Gene clustering analysis in human osteoporosis disease and modifications of the jawbone

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Gene

clustering

analysis

in

human

osteoporosis

disease

and

modifications

of

the

jawbone

Paolo

Toti

a,1

,

Carolina

Sbordone

b

,

Ranieri

Martuscelli

c

,

Luigi

Califano

b

,

Luca

Ramaglia

d

,

Ludovico

Sbordone

e,2,

*

a

PrivatePractice,ViaProvinciale87B,55041Camaiore(Lucca),Italy

b

ChairofMaxillo-FacialSurgery,SchoolofMedicine,UniversityofNaples‘‘FedericoII’’,ViaS.Pansini5,80100Naples,Italy

c_Clinical_Department_of_{Odontostomatology}_and_{Maxillo-Facial}_Surgery,_School_of_Medicine,_University_of_Naples_{‘‘Federico}_II’’,_Via_S._Pansini

5,80100Naples,Italy

d_Chair _of_Oral_Surgery,_Department_of_{Neurosciences,}_Reproductive_and_{Odontostomatological}_Sciences, _School_of_Medicine,_University_of

Naples‘‘FedericoII’’,ViaS.Pansini5,80131Naples,Italy

e

ChairofOdontostomatologicalDiseases,DepartmentofMedicineandSurgery,SchoolofMedicine,UniversityofSalerno,ViaS.Allende, 84081Baronissi(Salerno),Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Accepted28February2013 Keywords: Computationalbiology Genomics Bonerepair Boneremodelling Boneresorption Osteoporosis Maxilla Mandible

a

b

s

t

r

a

c

t

Objective: Ananalysisofthegenesinvolvedinbothosteoporosisandmodificationsofthe jawbone,throughtextmining, usingawebsearchtool,ofinformationregardinggene/ proteininteraction.

Design: The final set of genesinvolved in the present phenomenon was obtained by expansion-filtering loop. Using a web-available software (STRING), interactions among all genesweresearched for,and aclusteringprocedure wasperformedin whichonly high-confidencepredictedassociationswereconsidered.

Results: Twohundredforty-twogenespotentiallyinvolvedinosteoporosisandin modifica-tionsofthejawbonewererecorded.Seven‘‘leadergenes’’wereidentified(CTNNB1,IL1B, IL6,JUN,RUNX2,SPP1,TGFB1),whileanother10genesformedtheclusterBgroup(BMP2, BMP7,COL1A1,ICAM1,IGF1,IL10,MMP9,NFKB1,TNFSF11,VEGFA).Ninety-eightgeneshad nointeractions,andweredefinedas‘‘orphangenes’’.

Conclusions:Theexpansionofknowledgeregardingthemolecularbasiscausing osteopo-rotictraitshasbeenbroughtaboutwiththehelpofadenovoidentification,basedonthe data mining of genesinvolved in osteoporosis and inmodification ofthe jawbone. A comparisonofthepresentdata,inwhichnorolewasverifiedfor98genesthathadbeen previouslysupposedtohavearole,withthatoftheliterature,inwhichanother81genes,as obtainedfromGWASreviewsandmeta-analyses,appearedtobestronglyassociatedwith osteoporosis,probablyatteststoalackofinformationonosteoporoticdisease.

#₂₀₁₃_Elsevier_Ltd._All_rights_reserved.

*Correspondingauthor.Tel.:+393296332655;fax:+39089969642.

E-mailaddresses:lsbordone@unisa.it,ludovico.sbordone@med.unipi.it(L.Sbordone).

1 _Formerly_at_Department_of_Surgery,_University_of_Pisa,_Via_Paradisa_2,₅₆₁₂₄_Pisa,_Italy.

2 _Formerly_at_Implantology_and_{Periodontology,}_Department_of_Surgery, _University_of_Pisa,_Pisa,_Italy;_Complex_Operating_Unit_of

OdontostomatologyandImplantology,AziendaOspedaliero-UniversitariaPisana,Pisa,Italy.

Available

online

at

www.sciencedirect.com

journalhomepage:http://www.elsevier.com/locate/aob

0003–9969/$–seefrontmatter#₂₀₁₃_Elsevier_Ltd._All_rights_reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.02.013

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1. Introduction

Generally,osteoporosisisdiagnosedwhenadecreaseinbone massand bonequality,manifesting itselfasbone fragility, maylead toan increasedpropensity tofractures resulting fromminimaltrauma.1

Evenifsomeformsofosteoporosishavebeenclassifiedas primary, divided into ‘‘post-menopausal’’, or type I, and ‘‘senile’’,ortypeII,andassecondary,manifestingthemselves throughspecificsignsandaddedsymptomsstemmingfrom furthercauses,2,3 _osteoporotic _patients_have_been _generally screenedandfollowedthroughameasurementoftheirbone mineral density (BMD), which is widely considered as an indicatoroftheriskfactorforfracture(inconjunctionwithage andgender),andwhichhasorientedpreventivetherapeutic decisions.4,5

Thepreventionoffracturesobtainedthroughanincrease inboneformation,togetherwithinhibitionofboneresorption, istheultimateaimoftherapy;severalstrategiesareemployed, frommodificationofdietandlifestyle,6_to_the_utilization_of medications,7,8_each_of_which_may_be_quite_effective,_but_also associatedwithsevereside-effects.9_Moreover,_some unrelat-edBMDfactorsmayplayanimportantroleduetothefactthat mostindividualswithoutBMD-definedosteoporosisdevelop osteoporoticfractures.1

Despitethecommongeneticvariations,probablyeachof thethousandsofgeneticvariantsoutofapoolofthousands ofgenesinfluencesthe measuredBMDinan infinitesimal way.Experimental explorationsof raregenetic variations, helpful in improving our overall understanding of the pathophysiology and pharmacology necessary to prevent osteoporotic fractures, were started and developed using different genetic approaches, such as linkage analysis, candidate and genome-wide association studies (CGAS and GWAS, respectively), genome-wide sequencing and functionalstudies.10–12

Thejawisthesecondarytargetofosteoporosis,evenifthe basalpartofthejawremainsmoreintact,especiallyinthe malemandible,due toits densercorticalbase.So alveolar ridges and maxillary basal parts are mainly exposed to resorption,dueto thehigh percentageofcancellous struc-ture.13–15_Alveolar_ridge_resorption_can_be_induced_by_several metabolicbonediseases,suchasvitaminD-resistantrickets,16

primaryandsecondaryhyperparathyroidism,17,18_and_Paget’s disease.19,20

Forpatientswhohaveexperiencedanintenseprocessof resorption ofthe edentulous alveolarridge, an established rehabilitation strategy is that of bone reconstruction with successive insertionsofdental implants thatsupport fixed prostheses.Osteoporosisisnotanabsolutecontraindication toprosthetic-implanttherapy,althoughtheincreasedriskof fracture advises for the elimination of the risk factors associated with it.21 _Topic _areas _regarding _{osteoporosis,}_as summarizedbyErdoganetal.21_,_have_been_defined,_and_are listed as follows: changes in alveolar bone morphology in osteoporosis, bone repair and turnover in osteoporosis, alveolar bone regeneration/augmentation associated with low bonedensity, patients undergoingridge augmentation procedures.

Through aclusteranalysisofthegenesinvolved inthe present phenomenon, it was possible to identify certain genes (the so-called‘‘leader’’ genes), which can be safely assumedtoplaythemostessentialroleintheprocessasyet performedforotheranalyses.22–25_An_enormous_quantity_of data, including tens of thousands of simultaneous tran-scripts, had to be carefully sifted through in the highly complex analysis required in classical microarray experi-ments:theinformationobtainedregardingtheinvolvedsetof genescouldthenbeemployedforthedesign,analysis,and interpretation of microarray experiments that could be specificallytargeted,inwhichthe transcriptsamountedto onlyafewhundred.

Theprimaryaimofthisstudywasadenovoidentification ofrelevantgenesinosteoporosis,inparticularthoselinkedto proceduresofboneaugmentation.Oursecondaryaimwasto carryoutacomparisonbetweenresultingprimaeval informa-tionregardingtheobtainedfinalsetofgenesandthenewly putativegenessupposedasbeingassociatedwith osteoporo-sis,asresultedfromseveralgeneticanalyticalapproaches.

2. Materials

and

methods

2.1. Genesinvolvedinhumanosteoporosis

Abioinformatic/statisticalalgorithm,lesscomplexthanthat seeninthepristineanalysis,23_has_been_employed_and_utilized

Table1–Thenumbersoftheresultingintroductorysetofgenessortedbydifferentdatabasesused.

