor totargetedmanipulationsoftheepigeneticmachinery(forexample,enzymesresponsibleforDNAmethylation)toassesstheirroleintheaforementionedcomplexbehaviors.Second,likeothers,weareinterestedin improvingthedepthandresolutionatwhichwemeasureandanalyze epigeneticlandscapes.FocusingprimarilyonDNAmethylation,weare interestedinusinglong-readsequencingandnovelcomputationalapproachestoidentifymethylationpatternsatthelevelofsinglereads, i.e.,singlealleles.Thechallengewenowfaceistoefficientlydevelopand combinethesetwolinesofworkthatrequireabroadrangeofexpertise,fromexperimentalpsychologytobioinformaticsandcomputational biology.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour researchenvironment?

IhavehadtheprivilegetoworkduringmyPhDunderthesupervisionof ProfessorBrigitteKieffer(France)andmypost-docwithProfessorGustavoTurecki(Canada).FromBrigitte,Ivividlyrememberthetimeandenergyshewasreadytodevotetohertraineesforone-on-onesessions.To Gustavo,IamgratefulforthemanyopportunitiesIwasgiventodevelop asascientist,withthelibertytopursuemyintuitionsandinterests.Inow trytoreproducethese2aspectsinmygroup.

AtGenomicPress,weprioritizefosteringresearchendeavorsbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Arethereparticular culturalfacetswithinthescientificcommunitythatwarrant transformativescrutiny,oristhereacausewithinsciencethatdeeply stirsyourpassions?

Ibelieveitisimportantthatscientificresearchremainsadomainwhere criticalthinking,doubts,andhumilityarepracticalrealities.Therefore, Iamgenerallyinfavorofattitudesorinitiativesthatseektominimize tendenciestowardpersonalization,storytelling,oroverstatements.

Whatdoyoumostenjoyinyourcapacityasanacademicorresearch risingstar?

IcherishthelibertyIhavetoperiodicallydecideonthefocusofmyresearch.IalsoenjoythefactthatIhavetoconstantlyevolveandlearnnew thingstotrytoremaincreativeandproductive.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

Outsideofwork,Ispendmostofmytimewithmywifeandkids.Weenjoy goingtorestaurants,seeingclassicorfunnymovies,travelling,anddoing manyotherthings!

Part2:Pierre-ÉricLutz–SelectedquestionsfromtheProust Questionnaire1

Whatisyourideaofperfecthappiness?

Beingperfectlyhappyseemslikeadangerousidea.

Whatisyourgreatestfear? Politicalextremism.

Whichlivingpersondoyoumostadmire?

Iamafanofpeoplewhodaretosettheirownpathandareenthusiastic aboutwhattheydo.

Whatisyourgreatestextravagance?

Myaverageextravaganzalevelisrelativelylow,butIkeepgettingthose remarksaboutmyshoes.

Whatareyoumostproudof?

Itrynottotakeprideinmuchofanything.

Whatisyourgreatestregret?

IwishIhadlearnedtocodeearlierinmycareer.

Whatisthequalityyoumostadmireinpeople? Humility.

Whatisthetraityoumostdislikeinpeople? Selfishness.

Whatdoyouconsiderthemostoverratedvirtue? Pride.

Whatisyourfavoriteoccupation(oractivity)? Readingthenews.

Wherewouldyoumostliketolive?

Anyplacewherethetemperaturenevergetshigherthan25°C/77°F.

1 Inthelatenineteenthcentury,variousquestionnaireswereapopulardiversion designedtodiscovernewthingsaboutoldfriends.Whatisnowknownasthe35questionProustQuestionnairebecamefamousafterMarcelProust’sanswersto thesequestionswerefoundandpublishedposthumously.Proustansweredthequestionstwice,atages14and20.In2003Proust’shandwrittenanswerswereauctioned offfor$130,000.Multipleotherhistoricalandcontemporaryfigureshaveanswered theProustQuestionnaire,includingamongothersKarlMarx,OscarWilde,ArthurConanDoyle,FernandoPessoa,StéphaneMallarmé,PaulCézanne,VladimirNabokov, KazuoIshiguro,CatherineDeneuve,SophiaLoren,GinaLollobrigida,GloriaSteinem, Pelé,Valentino,YokoOno,EltonJohn,MartinScorsese,PedroAlmodóvar,Richard Branson,JimmyCarter,DavidChang,SpikeLee,HughJackman,andZendaya.The ProustQuestionnaireisoftenusedtointerviewcelebrities:theideaisthatbyansweringthesequestions,anindividualwillrevealhisorhertruenature.WehavecondensedtheProustQuestionnairebyreducingthenumberofquestionsandslightly rewordingsome.Thesecuratedquestionsprovideinsightsintotheindividual’sinner world,rangingfromnotionsofhappinessandfeartoaspirationsandinspirations.

Whatisyourmosttreasuredpossession? Allthosethatarenotmaterial.

Whenandwherewereyouhappiest?Andwhyweresohappythen? EverytimeIwasconfrontedwithadifficultconcept,readingorsetofexperimentaldata,Ihadtoslowlyacquireanddigestnewknowledgeinordertoseethebeginningofanunderstanding.Thereisfulfillmentinthese modestpersonaljourneys.

Whatisyourcurrentstateofmind? Iamworriedaboutsocioeconomicinequalities,climatechange,political risk,andtheworldwearepassingontothenextgeneration.

Whatisyourmostmarkedcharacteristic? Itrytoremainobjectiveandtempered.

Amongyourtalents,whichone(s)give(s)youacompetitiveedge? IdonotthinkIhaveanyparticulartalent.

Whatdoyouconsideryourgreatestachievement? Mytwosons,obviously,ateameffortwithmysignificantother.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe? Mycommunicationskills.

Whatdoyoumostvalueinyourfriends? Kindness.

Whoareyourfavoritewriters?

Louis-FerdinandCéline,ThomasMann,andJulienGracq.

Whoareyourheroesoffiction? TheCountofMonteCristo.

Whoareyourheroesinreallife? MilesDavis.

Whataphorismormottobestencapsulatesyourlifephilosophy? Mouronspourdesidées,d’accord,maisdemortlente (GeorgesBrassens).

Pierre-EricLutz1

1 FrenchNationalCentreforScientificResearch,CNRS,InstituteofCellularand IntégrativeNeuroscience,INCICNRSUPR3212,67000Strasbourg,France e-mail: pierre-eric.lutz@cnrs.fr

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

OpenAccess. ThisarticleislicensedtoGenomicPressundertheCreativeCommonsAttribution-NonCommercial-NoDerivatives4.0InternationalLicense(CCBY-NC-ND4.0).Thelicensemandates:(1)Attribution:Credit mustbegiventotheoriginalwork,withalinktothelicenseandnotificationofany changes.Theacknowledgmentshouldnotimplylicensorendorsement.(2)NonCommercial:Thematerialcannotbeusedforcommercialpurposes.(3)NoDerivatives: Modifiedversionsoftheworkcannotbedistributed.(4)Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulatedinthelicense.Public domainmaterialsorthosecoveredbystatutoryexceptionsareexemptfromthese terms.Thislicensedoesnotcoverallpotentialrights,suchaspublicityorprivacy rights,whichmayrestrictmaterialuse.Third-partycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunlessotherwisestated.Ifuseexceeds thelicensescopeorstatutoryregulation,permissionmustbeobtainedfromthe copyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/ licenses/by-nc-nd/4.0/.Thelicenseisprovidedwithoutwarranties.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

KerryJ.Ressler:Exploringthetranslationofamygdalafunctionatthecellularand genomiclevelstounderstandstress,fear,andtraumadisorders,suchas post-traumaticstressdisorder(PTSD)

©GenomicPress,2025.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry March2025;1(2):7–10;doi: https://doi.org/10.61373/gp025k.0005

Keywords: Amygdala,post-traumaticstressdisorder(PTSD),genomic, translation,circuit,fear,threat

Apioneeringforceinpsychiatricneuroscience,Dr.KerryRessler divideshistimebetweenservingasChiefScientificOfficeratMcLean Hospital,ProfessorofPsychiatryatHarvardMedicalSchool,and translationalneuroscientist.Drawingfrombothmolecularbiology andhumangenetics,hehasfundamentallychangedhowwe understandfearandanxietyinthebrain,especiallythroughhis innovativeresearchontheamygdala.Throughouthisremarkable career,whichincludesover500publishedpapers,hehasuncovered criticalinsightsintothegeneticandepigeneticbasisof post-traumaticstressdisorder(PTSD)andrelatedanxietydisorders. HisexpertisehasearnedhimmembershipintheNationalAcademyof MedicineandatermaspresidentoftheSocietyforBiological Psychiatry.Dr.Resslerco-directsthePsychiatricGenomicsConsortium PTSDWorkgroupandfoundedtheGradyTraumaProjectinAtlanta beforejoiningMcLeanHospital.ThisGenomicPressInterviewoffers anintimatelookatthepathandperspectivesofascientistwhohas shapedmodernpsychiatricresearchandtreatment.

Part1:KerryRessler–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience?

IgrewupintheDeepSouth–southernMississippi,Jackson,andthen OceanSprings,MS–first-generationcollege,anonlychildwithanincrediblesinglemom.Ihadafewfantasticteachersinhighschool,includinga greatscienceteacherandamathteacher,whohelpedmetounderstand therewereoptionsbeyondthelocalorregional.Iwasachildoftheearly personalcomputergenerationintheearly’80s,enjoyedcodingandthe local(phone-based)toolsandnetworksthatwerethepredecessortothe internet.

Withmyscienceteacher’s(Ms.PatDye)help,Iwasfortunatetobe admittedtoMITinCambridge,whereIhopedtostudycomputerscience andartificialintelligence.Alongtheway,Iwasexposedtothemolecular biologyrevolution(includinghavingasmallseminarclasswithSalvador Luriaandothermolecularbiologygurus)andtheearlydaysofsystems neuroscience–andmymindwasblown.Whileinthe’80s,artificialintelligencewasmerelyatheory,itwasveryclearthatthemolecular neurosciencerevolutionwastakingoff–andIwasmoreexcitedabout understandinghowbrainscreatelearning,memory,andemotionthanI wasabout’artificially’mimickingit.

IwasfortunatetobeabletoattendHarvard’sMD/PhDprogramin theearly’90s.Whiletherewerenotlabsfocusedasmuchonmyprimary interest–learning,memory,andemotion–IhadtheserendipitousopportunitytohearLindaBuckgiveherexcellentjobtalkaboutherdiscovery oftheodorantreceptorgenefamilyinratsinRichardAxel’slab.Ifound outthatitwasajobpresentationandthatshewouldsoonstarthernew

Received:20January2025.Accepted:23January2025. Publishedonline:4February2025.

labatHarvardMedicalSchool.Igotthenerveuptocallherandaskif Icouldworkinherlab–shegraciouslywelcomedme,andwithinafew weeks,wehadclonedthemouseodorantreceptorgenefamilybasedon thepriorratsequences.Itwasanextraordinarytimetobeinscience–focusedonadiscoverythatrapidlyledtoanewunderstandingof themolecularandanatomicallogicofapreviouslymysterioussensorysystembasedongenomicsadvances.InadditiontotheunbelievableopportunitytojoinLindainStockholmin2004fortheNobel Prizeceremony,thisworkinspiredmetospendmycareerusingmoleculargenomictoolstoasksystemsneurosciencequestionsrelatedto Psychiatry.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourmostrelevantleadershiprole.Whatdefiningmoments channeledyoutowardthatleadershipresponsibility?

IwenttoEmoryforresidencyinPsychiatryforseveralreasons:CharlieNemeroff,theEmoryChair,wasoneofthemostvisionaryandenergeticleadersinbiologicalpsychiatryinthe’90s;TomInsel,whowent ontobethenextNIMHdirector,initiatedtheCenterforBehavioral

Figure1. KerryJ.Ressler,MD,PhD.McLeanHospital,HarvardMedicalSchool, USA.

Figure2. KerryResslerandfamilyenjoyingahikeintheColoradoRockyMountains.Theylovetocamp,offthegrid,highupintheRockies,toescapesomeofthe chaosofthemodernworld,andenjoythebeautyandserenityofnature.

NeuroscienceinAtlanta–oneofthefirstandlargestcentersfocusedon neurosciencemechanismsofbehaviorwhichwasawonderfulcommunity tobeapartof;andMikeDavis,oneofthetrueleadersofourcurrentunderstandingofAmygdalafunction,hadbeenrecruitedbyCharlietomove fromYaletoEmory.AftertalkingwithMike,hislabofferedauniqueopportunityforcollaborationandtrainingforustoworkonthemolecularmechanismsofamygdalafunctioninfear,threat,andtrauma-related disorders;thiswasinparallelwithremarkablementorshipandcollaborationwithBarbaraRothbaum,aleaderinanxiety-relateddisordersand PTSD.

AsIgrewmycareeratEmory,runningabasicsciencelabfocusedon amygdalamechanismsoffearprocessinginmice,aswellasaclinicallab focusedonunderstandingthehumanbiologyofPTSD,IalsoservedonadmissionsandexecutivecommitteesfortheNeuroscienceGraduateProgramandMedicalSchoolandMD/PhDprograms,eventuallybecoming thedirectoroftheEmoryMedicalScientistTrainingProgram,andwriting/leadingEmoryPsychiatry’sNIMHR25foraresearchresidencytrack. Afteralmost20yearsatEmory,Imovedin2015toMcLeanHospital/HarvardMedicalSchoolto’tradein’mymultipleadministrativerolesforone relativelyfocusedontranslatingneurosciencetopsychiatryinmycurrentroleasChiefScientificOfficeratMcLeanHospital.McLean’sbreadth anddepthofpsychiatryclinicalexpertisewithanenormousamountof preclinicalandclinicalresearchprogramsfocusedonpsychiatricneuroscience–makeitanalmostuniqueandwonderfulplaceforworkingon translationalopportunitiesinpsychiatry.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteresearchorprofessionalfocusarea.

LearningfromMikeDavisandthebroaderamygdalafield,includingJoe LeDoux,MichaelFanselow,andtheiracademicoffspring,particularlyat thepreclinicallevel,howexcitingandwell-understoodtheamygdala,its connectivity,anditscausalassociationwithbehavior.Tome,thisseemed themosttractablecurrentcircuitforunderstandingatamolecularand cellularleveltoaddressspecificbehaviors(e.g.,fear/trauma)–I’vebeen workingonthisgeneralproblemeversince.

Whatimpactdoyouhopetoachieveinyourfieldbyfocusingon specificresearchtopics?

Mostproximally,Ihopethatourworkmayleadtonovelapproaches(pharmacological,neurostimulation,behavioral,etc.)tofear-andtraumarelateddisorders,perhapseventopreventPTSDdevelopmentinthe firstplace,bybetterunderstandingthebiologicalprocessesoffear-and trauma-memoryconsolidationandinterveningintheemergencydepartment,onthebattlefield,oraftermassdisasters.Beyondourspecific areas,thefieldofPsychiatryisindesperateneedofearly’wins’demonstratingthataneurobiologyand/orgenomicunderstandingofanyofour disordersaresufficientlymaturetoleadtoscience-drivenrationalinterventionapproachestoPsychiatricdisease.

Pleasetellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience.

Ourbroaderlabfocusesonbothtop-downandbottom-uptranslational approachestounderstandingfear-andtrauma-relateddisorders.Specifically,atthehumanlevel,Iamaco-PIofthePsychiatricGenomicConsortium–PTSDworkinggroup–workingwithCarolineNievergelt,Karestan Koenen,MurrayStein,andourmanyexcellentcollaborators–wehave recentlypublishedthelargestGWASto-dateofPTSD–including > 1M subjects,with ∼100GWASsignificantloci(Nievergeltetal., Nature Genetics,2024)–sothatweareindeedapproachingageneticarchitectureofPTSD.Inparallel,wehaverecentlypublished,withNikos Daskalakis,CharlieNemeroff,JoelKleinman,andcolleagues,amongthe largestpostmortembrainmultiomic(RNA,Protein,andepigenetic)along withsnRNAseqdatasetsofPTSDanddepressionvs.controls(Daskalakis etal., Science,2024).Thesedataprovidea’groundtruth’ofthehuman brainindisease,complementarytothelarge-scaleGWASdata,theextensiveneuroimagingandphysiologystudies,andlargeprospectivestudies ofPTSDfollowingtrauma–sothatthefieldisdevelopingtruehumanbiologytounderstandthesedisorders.Inparallel,wecontinuetoapplyallthe great’new’circuittoolsinmice,including,amongothers,cellularcalcium imaging,single-cellsequencing,intersectionalopto-andchemogenetics, tounderstandthemechanismsofthesecircuits,pathways,andmolecules

inamodelsystem(e.g.,Hartmannetal., PNAS,2024;Maddoxetal., MolecularPsychiatry,2024;Hiseyetal., BiolPsychiatry,2023;McCulloughetal., NatureComm,2020;Maddoxetal., Neuron,20195.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour researchenvironment?

Thegold-standardapproachesoflogic,precision,replication,testablehypotheses,andreductionisticquestionsholdupacrossmultiplelevelsof analyses.LindaBuckandMichaelDavis,mygraduateandpostdocmentors,areremarkable,thoughtful,creative,andprecisescientistswhoprovidedamazingexamplesandmentorshiptomeduringmydevelopment. Tothisday,ItrytothinkaboutcomplexproblemsinareductiveandlogicalwayasbestIcan,basedonobservingtheirinsightful,creative,and persistentapproaches.

AtGenomicPress,weprioritizefosteringresearchendeavorsbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Areparticularcultural facetswithinthescientificcommunitywarranttransformative scrutiny,oristhereacausewithinsciencethatdeeplystirsyour passions?

Mybiggestconcernregardingscienceandsocietyatthispointrelates tothecurrentsenseofananti-sciencemovement,particularlyinwhat weusedtoconsider’Westernliberaldemocracies’thatsuggesttremendousskepticismofscienceandthescientificapproachbythepublic.This isinpartrelatedtotheexponentialgrowthinsocialmedia,inwhich theloudestvoiceseemstobeseenasthe’mosttrue,’witharealdilutionoffact-anddata-basednarratives–beitglobalwarming,our fundamentalunderstandingofcommunicablediseases,amongothers. Thescientificcommunitymustfindabetterwaytocommunicate,educate,anddefendrationalthoughtanddata-drivendecision-makinginan increasinglyunpredictableworld.

Whatdoyoumostenjoyinyourcapacityasanacademicor researchleader?

Theabilitytobeanactivememberofoneofthemostexcitingareasof scienceanddiscoverydaily–Itisanhonortobepartoftheworldwide communitytryingtounderstandhowthebraincreatesbehaviorandis dysregulatedindisease.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

Quiettime,aswellasadventures,withfamilyandlovedones.

Part2:KerryRessler–Selectedquestionsfromthe ProustQuestionnaire1

Whatisyourideaofperfecthappiness?

Relaxingatthebeachorinthemountains,readingabook,beinglostin themoment.

Whatisyourgreatestfear? Beingadisappointment–notdoing(oratleasttryingtodo)mybest.

Whichlivingpersondoyoumostadmire? BarackObama.

Whatareyoumostproudof? Mychildrenandsomeamazingacademic‘offspring’!

Whatisyourgreatestregret?

WhileIworryalotaboutthefuture,Iamfortunatetonotregrettoomuch aboutthepast(forgoodorforbad!).

Whatisthequalityyoumostadmireinpeople? Intelligencewithgenerativity,grace,andhumility.

Whatisthetraityoumostdislikeinpeople? Self-absorptionandnarcissism.

Whatdoyouconsiderthemostoverratedvirtue? Independence.

Whatisyourfavoriteoccupation(oractivity)? Ienjoybeekeepingandhiking.

Wherewouldyoumostliketolive? Onabeachwithaviewofamountain.

Whatisyourcurrentstateofmind? Peace,occasionallyinterruptedbyangstaboutmissingsomanydeadlines!

Whatisyourmostmarkedcharacteristic? Easytolaugh,and(hopefully)slowtocriticize.

Amongyourtalents,whichone(s)give(s)youacompetitiveedge? Persistence,alongwithaflexibleperspective.

Whatdoyouconsideryourgreatestachievement?

Scientifically,itisatiebetweenmyearlycontributionsasateammember tothe2004NobelPrizeforunderstandingolfactionandmyworkover thelasttwentyyearshelpingthefieldofPTSDandfear/traumaachieve successintranslationacrossgenomicsandcircuits.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe? Iwouldliketobelesssociallyanxious.

Whatdoyoumostvalueinyourfriends? Kindness,grace,easy-going,andlaughter.

Whataphorismormottobestencapsulatesyourlifephilosophy? ‘Hetriedhisbesttodogood…’

Belmont,Massachusetts,USA 19January2025

KerryJ.Ressler1 1 McLeanHospital,HarvardMedicalSchool,115MillStreet,Belmont, Massachusetts02478,USA e-mail: kressler@mclean.harvard.edu

OpenAccess. The“GenomicPressInterview”frameworkiscopyrightedtoGenomicPress.Theinterviewee’sresponsesarelicensedtoGenomicPressundertheCreativeCommonsAttribution-NonCommercialNoDerivatives4.0InternationalLicense(CCBY-NC-ND4.0).Thelicensemandates: (1)Attribution:Creditmustbegiventotheoriginalwork,withalinktothelicense andnotificationofanychanges.Theacknowledgmentshouldnotimplylicensorendorsement.(2)NonCommercial:Thematerialcannotbeusedforcommercialpurposes.(3)NoDerivatives:Modifiedversionsoftheworkcannotbedistributed.(4) Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulatedinthelicense.Publicdomainmaterialsorthosecoveredbystatutoryexceptionsareexemptfromtheseterms.Thislicensedoesnotcoverallpotential rights,suchaspublicityorprivacyrights,whichmayrestrictmaterialuse.Thirdpartycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunless otherwisestated.Ifuseexceedsthelicensescopeorstatutoryregulation,permissionmustbeobtainedfromthecopyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/licenses/by-nc-nd/4.0/.Thelicenseisprovidedwithoutwarranties.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

CathyBarr:Geneticsandneurobiologyofchildhoodpsychiatricand cognitivedisorders

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry March2025;1(2):11–13;doi: https://doi.org/10.61373/gp024k.0097

Keywords: Genetics,cellbiology,childpsychiatricdisorders,depression, readingdisabilities,stemcellmodels

CathyBarrcompletedherPh.D.inmolecularbiologyattheUniversity ofTexas,GraduateSchoolofBiomedicalSciences(M.D.Anderson CancerCenter)inHouston,Texas,followedbypostdoctoraltrainingin thegeneticsofcomplexbehaviorsatYaleUniversityandtheHospital forSickChildreninToronto.CurrentlyservingasaSeniorScientistat boththeHospitalforSickChildrenandtheKrembilResearchInstitute (UniversityHealthNetwork)andasaProfessorintheDepartmentsof PsychiatryandPhysiologyatTheUniversityofToronto,Dr.Barr investigatesthegeneticfoundationsofbehavior,cognition,and psychiatricdisorderswithestablishedgeneticpredispositions.Her researchmainlyfocusesonchildhood-onsetconditions,including depression,attention-deficit/hyperactivitydisorder,reading disabilities,andTourettesyndrome,withspecialemphasison understandingsharedriskfactorsacrossdisorders–acriticalarea giventhatchildrenwithneurodevelopmentaldisordersfaceafivefold increasedriskofdepression.Throughinnovativeapproaches,Dr.Barr andherresearchteamhavesuccessfullyidentifiedrisk-contributing genes.TheyareinvestigatinghowDNAvariationsinthesegenes influencegenefunctionandneuralcellbehavior.InthisGenomic PressInterview,shegenerouslysharesinsightsfromher groundbreakingresearchintothegeneticunderpinningsofchildhood psychiatricandneurodevelopmentaldisorders.

Part1:CathyBarr–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience? WhenIwasaseniorinhighschool,wehadamedicalresidentcometoour advancedbiologyclass.Hetoldusaboutdeliveringababywithnobrain thatdiedimmediatelyafterbirth.Theythenfiguredoutthatthefather didnothavefullclosureofhisspinalcolumn,pointingtoageneticcause. Thatledmetothinkaboutgeneticsandthepotentialtounderstand humandisease.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourmostrelevantleadershiprole.Whatdefiningmoments channeledyoutowardthatleadershipresponsibility? TherewasmuchserendipityinwhereIendedup.Iletmyinterestsin molecularbiologyandgeneticsguidemycareerchoices,whichultimately ledmetomycurrentposition.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteresearchorprofessionalfocusarea.

MyinterestinthegeneticsofpsychiatricdisordersbeganwhenIhearda lecturebyBobWilliamson.Init,hementionedthatscientistswerebeginningtostudythegeneticsofschizophreniaandhowdifficultthatwould

Received:1June2024.Accepted:15June2024. Publishedonline:31December2024.

CathyBarr,PhD,HospitalforSickChildren,KrembilResearchInstitute,TheUniversityofToronto,Canada.

be.Atthatmoment,IknewthatIwantedtostudythis,notbecauseit wouldbedifficultbutbecauseofthepotentialofgeneticstounderstand thiscomplexanddebilitatingdisorder.

Whatimpactdoyouhopetoachieveinyourfieldbyfocusingon specificresearchtopics?

Ihopethattheknowledgegainedfromthestudyofgeneticsreducesthe stigmaforpsychiatricdisordersandleadstomorespecifictreatments.

Figure1.

Figure2. CathyBarr,standingnexttoherresearchpresentationposteratanAppliedBiosystemsreceptioninHawaii,associatedwiththeAmericanSocietyof HumanGenetics(ASHG)2009meetinginHonolulu,Hawaii,USA.Theposter,titled“ImprovingtheHumanCondition,”outlinesherworkasSeniorScientistatthe HospitalforSickChildrenandProfessorattheUniversityofToronto,focusingonchildhood-onsetpsychiatricandcognitivedisorders,particularlyherresearch ongeneticvariationinbraindevelopmentandfunction.

Pleasetellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience.

Afterdecadesofsearchingforgenescontributingtopsychiatricdisorders,wearenowblessedwithaplethoraofriskgenes.Thechallengeis tounderstandhowgeneticvariationaltersgeneandcellfunction.Weare usingmultiplemoleculartechniques(e.g.,CRISPR,CRISPRa,andCRISPRi) andstemcell-derivedneuralcells(gene-editedstemcellsandiPSCsfrom patients)tounderstandhowgeneticvariationalterscellfunction.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour researchenvironment?

Persistence!Ifsomethingdoesnotwork,tryanotherapproach.

Thedisparityinthetreatmentofwomenscientists.

Whatdoyoumostenjoyinyourcapacityasanacademicorresearch leader?

Planningprojectsandexperimentsandthenanalyzingdata.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

Ilovetotravel.Fortunately,scienceoffersexcellentopportunitiesthrough conferencesandcollaborations.

Part2:CathyBarr–SelectedQuestionsfromtheProust Questionnaire1

Whatisyourideaofperfecthappiness?

Havingapromisingscientificfinding!Outsideofscience,Iamhappiest whentravelingandseeingorlearningsomethingnew.

Whatisyourgreatestfear?

Cognitiveimpairmentthatwouldpreventmefromworking.

Whatisyourgreatestextravagance?

Besidesspendingmoneyontravel,Idonothaveanyextravaganthabits.

Whatareyoumostproudof?

Havingsurvivedinacademia.

1 Inthelatenineteenthcentury,variousquestionnaireswereapopulardiversion designedtodiscovernewthingsaboutoldfriends.Whatisnowknownasthe35questionProustQuestionnairebecamefamousafterMarcelProust’sanswersto thesequestionswerefoundandpublishedposthumously.Proustansweredthequestionstwice,atages14and20.In2003,Proust’shandwrittenanswerswereauctioned offfor$130,000.Multipleotherhistoricalandcontemporaryfigureshaveanswered theProustQuestionnaire,includingamongothersKarlMarx,OscarWilde,ArthurConanDoyle,FernandoPessoa,StéphaneMallarmé,PaulCézanne,VladimirNabokov, KazuoIshiguro,CatherineDeneuve,SophiaLoren,GinaLollobrigida,GloriaSteinem, Pelé,Valentino,YokoOno,EltonJohn,MartinScorsese,PedroAlmodóvar,Richard Branson,JimmyCarter,DavidChang,SpikeLee,HughJackman,andZendaya.The ProustQuestionnaireisoftenusedtointerviewcelebrities:theideaisthatbyansweringthesequestions,anindividualwillrevealhisorhertruenature.WehavecondensedtheProustQuestionnairebyreducingthenumberofquestionsandslightly rewordingsome.Thesecuratedquestionsprovideinsightsintotheindividual’sinner world,rangingfromnotionsofhappinessandfeartoaspirationsandinspirations.

Whatisyourgreatestregret? Ihavelotsofsmallregretsbutnosinglegreatestregret.

Whatisthequalityyoumostadmireinpeople? Compassionatehonesty.

Whatdoyouconsiderthemostoverratedvirtue? Intelligence.

Whatisyourfavoriteoccupation(oractivity)? Traveling,hiking,andreading.

Wherewouldyoumostliketolive? IlovelivinginTorontobutwouldbeOKwithabeachcottageinSanDiego forthewinter.

Whatisyourmosttreasuredpossession? MycondooverlookingTorontoHarbour.

Whenandwherewereyouhappiest?Andwhywereyousohappythen? TherewasnosingletimewhenIwashappiest.Ihavehadmanymoments ofgreathappiness.

Whatisyourmostmarkedcharacteristic? Stubborn.

Amongyourtalents,whichone(s)give(s)youacompetitiveedge? Hardwork.

Whatdoyouconsideryourgreatestachievement? Havingsurvivedinacademia.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe? Iwouldliketobemorepatient.

Whatdoyoumostvalueinyourfriends? Loyalty.

Whoareyourfavoritewriters? Therearetoomanytolistandthelistisalwayschanging.Myfavorites includeAbrahamVerghese,ToniMorrison,RohintonMistry,Margaret Atwood,andJaneAusten.

Whoareyourheroesoffiction? OwenMeanyandAtticusFinch.

Whoareyourheroesinreallife? RuthBaderGinsberg,RosalindFrankland,MalalaYousafzai,andMichelle Obama.

Whataphorismormottobestencapsulatesyourlifephilosophy? Keepgoing.

