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Genetic counselling and testing for Alzheimer's Disease and Frontotemporal Lobar

Degeneration:anItalianconsensusprotocol

RunningTitle:ItalianDIAfNProtocolforGeneticCounselling

MartinaBocchettaa,b,AnnaMegaa, LiviaBernardic, EmilioDiMariad, LuisaBenussie,GiulianoBinettif,

BarbaraBorronig,RosannaColaoc,GiuseppeDiFedeh, SilviaFostinellie,DanielaGalimbertii,Massimo

Gennarellij, Roberta Ghidonie, Irene Piacerik, Michela Pievania, Corinna Porteril, Veronica Redaellih,

Giacomina Rossih, Silvia Suardih, Claudio Babilonim, Elio Scarpinii, Fabrizio Tagliavinih, Alessandro

Padovanig,BenedettaNacmiask,SandroSorbik,GiovanniB.Frisonia,n,SINdem*,AmaliaC.Brunic.

*SINdemCollaborators:

MarcoBozzalio, Lucilla Parnettip, Carlo Ferrareseq, Stefano F. Cappar, CamilloMarras, CarloMasullot,

InnocenzoRainerou,VincenzoSilaniv,GiuseppeSorrentinow,GiuseppeBrunox,AnnachiaraCagniny.

a)LaboratoryofAlzheimer’sNeuroimagingandEpidemiology,IRCCSIstitutoCentroSanGiovannidiDio

Fatebenefratelli,Brescia,Italy;

b)DepartmentofMolecularandTranslationalMedicine,UniversityofBrescia,Brescia,Italy;

c)CentroRegionalediNeurogenetica,LameziaTerme,Italy;

d)DepartmentofHealthSciences,UniversityofGenovaandDivisionofMedicalGenetics,GallieraHospital,

Genova,Italy;

e)MolecularMarkersLaboratory,IRCCSIstitutoCentroSanGiovannidiDioFatebenefratelli,Brescia,Italy;

f)IRCCSIstitutoCentroSanGiovannidiDioFatebenefratelli,Brescia,Italy;

g)UniversityofBresciaandCentreforAgeingBrainandNeurodegenerativeDisorders,NeurologyUnit,

Brescia,Brescia,Italy;

h)IRCCSFondazioneIstitutoNeurologicoCarloBesta,Milan,Italy;

i)UniversityofMilan,FondazioneCàGranda,IRCCSOspedaleMaggiorePoliclinico,Milan,Italy;

j)GeneticUnit,IRCCSIstitutoCentroSanGiovannidiDioFatebenefratelli,Brescia,Italy;

k) Department of Neuroscience, Psychology, Drug Research and Child Health, University of Florence,

Florence,Italy;

l)BioethicsUnit,IRCCSIstitutoCentroSanGiovannidiDioFatebenefratelli,Brescia,Italy;

m)DepartimentofPhysiologyandPharmacology,UniversityofRome“LaSapienza”,Rome,Italy;IRCCS

SanRaffaelePisanaofRome,Italy;

n)MemoryClinicandLANVIE-LaboratoryofNeuroimagingofAging,UniversityHospitalsandUniversity

ofGeneva,Geneva,Switzerland;

o)NeuroimagingLaboratory,IRCCSSantaLuciaFoundation,Rome,Italy;

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p)SectionofNeurology,DepartmentofMedicine,CentreforMemoryDisturbances,UniversityofPerugia,

Perugia,Italy;

q)SchoolofMedicineandSurgery,MilanCenterforNeuroscience(NeuroMI),UniversityofMilanoBicocca,

Italy;NeurologyUnit,SanGerardoHospital,Monza,Italy;

r)NeTSCenter-IstitutoUniversitariodiStudiSuperiori(IUSS),Pavia,Italy;

s)InstituteofNeurologyandCenterforNeuropsychologicalResearchofthePoliclinicoGemelli,Catholic

UniversityofRome,Italy;

t)DepartmentofNeuroscience,InstitutesofNeurology,CatholicUniversityoftheSacredHeart,Rome;

u)NeurologyI-HeadacheCenter,DepartmentofNeuroscience"RitaLeviMontalcini,UniversityofTorino,

Torino,Italy;

v) Department of Neurology-Stroke Unit and Laboratory of Neuroscience, IRCCS Istituto Auxologico

Italiano, "Dino Ferrari" Centre, Department of Pathophysiology and Transplantation, Universita' degli

StudidiMilano,Milan,Italy;

w)UniversityofNaplesParthenope,Naples,Italy;

x)MemoryClinic,DepartmentofNeurologyandPsychiatry,UniversityofRome"Sapienza",Italy;

y) Department of Neurosciences (DNS), University of Padova, Padua; IRCCS, San Camillo Hospital

Foundation,Venice,Italy.