Database Researchfield Resultedgenes

(number)

GenBank Acollectionofpubliclyavailableannotatednucleotidesequences,includingmRNA sequences

withcodingregions,segmentsofgenomicDNAwithasinglegeneormultiplegenes,and ribosomalRNAgeneclusters

109

OMIM Acomprehensive,authoritative,andtimelycompendiumofhumangenesandgenetic phenotypes

65 PubMed AccesstoallcitationsfromMEDLINEdatabaseandadditionallifesciencesjournals 188 Genatlas Containsrelevantinformationwithrespecttogenemappingandgeneticdiseases 15 Kegg Databaseofbiologicalsystems,consistingofgeneticbuildingblocksofgenesandproteins

(KEGGGENES)

5

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Fig.1–Dataanalysisforosteoporosisprocess:(a)Finalmapofinteractionsof242genesinvolvedinthepresent

phenomenonaccordingtoSTRING.LeadergenesandclusterBgenesarered.Thelinesthatconnectsinglegenesrepresent predictedfunctionalassociationsamongproteins,withahighdegreeofconfidence;(b)genesbelongingtotheleader clusterindifferentk-meanclusteringexperimentswithanincreasingnumberofclusters;(c)plotofthegapstatisticmethod forestimatingthenumberofclusters ˆk;(d)WNLforgenesinvolvedintheprocess.OpencirclesareclusterBgenes.Grey

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inthepresentstudy.24,25_This_approximate_clustering_analysis wasmadeupofseveralsteps,andforeachofthemadifferent softwareprogramwasused.

Thefirststepwastheestablishmentofanintroductoryset of genes by using the web-available engine Entrez (www.ncbi.nlm.nih.gov/sites/gquery, thatwas linkedtothe following list of integrated experiment cross-databases: PubMed,Genbank,Kegg,Omim,Genatlas),andviascanning ofthecommerciallyavailableDNA-microarraygenelists,as reportedinTable1.

Thesearchstrategyincludedthefollowingpertinentkey words,obtainedfromareviewpaperdealingwith osteoporo-sisandmodificationsofthejawbone,21combinedusingproper booleanlogicoperators(AND,OR,NOT)foreachsearchinthe above-mentioneddatabases:

(1) Gene,human,osteoporosis; (2) Bonechanges;

(3) Bonealveolarregeneration; (4) Bonealveolaraugmentation; (5) Ridgeregenerationprocedure; (6) Ridgeaugmentationprocedure; (7) 2OR3OR4OR5OR6 (8) repair; (9) turnover; (10)resorption; (11)remodelling; (12)lowdensity; (13)8OR9OR10OR11OR12

(14)exclusionsearchstrategy:NOTcancer (15)1AND7AND13AND14

Onceanewgenewasobtained,itsnamewasverifiedby means oftheofficialHumanGenomeOrganization (HUGO) GeneNomenclatureCommittee,orHGNC(availableathttp:// www.genenames.org/),and the approvedgene symbolwas applied erasing previous symbols or aliases. In the second step,thelistofgeneswascompleted;asalreadydescribed,25 the new gene dataset was expanded with web-available softwareSTRING(SearchToolfortheRetrievalofInteracting Genes/Proteins:version8.3availableathttp://string-db.org/), then false positives were filteredby a further searchwith PubMed.Consecutiveexpansion-filteringloopswerestopped attheachievementoftheconvergence,thatiswhennonew gene, potentially involved in the present process, was identified.Beforefurtheranalysis,thenameofeachgeneof thefinalconvergencesetinvolvedinosteoporosisprocesses wasnewlychanged,whennecessary,applyingthatusedinthe STRINGsoftware.

2.2. Identificationofleadergenes

Foreachgene(1,2,3,...)oftheoverallfinalconvergenceset, STRINGpresentedallPredictedFunctionalPartners(a,b,x, ...);theSTRINGScoresrepresentedthelevelofconfidenceof thecombinedpredictedassociationsforcouples1a,1b,1x, ...Foreachgeneofthefinalconvergenceset,onlycouples forwhichtherewasahighlevelofconfidence(resultswitha score0.9)wereconsidered,andthescoreswereextracted, allowing the obtaining of a numerical variable called ‘‘WeightedNumberofLinks’’(WNL),thatis,thearithmetical sum of the high level combined predicted association scores.

Fig.1.(Continued ).

circlesareclusterleadergenes.Squaresarethecentroidsoftheclustergroups;Mapofinteractionforgenesinvolvedin osteoporosis,sortedbydifferentpathwaysaccordingtoSTRING,sothatleadergenesand/orclusterBgenes(bothinred) wereexposed.Thelinesthatconnectsinglegenesrepresentpredictedfunctionalassociationsamongproteins,withahigh degreeofconfidence:(e)oestrogenendocrine-andvitaminDendocrine-pathway;(f)canonicalWnt/b-cateninsignalling pathway;and(g)RANKL/RANK/OPGpathway.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderis referredtothewebversionofthisarticle.)

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Allgene-relateddatawereenteredintoamatrix laborato-ry,3allowingcalculationstobeperformedautomatically.A k-mean algorithmwas applied totheinput variableWNL,or ‘‘WeightedNumberofLinks’’,andapartitioningoftheoverall datasetofgenesintomutually-exclusiveclusterswas auto-maticallyperformed.

Aswas already described,a‘‘gapstatistic’’methodwas appliedtothesetofdataforestimatingtheoptimalnumberof clusters ˆk.25 _A _null _reference _{distribution,} _required _for _an accurate application of the gap statistic method, was generated, i.e. a uniform distribution throughout the data dimension, as described by Tibshirani et al.26_: _reference datasetswereobtainedbydrawingmsamples(m=number ofgenes),repeated5times.26_Comparisons_were_performed betweenthe logarithmicvalueofthe pooledwithin-cluster sum of squares around cluster means [log(Wk)] and its

logarithmic expectation according to the null reference distribution ofthe data for the clusters from2 to 16.The estimateofthenumberofclusterswasgivenbythevalueof ˆk forwhichlog(Wk)fellthefarthestfromthereferencecurve.26

Significant differences among WNLs of cluster groups obtained by the gap statistic method, were found by Kruskal–Wallis test (statistical significance at a level of a=0.01), verifying the accurate estimate ofthe number of clusters.

Thefirstcluster,constitutedbythegeneswiththehighest WNL, was named ‘‘leader’’, or A: so ‘‘leader genes’’ were assumed to play a leading role in each analyzed process. Accordingtotheirprominence,allotherrankedgeneclasses werenamedasB,C,D,andsoon,untilthelastgeneclass,in which genes that had no identified predicted associations (WNL=0)werecalled‘‘orphans’’.

Genescorecitations(gsc)werealsoevaluated,asdescribed inapreviouspaper,25bysearchingforcitationsinPubMed usingBooleanlogiconpertinentkeywordsinallintroduced combinations, as above described,with sequential scoring strategies:linearweightapproachwasapplied:

no impact-factor value restriction27 was applied to the searchforcitations;

nodiscriminationwasappliedbetweeninvitroorinvivo data;

increment of the gene score citation (gsc) by 1 for first citationinanarticle,obtained bythe searchforcitations regardingtheanalyzedprocessandpublishedinajournal citedintheJournalCitationReports;

incrementofthegenescorecitation(gsc)by0.5incaseofthe multiplereferences, aftertheinitial reference,that incre-ments gsc by 1, and published by the same group of researchers (at least two of the same researchers) on a specificgene.

Foreachoftheupperclustergenes(LeaderandBclass)a Specificity Score (SS) was assigned, computed as the ratio betweenthenumberofhighlevelpredictedinteractionsofthe presentanalysisandthetotalnumberofpredicted interac-tions with a higher level of confidence (results with a

score0.9) in the whole STRING database. The research concludedonDecember8,2011.

3. Results

Afteraninitialcanvassingusingthepresentedkeywords,199 genes potentially involved in osteoporosis disease were numbered. The searches carried out among the several databases employed are shown in Table 1. Repetitions of the same genes found indifferent databases were erased, leading,atthemomentoftheconvergence(four expansion-filteringloops),to242geneswhichconstitutedtheoverallfinal convergenceset.