Toronto,Ontario,Canada 1June2024

CathyBarr1 1 HospitalforSickChildrenandKrembilResearchInstitute, UniversityHealthNetwork,DepartmentsofPsychiatryandPhysiology, TheUniversityofToronto,60LeonardAvenue,8KD412,TorontoWestern Hospital,UniversityHealthNetwork,Toronto,Ontario,CanadaM5T0S8 e-mail: Cathy.Barr@UHN.ca

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

YogeshDwivedi:Pre-clinicalandtranslationalresearchfocusingongeneregulation throughepigeneticandepitranscriptomicmechanismsandtheirimplicationsin mooddisordersandsuicidalbehavior

©TheAuthor(s),underexclusivelicensetoGenomicPress2024

GenomicPsychiatry March2025;1(2):14–16;doi: https://doi.org/10.61373/gp024k.0025

Keywords: Depression,suicide,microRNA,non-CodingRNA,biomarkers Dr.YogeshDwivediistheElesabethRidgelyShookEndowedChairand ProfessorintheDepartmentofPsychiatryandBehavioral NeurobiologyattheUniversityofAlabamaatBirmingham(UAB).He joinedUABin2013afterworkingattheUniversityofIllinoisat Chicagoforabout20years,whereherosetotenuredProfessor.Heis theViceChairforFacultyAffairsandFacultyDevelopment, Co-DirectoroftheUABDepressionandSuicideCenter,Directorof TranslationalResearchoftheUABMoodDisorderProgram,andthe DirectoroftheAlabamaBrainBank.Hehaspublishedover160papers inpeer-reviewedjournalsandservesontheeditorialboardofseveral scientificjournals.HeleadsmultipleNationalInstituteofMental Health-fundedstudiesandhasalsoeditedabookentitled The NeurobiologicalBasisofSuicide.Therecipientofseveralnationaland internationalawards,Dr.DwivediisamemberoftheScientificCouncil oftheAmericanFoundationforSuicidePreventionandaFellowofthe AmericanCollegeofNeuropsychopharmacology(ACNP),International CollegeofNeuropsychopharmacology(CINP),andInternational NeuropsychiatricAssociation.Hisresearchbroadlyelucidatesthe molecularandcellularmechanismsassociatedwithearly-lifestress, mooddisorders,andsuicidalbehaviorbyintegratingbasicand clinicalneuroscience.Morespecifically,hisstudiesfocusongene regulationthroughepigenetic,epitranscriptomic,andnon-coding RNAsandwhetherthesemechanismsplayaroleinmoodregulation andsuicidalbehavior.Heisalsoworkingontranslatingthesefindings intobiomarkersfordepression,suicidality,andtreatmentresponse. Dr.YogeshDwivedikindlyagreedtoengageintheGenomicPress Interview,sharinghisdistinctiveblendofpersonalandprofessional narratives.

Part1:YogeshDwivedi–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience?

IwasbornandeducatedinasmalltowninnorthernIndia.Ifirstdiscovered organicchemistryinhighschool,andmypassionforsciencewaskindled. MyChemistryteachertaughtthesubjectwithreferencetoday-to-daylife insucharelatablemannerthatbytheendofmyfirstyearinhighschool, IhadamakeshiftChemistrylabofmyownintheshedofmyhome.After school,thisiswhereIcouldbefound,mixingsolventsandreplicatingthe lessonslearnedinschoolwiththematerialsIcouldaccessathome.The realfascinationlayintheapplicationofthesubjecttohumanlife.ChemistrywasmymajorinBS,andIsecuredthehighestmarksinmygraduating class.MyinterestinChemistrytookmeawayfrommysmalltownsinceadvanceddegreeswereunavailable,andIhadgraduatedearly.Duringthose days,onlyfiveuniversitiesinIndiaofferedaMaster’sprograminBiochemistry.Theselectionwashighlycompetitiveduetothelimitednumberof seats;Iwasfortunatetosecureaseat,andtheeducationwastransformative.IcommittedtobiochemistryduringmyMSdegree.Mypassionfor

Received:1April2024.Accepted:3April2024. Publishedonline:5April2024.

BiochemistryanditsapplicationtohumankindledmetopursuemyPh.D. attheCentralDrugResearchInstitute,aprestigiousresearchorganizationinIndiafocusingondrugdiscovery.ThisiswheremyloveforNeurosciencewasborn;oneofmymentorswasaninternationallyrenowned neuroscientistandneuropharmacologist.Withhisrecommendationand support,IjoinedapostdoctoralpositionattheIllinoisStatePsychiatric Institute(ISPI),Chicago,whichwasattheforefrontofresearchinbiologicalpsychiatry.

Mypassionforsciencetookmefartherandfartherawayfrommysmall townuntilfinally;everythingcametogetherduringmypostdoctoralresearchatISPI;inthechallengingnewdomainofmentaldisorders,Icould utilizemyeducationinBiochemistryandMolecularBiologytomakeadifference.WhileworkinginChicago,IcameincontactwithseveralprominentresearchersattheNationalInstituteofMentalHealth,whichfurther shapedmyideasandbecameinstrumentalinestablishingmylabasPrincipalInvestigator.ThejourneyfromtheshedthathousedmyfirstChemistrylabtoestablishingmyownlabasafacultymemberinadifferent countrywasarduous.However,suchwasmycommitmenttosciencethat noboundariescouldrestrictme.

Figure1. YogeshDwivedi,UniversityofAlabamaatBirmingham(UAB),USA.

Figure2. ApatchofDriftRosesembellishesYogeshDwivedi’sgardenin Birmingham,Alabama.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourmostrelevantleadershiprole.Whatdefiningmoments channeledyoutowardthatleadershipresponsibility?

AsafacultymemberattheUniversityofIllinoisatChicago,IwascontentwiththespaceandresponsibilityIwasgivenwhereIwasengagedin harnessingmyideasandmakingmywayintothefieldofbiologicalpsychiatry.WhileIhadenvisionedleadershipinthefieldofresearchinthe distantfuture,IhadnotanticipatedaleadershiproleinacademicpsychiatryuntilIwasgivenachancetohelpestablishtheDepressionandSuicide CenterwhenIjoinedUAB.Itgavemeanexcellentopportunitytotranslate myideasintoexpandingthebasicandtranslationalresearcharenawithin theconfinesoftheCenter.Thisledtootheropportunitiesforleadership bothwithinUABandinthelargerscientificcommunity.Asthedirectorof theBehavioralNeuroscienceDivisionatUABandthedirectoroftranslationalresearchoftheUABmooddisordersprogram,Ihadtheopportunitytomentorandcollaboratewithawiderangeoffacultyandimpact thefuturecourseofresearch.Theserolespreparedmeformyposition asViceChairforFacultyAffairsandDevelopmentandDirectoroftheAlabamaBrainBank.Theseleadershipopportunitieshaveprovidedmewith asenseoffulfillmentinmakingadifferenceandevolvingbylisteningto otherpeople’sideas.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteareaofresearchorprofessionalfocus. Whileworkingintheareaofgeneregulation,Ibecamehighlyinterestedin thefieldofnon-codingRNAs.Notonlydonon-codingRNAsoccupymostof thegenome,buttheyalsoallowventuringintoaterritorywithmanyunknowns.IwasprimarilyinterestedinmicroRNAs,aspecificclassofsmall non-codingRNAswhosemodeofactionisunique,wheremultiplemiRNAscouldregulateasinglegeneormultiplegenescouldberegulatedby asinglemiRNA,moresoinahighlycoordinatedfashion.Thishasledme todeeplyexploreothernon-codingRNAs,theircontributiontoepigenetic modifications,and,ultimately,theirrelevancetopsychiatricillnesses.

Whatkindofimpactdoyouhopetoachieveinyourfieldthroughyour focusonyourspecificresearchtopics?

Ihopethatmyworkwillhelpinunderstandingthebiologicalbasisofdepressionandsuicide,whichwillnotonlyleadtoidentifyingthecausal

factorsbutalsohelpindevelopingtargetedtherapies.IalsohopetodevelopCNS-basedbiomarkerstohelpcliniciansaccuratelydiagnosepatientsandgaugethetreatmentresponse.

Couldyoutellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience?

Currently,Iamworkingtounderstandtheroleofnon-codingRNAsin generegulationatmultiplelevelsfortheircontributiontopsychiatricillnesses,particularlydepressionandsuicidalbehavior.Theseincludethe roleofnon-codingRNAsinepigeneticandepitranscriptomicmodificationsofgenesusingthehumanpostmortembrain,rodentmodels,peripheralcellsfromhumanbloodsamples,andhumaninducedpluripotent stemcells(iPSCs).Simultaneously,Iamresearchingifthesemodifications canbeusedasbiomarkersfordiagnosisandtreatmentresponse,particularlyusingextracellularvesiclesderivedfromthebrain.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiences,thatyouupholdwithinyour ownresearchenvironment?

Throughmyacademicstudiesandpostdoctoraltraining,thehabitsIdevelopedandthevaluesthatshapemyresearchenvironmentarethoseof persistence,patience,constantquestioning,andpreparednessforchallengesandfailures.

AtGenomicPress,weprioritizefosteringresearchendeavorsbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Arethereparticular culturalfacetswithinthescientificcommunitythatyouthinkwarrant transformativescrutiny,oristhereacausewithinsciencethatdeeply stirsyourpassions?

Iampassionateaboutsciencebeingfreeofallpoliticalorsociocultural restraintsandderivingonlyfromfacts.

Whatdoyoumostenjoyinyourcapacityasacademicorresearch leader?

Ilovetodiscussnewideasandhowtoimplementthemwithinnovative techniquesthatcantransformthefield.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

GardeningiswhatIcherishmostinmyleisuremoments.MywifeandIare habitualgardeners,alwaysplanting,replanting,pruning,fertilizing,and, morethanallelse,growingwithourplants.

Part2:YogeshDwivedi–SelectedquestionsfromtheProust Questionnaire1

Whatisyourideaofperfecthappiness?

Perfecthappinessformeisbeingatpeacewithmyself,knowingthatI havedonemybitandamcherishedbymylovedones.

Whatisyourgreatestfear? Mygreatestfearisthesuddenlossofalovedone.

Whichlivingpersondoyoumostadmire? ThereareseveralpeopleIadmirefromvariousspheres.

Whatisyourgreatestextravagance?

Planningtripswithmywifeasweconstantlyseeknewadventures;our latestadventurewasatripaboardtheArcticCircletoseetheAurora BorealisinAbisko.

Whatareyoumostproudof?

Iammostproudofmyson—notjustofhisachievementsbutalsoofthe sensitiveandkindpersonheisandthewonderfulmanheisbecoming.

Whatisyourgreatestregret?

Apartfromthesmalloneslitteredacrossmylifespan,Ihavedonemybest tolivewithoutsignificantregrets.

Whatisthequalityyoumostadmireinpeople? Honestyandintegrity.

Whatisthetraityoumostdislikeinpeople? Pretentiousness.

Whatdoyouconsiderthemostoverratedvirtue? Virtuesarevirtues—Itrynottooverthinkthembutfocusontryingtobe agoodhumanbeing.

Whatisyourfavoriteactivity(physicalorintellectual)? Atthetopofthelistaregardeningandreading.

Wherewouldyoumostliketolive?

MukteshwarisasmalltownnestledintheHimalayasinIndia.Ihavetried torecreatethissettinginmyhomeinthehillsofBirmingham,Alabama.

Whatisyourmosttreasuredpossession?

Myfather’swatch,whichhegavetomewhenIlefthometopursuemy educationattheageof19,hasbeenaconstantreminderofhispresence evenafterhewasgone.

Whenandwherewereyouhappiest?Andwhyweresohappythen? Thankfully,therehavebeenmanysuchinstances;itishardtochooseone specificmoment.

Whatisyourcurrentstateofmind? Calm:Idomybesttostaybalanced.

Whatisyourmostmarkedcharacteristic? Iamhighlydriven.

Amongyourtalents,whichonedoyouthinkgivesyouacompetitive edge?

Gardeninghasinstilledinmethevaluesofhardwork,persistence,and patience,whichgivemeanedgeinscience.

Whatisapersonality/characteristictraityouwishyouhad?

Wearewhoweare,butwecanconstantlybetterourselves.Iwouldliketo cultivateasharpersenseofhumor.

Whatdoyouconsideryourgreatestachievement?

ThatIhaveplayedasmallpartinthefieldofdepressionandsuicide studies.

Whatdoyoumostvalueinyourfriends?

OpennessandtrustarewhatIvaluemostinmyfriendships.

Whoareyourfavoritewriters?

IreadextensivelyinEnglishandHindi,aswellasfictionandnonfiction,so itisnoteasytochoosefavorites.MyfavoritesareIndian-Americanwriters:AmartyaSen,JhumpaLahiri,SiddharthaMukherjee,AshwiniSanghi, ChitraDivakaruni,andothers.

Whoareyourheroesoffiction?

Myheroesoffictionareprimarilyhumanswhoareoftenflawedbutheroic intheireffortstosurvive.

Whoareyourheroesinreallife?

Peoplewhoinspiremedailyarealwaysatit,tryingtheirbesttolivemeaningfully,onedayatatime.

Whataphorismormottobestencapsulatesyourlifephilosophy? Nevergiveup!

YogeshDwivedi1 1 DepartmentofPsychiatryandBehavioralNeurobiology,Universityof AlabamaatBirmingham,Birmingham,Alabama35294,USA e-mail: yogeshdwivedi@uabmc.edu

OpenAccess. ThisarticleislicensedundertheCreativeCommons Attribution-NonCommercial-NoDerivatives4.0InternationalLicense (CCBY-NC-ND4.0).Thelicensemandates:(1)Attribution:Creditmustbegiventothe originalwork,withalinktothelicenseandnotificationofanychanges.Theacknowledgmentshouldnotimplylicensorendorsement.(2)NonCommercial:Thematerial cannotbeusedforcommercialpurposes.(3)NoDerivatives:Modifiedversionsofthe workcannotbedistributed.(4)Noadditionallegalortechnologicalrestrictionsmay beappliedbeyondthosestipulatedinthelicense.Publicdomainmaterialsorthose coveredbystatutoryexceptionsareexemptfromtheseterms.Thislicensedoesnot coverallpotentialrights,suchaspublicityorprivacyrights,whichmayrestrictmaterialuse.Third-partycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunlessotherwisestated.Ifuseexceedsthelicensescopeorstatutory regulation,permissionmustbeobtainedfromthecopyrightholder.Forcomplete licensedetails,visit https://creativecommons.org/licenses/by-nc-nd/4.0/.The licenseisprovidedwithoutwarranties.

GenomicPsychiatry

VIEWPOINT

Theassociationbetweentraceamine-associatedreceptor1(TAAR1)genetic mutationsandneuropsychiatricdisorders

©TheAuthor(s),2024.ThisarticleisunderexclusiveandpermanentlicensetoGenomicPress

GenomicPsychiatry March2025;1(2):17–20;doi: https://doi.org/10.61373/gp024v.0058

Keywords: Geneticvariants,traceamines,ulotaront,amphetamine, signalling,personalisedtherapeutics,neuropsychiatry

Traceamine-associatedreceptor1(TAAR1)isanemergingdrug targetforthetreatmentofneuropsychiatricconditions.Several TAAR1-targetedtherapeuticsarecurrentlyinclinicalandpreclinical development.EmergingstudieshighlightlinksbetweenTAAR1 single-nucleotidevariants/polymorphismsandneuropsychiatric disorders.AnimprovedunderstandingofTAAR1geneticvariantsand theirfunctionalimpactwillinformthepotentialroleoftheTAAR1 systeminthepathophysiologyofneuropsychiatricconditionsandfor bettertherapeuticdosing.Thisviewpointexaminesclinicaland molecularstudiesinvolvingTAAR1geneticvariantsandtheir associationwithneuropsychiatricdisorders.

Neuropsychiatricconditionssuchasschizophrenia,bipolardisorder,and majordepressionhavegeneticheritabilitywithseveralsharedclinical symptoms.Thesedisordersareoftendisabling,andtogethertheycontributetoasubstantialglobalhealthburdenofdisease.Thoughthese diseasesareclassifiedasdistinctconditions,theoverlapbetweenthe cognitive,emotional,andbehavioralsymptomologiesoftenpresents challengesinaccurateandearlydiagnosis(1).Severalstudieshavebeen conductedtodistinguishthesebetter,rangingfromanimalmodelsto neuroimagingtogenome-wideassociationstudies(GWAS).Giventheir highlyheritablenature(e.g.,schizophrenia,approximately80%),GWAS hasbeenpresentedasapowerfulwaytoinvestigatethecontributions ofgeneticvariantstoaspecificdisorder.Further,geneticmappinghas beenusefulinidentifyingsharedgeneticfactorsinbipolardisorderand schizophrenia(2, 3).However,thepolygenicnatureofmostneuropsychiatricconditionsraiseschallengesandreliablepredictionscannotbemade fromasingle-nucleotidevariantoralownumberofvariants.Assuch, researchersdevelopedaggregatescoressuchaspolygenicriskscores. Thesescoresassessthetotalnumberofriskvariantsandpredictthe likelihoodofdevelopingassociatedconditions.Suchstudiesunveileda largedegreeofgeneticpleiotropyandcommonlyshareddysregulated systemsbetweenmanyoftheseconditions.Forinstance,dysfunctionof theGABAergicsystemisobservedinmanyneuropsychiatricandneurobiologicalconditionsincludingschizophrenia,bipolardisorder,majordepression,andothers,while,dysregulateddopaminergiccircuitrieshave beenassociatedwithschizophrenia,attention-deficithyperactivitydisorder(ADHD),substanceabusedisorders,anddepression(4, 5).Further, geneticvariantsinthedopamine2receptor(D2receptor)andserotonin 1Areceptorhavebeenassociatedwithaddiction,andincreasedriskfor depressiveepisodesandtreatmentresponse,respectively(6, 7).Polymorphismsindopaminergicgeneshavebeenassociatedwithantipsychoticdrugsensitivity,adverseeffects,andmotordeficits(8).Asaresult,allmajorneuropsychiatricconditionssuchasschizophrenia,bipolar disorder,andmajordepressionhavebeenlinkedtomultiplegenes(2, 9, 10).Schizophrenia,forexample,hasbeenlinkedtomorethan200genes (9).Further,anincreasedriskforbipolardisorderhasbeenobservedin patientswithultra-rareprotein-truncatingvariantsofA-kinaseanchor-

ingprotein11(AKAP11)(2).Thus,neuropsychiatryresearchestablishing geneticassociationswithpsychopathologyofneuropsychiatricdisorders isanareaofsignificantinterest.

Traceamines(TAs)arebiogenicaminesthatexistinlowconcentrations(1–100ng/goftissue).Theyarestructurallysimilartotypicalneurotransmitterssuchasdopamineandserotoninbutdisplayadistinctpotencyfortraceamine-associatedreceptors,anovelfamilyofaminergic receptorsdiscoveredovertwodecadesago(11).TheTAARfamilyencodes forsixfunctionalgenes,withisoform-specificexpressioninneuronaland non-neuronaltissues.Amongall,TAAR1showsthehighestexpression inthebrain,especiallyatthepresynapticandpostsynapticterminalsof monoaminergicnuclei,respondingtointracellularandextracellulartrace amines,monoamines,andsecondarymetabolites(11, 12).Incelllines, stimulationbysuchligandsprimarilyincreasestheintracellularcyclic adenosinemonophosphate(cAMP)levels,whichpromptsfunctionalinteractionwithothermonoaminergicsystems,influencingtheiractivity. Suchobservationsextendtoinvivosystems,especiallyinTAAR1overexpressed(TAAR1-OE)andTAAR1knockout(TAAR1-KO)models,which broadlydisplayhyposensitivityandhypersensitivitytoamphetamines,respectively(13).Similartopatientswithschizophrenia,TAAR1-KOmodels exhibitincreasedlevelsofdopamineandserotonin,inadditiontospontaneousactivationofassociatedneuralcircuitriesinthemidbrain.Bycontrast,theTAAR1-OEmodelshowshyposensitivitytoamphetamineand adecreaseinbaselinelocomotion.Indrug-inducedpsychoticmodelsof mice,theadministrationofTAAR1agonistsprovidessymptomreliefsuggestingthatTAAR1hasaneuromodulatoryeffect(13).Assuch,TAAR1is consideredanendogenousrheostat,anditsfunctioniscriticalfornormal neurotransmission.

AwealthofstudiesthereforeexploredtheutilityofTAAR1asa druggabletargetfortreatingschizophreniaandseveralotherneuropsychiatricdisorders(13).Emergingantipsychoticagents,TAAR1agonists, showpromisefortreatingschizophreniaandotherneuropsychiatricdisorders.ThenonselectiveTAAR1agonistulotarontreceivedaBreakthroughTherapyDesignationfromtheU.S.FDAfortreatingschizophrenia.However,ulotarontfailedtwopivotalphaseIIIclinicaltrials.This clinicalcandidateiscurrentlyinphaseII/IIItrialsforotherconditionsincludingsleepdisorders,Parkinson’sdiseasepsychosis,majordepression, andgeneralizedanxietydisorders(ClinicalTrials.govIDsNCT05015673, NCT02969369,NCT05593029,andNCT05729373,respectively).Moreover,severalnewTAAR1agonistsareinpreclinicaldevelopment(14). However,studiesconsideringTAAR1asapotentialinstigatorofdisease statesremainunderstudied.Meanwhile,othermembersoftheaminergicfamilywhicharealsomajorpharmaceuticaltargets,havebeenassociatedwithmultipleneurobiologicalandneuropsychiatricconditions, asdescribedpreviously.Inthesamecontext,immediatefamilymembers suchasTAAR2,4(pseudo-gene),5,and6havebeenstudied,withassociationsdescribedbetweenschizophreniaandbipolardisorders(15–17).Notably,theTAAR1geneismappedtochromosome6q23.2,which coincideswithsusceptibilitylocusforschizophrenia,bipolarandaffectivedisorders(16).ThepotentialclinicalconsequencesofTAAR1genetic

Received:11June2024.Revised:9August2024and13August2024.Accepted:14August2024. Publishedonline:30August2024.

Figure1. TheassociationbetweenTAAR1variants,neuropsychiatricdisorders,andtherapeuticresponse.TAAR1mutations(onlyselectedmutationsareshown forclarity)inpatientswithneuropsychiatricdisorders(toppanel).AmodelproposingalteredsignalingoftraceaminesinvariantTAAR1observed inneuropsychiatricdisorders(bottomleft).TreatmentoptionsaretailoredtopatientswithTAAR1mutations(bottomright).Thefigurewascreatedwith Biorender.com variantsindiversepopulationsmaythereforebequestioned.TAAR1gene knockoutstudiesdemonstratedthecriticalnatureofnativeTAAR1functionanddemonstratedlinkswithschizophreniaandotherneuropsychiatricdisorders.ItcanthusbehypothesizedthatTAAR1genevariantsthat causeaberrationsinitsfunctionmayberesponsiblefordisordersassociatedwithdysregulatedneurotransmission.Moreover,theoccurrenceof TAAR1variantsmaydisplayvariabilityintherapeuticresponseinpatients targetedwithTAAR1-basedmedications.

OurrecentworkonTAAR1identifiedvarianceingeneticdifferences indiversegeographicalpopulationsthatcaninfluencethestructureand functionofTAAR1protein(18).Wemappedover40raremutationsthat mayinfluencetheligand-activatedmechanismsofTAAR1including,variantsinthebindingpocket,microswitchregions,andsignalingdomains. Specifically,theD103NvariantfoundinsoutheastAsianregionsand WesternPacificregionscompletelyablatethereceptoractivity,withfew othersshowingasignificantimpactonreceptorfunctioning.However, thedensityand/orpresenceofsuchTAAR1variantsinneuropsychiatric patientsremainunknownandrequirefurtherstudy.Inthisviewpoint, weconsolidatedallliteratureonTAAR1nonsynonymousvariantsfrom clinicalstudiesanddiscussedthepotentialimplicationsofTAAR1mutationsondiseasemanifestationandTAAR1-targetedtherapeuticsin neuropsychiatry.

OnlyafewstudieshavebeenreportedonTAAR1mutationsinclinicalsamples,someofwhichhavebeenreviewedpreviously(19).Despitelimitednumbers,thesestudiesprovidesignificantinsightslinking rareTAAR1mutationswithneuropsychiatricconditions(Figure1).The firststudyconductedbyJohnandcolleaguesonpatientswithsporadic schizophreniaidentifiedseveralnonsynonymousvariantsinunrelated patientsofIndian(S47C,F51L,Y294∗ ,andL295S)andAmericanorigin (A109TandV250A).Inparticular,theIndiancohortreportedakeyvariant,C182F,whichdisruptsthedisulfidebridgethatiscriticalforthestabilityandfunctionofthereceptor.Thisvariantwasfoundinamother andhertwochildren,allofwhomhadadiagnosisofschizophrenia.Notably,theunaffectedsiblingswerenegativeforthisvariantoranyother TAAR1mutations,includingthecontrolsubjects(20).Thismaternallink suggeststhatinheritedTAAR1variantsmaycontributetoschizophrenia. Inanycase,disruptionofthedisulfidebridgemaycausetheproteinto misfoldordestabilize,duetoalossofstructuralpropertiesfacilitated bythecysteinesidechain.Whileatruefunctionalvalidationofthisvariantawaits,severalbioinformaticfunctionalpredictiontoolshavepredictedthisalleletobedamaging.Meanwhile,previouscelllinestudies demonstratedtyrosinevariantatthisposition(C182Y)producesafunctionalknockoutofthereceptor,withoutaffectingitsexpressionlevelsand cellularlocalization(21).OthervariantssuchasS47C,F51L,A109T,and

L295SwerepredictedtoreduceTAAR1activity,withnochangeinactivitypredictedforV250A(20).ThevariantofY294∗ introducesaprematurestopcodonthuslikelytoinfluencereceptorfunction.Asimilarphenotypeinmiceexpressingfunctionallyknocked-outTAAR1genethrough P77Tmutationdemonstratedincreasedmethamphetamineconsumption andreducedsensitivitytomethamphetamine-inducedhypothermia(22). Supporting,invitrostudiesalsodemonstratedthattheP77Tvariantproducesafunctionalknockoutofthereceptorwithoutimpairingexpressioncapabilities(22).Whilesomemayagreethatfunctionalknockouts certainlyencapsulatetheessenceofatraditionalTAAR1-KOmodel,itis unlikelythatTAAR1’sfunctionalmechanismismerelyreservedforligandactivatedsignalingalonebutmayinvolveinteractionswithotherreceptors(suchasD2)asdemonstratedinpreviousstudies(23).Assuch,functionalknockoutsmayalsohavemoreunidentifiedrogueeffects,which needfurtherinvestigation.

ThestudybyMühlhausandcolleaguesreportedthreenonsynonymous mutationsinTAAR1(24).Here,themutationswereidentifiedfroman unrelatedpatientcohortconsistingofobese/overweightsubjectswith impairedglucosehomeostasis.Notably,individualmutationswerefound inseparatepatients,whereinpatients1,2,and3carriedthevariants R23C,I171L,andS49L,respectively.Invitro,studiesdemonstratedthat twovariants,R23CandS49L,significantlyimpairreceptorresponsetoits ligands.Inahomozygousstate,R23Cdemonstratedacompletelossof activityandtheS49Lvariantdemonstrateda40%reductioninmaximal responseto3-Iodothyronamine(T1AM).Inaheterozygousstate,maximalsignalingforR23CandS49Lpeakedat58%and55%,respectively (relativetowildtype).Inaddition,stimulationwith β -Phenylethylamine (PEA)alsodemonstratedacompletelossfunctionfortheR23Cvariantandapproximately70%lossforS49L(24).Carriersofthevariants exhibitedsignsoflowcognitionandpsychiatricabnormalities,respectively.Patient1hadalowIQ(71)attheageof7,meanwhile,authors describedpatient3(variantS49L)ashaving“psychiatricproblems”.Conversely,infunctionalstudies,I171Lretainedmostofitsfunctionandwas describedtomimictheactivityofwildtypeandnoobservationswere associatedwithdeclinedcognitivefunctionorpsychiatricillness.Notably,thefrequencyofS49Lincontrolsampleswasapproximately0.38% (27/7158),meanwhile,theR23Cvariantonlyhadafrequencyof0.056% (4/7181).Thissuggestsadichotomywhereaslightlossoffunctionmay betoleratedandanysignificantlossmaybeassociatedwithdisease augmentation(24).

Inanotherstudy,Rutiglianoandcolleaguesidentified16TAAR1variantsinthecodingregionsofmentalhealthpatients(25).Threeweresynonymous(C265C,V288V,andR312R)withtheremaining13missense.Of these,threewerefurtherfunctionallyevaluatedincelllines,whichconsistedofR23C,Y131C,andC263R.Allthreepatientshadauniquediagnosis,wherethecarrieroftheR23Cvariantwassymptomaticforschizoaffective,bipolartypeandobsessive-compulsivedisorders,thecarrierof theY131Cvariantwasdiagnosedwithtype1bipolardisorderandthe carrieroftheC263Rvariantwasdiagnosedwithtype2bipolardisorder. Notably,allthreepatientsalsoreportedfamilyhistoriesofschizophreniaspectrumdisorders(carrierofR23C),depressiveandanxietydisorders(Y131C)andageneralhistoryofmentaldisorders(C263R).Incellline studies,theheterozygousstate,R23C,Y131C,andC263RshowedsignificantlydecreasedmaximalcAMPaccrualinresponsetoPEA,RO5166017, andT1AM.Meanwhile,thehomozygousstateisdescribedtorenderthe proteinfunctionallyinoperative.Notably,Mühlhausandcolleaguesfound theR23CTAAR1variantinobesepatientswithlowIQandmetabolicdisorders(24),whereasRutiglianoandcolleaguesfoundthesamemutationin patientswithneuropsychiatricconditions(25).Therefore,TAAR1variants mayindicategeneticpathwaysorpredisposingfactorsthatlinkmetabolic disordersandneuropsychiatricconditions.