Corresponding Author: Amalia Cecilia Bruni, MD; Centro Regionale di Neurogenetica, Viale Arturo

Perugini, 88046 Lamezia Terme, Italy; phone: +39 0968 208080; fax: +39 0968 208032; e-mail:

bruni@arn.it

Keywords:Alzheimerdisease;frontotemporaldegeneration;geneticcounselling;genetictesting

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BACKGROUND Genetic testing of familial Alzheimer’s disease (AD) and frontotemporal lobar

degeneration(FTLD)isattractinginterestthankstoinnovativeprimarypreventionclinicaltrialsand

increased request for information by at-risk individuals. However, ethical, social and psychological

implicationsareparamountandgenetictestingmustbesupportedbystructuredgeneticcounselling.In

Italy,practiceparametersandguidelinesforgeneticcounsellingindementiaarenotavailable.

AIMTodevelopanationallyharmonizedprotocolforgeneticcounsellingandtestingoffamilialADand

FTLD.

METHODSActivitieswerecarriedout inthecontextof theItalianDominantlyInheritedAlzheimer’s

and Frontotemporal Network (IT-DIAfN) project, a national network of centres of excellence with

expertise in managing patients with familial AD and FTLD. A survey of the literature on genetic

counselling protocols and guidelines was conducted. Local protocols for genetic counselling were

surveyed.Differencesandcommonalitiesamongprotocolswereidentifiedanddiscussedamongproject

partners.Consensuswasreachedfollowingimplicitaggregationmethods.

RESULTSConsensuswasreachedonaprotocol forpatientswithclinicallydiagnosed familialADor

FTLDandadistinctprotocol for their at-risk relatives.Genetic counselling shouldbeprovidedby a

multidisciplinary team including a geneticist, a neurologist/geriatrician, and a

psychologist/psychiatrist, according to the following schedule: (i) initial consultation with tailored

informationon thegeneticsof thedementias; (ii) clinical,psychologicalandcognitiveassessment; if

deemedappropriate(iii)genetictestingfollowingastructureddecisiontreeforgenemutationsearch;

(iv)genetictestingresultdisclosure;(v)psychologicalsupportfollow-up.

CONCLUSIONSThisgeneticcounsellingprotocolprovidesItaliancentreswithalineofsharedpractice

for dealingwith the requests for genetic testing for familialAD andFTLD frompatients and at-risk

relatives,whomayalsobeeligibleparticipantsfornovelpreventionclinicaltrials.

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1.INTRODUCTION

Alzheimer’sDisease(AD)andfrontotemporallobardegeneration(FTLD)aretwoofthemostcommon

formsofdementia.Althoughthemajorityofcasesaresporadic(i.e.withoutafamilyhistory),amarkedly

familialcomponenthasbeenreportedin60%ofearlyonset(<65years)ADpatients[1]andin25-50%

ofFTLDpatients[2,3].Anautosomaldominantmodeofinheritanceisfoundinabout1-5%ofADand

10-50% of FTLD cases [4,5]: specifically, pathogenic AD mutations were identified in the Amyloid

precursorprotein(APP)[6]andthePresenilin(PSEN1andPSEN2)genes[7,8],whilethegenesmainly

involved in FTLD are theMicrotubule associated protein tau (MAPT) [9-11], the Progranulin (GRN)

[12,13] and Chromosome 9 open reading frame 72 (C9orf72) [14,15]. Since the majority of GRN

pathogeneticmutationscauseproteinhaploinsufficiency,thedosageofcirculatingprogranulinhasbeen

proposedasausefultoolforaquickandinexpensivelarge-scalescreeningofGRNmutationscarriers

[16-19].