TheSTRINGmapofinteractionsisrepresentedinFig.1Afor the present phenomenon. As the number of clusters increased, the k-mean clustering gave a number of genes belongingtotheleadercluster7(Fig.1B).The ˆknumberof clusters estimatedbythe ‘‘Gapstatistic’’methodwas9,as shown in Fig. 1C: excluding the leader- and the orphan-clusters,theotherclusterswerenamedB,C,andsoontoH. For the analyzed process, seven genes belonged to the clusterof‘‘leadergenes’’(Fig.1DandTable2),andresultswere validated by Kruskal–Wallis test, which computed statisti-cally-significantdifferencesamongdifferentclusters:leader genes,B,C,andsoon.Another10geneswereidentifiedinthe classBgroup.The‘‘leadergenes’’wereCTNNB1,IL1B,IL6,JUN, RUNX2, SPP1, TGFB1, while the class B gene group was constitutedbyBMP2,BMP7,COL1A1,ICAM1,IGF1,IL10,MMP9, NFKB1,TNFSF11,VEGFA.Table2presentsacronyms,official names,andintroducedvariablesoftheupperclustergenes. Theliteraturewascarefullyexaminedinordertodetermine whichgenesshowedhighergenescorecitations(gsc)(Table3). Among genes belonging tothe two highest clusters (leader genesandclusterBgenes),only5werewellknowntobelinked toosteoporosisandmodificationsofthejawbone(withavalue ofgsc>100).Amongupperclustergenes,allbelongingtoclass B,only4presentedrelativelylowvaluesofgenescorecitations (withavaluecloseto12):BMP7(11),IL10(10.5),ICAM1(10),and MMP9(5).ThespecificityscoresforeachleaderandclassBgene areshowninTable2.Inbrief,themeanspecificityscoreswere 20.4%and21.1%forleaderandclassBgenes,respectively.

InTable4ispresentedtheoverallsetofremaininglower clustergenes(uptoHcluster),withtherespectivegenenumber, acronyms,officialnames,andtheprimaryfunctionasreported bySTRING.The98genesthathadnointeractions,definedas ‘‘orphan genes’’, are rankedin Table 5, in whichthere are reportedafurther81genesrobustlyassociatedwithareduced BMDvalue(resultingfromseveralmeta-analysisandreviews), thathadnohighlevelcombinedpredictedassociationscores relatedtoanygenefromtheoverallfinalconvergenceset.

4. Discussion

The measurement of BMD is still the best predictor of osteoporoticfractures,althoughnotallindividualsexperience lossofBMDwithage;theaetiologyofosteoporosisisseentobe multifactorial andincombinationwithclinical riskfactors, whiletherateofbonelossseemstobegeneticallydetermined,

3 _{Bioinformatic} _and _Statistics _Toolboxes, _MatLab _7.0.1, _The

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Table3–Genesclassifiedaccordingtonumberofcitationsamong199genesspecificallyinvolvedintheosteoporosis phenomenon.

Genename Officialname Genescore

citations(gsc)

Interactioninthe analyzedphenomenon

Class

PTH Parathyroidhormoneprecursor >100 10 C

GH1 Somatotropinprecursor >100 2 G

TNFRSF11B Tumournecrosisfactorreceptorsuperfamily member11Bprecursor(osteoprotegerin)

>100 6 E

TNF Tumournecrosisfactorprecursor(TNF-a) >100 3 F

TNFSF11 Tumournecrosisfactorligandsuperfamily member11

>100 15 B

CYP19A1 CytochromeP45019A1 >100 1 H

IL6 Interleukin-6precursor >100 21 Leader

CA10 Carbonicanhydrase-relatedprotein10 >100 0 Orphan

VDR VitaminD3receptor >100 4 F

IGF1 Insulin-likegrowthfactorIAprecursor >100 17 B

ESR1 Oestrogenreceptor >100 8 D

TNFRSF11A Tumournecrosisfactorreceptorsuperfamily member11Aprecursor

>100 2 G

TGFB1 Transforminggrowthfactorbeta-1precursor >100 19 Leader

CALCR Calcitoninreceptorprecursor >100 2 G

BMP2 Bonemorphogeneticprotein2precursor >100 17 B

SHBG Sexhormone-bindingglobulinprecursor >100 1 H

Table2–LeadergenesandclassBgenesaccordingtoclusteringexperimentswithgenescorecitationsinPubMed,

numberofpredictedinteractions,andSpecificityScoreforosteoporoticphenomenon.

Genename Officialname Gene

number Genescore citations(n) Interactionsin theanalyzed phenomenon Interactionsin wholeSTRING database(n) Specificity score(%) Leadergenes

IL1B Interleukin-1betaprecursor 35 31.5 18 115 15.7

IL6 Interleukin-6precursor 137 340.5 21 142 14.8

JUN TranscriptionfactorAP-1 (activatorprotein1)

139 37.5 22 229 9.6

RUNX2 Runt-relatedtranscription factor2

141 78.5 18 55 32.7

SPP1 Osteopontinprecursor 187 87.5 19 45 42.2

TGFB1(CED) Transforminggrowthfactor beta-1precursor

201 146.5 19 110 17.3

‘CTNNB1’ Cateninbeta-1 208 38 19 181 10.5

ClassBgenes

BMP2 Bonemorphogeneticprotein 2precursor

31 108.5 17 75 22.6

BMP7 Bonemorphogeneticprotein 7precursor

64 11 14 47 29.8

COL1A1 Collagenalpha-1(I)chain precursor

81 85 14 58 24.1

ICAM1 Intercellularadhesion molecule1precursor

132 10 14 79 17.7

IGF1 Insulin-likegrowthfactor IAprecursor

133 229 17 125 13.6

IL10 Interleukin-10precursor 134 10.5 13 79 16.5

MMP9 Matrixmetalloproteinase-9 precursor

152 5 12 67 17.9

NFKB1 NuclearfactorNF-kappa-B p105subunit

158 28.5 15 130 11.5

RANKL(TNFSF11) Tumournecrosisfactorligand superfamilymember11

222 413 15 33 45.5

VEGFA Vascularendothelialgrowth factorAprecursor

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Table4–Alllowerclustergeneswithrespectivegenenumber,clustergroupandprimaryfunctionasreportedinSTRING.

Genename Gene

number

Officialname ProteinprimaryfunctioninSTRING Cluster

RHOA 33 Rashomologgenefamily,memberA Regulatesasignaltransductionpathwaylinking plasmamembranereceptorstotheassemblyof focaladhesionsandactinstressfibres

C

FADD 108 Fas(TNFRSF6)-associatedviadeath domain

Apoptoticadaptormoleculethatrecruitscaspase-8 orcaspase-10totheactivatedFas(CD95)orTNFR-1 receptors

C

IL1A 136 Interleukin1,alpha; Producedbyactivatedmacrophages,IL-1stimulates thymocyteproliferationbyinducingIL-2release, B-cellmaturationandproliferation,andfibroblast growthfactoractivity

C

PPARG 179 Peroxisomeproliferator-activated receptorgamma

Receptorthatbindsperoxisomeproliferatorssuchas hypolipidemicdrugsandfattyacids

C PTH 181 Parathyroidhormone PTHelevatescalciumlevelbydissolvingthesaltsin

boneandpreventingtheirrenalexcretion

C STAT1 203 Signaltransducerandactivatorof

transcription1

Signaltransducerandactivatoroftranscriptionthat mediatessignallingbyinterferons(IFNs)

C PTHLH 212 Parathyroidhormone-likehormone Neuroendocrinepeptidewhichisacriticalregulator

ofcellularandorgangrowth,development, migration,differentiationandsurvivalandof epithelialcalciumiontransport

C

TRADD 223 TNFRSF1A-associatedviadeath domain

AdaptermoleculeforTNFRSF1A/TNFR1that specificallyassociateswiththecytoplasmicdomain ofactivatedTNFRSF1A/TNFR1mediatingits interactionwithFADD

C

TRAF6 224 TNFreceptor-associatedfactor6 Adapterproteinandsignaltransducerthatlinks membersofthetumournecrosisfactorreceptor familytodifferentsignallingpathwaysby associationwiththereceptorcytoplasmicdomain andkinases

C

BMPR1B 30 Bonemorphogeneticproteinreceptor, typeIB

Onligandbinding,formsareceptorcomplex consistingoftwotypeIIandtwotypeI transmembraneserine/threoninekinases

D

WNT3A 36 Wingless-typeMMTVintegrationsite family,member3A

Ligandformembersofthefrizzledfamilyofseven transmembranereceptors

D

FZD1 37 Frizzledhomolog1 ReceptorforWntproteins D

LRP6 39 Lowdensitylipoprotein receptor-relatedprotein6

EssentialfortheWnt/betacateninsignalling pathway,probablybyactingasacoreceptor togetherwithFrizzledforWnt

D

ESRRG 41 Oestrogen-relatedreceptorgamma Orphanreceptorthatactsastranscriptionactivator intheabsenceofboundligand

D BMP4 65 Bonemorphogeneticprotein4 Inducescartilageandboneformation D COL2A1 83 Collagen,typeII,alpha1 TypeIIcollagenisspecificforcartilaginoustissues D CXCL10 89 Chemokine(C-X-Cmotif)ligand10 ChemotacticformonocytesandT-lymphocytes D