InadditiontogeneticvariantsinTAAR1,studieshavealsoshownalteredlevelsoftraceamines(TAAR1agonists)inpatientswithbraindisorders.HigherlevelsofPEAinplasmaandurinesampleshavebeennoted inpatientswithschizophrenia(13, 26).Contrastingly,patientswithADHD werereportedtohavesignificantlylowerlevelsofPEAinurinesamples comparedwithcontrolsubjects(27).Similarly,decreasedlevelsofPEA werealsoreportedinpatientssufferingfromdepressionandParkinson’s

disease(28).Lowconcentrationandrapidturnoveroftraceamines,coupledwithalackofsensitivityandspecificityofthetechniquesutilized poseddifficultiesinaccuratelymeasuringTAsinbiologicalfluidsandtissues,resultingindifferencesintraceaminelevelsbetweenstudies(13, 26).AlterationsinTAAR1signalingthatresultfromlowambientagonistlevelsmayalsocontributetosuchstates,whichcanfurtherbeinfluencedbygeneticvariantsintheprotein(Figure1).Furthermore,thecurrentstateandfocusofTAAR1therapeuticsmaybechallengedbygenetic variations,whichisseeminglyanunexploredarea.Ifpatients,whoarerecipientsofthesetherapiesarepronetogeneticvariantsofTAAR1,itmay bringsignificantchallengestothetherapy(Figure1).Recentmolecular studiesdemonstratedtheinfluenceofpointmutationsonreceptorfunctioningandsignalingcascades(29, 30).Residue-specificinteractionsat thebindingpocketsserveasakeythatinfluencesthesignalingpathways, whichiscriticaltoacompound’sefficacyandtomitigateanyunforeseen activities(29).Moreover,theinfluenceofTAAR1mutationsonitsinteractionswithotheraminergicsystemsremainsunknown.Previousaccounts attributedligand-inducedactivityasthekeymechanismofdimerization betweentheD2receptorandTAAR1,whichappearedtohaveasignificant influenceontheefficacyofantipsychotics(23).Incelllines,responsesto antipsychoticssuchashaloperidol,raclopride,andamisulpridewereamplifiedwhentheD2receptorandTAAR1werecoexpressed.InTAAR1-KO micemodels,haloperidoltreatmentactivated30%fewerneurons,and themagnitudeofhaloperidol-inducedcatalepsywassignificantlylower comparedwiththewildtypesuggestingthatthereisafunctionalinteractionbetweenboth(23).Suchindicatorsmayprovideopportunities forexploringpersonalizedTAAR1medicationswithstudiesinpsychotic micemodelsdemonstratingthatactivationofeitherGsorGqpathwaysis equallybeneficialinalleviatingschizophrenia-likesymptoms(30).Such findingsaidrationaldrugdevelopmentalapproachesandfindingalternativeTAAR1therapeuticsthatmayfindutilityinpatientswithTAAR1 mutations.

TAAR1disruptionsmaycontributetoneuropsychiatricdisorders,as observedinanimalknockoutstudiesandfromgeneticvariationsinpatients.Fromageneticperspective,furtherresearchisneededtodeterminewhethercertainraregeneticmutationsinTAAR1maypredispose thedevelopmentofspecificpsychiatricdisorders.Beingrarevariants, largersamplesizesarerequiredtoassessthetruesignificanceofsuch mutations.Moreover,invitroandinvivostudiesshowthatTAAR1canregulateD2-receptoractivityviaheterodimerization;therefore,itisessential tounderstandhowTAAR1variantsmayinfluencedopaminergicsignaling.Ontheotherhand,pointmutationsinTAAR1havebeenshowntoinfluencetheselectionofsignalingpathways;whetherthistranslatestoin vivoremainsunknown.Aberrationsassuchmaywelcomeunwantedside effectsfromTAAR1therapeutics.TherationaldevelopmentofTAAR1basedtherapeuticswillcontinuetogrowwiththeemergenceofexperimentalstructuresofTAAR1andthesignificantneedforbettertherapies forneuropsychiatricdisorders.Futurestudiesshouldfocusonevaluating TAAR1mutationsinclinicalsubjects,specificallytoassesstheeffectsof thesemutationsontherapeuticefficacy,andadverseeffects.Inthefuture, theuseofpharmacogenetictestingwillfacilitatedeterminingtheprevalenceofmutationsinTAAR1amongneuropsychiatricpatients,thussupportingthedevelopmentofpersonalizedtreatmentsforemergingTAAR1 therapeutics.

Acknowledgments

P.C.N.andT.B.acknowledgeFlindersUniversityandSouthernAdelaideLocalHealthNetworkforInnovationPartnershipSeedFunding.

BrittoShajan1 ,TarunBastiampillai2 , 3 ,andPramodC.Nair1 , 4 , 5 , 6

1 DisciplineofClinicalPharmacology,CollegeofMedicineandPublicHealth, FlindersUniversity,Adelaide,SouthAustralia,Australia; 2 Departmentof Psychiatry,MonashUniversity,Parkville,Melbourne,Victoria,Australia; 3 Discipline ofPsychiatry,CollegeofMedicineandPublicHealth,FlindersUniversity,Adelaide, SouthAustralia,Australia; 4 FlindersHealthandMedicalResearchInstitute (FHMRI)CollegeofMedicineandPublicHealth,FlindersUniversity,Adelaide,South Australia,Australia; 5 SouthAustralianHealthandMedicalResearchInstitute, UniversityofAdelaide,Adelaide,SouthAustralia,Australia; 6 Disciplineof

Medicine,AdelaideMedicalSchool,UniversityofAdelaide,Adelaide,South Australia,Australia e-mail: pramod.nair@flinders.edu.au

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GenomicPsychiatry

THOUGHTLEADERSINVITEDREVIEW

CirculatinglongnoncodingRNA:Newfrontiersinbiomarkerresearch formooddisorders

BhaskarRoy1 ,AnujKumarVerma1 ,EllieMarieHulwi1 ,andYogeshDwivedi1

Unipolarandbipolardepression[majordepressivedisorder(MDD)andbipolardisorder(BD)]arecomplexpsychiatricdisorderscharacterized bydisturbancesinmood,affect,andcognition.Increasingevidencehasconfirmedepigeneticmalfunctioningatthecoreofthesetwomental conditions;however,theexactmolecularnatureofthatepigeneticmaladaptivityislessknown.Lately,longnoncodingRNAs(lncRNAs)have emergedasessentialepigeneticregulatorsofgeneexpressionandcellularprocesses,offeringnewavenuesforexploringthepathophysiology ofmooddisorders.Inthisreport,wepresentacomprehensivereviewofrecentclinicalstudiesinvestigatingtheinvolvementoflncRNAinMDD andBD,andemphasizingtheirdisease-specificcontributionaspotentialbiomarkers.WeexplorethedysregulationofspecificlncRNAs detectedinperipheralbloodsamplesofindividualswithmooddisorders,whileunderscoringtheirsignificanceforclinicaldiagnosis,prognosis, andpredictingtreatmentresponse.Additionally,weprovidefuturedirectionsforlncRNAresearchinthecontextofmooddisorders.

GenomicPsychiatry March2025;1(2):21–33;doi: https://doi.org/10.61373/gp024i.0046

Keywords: lncRNA,mooddisorders,depression,bipolardisorder,epigenetic,blood

Introduction

Majordepressivedisorder(MDD)andbipolardisorder(BD)remainsignificantpublichealthchallengesaffectingmillionsofindividualsworldwide. AccordingtoDSM-5,MDDandBDarethetwomostcommontypesofmood disordersrepresentedbymoodinstability,whichresultsfromdysfunctionalityinemotional,cognitive,andbehavioraldomains(1).According tothereport,thismayaffectawiderangeofthepopulation,including adolescentsandadults.Anestimated21.4%ofU.S.adultsexperiencea mooddisorderatsometimeintheirlives,andithasbeensuggestedthat theprevalenceofmooddisorderishigherinfemalesthanmales(2, 3). BDisamongthemostcommonmajorpsychiatricdisorders,witha1%–4% prevalencerate(4).Ontheotherhand,MDDisacommonanddebilitatingpsychiatricdisorderaffectingasmanyas12%ofadultsglobally,with itsprevalenceintheUnitedStatesbeinghighestamongyoungadults, women,andtheelderly(5, 6).

BothMDDandBDareassociatedwithanincreasedriskofsuicide.Data suggestthatanestimated31%ofMDDand34%ofBDsubjectshadat leastonesuicideattemptintheirlifetime(7, 8).Itisnowincreasinglyevidentthatacombinationofgenetic,environmental,andpsychologicalfactorsislikelytobethecauseofmooddisorders,embracingalltheMDDand BDcasesworldwide(9, 10).Impairedstressresponsehasbeenstrongly implicatedintheetiopathogenesisofbothMDDandBD.Overthepast years,molecularstudiesinthebrainhaveunderscoredtheimportance ofalteredgeneexpressiondynamicsinthedevelopment,manifestation, andprogressionofMDDandBD(11, 12).Inaddition,increasingknowledgehashighlightedtheroleofstress-associatedenvironmentalinfluencesontranscriptomicperturbationofMDDandBDbrainsintheface ofcompromised“epigeneticplasticity”(13).Despiteextensiveresearch inthepastseveralyears,themaladaptiveepigeneticchangesinMDD andBDbrainsremainpoorlyunderstood.LongnoncodingRNA(lncRNA) hasemergedasanewmasterepigeneticregulatorandhasshownenormouspotentialforconnectingthemissingdotsinthecompromisedgene regulatorymapofMDDandBDbrains(14–16).Inourunderstanding,as essentialregulatorsofgeneexpressionanddiversecellularprocesses, lncRNAmayprovidenewopportunitiesforexploringthepathophysiology ofmooddisorderswiththeaddedbenefitofbeingusedasdiagnosticand treatmentresponsebiomarkersinperipheralcirculation(14).Agrowing

bodyofknowledgefrombothclinicalandpreclinicalstudieshasprovided valuablemechanisticinsightintothefunctionalrolesoflncRNAsinmoodrelatedbehaviorsandneurobiologicalprocesses.Althoughincreasingevidenceregardingtheiravailabilityinperipheralcirculationhasreinforced theirpotentialuseasclinicalbiomarkers,additionalresearchiswarranted tountiethecomplexregulatoryroleoflncRNAsandtheirimplicationsfor thediagnosis,prognosis,andtreatmentofmooddisorders.

Thisreportoffersanoverviewofrecentstudiesexploringtheroleof lncRNAsinbothbipolarandunipolardepression,whileencompassinginsightscollectedfromclinicalsamples.Wediscussthedysregulationof specificlncRNAsobservedinperipheralbloodsamplesofindividualswith mooddisorders,whilehighlightingtheirpotentialasbiomarkersfordiagnosis,prognosis,andtreatmentresponseprediction.Furthermore,we lookintotheprobablefutureimplicationsofthesefindingsinclinical practiceforearlydiseaseprognosisandtreatmentmanagement.

ChroniclesoflncRNA

Inthebrain,lncRNAisaheterogeneousclassoftranscriptsthatcontributes30%to70%oftheexpressedtranscriptome;however,thepercentagecanvarydependingonbrainregionsandcelltypes(17, 18).Arbitrarily,lncRNAsaremorethan200-nucleotide-longRNAtranscriptswith limitedcodingpotentialandlowlevelsofexpressionandsequenceconservation(18, 19).Coherencyinexpressioncorrelationbetweengenes andlncRNAsinbothpositiveandnegativefashionhasledtodeciding theirroleinspatiotemporaltranscriptomicregulation,unlikeothernoncodingRNAfamilymembers[e.g.,microRNAs(miRNAs)].Despitethe standardstructuralfeaturesof5’methylcapping,polyadenylatedtail, andcapabilityofproducingsplicevariants,lncRNAsdifferfromstandardprotein-codingmRNAsinseveralattributes.Withlimitedcodingpotential,lncRNAsremaincontroversialbecauseopenreadingframesare sparselyfoundonthem.Mostoften,thelncRNAisfoundtohavefewer andlongerexonlengthswithlessprimarysequenceconservationpattern(18).Regardlessofthelessclassifiedstructuralconspicuityandinadequatelydocumentedbiogenicorigin,manylncRNApopulationshave showndiscerniblefunctionalrelationshipsinregulatingcomplexcellularprocesses(20).Consideringthemodeoffunctionatthecellularlevel, lncRNAsaremostlyrecognizedasepigeneticmediatorsthatmodulate

1 DepartmentofPsychiatryandBehavioralNeurobiology,UniversityofAlabamaatBirmingham,Birmingham,Alabama35294,USA

CorrespondingAuthor: YogeshDwivedi,PhD,DepartmentofPsychiatryandBehavioralNeurobiology,UniversityofAlabamaatBirmingham,SC711SparksCenter,17207th AvenueSouth,Birmingham,Alabama,USA.Phone:01-205-975-8459.E-mail: ydwivedi@uab.edu Received:1May2024.Revised:23May2024.Accepted:18June2024. Publishedonline:18July2024.

Figure1. Thenetworkplotillustratestheinterconnectednessofthreedistinct mooddisorders(majordepressivedisorder:MDD,bipolardisorder:BD,andsuicidalbehavior:SB)basedonsharedlncRNAprofilesdetectedintheperipheral circulation.EachnoderepresentsaspecificlncRNA,andedgesbetweennodes indicatesignificantassociationsbetweenthemanddisordersacrosstheanalyzedstudies.Nodecolorandshapecorrespondtodifferentmooddisorders, facilitatingvisualizationandinterpretationofthefindings.

informationprocessingpathwaysbytargetingalmosteverystageofthe centraldogma(21).Theirbimodalroleinachievingbothcisandtransregulationcomesfromtheabilitytoactasscaffolds,decoys,andantisense interference(19, 22).Altogether,theepigenomiccomplexityachievedby lncRNAsduetotheirdiversenatureofregulatoryfunctionmakesthem anobviouschoicetobeusedaspotentialtherapeutictargetstomodulategenefunction(Figure1).

Ithasbeensuggestedthatapproximately40%ofidentifiedlncRNAs exhibitbrain-specificfunctions(23).TheselncRNAs,consideredamong themostevolutionarilyconservedtranscripts,demonstratespecificexpressionpatternsinkeybrainregionssuchasthecortex,cerebellum, andhippocampus(24, 25).RecentstudieshaveunderscoredtheemergingsignificanceoflncRNAsinregulatingcrucialneuronalprocesseslike synaptogenesis,plasticity,neuritedevelopment,andneuronaldifferentiation(23, 26, 24).However,furtherexplorationoftheirroleinneuropsychiatricdisordersiswarranted.Brain-expressedlncRNAsexhibitgreater spatiotemporalandcell-typespecificitywithinterestingsubcellularexpressionprofilesthananyotherprotein-codinggenes(27).Moreover, theirexpressionisdynamicallyregulatedinresponsetoneuronalactivity.RecentRNAsequenceanalyseshavehighlightedthetranscriptional deregulationoflncRNAsinthepostmortembrainsofindividualswithpsychiatricdisorders;thisalignswithfindingsindicatingthatmanymutationsassociatedwithneuropsychiatricconditionsoccurinnoncodingregionsofthegenome(28).Genome-wideanatomicaldetailshaverevealed theirpreferentialgenomicpositioningnearcodinggenesspecifictothe brain,whichoftenshareasimilarexpressionpatternwiththesegenes (23, 29).ThisdemonstratesauniquefeatureoflncRNAs,whichlikelyplays apivotalroleinfinelymodulatinggenelevelsessentialforneurogenesis, akeyeventoftenseentobeputurbedinmooddisorders.

ThePotentialoflncRNAasaCirculatingBiomarkerinMoodDisorders Thesignificanceoftheprecedingdiscussionliesinitsclinicaltranslationalvalue,particularlyinestablishingaconnectionbetweenthe

mechanicalrelevanceobservedinpsychiatricbrainsandthedevelopmentofcirculatinglncRNAsasperipheralbiomarkersformooddisorders.ThesecirculatinglncRNAs,detectableinbodilyfluidssuchasblood, cerebrospinalfluid,andsaliva,holdimmensepotentialasnoninvasive toolsfordiagnosingmooddisorders,predictingtherapeuticresponse, andmonitoringtreatmentoutcomes.Theiraccessibilityandstability makethemattractivecandidatesforbiomarkerdiscovery,providinginsightsintodiseasepathogenesisandprogressionwithoutrequiringinvasiveprocedures(30).ExpandingtheclinicalutilityoflncRNAsholds significantpromise,especiallyinthedomainofmooddisorders.While currentresearchhasshownpreliminaryevidenceoflncRNAs’clinicalrelevance,thereisaburgeoninginterestinexploringtheirbroaderapplicationsinpsychiatricpractice,particularlyindiagnosing,prognosticating, andtreatingmooddisorders.

Diagnosticbiomarkers,suchaslncRNAMALAT1,haveshownpromise indiscerningdistinctexpressionpatternsinindividualswithmooddisorders,suggestingtheirpotentialforearlydetectionandintervention strategies.Similarly,lncRNAslikeHOTAIRmayserveasprognosticindicators,offeringinsightsintotreatmentresponsesanddiseaseprogression. Additionally,targetingdysregulatedlncRNAs,suchasNEAT1,presents therapeuticavenuesforrestoringneuralcircuitryfunctionandalleviating symptomsassociatedwithmooddisorders.Despitethesepromisingapplications,severalchallengesmustbeaddressedtofacilitatethebroader clinicalintegrationoflncRNAsinmooddisorders.Standardizationofassays,encompassingsamplecollection,RNAisolationmethods,anddata analysispipelines,iscrucialforensuringreproducibilityandreliability acrossstudies.Collaborativeeffortsamongmultidisciplinaryteamsare vitalforelucidatingthemolecularmechanismsunderlyinglncRNAinvolvementinmooddisordersandovercomingbiologicalcomplexities. Ethicalandregulatoryconsiderationsalsoplayasignificantroleinthe clinicalintegrationoflncRNAs.Upholdingpatientprivacy,obtaininginformedconsent,andadheringtodata-sharingprotocols,areessentialfor ethicalclinicaltranslation.Transparencyinreportingstudyfindingsand adherencetoregulatoryguidelinesarevitalformaintainingpublictrust inlncRNA-basedapproaches.Inconclusion,theexpandedclinicalusage oflncRNAsinmooddisorderspresentsanexcitingfrontierinpsychiatric practice.ByleveraginglncRNAbiomarkersasdiagnostictools,prognosticindicators,andtherapeutictargets,clinicianscanadoptpersonalized approachestopatientcare.Addressingchallengesrelatedtostandardization,biologicalcomplexity,andethicalconsiderationsiscriticalfor realizingthefullclinicalpotentialoflncRNA-basedstrategiesinmood disorders.

Theproceedingsectionswillexaminetheseaspects,ensuringalignmentwiththescopeandinterestofthisreviewarticle.Asummaryofthe discussedreportsisprovidedin Table1

lncRNAsinMDD

UnderstandingthemolecularunderpinningsofMDDiscrucialforadvancingdiagnosticandtherapeuticstrategiesinpsychiatry.Overtheyears, researchershaveincreasinglyfocusedontheroleoflncRNAsinMDD pathogenesis,aimingtoelucidatetheirregulatoryfunctionsandpotentialasbiomarkersortherapeutictargets.Inthisnarrative,wedigdeeply intostudiesinvestigatingthedysregulationoflncRNAsinpatientswith MDD.Thesestudiesencompassinvestigationsintogeneticassociations, expressionprofiling,andthefunctionalimplicationsoflncRNAsinaspectssuchasdiagnosis,suiciderisk,andtreatmentresponse.However, weaimtoexplorethediagnosticandtherapeuticopportunitieslncRNAs presentinMDD,particularlyemphasizingtheirpotentialroleasperipheralbiomarkers,especiallyindiseasediagnosisandtreatmentresponse.

Tobegin,oneofthepolymorphicstudieshasinvestigatedthepotentialinvolvementofintergenicgeneticvariantsinMDDpathogenesisby regulatinglncRNAlocatedwithintheseregions(31).Genome-wideassociationstudieshaveidentifiednumerousgeneticvariantsforMDD,with asignificantportionlocatedinintergenicregionswhereapproximately 54%oflncRNAsarefound.ThehypothesisisthattheseintergenicvariantsmightinfluenceMDDsusceptibilitybymodulatingtheexpression oflncRNAsintheirvicinity.SeveralMDD-associatedsingle-nucleotide polymorphisms(SNPs)withinthreeknownintergeniclncRNAswereini-

Table1. ThelncRNAprofilingandtheirclinicalutilityinhumanperipheralbloodsamplesfromsubjectswithMDD,BD,andSBforbiomarker analysis

SourcelncRNAslncRNAFindingOutcomeTechniquesUsedReferences

Peripheralbloodchr10:874695-874794, chr10:75873456-75873642, chr3:47048304-47048512

PBMCTCONS_00019174, ENST00000566208, NONHSAG045500, ENST00000517573, NONHSAT034045,and NONHSAT142707

MajorDepressiveDisorder

Expression profiling

Peripheralblood cells LINC01108 Expressionand SNPanalysis

AuthorsidentifiedlncRNAthat areaberrantlyexpressedinMDD andcontributedinthemolecular pathogenesisofthisdisorder

Microarray-based high-throughputgene expressionprofiling 36

ExpressionofsixlncRNAsin PBMCsmayserveaspotential biomarkersfordiagnosisand therapeuticresponseinMDD Microarrayand quantitativePCR

Thisstudyprovidespreliminary evidencethatintergenicvariants mightcontributetothe pathogenesisofMDDthrough regulatingtheexpressionof lncRNAswherethesevariantsare located

Peripheralblood

RMRPExpression profiling

Y5,MER11C,PCAT1,andPCAT29Expression

PeripheralbloodXIST,RP11-706O15.3, RP11-706O15.5,RP11-415F23.2, RP11-1250I15.1,CTC-523E23.11, RP11-706O15.7,AL122127.25, TNRC6C-AS1,RP4-575N6.4

RP11-51J9.5

WholebloodANRIL-associatedSNPrs1333045 andrs1333048

Peripheralblood25DElncRNAs(MALAT1, LINC00504,HCG18,LINC02503, AL590867.1,andSMIM25)

Outcomeofthisstudysuggests thatfivelncRNAsareDEin patientswithMDDcomparedto healthysubjectsandlowerRMRP levelmayserveasapotential biomarkerforMDD

Expression profiling DElncRNAmoleculesare attractivebiomarkersto diagnosedepression

TaqMangenotyping

SNPanalysisSNPswithinselectedlncRNAs suchas ANRIL mightconferrisk ofneuropsychiatricdisorders

Expression profiling ResultssuggeststhatlncRNAs determinedinperipheralblood mayaffectmetabolism, inflammation,immunity,and oxidativephosphorylationand matbeinvolvedinthe pathogenesisofMDD

WholebloodFEDORA(RP11-298D21.1)Expression profiling lncRNAFEDORAmayplayan importantroleinshapingthe sex-specificlandscapeofthe brainandcontributetosex differencesinMDD

BipolarDisorder

PeripheralbloodCCAT2,TUG1,PANDAExpression profiling

Resultsdemonstratesthe possibleroleofcertainlncRNAs inthepathogenesisofbipolar disorderandtheirpotentialuse asdiagnosticmarkersinthis disorder

Genotypingusing tetra-primer amplification refractorymutation systemPCRmethod 43

Wholetranscriptome sequencing

QuantitativePCR (qPCR)-based expressionprofiling

Table1.(Continued)

SourcelncRNAslncRNAFindingOutcomeTechniquesUsedReferences

PeripheralbloodOIP5-AS1Expression profiling

WholebloodANRILassociatedSNPrs1333045 andrs1333048

SNPanalysisSNPswithinselectedlncRNAs suchas ANRIL mightconferrisk ofneuropsychiatricdisorders

PeripheralbloodMALAT1Expression profiling

PBMC SCAL1 (LUCAT1), RMST, MEG3 Expression profiling

WholebloodGAS5andFOXD3-AS1Expression profiling

Peripheral venousblood MALAT-1, GAS-5 Expression profiling

ExpressionlevelofMALAT1can serveasapotentialbiomarkerfor bipolardisorder

Peripheralexpressionofcertain lncRNAsmaybeusedaspotential biomarkersforbipolardisorder

Theresultshighlightthat dysregulationofFOXD3-AS1and GAS5maybeassociatedwithan increasedriskofbipolardisorder

Thisstudyprovidesevidencefor theuseoflncRNAsasbiomarkers forthediagnosisandmonitoring theeffectivenessoftherapiesof bipolardisorder

SuicidalBehavior

PBMCTCONS_00019174, ENST00000566208, NONHSAG045500, ENST00000517573, NONHSAT034045,and NONHSAT142707

Expression profiling

tiallygenotypedamongacohortof978patientswithMDDand1,176 controlstoexplorethishypothesis.Subsequently,quantitativereverse transcriptasePCR(qRT-PCR)assayswereconductedtoquantifytheexpressionlevelsoftwospecificlncRNAs,LINC01108andLINC00578,in peripheralbloodcellsfromasubsetof20patientswithMDDand20 controls.Theresultsrevealedastrongassociationbetweenrs12526133 withinLINC01108andMDD,withsignificantlyhigherexpressionlevels ofLINC01108inthepatientgroupcomparedtocontrols.Additionally, theanalysisofLINC00998expressionviamicroarrayshowedasignificantlylowerlevelinpatientswithMDDthanincontrols,withfurther genotypingrevealinganassociationbetweenrs2272260inLINC00998 andMDD.Thesefindingssuggestthepotentialroleofnoncodingvariants,particularlythosewithinintergenicregions,incontributingtothe riskforMDD.Byelucidatingtheinterplaybetweengeneticvariantsand lncRNAexpression,thisstudyprovidesvaluableinsightsintothecomplex molecularmechanismsunderlyingMDDpathogenesis,openingavenues forfurtherresearchintothefunctionalsignificanceofnoncodingvariantsandtheirpotentialastherapeutictargetsordiagnosticmarkersfor MDD(31).

AnotherstudyaddressedsuicideinthecontextofWorldHealthOrganizationreportingnearly1millionsuicidesannuallyworldwide,with 40%ofsuicidecompletersexperiencingmajordepression(32).FocusingonpatientswithMDD,theprimaryobjectiveofthisstudywastoinvestigatetheassociationbetweenlncRNAexpressioninperipheralblood mononuclearcells(PBMCs)andsuiciderisk(33).UtilizingHumanLncRNA 3.0microarrayprofiling,encompassing30,586humanlncRNAs,andemployingRT-PCR,thestudyidentifiedsixdownregulatedlncRNAsdifferentiallyexpressed(DE)inpatientswithMDD.Basedonsuicidalideationand pastsuicidalattempts,patientswithMDDwerecategorizedintogroups: suicidalideation,nosuicidalideation,pastsuicideattempt,andnopast suicideattempt.RT-PCRanalysisrevealedsignificantdifferencesinthe

Expressionofsixdownregulated lncRNAshadanegative associationwithsuicideriskin MDD.

Genotypingusing tetra-primer amplification refractorymutation system(ARMS)PCR method

(qPCR)-based expressionprofiling

MicroarrayandqPCR34

expressionofthesixlncRNAsbetweenthesuicidalideation,nosuicidalideation,andcontrolgroupswhilecorrespondinglncRNAsassociatedwithsuicidalattemptexhibitednotabledifferencesbetweenpast attempt,nopastattempt,andcontrolgroups.Interestingly,onlytheexpressionoflncRNAsinthesuicidalideationandpastattemptgroups significantlydecreasedcomparedtocontrols.Thesefindingssuggesta negativeassociationbetweentheexpressionofthesixdownregulated lncRNAsandsuicideriskinpatientswithMDD.Importantly,thestudyunderscoresthepotentialoflncRNAexpressioninPBMCsasabiomarker forassessingsuicideriskinpatientswithMDD,therebyenablingclinicianstodelivertimelyinterventionsandpreventsuicide.Thisresearch contributesvaluableinsightsintothemolecularmechanismsunderlying suicideriskinMDD.IthighlightstheclinicalutilityoflncRNAsaspotentialdiagnosticmarkersforsuicideriskassessmentinpsychiatricpractice. However,furtherinvestigationsarewarrantedtovalidatethesefindings andexplorethetherapeuticimplicationsoflncRNA-basedinterventions insuicidepreventionstrategiesforpatientswithMDD(33).

Inaseparatestudy,theroleofanewlydiscoveredlncRNA,FEDORA, wasfoundinbloodsamplesofwomendisproportionatelyaffectedbyMDD (34).ThebloodlevelsofFEDORAexhibitdiagnosticimplicationsfordepressedwomenandarelinkedtotheclinicalresponsetoketaminetreatment.ThesefindingsnotonlyunderscorethepivotalroleoflncRNAs,particularlyFEDORA,inshapingthesex-specificlandscapeofthebrain,but alsoitscontributiontoperipheraldiagnosistodeterminesexdifferences inMDD.ThishighlightsthepotentialdiagnosticandtherapeuticimplicationsoflncRNAslikeFEDORAinpavingthewayforpersonalizedinterventionstailoredtoaddresssex-specificvulnerabilitiesindepression(34).

AnotherstudyexaminedtheemergingroleofalargepaneloflncRNAs inmooddisorders,particularlyinMDD(35).Toaddressthis,theresearch employedmicroarraytechnologytoprofiletheexpressionof34,834 lncRNAsand39,224mRNAsinperipheralbloodsamplesobtainedfrom

patientswithMDDanddemographicallymatchedcontrols.Amongthese transcripts,2,007lncRNAsand1,667mRNAswerefoundtobeDE,including17previouslydocumenteddepression-relatedgenes.GeneOntology (GO)andpathwayanalysesrevealedthatthebiologicalfunctionsofDE mRNAswereassociatedwithfundamentalmetabolicprocessesandneurodevelopmentaldiseases.Toelucidatethepotentialregulatoryrolesof DElncRNAsonmRNAs,coexpressionnetworkscomprisinglncRNAsand mRNAswereconstructed,revealingsignificantlycorrelatedexpression patterns.Notably,theMDD-derivednetworkexhibitedagreaternumberofnodesandconnectionscomparedtothecontrol-derivednetwork. Specifically,lncRNAslocatedatchr10:874695-874794,chr10:7587345675873642,andchr3:47048304-47048512wereidentifiedaspotential regulatorsofmRNAexpression,havingpreviousassociationswithMDD. Thispioneeringstudyrepresentsthefirstexplorationofgenome-wide lncRNAexpressionandcoexpressionpatternswithmRNAinMDD.IdentifyingaberrantlyexpressedcirculatinglncRNAsinMDDsuggeststheirpotentialcontributiontothemolecularpathogenesisofthedisorder.These findingsoffervaluableinsightsintotheregulatorynetworksunderlying MDDandhighlightthesignificanceoflncRNAsaspotentialbiomarkers andtherapeutictargetsforMDD.Furtherresearchiswarrantedtovalidatethesefindingsandelucidatethefunctionalrolesofdysregulated lncRNAsinMDDpathophysiology(35).