GenetictestingforADandFTLDischangingrapidlyduetotheincreasingavailabilityofnewandfaster

technologiesforDNAtest,anditisattractinginterestfrompatientsandfamiliesthankstoinnovative

primarypreventionclinicaltrialstargetinggeneticdementiaandtothegrowingrequestforinformation

by at-risk individuals. Two recent innovative clinical trials targeting genetic dementia are the

Dominantly Inherited Alzheimer Network Trial for AD (DIAN-TU,

www.clinicaltrials.gov/show/NCT01760005) and the Presymptomatic Neurodegeneration Initiative

for FTLD (PreNI, www.neurodegenerationresearch.eu/initiatives/jpnd-alignment-

actions/longitudinal-cohorts/call-for-working-groups/call-results/). Genetic mutation carriers, both

symptomaticandasymptomatic,couldthereforebeeligibleparticipantsforthesenovelclinicaltrials.

Genetictestinginsymptomaticpatientsisusedtoconfirmtheclinicaldiagnosis(diagnosticDNAtest);

genetictestingincognitivelyunimpairedrelativesofpatientswithamutationidentifiesthosewhowill

develop the disease in the future (predictive DNA test) [20-23]. In at-risk asymptomatic relatives,

predictivegenetictestingoffersthepossibilityofassessingtheirpersonalrisk,thusallowingthemto

organizetheirlivesandmakeinformedcareerorreproductivechoicesanddecision[23].Undergoing

predictive testsmayhelpat-risk individuals to copeemotionallywith theirgenetic riskby reducing

uncertaintyabouttheirstatusandfocusingonplanningforthefuture[24],assuggestedbyaseminal

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studyinHuntington'sdisease[25].AscomparedtoAmericans,Italianat-riskrelativesexpressedhigher

intentionstoundergogenetictesting[20].

However, togetherwiththesepotentialadvantages,genetic testshave importantethical,social, legal

and psychological implications for the patients and thewhole family. In fact, the identification of a

geneticmutation inapatient implicitlydefines the risk for theother familymembers,withpossible

implicationfortheirhealthandtheirfuture.Ontheotherhand,informationongeneticstatuscannotbe

sensiblyusedfortherapeuticpurposes,asnoapproveddisease-modifyingtreatmentisavailabletodate.

Inhealthyat-riskindividuals,thereareconcernsthatapositivegenetictestingmaytriggeranegative

psychologicalresponse,suchasseveredepression,anxiety,helplessness,orevensuicidalideation[26].

Fromasocial/legalperspective,theremaybeissuesofpotentialgeneticdiscrimination,difficultiesin

findingajoborserviceassistance(includinginsuranceimplications),andincreatingrelationships[21].

Current guidelines for AD and other inherited dementia recommend that genetic testing should be

offeredwithinapropergeneticcounsellingprocedure[21].Forasymptomaticat-riskindividuals,the

protocolestablishedforHuntington’sdiseaseandrecentlyrevised[27]isrecommended.

Withtheadventofthepreventionclinicaltrialsindementia,suchasDIAN-TUforADinpre-symptomatic

mutationcarriers,disclosureofgeneticstatusisapre-conditiontoparticipateinatrialinItaly.Such

disclosurecannotbemadewithouteffectivepre-andpost-genetictestingsupport[28].

To date, in Italy no practice standards and consensus-based guidelines are available for genetic

counsellingoffamilialADorFTLD.TheIT-DIAfNproject,anetworkofItaliancentresofexcellencewith

recognized experience in managing patients with familial AD and FTLD, aimed at developing a

harmonized and structured protocol for genetic testing and counselling for those families, both for

symptomaticpatientsandat-riskrelatives.AsinheritedADandFTLDaregeneticallyheterogeneousand

symptomsoftenoverlap,wealsogeneratedadecisiontreetoassistclinicians/researchersinmutations

search.

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2.METHODS

Thisstudywasconductedaspartofthe"ItalianDominantlyInheritedAlzheimer’sandFrontotemporal

Network(IT-DIAfN)",anationalproject fundedby the ItalianMinistryofHealth (RF-2010-2319722,

BandoRicercaFinalizzata2010).ItgatherssixcentresofexcellenceinthestudyofgeneticADandFTLD:

IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli of Brescia, IRCCS Fondazione Istituto

NeurologicoCarloBestaofMilan,UniversityofFlorence,CentroRegionalediNeurogeneticaofLamezia

Terme,UniversityofBrescia,andFondazioneIRCCSCàGrandaOspedaleMaggiorePoliclinicoofMilan.

Aworkinggroupwascomposedbygeneticists,psychologists,neurologists,andbioethicists,withatleast

one representative from each centre, with the collaboration of the Department of Health Sciences,

UniversityofGenovaandDivisionofMedicalGenetics,GallieraHospital,Genova.

