TwoGenesBehindPLOSL: MolecularandPathologicalCharacteristicsoftheDisease by JuhaPaloneva DepartmentofMolecularMedicine,NationalPublicHealthInstitute,Helsinki,Finland andDepartmentofMedicalGenetics,UniversityofHelsinki,Helsinki,Finland and DepartmentofPathology,UniversityofHelsinki,Helsinki,Finland and DepartmentofHumanGenetics,DavidGeffenSchoolofMedicineatUCLA, LosAngeles,California,USA. AcademicDissertation HelsinkiUniversityBiomedicalDissertationsNo.32 HelsinkiBiomedicalGraduateSchool,Helsinki,Finland TobepubliclydiscussedwiththepermissionoftheMedicalFacultyoftheUniversityof Helsinki,intheauditorium2oftheBiomedicumHelsinki,Haartmaninkatu8, onJune6th,2003at12o'clocknoon Helsinki2003 Supervisedby ProfessorLeenaPeltonen-Palotie NationalPublicHealthInstitute andUniversityofHelsinki, Helsinki,Finland and DavidGeffenSchoolofMedicineatUCLA, LosAngeles,California,USA Reviewedby DocentPenttiTienari ProfessorJohnHardy HelsinkiUniversityCentralHospitalNationalInstituteonAging UniversityofHelsinki NationalInstitutesofHealth Helsinki,Finland Bethesda,USA JulkaisijaUtgivare Publisher KansanterveyslaitosFolkhälsoinstitutetNationalPublicHealthInstitute Mannerheimintie166Mannerheimvägen166Mannerheimintie166 00300Helsinki00300HelsingforsFIN-00300Helsinki,Finland puh.vaihde(09)47441tel.växel(09)47441telephone+358947441 telefax(09)47448408telefax(09)47448408telefax+358947448408 PublicationsoftheNationalPublicHealthInstitute,KTLA8/2003 CopyrightNationalPublicHealthInstitute ISSN0359-3584 ISSN1458-6290(PDF) ISBN951-740-352-6 ISBN951-740-353-4(PDF) HelsinkiUniversityBiomedicalDissertationsNo.32 ISSN1457-8433 2 Thereisalwaysabiggerfish -unknown- ToPetra 3 4 TABLEOFCONTENTS LISTOFORIGINALPUBLICATIONS..............................................................................7 ABBREVIATIONS.............................................................................................................8 SUMMARY ........................................................................................................................9 REVIEWOFTHELITERATURE ....................................................................................11 TheHumanGenome...................................................................................................11 GeneticDiversityinHumans ..................................................................................12 Modelorganisms ....................................................................................................13 GeneticFactorsandDisease......................................................................................13 IdentificationofHumanDiseaseGenes......................................................................15 TheFinnishDiseaseHeritage.....................................................................................16 PLOSL(Nasu-Hakoladisease)...................................................................................21 SymptomsofPLOSL ..............................................................................................21 IdentificationoftheFirstPLOSLLocusonChr.19q13.1 .......................................23 Dementia.....................................................................................................................23 InheritedDementias................................................................................................23 DisordersoftheBone .................................................................................................27 AIMSOFTHESTUDY ....................................................................................................29 MATERIALSANDMETHODS........................................................................................30 PatientsandEthicalaspects.......................................................................................30 SequenceandMutationAnalysis................................................................................30 RNAAnalyses .............................................................................................................30 PhenotypicAnalysisofPBMC ....................................................................................31 CytotoxicityAssay.......................................................................................................31 ImmunoprecipitationandWestern-blotAnalysis.........................................................31 Neuroimaging..............................................................................................................32 Neurophysiology .........................................................................................................32 HistologyandImmunohistochemistry .........................................................................32 InductionofOsteoclasticCells....................................................................................33 Invitroandinvivoanalysisofboneresorption..........................................................33 HistochemistryandImmunofluorescenceStainings..................................................34 GenBankAccessionNumbers ....................................................................................34 Dap12knockoutMouse ..............................................................................................34 RESULTSANDDISCUSSION .......................................................................................35 IdentificationoftheFirstPLOSLgene(I)....................................................................35 AnalysisoftheCriticalPLOSLRegion ...................................................................35 IdentificationoftheFirstPLOSLGeneasDAP12..................................................36 ExpressionofDAP12andDAP10inFinnishPLOSLpatients ...............................37 PhenotypicandFunctionalAnalysisofPBMCinPLOSLpatients .........................37 IdentificationofthesecondPLOSLgeneasTREM2(II)............................................38 ExpressionofDAP12andTREM2 .........................................................................40 5 DAP12andTREM2MutationsResultinanIdenticalPhenotype ..........................41 NaturalCourseofPLOSL(III).....................................................................................41 ClinicalPicture ........................................................................................................41 Neuroimaging .........................................................................................................43 Neurophysiology.....................................................................................................43 MacroscopicalAutopsyFindings ............................................................................44 HistologicalandImmunohistochemicalFindings....................................................44 OsteoclastDifferentiationandFunctioninPLOSLPatients(IV) ................................45 DAP12-andTREM2-DeficientMonocytesInefficientlyDifferentiateinto Osteoclasts .............................................................................................................46 AberrantMorphologyofDAP12-andTREM2-DeficientOsteoclasts.....................47 QuantitativeRT-PCRAnalysis ...............................................................................47 DAP12-DeficientOsteoclastsResorbBone ...........................................................48 DAP12-andTREM2-DeficientPBMCMigrateEfficiently.......................................50 OsteoclastsinPLOSLPatientsAreMultinucleatedandResorptive ......................50 AnalysisofDAP12KnockoutMouse(unpublished) ...................................................51 GENERALDISCUSSION ...............................................................................................52 ThePowerofDifferentApproachesforFindingDiseaseGenes................................52 StructureandFunctionofDAP12/TREM2SignalingComplex...................................52 MutationsinGenesoftheDAP12SignalingPathway................................................54 ITAMDomaininHematopoieticCellSignaling ...........................................................56 DAP12-MediatedSignalTransductionComplex .......................................................56 SignificanceofDAP12andTREM2............................................................................60 DAP12KnockoutMiceShowContradictoryManifestations .......................................60 ImmunologicalPhenotypeofMicewithManipulatedDap12Genes ..........................62 PathogenicMechanismsinPLOSL ............................................................................63 DevelopmentoftheBoneLesions..........................................................................64 ClassificationoftheBoneLesions ..............................................................................65 NeuropathologicalMechanismsinDifferentFormsofDementia ...............................66 PsychiatricSymptomsinDementingDisorders..........................................................66 DifferentialDiagnosisofPLOSL .................................................................................66 TreatmentandClinicalCounselling ............................................................................67 CONCLUDINGREMARKS.............................................................................................68 ELECTRONICDATABASEINFORMATION .................................................................69