OnestudyutilizedanintegratedanalysistoexaminethedifferentialexpressionofmiRNAs,lncRNAs,circularRNAs(circRNAs),andmRNAs betweenMDDandhealthycontrols(HCs)(36).Wholetranscriptomesequencingonperipheralbloodsamplesfrom15patientswithMDDand15 matchedHCs,followedbyweightedgeneco-expressionnetworkanalysis (WGCNA),wasemployedtoidentifyRNAcoexpressionmodulesassociated withMDD.Additionally,acompetitiveendogenousRNA(ceRNA)network wasconstructedtointerpretinteractionsbetweendifferentRNAspecies. KyotoEncyclopediaofGenesandGenomes(KEGG)pathwayanalyseswere conductedtoexplorepotentialbiologicalmechanismsassociatedwith MDD.TheresultsrevealedsignificantdysregulationofmultipleRNAsand coexpressionmodulesinMDDcomparedtoHCs.Furthermore,aceRNA network(alsoknownasatripartitetranscriptionalregulatorynetwork, thisnetworkisbasedonthecomplexinteractionsamonglncRNA,miRNA, andmessengerRNAs(mRNA),anditfunctionstomediateaceRNAinhibitionenvironmentwithincellularconditions)comprisingdysregulated RNAsinMDDwasconstructedbasedontheidentifieddifferentialRNAs. PathwayanalysishighlightedassociationsbetweenMDDandprocesses relatedtooxidativephosphorylationandchemokinesignaling,suggestingthepotentialinvolvementofenergymetabolismandinflammationin thepathophysiologyofMDD.Asexaminedinperipheralcirculation,these findingsofferinsightsintothecomplexregulatorynetworksconstructed bylncRNA-miRNAandmRNAinteractionunderlyingMDDpathogenesis, andprovidepotentialtargetsforfurtherinvestigationaswellastherapeuticintervention(36).

MDDoftenco-occurswithsignificantlevelsofanxiety,withgreaterillnessseverityandfunctionalimpairmentobservedinpatientsexperiencingbothconditions.However,theunderlyingpathogenesisofthiscomorbidityremainsuncertain.Toexplorepotentialmolecularlinksbetween MDDandgeneralizedanxietydisorder(GAD),lncRNAmicroarrayprofilingandreversetranscriptionpolymerasechainreaction(RT-PCR)wasutilizedtoidentifysixdownregulatedlncRNAsaspotentialbiomarkersfor MDDandthreeupregulatedlncRNAsforGADinbloodmononuclearcells (PBMCs)(37).TheselncRNAswerethencross-checkedin40patientswith MDD,40patientswithGAD,and40normalcontrols.Theresultsrevealed thatthesixdownregulatedlncRNAsassociatedwithMDDexhibitedsignificantlylowerexpressionlevelsinGADthannormalcontrols,withno significantdifferencebetweenGADandMDDgroups.Conversely,three upregulatedlncRNAsinGADshowednosignificantdifferenceinexpressionlevelscomparedtoMDD,butexhibitedaremarkabledifferencebetweenMDDandGADgroups.ThesefindingssuggestthatlncRNAsinPBMC couldserveaspotentialmolecularlinksbetweenMDDandGAD,providingnewinsightintothesharedpathogenesisofthesedisorders.Furthermore,theresultsimplythatanxiousdepressioncouldrepresentadistinctdiagnosticsubtypeofMDD.Overall,thisstudycontributestoour understandingofthemolecularmechanismsunderlyingthecomorbidity

betweenMDDandGAD,potentiallypavingthewayfordevelopingmore targeteddiagnosticandtherapeuticapproaches(37).

Inaseparatestudy,onegroupofinvestigatorsaimedtoexamine lncRNAsinPBMCfromdepressedpatientsandperformedbioinformaticanalysisforlncRNAtargetgenepredictionandfunctional annotation(38).Initially,microarrayanalysiswasconductedtoidentify transcriptome-widedysregulatedlncRNAs.Subsequently,10lncRNAs withthehighestexpressionchangeswereselectedforvalidationusing qRT-PCRandbioinformaticsanalysis.Theresultsrevealedsignificant downregulationintheexpressionlevelsofninelncRNAs(TCONS_L2_ 00001212,NONHSAT102891,TCONS_00019174,ENST00000566208, NONHSAG045500,ENST00000591189,ENST00000517573,NONHSAT034045,NONHSAT142707)comparedtocontrols(p < 0.05).Furthermore,lncRNAtargetgenepredictionandfunctionalannotationanalysis indicatedsignificantenrichmentinGObiologicalprocesses(BP)and KEGGpathwaysassociatedwiththenervoussystemandbrainfunctions, suggestingthepotentialinvolvementofthedysregulatedlncRNAs inthepathogenesisofMDD.Additionally,Cytoscape-basednetwork constructionprovidedfurthercluestotheassociationoftheselncRNAs withMDD.Inconclusion,thestudysuggeststhatalteredexpressionof theidentifiedlncRNAsmayplayaroleinthepathogenesisofMDDand couldserveasnoninvasivebiomarkersforMDDdiagnosis.Thesefindings furthercontributetoourunderstandingofthemolecularmechanisms underlyingMDDandmayfacilitatethedevelopmentofnoveldiagnostic strategies(38).

TofurtherunderstandaberrantlncRNAexpressionandMDDpathophysiology,thenextstudyaimedtoassessthepotentialoflncRNAsin peripheralbloodleukocytesasbiomarkersforMDD(39).Inthisstudy, theauthorsperformedqRT-PCRanalysistomeasuretheexpressionlevelsof83lncRNAsintheperipheralbloodleukocytesof29patientswith MDDand29age-andgender-matchedHCs.ThefindingsrevealeddistinctexpressionsignaturesinpatientswithMDD,withlowerexpressionof onelncRNA(RMRP)andhigherexpressionoffourlncRNAs(Y5,MER11C, PCAT1,andPCAT29)comparedtoHCs.Notably,theexpressionlevelof RMRPcorrelatedwithdepressionseverity,asmeasuredbytheHamiltonDepressionRatingScale(HAM-D).Furthermore,theauthorstested theirhumanfindingsinanimalsanddetectedlowerRMRPexpressionin amousemodelofdepression,supportingthefindingsfrompatientswith MDD.TheseresultssuggestthatlowerRMRPlevelsmayserveasapotentialbiomarkerforMDD.Thisstudycontributestoourunderstandingofthe molecularmechanismsunderlyingMDDandofferspromisingavenuesfor developingbiomarkersforbetterdiseasediagnosis(39).

SNPsoflncRNAhaveemergedaspotentialcontributorstodepressionsusceptibility.InaChinesepopulationstudy,fourlncRNASNPs (rs2242385,rs155979,rs3762983,andrs3762984)werefoundtobe associatedwithlncRNA,NONHSAT102891,ondepressionsusceptibility (40).Throughacase–controlstudyinvolving480depressionpatientsand 329HCs,genotypingwasperformedusinggenesequencing.Thefindings revealedthatthers155979GCgenotypeexhibitedasignificantassociationwithanincreasedriskofdepressioncomparedtoHCs.Thestratified analysisfurtherindicatedanapproximately2-foldelevatedriskofsuicide amongpatientswiththers155979GCorGGgenotype.However,nosignificantassociationswereobservedbetweenthers2242385,rs3762983, andrs3762984polymorphismsanddepressionrisk.Haplotypeanalysisunveiledlinkagedisequilibriumbetweenrs155979,rs3762983,and rs3762984,withtheCCGhaplotype,demonstratingareducedriskof depression.Despitethestudy’slimitations,suchasarelativelysmall samplesizeandrestrictiontopatientsfromaspecificpopulation,these findingsshedlightonthepotentialassociationofthers155979polymorphismwithdepressionoccurrenceintheChinesepopulation.Furtherinvestigationsinvolvinglargercohortsanddiverseethnicgroupsarewarrantedtovalidatethesefindingstoascertaintheirreliability.Thisstudy underscoresthesignificanceofexploringgeneticvariations,particularly lncRNASNPs,fromperipheralcirculationwithapromisetounravelthe complexetiologyofdepressionandadvancepersonalizedtreatmentapproaches(40).

Thediagnosisofdepressionreliesprimarilyonbehavioralobservationandself-reportingofsymptoms,lackingbiologicalvalidation.

Toovercomethis,onestudyaimedtoidentifylncRNAsinperipheral PBMCsasbiomarkersfordiagnosingandpredictingtreatmentresponse inMDDcases(30).HumanlncRNA3.0microarrayprofiling,covering 30,586humanlncRNAs,wasusedinPBMCsfrom5patientswithMDD andfivecontrols.DElncRNAsinPBMCsofpatientswithMDDwere identified,and10candidatelncRNAswereselectedforfurthervalidationusingRT-PCRanalysisinalargervalidationcohortof138patientswithMDDand63HCs.Additionally,amongthe138patientswith MDDreceivingstandardantidepressanttreatment,30wererandomly selectedforlncRNAexpressionretestingandsymptomatologyassessmentsafter3and6weeksoftreatment.Thefindingsrevealedthat sixlncRNAs(TCONS_00019174,ENST00000566208,NONHSAG045500, ENST00000517573,NONHSAT034045,andNONHSAT142707)weresignificantlydownregulatedinpatientswithMDDcomparedtocontrols.The combinedexpressionofthesesixlncRNAsexhibitedanareaunderthereceiveroperatorcurve(ROC)of0.719[95%confidenceinterval(CI):0.617–0.821],suggestingtheirpotentialasdiagnosticbiomarkersforMDD.Importantly,therewerenodifferencesintheexpressionoftheselncRNAs basedongenderorage.Inconclusion,thecombinedexpressionofsix lncRNAsinPBMCsholdspromiseasapotentialbiomarkerfordiagnosingandpredictingtherapyresponseinMDD,offeringavaluabletoolfor clinicalpractice(30).

Perinataldepression(PD)posessignificantchallengesindiagnosis andtreatment,especiallyinregionswithlimitedaccesstomental healthprofessionals.UsingRT-PCR,sixdownregulatedlncRNAs,found tobeassociatedwithMDD(NONSUSG010267,NONHSAT140386, NONHSAG004550,NONHSAT125420,NONHSAG013606,andNONMMUG014361),wereassessedin39pregnantwomenwithPD(PDgroup), 20PDpatientsundergoingmindfulness-integratedcognitivebehavior therapy(MiCBT)[treatmentgroup(TG)],and51normalpregnantwomen [normalcontrol(NC)group]duringthesecondtrimesterandat42days postpartum(41).TheresultsshowedthatthesesixlncRNAsweresignificantlydownregulatedinthePDgroupduringthesecondtrimesterandat 42dayspostpartumcomparedwiththeNCgroup.AfterMiCBTtherapy, theexpressionofNONHSAG004550andNONHSAT125420wassignificantlyupregulatedintheTG,withnosignificantdifferencesobserved betweenTGandtheNCgroupat42dayspostpartum.Furthermore, NONHSAG004550andNONHSAT125420exhibitedsignificantdifferentialexpressioninthePDgroup,andthisexpressionpatternchanged withtheimprovementofdepressivesymptoms.TheROCcurveanalysis revealedthatthecombinationofthesetwolncRNAshadgoodpredictive valueforPD,withanareaunderthecurve(AUC)of0.764(95%CI:0.639–0.888).Inconclusion,thecombinationoflncRNAsNONHSAG004550and NONHSAT125420showspromiseasanoveldiagnosticbiomarkerforPD inperipheralcirculation(41).

Inanotherstudy,theresearchersfocusedontwoSNPs,rs1333045 andrs1333048,withintheANRILgenelocusandtheirassociationwith BDandMDD(42).ANRILisanantisensenoncodingRNAintheINK4 locus(ANRIL)andhasbeenfoundtobeimportantinmentaldisorders.InmethamphetamineaddictioninanIranianpopulationstudy, thepolymorphicanalysisrevealedintriguingassociationsbetweenthese SNPswithMDDandBD.Forinstance,rs1333045showedassociations withmethamphetamineaddictioninrecessiveandmultiplicativemodels, whilers1333048exhibitedassociationswithmethamphetamineaddictioninthecodominantmodel.Moreover,rs1333048showedassociations withBPIinthecodominantmodelandotherinheritancemodels,whereas rs1333045wasnotassociatedwithBPIinanyinheritancemodel.Notably, significantassociationswereobservedbetweenbothSNPsandBPIIinall inheritancemodels.Interestingly,thestudyalsouncoveredassociations betweentheselectedSNPsandMDD,withrs1333045beingassociated intherecessivemodelandrs1333048indominant,recessive,andmultiplicativemodels.Additionally,haplotypeanalysesindicatedthatcertain haplotypeswereassociatedwithdecreasedorincreasedriskofaddiction, BPI,BPII,andMDD(42).

Separately,inaninterestingstudy,agroupofresearchersaimedtouncovermoleculardisparitiesbetweentype2diabetesmellitus(T2DM)and T2DMwithdepressionbyinvestigatingtheexpressionprofilesoflncRNA, mRNA,andcircRNAinpatients’blood(43).Throughmeticulousscreen-

ingandprofiling,thestudyidentified28lncRNAs,107circRNAs,and89 mRNAsthatexhibiteddifferentialexpressionindepressedpatientscomparedtothosewithT2DMalone.Furtheranalysis,includingbioinformaticsassessment,shedlightonthefunctionalrolesoftheseDEgenes, revealingtheirinvolvementinvariousBPandpathwaysassociatedwith depression.Notably,genesimplicatedinneuropsychiatricsystemdevelopment,immunity,andinflammationwerefoundtobedysregulatedin thedepressedgroup,underscoringthecomplexinterplaybetweenmolecularpathwaysandpsychiatricdisorders.ValidationofkeyDElncRNAsand mRNAsthroughRT-PCRexperimentscorroboratedthefindingsfromsequencing,affirmingtheexistenceofdistinctexpressionprofilesinpatientswithMDDcomparedtothosewithT2DMalone.Additionally,the constructionoflncRNA-mRNAregulatorynetworkselucidatedregulatory mechanismsgoverninggeneexpressioninMDDandprovidedaframeworktoconsidertheuseoflncRNAexpressioninbloodfordiagnostic screening(43).

InapromisingsteptowardidentifyingexosomallncRNAsasbiomarkersforadolescentdepression,astudyenrolledasignificantnumberof adolescentsubjectsandusedmicroarrayassaystoscreenfordifferential expressionsoflncRNAsandmRNAsinplasmaexosomes(44).BygeneratingtwosetsofceRNAnetworkscomprisinglncRNAs,miRNAs,andmRNAs,thestudydelvedintotheintricatemolecularinteractionsunderlying depression.Theidentificationofcandidategenes,includingAC156455.1, miR-126-5p,AAK1,CCDC18AS1,miR-6835-5p,andCCND2,fromthese networks,highlightedpotentialtargetsforfurtherinvestigation.Importantly,thedifferentialexpressionofthesegenesbetweenpatientswith MDDandHCs,aswellasbeforeandafterantidepressanttreatment,suggestedtheirutilityasdiagnosticandtherapeuticbiomarkers.Ofparticularinterestarethefindingsregardingtheexpressionlevelsofgenes, suchasAAK1,CCDC18AS1,andmiR6835,whichvariedinefficacyfollowingsertralinetreatment.Thisunderscoresthepotentialofthesegenes asindicatorsoftreatmentresponseandhighlightstheimportanceofpersonalizedmedicineinmanagingMDDintheadolescentpopulation.Moreover,identifyingbaselineexpressionlevelsofCCDC18AS1,miR-6835-5p, andCCND2aspredictorsofantidepressantefficacyisasignificantadvancementinthefield.Theproposedmediationofantidepressantefficacy throughthereductionofsuicidalideationandimprovementofcognitive functionaddsdepthtoourunderstandingofthemechanismsunderlying MDDanditstreatment(44).

Anotherexosomalstudyshedslightonapotentiallygroundbreaking avenuefordiagnosingandunderstandingthelong-termeffectsofrepetitivemildtraumaticbraininjuries(rmTBI),particularlyinmilitaryservice membersfollowingthescreeningofexosomallncRNAfromblood(45). IdentifyingspecificlncRNAsinserumsamples,particularlythoseofcentralnervoussystem(CNS)origin,opensupanewfrontierinbiomarkerresearchforneurologicaldisorders.Oneofthekeyfindingsofthisstudyis theconsistentpresenceoffourlncRNAsinserumsamplesobtainedfrom individualswithandwithoutrmTBI.Amongthese,VLDLR-AS1emerged asasignificantcandidate,withlowerlevelsdetectedinindividualswith rmTBIthanthosewithoutTBIhistory.ThisobservationsuggeststhepotentialroleofVLDLR-AS1asabiomarkerforidentifyingchronicrmTBI. TherobustnessofthefindingsisfurthersupportedbytheROCanalysis, whichyieldedanAUCof0.74,indicatingareasonablyhighdiscriminatorypowerofVLDLR-AS1indistinguishingbetweenindividualswithand withoutrmTBI.TheidentifiedoptimalcutoffforVLDLR-AS1levelsprovidesapracticalthresholdforpotentialclinicalapplications.Moreover, thestudyprobesintotheassociationbetweenlncRNAsandpsychological symptomburden,particularlydepression,amongindividualswithrmTBI. ThecorrelationbetweenVLDLR-AS1andMALAT1levelswithsymptoms ofdepressionunderscoresthepotentialutilityoftheselncRNAsasnot onlydiagnosticmarkersforrmTBI,butalsoasindicatorsofassociated psychiatriccomorbidities.Thesefindingshavesignificantclinicalimplications.TheabilitytodetectandmonitorrmTBIusingblood-basedbiomarkersoffersanoninvasiveandpotentiallycost-effectiveapproachforearly diagnosisandintervention.Additionally,theidentificationoflncRNAsassociatedwithpsychiatricsymptomsprovidesinsightintothecomplex interplaybetweenneurologicalandpsychologicalaspectsofTBIsequelae.However,furtherresearchiswarrantedtovalidatethesefindingsin

largercohortsanddiversepopulations.Additionally,elucidatingthefunctionalrolesoftheidentifiedlncRNAsinthepathophysiologyofrmTBI andassociatedcomorbiditieswouldenhanceourunderstandingandpave thewayfortargetedtherapeuticinterventions.Insummary,thestudy highlightsthepromiseoflncRNAsinextracellularvesicles(EVs),particularlyVLDLR-AS1,asbloodbiomarkersforidentifyingchronicrmTBIand associatedpsychiatricsymptoms.Embracingsuchmolecularapproaches holdsthepotentialtorevolutionizethediagnosis,prognosis,andmanagementofneurologicaldisorders,particularlythosearisingfromtraumaticbraininjuries(45).

Inconclusion,theexplorationoflncRNAsinthecontextofMDDrepresentsapromisingavenueforunderstandingthemultifacetednature ofthisdebilitatingdisorder.ThecollectivefindingsunderscoretheintricateinterplaybetweenlncRNAdysregulationandMDDpathophysiology, fromgeneticassociationstofunctionalimplicationsinbrainandtreatmentresponse.IdentifyinglncRNAsaspotentialbiomarkersfordiagnosis,prognosis,andtreatmentresponseholdsgreatpromiseforpersonalizedpsychiatry,offeringnewavenuesfortargetedinterventionsand precisionmedicineapproaches.However,furtherresearchiswarrantedto validatethesefindings,elucidateunderlyingmechanisms,andtranslate themintoclinicalpractice,ultimatelyimprovingoutcomesforindividuals affectedbyMDD.

lncRNAsandBD

BDisacomplexpsychiatricconditioncharacterizedbyrecurrentepisodes ofmaniaanddepression,posingsignificantchallengesindiagnosisand treatment(46).Despiteextensiveresearch,theprecisemolecularmechanismsunderlyingBDremainelusive.Recently,severalstudieshaveinvestigatedthedysregulatedexpressionoflncRNAsinpatientswithBD, aimingtoelucidatetheirpotentialasdiagnosticbiomarkersandtherapeutictargets.Here,weprovideacomprehensiveoverviewofthesefindingsinBD,highlightingtheirinvolvementindiversepathwaysandtheir diagnosticutility.

Theveryfirststudyinthisseriesinvestigatedtheexpressionpatternsofsixapoptosis-relatedlncRNAsinpatientswithBDcomparedto healthyindividuals,sheddinglightontheirinvolvementindiseasepathogenesisandpotentialdiagnosticutility(47).Notably,CCAT2,TUG1,and PANDAwerefoundtobeupregulatedinpatientswithBD,whileOIP5AS1wasdownregulated.Gender-specificdifferencesinexpressionwere observed,withCCAT2andTUG1alterationsexclusivelyinmalepatients withBD,whilePANDAinbothmaleandfemalepatientscomparedto theirrespectivecontrolgroups.Nonetheless,thestudyhighlightsthediagnosticpotentialoflncRNAsinBD,withTUG1emergingasapromising candidatebiomarkerandthecombinationofmultiplelncRNAtranscripts significantlyenhancingdiagnosticaccuracy.UnderstandingthefunctionalrolesofdysregulatedlncRNAsfromthisstudycouldalsoofferinsightintonoveltherapeuticstrategiesforBD,potentiallytargetingthese lncRNAsortheirdownstreameffectors.However,severallimitationswere notedinthisstudy,includingthesmallsamplesizeandtheneedforvalidationinindependentcohorts.Futureresearchdirectionsmayinvolve exploringthefunctionalsignificanceofdysregulatedlncRNAsusingin vitroandinvivomodels,elucidatingtheirinteractionswithothermolecularpathwaysimplicatedinBD,andinvestigatingtheirpotentialastherapeutictargetsorbiomarkersinlarger,well-characterizedpatientcohorts. Nevertheless,thisstudyunderscorestheimportanceofapoptosis-related lncRNAsinBDpathogenesis.Italsohighlightstheirpotentialasdiagnosticmarkers,callingforfurtherresearchtovalidatetheirclinicalrelevance andexploretheirtherapeuticimplications(47).

EmergingevidencesuggeststhatlncRNAscouldplayapivotalrolein keysignalingpathways,suchasthePI3K/AKT,implicatedinBD(48).The expressionlevelsofPI3K/AKTpathway-relatedlncRNAs,namelyTUG1, GAS5,andFOXD3-AS1,wereassessedinPBMCfrom50patientswithBD and50HCs(49).TheresultsrevealedsignificantexpressiondownregulationofFOXD3-AS1andGAS5inpatientswithBDcomparedtoHCs.Importantly,afteradjustingforpotentialconfounders,theresultsremained statisticallysignificant(q value < 0.0001).Furthermore,ananalysisof ROCindicatedthatGAS5andFOXD3-AS1hadthepotentialtoserveas candidatediagnosticbiomarkersforBD,asevidencedbytheirhighAUC,

specificity,andsensitivity.Thesefindingssuggestthatthedysregulation ofFOXD3-AS1andGAS5maybeassociatedwithanincreasedriskofBD, sheddinglightonpotentialmolecularmechanismsunderlyingthedisorderandofferingopportunitiesfordevelopingdiagnostictools.Overall, thestudyunderscorestheimportanceoflncRNAsinBDpathogenesisand highlightstheirpotentialasdiagnosticbiomarkers,callingforfurtherresearchtoelucidatetheirfunctionalrolesandclinicalimplicationsinBD management(48).

BDandpanicdisorder(PD)arechronicmooddisordersthatareoftencomorbid,suggestingapotentiallysharedgeneticandpathophysiologicalbackground(49).Astudyexaminedtheexpressionlevelsof MALAT1,PANDA,GAS5,HOTAIRlncRNA,andmiR-221-5p,whicharehighly expressedintheCNSindrug-naïve/drug-freepatientswithBDandPD. Sixteenpatientswithafirstdiagnosisoftype1ortype2BDand10patientswithPDwererecruited,excludingthosewithmedicalorpsychiatric comorbidities.Peripheralvenousbloodwascollectedfrompatientsand HCs,witheachpatientreceivingtherapy.SerumncRNAlevelsweremeasuredbeforeandafter5monthsoftherapy.TheresultsrevealedsignificantupregulationofMALAT-1,GAS-5,andmiR-221-5pinpatientswith BDaftertherapy,whileallinvestigatedncRNAsweredownregulatedin thePDgroupposttherapy.Thesefindingsprovidenovelinsightintothe dysregulationofncRNAsinBDandPD,suggestingtheirpotentialroleas biomarkersandtherapeutictargetsinthesedisorders(49).

Inaseparatestudyreport,Illuminahigh-throughputsequencingwas employedtoidentifyDEgenesinpatientswithBD(50).ValidationofDERNAswasconductedusingqRT-PCRinafirstcohortcomprising50BD and50controlsubjects.FunctionalpredictionsofDE-RNAsweremade usingGOandKEGGpathwayanalyses,alongwithlncRNA-mRNAcoexpressionandlncRNA-miRNA-mRNAcompetingceRNAnetworkanalyses. ROCanalysisandlogisticregressionwereemployedtoevaluatediagnosticperformanceinanadditionaltestinggroupcomprising80BDand 66controlsubjects.Atotalof576significantlyDElncRNAsand262 DEmRNAswereidentifiedinpatientswithBD,andaceRNAregulatorynetworkcomprising95lncRNA—miRNA–mRNAinteractionswasconstructed.Analysisofthefirstcohortrevealeddifferentialexpressionof sixRNAs(NR_028138.1,TCONS_00018621,TCONS_00002186,TNF,PID1, andSDK1)intheBDgroup.NR_028138.1emergedasacentralelementin BDtranscriptionalregulationandapotentialbiomarker,withadiagnostic modelshowinghighaccuracy(areaundertheROC0.923, P < 0.004,95% CI:0.830–0.999).VerificationinthesecondcohortdemonstratedconsistentsignificantdifferencesinNR_028138.1(P < 0.0001).Thisstudynot onlyconstructedaceRNAregulatorynetworkbutalsoproposedahypothesisforBDpathogenesis,withNR_028138.1identifiedasakeyelement involvedintranscriptionalregulationandapromisingbiomarkercandidate(50).

Inthenextstudy,researchersinvestigatedtheexpressionlevels ofthreelncRNAs—lincRNA-p21,lincRNA-ROR,andlincRNA-PINTinthe PBMCofpatientswithBD(n = 50)andhealthyindividuals(n = 50)(51). TheresultsshowedthatexpressionlevelsofallthreelncRNAsweresignificantlyreducedinpatientswithBDcomparedtocontrols.Interestingly,in sex-basedanalyses,downregulationoftheselncRNAswasobservedonly inmalepatientswithBDcomparedtomalehealthysubjects.Additionally, inpatientswithBD,allthreelncRNAsexhibitedsignificantpairwisepositivecorrelationsinexpressionlevels.TheAUCvaluesforlincRNA-p21, lincRNA-ROR,andlincRNA-PINTwere0.66,0.75,and0.66,respectively, indicatingmoderatediagnosticpotential.Particularly,theROCanalysis suggestedthatlincRNA-RORmightserveasadiagnosticbiomarkerfor distinguishingbetweenpatientswithBDandcontrols.Overall,thisstudy proposesaroleforlincRNA-p21,lincRNA-ROR,andlincRNA-PINTinthe pathogenesisofBD.Furthermore,theperipheralexpressionoftheselncRNAsmightbeusefulaspotentialbiomarkersforBD(51).

TheexpressionlevelsofMALAT1andUCA1lncRNAshavebeenevaluatedinPBMCsobtainedfrom50bipolarpatientsand50HCsusingrealtimePCR(52).AdditionalanalysesfocusedontheROCandcorrelation analysisbetweenthegeneexpressionlevelsandsomeclinicalfeaturesof bipolarindividuals.TheresultsrevealedasignificantdeclineinMALAT1 expressionlevelsinpatientscomparedtocontrols,whilenosignificant differencewasobservedintheexpressionlevelsofUCA1betweenthetwo

groups.TheROCanalysisshowedthattheAUCforMALAT1was0.80,suggestingitspotentialasadiagnosticbiomarkerforBD.Inconclusion,these findingssuggestthattheexpressionlevelofMALAT1couldserveasapotentialdiagnosticbiomarkerforBD;however,furtherresearchisneeded tovalidatetheseresultsandexploretheunderlyingmechanismsinvolved inthedysregulationofMALAT1inthisdisorder(52).

Altogether,theemergingroleoflncRNAsinBDpathogenesisofferspromisingavenuesfordiagnosticandtherapeuticadvancements. ThroughcomprehensiveanalysesoflncRNAexpressionpatterns,several studieshaveidentifiedpotentialbiomarkersassociatedwithBD,sheddinglightonunderlyingmolecularmechanisms.DysregulatedlncRNAs, suchasTUG1,GAS5,andFOXD3-AS1,havebeenimplicatedincritical signalingpathways,includingapoptosisandthePI3K/AKTpathway,providinginsightsintodiseasepathophysiology.Moreover,lncRNAssuchas NR_028138.1andIFNG-AS1haveshowndiagnosticpotential,emphasizingtheirutilityascandidatebiomarkersforBD.However,furtherresearch isneededtovalidatethesefindingsinlargercohortsandelucidatethe functionalsignificanceofdysregulatedlncRNAsinBD.Ultimately,unravelingthecomplexinterplaybetweenlncRNAsandBDpathogenesisusing ablood-basedscreeningstrategyholdspromisefordevelopingpersonalizeddiagnostictoolsandtargetedtherapeuticinterventions,ultimately improvingpatientoutcomesinthisdebilitatingdisorder.

lncRNAsandPosttraumaticStress

Posttraumaticstress(PTSD)presentsacomplexarrayofsymptoms,includingimpairedfearextinction,excessiveanxiety,anddepression.Inrecentyears,ncRNAs,particularlylncRNAs,havegarneredsignificantattentionaspotentialregulatorsofgeneexpressionandkeyplayersin PTSDwithadditionalinterestinexploringtheirpotentialasperipheral biomarkers(53).Oneofthestudiessetsouttounravelthealterations inlncRNAsandtheircoexpressionwithmRNAsinPTSD,aimingtoidentifybiomarkersandelucidatepathwayscrucialtothisdisorder(54).Part oftheresearchstrategyincludedgeneexpressionprofilesbydownloadingdatafromtheGSE68077-GeneExpresssionOmnibusdatabase. FollowingGO,KEGGpathwayenrichment,andprotein–proteininteractionnetworkanalysis,alncRNAs-mRNAscoexpressionnetworkwas constructed,andcorepairlncRNAsinvolvedinPTSDwereidentified. Atotalof45DElncRNAsand726DEmRNAswereidentified,with17 lncRNAsand86mRNAsbeinginter-regulated.MostlncRNA-mRNAcoexpressionshowedpositivecorrelations,suggestingtheirpotentialrolesin PTSD.ThecoexpressednetworkhighlightedthefunctionalrolesoflncRNAs,regulatedmRNAs,andrelatedpathways.CorepairnetworkanalysisrevealedthatlncRNA-NONMMUT010120.2synergisticallyupregulatedPpargc1aanddownregulatedCir1,Slc38a9,Atp6v0a2.Additionally, lncRNA-NONMMUT023440.2,NONMMUT034155.2,NONMMUT105407.1, andNONMMUT149972.1werecoexpressedwith10mRNAs,indicating theirinvolvementinregulatingcoexpressedmRNAsinPTSD.ThesefindingsshedlightonthepotentialmechanismsunderlyingPTSDandprovide insightsintopotentialbiomarkersandtherapeutictargetsforthedisorder.Furthervalidationandexplorationofthesefindingsarewarrantedto betterunderstandthepathophysiologyofPTSD(54).