Asafirststep,thegroupconductedasurveyofgeneticcounsellingprotocolsapprovedbythelocalethics

committees and in use at each centre. In particular, the group collected information regarding the

professionals involved in the genetic counselling team, tests used for the assessment, phases,

procedures,timelines,informedconsentforms,accesscriteriaandspecificrequestsfromthelocalethics

committees.Moreover,otherspecificissueswereaddressed,suchastheinformedconsent/refusalfor

storageofthebiologicalsamplesinabiobankforpossiblefutureresearchstudies.Literaturesearchand

experiencefromcentreswerecombinedtodevelopadecisiontreetoassistinthesearchofthemutation,

tobemoreaccurate,andtoreducetimeandcost.

Aliteraturesearchwasconductedtoanalyseguidelinesorrecommendationsavailableforothergenetic

diseases,suchasHuntington'sdisease[27,29,30],or inuse inothercountries. Italianregulationsfor

data protection were considered (The Italian Data Protection Authority, General Authorisation No.

8/2014fortheProcessingofGeneticData,doc.webNo.3632835).

InFebruary-March2013theworkinggroupparticipatedinthefirstmeetings(onein-personandonea

remoto, via teleconferences) aimed at identifying and discussing the surveyed differences and

commonalitiesamongprotocols.Thefirstdraftwasdiscussedduringanin-personmeetinginJuly2013

and circulated to the working group in the ensuing months. All comments and feedbacks were

periodicallysummarizedandcollatedbytheProjectCoordinator,whowasinchargeofmoderatingthe

discussion via electronic communication and of reconciling the different viewpoints to reach a

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consensus.ThefinalconsensuswasreachedinOctober2014followingimplicitaggregationmethods

[31]:thegroupexpertsineachfieldguidedthedecision-makingprocessandthefinalconsensuswas

defined by majority position. Further comments from SINdem (Italian Society for the study of

Dementias)werealsotakenintoaccount.

The harmonized protocol for genetic counselling was approved in February 2015 by the Ethics

CommitteeoftheCoordinatingCentreoftheIT-DIAfNproject(IRCCSIstitutoCentroSanGiovannidiDio

FatebenefratelliofBrescia).

3.RESULTS

3.1Surveyforlocalprotocols

TheresultsofthesurveyarereportedinTable1.Inallcentresgeneticcounsellingwasprovidedbya

multidisciplinary team, including a geneticist, a neurologist, a psychologist or a psychiatrist. The

schedule of the consultation and the procedures varied among centres. Three centres divided the

procedureintothreeconsultations:thefirstwasaninformationalmeeting,wherethefamilyhistorywas

collectedandthesubjectsunderwentaneurologicalandapsychologicalexamination;thesecondwas

thebloodsamplecollection,whilethethirdwasthedisclosureofthegenetictestresults.Theotherthree

centresdividedthepre-testphaseintotwovisits:oneinformationalandonefortheassessmentofthe

patient. The timetable for the visits and for the supportive follow-up varied from centre to centre.

Moreover,somecentresconsideredthefollow-upasnonmandatoryortheyoffereditonlyincaseof

positivityatthetest.Differenttestswereusedtoassessdifferentdomainslocally(Table1).Onlytwo

centres had a specific protocol for symptomatic subjects (diagnostic test), while all centres had a

protocolforat-riskrelatives.

3.2Harmonizedprotocolforgeneticcounselling

3.2.1Targetpopulation

Genetic counselling can be requested (and eventually stopped or suspended at any stage) by (i)

symptomaticpatientswithapositivefamilyhistorysuggestiveofanautosomaldominantgeneticcause

forADandFTLDand(ii)relativesofpatientswithprovengeneticADorFTLD.Eachsubjecthastobe≥

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18yearsold.Apositivefamilyhistoryisdefinedwithinthegroupbasedonthepresenceofoneofthe

followingcriteria:i)atleastthreeaffectedfirst-degreerelativesintwogenerations,irrespectivelyofthe

ageatonset; ii)at least twoaffectedfirst-degreerelatives intwogenerations,withat leastonewith

onsetat≤65years;oriii)oneaffectedfamilymemberwithonsetat≤60yearsorwithasuggestive

clinicalphenotype(e.g.dementiawithatypicalpresentation[32],recurringpresenceinotherrelatives,

peculiargeographicorigin).