ESR1 103 Oestrogenreceptor1 Nuclearhormonereceptor D

FASLG 110 Fasligand(TNFsuperfamily,member 6)

CytokinethatbindstoTNFRSF6/FAS,areceptorthat transducestheapoptoticsignalintocells

D IRAK1 140 Interleukin-1receptor-associated

kinase1

BindstotheIL-1typeIreceptorfollowingIL-1 engagement,triggeringintracellularsignalling cascadesleadingtotranscriptionalup-regulation andmRNAstabilization

D

NFATC1 157 NuclearfactorofactivatedT-cells, cytoplasmic,calcineurin-dependent1;

Playsaroleintheinducibleexpressionofcytokine genesinT-cells,especiallyintheinductionofthe IL-2orIL-4genetranscription

D

NR3C1 167 Nuclearreceptorsubfamily3,groupC, member1

Receptorforglucocorticoids(GC) D SOX9 174 SRY(sexdeterminingregionY)-box9; Playsanimportantroleinthenormalskeletal

development

D SERPINE1 192 Serpinpeptidaseinhibitor,cladeE

(nexin,plasminogenactivator inhibitortype1),member1

Thisinhibitoractsas‘bait’fortissueplasminogen activator,urokinase,andproteinC

D

MEF2C 10 Myocyteenhancerfactor2C Transcriptionactivatorwhichbindsspecificallyto theMEF2elementpresentintheregulatoryregions ofmanymuscle-specificgenes

E

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Table4 (Continued)

Genename Gene

number

AR 59 Androgenreceptor Steroidhormonereceptorsareligand-activated transcriptionfactorsthatregulateeukaryoticgene expressionandaffectcellularproliferationand differentiationintargettissues

E

CD40LG 75 CD40ligand MediatesB-cellproliferationintheabsenceof co-stimulusaswellasIgEproductioninthe presenceofIL-4

E

COL1A2 82 Collagen,typeI,alpha2 TypeIcollagenisamemberofgroupIcollagen (fibrillarformingcollagen)

E FAS 109 Fas(TNFreceptorsuperfamily,

member6)

ReceptorforTNFSF6/FASLG E

HMOX1 129 Hemeoxygenase(decycling)1; Hemeoxygenasecleavesthehemeringatthealpha methenebridgetoformbiliverdin

E

MSX2 153 Mshhomeobox2; Probablemorphogeneticrole E

NOG 161 Noggin; Essentialforcartilagemorphogenesisandjoint

formation

E NOS3 162 Nitricoxidesynthase3(endothelial

cell);

Producesnitricoxide(NO)whichisimplicatedin vascularsmoothmusclerelaxationthrougha cGMP-mediatedsignaltransductionpathway

E

POMC 176 Proopiomelanocortin ACTHstimulatestheadrenalglandstorelease cortisol

E SMAD6 197 SMADfamilymember6 AntagonistofsignallingbyTGF-beta(transforming

growthfactor)type1receptorsuperfamilymembers E SP1 199 Sp1transcriptionfactor Transcriptionfactorthatcanactivateorrepress

transcriptioninresponsetophysiologicaland pathologicalstimuli

E

SP7 200 Sp7transcriptionfactor Transcriptionalactivatoressentialforosteoblast differentiation

E TGFBR1 209 Transforminggrowthfactor,beta

receptor1

Onligandbinding,formsareceptorcomplex consistingoftwotypeIIandtwotypeI transmembraneserine/threoninekinases

E

TNFRSF10A 216 Tumournecrosisfactorreceptor superfamily,member10a

ReceptorforthecytotoxicligandTNFSF10/TRAIL E TNFRSF11B 218 Tumournecrosisfactorreceptor

superfamily,member11b

ActsasdecoyreceptorforRANKLandthereby neutralizesitsfunctioninosteoclastogenesis

E TNFRSF1B 220 Tumournecrosisfactorreceptor

superfamily,member1B

ReceptorwithhighaffinityforTNFSF2/TNF-alpha andapproximately5-foldloweraffinityfor homotrimericTNFSF1/lymphotoxin-alpha

E

TNFSF10 221 Tumournecrosisfactor(ligand) superfamily,member10

CytokinethatbindstoTNFRSF10A/TRAILR1, TNFRSF10B/TRAILR2,TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4andpossiblyalsoto TNFRSF11B/OPG.Inducesapoptosis

E

ALPL 54 Alkalinephosphatase,liver/bone/ kidney

Thisisozymemayplayaroleinskeletal mineralization

F

CALCA 69 Calcitonin-relatedpolypeptidealpha CGRPinducesvasodilation F

CYP19A1 93 CytochromeP45019,subfamilyA1, polypeptide1

CytochromesP450areagroupofheme-thiolate monooxygenases

F

ESR2 104 Oestrogenreceptor2(ERbeta) Nuclearhormonereceptor F

FST 116 Follistatin Bindsdirectlytoactivinandfunctionsasanactivin antagonist

F GHRH 124 Growthhormonereleasinghormone GRFisreleasedbythehypothalamusandactsonthe

adenohypophysetostimulatethesecretionof growthhormone

F

GSTM1 127 GlutathioneS-transferasemu1 Conjugationofreducedglutathionetoawidenumber ofexogenousandendogenoushydrophobic electrophiles

F

GSTT1 128 ENSG00000239562-GSTT1protein Unknown F

IBSP 131 Integrin-bindingsialoprotein Bindstightlytohydroxyapatite F

PDGFA 171 Platelet-derivedgrowthfactoralpha polypeptide

Platelet-derivedgrowthfactorisapotentmitogen forcellsofmesenchymalorigin

F SOST 198 Sclerosteosis Negativeregulatorofbonegrowth.Interfereswith

theWntsignallingpathway

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Table4 (Continued)

Genename Gene

number

TNF 215 Tumournecrosisfactor(TNF superfamily,member2)

CytokinethatbindstoTNFRSF1A/TNFR1and TNFRSF1B/TNFBR.Itismainlysecretedby macrophagesandcaninducecelldeathofcertain tumourcelllines

F

VDR 233 VitaminD(1,25-dihydroxyvitaminD3) receptor

Nuclearhormonereceptor F

SFRP4 241 Secretedfrizzled-relatedprotein4 Solublefrizzled-relatedproteins(sFRPS)functionas modulatorsofWntsignallingthroughdirect interactionwithWnts

F

FLT1 28 Fms-relatedtyrosinekinase1 (vascularendothelialgrowthfactor/ vascularpermeabilityfactorreceptor)

ReceptorforVEGF,VEGFBandPGF G

APC 34 Adenomatouspolyposiscoli Tumoursuppressor.Promotesrapiddegradationof CTNNB1andparticipatesinWntsignallingasa negativeregulator

G

ACVR2B 43 ActivinAreceptor,typeIIB Onligandbinding,formsareceptorcomplex consistingoftwotypeIIandtwotypeI transmembraneserine/threoninekinases

G

ADAM17 44 ADAMmetallopeptidasedomain17 Cleavesthemembrane-boundprecursorof TNF-alphatoitsmaturesolubleform

G ADH1B 46 Alcoholdehydrogenase1B(classI),

betapolypeptide

G

ADM 47 Adrenomedullin AMandPAMParepotenthypotensiveand

vasodilatatoragents

G ALOX15 53 Arachidonate15-lipoxygenase Convertsarachidonicacidto

15S-hydroperoxyeicosatetraenoicacid

G

CALCR 70 Calcitoninreceptor Thisisareceptorforcalcitonin G

CTSK 88 CathepsinK Closelyinvolvedinosteoclasticboneresorptionand mayparticipatepartiallyinthedisorderofbone remodelling

G

DKK1 96 Dickkopfhomolog1(Xenopuslaevis) InhibitorofWntsignallingpathway G ENPP1 99 Ectonucleotidepyrophosphatase/

phosphodiesterase1

InvolvedprimarilyinATPhydrolysisattheplasma membrane

G ESRRA 105 Oestrogen-relatedreceptoralpha BindstoanERR-alpharesponseelement(ERRE)

containingasingleconsensushalf-site, 50_-TNAAGGTCA-30

G

FOXA1 115 ForkheadboxA1 Transcriptionactivatorforanumberoflivergenes suchasAFP,albumin,tyrosineaminotransferase, PEPCK,etc.