Basedonthereviewedliteraturethusfar,wecreatedanetworkplot illustratingtheconvergenceofMDD,BD,andsuicidalbehavior(SB)and theirinterrelatednessregardingsharedanduniquelncRNAsdetectedin theperipheralcirculation(Figure2).Thenetworkdiagramrepresentsthe relationshipsbetweenlncRNAsandthreedifferentdisorders:MDD,BD, andSB,asindicatedbythethreecentralnodesdifferentiatedbycolor. SharedlncRNAs,connectedtomorethanonedisorder,suggestarole incommonpathogenicpathwaysormolecularmechanismsacrossthese conditions.OuranalysisidentifiesANRILandHOTAIRassharedlncRNAs betweenMDDandBD.HOTAIR,implicatedinvariouscancersanddisorders,isconnectedtotheMDDandBDnodes,highlightingitspotentialdualassociation.ANRIL,partoftheINK4b-ARF-INK4agenecluster andassociatedwithcardiovasculardiseases(57),alsolinksMDDandBD, suggestingasharedroleindiseasepathogenesis.Additionally,sixlncRNAs,includingTCONS_00019174,ENST00000566208,NONHSAG045500, ENST00000517573,NONHSAT034045,andNONHSAT142707,appearto convergeinthepathophysiologyofMDDandSB.Uniqueassociationswere alsofound:XIST,NEAT1,FEDORA,andPCAT1weredistincttoMDD,while MEG3,PANDA,TUG1,andMALAT1weresolelylinkedtoBDinournetwork. Thisspecificityhighlightsthepotentialfortargetedtherapeuticinterventionsanddiagnostics.WehavealsopresentedsomeofthebasiccellularfunctionalitythatcanbethedirecttargetofafewlncRNAsthatwe foundaspartofbiomarkerdiscoveryacrosstheliteraturewesearchedto constructthisreview.WehaveincludedANRIL,GAS-5,HOTAIR,MALAT1, NEAT1,RMRP,andXISTlncRNAtranscriptandtheirtargetedgeneontological(GO)functionsregardlessofdiseasestates.Thechorddiagram constructedin Figure3 displaystheinteractionsbetweenallsevenlncRNAsandvariousGOprocesses,eachrepresentedbyauniquecolor.The ribbonsconnectthelncRNAswithGOcategories,indicatingthestrength andfrequencyoftheirrelationships,withthickerribbonssignifyingmore substantialinteractions.Overall,theplotsuggeststhatsomeofthemost crucialandsensitivecellularfunctions—includingnuclearbodyorganization,mitochondrialRNAprocessing,chromatinassembly,growtharrest, andalternativesplicing—couldbecompromisedifanyorallofthelncRNAsfailtoperformattheiroptimumlevels(58).

Toconclude,theabovestudies,investigatingdysregulatedlncRNAsin individualswithMDDandBD,supportthehypothesisthatlncRNAsmay playacrucialroleinthepathophysiologyofthesedisorders.Examining lncRNAexpressioninperipheralbloodsamplesandtheirfunctionalroles inmood-relatedbehaviorsandneurobiologicalprocessesoffersvaluable insightintothemechanismsunderlyingMDDandBD.Findingsindicate thatidentifyingdysregulatedlncRNAsinindividualswithmooddisordersmayserveasreliablediagnosticbiomarkers.Additionally,lncRNAs mayalsoserveasbiomarkersfortreatmentresponse.Altogether,these findingsunderscoretheimportanceoffurtherresearchintotheregulatoryroleoflncRNAsinmooddisordersandtheirimplicationsinclinicalpractice.UtilizinglncRNAsasbiomarkerscouldleadtoearlydisease prognosisandimprovedtreatmentmanagementforindividualswithMDD andBD.

FutureDirections

FuturedirectionsinstudyinglncRNAsascirculatorybiomarkersinmood disordersholdconsiderablepromiseforadvancingdiagnosticandtherapeuticapproaches;however,severalchallengesremain(59).Firstly,furtherresearchisneededtoelucidatethespecificlncRNAsignaturesassociatedwithdifferentsubtypesandstagesofmooddisorders,including

PTSD,oftenfollowingapsychologicallytraumaticevent,ischaracterizedbyheightenedinflammation,withindividualsalsoexperiencing variouscomorbidclinicalandbehavioraldisorderslinkedtochronicinflammation(55).Inthisconnection,astudyexaminedtheroleoflarge interveningnoncodingRNAs(lincRNAs)inregulatinginflammationinindividualsdiagnosedwithPTSD(56).TheresearchersnotedanupregulationoftheWNTligand,WNT10B,inPBMCsofpatientswithPTSD.This upregulationwasassociatedwithhigherH3K4me3signalsaroundthe WNT10BpromoterinpatientswithPTSDcomparedtothosewithoutPTSD. TheincreasedH3K4me3wasattributedtoLINC00926,whichwasfoundto beupregulatedinthePTSDsubjects.Moreover,addingrecombinanthumanWNT10BtopreactivatedPBMCsincreasedtheexpressionofinflammatorygenessuchasIFNGandIL17A,suggestingWNT10B’sinvolvement intheirupregulation.ThedataindicatedthatLINC00926physicallyinteractswithMLL1,therebyregulatingtheexpressionofIFNGandIL17Avia WNT10B.ThisstudypresentsthefirstevidenceofalincRNAregulating theexpressionofWNT10Bandsubsequentinflammation.Thesefindings holdsignificantrelevanceforunderstandingthediseasemechanismsunderlyingPTSD(56).Altogether,thestudyunveiledanetworkoflncRNAmediateddysregulatedgenesandpathwaysimplicatedinPTSD.Through theidentificationofDElncRNAsandmRNAs,aswellastheircoexpression patterns,thestudyhighlightedpotentialbiomarkersandtherapeutictargetsforPTSD.ThesynergisticregulationofkeygenesbycorepairlncRNAsunderscoredtheintricateinterplaybetweennoncodingRNAsand protein-codinggenesinmodulatingthepathophysiologyofPTSD.These findingsnotonlyenhanceourunderstandingofthemolecularmechanismsunderlyingPTSD,butalsoholdpromiseforthedevelopmentofpersonalizedtreatmentstailoredtoaddresstheuniquemolecularsignatures ofindividualsaffectedbythisdebilitatingdisorder.

Figure2. Thenetworkplotillustratestheinterconnectednessofthreedistinctmooddisorders(majordepressivedisorder:MDD,bipolardisorder:BD,andsuicidal behavior:SB)basedonsharedlncRNAprofilesdetectedintheperipheralcirculation.EachnoderepresentsaspecificlncRNA,andedgesbetweennodesindicate significantassociationsbetweenthemanddisordersacrosstheanalyzedstudies.Nodecolorandshapecorrespondtodifferentmooddisorders,facilitating visualizationandinterpretationofthefindings.

BDanddepression(60, 61).Thiswouldinvolvelarge-scale,multicenter studiestovalidatethediagnosticutilityoflncRNAbiomarkersandestablishtheirprognosticvalueinpredictingtreatmentresponseanddiseaseprogression(62).Additionally,longitudinalstudiesareessentialto investigatedynamicchangesincirculatinglncRNAlevelsovertimeand theircorrelationwithclinicaloutcomes(62).

Regardingclinicaltranslation,developingminimallyinvasivemethodsfordetectingcirculatinglncRNAs,suchasliquidbiopsyassays,holds greatpotentialforroutineclinicaluse(63).Standardizationofsamplecollection,storage,andprocessingprotocolsiscrucialtoensurereproducibilityandreliabilityofbiomarkerassays(64).Additionally,collaborationbetweenresearchers,clinicians,andindustrystakeholdersis essentialtoacceleratethetranslationofresearchfindingsintoclinically actionabletoolsandtherapiesforpersonalizedmedicineapproachesin mooddisorders.Overall,thefutureoutlookforstudyinglncRNAsascirculatorybiomarkersinmooddisordersispromising,withthepotential torevolutionizediagnosticandtherapeuticstrategies,improvepatient outcomes,andadvanceourunderstandingofthebiologicalunderpinningsofthesecomplexpsychiatricconditions(65, 66).However,thepotentialofEVs,includingexosomes,asatooltostudylncRNAsascirculatingbiomarkersinMDDandBDsisparticularlyassuring(67).EVs, includingexosomes,aremembranousvesiclesreleasedbyvariouscell typesintotheextracellularspace,carryingacargoofproteins,lipids, andnucleicacids,includinglncRNAs(68).TheseEVscantraversebiologicalbarriers,suchastheblood–brainbarrier,allowingfortheexchange ofmolecularinformationbetweendifferentcelltypesandtissues(69). UtilizingEVsascarriersoflncRNAsoffersseveraladvantagesforstudyingcirculatingbiomarkersinmooddisorders.Firstly,EVsprotectlncRNAsfromdegradationbyRNases,preservingtheirstabilityincirculation andenhancingtheirdetectionsensitivity(68).Additionally,EVsprovide ameansforcell-to-cellcommunication,facilitatingthetransferoflncRNAsbetweenneurons,glialcells,andperipheraltissues,therebyreflectingthepathophysiologicalchangesoccurringinthediseaseprocess(68, 70).Furthermore,EVscanbeisolatedfromvariousbiologicalfluids,includingblood,cerebrospinalfluid,andsaliva,offeringaminimallyinvasiveandeasilyaccessiblesourceforbiomarkeranalysis(71).Inmood disorders,EV-associatedlncRNAsholdpotentialasdiagnosticandprognosticbiomarkers,reflectingdisease-specificalterationsincellularsignalingpathwaysandneuralcircuits(72, 73).Moreover,theanalysisof

EV-lncRNAsmayprovideinsightsintodiseasemechanismsandtreatmentresponse,guidingthedevelopmentofpersonalizedtherapeutic interventions.

OneinnovativeapproachinlncRNA-basedbiomarkerdiscoveryfor MDDandBDinvolvestheintegrationofcutting-edgetechnologiesand multidimensionaldataanalysismethods(74).Thisapproachleverages advancesingenomics,transcriptomics,proteomics,andmetabolomicsto comprehensivelycharacterizethemolecularlandscapeofmooddisorders andidentifynovelbiomarkerswithdiagnosticandprognosticutility(75). Oneaspectofthisapproachistheuseofmultiomicsprofilingtechniques togeneratehigh-dimensionaldatasetsfromdiversebiologicalsamples, includingbraintissues,blood,andcerebrospinalfluid(76).Integrative analysisofthesemultiomicsdatasetsallowsforidentifyingdysregulated pathwaysandnetworksunderlyingmooddisorders,includingtheinvolvementoflncRNAsingeneregulatorynetworks(77, 78).Furthermore,integratingmachinelearningandartificialintelligencealgorithmscanenabletheminingofcomplexmultiomicsdatatoidentifyrobustbiomarker signaturesassociatedwithmooddisorders(79, 80).Combininginformationfrommultiplemolecularlayers,includinglncRNAexpressionprofiles, geneticvariants,proteinabundance,andmetabolicprofiles,thesealgorithmscanuncoversubtlepatternsandassociationsthatmaynotbeevidentthroughtraditionalstatisticalmethods.

Anotherinnovativeapproachinvolvestheexplorationofwearable biosensorsanddigitalhealthtechnologies(81).Thesetechnologiesofferthepotentialtocontinuouslymonitorphysiologicalandbehavioral parametersinreal-time,providingvaluableinsightsintothedynamic changesassociatedwithmooddisordersandfacilitatingtheidentificationoflncRNAbiomarkerscorrelatedwithdiseasestates(82).Thismay involvetheintegrationofwearablebiosensorssuchassmartwatchesor fitnesstrackers,withmobilehealthapplicationsdesignedtocapturevariousbiometricdata,includingheartratevariability,sleeppatterns,physicalactivitylevels,andmoodfluctuations(83).Thesedevicescanpassively collectdatafromindividualswithmooddisordersintheirnaturalisticenvironments,offeringaholisticviewoftheirdailyactivitiesandphysiologicalresponses.ByincorporatinglncRNAexpressionprofilingintothese digitalhealthplatforms,researcherscancorrelatechangesincirculating lncRNAlevelswithfluctuationsinmoodandbehavioralpatternsobserved inindividualswithMDDandBD(84).Forexample,wearablebiosensors candetectchangesinphysiologicalparametersassociatedwithstressor

Figure3. ThechorddiagramdisplaystheinteractionsbetweenaspecificsetoflncRNAsandvariousGOprocesses,eachrepresentedbyauniquecolor.The ribbonsconnectthelncRNAswithGOcategories,indicatingthestrengthandfrequencyoftheirrelationships,withthickerribbonssignifyingmore substantial interactions.ThisvisualizationhelpshighlighttheinterconnectednessofseveralimportantlncRNAswithprominentBPcontrollingmanyaspects ofcellular functionality.

mooddysregulation,whileconcurrentanalysisofcirculatinglncRNAscan identifymolecularsignaturesindicativeofdiseasestatesortreatmentresponses.Byleveragingadvanceddataanalyticstechniques,researchers canuncovernovelassociationsbetweencirculatinglncRNAsandbehavioralphenotypes,facilitatingearlydetectionandinterventionstrategies (84).Overall,theintegrationofwearablebiosensorsanddigitalhealth technologieswithlncRNA-basedbiomarkerdiscoveryoffersaninnovativeapproachtoadvancingdiagnosticandtherapeuticstrategiesinmood disorders(85).Bycombiningcontinuousmonitoringofphysiologicaland behavioralparameterswithmolecularprofilingofcirculatinglncRNAs, researcherscandeveloppersonalizeddiagnostictoolsandinterventions tailoredtoindividualpatient’sneeds,ultimatelyimprovingoutcomesand qualityoflifeforindividualswithMDDandBD.

ExploringlncRNAsascirculatorybiomarkersinmooddisordersholds immensepotentialforimprovingdiagnosticsandtreatments.Addressingcurrentchallenges,withafocusonidentifyingmooddisorder–specific lncRNAsignatures,conductinglarge-scalemulticenterandlongitudinal studies,anddevelopingminimallyinvasivedetectionmethodswillenhancetheunderstandingandmanagementofmooddisordersandwill pavethewayforpersonalizedmedicine.Weproposethefollowingrecommendationsforfuturestudies.

IdentifyingSpecificlncRNASignatures

FutureresearchshouldaimtoidentifylncRNAsignaturesspecificto differentsubtypesandstagesofmooddisordersthroughlarge-scale,

multicentercohortstudies.Techniqueslikesingle-cellRNAsequencing cancapturecellular-levelheterogeneityoflncRNAexpression,revealing patternsspecifictoBDanddepression.

Large-scale,MulticenterStudies

Establishinginternationalconsortiatoconductlarge-scale,multicenter studieswillensurediversesamplesizesandstandardizedprotocols.This approachwillhelpvalidatelncRNAbiomarkers’diagnosticandprognostic utility,ensuringconsistencyandreproducibilityacrossdifferentresearch centers.

LongitudinalStudies

ItiscrucialtodesignlongitudinalstudieswithfrequentsamplingtomonitordynamicchangesincirculatinglncRNAlevelsovertime.Bycollecting comprehensiveclinicaldataalongsidelncRNAprofiling,researcherscan correlatethesechangeswithtreatmentresponsesandclinicaloutcomes, usingadvancedstatisticalmodelstoanalyzetheserelationships.

MinimallyInvasiveDetectionMethods

DevelopingliquidbiopsyassaystodetectspecificlncRNAsusingaccessiblebiologicalfluidswithhighsensitivityandspecificityisessential.Innovationsinpoint-of-caretestingdeviceswillenablerapidmeasurementof circulatinglncRNAsinclinicalsettings.Standardizingprotocolsforsamplecollection,storage,andprocessingwillensurethereliabilityofthese methods.

ClinicalTranslationandCollaboration

Forgingpartnershipswithbiotechandpharmaceuticalcompaniesto commercializelncRNA-baseddiagnostickitsisacriticalstep.Engagingregulatorybodieswillhelpnavigateapprovalpathwaysandensure compliancewithclinicalandsafetystandards.

Conclusion

Asmentionedabove,byfocusingondetailedmethodologiesandcollaborativeefforts,researcherscansignificantlyadvancethestudyoflncRNAs ascirculatorybiomarkersinmooddisorders.Theseadvancementswillimprovediagnosticandtherapeuticapproaches,enhancestudyoutcomes, anddeepenourunderstandingofthesecomplexpsychiatricconditions. Toidentifybiomarkerslinkedtomoodandbehavioralpatterns,weproposecombiningexistingdatawithlncRNAprofilingandusingadvanced analyticstouncoverassociationsthatfacilitateearlydetectionandpersonalizedinterventions.Advancedbioinformaticstechniques,suchasmachinelearningmodels,canintegratemultiomicsdatatopredictdiseasestatesandtreatmentresponsesbasedonlncRNAprofiles.Creating predictivemodelsthatcombinelncRNAexpressionwithclinicaland demographicdatawillalsohelpidentifyhigh-riskindividuals.Forexperimentalvalidation,functionalstudiesusingCRISPR-Cas9technologyarenecessarytoelucidatetherolesofcandidatelncRNAs.ImplementingclinicaltrialstotestlncRNA-basedinterventionswillvalidate thesebiomarkersforclinicaluseandenhancediagnosticandtherapeuticstrategies.Thesetargeteddirectionswillsignificantlyadvance thestudyoflncRNAsascirculatorybiomarkersinmooddisorders,improvingdiagnosticandtherapeuticapproaches,enhancingpatientoutcomes,anddeepeningourunderstandingofthesecomplexpsychiatric conditions.

Glossary

Epigeneticplasticity: Epigeneticplasticityreferstotheabilityofanorganism’sepigenometoadaptandchangeinresponsetoenvironmental factors,therebyinfluencinggeneexpressionwithoutalteringtheunderlyingDNAsequence.

Exon: CodingsegmentofDNAthatcantranscribeintoRNAtoparticipate inproteintranslation.

Centraldogma: ThisoutlinestheprocessinwhichDNAcodesforRNAand RNAtranslatedintoprotein.

Synaptogenesis: Formationofsynapsesduringthedevelopmentofnervoussystem.

Neurites: Neuriteisatypeofprojectionsextendingoutfromneuronalcell body.

Neuralplasticity: Aprocessthatcausesbraintoadaptfunctionallyand structurally.

Peripheralbiomarkers: Circulatingbiomoleculewhicharefoundinblood, otherbodyfluidsandtissuethatreflectspathologicalandphysiological stateofthebody.

IntergeniclncRNA: lncRNAtranscribedfromthestretchofDNAsequencesthatpresentbetweenthegenes.

Geneontology: GeneOntology(GO)categorizesgeneproductsbybiologicalprocesses,cellularcomponents,andmolecularfunctions,aiding standardizedfunctionalannotationsacrossorganismsforsystematicbiologicalanalysis.

PBMC: BloodcellshavingsingleroundnucleuscalledPBMC.Theseincludeslymphocytes,macrophages,monocytesanddendriticcells.

Microarray: Itisahighthroughputgeneexpressionprofilingplatform withanabilitytodetectseveralthousandgenessimultaneously.

Alternativesplicing: AlternativesplicingisamechanismthatallowspremRNAtobecutandrearrangedinvariouscombinations,resultinginthe productionofmultiplemRNAisoforms.Thisprocessenhancesproteindiversitywithincells.

Exosomes: Invariouspathophysiologicalprocesses,cellsreleasesmall vesiclescalledexosomes,whichplayrolesincell-to-cellcommunication.

Extracellularvesicles(EVs): Aheterogenousgroupofsmalllipidbound vesicles,servingasanimportantfacilitatorofvariouspathophysiological processes.

Glialcells: Atypeofnon-neuronalcellsthatprovidechemicalandphysicalsupporttoneurons.

Cerebrospinalfluid: Itisaplasmacontainedultrafiltratefluidthatflows aroundthesubarachnoidspacesofspinalcordandbrain.

DeclarationofPossibleConflictsofInterests

Allcontributorshaveconfirmedthatnoconflictofinterestexits.

AuthorContributions

Conceptualization:Y.D.Co-written:A.K.V.,B.R.,E.M.H.,andY.D.Fundingacquisition:Y.D.Reviewandediting:Y.D.Allauthorshavereadandagreed topublishthecurrentversionofthemanuscript.

FundingSources

ThisworkwassupportedbygrantsfromtheNationalInstituteofMental Health(R01MH130539,R01MH124248,R01MH118884,R01MH128994, R01MH107183),toY.D.

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THOUGHTLEADERSINVITEDREVIEW

Rolesofalternativepolyadenylationinpsychiatricdisorderrisk

MichellePaff1 ,StevenF.Grieco2 , 3 ,YaCui3 ,OleA.Andreassen4 ,AlbertLaSpada6 , 7 , 8 ,WeiLi,PhD6 ,andXiangminXu2 ,

, 11

Alternativepolyadenylation(APA)isapervasiveregulatorymechanisminthehumanbrainthatcontrolsthestabilityandcellularlocalization ofmRNAtranscripts.Single-nucleotidepolymorphismsassociatedwithpsychiatricdisordersmayexerttheirdeleteriouseffectsbyaltering 3’untranslatedsiteusage,whichmaychangethestabilityandprocessingofmRNAtranscripts.Theauthorspreviouslyperformeda3’APA transcriptomic-wideassociationstudyusingtheDePars2frameworkandtheGTExv8,PsychENCODE,andROS/MAPdatasetstoidentify APA-linkedgenesassociatedwithelevenbraindisorders.Herewefocuson3’APA-linkedgenesassociatedwiththemajorpsychiatricconditions: schizophrenia,bipolardisorder,anddepression.Thereare286APA-linkedgenesassociatedwiththesepsychiatricdisorders,and60%–65%of thesegeneshavenotbeenassociatedwiththemajorpsychiatricdisordersthroughtheirexpressionand/orsplicing.Protein–proteininteraction networksindicatethatAPA-linkedgenesassociatedwithschizophreniaareinvolvedinintracellulartransportandcellularlocalization pathways.Futureresearchisneededtoelucidatetheroleofalternative3’untranslatedregionusageofAPA-linkedgenesonneuronalfunction andphenotypicexpressioninpsychiatricdisorders.

GenomicPsychiatry March2025;1(2):34–41;doi: https://doi.org/10.61373/gp024i.0049

Keywords: Alternativepolyadenylation,transcriptome-wideassociationstudies,psychiatricdisorders,schizophrenia,bipolardisorder,depression

Introduction

Overthepast15years,genome-wideassociationstudies(GWAS)have uncoveredhundredsofsingle-nucleotidepolymorphisms(SNPs)associatedwiththemajorpsychiatricdisorders.However,translatingthesegeneticassociationstobiologicallyrelevantmechanismsremainsamajor challenge.ThemajorityofGWAS-significantSNPslocalizetonon-coding regionsofthegenomeandoftenresidewithinlocidemonstratingahigh degreeoflinkagedisequilibrium.Thus,identifyingcausalgenesresponsibleforthephenotypicexpressionofpsychiatricconditionscanbeanarduousprocess.Transcriptome-wideassociationstudies(TWAS)serveasone methodofnominatingputativecausalgenesbyintegratingpopulationleveltranscriptomicdatasetsandGWASsummarystatistics(1–4).Leveragingpopulation-leveldatasetswithbothgenotypeandgeneexpression information,TWASimputethe cis-componentofgeneexpressionofcommonvariantstoprioritizegenesattrait-associatedloci,referredtoas quantitativetraitloci(QTL).

WhilethemajorityofTWAShavefocusedontotalgeneexpression,it hasbecomeincreasinglyrealizedthatdisease-relevantSNPsmayhave regulatoryeffectsthataltertranscriptsplicing,epigeneticregulation, andproteinexpression,amongmanyotherpotentialmechanisms(5, 6). Recently,alternative3’untranslatedpolyadenylationsiteusagehasbeen recognizedasamechanismbywhichGWASsignificantSNPsmayaffect theabundanceoftranscriptisoforms(7, 8).Alternativepolyadenylation (APA)isapervasiveregulatorymechanismofmRNAtraffickingandtranslationthatisparticularlycriticalinthecentralnervoussystem(CNS). AberrantAPAplaysaroleinmultipleneurologicaldisorders,includingParkinson’sdisease(PD)(9),Huntington’sdisease(10),andcertain formsofintellectualdisability(11).UnliketotalexpressionlevelsoralternativelysplicedmRNAisoforms,theimpactofAPAondisease-related

geneexpressionhasnotbeenaccountedforinthemajorityofTWAS analyses.

Polyadenylationisanimportantstepintheproductionofmature mRNAspecies.Followingtranscription,the3’endofamRNAiscleaved ateitheraproximalordistal3’APAsite,andapoly(A)tailissynthesized atthe3’terminus.APAsitesarelocatedwithinthe3’untranslatedregions (3’UTRs)ofgenes.Approximately70%ofhumangenesincludemultiple APAsitesthatproduce3’UTRsofdifferentlengths(12).UseofaproximalAPAsiteproducesashortermRNAwithashort3’UTR,whileuseof adistalAPAsiteproducesalongermRNAwithalong3’UTR(Figure1A). 3’UTRscontributetoposttranscriptionalregulationofgeneexpressionin multipleways.Regulatoryelementsresidingwithin3’UTRsaffectmRNA stability,translationefficiency,andcellularlocalization(13).3’UTRsproducedbydistalAPAsitestendtocontainmoretargetsequencesforRNAbindingproteinsandmicroRNAs(miRNA),whichcandestabilizemRNAs andpromotedegradation(Figure1B)(14, 15).Aslonger3’UTRstendto containagreaternumberofregulatoryelements,geneticmutationsthat leadtoachangeinAPAsiteusagecanaltermRNAstability.

APAmayserveasaregulatorymechanismwithimportantdevelopmentalimplications.Ascellsandtissuesevolvefromprimitivetofullydifferentiatedforms,theAPAoftheirexpressedtranscriptsoftenchanges. Transcriptomicstudiesofembryonicmousetissuehaveshownthatmouse genestendtoexpresstranscriptswithlonger3’UTRsasembryonicdevelopmentprogresses(16).Additionally,3’UTRlengthvariesaccording tocelltype.Amongstthemajorcelltypes,stromalcellsandneuronalcell typesexpresstranscriptswiththelongest3’UTRs,whilebloodcells,hepatocytes,chondrocytes,andosteoblastsexpresstranscriptswiththeshortest3’UTRs(17).Furthermore,amongcellsofagivenlineage,useoflong 3’UTRstendstoincreaseasprimitivecellsdifferentiatetomaturecell

1 DepartmentofNeurosurgery,SchoolofMedicine,UniversityofCalifornia,IrvineMedicalCenter,Orange,California92868,USA; 2 DepartmentofAnatomyandNeurobiology,SchoolofMedicine,UniversityofCalifornia,Irvine,California92697,USA; 3 CenterforNeuralCircuitMapping,UniversityofCalifornia,Irvine,California 92697,USA; 4 DivisionofComputationalBiomedicine,DepartmentofBiologicalChemistry,SchoolofMedicine,UniversityofCalifornia,Irvine,Irvine,California92697, USA; 5 NorwegianCentreforMentalDisordersResearch(NORMENT),DivisionofMentalHealthandAddiction,OsloUniversityHospital,Oslo0450,Norway; 6 Departmentof BiologicalChemistry,SchoolofMedicine,UniversityofCalifornia,Irvine,California92697,USA; 7 DepartmentsofPathologyandLaboratoryMedicine,Neurology,andBiologicalChemistry,UniversityofCaliforniaIrvine,Irvine,California92697,USA; 8 UCIInstituteforNeurotherapeutics,UniversityofCaliforniaIrvine,Irvine,California92697, USA; 9 DepartmentofMicrobiologyandMolecularGenetics,UniversityofCalifornia,Irvine,California92697,USA; 10 DepartmentofBiomedicalEngineering,Universityof California,Irvine,California92697,USA; 11 DepartmentofComputerScience,UniversityofCalifornia,Irvine,California92697,USA

CorrespondingAuthor: MichellePaff,MD;UniversityofCalifornia,Irvine,DepartmentofNeurologicalSurgery,200SouthManchesterAvenue,Suite210,Orange,CA92868, Phone:714-456-6966,Fax:714-456-6966.E-mail: mpaff@hs.uci.edu

Received:15April2024.Revised:28May2024.Accepted:19June2024. Publishedonline:18July2024.

Figure1. Alternativepolyadenylation(APA).(A)SplicingcanleadtomRNAisoformswithdifferent3 UTRsand/orAPAbasedonispolyadenylationsite(PAS) usageinvariouscombinations.(B)TwomRNAisoformswithdifferent3 UTRsduetoAPA.OnlytheconstitutiveUTR(cUTR)ispresentintheshortisoform, whereasthethealternativeUTR(aUTR)isalsopresentinthelongisoform.InteractionsbetweentheUTRsandRNA-bindingproteins(RBPs),miRNAsand long non-codingRNAs(lncRNAs)canhavefunctionalconsequences.(C)BDNF3 UTR-APAisoformlocalizationinneuronssupportsdendriticproteinsynthesis—the longisoformlocalizestodendritesmorethantheshortisoform(adaptedfromTianandManley,2017,Nat.Rev.Mol.CellBiol).

types.Forexample,inthehematopoieticlineage,theswitchfromshortto long3’UTRsisexplainedbytheevolutionfromprimitivetodefinitiveerythropoiesis(17).Thus,APAisadynamicprocessthatisparticularlycritical duringprenataldevelopment.

ChangesinAPAsiteusagemayalterthedistributionandlocalization oftranslatedproteins.Forexample,theshortisoformofbrain-derived neurotropicfactor(BDNF)isrestrictedtotheneuralcellbodywhilethe longisoformlocalizestodendrites(Figure1C)(18).Theroleof3’UTRsin influencingthecellularlocalizationofmRNAisparticularlyconsequential forneuronssincedendrites,axonterminalsandcellbodieshavedistinct rolesinhealthanddisease.APAofthealphasynuclein(aSyn)transcript wasimplicatedinafamilialformofPD(9).Forexample,SNPsassociated withafamilialformofPDwerefoundtoincreasetheabundanceofthe long3’UTRisoformofalphasynucleinrelativetotheshort3’UTRisoform. Overproductionofthelong3’UTRisoformwasfoundtoleadtotheaccumulationofalphasynucleinproteinwiththemitochondriaofneurons, leadingtoneuronaldysfunction.Thus,geneticvariantsthatalterAPAsite usagemayhaveasignificantimpactoncellularhomeostasis,particularly inthebrainwherethemajorityoftranscriptsareregulatedbyAPA.