Cognitivelyunimpairedat-riskrelativesenterthecounsellingandareeligibletoundergothegenetic

testingonlyaftertheprovenpresenceofthemutationinafirst-degreesymptomaticrelative.

Twospecificgeneticcounsellingprotocolsweredefined:oneforsymptomaticpatients(Section3.2.4)

andoneforat-riskrelatives,withamorerelaxedandprolongedschedule(Section3.2.5).

3.2.2GeneticCounsellingTeam

Geneticcounsellingisprovidedbyamultidisciplinaryteamofhealthprofessionalswhoworktogether

toprovideanindividualorafamilywithcurrentinformationandsupportivecounsellingaboutgenetic

testing.GeneticcounsellingisstructuredandguidedbyaGeneticistandbyaSpecializedMedicalDoctor

with specific competences in neurodegenerative diseases. The Geneticist is a specialist in medical

geneticswithexpertiseinneurodegenerativediseases,whohadeducationingeneticcounselling.The

SpecializedMedicalDoctor(Neurologist,Geriatrician,and/orPsychiatrist)istheclinicalcontactperson

forthepatient:thisrolecanbealsoplayedbythegeneticist,butonlyifhe/sheisamedicaldoctorwith

a consolidated clinical background for the aforementioned diseases. Beside the Geneticist and the

Specialized Medical Doctor, the multidisciplinary team is composed by other professionals (i.e. a

Psychologistand/oraPsychiatrist),whohaveexpertiseincounselling,andhaveskillsatprovidingon-

goingsupporttoindividualswithpossiblesocio-psychologicalconsequencesassociatedwiththeriskof

beingamutationcarrier.

3.2.3EvaluationTests

Acommonbatteryofclinical,psychologicalandcognitivetestswasdefinedforsymptomaticpatients

andtheirat-riskrelatives.Specifically,thebatteryincludestheMini-MentalStateExamination(MMSE)

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[33]andtheClinicalDementiaRatingScale(CDR)[34],theQuestionnaireontheHealthStatus-12(SF-

12)[35]andtheWorldHealthOrganizationQualityofLife(WHOQOL)[36],fortheassessmentofquality

oflifeandhealthstatus;theStateTraitAnxietyInventory(STAI-Y)[37],theBeckDepressionInventory

(BDI) [38] and the Hamilton Rating Scale for Depression (HRSD) [39], for anxiety and depression

measurement;theBriefCOPE(BC)[40],theResilienceScaleforAdult(RSA)[41]andtheHealthLocus

ofControl(HLC)[42,43],fortheevaluationofthecopingstyleandthelocusofcontrols.Moreover,a

depth evaluation of the risk of suicide is recommended, if the subject showed suicidal thoughts or

attitudesinthepastoratpresent(item9ofBDIand/oritem3ofHRSD).OnesuggestedscaleistheBeck

HopelessnessScale[44,45].Inadditiontothesetests,theat-riskrelativesarealsoassessedwiththeBig

FiveQuestionnaire(BFQ)forpersonalityassessment[46,47].

Thesubject isassessedatapre-testphase, to investigatehis/herabilitytocopewiththetestresult.

ExceptfortheMMSE,theCDRandtheBFQ,theassessmentwiththeothertestsisrepeatedpost-testat

differenttime-points,tomonitorthepsychologicalstatusofthesubject,asspecifiedlaterinthetextand

inFigure1A-B.

Basedontheprofileofthesubject,themultidisciplinaryteamcansuggesttopostponethegenetictest.

Theteamcontinuestofollowandsupporttheindividualaslongasitisdeemednecessarybytheteam

orrequestedbythesubject.

3.2.4ProtocolforSymptomaticSubjects

Thegenetictestingforsymptomaticsubjectshasadiagnosticpurpose,i.e.toidentifythegeneticcause

ofthediseaseandconfirmthediagnosisofADorFTLD.TheproceduresareillustratedinFigure1Aand

include:atleastonepre-testconsultation,onemeetingforbloodwithdrawalforthegenetictesting,a

post-testconsultation,wherethegeneticstatusisdisclosed(forthosewhowishtoknow)andthenthree

follow-ups.Thenumberoftheconsultationscanbeincreasedandthetimespanrelaxed,ifrequiredby

the patients of if considered appropriate by the team.Genetic counselling for symptomatic patients

shouldbeperformedinthepresenceofthecaregiveraccordingtopatient'swill,andtheindividual’s

legalguardianifappointedfortheindividualpatient,tohelpthepatientinthedecision-makingprocess,

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specificallytohelpinunderstandingandappreciatingthepurposeandpossibleresultsofthegenetic

testandinexpressingachoice[21,48].