G

GH1 123 Growthhormone1 Playsanimportantroleingrowthcontrol G

GSR 126 Glutathionereductase Maintainshighlevelsofreducedglutathioneinthe cytosol

G IL11 135 Interleukin11 Directlystimulatestheproliferationof

hematopoieticstemcellsandmegakaryocyte progenitorcellsandinducesmegakaryocyte maturationresultinginincreasedplateletproduction

G

IL1RN 138 Interleukin1receptorantagonist InhibitstheactivityofIL-1bybindingtoitsreceptor. HasnoIL-1likeactivity

G KIT 142 v-KitHardy-Zuckerman4feline

sarcomaviraloncogenehomolog

Thisisthereceptorforstemcellfactor(mastcell growthfactor).Ithasatyrosine-proteinkinase activity

G

LEPR 144 Leptinreceptor Receptorforobesityfactor(leptin).Onligand binding,mediatessignallingthroughJAK2/STAT3

G LRP5 148 Lowdensitylipoprotein

receptor-relatedprotein5

InvolvedintheWnt/betacateninsignalling pathway,probablybyactingasacoreceptortogether withFrizzledforWnt

G

NPY 165 NeuropeptideY; NPYisimplicatedinthecontroloffeedingandin secretionofgonadotrophin-releasehormone

G NQO1 166 NAD(P)Hdehydrogenase,quinone1 Theenzymeapparentlyservesasaquinone

reductaseinconnectionwithconjugationreactionsof hydroquinonsinvolvedindetoxificationpathways

G

PELO 173 Pelotahomolog Requiredfornormalchromosomesegregation

duringcelldivisionandgenomicstability (Bysimilarity)

(10)

Table4 (Continued)

Genename Gene

number

PPARGC1A 180 Peroxisomeproliferator-activated receptorgamma,coactivator1alpha

Transcriptionalcoactivatorforsteroidreceptors andnuclearreceptors.Greatlyincreasesthe transcriptionalactivityofPPARGandthyroid hormonereceptorontheuncouplingprotein promoter

G

RHOU 185 Rashomologgenefamily,memberU ActsupstreamofPAK1toregulatetheactin cytoskeleton,adhesionturnoverandincreasecell migration

G

RUNX1 186 Runt-relatedtranscriptionfactor1 CBFbindstothecoresite,50_-PYGPYGGT-30_,_of_a

numberofenhancersandpromoters

G TANK 206 TRAFfamilymember-associatedNFKB

activator

ActsasaregulatorofTRAFfunctionbymaintaining theminalatentstate

G TGFBR3 210 Transforminggrowthfactor,beta

receptorIII

BindstoTGF-beta.Couldbeinvolvedincapturing andretainingTGF-betaforpresentationtothe signallingreceptors

G

TNFRSF11A 217 Tumournecrosisfactorreceptor superfamily,member11a,NFKB activator

ReceptorforTNFSF11/RANKL/TRANCE/OPGL; essentialforRANKL-mediatedosteoclastogenesis

G

TWIST1 230 Twisthomolog1 Probabletranscriptionfactor,whichseemstobe involvedinthenegativeregulationofcellular determinationandinthedifferentiationofseveral lineagesincludingmyogenesis,osteogenesis,and neurogenesis

G

TYROBP 232 TYROproteintyrosinekinasebinding protein

Non-covalentlyassociateswithactivatingreceptors oftheCD300family

G HDAC5 9 Histonedeacetylase5 Responsibleforthedeacetylationoflysineresidues

ontheN-terminalpartofthecorehistones(H2A, H2B,H3andH4)

H

SOX6 15 SRY(sexdeterminingregionY)-box6 Transcriptionalactivator.Bindsspecificallytothe DNAsequence50_-AACAAT-30_._Plays_a_key_role_in

severaldevelopmentalprocesses,including neurogenesisandskeletonformation

H

PBX1 21 Pre-B-cellleukaemiahomeobox1 Bindsthesequence50_-ATCAATCAA-30_._Acts_as_a

transcriptionalactivatorofPF4incomplexwith MEIS1.

H

ITGA1 23 Integrin,alpha1 Integrinalpha-1/beta-1isareceptorforlamininand collagen

H IGFBP2 26 Insulin-likegrowthfactorbinding

protein2

IGF-bindingproteinsprolongthehalf-lifeoftheIGFs andhavebeenshowntoeitherinhibitorstimulatethe growthpromotingeffectsoftheIGFsoncellculture

H

UGT2B17 40 UDPglucuronosyltransferase2family, polypeptideB17

UDPGTsareofmajorimportanceintheconjugation andsubsequenteliminationofpotentiallytoxic xenobioticsandendogenouscompounds

H

ACP1 42 Acidphosphatase1,soluble Actsontyrosinephosphorylatedproteins,low-MW arylphosphatesandnaturalandsyntheticacyl phosphates

H

ADRB3 48 Adrenergic,beta-3-,receptor Beta-adrenergicreceptorsmediatethe catecholamine-inducedactivationofadenylate cyclasethroughtheactionofGproteins

H

ALDH2 51 ALDH2–aldehydedehydrogenase2 family

Mitochondrial H

ALOX12 52 Arachidonate12-lipoxygenase Oxygenaseand14,15-leukotrieneA4synthase activity

H

AOX1 57 Aldehydeoxidase1 H

ARHGEF12 61 Rhoguaninenucleotideexchange factor(GEF)12

MayplayaroleintheregulationofRhoAGTPaseby guaninenucleotide-bindingalpha-12(GNA12)and alpha-13(GNA13)

H

ARHGEF3 62 Rhoguaninenucleotideexchange factor(GEF)3

Actsasguaninenucleotideexchangefactor(GEF)for RhoAandRhoBGTPases

H CASR 71 Calcium-sensingreceptor; Senseschangesintheextracellularconcentrationof

calciumions

H

CBS 73 Cystathionine-beta-synthase H

CD38 74 CD38molecule SynthesizescyclicADP-ribose,asecondmessenger forglucose-inducedinsulinsecretion

(11)

Table4 (Continued)

Genename Gene

number

CLCN7 77 Chloridechannel7 Mediatestheexchangeofchlorideionsagainst protons

H COL3A1 84 Collagen,typeIII,alpha1 CollagentypeIIIoccursinmostsoftconnective

tissuesalongwithtypeIcollagen

H COMT 86 Catechol-O-methyltransferase CatalyzestheO-methylation,andtherebythe

inactivation,ofcatecholamineneurotransmitters andcatecholhormones

H

CXCL2 90 Chemokine(C-X-Cmotif)ligand2 Producedbyactivatedmonocytesandneutrophils andexpressedatsitesofinflammation

H CYP17A1 91 CytochromeP450,family17,

subfamilyA,polypeptide1

Conversionofpregnenoloneandprogesteroneto their17-alpha-hydroxylatedproductsand subsequentlytodehydroepiandrosterone(DHEA) andandrostenedione

H

FABP4 107 Fattyacidbindingprotein4,adipocyte Lipidtransportproteininadipocytes H GBP1 118 Guanylatebindingprotein1,

interferon-inducible

BindsGTP,GDPandGMP H

GGCX 122 Gamma-glutamylcarboxylase MediatesthevitaminK-dependentcarboxylation ofglutamateresiduestocalciumbinding gamma-carboxyglutamate(Gla)residueswiththe concomitantconversionofthereduced

hydroquinoneformofvitaminKtovitamin Kepoxide

H

HSD11B1 130 Hydroxysteroid(11-beta) dehydrogenase1

Catalyzesreversiblytheconversionofcortisoltothe inactivemetabolitecortisone

H MEPE 150 Matrixextracellular

phosphoglycoprotein

Seemstoplayaroleinmineralization H MITF 151 Microphthalmia-associated

transcriptionfactor

Transcriptionfactorfortyrosinaseand tyrosinase-relatedprotein1

H MTHFR 155 5,10-Methylenetetrahydrofolate

reductase(NADPH)

Catalyzestheconversionof 5,10-methylenetetrahydrofolateto

5-methyltetrahydrofolate,aco-substratefor homocysteineremethylationtomethionine

H

NNMT 160 NicotinamideN-methyltransferase; CatalyzestheN-methylationofnicotinamideand otherpyridinestoformpyridiniumions

H NPPB 163 NatriureticpeptideprecursorB; Actsasacardiachormonewithavarietyof

biologicalactionsincludingnatriuresis,diuresis, vasorelaxation,andinhibitionofreninand aldosteronesecretion

H

NPR2 164 Natriureticpeptidereceptor B/guanylatecyclaseB

Receptorforatrialnatriureticpeptide(ANP),brain natriureticpeptide(BNP),andC-typenatriuretic peptide(CNP)

H

PON1 177 Paraoxonase1 Hydrolyzesthetoxicmetabolitesofavarietyof organophosphorusinsecticides