APAinPsychiatricConditions

TheCNSharborsthelongest3’UTRsofanytissue,suggestingthatregulatoryelementswithin3’UTRsplayanimportantroleinproteinexpressionthroughoutthebrain.WhiletraditionalTWASmethodshaveidentifiedmRNAexpressionlevelsandisoformlevelchangesduetoalternative splicing,theyhavenotinvestigatedAPA.Toaddressthisgap,Cui etal (8)performeda3’UTRTWAStoidentifylocalgeneticeffectsassociated withvariationof3’UTRusageamongtheGTExv8(19),ROS/MAP(20), andpsychENCODE(21)datasets.TheauthorsimplementedtheDaPars2 framework(22)tocalculatethepercentageofdistalpoly(A)siteusage andtoidentify3’UTRlengtheningandshorteningevents.TheDePars2 frameworkcalculatesa3’UTRusagevalueforeachtranscriptacrosssamples.Alinearregressionframeworkisthenappliedtotesttheassociationbetweennormalizedvaluesof3’UTRusageandSNPswithinan intervalof1Mbpofthe3’UTRregion,adjustingforcovariates.Theyidentified cis-SNPsassociatedwith3’UTRusageandexaminedtheassociation betweenGWASsummarystatisticsand3’UTRusage.Transcriptomeand individual-matchedgenotypedatafromtheROS/MAP,PsychENCODE,and GTExConsortiawasthenimplementedtoestablish3’aTWASsingle-tissue predictionmodelsfor3’UTRusageusingFUSION(2).Theydiscovered354 APA-linkeddiseasesusceptibilitygenesidentifiedamong11braindisorders,includingthemajorpsychiatricdisorders,schizophrenia(SCZ),bipolardisorder(BD),anddepression(DEP)(Figure2A–C).Thelargestnumberof3’aTWAS-signficantgeneswasfoundforSCZ,with281non-HLA

APA-linkedgenesassociatedwiththedisorderamongthethreedatasets. AcomprehensivelistofAPA-linkedgenesforSCZ,BD,andDEPfromCui etal.canbefoundinSupplementaryTableS1.

SomeofthegeneticrisklociassociatedwithAPA(3’aQTLs)werealso implicatedinexpressionandsplicingTWAS.Many3’aTWASgeneshada moresignificant3’aQTLsignalthaneQTLorsQTLsignals,indicatingthat theirGWASsignalisbetterexplainedbytheireffectonAPA.Onevalidated exampleofthisisthedetectionofaknownAPA-susceptibilitygene,SNCA (encodingaSyn)associatedwithPD.SNCAwasthemostsignificantgene identifiedinallthreereferencepanels,andlonger3’UTRusagewasassociatedwithincreasedPDrisk,consistentwithpriorevidence(9).The leadingPDGWASSNPnear SNCA islessstronglyassociatedwithdifferentialexpressionandsplicing,supportingthat3’UTRusageisprimarily responsibleforPDrisk.Amongthemajorpsychiatricdisorders,49of151 (32%)non-HLA,APA-linkedgenesassociatedwithSCZwerealsoimplicatedinexpressionTWAS,splicingTWAS,orboth.Eighty-sevengenes,including ZFN592, PBX2 and RBX1,wereassociatedwithSCZonlythrough APAandnotpreviouslyimplicatedinotherTWAS(Figure3A).Seventeen of71(24%)APA-linkedgenesassociatedwithBDwerealsoimplicated inexpressionandsplicingTWAS(Figure3B),and24of64(37.5%)APAlinkedgenesassociatedwithDEPwerealsoimplicatedthroughexpressionand/orsplicing(Figure3C).

Inthefollowingparagraphs,wewilldiscusstheimplicationsofsome APA-linkedgenes,identifiedinCui etal.associatedwiththemajorpsychiatricconditions,SCZ,BD,andDEP.

DDHD2 (DDHD-Domain-Containing2)

The DDHD2 geneonchromosome8pencodesatriacylglyceridehydrolase thatisinvolvedinmembranetraffickingbetweentheendoplasmicreticulumandGolgibody. DDHD2 isubiquitouslyexpressedinthebrain,and multipletranscriptvariantsresultfromalternativesplicing.Missensemutationsin DDHD2 causeanautosomalrecessiveformofhereditaryspastic paraplegia,whichincludesintellectualdisabilityamongitsclinicalfeatures(23).Furthermore, DDHD2 knockoutmicedemonstratemotorand cognitivedeficitsaswellaslipidaccumulationwithinneurons(24).Althoughtheroleof DDHD2 incognitivefunctionhasnotbeenfullyelucidated, DDHD2 hasbeenlinkedtocaudate,putamen,andpallidumvolume andisdownregulatedinthedorsolateralprefrontalcortex(DLPFC)ofpatientswithSCZ(25).The8p12genomicregion,nearthe DDHD2 gene,has beenidentifiedasasignificantrisklocusforSCZamongHanChineseand Europeanpopulations(26, 27)aswellasforautismspectrumdisorder (28)andBD(6, 29).Decreasedexpressionof DDHD2 hasbeenassociated withSCZinmultipleTWAS(6, 30),includingeQTLsderivedfromprenatal brainanddopaminergicneurons(6, 8, 29, 31).

Figure2. Psychiatric3 aTWAS.(A1)RNAsequencingandmatchedgenotypedatawerecollectedfromtheGTEx,ROS/MAP,andPsychENCODEcohortsasreference panels.(A2)3 aQTLanalysiswasperformed,andthena3 aTWASmodel(A3)wasbuilttopredicttheAPAusageoftargetgeneswithcis-SNPsinthereference panels.(B1)WeusedGWASsummarystatisticsandthe(B2)3 aTWASmodelsforeachreferencepanelto(B3)perform3 aTWASanalysistonominatesusceptibility genesinbraindisorders.(C1)APA-linkedsusceptibilitygenesinbraindisordersidentifiedby3 aTWAS(onlyPsychENOCDEdatashown).(C2)Barplotshowsthe numberof3 aTWASsignificantgenesfor11braindisordersinPsychENCODEDLPFC.(C3)Venndiagramshowstheoverlapof3 aTWASsignificantgenesfor11 braindisorderswithexpressionandsplicingTWAS(includestheGTEx,ROS/MAP,andPsychENCODEcohorts)(adaptedfromCui etal.,2023,NatCommun).

Figure3. Venndiagramsdemonstratingtheoverlapbetweensignificantgenesimplicatedintheexpression,splicingand3’aTWASinCui etal.(2023)forSCZ (A),BD(B),andDEP(C).

Inthe3’aTWASanalysis,theshort3’UTRisoformof DDHD2 wassignificantlyassociatedwithSCZintheGTExv8,ROS/MAP,andpsychENCODEdatasets.Thisisconsistentwithpreviousanalyseswhichidentified aGWAS-significantSNPwithinthe3’UTRofthe DDHD2 mRNAtranscript thatdisruptsbindingofthequakingRNA-bindingprotein(32).Interestingly,downregulationofthequakingRNA-bindingproteinwasassociated withriskofSCZinalargeSwedishpedigree,indicatingthattheRNAbindingproteintargetsequencewithinthe3’UTRof DDHD2 playsarole inriskofSCZ(33).EliminationoftargetsequencesforRNA-bindingproteins,suchasthatforthequakingRNA-bindingprotein,mayoccurwith useofproximal3’UTRsitesthatomitportionsoftheextended3’UTR, yieldingasimilarresult.

ARL3 (ADPRibosylationFactor-LikeGPTase3)

Theproductofthe ARL3 geneonchromosome10qisaGTP-binding proteinthatlocalizestociliaandmicrotubulesandplaysaroleinthe formationofaxonsandcilia.Mutationsin ARL3 canresultinciliopathies, includingneurodevelopmentaldisorderssuchasJoubertsyndrome,characterizedbyhypoplasiaofthecerebellarvermis,brainstemabnormalities,psychomotordelay,hypotonia,andretinalabnormalities(34).ASNP locatedwithinanintronof ARL3 wassignificantlyassociatedwithSCZina HanChinesepopulation(35),anddecreasedexpressionof ALR3 hasbeen associatedwithSCZinTWAS(6, 31)andinaproteome-wideassociation study(36).Theshort3’UTRisoformwasassociatedwithSCZinallthree datasetsexaminedinCui etal.,however,thebiologicalsignificanceofincreasedshort3’UTRusagebythe ARL3 geneinregardstoeitherciliary functionorneuronaldevelopmenthasyettobeexplored.

SNX19 (SortingNexin19)

SNX19 onchromosome11qencodesaproteinthatbelongstoafamily ofsortingnexinsthatfunctioninendosomaltraffickingregulationand sorting.Expressionandalternativesplicingof SNX19 hasbeenassociated withSCZinmultipleTWAS(6, 37–39),whileshort3’UTRusageof SNX19 wasassociatedwithSCZintranscriptomicdatafrommultiplebrainregionsoftheGTExv8dataset.Greaterexpressionofanisoformwithskippingofexon9wasassociatedwithadownstreamSCZrisklocus,withmost unaffectedindividualsexpressingverylowlevelsofthistranscriptisoform.Skippingofexon9producesaframeshiftthatispredictedtoresult intheabsenceofthesortingC-terminaldomain(38).Insituhybridizationstudiessupportthat SNX19 islocalizedtoglutamatergicneuronsin theDLPFC(40). SNX19 maybeassociatedwiththesodium-coupledneutralaminoacidtransporter1(SLC38A1),whichsuppliesneuronswithglutamineforsynthesisofneurotransmitters(40).Theimpactofshort3’UTR usageof SNX19 onneuronalfunctionhasyettobeinvestigated.

ZNF592 (ZincFingerRNA-bindingProtein2)

The ZNF592 geneencodesa1,267aminoacidzincfingerproteinthatis expressedintheCNS(41).Zincfingerproteinsfunctionastranscriptional regulators,mediatinginteractionsbetweenDNAandproteins.Missense mutationsin ZNF592 causecerebellarataxiawithmentalretardation,opticatrophy,andskinabnormalities(41).Inthe3’aTWAS,short3’UTRusageof ZNF592 wasassociatedwithbothSCZandBDintheROS/MAP andpsychENCODEdatasets.Importantly, ZNF592 hasnotbeenpreviously identifiedinexpressionorsplicingTWAS,indicatingthattheGWASsignal atthislocusisalmostentirelyexplainedbyalternativepolyadenylation.

FADS1 (FattyAcidDesaturase1)

FADS1 encodesafattyaciddesaturase,whichisaratelimitingenzyme indesaturationoflong-chainpolyunsaturatedfattyacids.The FADS1 and FADS2 genesin11q12.2locusareintightLD,andthislocushasbeenassociatedwithBDinmultiplepopulations.AGWASsignificantSNPatthe 11q12.2locus,containingthe FADS1/2 gene,wasassociatedwithBPin Asianpopulations(42, 43)andthenreplicatedinGWASofEuropeanpopulations.(44, 45)InCui etal.,expression,alternativesplicing,andshort 3’UTRusageof FADS1 wasassociatedwithBPintheROS/MAPdataset. MultiplemRNAisoformsof FADS1 aregeneratedbyalternativetranscript initiation,alternativepolyadenylationsiteusage,andinternalexondeletions(46).Therearesevenpossibleisoformsof FADS1 thatdifferonlyin thelengthoftheirpoly(A)tailduetouseofalternativepoly(A)sites.

Sincethecommencementofcropagricultureleadingtoincreasedintakeofgrainoils,therehasbeenanincreaseintheproportionofindividualscarryingahaplotypeassociatedwithgreater FADS1/2 activitythat hasaprotectiveeffectagainstBD(47).Transgenicmicewithdecreased FADS1/2 activitydemonstratebehavioralchangesincludingboutsofhyperactivityinterspersedwithperiodsofdepressive-likehypoactivityas wellasabnormalcircadianrhythms,suggestingthatreducedFADS1/2 activityproducesarobustanimalmodelofBD(48).Furthermore,these behavioraldeficitswererescuedwithdietarysupplementationofpolyunsaturatedfattyacids(48).However,dietarysupplementationwith omega-3fattyacidstotreatmoodfluctuationsinBDhavedemonstrated mixedresults(49, 50).

GABRA2 (Gamma-AminobutyricAcidTypeAReceptorAlpha2) GABRA2 encodesthealphasubunitoftheGABAAreceptor,whichmediatesanxiolytic-like,reward-enhancing,andanti-hyperalgesicactions ofbenzodiazepines(51). GABRA2 hasbeenimplicatedinalcoholismand substanceabusedisorders(52, 53),andSNPsresidingwithinGABAAreceptorsubunitgeneshavebeenassociatedwithriskofBD(54–56).GABA receptormRNAsdisplaylong3’UTRs,andincreased3’UTRlengthislinked toreducedtranslation(57, 58).Inour3’aTWAS,short3’UTRusagewas associatedwithBDintheGTExv8,ROS/MAP,andpsychENCODEdatasets. Strong3’aQTLsbutweakeQTLswerefoundfor GABRA2 in3’aTWAS,indicatingthatthe3’aQTLassociationwith GABRA2 almostentirelyexplains theBDGWASsignalatthislocus.

CACAN1B (CalciumVoltage-gatedChannelSubunitAlpha1B) Genesencodingsubunitsofcalciumchannelshavebeenrepeatedly implicatedinBDGWAS(44, 59, 60). CACNA1B encodesthealpha-1B subunit;thepore-formingsubunit,ofthepresynapticN-typevoltagedependentcalciumchannel(CaV 2.2).TwomRNAisoformsof CACNA1B areproducedbyalternativesplicingbasedontheinclusionofone oftwomutuallyexclusiveexons,e37aande37b,whichencodesequencesoftheC-terminus(61).Comparedtochannelscontaininge37b, channelscontaininge37aaretraffickedmoreefficientlytothecell membraneandareinhibitedmorestronglybyG-protein–coupledreceptors(62, 63).E37a-richchannelsareabundantincalcium/calmodulindependentexcitatoryprojectionneurons,includingthosecomprisingexcitatorycortico-hippocampalsynapses(64).Miceexpressingonlythe e37bisoformdisplaydecreasednovelty-inducedanxiety,suggestingthat alternativesplicingof CACN1B influenceswithdrawalandanxiety-related behaviors.

Ithasbeendemonstratedinsympatheticneuronsthatthehalf-life ofthealpha-1BsubunitmRNAisregulatedbyits3’UTRandmodulated byvoltage-dependentcalciumentry(65).IntheanalysisbyCui etal., short3’UTRusage,expression,andsplicingof CACNA1B wereallassociatedwithBDintheGTExv8andROS/MAPcohorts.

Thereplicatedassociationofmultiplecalciumchannelsubunitgenes withBDhaspromptedtheinvestigationofcalciumchannelblockersas adjunctivetherapyinthetreatmentofBD,however,withabouthalfof patientsdemonstratingaclinicalbenefit(66).Inasmallcohortof38 patientswithBD,twoSNPsinthe CACNA1B locuswereassociatedwith treatmentresponsetocalciumchannelblockers.However,nosignificant differenceinthepredictedexpressionof CACNA1B wasfoundbetween respondersandnon-responders(67).

ARL17A (ADPRibosylationFactorLikeGTPase17A)

ARL17A encodesaGTP-bindingproteinwhichisamemberoftheADPribosylationfactorfamily.ADPribosylationfactorlikeGTPase17Aplays aroleinvesicle-mediatedintracellularproteintransportbetweenthe endoplasmicreticulumandtheGolgiapparatusandisimportantfor neuronaldevelopment.Long3’UTRusageofARL17Awassignificantly associatedwithbothSCZandDEPamongtheGTExv8,ROS/MAP,andpsychENCODEdatasets.Althoughthecontributionof ARL17A topsychiatric conditionshasyettobefullyelucidated,expressionof ARL17A inthe DLPFC,putamen,andcerebellumhasbeenimplicatedinSCZ(68). ARL17A expressionhasadditionallybeenassociatedwithintracranialbrainvolume(69),thalamicvolumeinchildhood(68),andreactiontimeandcognitivefunction(69).

Figure4. Protein–proteininteractionnetworksfor3’aTWASsignificantgenesforSCZ(A),BD(B),andDEP(C).Pathwayanalysisdemonstratesthat3’aTWAS significantgenesassociatedwithSCZareenrichedinintracellulartransportandcellularlocalizationpathways.

MTCH2 (MitochondrialCarrierHomolog2)

MTCH2 encodesamemberoftheSLC25familyofnuclear-encodedtransporters,whichislocalizedtotheoutermitochondrialmembraneandplays animportantroleinoxidativephosphorylation(70). MTCH2 hasbeen associatedwithbody-massindexinmultipleobesityGWAS(71, 72)as wellaswithneuroticism(73)andsusceptibilitytoloneliness(74).The SNPimplicatedinneuroticismwasfoundtoregulate MTCH2 inthecerebellum(73).Long3’UTRusageby MTCH2 wassignificantlyassociated withDEPinallthreereferencepanels.Manjunathandcolleagues(75) demonstratedthattranscriptionof MTCH2 issubjecttostopcodonreadthroughinwhichthreedifferenttranscriptisoformsmaybeproduceddependingonwhichstopcodonisused.Thelongisoformlocalizestothe cytoplasmwhereitisrapidlydegraded,andthisleadstoreducedmitochondrialmembranepotentialanddecreasedproductionofreactiveoxygenspecies(75).WhileAPAofthe MTCH2 hasnotbeenstudied,APAmay similarlyregulatethecellularlocalizationofthe MTCH2 transcriptand consequentlyaltermitochondrialfunctioninneurons.

ProteinInteractionNetworks

WeusedSTRING(76)toevaluatetheinterconnectivityofnon-HLA3’aTWASgenesbyphysicalprotein–proteininteractions(PPIs)andCytoscape (v3.3.0)(77)tovisualizethePPInetworksforAPA-linkedgenesassociatedwithSCZ,BD,andDEP(Figure4).APA-linkedgenesassociated withthemajorpsychiatricconditionsareenrichedinbiologicalpathways,includingintracellulartransport(p = 0.0152)andestablishment oflocalizationinacell(p = 0.0152).NodesintheSCZPPInetwork,the

largestnetwork,includegenesinvolvedinpre-mRNAsplicing,suchas SNU1, SMU1, TXNL4A,and ZMAT2.Anotherprominentnodeiscentered on RBX1,anE3ubiquitinproteinligase,whichinteractswith GLMN,an ubiquitinligaseinhibitor,and CUL3,acomponentofubiquitinproteinligasecomplexes.FortheDEPPPInetwork,themostprominentnodeiscenteredon MAPT,microtubule-associatedtauprotein,whichfunctionsin promotingmicrotubuleassemblyandstability.While3’aTWASsignificant genesrepresentasmallportionofSCZQTLs,pathwaysarenotablydistinctfromthosethathavebeenassociatedwithdifferentiallyexpressed andsplicedgenes,suchasmetabolicpathways,synapticplasticity,excitatorysynapses,andimmune-relatedpathways(5, 78).Asummaryofthe prominentprotein–proteininteractionnetworksidentifiedinthisanalysis isprovidedinSupplementaryTableS2.

ConclusionsandFutureDirections

Inthepost-GWASera,thefieldofpsychiatricgenomicsischallengedwith interpretingthebiologicalsignificanceofhundredsofriskloci.TWAS hasbecomeawidelyimplementedmethodofnominatingputativecausal genesbyleveragingrelativelysmall transcriptomicdatasets.However, therearemultiplemechanismsbywhichSNPsmaycontributetoabiologicalphenotype,whicharenotallconsideredinmostTWASanalyses.The discoveryof3’aQTLshelpsexplainsomeGWAS-significantSNPsthatare notassociatedwithdifferentialexpressionorsplicingintraditionalTWAS analyses.WhileapercentageofAPA-linkedgenesarealsoimplicatedin expressionandsplicingTWASanalyses,approximately60%–75%ofAPAlinkedgenesforSCZ,BD,DEPhavenotbeenassociatedwithtotalgene

expressionorisoformexpressioninthelargestTWAS.Forexample,GWASsignificantSNPs,rs2024566andrs5751204,onchromosome22nearthe SNU13 genedidnotreachsignificanceinalargeisoform-levelTWAS(6); however,alternative3’UTRusageofthisgenewasassociatedwithSCZin theanalysisofCui etal.,whichutilizedthesametranscriptomicdatasets. Theassociationofgeneswithdifferential3’UTRusagein3’aTWASsuggeststhataberranciesinmRNAtranslocationordegradationmayplaya roleindiseaserisk.However,furtherconfirmatorystudiesareneededto determinetheimpactofdifferential3’UTRusageof3’aTWASsignificant genesonneuronalfunctionandhomeostasis.Astranscriptomicreference setscontinuetogrow,itmayeventuallybecomepossibletodirectlymeasuredifferentialexpression,splicing,3’UTRusagebetweencasesandcontrols.ThiswillbecriticalforvalidatingthefindingsofTWASandguiding focusedeffortstodecipherdownstreammechanisms.

InadditiontoAPAofSNCAinPD,thereseveralwell-studiedexamplesofAPAalterationshavingsignificantneurologicalconsequencesthat helpexplainphenotypictraits.Arelativelywell-characterizedexampleis theMECP2gene,whichencodesmethyl-CpGbindingprotein2(MeCP2), involvedintheregulationoftranscriptionofmanydifferentgenes. Loss-of-functionmutationsofMECP2resultinRettsyndrome,whichis characterizedbydevelopmentalregressionandintellectualdisabilitybeginningataround18monthsofage(79).TheMECP2transcriptexistsin twoisoformswitheitheralong3’UTRorshort3’UTR(80).Theproximal APAsiteofthegene,whichproducestheshortisoform,isincreasingly usedthroughoutpostnataldevelopmentandisassociatedwithincreased proteinabundance(81).ThelongMECP2isoform,ontheotherhand,is translatedlessefficientlycomparedtotheshortisoformandleadstodecreasedproteinabundance.MutationsingeneproductsthatregulateAPA ofMECP2canalsocauseformsintellectualdisabilityandautismspectrum disorders.Anexampleofthisiscopynumbervariants(CNV)duplications ofNUDT21(11).TheproteinproductofNUDT21,acomponentofmammaliancleavagefactor1complex(CFIm25),bindstothedistalAPAsites ofgenes,includingMECP2,andfacilitatesproductionoftranscriptswith long3’UTRs(82).Lymphoblastoidcelllinesderivedfrompatientswith NUDT21duplicationshave ∼50%lessMeCP2proteinandincreasedabundanceofthelongMECP2transcript(82).Inthisscenario,thereductionin MeCP2proteinisnotquiteassevereasinRettsyndrome,yetitissufficienttoproduceintellectualdisability(11).

MoredetailedinvestigationsasdescribedaboveareneededtoexaminetheimpactofAPAofgenesassociatedwithpsychiatricdisorders.As welearnmoreabouthowAPAalterationsaffectthebrain,wewillbetterdefinethemolecularandbiologicalunderpinningsofneuropsychiatric disorders,whichwillguidethedevelopmentoftreatmentstrategies.EffortstodeterminethecriticalstagesofdevelopmentduringwhichAPA alterationscontributetophenotypicexpressionwillalsobeinformative foridentifyingopportunitiesfortherapeuticintervention.

AuthorContributions

M.P.performedtheliteraturereviewandwrotethemanuscript.S.G.conceptualizedthereview,editedthemanuscriptandproducedthefigures forthemanuscript.Y.C.contributedcontentforthereviewandeditedthe manuscript.O.A.A.reviewedandeditedthemanuscript.A.L.S.supervised preparationandeditedthemanuscript.W.L.conceptualizedandedited themanuscript.X.X.supervisedandeditedthemanuscript.

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RESEARCHARTICLE

Sexdifferencesinalcoholandtobaccousedisordersamongindividualswithpanic disorder:Across-sectionalanalysisfromthegenomicpsychiatrycohort

MichaelJ.Chung1 ,PenelopeGeorgakopoulos2 ,JacquelynMeyers3 ,ShivangiSharma2 ,CarlosN.Pato2 ,andMicheleT.Pato2

Anxietydisordersandsubstanceusefrequentlyco-occur,yetthemoderatingeffectsofsexandancestryontheserelationshipsremain understudied.Thisinvestigationexaminedassociationsbetweenpresumedpanicdisorder(pPD)andbothpresumedalcoholusedisorder (pAUD)andtobaccousedisorder(pTUD)in10,953individualsfromtheGenomicPsychiatryCohortscreenedagainstseverementalillness.Our samplewasdemographicallydiverse(56%female;55%EuropeanAncestry,45%AfricanAncestry),allowingrobustcomparisonacrossthese groups.IndividualswithpPD(n = 342)demonstratedsignificantlyhighermeanseverityscoresforbothpAUD(1.26vs.0.33, p < 0.05)andpTUD (1.65vs.0.93, p < 0.05)comparedwiththosewithoutpPD.WhilefemalesexwasassociatedwithdecreasedriskforpAUD(B: 0.351, p < 0.05) comparedwithmales,weobservednosignificantancestry-baseddifferencesinsubstanceusepatternsamongthosewithpPD.Two-way interactionanalysesrevealedthatsexsignificantlymoderatedtherelationshipbetweenpPDandpAUD(B: 0.97, p < 0.001),withthe associationbeingstrongeramongmalesthanfemales.Additionally,comorbidpresumedposttraumaticstressdisorderwassignificantly associatedwithincreasedriskforbothpAUD(B:0.650, p < 0.05)andpTUD(B:0.825, p < 0.05)butdidnotinteractsignificantlywithpPD.These findingsadvanceourunderstandingofhowbiologicalsexinfluencesthemanifestationofcomorbidpanicandsubstanceusedisorders,offering clinicalimplicationsforassessmentandtreatmentstrategiesthatacknowledgesex-specificvulnerabilitypatternswhilehighlightingthe consistentrelationshipbetweentheseconditionsacrossancestralgroups.

GenomicPsychiatry March2025;1(2):42–48;doi: https://doi.org/10.61373/gp025a.0020

Keywords: Alcoholusedisorder(AUD),panicdisorder(PD),post-traumaticstressdisorder(PTSD),tobaccousedisorder(TUD)

Introduction

Panicdisorder(PD)isadebilitatingconditioncharacterizedbyrepeated episodesoffearthatlastforseveralminutesorlonger.Physicalsymptomsmayincludeheartpalpitations,sweating,chills,trembling,breathingproblems,weakness,dizziness,and/orfeelingofbeingoutofcontrolorimpendingdoom.TheetiologyofPDismultifactorial.Current researchsuggeststhatbiological,psychological,and/orenvironmental factorsmaycontributetowhysomeindividualsdevelopthisdisorder comparedwithothers.Fromagenomicperspective,withincertainfamilies,somesuffermorefromPDthanothers.AccordingtotheWorldMentalHealthSurveys(WMHS),theprevalenceofPDacrossvariouscountries isestimatedatapproximately1.7%.Typically,theageofonsetis32,with mostcasesemergingbetweentheagesof20and47(1).PDafflictsabout 4.7%ofU.S.adultsatsometimeintheirlivesandbasedondiagnostic interviewdatafromtheNationalComorbiditySurveyReplication,anestimated2.7%ofU.S.adultswerediagnosedwithPDin2017(2).Interms ofsexdifferences,PDtendstoafflictmorewomenthanmen.Theprevalenceishigherinfemales(3.8%)thaninmales(1.6%)(2).Further,theNationalSurveyofAmericanLife(NSAL)andtheNationalComorbidityStudy (NCS)showedthatnon-HispanicWhiteparticipantsreportedhigherrates ofPDthannon-HispanicBlackparticipants(4.8%vs.3.5%)(3).WMHSalso foundthat80.4%oflifetimePDcaseshadcomorbiditywithotherpsychiatricillnesses(1).Lifetimecomorbiditywithothermentaldisordersincludesotheranxietydisorders,affective,substanceusedisorders(SUD), andimpulse-controldisorders(1, 4–6).PDisalsoassociatedwithphysicalandemotionaldistress,detrimentsinsocialandromanticrelationships,suicideattempts,financialdifficulties,andSUD(7–10).SeveraltheoriesforthecomorbidrelationshipsbetweenanxietydisorderssuchasPD andSUDhavebeenposited.Forexample,“self-medication”or“tensionreduction”theorystatesthatpatientswithanxiousdisordersusesubstancestorelievetheirpanicsymptomstemporarily,thusdevelopingSUD (11).Physiologicand/orenvironmentalstressorsfromchronicsubstance

usecreatesituationsorcircumstancesinwhichanxietysymptomsare morelikelytoemergeorworsen(12).Asnoted,theself-medicationhypothesishighlightsakeymechanismbywhichindividualswithPDmayuse substanceslikealcoholandtobaccotomanagetheirsymptoms,leading tohigherratesofAUD(alcoholusedisorder)andTUD(tobaccousedisorder).WhilethereisarobustrelationshipbetweenPDwithTUDandAUD (13, 14).therearefewstudiesthatexaminesexdifferencesinPDandSUD comorbidity(15)andevenfewerreportingonancestry/race(orancestryby-sex)differencesinthecomorbidityofPDwithcommonSUD(AUDand TUD)inlarge,diversesamples.Sexandancestry/racemayinfluencecomorbiditypatternsinPDandSUD.Forexample,socioculturalfactorsmay shapesubstanceusebehaviors,potentiallyleadingtovaryingprevalence ratesofcomorbidPDandSUDacrossthesegroups.

IndividualswithPDaremorelikelytosmokeandmisusealcoholcomparedtoindividualswithanyotheranxietydisorders(16–20).Multiple population-basedstudieshavedemonstratedthestrongassociationbetweenPDwithTUDandAUD,includingtheEpidemiologicCatchmentArea (ECA)survey,NCS,andtheNationalEpidemiologicalSurveyofAlcoholRelatedConditions(NESARC).WithregardtosexdifferencesobservedinrecentstudiesonPDcomorbiditywithDSM-5AUDandTUD,dataderived fromtheNESARCfoundthatfemaleswithPDwere4.5timesmorelikely tohaveTUDcomparedwithmaleswithPD(21).Regardingcomorbidity, therearethreemainhypothesesofthemechanismunderlyingtheassociationbetweensmokingandPD,thoughnonecomparebyancestry/raceas wewill.(1)SmokingmayincreasetheriskofdevelopmentofPDthrough impairedrespirationandincreasedvulnerabilityforpanicattacks(22, 23) andthroughtheexpectedcalmingeffectsofnicotine(24, 25);(2)Stimulantpropertiesofnicotinemayalsoreducethethresholdforexperiencing panicattacks,especiallyasnicotineismetabolizedinthebloodstream, andthenicotinelevelsdrop(23, 26–28);(3)Theassociationmaybemoderatedbyothervariablessuchasanxietysensitivity,ariskfactorfor developingPD(11).