3.2.4.1Firstconsultation:Pre-test

During the first informational visit, the whole multidisciplinary team provides general information

regardingthefamilialformsofdementiaanddetailsaboutgeneticcounselling(i.e.timespan,teamroles,

aimsandprocedures),thereliabilityofthetestandtheinterpretationoftheresults.Theteamshould

explainmedical andgenetic termsandavoid technical jargon.The implicationsofbothpositiveand

negativetestresultarediscussed,aswellas ifandhowtheseresultswillbecommunicatedtoother

familymembers.Patientsandtheirfamiliesshouldunderstandthatmultiplepossibleresultsmaybe

revealed:acausativemutationinoneofthegenes,nomutationinanyofthegenes,avariantofunknown

significance,orevenmutationsorvariants inmore thanonegenes. Supportive information is given

regarding the diagnosis and risk for the genetic disease in the family and its clinical, psychological,

sociological and ethical implications, togetherwith the impact on the familymembers. The specific

implications,prospectivebenefitsandrisksofundergoinggenetictestingarediscussed.Theaimisto

provideasmuchclearinformationaspossible,anddiscussanyquestionordoubt,toallowsubjectsto

takeanautonomousandinformeddecisionwhethertheyarewillingtoundergogeneticcounsellingand

testingandtoknowtheirgeneticstatus.Theteamavoidstoinfluencethedecisionoftheindividualand

thefamily,andaimsatfacilitatingtheirautonomousdecisionmaking.

If agreed, the subject, and/or his/her legal guardian on his/her behalf if appropriated, signs the

informedconsentformforgeneticcounselling.Thecaregiver,ifpresentaccordingtopatient'swishes,

canalsosigntheinformedconsenttoexpressagreement[48].Thefamilyhealthhistoryiscollectedto

constructthefamilypedigreeand,wherepossible,acompletefamilyhistoryshouldbecollectedusing

a structuredquestionnaire.Personal clinicalphenotypeand familyhistorywill beused toguide the

algorithm formutation search (see3.2.6Flowchart for genetic testing) and to evaluatewhether the

genetictestisappropriate(asaguide:[49,50]).

Finally,ifnottooseverelyimpaired,thesubjectisassessedusingthetestdescribedin3.2.3Evaluation

Tests.

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Aftertheconsultation,theteamprovidesthepatientwithawrittensummaryorbrochurewithallthe

general information discussed in person, to allow their elaboration and comprehension. In case of

doubts,asecondinformationalvisitcanbescheduled.

3.2.4.2Secondconsultation:BloodSampleCollection

At the beginning of the second consultation, thewhole team evaluateswhether all the information

provided during the first visit was fully understood, and it clarifies any remaining doubts or new

questionsthatmayarise.Theresultsoftheassessment(ifdone)arediscussed.Ifthepatientwishesto

proceedwiththetest,thesubject,and/orhis/herlegalguardianonhis/herbehalfifappropriated,signs

the informed consent for genetic testing and blood sample is collected. The caregiver, if present

accordingtopatient'swishes,canalsosigntheinformedconsenttoexpressagreement.Atthisstage,

thepatientcandecidewhetherhis/hersamplecanbestoredinabiobankforpossiblefutureresearch

studiesongeneticformsofdementiaordestroyedafterthecompletionoftheanalyses.

The team informs the patient about the timespan for the completion of molecular genetic analysis

(aroundtwomonths).

3.2.4.3Thirdconsultation:Post-test

Withintwomonthsfromthebloodsamplecollection,theresultsaremadeavailable.Ifthepatientstill

wishes toknow, the genetic test results aredisclosedanddiscussedwith thepatient, andawritten

reportisgiven.Thewholeteamshouldassesstheindividual’sunderstandingofhis/hersituationand

adapt the language to the patient in order to clearly explain themeaning and the implications of a

positive,negative,oruncleartestresult.

Theteamshouldsupportandassistthepatientandthefamilyinexpressingtheiremotionalreactions

to their genetic status. Before leaving the visit, the presence of psychological distress should be

informallyassessed.