H SFRP1 193 Secretedfrizzled-relatedprotein1 Solublefrizzled-relatedproteins(sFRPS)functionas

modulatorsofWntsignallingthroughdirect interactionwithWnts

H

SHBG 194 Sexhormone-bindingglobulin Functionsasanandrogentransportprotein,butmay alsobeinvolvedinreceptormediatedprocesses

H TCIRG1 207 T-cell,immuneregulator1,ATPase,

H+transporting,lysosomalV0 subunitA3

PartoftheprotonchannelofV-ATPases (Bysimilarity)

H

TIMP2 211 TIMPmetallopeptidaseinhibitor2 Complexeswithmetalloproteinases(suchas collagenases)andirreversiblyinactivatesthem

H TREM2 225 Triggeringreceptorexpressedon

myeloidcells2

Mayhavearoleinchronicinflammationsandmay stimulateproductionofconstitutiveratherthan inflammatorychemokinesandcytokines

H

WNT10B 238 Wingless-typeMMTVintegrationsite family,member10B

Ligandformembersofthefrizzledfamilyofseven transmembranereceptors.Probabledevelopmental protein

H

ZNF384 240 Zincfingerprotein384 Transcriptionfactorthatseemstobindandregulate thepromotersofMMP1,MMP3,MMP7andCOL1A1 (Bysimilarity)

(12)

Table5–Listof‘‘orphan’’genesandnewgenesstronglyassociatedwithosteoporosis,obtainedfromGWASreviewsand meta-analyses.

Genename Genenumber Officialname

Orphangenes

RTP3 1 Receptor(chemosensory)transporterprotein3

PLCL1 2 PhospholipaseC-like1

C17orf53 3 UncharacterizedproteinC17orf53

CRHR1 4 Corticotropinreleasinghormonereceptor1

DCDC1 5 Doublecortindomaincontaining1

DCDC5 6 Doublecortindomaincontaining5

ENSG00000197851 7 PutativeuncharacterizedproteinFLJ42280

STARD3NL 8 STARD3N-terminallike

IL21R 11 Interleukin21receptor

MARK3 12 MAP/microtubuleaffinity-regulatingkinase3

ZBTB40 13 ZincfingerandBTBdomaincontaining40

FAM3C 14 Familywithsequencesimilarity3,memberC

CCDC27 16 Coiled-coildomaincontaining27

SSU72 17 SSU72RNApolymeraseIICTDphosphatasehomolog

ISG15 18 ISG15ubiquitin-likemodifier

RERE 19 Arginine-glutamicaciddipeptide(RE)repeats

LTBP2 20 Latenttransforminggrowthfactorbetabindingprotein2

CLDN14 22 Claudin14

FLNB 24 FilaminB,beta

HMGA2 25 HighmobilitygroupAT-hook2

TASP1 27 Taspase,threonineaspartase,1

DMP1 29 Dentinematrixacidicphosphoprotein1

FOXC2 32 ForkheadboxC2(MFH-1,mesenchymeforkhead1)

ADAMTS18 45 ADAMmetallopeptidasewiththrombospondintype1motif,18

ADRBK2 49 Adrenergic,beta,receptorkinase2

AHSG 50 Alpha-2-HS-glycoprotein

ANGEL1 55 Angelhomolog1

ANKH 56 Ankylosis,progressivehomolog

APOE 58 ApolipoproteinE

ARHGEF1 60 Rhoguaninenucleotideexchangefactor(GEF)1

B3GAT3 63 Beta-1,3-glucuronyltransferase3(glucuronosyltransferaseI)

CA10 66 CarbonicanhydraseX;Doesnothaveacatalyticactivity

CA8 67 CarbonicanhydraseVIII

CADM1 68 Celladhesionmolecule1

CAT 72 Catalase

CEACAM1 76 Carcinoembryonicantigen-relatedcelladhesionmolecule1(biliary glycoprotein)

CNP 78 20_,30_-Cyclic_nucleotide₃0_{phosphodiesterase}

CNR2 79 Cannabinoidreceptor2(macrophage)

COL15A1 80 Collagen,typeXV,alpha1

COL5A2 85 Collagen,typeV,alpha2

CP 87 Ceruloplasmin(ferroxidase)

CYP19A1 92 CytochromeP450,family19,subfamilyA,polypeptide1

DBP 94 Dsiteofalbuminpromoter(albuminD-box)bindingprotein

DCXR 95 Dicarbonyl/L-xylulosereductase

EBP 97 Emopamilbindingprotein(sterolisomerase)

ENPEP 98 Glutamylaminopeptidase(aminopeptidaseA)

ARHGAP1 100 RhoGTPaseactivatingprotein1

AXIN1 101 Axin1

CYLD 102 Cylindromatosis(turbantumoursyndrome)

EIF6 106 Eukaryotictranslationinitiationfactor6

ENPP1 111 ectonucleotidepyrophosphatase/phosphodiesterase1

ERC1 112 ELKS/RAB6-interacting/CASTfamilymember1

F2 113 CoagulationfactorII(thrombin)

FGFR2 114 Fibroblastgrowthfactorreceptor2

GPR177 117 WLS–Gprotein-coupledreceptor177

HOXA13 119 HomeoboxA13

RARG 120 Retinoicacidreceptor,gamma

WNT16 121 Wingless-typeMMTVintegrationsitefamily,member16

WNT4 125 Wingless-typeMMTVintegrationsitefamily,member4

WNT5B 143 Wingless-typeMMTVintegrationsitefamily,member5B

LIPC 145 Lipase,hepatic

(13)

Table5 (Continued)

LRP4 147 Lowdensitylipoproteinreceptor-relatedprotein4

MAPK8IP2 149 Mitogen-activatedproteinkinase8interactingprotein2

MT1G 154 Metallothionein1G

MTRR 156 5-Methyltetrahydrofolate-homocysteinemethyltransferasereductase

NKRF 159 NFKBrepressingfactor

OSCAR 168 Osteoclastassociated,immunoglobulin-likereceptor

P2RX7 169 PurinergicreceptorP2X,ligand-gatedionchannel,7

PDE4D 170 phosphodiesterase4D,cAMP-specific(phosphodiesteraseE3dunce

homolog)

PDLIM4 172 PDLIM4–PDZandLIMdomain4

PLOD1 175 Procollagen-lysine1,2-oxoglutarate5-dioxygenase1

PON2 178 Paraoxonase2

PTN 182 Pleiotrophin

QPCT 183 Glutaminyl-peptidecyclotransferase

RAGE 184 Renaltumourantigen

RXFP2 188 Relaxin/insulin-likefamilypeptidereceptor2

SATB2 189 SATBhomeobox2

SBDS 190 Shwachman-Bodian-Diamondsyndrome

SCARA3 191 ScavengerreceptorclassA,member3

SLC34A1 195 Solutecarrierfamily34(sodiumphosphate),member1

SLPI 196 Secretoryleucocytepeptidaseinhibitor

SPP2 202 Secretedphosphoprotein2,24kDa

STC2 204 Stanniocalcin2

SULT2B1 205 Sulfotransferasefamily,cytosolic,2B,member1

TMSL1 213 Thymosin-like4(pseudogene)

TMSL2 214 Thymosin-like4(pseudogene)

TNFRSF18 219 Tumournecrosisfactorreceptorsuperfamily,member18

TRPM7 226 Transientreceptorpotentialcationchannel,subfamilyM,member7

TRPV5 227 Transientreceptorpotentialcationchannel,subfamilyV,member5

TRPV6 228 Transientreceptorpotentialcationchannel,subfamilyV,member6

TSC22D3 229 TSC22domainfamily,member3

TXNRD1 231 Thioredoxinreductase1

VPS13B 235 Vacuolarproteinsorting13homologB

WASF2 236 WASproteinfamily,member2

WIPF1 237 WAS/WASLinteractingproteinfamily,member1

WRN 239 Wernersyndrome,RecQhelicase-like

SPTBN1 242 Spectrin,beta,non-erythrocytic1

GenesrobustlyassociatedwithBMDonGWASmeta-analysesandreviews

AAAS – Achalasia,adrenocorticalinsufficiency,alacrimia(Allgrove,triple-A)