1 DepartmentofChild&AdolescentPsychiatry,NewYorkUniversityLangoneMedicalCenter-BellevueHospitalCenter,NewYork,NY10016; 2 DepartmentofPsychiatry, RutgersUniversity,Piscataway,NJ08854; 3 DepartmentofPsychiatryandBehavioralHealth,StateUniversityofNewYorkatDownstate,Brooklyn,NY11203

CorrespondingAuthor: MicheleT.Pato,DepartmentofPsychiatry,RutgersUniversity,Piscataway,NJ08854.Phone:626-676-9108.E-mail: m.pato@rutgers.edu

Received:30May2024.Revised:31March2025and2April2025and4April2025.Accepted:7April2025. Publishedonline:22April2025.

Table1. ControlswithandwithoutpPD

UsingdatafromtheNCS,Kesslerdescribedthateventhoughmales hadahigherincidenceofAUD,femalesstillhadhigherincidencesofcomorbidPDandAUDthanmales.FemalesweremorelikelytohaveadiagnosisofPDbeforeAUD,whereasmalesweremorelikelytohaveadiagnosisofAUDbeforePD,andthismayexacerbatealcoholproblemsin femaleswithPDsinceitmaybeaformofself-medication(29).InanotherstudythathypothesizedthereweresexdifferencesbetweenPD individuals,169patientswithPDandestablishedalcoholdependence (89malesand80females)intheNHIRD[TaiwanNationalHealthInsuranceResearchDatabase]werenestedwithother9480patientswith onlyPD,thestandardizedincidentratio(SIR)forAUDwassignificantly higherforfemaleswithPD(SIR = 6.29)ascomparedwithmaleswithPD (SIR = 3.36)(15).

PreviousstudieshaveconsistentlydemonstratedthatPDismore prevalentamongWhiteAmericansincomparisontotheirAfricanAncestry(AA)counterparts(30–33).Surprisingly,panicdisorderwithcomorbid AUDandTUDwithafocusonancestry/racehasbeensparselyresearched. InastudypoolingdatafromtheNESARC,Smith etal.analyzedracialdifferencesinthe12-monthprevalenceandco-occurrencebetweenDSMIVAUDandPD.Inthestudy,AAhadhigherratesofcomorbidAUDand PDwithandwithoutagoraphobia(OR = 4.2andOR = 3.5,respectively) comparedwithEAcounterparts(OR = 2.7and2.1,respectively)(30).This findingwasconsistentwithGrant’spreviousNESARCpublication(21).Ina separatestudy,Huang etal.comparedtheprevalenceandco-occurrence ofDSM-IVAUDalongwithPDamongEuropeanCaucasianAncestry(EA) andAAinalargerepresentativesampleoftheU.S.populationbetween 2001and2002.Consistentwithpaststudies,Blacks(AA)(OR = 2.5)were morelikelythantheirwhitecounterparts(OR = 1.9)tohaveco-occurring PDandAUD(34).

Inthisstudy,weexaminedratesof“presumed”alcoholandtobacco usedisorders(pAUD,pTUD)andtheirassociationwith“presumed”panic disorder(pPD)basedonscreeningitemsforeachdisorderandthemoderatinginfluenceofsexandancestry/race.Asmentionedearlier,PDhas manypsychiatriccomorbidities,includingPTSD(posttraumaticstressdisorder).BerensconductedafurtheranalysisoftheNESARCstudy,finding thatapproximately27.4%ofindividualswithprimaryPDalsohavelifetimePTSD(35).Thesefindingsaligncloselywiththoseofotherepidemiologicalstudies,suchastheNCS,whichsimilarlyreportedacomorbidity rateof27%(36).Therefore,wealsoexaminedtheroleofpPTSD(presumedPTSD)intheassociationsofpPDwithpAUDandpTUDanddifferencesbysexandancestry/race.Thus,ourresearchquestionsforthisanalysisoftheGPCcohort,basedonrecentfindings,are:

1.Doessexandancestry/racehaveanassociationintheprevalenceofindividualswhoexperiencecomorbidpPDandpAUDandpTUDcompared withthosewithoutPD(nopPD)?

2.Howdosexandancestry/raceworkseparatelyandincombinationwith oneanothertoinfluencetheprevalenceratesofcomorbidpPDand pAUDand/orpTUD?

3.WhataretheeffectsizesofpAUD/pTUDinpPDaloneversuswithcomorbidpPD + pPTSD?IsthereaninteractionbetweenpPDandcomorbidpPTSDand/orpAUDandpTUD?

Results

Amongallparticipantsintheanalyticsample(n = 10,953),342screened positivelyforpPD,and10,611didnot(nopPD).Asshownin Table1, ahigherproportionofparticipantswerefemale;however,ratesofpPD ascomparedwithnopPDweresimilaramongfemales(pPD:57.9%,no pPD:56.0%)andmales(pPD:42.1%,nopPD:44.0%).Similarly,while therewasagreaternumberofparticipantsofEAthanofAA,ratesof pPDwerenotstatisticallysignificantlydifferent(EA:55.6%;AA:44.4%) (Table1).

Asshownin Table2 and Figure2,meanalcoholproblemscoreswere significantlyhigheramongthosewhoscreenedpositiveforpPDwhen comparedwiththosewithnopPD(pPD:M = 1.26,SD = 1.923;nopPD: M = 0.33,SD = 1.940; p < 0.05).Similarly,tobaccousescoresweresignificantlyhigheramongthosewhoscreenedpositiveforpPDwhencompared withthosewithnopPD(pPD:M = 1.65,SD = 1.675;nopPD:M = 0.93, SD = 1.435, p < 0.05).Femalegenderwasassociatedwithadecreased riskforAUD(B: 0.351, p < 0.05)andTUD(B: 0.520, p < 0.05)ascomparedwithmales.ScreeningpositiveforpPTSDwasassociatedwithincreasedriskforpAUD(B:0.650, p < 0.05)andpTUD(B:0.825, p < 0.05) (Figure3).

Two-wayinteractions(pPDxancestry/race;pPDxsex;pPDxpPTSD), andthree-wayinteractions(pPDxsexxancestry/race)werealsoexamined(Table2).Theseinteractioneffectswereincludedtoexplorewhether therelationshipsamongpPD,pAUD,andpTUDvariedbydemographicfactorssuchassex,ancestry/race,andpPTSDstatus.Significantinteraction termsindicatethattheassociationbetweenpPDandsubstanceuseoutcomeswasmodifiedbythesefactors,suggestingdifferencesincomorbiditypatternsacrossdemographicgroups.Theonlysignificantmoderating influencesofsexontheassociationofpPDandalcoholuseproblemswere observed(B: 0.97, p < 0.001).Thatis,theinfluenceofpPDonalcoholuse problemsisgreateramongmales(see Figure3)ascomparedwithfemales (Table2; Figure3).

Table2. TheassociationofpPDwithalcoholuseandtobaccouse disordersandinteractionswithsexandrace

∗∗ Significance p < 0.05.

Figure1. GPCscreenerquestions.ThisfigureillustratesthescreeningquestionnaireitemsusedintheGPCstudytoidentifypPD,pAUD,pTUD,andpPTSD.The screenerincludesspecificquestionsforeachconditionwithyes/noresponseoptions.ForpPD,participantswereclassifiedaspositiveiftheyanswered“yes”to bothitems11and11a,indicatingexperienceofsuddenanxietyattackswithphysicalsymptomsandsubsequentworryorbehaviorchanges.Thealcoholsection includessixquestions(items12–17)addressingriskydrinkingpatternsandconsequences,whilethetobaccosectionincludesfouritems(18–21)addressinguse patternsandquitattempts.PTSDscreening(items32–32c)focusesontraumaticexperiencesandsubsequentsymptoms.

Figure2. Meansumscoresforalcoholandtobaccoriskuseforparticipantswithpresumedpanicdisorder(pPD)andwithoutpresumedpanicdisorder(nopPD). ThisbargraphcomparestheseverityofalcoholandtobaccouseproblemsbetweenindividualswithandwithoutpPD.ParticipantswithpPDdemonstrated significantlyhighermeanseverityscoresforbothalcoholuse(1.26vs.0.33, p < 0.05)andtobaccouse(1.65vs.0.93, p < 0.05)comparedwiththosewithout pPD.ThebluebarsrepresentindividualswithpPD,whiletheorangebarsrepresentthosewithoutpPD.ErrorbarsindicateSDs.

Figure3. Meansumscoresforriskyalcoholandtobaccouseamongmaleandfemaleparticipantswithpresumedpanicdisorder(pPD)andwithoutpresumed panicdisorder(nopPD).ThisfigureillustratesthesexdifferencesinalcoholandtobaccouseproblemsamongindividualswithandwithoutpPD.Thedataare stratifiedbybothsexandpPDstatus,revealingsignificantinteractions.AmongmaleswithpPD,meanalcoholriskscores(2.10)weresubstantiallyhigherthan amongmaleswithoutpPD(0.53).Similarly,maleswithpPDshowedhighertobaccoriskscores(2.06)comparedwithmaleswithoutpPD(1.23).Forfemales, thosewithpPDalsodemonstratedhigheralcoholriskscores(0.64vs.0.18)andtobaccoriskscores(1.34vs.0.69)comparedwithfemaleswithoutpPD. All comparisonswerestatisticallysignificant(p < 0.05).Notably,maleswithpPDreportedhigheralcoholriskscoresthanfemaleswithpPD(2.10vs.0.64).

Chungetal.

Discussion

ThisstudystandsoutasoneofthefewthatinvestigatestheintersectionofpAUDandpTUDwithinindividualswithpPD.Moreover,itaimsto delineatethesegroupsmorepreciselybyconsideringfactorssuchassex andancestry/race,addingdepthtoourunderstandingofthesecomplex relationships.Toourknowledge,thisisthefirstpaperthatlooksatcomorbidpPTSDinpatientswithpPDalongwithpAUDandpTUD.Asaforementioned,theprevalenceofpeoplewithPDintheUnitedStatesisabout 2.7%,whichissimilartooursample’sprevalencerateof3.22%.Females aretypicallymoreafflictedwithPDthanmales,whichisconfirmedinour dataaswellsinceinouroverallcasesample,about58%ofourcaseswith pPDwerefemale,and42%ofourcaseswithpPDweremale.Wehavealso found,likeothers,thatindividualswithpPDhavesignificantlymorecomorbidities,includingpTUD,pAUD(Figures2 and 3),andpPTSD(Tables1 and 2).

PriorstudiesreportthatindividualswithPDaretwotofourtimes morelikelytosmokeanddrinkcomparedwiththegeneralpopulation (37).Whileourstudydoesnotmeasureprevalencedirectly,wefound thatamongthosewhoengageinthesebehaviors,individualswithpPD hadahighermeanseverityscoreforpTUD(1.65/4.0)comparedwith thosewithnopPD(0.93/4.0).Similarly,individualswithpPDhadahigher meanseverityscoreforpAUD(1.26/6.0)comparedwiththosewithnopPD (0.33/6.0).ThissuggeststhatindividualswithpPDmayexperiencemore problematicuseratherthanjusthigherratesofuse.Ouranalysisconfirms thecurrentliteraturethatindividualswithPDdosmokemoretobaccoand drinkmorealcohol(Figures2 and 3)(16–19, 20, 23, 38–40).

AncestryDifferencesinpPDandComorbidpTUDorpAUD

Althoughpaststudieshavereportedancestry/racedifferencesinindividualswithcomorbidpPD + pAUDwithAAindividualshavinghigherprevalencethanEAindividuals,wefoundalowerprevalenceinourAAsample (21, 30, 34, 41).Initially,itappearedthatancestry/racehadaneffectwhen lookingatseveralcovariatesatonce;however,thiswasnotthecasewhen weperformedtwo-wayandthree-wayinteractions(Table2).OnepossibleexplanationisthatalcoholmisuseandAUDsarelessprevalentamong AAindividualsthanthoseofEA,andthereforethismaybedrivingthese results.Ransome etal. (2017)(41)demonstratedthatBlackwomenwith AUDhaddifferenthealthoutcomesanddrinkingpatternsthantheirWhite counterparts,potentiallyinfluencedbysocioculturalfactorsandsystemic barrierstocare.WhilethisstudydidnotspecificallyexaminePDorPTSD, itsfindingsonracialdisparitiesinalcoholusealignwithourobservation oflowerpAUDprevalenceinAAindividuals.

AnalternativehypothesiscouldrelatetotheinherentnatureofstructuralracismintheUnitedStates.Forexample,arecentstudyhashighlightedtheroleofdiscriminationininfluencingmentalhealthandsubstanceuseoutcomes.Vu etal. (2019)(42)demonstratedthatintersecting identities,suchasancestry/raceandsexualorientation,andexperiences ofdiscriminationsignificantlyimpactmentalhealthoutcomes,including depressivesymptomsandsubstanceuse.ThiscouldfurtherexplaindifferencesincomorbidsubstanceuseandPDacrossracialgroups(42).

Additionally,AAchildrenaremorelikelysocializedtoexpecthostility, irrationalrestrictions,insults,andunfairtreatmentbasedonthecolorof theirskin.Thus,toprotectagainstthis,AAchildrenaretaughttodevelop highlevelsoftoleranceforunfairacts(43).Thistypeofsocializationhas beenshowntobeprotectiveofmentalhealthsymptoms(44).Although thissocializationwasnotexplicitlytiedwithPD,itcouldbepostulatedas afactorforthelowerprevalenceofPD.Whilethishypothesisisspeculative,itwarrantstheneedforfurtherinvestigation.

Anotherpossibleexplanationcouldbethatlimitedaccesstomental healthcareandpoorqualitycarewhenaccessingcaremaybeafactorfor thelowerprevalenceofPDdiagnoses(45, 46).Third,stigmaandjudgmentalsoplayaroleinthelowerratesofAAindividualsreceivingtreatment.InaqualitativestudybyAlvidrez,one-thirdofblackindividuals regardedreceivingtreatmentformilddepressionandanxietyas“crazy” (47).Inregardstocomorbidsubstanceuse,studieshaveshownthateven inprecariouslivingsituationscomparablytotheirwhitecounterparts, BlackindividualswithPDengagedinfewerunhealthybehaviors,suchas tobacco,alcohol,andsubstanceuse(48, 49).Thus,thereasonsforade-

creasedprevalenceofPDandTUDorAUDinAAaremultifactorialandare likelyunderreportedinoursample(Table1).

SexDifferencesinpPDandComorbidpTUDorpAUD Inouranalysis,pPDisassociatedwithincreasedpAUDandpTUDwhen lookingatthemaineffect,adjustingforage,sex,ancestry/race,and pPTSD.Whenexamininginteractionsbetweensex,ancestry/race,and pPTSD(two-wayinteractions),andsexandancestry/race(three-wayinteractions)forpAUDandpTUD,onlytheinteractionofsexwithpAUDwas significant,withitbeinggreateramongmalescomparedwithfemales (Table2; Figure3).Thisisinagreementwithdecadesofresearchdemonstratingagreaterriskforalcoholuseproblemsamongmalescompared withfemales(50–52).Generalexplanationsforthismalebiastowardalcoholuseproblemsincludetrendsofmaledrinkersconsumingalcohol moreoftenandinlargerquantitiesthanfemaledrinkersand,historically, limitedopportunitiesforfemalestodrinkheavilyduetosocietalnorms, pregnancy,andchild-rearing(53).

However,weshouldnotethatthiscontrastswithsomerecentstudiesthathaveshownthatwomenwithanxietydisorders,includingbutnot limitedtoPD,drinkmorealcoholcomparedtomen.Forexample,ina studyinGermany,womenwithanxietydisorderstendedtohaveanearlier onsetofdrinking,regulardrinking,andoccurrenceofwithdrawalsymptomswhencomparedwithwomenwithoutanxietydisorders(54).They alsonotedtheremaybemoreseverephysicalsymptomsamongthese women(54).Anotherstudyalsofoundthatwomenhavemoresevere respiration-relatedsymptomsandmoresevereagoraphobicavoidance symptomologycomparedwithmen(55).Withbothstudiesinmind,(54, 55)itcouldbehypothesizedthatfemaleswithanxietydisordersarealreadysusceptibletosignificantlydistressingwithdrawalsymptomsthat couldpotentiatethedesiretoself-medicatewithalcoholasatemporarymeasuretodampenpanicdisordersymptomology.However,Schneider’s(54)studydoesnotexplicitlydifferentiatefemaleindividualswith PDfromfemaleindividualswithotheranxietydisorders.Therefore,the differencewefound,whereinwomenwithpPDweredrinkinglessthan menwithpPD,isinagreementwiththegeneralliteratureonincreased alcoholuseproblemsinmalescomparedwithfemalesandmaynotbe incontradictiontothesepreviousstudiesofwomenwithanxietyincludingPD,sincetheyhadnotseparatedwomenspecificallywithPDfrom othermorecommonanxietydisorders(e.g.,generalizedanxietydisorder) (54, 55).

WhilestudieslikeSchneider etal. (54)andSheikh etal. (55)explored broaderanxietydisordersratherthanPDspecifically,ourfindingsalign withtheirobservationsregardingdifferencesindrinkingbehaviors.However,itisimportanttonotethatthesestudiesutilizedmorecomprehensivedatacollectionmethods.Forinstance,White etal. (2015)(53) capturedlongitudinaldrinkingpatternsanddetailedalcoholusehistory, whereasourstudyreliedonalifetimescreenertoassessdrinkingbehaviorsacrossbothcasesandcontrols.Thisdifferenceinmethodologycould contributetotheobserveddiscrepanciesbetweenstudies.

Withregardtotobaccouse,therehasbeenmuchresearchdedicatedto understandinghowsubstancesmaybeusedtoalleviatefeelingsofanxiety,suchasthosethatariseinPD.Inanintegrativemodelofsmokinganxietycomorbidity,smokerswithanxietydisorderstendtobefearfulof anxiety-relatedsymptomsandbodilysensations,andtheycopebyusingnicotinetoachievethe“positive”sensationsofnicotineinhopesof relaxingthemselves.Alternatively,individualsmayalsousenicotineto escapeandavoidemotionallydistressingevents(56, 57).Thiscreatesa feed-forwardcyclethatisdifficulttobreakbecausetheindividualwillinevitablyfeelnicotinewithdrawal-relatedaversiveinteroceptivecuesthat occurroutinelyandcopebysmokingsomemore(58, 59).

Historically,intheUnitedStates,maleshavealwayssmokedmorethan females.Thus,atfirstglance,ourfindingoflesssmokingamongwomen withpPDthanmenmightseemsupportedbytheliterature.However, whilesmokingrateshavedecreasedthroughoutmostagegroups,ratesof smokingcessationinwomenhavenot.Fromaphysiologicalstandpoint, thismaybeduetothedifferencesinnicotinemetabolismobservedbetweenfemalesandmales.InastudybyBenowitz,intravenousinfusions ofnicotineandcotinineshowedhigherclearanceratesinfemalesthan

inmales,andoralcontraceptivesfurtheracceleratedthisclearance(60). Thus,oneexplanationofthenotablehighersmokingratesinfemaleswith PDversuswomenwithoutPDcouldbeacombinationofthisfeed-forward cycleandthemorerapidmetabolicclearanceofnicotine.Goodwinsubstantiatesthehypothesisthatfemalesmokerslikelysmokeduetounderlyinganxietyspectrumdiagnoses.Inasampleof4149individuals,Goodwinexaminedtheprevalenceofmoodandanxietydisordersamongmale andfemalecurrentsmokersintheUnitedStatesusingdatafromNCSand NCS-RandfoundthatPDwassignificantlymorecommonamongfemale currentsmokersthaninmalesin2001comparedwith1990(61).

WedidnotseethisTUDinteractioninourfemalesample(Table2) andwonderwhetherthisissimplyduetothepowerlimitsofoursamplesizesince,as Table1 indicates,theoverallfemalesamplewasonly 198pPDversus5942withoutpPD,andformales.Atotalof144withpPD versus4668withoutpPD.Anotherlimitationincludesthediagnosesfor PD,PTSD,AUD,andTUD,whicharebasedonscreener/self-reportitems andnotonafulldiagnosticassessmentforaDSMdiagnosis.Forthisreason,AUDandTUDweretreatedassumscores,representingthesumof thenumberofscreeningitemsendorsed.Althoughitmaybeconsidereda limitation,inourstudy,only342participantsoutof10,611answeredyes toitems11and11a.Theuseoffewerquestionscouldbeimplemented inclinicalsettings,wheresometimesthesequestionsarenotasked,to screenoutparticipantsanddirectthemtoappropriateservicesbriefly.

Limitations

Findingsshouldbeconsideredwithinthecontextofthisstudy’slimitations.ThediagnosisforPDandPTSDwerebasedonscreeneritemsand notonafullDSMdiagnosis.Further,screenerswereself-reportedand, therefore,subjecttobias.However,evenwiththelimitednumberofquestions,onlyaverysmallportionmetthecriteriaforPDorPTSD,andrates werecomparabletothoseinthegeneralpopulation.IntermsofpAUDand pTUDdiagnosesinoursample,tomeasureseverity,weusedsumscores torepresentthesumofthenumberofscreeningitemsendorsed.Future analysisshoulddeterminewhetherthesefindingsextendtoDSMdiagnosesofAUDandTUD(e.g.,DSM-VAUD,TUD).Alargersamplesizewith PDinfuturestudieswouldalsostrengthenourfindings.Anotherlimitationisthatthecross-sectionalstudydesignforalifetimeofillnessdid notallowustodeterminewhetherAUDorTUDprecededorfollowedPD orPTSD.FuturestudiesofamorelongitudinalnaturecouldexamineSUD diagnosesthatprecedeorfollowadiagnosisofPD.Intermsofmissing data,theoveralldatasethadminimalmissingentries.Participantswith incompletedatawereexcludedfromanalysesinvolvingthosevariables. Giventhelowvolumeofmissingdata,imputationtechniqueswerenot necessary,andtheresultswerenotsignificantlyimpacted.

Conclusion

Inourstudy,wefoundsignificantlymorealcoholandtobaccouseinthose withpPD.Bysex,femalesweremorelikelythanmalestohavepPD(Table 1),butwerelesslikelytohavealcoholuseproblemscomparedwithmales (Figure3; Table2).Wedidnotfindthatancestry/race(AAvs.EA)hada moderatingeffectonalcoholandtobaccouseproblemsamongindividualswithpPD.Wedidnotfindanydifferencesinalcoholortobaccouse problemswhenwelookedatinteractionswithcomorbidpPTSD(Table2). However,wedidfindsignificantcomorbidityofpPTSDinthepPDsample comparedwithcontrols(Table1).Continuingtodocumentdifferencesin riskfactorsformentalhealthconditionsbyancestry/raceandsexisimportant,asthiscanimpactandguidenationalhealthcarepolicyinthe UnitedStates.FutureresearchshouldlookatspecificgeneticorenvironmentalfactorsthatreinforcethecomorbiditiesofPD-AUDandPD-TUD. Thiswillhelpguidefuturemanagementandtreatments.Insummary,this studyadvancesourunderstandingoftheinterplaybetweenanxiety,SUD, andPD,particularlyinthecontextofsexandancestry/racedifferences, offeringafoundationforfutureresearchandtailoredclinicalapproaches.

MaterialsandMethods

Sample

TheanalyticsampleisderivedfromtheGenomicPsychiatryCohort(GPC), alargeanddiversesampleofindividualsscreenedforneuropsychiatric disorders.TheprimaryfocusoftheGPCisonseverementalillnessessuch

asschizophreniaandbipolardisorder.However,italsoincludesalargecohortofparticipantswhoareunaffectedbythesedisorders(N = 10,953), definedasindividualsnotdiagnosedwithschizophreniaorbipolardisorderandwithnofirst-orsecond-degreerelativeswiththesedisorders. Controlsalsohavenohistoryoftraumaticbraininjuryandnohistoryof multipledepressiveepisodes(>4)(62).Further,individualswereidentifiedasmaleandfemalebasedongenome,XXorXY,andself-identified asAfricanorEuropeanAncestry(AA,EA,respectively)wereincludedin thisanalysisgiventhelimitednumberofparticipantsofLatinoormixed ancestry.Thisanalyticsampleis56%female,55%ofEA,and45%ofAA (Table1).Oneuniquefeatureofthesampleisacloseto ∼1:1ratioofindividualsofAAandEAincasesandcontrolsamples,makingitavaluable datasetforthestudyofancestry/race(andsex)differencesinthecomorbidityofthesedisorders.ParticipantsaredrawnfromacrosstheUnited States,Canada,Mexico,Portugal,andotherregionsofSouthAmericaand Europe;however,onlythosewhoidentifiedasAAorEAwereincludedin theanalyticsample.

Measures

IndividualswhoendorsedbothPDscreeningitems(i.e.,responded“Yes” toitems11and11a)(Figure1)aredescribedasaffectedwithpPDinthis study,andindividualswhoendorsed0–1items,weredescribedasunaffectedwithpanicdisorder(nopPD).Usingtheseparameters,weidentified342individualswithapPDandcomparedtheseindividualswiththose unaffectednoPD(Table1).Thescreeningquestionnairealsoincludessix items(items12–17)regardingalcoholuseproblemsandfouritems(items 18–21)regardingtobaccouseproblemsadaptedfromthe“Cuttingdown, Annoyancebycriticism,Guiltyfeeling,andEye-openers”(CAGE)questionnaire(63).Forbothalcoholandtobaccouseproblems,screeningitems, sumscoresforseveritywerecomputed,AUDrangedfrom0to6andTUD from0to4.Thesespecificcut-offpointswerechosenbasedonSAMHSA’s TreatmentImprovementProtocolrecommendations,whichaimtoidentifyindividualsatriskforSUDbycastingawidernet.Thegradedscoringsystemallowedustocaptureaspectrumofseverity,wherehigher scoresreflectedagreatercumulativeburdenofalcoholortobaccouse.Of note,whilePTSDscreeningitemswereincludedonthescreeningquestionnaire,otheranxietydisorders,includinggeneralizedanxiety,social phobia,separationanxiety,andspecificphobiawerenotassessed.pPTSD wasdefinedasrespondingyestoitem32endorsingatraumaticevent andthenyestoatleasttwoofthethreescreenerquestions32a,32b,32c (Figure1).

StatisticalAnalysis

First,Chi-squaredandindependent t testwereusedin Table1 toassess theassociationsbetweenpPDanddemographicvariables(Table1).Significancewasdefinedasa p < 0.05.Then,linearregressionmodelswere usedtoexaminetheassociationofpPDwithpAUD,pTUD,andpPTSD. Thesemodelswerecomputedseparatelyforalcoholandtobaccouse problemsandincludedthefollowingcovariates:age,sex,self-reported ancestry/race,andpPTSD(Table2).Further,allindividualsidentifiedas maleandfemaleandofAAandEAwereincludedthroughoutthisanalysis,giventhelimitednumberofparticipantsinothergroups.Allanalyses weredoneusingSPSSsoftware(version24).

StudyApproval

TheGPCstudyisapprovedbytheRutgersInstitutionalReviewBoard–HealthSciences,NewBrunswick/Piscataway,locatedat335GeorgeSt., LibertyPlazaSte.3100,NewBrunswick,NJ08901.Thisstudyinvolveshumansubjectresearch,andwritteninformedconsentwasobtainedfollowingathoroughexplanationofthestudy’snatureandpotentialoutcomes.

Acknowledgment

WearegratefultotheGenomicPsychiatryCohortConsortium.

AuthorContributions

M.J.Chung:conceptualization,writing–originaldraft.P.Georgakopoulos: formalanalysis,projectadministration.J.Meyers:formalanalysis,writing–originaldraft.S.Sharma:writing,projectadministration.C.N.Pato MD:conceptualization,fundingacquisition,investigation,methodology.

M.T.Pato:conceptualization,fundingacquisition,investigation,methodology,writing–originaldraft.

FundingSources

ThisworkwassupportedbyNationalInstituteofMentalHealth(NIMH).

AuthorDisclosures

Theauthorsdeclarenoconflictofinterest.

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GenomicPsychiatry

OPEN RESEARCHREPORT

Protectiveinheritedmutationsin activity-dependentneuroprotective protein(ADNP):thegood,thebad,and theugly

IllanaGozes1 ,ShulaShazman2 ,EliezerGiladi1 ,and JosephLevine1 , 3

1 TheEltonLaboratoryforMolecularNeuroendocrinology,Department ofHumanMolecularGeneticsandBiochemistry,FacultyofMedicine, SagolSchoolofNeuroscienceandAdamsSuperCenterforBrainStudies, TelAvivUniversity,TelAviv6997801,Israel

2 DepartmentofMathematicsandComputerScience,TheOpen UniversityofIsrael,Ra’anana4353701,Israel

3 PsychiatricDivision,BenGurionUniversityoftheNegev, Beersheba8410501,Israel

CorrespondingAuthor: IllanaGozes.TheEltonLaboratoryforMolecular Neuroendocrinology,DepartmentofHumanMolecularGeneticsand Biochemistry,FacultyofMedicine,SagolSchoolofNeuroscienceand AdamsSuperCenterforBrainStudies,TelAvivUniversity,TelAviv 6997801,Israel;E-mail: igozes@tauex.tau.ac.il

GenomicPsychiatry March2025;1(2):49–55; doi: https://doi.org/10.61373/gp024r.0079

Activity-dependentneuroprotectiveprotein(ADNP),essentialfor brainformation/function,revealsmultiplecytoplasmicandchromatin interactingsites.Computationalmodeling,alongsidetheVineland AdaptiveBehaviorScales,aleadinginstrumentsupportingthediagnosisofintellectual/developmentaldisabilities,nowrevealeda protectiveframeshift/stopmutationinADNP.Thus,awomanwith inheritedmutation,ADNP_Glu931Glyfs∗ 12(VB),showedaboveaverageVinelandperformance.Bioinformatics/insilico proteinmodelingindicatedthatwhileADNPcontainsfour14-3-3proteininteractionsites(instrumentalforADNPnuclear/cytoplasmicshuttling), ADNP_Glu931Glyfs∗ 12containsanadditionalfifth14-3-3interaction site,implicatingstrongerassociations.Furthermore,theendogenous neuroprotective(investigationaldrug,davunetide)NAPVSIPQ(NAP) sitewasinvolvedintheADNPandADNP_Glu931Glyfs∗ 12-14-3-3interactions.Inthisrespect,themutationalsoenhancedADNP-SH3associations(anotherNAPVISPinteractionsite354-361aaonADNP, criticalforcytoskeletal/cellularsignaling).HB,the8-year-oldVB’s son,whileinheritingthemother’sADNPmutation,furtherpresented aheterozygouspathogenic denovo mutationADNP,p.Arg730Thrfs∗ 5. However,incomparisontocarriersofasimilarp.Arg730∗ mutation (partoftheautistic/intellectualdisabilityADNPsyndrome),HBexhibitedoverallbetterVineland3standardscoreof70–80forallmeasures, comparedtothenominalscoreof20ina27-year-oldADNP,p.Arg730∗ subjectandthe100 ± 15norm,corroboratingADNP_Glu931Glyfs∗ 12 protection.