ABCF2 – ATP-bindingcassette,sub-familyF(GCN20),member2

ADRA1D – Adrenergic,alpha-1D-,receptor

AKAP11 – Akinase(PRKA)anchorprotein11

ALDH7A1 – Aldehydedehydrogenase7family,memberA1

C11orf49 – UPF0705proteinC11orf49

C12orf10 – UPF0160proteinMYG1,mitochondrialPrecursor

C12ORF23 – UPF0444transmembraneproteinC12orf23

C16ORF38 – Neuronalpentraxin-likeproteinC16orf38Precursor

C18ORF19 – UncharacterizedproteinC18orf19

C6orf97 – Coiled-coildomain-containingproteinC6orf97

CCDC55 – CCDC55

CDK5RAP2 – CDK5regulatorysubunitassociatedprotein2

CDKAL1 – CDK5regulatorysubunitassociatedprotein1-like1

CKAP5 – cytoskeletonassociatedprotein5

CLCN7 – Chloridechannel7

CPN1 – CarboxypeptidaseN,polypeptide1

DHH – Deserthedgehoghomolog

DNM3 – Dynamin3

EFCAB5 – EF-handcalciumbindingdomain5

ERLIN1 – ERlipidraftassociated1

ESPL1 – extraspindlepolebodieshomolog1

FAM9B – Familywithsequencesimilarity9,memberB

FLJ42280 – PutativeuncharacterizedproteinFLJ42280

FOXE1 – ForkheadboxE1(thyroidtranscriptionfactor2)

FOXL1 – ForkheadboxL1

(14)

eveniffurtherinheritablemechanisms,partlyindependentof BMD, are seen to contribute to an increased incidence of fractures.28

Fromthis perspective, osteoporoticclinical phenotyping thatnowappearsanomalousmaybeexplainedthroughthe

discoveryofheretoforeunknowngeneticinformation.Agreat many genes regulating the BMD have been identified by severalgeneticapproaches,suchaslinkageanalysis (discov-ering inherited monogenic Mendelian human disease) or associationstudiesforidentifyingsusceptibilitygenes,such

Table5 (Continued)

GALNT3 – UDP-N-acetyl-alpha-D-galactosamine:polypeptide

N-acetylgalactosami-nyltransferase3(GalNAc-T3)

GPATCH1 – Gpatchdomaincontaining1

IDUA – Iduronidase,

alpha-L-IFT80 – Intraflagellartransport80Homolog

INSIG2 – Insulininducedgene2

JAG1 – Jagged1(Alagillesyndrome)

KAL1 – Kallmannsyndrome1sequence

KCNMA1 – Potassiumlargeconductancecalcium-activatedchannel,subfamilyM,

alphamember1

KIAA2018 – KIAA2018

KLHDC5 – kelchdomaincontaining5

KPNA4 – karyopherinalpha4(importinalpha3)

KPRP – Keratinocyteproline-richprotein

LCE2A – Latecornifiedenvelope2A

LCE2B – Latecornifiedenvelope2B

LCE2C – Latecornifiedenvelope2C

LCE4A – Latecornifiedenvelope4A

LEKR1 – PutativeuncharacterizedproteinENSP00000407071

LIN7C – Lin-7homologC(C.elegans)

MBL2 – Mannose-bindinglectin(proteinC)2,soluble(opsonicdefect)

MFSD5 – Majorfacilitatorsuperfamilydomaincontaining5

MHC – ENSG00000233904–HLAclassIhistocompatibilityantigen,alphachainE

Precursor(MHCclassIantigenE)

MPP7 – Membraneprotein,palmitoylated7(MAGUKp55subfamilymember7)

MTHFR – 5,10-Methylenetetrahydrofolatereductase(NADPH)

NTAN1 – N-terminalasparagineamidase

OSBPL8 – Oxysterolbindingprotein-like8

PBX1 – Pre-B-cellleukaemiahomeobox1

PFDN5 – prefoldinsubunit5

PKDCC – Proteinkinasedomaincontaining,cytoplasmichomolog

RPS6KA5 – RibosomalproteinS6kinase,90kDa,polypeptide5

RSPO3 – R-spondin3homolog

SHFM1 – splithand/footmalformation(ectrodactyly)type1

SLC25A13 – Solutecarrierfamily25,member13(citrin)

SMC4 – Structuralmaintenanceofchromosomes4

SMG6 – Smg-6homolog,nonsensemediatedmRNAdecayfactor(C.elegans)

SOX4 – SRY(sexdeterminingregionY)-box4

SUPT3H – SuppressorofTy3homolog

TBC1D1 – TBC1(tre-2/USP6,BUB2,cdc16)domainfamily,member1

TRIM59 – Tripartitemotif-containing59

XKR9 – XK,Kellbloodgroupcomplexsubunit-relatedfamily,member9

ZNF408 – zincfingerprotein408

ENSG00000163286 – Alkalinephosphatase,placental(Reganisozyme) ENSG00000206292 – Majorhistocompatibilitycomplex,classII,DOalpha

ERAP1 – Endoplasmicreticulumaminopeptidase1

FABP3 – Fattyacidbindingprotein3,muscleandheart(mammary-derivedgrowth

inhibitor)

FCGR2A – FcfragmentofIgG,lowaffinityIIa,receptor(CD32)

FKBP2 – FK506bindingprotein2

FLT3 – Fms-relatedtyrosinekinase3

FOSB – FBJmurineosteosarcomaviraloncogenehomologB

FTH1 – Ferritin,heavypolypeptide1

GC – Group-specificcomponent(vitaminDbindingprotein)

GCM1 – Glialcellsmissinghomolog1

GDF15 – Growthdifferentiationfactor15

GRPEL1 – GrpE-like1,mitochondrial

(15)

asCGAS(analyzingpolymorphicvariantsincandidategenes andrelatinghaplotype),orGWAS(genotypinglargenumbers of SNPs across the genome, rather than a few candidate genes),withresultsthatarenotperfectlyinagreement,and aresometimesconflicting.11

Numerous genes of at least three metabolic pathways, these being the estrogenic endocrine pathway, the Wnt/ Cateninpathway,andtheRANKL/RANK/OPGpathway,have beenidentifiedassurelyassociatedtoosteoporotictraits,and other genes, instead, have been indicated as being either promising,29_or_robustly_associated_with_BMD,10,28,30_whereas novel genome-wide-significant loci were supposed to be stronglyassociated.1,11,12,31

Thepreliminarysetcounted199genes;afterfour expan-sion-filtering loops basedon boththe database-searchand microarraygenelistscanning,thetotalnumberwasincreased to242genes,showingthatSTRINGexpansion-andPubMed filtering-process, combined into an iterative loop, were essentialto reachthe convergenceofdata and toperform anaccurateanalysis.STRINGwasemployedforacombination ofdatabasesregardingdirectphysicalprotein–protein inter-action, experimental gene expression, an active role in particularmetabolicpathways,andindirectfunctional asso-ciations.

Although previous studies regarding the cell cycle of human T lymphocytes, human periodontal disease, and humanboneaugmentation/remodellingestablishedthatthe numbersofgenesinthehigherclustersweresmall,ourstudy revealedthat17genesbelongedtoleader-orB-cluster;this resultingnumberofhigherclustergeneswasinaccordance withtheincreasingsetofgeneswhichwereeithercandidates tobeassociatedorwerefoundtoberobustlyassociatedwith osteoporoticdisease.

Mostofthediscovered‘‘leadergenes’’werethesameas those involved in the already examined augmentation/ remodellingprocess25_:_these_were_divided_into_cytokines,_such as interleukins, in particular IL1B, which is produced by activatedmacrophagesthatareimportant mediatorsofthe inflammatory response, whereas IL6 has the capacity to stimulatethedevelopmentofosteoclastsfromtheir hemato-poietic precursors,32 _or_such_as_the _growth_factor _TGFB1,_a multifunctionalpeptidethatregulatesosteoblastchemotaxis, proliferationanddifferentiation,33,34_and_{transcription}_factors, likeRUNX2,thatplaysanimportantroleinthematurationof osteoblasts aswellas inbothintramembranousand endo-chondralossification,withits ‘‘masterswitch’’activity,35–37 andJUN,whichisinvolvedinregulatinggeneexpression.38

The two adjunctive ‘‘leader genes’’ were CTNNB1, an essentialmoleculeofthecanonicalWnt/b-cateninsignalling pathway, and SPP1, also known as osteopontin, which is bounded tightly tohydroxyapatite, performinginteractions betweenthematrixandcells,39_and_these_two_genes_resulted as being associated with BMD at genome-wide-significant levelsinseveralmeta-analysesandreviews.1,11,31