Keywords: Activity-DependentNeuroprotectiveProtein(ADNP),ADNP Syndrome(HelsmoortelVanDerAaSyndrome),Davunetide(NAP),In SilicoModeling,VinelandAdaptiveBehaviorScales.

Introduction

Discoveredinourlaboratory,activity-dependentneuroprotectiveprotein(ADNP)(1, 2)isessentialforbrainformation(3).Assuch,aberrationsinADNPareassociatedwithneurodevelopment(4),neuropsychiatry(5),andneurodegeneration(6, 7).TheADNPsyndrome(alsoknown

Received:15October2024.Revised:30October2024.Accepted:1November2024. Publishedonline:14November2024.

asHelsmoortelVanDerAasyndrome)iscausedby denovo mutations inADNP(8–10).Overthelastyears,wehaveestablishedcellular(6, 11–14)andanimalmodels(4, 15, 16)tocharacterizeADNPfunctionandthe potentialprotectionbytheADNP-derivedinvestigationaldrug,davunetide(alsoknownasNAP,AL-108,CP201).Ourhaplo-insufficient Adnp+/ mousemodeloriginallyshowedADNP’s/davunetidecrucialfunctionin cognitiveprotectionthroughmicrotubulefortification,enhancementof axonaltransport(17)andsynapseformation(16),aswellasinhibition oftauopathy(3).Wehavefurthershownagainoftoxicfunctionwith ADNPpathologicalmutationlikethep.Tyr719∗ (p.Tyr718∗ inmice)associatedwiththeADNPnuclearlocalizationsignal(4).Thus,micemodelingthisheterozygoustruncationmutationexhibitedmoreextensive motor(gait)impedimentsaswellasearlyTaudepositioncomparedto the Adnp+/ mousemodel.Davunetideprovidedsignificantprotection inbothcases(4).Mechanistically,throughdirectinteractionwithmicrotubuleendbindingproteins(EB1andEB3)SH3bindingandassociation withWntsignalingthroughthearmadillodomainofbetacatenin,davunetideenhancesTaubindingtothemicrotubules,protectingthecytoskeletonwhileinhibitingtauopathy.Thiscytoskeletalfortificationisfurther requiredtomaintainnuclearenvelopeintegrityprotectingagainstacceleratedaging/death(whichisadditionallyaccentuatedwiththep.Tyr718∗ mutationinmousecellcultures)(13, 14).Thecorrectionbydavunetide hasbeenfurtherextendedtoahumanmutation,anoveldavunetide (NAPVSIPQQtoNAPVSIPQE)pointmutationinADNPcausingamilddevelopmentalsyndromeinayoungchild(9).Mostimportantly,davunetidehas recentlyshownefficacycoupledwithneuroprotectioninwomensufferingfromtheneurodegenerativedisorder,progressivesupranuclearpalsy, alate-onsettauopathy(18)aswellassex-dependentmemoryboost inelderlyindividualspresentingamnesticmildcognitiveimpairments, precedingAlzheimer’sdisease(19).

Therefore,fromatranslationalmedicinepointofview,wehavebeen characterizingADNPsyndromeindividualsutilizingtheVinelandAdaptiveBehaviorScales,aleadinginstrumentforsupportingthediagnosis ofintellectualanddevelopmentaldisabilities(20–22).Givenpotential genotype-phenotypedifferencesandage-dependency,wehaverecently assessedacohortof15individuals(1-to27-year-old),using1–3longitudinalparent(caretaker)interview/s(Vineland3questionnaire)overseveralyears(21).Ourresultsindicateddevelopmentaldelays,andpotential developmentalarrests.Wehavepositivelycorrelatedsymptomseverity (e.g.,communicativeproblems)withtheheterozygouspathogenicADNP alleleproteinsizeaswellaswithage(withallindividualsseemtoacquireanageequivalentof1–6years).Additionally,correlationswere discoveredbetweenthetwopreviouslydescribedmousephenotypesalludedtoabove(4),whichinhumanstranslatedtotwoepigeneticsignaturesinADNPemphasizingaberrantacquisitionofmotorbehaviors,with truncatingmutationsaroundthenuclearlocalizationsignalbeingmostly affected(21).

FurtherdetailedanalysisofADNPmutationsintransfectedcellculturesrevealedthatthe denovo additionofproteinsequencesinADNP frameshiftmutationsmayprovideprotection,suchastheadditionof SH3bindingsite(12).Here,by insilico modelingcoupledwithVineland3 questionnaire,weinvestigatedararecaseofinherited(mother,VB)and denovo plusinheritedmutationinason(HB),withonlythesonexhibiting ADNPsyndromecharacteristics.

Thecurrentpaperreviews,cites,andfocusesonourworkonADNP. OuroriginalfindingsonADNPstemmedfromstructureanalysisofprotectiveproteinsstimulatedbyvasoactiveintestinalpeptidetoprovide protectionandenhancesynapseformationbyneuroglialinteraction(23–25).Wehaveinitiallyidentifiedactivity-dependentneurotrophicfactor (23)andbymolecularcloning,structure,andfunction(neuroprotection) analysiscoupledtoareductionistapproachofsmallactivepeptidesidentifiedADNPanditsinvestigationaldrug,davunetide(NAP)(1).Byfurtherstructuralanalysis,wehaveidentifiedADNP2,whichpartlymimics ADNP,butdoesnotcontaintheNAP(davunetide)motif(2).Together,this workconnectsstructurepredictions(currentlyawardedtheNobelprize

Figure1. ADNPlinearstructurehighlightingmultipleproteininteractionsitesandpresumptiveadditionaldomainsoftwodifferentframeshiftmutations.(A) ADNPlinearstructure,highlightingproteininteractiondomains.Thestructurewasassembledbasedonourpreviouspublications(1–7, 14, 16, 17, 28–33).(B) Ontheleft,thepotentialadditionaldomainsaddedforADNPwiththemutationp.Arg730Thrfs∗ 5inthesequence725-733aaaredisplayed.Ontheright,the domainspresentinthecompatiblewild-typeADNPsequence725-733aacanbefound.(C)Ontheleft,thepotentialadditionaldomainsaddedforADNPwith themutationADNP,p.ADNP_Glu931Glyfs∗ 12inthesequence926-941aaaredisplayed.Ontheright,thedomainspresentinthecompatiblewild-typeADNP sequence926-941aacanbefound.

inchemistry(26, 27))tofunctionalbehaviorimpactingourdailylivesand illuminatingthebeautyofgenomicpsychiatry.

Results

TheADNPp.Arg730Thrfs∗ 5FrameShift deNovo MutationDoesnot DuplicateaKnownADNPMotif

Figure1A showsselectedmolecularinteractionmotifsonADNPindicatingmultiplepartnersandinvolvementinkeycellularpathways.A mostprevalentpathogenicmutationintheADNPsyndromeisp.Arg730∗ (34, 35).Here,achildpresentingdevelopmentaldelayswassubjectedtowholeexomesequencingdiscoveringauniqueADNPmutation p.Arg730Thrfs∗ 5(Figure1B).Theadditionalaminoacidsinthiscaseare associatedwithproteasomalturnover(DEG_Cend_FEM1AC_1)aswellas neuropilin(LIG_NRP_CendR_1),whichislinkedtoprotectionagainstviralinfection(Figure1B).Thepotentialadditionaldomains,whilenotdirectlyfoundinthenativeADNPsequence,arelinkedtoADNP’sfunction, withADNPsyndromechildrenbeingmoresusceptibletoinfectionswith slowerrecuperationtime(34)andwithADNPregulatedgeneslinkedwith theproteasomalsystem(35).

PotentiallyAcceleratedMutatedProteinDegradation(http://elm.eu. org/ ). Morespecifically,regardingtheadditional DEG_Cend_FEM1AC_1 motif:“C-degronsplayvitalfunctionsintargetingreceptorsofseveral cullin-RINGE3ligasecomplexes(CRLs)toinitiateproteindegradation. FEM1proteins,includingFEM1A/B/C,actasthereceptorstospecifically recognizeCdegronsendingwitharginine(Arg(R)/C-degron)toenable CRL2-mediatedproteasomalturnover.Cul2ligasecomplexesareresponsiblefortargetingsubstrateswitharginineastheirC-terminalresidue (36)withsomeoftheknownsubstrateshavinganativeC-terminiendinginArg(R).”Indeed,inHBcase,aC-terminalRhasbeenaddedbecause oftheframeshiftmutation.

PotentialProtectionAgainstViralInfection(http://elm.eu.org/ ). Regardingthe LIG_NRP_CendR_1 –“CendRMotifBindingtoNeuropilinReceptors,neuropilins(NRPs)arevitalmultifunctionalcellsurfacereceptorsplayingimportantrolesinvariouscellularsignalingpathwaysthat includeVEGF-dependentvascularpermeability,semaphorin-dependent

axonguidance,angiogenesis,immunity,cellsurvival,migration,andinvasion.NRPsspecificallyrecognizeaC-terminalmotif,sometimesata polybasicFurincleavagesite,knownastheCendRmotif.PhysiologicalligandssuchasVEGF-165andsemaphorin3Ainteractwiththeb1domain ofNRP1andpromotecellularinternalization.SeveralvirusessuchasEBV, HTLV-1,andLujoalsouseNRP1forcellularentry.RecentlyNRP1hasbeen identifiedasanentrypointofSARS-CoV-2viaaFurin-generatedCendR motifpresentintheviralS1protein.LossofNRPfunctionresultsinsignificantcardiovascularandneuronalphenotypesandisalsoassociated withembryoniclethality.Thus,NRPsplaycriticalrolesinbothphysiologicalandpathologicalcontextsandarepotentialtherapeutictargetsfor viralinfection.”Withperipheralandcirculating(37)ADNPdirectlylinked tospleenproteinexpression(4)andimmuneresponse(38),thesefindingsareoffurtherinterest.

ADNPGlu931Glyfs∗ 12InheritedMutationContainsAdditional14-3-3 BindingSite(NuclearCytoplasmShuttle)andaPhosphotyrosineBinding Domain–TRAIL(TNF-RelatedApoptosisInducingLigand) FouridentifiedproteinmotifsaddedtoADNPbecauseofthe Glu931Glyfs∗ 12frame-shift-STOPmutation(Figure1C)areofinterest, asfollows. LIG_MSH2_SHIPbox_1,mismatchrepaircontributingtothe overallfidelityofDNAreplication;N-linkedglycosylation(MOD_N-GLC_1) isaco-translationalprocessinvolvingthetransferofaoligosaccharide chaintoasparagineresidueintheprotein;14-3-3proteinassociation (LIG_14_3_3_CanoR_1),with14-3-3formerlyfoundtobeinvolvedin ADNPnuclear-cytoplasmicshuttling(28);andaPTB_Apo_2Phosphotyrosinebinding(LIG_PTB_Apo_2)domainsrecognizingshortpeptideswith acoreAsn-X-X-Tyr.Theclassicalphosphotyrosinebinding(PTB)domains bindthemotifwhenitisphosphorylatedontheTyrresidue(Figure1C). Importantly,thelasttwoaddedmotifsareadditionaltosimilarinternal ADNPmotifs(Figures2 and 3).

Concentratingon LIG_14_3_3_CanoR_1 Associatingwiththe14-3-3Proteins,aFamilyofConservedRegulatoryMoleculesThatareInvolved inDiverseCellularProcessesThroughtheInteractionwithHundredsof DifferentProteins(http://elm.eu.org/ ). “Inmammals,sevenisoforms arepresent.14-3-3proteinsformeitherhomo-orheterodimersthat

Figure2. ADNPGlu931Glyfs∗ 12interactionwith14_3_3andwithanSH3domain.(A)TheresultsofdockingADNP_Glu931Glyfs12(lightorange)tothe14-3-3 protein(PDBcode3IQJ,darkpurple)inthe936-941aminoacidregion(red)—oneofthe14-3-3bindingsiteswithinADNP_Glu931Glyfs12—presentedhere.The dockingresultsindicatethattheinternalNAP(cyan)interactswiththe14-3-3proteinthroughresidues354and361.(A1)Toevaluatetheaccuracyof thedocking results,threemeasureswereused.Thedockingscoreisashape-basedpairwisescoringfunction,wherealowervalueindicatesbettershapecomplementarity betweenthetwodockingelements.Theconfidencescorereflectsthereliabilityofthepredictedbindingmode.Thisscoretypicallyrangesfrom0to1, withhigher valuesindicatinggreaterconfidenceinthepredictedinteraction.Finally,theLigandRMSDmeasuresthedeviationoftheprotein’spredictedpositionfromother models.AsmallerRMSDvalueisassociatedwithgreaterconfidence,asitsuggeststhatthecurrentmodelislikeotherdockingmodels,therebysupportingits validity.(B)TheresultsofdockingADNP_Glu931Glyfs12(lightorange)tothe14-3-3protein(PDBcode3IQJ,darkpurple)inthe391-395aminoacidregion (red)—oneofthe14-3-3bindingsiteswithinADNP_Glu931Glyfs12—presentedhere.ThedockingresultsindicatethattheinternalNAP(cyan)doesnot interact withthe14-3-3protein.(B1)Threemeasureswereusedtoevaluatethedockingin Figure2B:thedockingscore,theconfidencescore,andtheligandRMSD,as explainedinA1.(C)TheresultsofdockingADNPwildtype(lightorange)tothe14-3-3protein(PDBcode3IQJ,darkpurple)inthe391-395aminoacidregion (red)—oneofthe14-3-3bindingsiteswithinADNPwildtype—presentedhere.ThedockingresultsindicatethattheinternalNAP(cyan)interactswith 14-3-3 throughresidues354,356and359-361.(C1)Threemeasureswereusedasexplained(A1).(2D)TheresultsofdockingADNP_Glu931Glyfs12(lightorange)tothe 14-3-3protein(PDBcode3IQJ,darkpurple)inthe391-395aminoacidregion(red)—oneofthe14-3-3bindingsiteswithinADNP_Glu931Glyfs12—presented here.ThedockingresultsindicatethattheinternalNAP(cyan)doesnotinteractwiththe14-3-3protein.Inaddition,SH3domain(darkgreenisdockedtoADNP). (D1)Acloserlookatthedocking(D).(D2)ThreemeasureswereusedtoevaluatethedockingasA1.(E)TheresultsofdockingADNPwildtype(lightorange)to 14-3-3(PDBcode3IQJ,darkpurple)inthe391-395aminoacidregion(red)—oneofthe14-3-3bindingsiteswithinADNPwildtype—arepresentedhere.The dockingresultsindicatethattheinternalNAP(cyan)interactswiththe14-3-3proteinthroughresidues354,356and359-361.Inaddition,SH3domain(dark greenisdockedtoADNP).(E1)AcloserlookatthedockingisprovidedforE.(E2)Threemeasureswereusedtoevaluatethedockingin Figure2E asabove(A1).

targetcertainphosphoserine/threonine-containingmotifswithalow micromolaraffinity.Bindingtoasmallsetofunmodifiedproteinshas alsobeenreported.Phosphorylation-dependentand-independentbindingoccursviathesamedeepligand-bindinggroove.Therearecanonicalarginine-containingmotifsandanoncanonicalmotifgroupthatare difficulttoclassifybututilizeadditionalhydrophobicinteractions.The canonicalArg-containing14-3-3bindingpeptidesarephosphorylatedby membersofbasophilickinases.”

SpecificallyconcentratingonADNP–14-3-3proteinassociation (Figure2A–C)weshowedthattheadditionalADNPmutatedsite (aa936-941)interacted insilico withfurtherassociationofaa354,361 inNAPVSIPQ(spanningaa354-361inADNP, Figure2C).Inthe Glu931Glyfs∗ 12mutatedADNP,theinternalaa391-395/14-3-3interaction,didnotshowfurtherinteractionwithNAPaa(Figure2B),contrasting thecontrol/nonmutatedADNPshowingextensiveinteractionwithNAP aa354,356,359-361.Themostfavorabledockingscorewaswiththe internalsiteonthemutatedADNP( 254.02)(Figure2A1),withsecond bestbeingontheadditionalsiteinthemutatedADNP(Figure2A2), andtheleastfavorableonthenativeADNP(Figure2A3).WithSH3

domainsinteractingwiththeNAPsequence,dockingSH3togetherwith 14-3-3resultedinamuchfavorableinteractionwiththemutatedprotein internalinteractionsite( 255.51)(Figure2D,D1,D2)versus( 198.6) forthenativeprotein,includingamoreextensiveNAPinvolvement (Figure2E,E1,E2).

Focusingon LIG_PTB_Apo_2,PTBDomainsRecognizingShortPeptides withaCoreAsn-X-X-TyrMotifPrecededbyaShortPeptideSegmentThat DocksbyBetaAugmentation(http://elm.eu.org/ ). “TheclassicalPTB domainsbindthemotifwhenitisphosphorylatedontheTyrresidue.HoweverotherPTBsrecognizeessentiallythesamemotifwhenunmodified.”

FurtherfocusingonPTBandADNPalsoresultedinamuch-preferred mutatedproteininteractionwithPTB(Figure3, 264.85vs. 133.06). Similarly,theadditionalsiteshowedpreferredinteraction 268.88. Glu931Glyfs∗ 12isaProtectiveMutationandp.Arg730Thrfs∗ 5Maybe LessDeleteriousthanp.Arg730∗ Vineland3analysisindicatedthatVB(aged43years)Vineland3standardscores(SS)wereaboveaverageintermsofperformance,withSS ofcommunicationdomainof106,dailylivingskillsof107,socialization

Figure3. DockingofADNP_Glu931Glyfs12tothePTBprotein.(A)TheresultsofdockingADNP_Glu931Glyfs12(lightorange)tothePTBprotein(PDBcode 3DXC,navyblue)inthe934-941aminoacidregion(red)—oneofthePTBbindingsiteswithinADNP_Glu931Glyfs12—arepresentedhere.Thedockingresults indicatethattheinternalNAP(cyan)interactswiththePTBproteinthroughresidue354.(A1)Threemeasureswereusedtoevaluatethedockingasexplainedin Figure2A1.(B)TheresultsofdockingADNP_Glu931Glyfs12(lightorange)tothePTBprotein(PDBcode3DXC,navyblue)inthe450-457aminoacidregion(red)— oneofthePTBbindingsiteswithinADNP_Glu931Glyfs12—arepresentedhere.ThedockingresultsindicatethattheinternalNAP(cyan)interactswith thePTB proteinthroughresidues354-357and361.(B1)Threemeasureswereusedtoevaluatethedockingasin Figure2A1.(C)DockingADNPwildtype(lightorange) tothePTBprotein(PDBcode3DXC,navyblue)inthe450-457aminoacidregion(red)—oneofthePTBbindingsiteswithinADNPwildtype—arepresented.The dockingresultsindicatethattheinternalNAP(cyan)doesnotinteractwiththePTBprotein.(C1)Asabove,threemeasureswereusedtoevaluatethedockingin Casexplainedin Figure2A1

domainof107,andtheAdaptiveBehaviorComposite(ABC)of108. Figure4 showsHBresults(aged8years),withSSaveraging70–80,much higherthanscoresobservedbeforeforADNPsyndrome(21),(averaging 35–53for10ADNPsyndromerandomizedindividualswithsometested longitudinally).Regardless,whenusingtheRandomForestalgorithm (withWeka3.8.6)toclassifythetwoinstancesofHBandVB,basedonthe Vineland3datapresentedabove(21)andcontrolsetofanequalnumber ofinstanceswithvaluesrandomlygeneratedbetween85and115,the resultsclearlyclassifiedthemother(VB)asnormal,andthechild(HB)as havingADNPmutationdeficits,albeitwithinthehigherperformingADNP syndromeindividuals.

Discussion

WediscoveredherecorrelationsbetweenADNPstructureandfunction ontheadaptivebehaviorscalelookingatyetundescribedADNPmutations.Ourpaperunderscorestheimportanceofdivingintoprecisedetails.Assuch,VB’sinheritedmutation,ADNPp.Glu931Glyfs∗ 12,showsa Vineland3SSslightlyaboveaverageresultsof100,whileourprevious study(21)showed50–60SSresultsforachildwithanADNPmutation

atp.955Argfs∗ 36.Thus,theinheritedADNPp.Glu931Glyfs∗ 12isapparentlyprotectiveagainstthepotentialp.Arg730Thrfs∗ 5effects,possibly throughtheacquiredinteractionwith14-3-3,importantforADNPcytoplasmiclocalization(28).

Indeed,HBmutationanalysisidentifiedthepathologicalmutation, p.Arg730Thrfs∗ 5.Interestingly,anothervariationoftheADNPsyndrome prevalentp.Arg730∗ mutation,wasalsofoundinpostmortemAlzheimer’s diseasebrains,namely,p.Arg730Thrfs∗ 4withthissomaticmutationfrequencycorrelatedtoBraakstage(tauopathy)andaging(6).Interestingly thosemutationspresentalostcaspasebindingsiteon(ADNPaa733-738) (39).However,aselaboratedinthe Results,thep.Arg730Thrfs∗ 5contains aC-terminalArg(R)servingasaproteindegradationsignal(36).Indeed, theacquiredC-terminalRcouldcontributetotherapideliminationofthe mutatedprotein.WithfurtherADNPautocrineregulationofitsexpression (29),thiscouldresultinincreasedADNPp.Glu931Glyfs∗ 12,encompassing theapparentbetterclinicaloutcomes.

Tofurtherputfindingsintocontext,wehavepreviouslyreviewedthe ADNPliteraturediscussinggenotype/phenotypecorrelations(19, 40), expandingonskin-relatedabnormalities(40).Here,summarizingnew

Figure4. Vinlandadaptivebehaviorquestionnaire.ThisfigurepresentstheresultsofthecomprehensiveversionoftheVinelandAdaptiveBehaviorquestionnaire forHB(8-year-old)includingthatoftheAEscores,theGSVscoresbothforthedifferentsubdomainsofthisquestionnaireaswellastheSSforthedifferent domainsofthequestionnairealongwiththeSSfortheABC.Confidenceintervalwastakenatthelevelof90%.Noticethatallstandardscorevaluesareabove 70whiletheageequivalentinthedomesticsubdomainisevenabovethatofachildof8years. ResearchReport Gozesetal.

discoveriesin2024,wewouldliketohighlightaphenotypeofnarrow eyeopenings,droopyeyelids,andfoldsofskinontheinnerpartofthe eyesgoingfromthebottomtotheuppercornercalledblepharophimosisandassociatedwithpathologicalproteintruncatingmutationsaround thenuclearlocalizationsignalofADNP,comparedtoothertruncatingmutations(41).AnotherstructureandfunctionmutationinADNP,namely, (Gln423Serf∗ 17)exhibitsrenalabnormalitiesandpolycysticovariandisease,whichmaybespecificallyassociatedwiththisvariant(42).Indeed, theadditionalaminoacidsequencehereisSVQFQTCCSCHRPSPR,which includesthesequenceSVQF,interactingwithcaspasesassociatedwith tissuewell-being(ELME000285),forexample,ref. 39

Moreover,thegeneticbackgroundaswellasenvironmentaleffects areinstrumentalinthemanifestationoftheADNPmutationphenotype. Forexample,modelsofzebrafishofdifferentstrains(43),mayshowvariablephenotypicoutcomes(44, 45),aswellasdifferentialsusceptibilityto environmentalstress(45),furthercapitulatedinmousemodelsofADNP deficiency(4, 46).

Importantly,additionalstudylimitationsincludeinterventionsmethods,whichmayacceleratedevelopmentandcontributetotheapparently improvedoutcomeintheVinelandquestionnaireresults.Inthecaseof HB,giventhefactthattheADNPsyndromegenotypeisassociatedwith microbiomealteration(4, 47),afathersonfecaltransferwasperformed, whichresultedinanapparentimprovement(VB,personalcommunication).Regardless,itshouldbekeptinmindthatthisisacasestudyof uniquemutationswithworkfocusingonstructureandfunctionrelations intheclinicalscenario.Futurestudiesshouldaimatbiochemical,cellular, andanimalmodeling,gainingfurtherinsightsandbetterunderstanding.

Thefindingsfromthisstudyandrelatedstructural/functionalanalyses(refs. 9, 12, 21)areparticularlysignificantfortherapeuticdevelopmentbecauseintellectualdisabilityrepresentsamajorchallengein ADNPpatients,bothchildrenandadults.Thismakesthedemonstrated effectsofdavunetide(NAP)–notablyitsabilitytoboostmemoryandprotectdailyfunctionalactivitiesinmiceandhumanswithADNP-regulated pathology(refs. 16, 18, 19, 48–50)–especiallypromisingasatherapeuticintervention.Ofparticularinterestinthiscase,davunetide(NAP)has showncorrectiveeffectsonalteredADNPsyndrome-likegutmicrobiota compositionintwoindependentADNPmousemodels(4, 47),correlating withbehavioralimprovement,whichareofdirecttranslationalefficiency inHB’scase,enjoyingthebenefitsoffecaltransfer,andmuchbeyond.For moreinformation,fromapersonalperspective,pleaseseeanoverviewof ADNPanddavunetidediscoveryanddevelopment(51).

MaterialsandMethods

Subjects

Twosubjectsarediscussedhere,amother(VB)withaninheritedmutationADNPp.Glu931Glyfs∗ 12,thatis,ADNP921SESEEKLDQKGWFKIRN YSFD∗ 942(singleaminoacidcode)versuscontrol,921SESEEKLDQKEDGS KYETIH939andan8-year-oldson(HB)withtheinheritedmutationas wellasa denovo mutation,ADNPp.Arg730Thrfs∗ 5,thatis,721QMEFP LLKKTKVR∗ 737,versuscontrol,721QMEFPLLKKRKLDDDSDSPS740.

InSilico Modeling

Weusedtheeukaryoticlinearmotif(ELM)resourceforassessementof functionality(52).

I-TASSER(https://zhanggroup.org/I-TASSER)wasusedforprotein structuremodelingandHDOCK(http://hdock.phys.hust.edu.cn/)was usedfor insilico protein/proteindocking. PyMOLsoftwarewasusedtocreatefigures.

Vineland3Questionnaire

TheVineland3(53)isastandardizedmeasureofadaptivebehavior— describingwhatsubjectsactuallydotofunctionintheireverydaylives. Aprecisedescriptionoftheassessmentanddataanalysiswasdescribed previously(21, 22).Inshort,thisquestionnaireincludesseveraldomains asfollows.1)TheCommunicationdomaincontainsthreesubdomains:receptive,expressiveandwritten.2)TheDailyLivingSkillsdomaincontainingthreesubdomains:personal,domestic,andcommunity.3)TheSocializationdomaincontainsthreesubdomains:interpersonalrelationships, playandleisureandcopingskills.4)TheMotorSkillsdomaincontaining

twosubdomains:GrossmotorandFinemotor.Thisdomainisnormedonly throughtheageof9years.

Theassessmentsofadaptivebehaviorswerecarriedoutinaquiet comfortableplacebyinterviewingtheparentsusingtheZoomplatform. Individualswereassessedatthreelevels(1–3,below)asperthedefinitionsofthecomprehensiveversionoftheVinelandAdaptiveBehavior Scales.

1)SSsprovideastandardizedmeasureoftheindividual’sadaptivebehaviorrelativetotheirsame-agedpeers.TheVineland3questionnaireusesaSSrangeof1–160,withameanscoreof100anda standarddeviationof15.ASSof100isconsideredaverage,with scoresbelow70indicatingsignificantdeficitsinadaptivebehavior.

2)GrowthScaleValues(GSV)aredesignedtomeasurechangeover time.Likearawscore,theGSVscoreisanindicatorofabsolute, notrelative,performance.

3)Age-equivalent(AE)scoresrepresenttheageatwhichtheindividual’sadaptivebehavioristypicallyobserved.Itisimportanttonote thatAEscoresshouldnotbeinterpretedastheindividual’sdevelopmentalageorintellectualability.

ItisimportanttointerpretbothAEsandSSstogether,astheyprovidedifferentbutcomplementaryinformationaboutanindividual’sadaptivebehavior.WhileAEsgiveageneralsenseoftheindividual’sadaptive functioningindifferentsubdomains,SSsofferamoreprecisemeasureof theindividualstrengthsandweaknessescomparedtohis/hersame-aged peersinthedifferentdomainsaswellasanABC,whichisbasedonthe SSsforthreespecificadaptivebehaviordomains:Communication,Daily LivingSkills,andSocialization.

GraphPadPrism7.0softwareincludedD’AgostinoandPearsontestfor normalitycoupledwithlog10transformationtoachievenormaldistribution.

RandomForestAlgorithm(withWeka3.8.6)

Giventhesmallsamplesize,RandomForestusingbootstrapping,aspart ofitsensemblelearningprocess,wasapplied.Inarandomforest,multiple decisiontreesaretrainedondifferentsubsetsofthetrainingdata.These subsetsarecreatedusingbootstrapping,whichinvolvesrandomlysamplingthedatawithreplacement.Thismeansthatsomesamplesmaybe repeatedinasubset,whileothersmaybeleftout.Thistechniquehelps toincreasethediversityamongthetrees,whichcanimprovetheoverallperformanceofthemodel.Thus,tofurthercompareHBtothenormal (SS85-115)versustheADNPsyndromepopulation,arandomlyselected previousdatawereused(21)alongsidewithacontrolsetofanequal numberofinstanceswithSSvaluesrandomlygeneratedbetween85 and115.

Acknowledgments

WethanktheVBfortheVinelandinterviewandforsharingtheprecise mutations.WethankJasonBlattandMaramGanaiemfortheirhelpwith Figure4 generation.

AuthorContributions

I.G.initiatedandledthestudy.E.G.mappedtheframeshiftadditional sequences.S.S.providedthemolecularmodelsinconsultationwithI.G., Y.L.implementedtheVinelandquestionnaire.I.G.wrotethepaperwith editorialinputsfromallauthors.

FundingSources

Thisstudywaspartiallysupportedbygrantsanddonationstoresearch conductedatthelaboratoryofI.G.fromDr.RonithandDr.ArmandStemmer(FrenchFriendsofTelAvivUniversity)andAnneandAlexCohen (CanadianFriendsofTelAvivUniversity).I.G.isDirectoroftheEltonLaboratoryforMolecularNeuroendocrinologyandtheformerfirstincumbentoftheLilyandAvrahamGildorChairfortheInvestigationofGrowth Factors.

AuthorDisclosures

I.G.servesasVPDrugDevelopmentatExonavisTherapeuticsLtd.DevelopingdavunetidefortheADNPsyndrome(underpatentprotection,I.G. inventor).Othercontributorshaveconfirmedthatnoconflictofinterest exists.Themanuscripthasbeenreadandapprovedbyallauthors.

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