TheclassBgenegroupisconstitutedbygenes function-ally and structurally divided into architectural proteins, non-collagen matrix molecules, and transcription- and cytokines/growth-factors. COL1A1 and ICAM1 belong to the first group. COL1A1 encodes the a-1 chain of type I collagen, the principal component of bone extracellular

matrix:thestudiesperformedontheeffectsonBMDofthe three polymorphisms suggested that homozygotes for different haplotypes showed reduced (haplotype2) or in-creased (haplotype3) BMD.40 _Although _COL1A1 _has _been extensively studied in osteoporosis,11,12,28 _no _information hasbeenobtainedinconnectiontoICAM1,whichplaysan active role in the inflammatory process due to contact between osteoblasts(Obs) andosteoclasts(Ocs),occurring throughtheObmembranecomplex,inwhichthemembrane protein icam1bindsthecounter-receptorexpressedatthe surfaceofosteoclasts.41_Also_MMP9,_an_enzyme_involved_in degradationoftypesIVandVcollagens,andNFKB1,which hasaprominentrolein(antibody)clearance,phagocytosis, pathogen recognition, and inflammatory response, have been little examined in more recent meta-analyses and reviews,evenif,togetherwithCOL1A1,theyhavebeenseen to be significantly down-regulated in the bone tissue of osteoporoticwomen.42,43

The analysis of gene-coding cytokines resumed with 5 growth factors, such as BMP2, BMP7, IGF1, VEGFA and TNFSF11,andoneinterleukin,IL10.Interleukin10,primarily produced by monocytes and bone morphogenetic proteins (BMPs) belonging to the group of multifunctional peptides (TGFbsuperfamily)thatcontrolproliferationand differentia-tioninmanycelltypes,yieldedinconclusiveresultsregarding an association with osteoporosis44,45 _as _attested _to _by _the absenceofthesetwogenesfromallthemorerecentreviews andmeta-analysesregardinggenome-wideassociation stud-ies.1,12,31

Anothertwogenes,althoughbelongingtoupperclusters, appearednottobeassociatedtoosteoporosisafterresearch performed among genome-wide-significant loci of GWAS meta-analyses1,12,31_: _IGF1, _encoding _a _protein _similar _to insulin in function and structure, which, interacting with somevariantsofESR2(OestrogenReceptor-b),influencesthe riskoffractureinpostmenopausalwomen,46_and_the_growth factor encoded by VEGFA, that shows several effects on osteoclastdifferentiation,angiogenesis,and monocyte/mac-rophagemigration.47

ThelastclassBgenewasTNFSF11,amemberofthetumour necrosisfactor (TNF) cytokinefamily,which isaligandfor osteoprotegerinandfunctionsasakeyfactorforosteoclast differentiationandactivation.48

Thepreventionofbonelossinpostmenopausalwomenvia oestrogenreplacementtherapyhasshownthatanactiverole is played by the oestrogen endocrinesystem in regulating bonemassandtheoccurrenceofosteoporosis.Thebinding between oestrogen and its receptors, such as the most extensively studied oestrogen receptors 1 and 2 (ESR1, ESR2), and oestrogen-related receptors ERR-a (ESRRA) and ERR-g(ESRRG),resultedintheup-regulationoftheexpression of many genes. Experiments and link connection analysis showedthatoestrogenreceptorsinfluencedtheoccurrenceof osteoporosis,bothontheirown,andbyinteractingwiththe higherclustergenesCTNNB1and IGF1,aswellaswiththe leader SPP1geneforESRsandforESRRs,respectively,asis seeninFig.1E.29

AsregardsthevitaminDendocrinepathwayand mainte-nance of calcium homeostasis, effects of vitamin D are mediatedthroughitsnucleartranscriptionfactor(VDR),but

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theassociationbetweenVDRpolymorphisms(Cdx2,FokI,BsmI, ApaI,andTaqI)andBMDwereuncertain;evenifvitaminD bindingprotein(DBP) binds and transportsvitaminD, and mediatesbone resorptionby directly activating osteoclasts actingasDBP-macrophageactivatingfactor,thepresenceof othergeneticandenvironmentalfactorswasanindispensable conditionfor revealingthe pooreffect ofthe DBP geneon fracturerisk.49DBP was anorphan gene, whereas,at third knot,VDRresultedasconnectedthroughoutthe oestrogen-relatedreceptorgamma(ESRRG)toCTNNB1,SPP1,JUN,IL6, MMP9,andIGF1(Fig.1E).

The oestrogen endocrine system and the vitamin D endocrinepathwayseem tobeinterconnectedthroughthe ESRRG, which isa candidate geneforosteoporosis, due to consistentassociationsdiscoveredbyrecentanalysis.50

ThemechanismofthecanonicalWnt/b-cateninsignalling pathway has been well known, and its importance in the regulationofbonemassisattestedtointheliterature.51The proteinencodedbyCTNNB1(b-catenin)ispartofacomplexof proteinsthatisresponsiblefortransmittingsignals through-outthenucleartranscriptionalactivityofb-catenin,whenthe receptor complex consisting of frizzled family proteins (encoded by frizzled precursors, FZDs) and low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6 co-receptorbindsasligandtheWnt.52_In_Fig._1F,_all osteoporo-sis-associated genes reviewed by Li and co-workers are presented with all their connections at first knot29_; _the presenceofthetwoleadergenes,RUNX2,whichisthatacting in the proliferation of the osteoprogenitor cell via the canonical Wnt/b-catenin signalling pathway, and CTNNB1, givesevidencethatthegeneencodingb-catenin,asreported in several GWAS meta-analyses, is strongly associated to osteoporoticdisease.1,11,29,31

Associationsbetweenosteoporosisandthegenes belong-ingtoRANKL/RANK/OPGpathwaywereperfectlyestablished. Thethreegenesofthetumournecrosisfactor superfamily, TNFSF11,TNFRSF11A,andTNFRSF11B,encodedligandrankl (receptoractivatorofnuclearfactor-kBligand),receptorrank (receptoractivatorofthenuclearfactor-kB),andopg (osteo-protegerin or tumour necrosis factor receptor superfamily member 11B). The formation, activation and survival of multinucleatedosteoclastsinnormalboneremodellingwere promotedbythenormalbindingbetweenranklandrank;opg actedasadecoyreceptorforrankl,and,thereby,neutralized itsfunctioninosteoclastogenesis.48InFig.1G,rankl,rankand opgarepresentedwithallconnectionsatfirstknot;mostof thelinksinFig.1Gwereduetorankl,indicatingthatthisligand maybeaffectedbyotherpathways.

Thepresent insilico studyfurnished alistof242genes potentiallyinvolvedinosteoporosislinkedtoproceduresof jawboneaugmentation.Table5reports98orphangenesalong withafurther81 genes,obtained fromGWAS reviewsand meta-analyses.1,10–12,28,30,31_The_majority_of_these_latter _also resultedas‘‘orphans’’,whiletheremainingonesaremembers ofthelowestclass,thatis,with,atmost,justonesignificant interactionwithpresentgenesbelongingtothefinal conver-genceset.

Thestrengthofthepresentanalysis,whichaimedatthe identificationofgenesplayingarelevantroleinosteoporosis linkedtojawbonereconstruction,wasbasedonacombination

ofdatabasesregardingdirectphysicalprotein–protein inter-action, experimental gene expression, an active role in particularmetabolicpathways,andindirectfunctional asso-ciations, could also be its weakness, owing to possible ‘‘publication bias’’. Some genes of the lower class and, moreover,the‘‘orphans’’,forwhichinformationisveryscant, couldbefoundtohaveasignificantroleinthefuture,ascanbe seen from the wide number of genes for which a strong association wasindicated,albeitwiththerebeingalack of informationregardingtheirrole.

Severalmicroarrayexperimentswereperformedinorder toidentifydifferentiallyexpressedgenesinosteoporoticbone; moreover, genes identified and implicated in osteoporosis pathogenesiswerevariableindifferentexperimental analy-ses.53,54_The_set_for_microarray_analysis_that_is_commercially available presents lists specifically addressed to different pathways(SuperArrayBioscienceCorporation:7320Executive Way Frederick, MD 21704 USA), but the roles of several promising genes (lower class and orphan genes) are not considered.Thelackofinformationconcerningosteoporosis wasborneoutbytheroleplayedbybothassociatedgenes,and bythenumerousoldandnew‘‘orphangenes’’,notpartofthe known and well-analyzed pathways; further targeted DNA microarray experiments might help to clarify these still obscurebiomolecularmechanisms.

Funding

MIUR Italian Dept. of Education, University and Scientific Research: PRIN 2008 2008K4XXF8_02(CUPG51J10000030001) and2008K4XXF8_03(CUPE61J100000200001).

Competing

interest

Noconflictofinterest.

Ethical

approval

Noethicalapprovalisrequired.

Acknowledgments

Thepresent studyhasbeen supported inpartthrough the Ministero dell’Istruzione, Universita` e Ricerca Scientifica (MIUR Italian Dept. of Education, University and Scientific Research)PRIN20082008K4XXF8_02(CUPG51J10000030001)to Prof. Ludovico Sbordone, and 2008K4XXF8_03 (CUP E61J100000200001)toProf.RanieriMartuscelli.

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