3.2.4.4Follow-up

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Afterthedisclosureoftheresults,theteamofferson-goingsupportandinformation.Threedifferent

timepoints for the follow-upweredefined: after1,6and12months from thedisclosure.The team

collectsinformationaboutanypossiblechangeoccurredinthefamilyandinhis/hereverydaylife.The

patientisqualitativelyassessedabouthis/herpsychologicalconditions,andthetestsdescribedin3.2.3

EvaluationTestsareadministered,wheneverpossible.

3.2.5ProtocolforAt-riskSubjects

Thepredictivetestingeneticcounsellingaimsatconfirmingwhetherthegeneticmutationidentifiedin

thefamilyhasbeeninheritedbytheat-riskrelative,atastagewheretheindividualdoesnotpresent

anysymptom.Thisprotocolarisesmuchmoreethicalandpsychosocialissuesthatthediagnostictestin

symptomaticsubjects,becausethepotentialidentificationasbeingcarrierofanautosomaldominant

mutation almost certainly predicts the future development of the disease in an individual currently

healthy.Undergoingapredictivetestisahighlypersonaldecisionthatdemandsextensivecounselling.

TheproceduresareillustratedinFigure1Banddetailedbelow.Theschedulemainlydiffersfromthe

diagnostictestinprescribingatleasttwopre-testconsultationsbeforethegenetictesting,andinhaving

aphonecalloneweekafterthedisclosureofthegeneticstatus.

3.2.5.1Firstconsultation:Pre-test

Asforthesymptomaticpatients,thefirstconsultationisaninformationalandsupportingvisit,when

thewholeteamcoversspecificissuesofthefamilialformsofdementia,geneticcounsellingandtesting,

and the implications of the results (for further details see 3.2.4.1 First consultation: Pre-test). The

presenceofasupportpersonisencouraged.

If agreed, the subject signs the informed consent form for genetic counselling. Then, there is an

evaluationofthepersonalandfamilialmedicalhistory.

Aftertheconsultation,theteamprovidesthesubjectwithawrittensummaryorbrochurewithallthe

generalinformationdiscussedinperson,toallowtheirelaborationandcomprehension.

3.2.5.2Secondconsultation:Pre-test

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Afteronemonthfromthefirstvisit,thesecondoneisscheduled,tofurtherdiscussthemotivationfor

thepredictivetestandclarifyanydoubtswhichmayhavearisen.

Thesubjectisassessedusingthetestsdescribedin3.2.3EvaluationTests.

3.2.5.3Thirdconsultation:BloodSampleCollection

Theproceduresarethesameasforthesymptomaticprotocol(see3.2.4.2Secondconsultation:Blood

SampleCollection).

3.2.5.4Fourthconsultation:Post-test

Theproceduresarethesameasforthesymptomaticprotocol(see3.2.4.3Thirdconsultation:Post-test).

3.2.5.5Follow-up

Oneweekafterthedisclosureoftheresults,theteam(specificallythePsychologistand/orPsychiatrist),

contactsthesubjectbyphoneandofferson-goingsupportandinformation.Theotherfollow-upvisits

arethesameasforthesymptomaticprotocol(see3.2.4.4Follow-up).

3.2.6Flowchartforgenetictesting

Adecisiontreewasdevelopedtoassistinthesearchofthemutationinsymptomaticpatients(Figure

2).

IfthepatienthasrecentlyundergonealumbarpunctureandAβandtaulevelsinthecerebrospinalfluid

(CSF)havebeenanalyzed,thismeasureguidesthegeneticscreening.IftheCSFtauandAβ42levelsare

abnormal(i.e.tau/Aβ42>0.52)[51],geneticmutationslinkedtoADaresearchedasfirst.Todetermine

which gene should be sequenced first (i.e., APP, PSEN1, or PSEN2), patient’s age at onset (AAO) is

considered:ifthepatienthasanearlyageatonset(≤65years),APPgene(exons16-17)issequenced;if

negative: all PSEN1 exons and flanking regions; if negative: all PSEN2 exons and flanking regions.

Instead,ifthepatienthasalateageatonset,PSEN2geneisthefirstchoiceforsequencing,andthenAPP

and PSEN1. If all AD genes aremutation-negative, but there is a strong evidence for an autosomal

dominantpattern, thengenes linked toFTLDare screened.Plasmaprogranulindosage forGRN null

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mutationscreeninghasthepriority:ifthelevelis<61.55ng/ml[19],thepresenceofthecommonItalian

p.L271fs(exon8:c.811_814del,refsequenceNM_002087)mutationisassessed[52];ifnomutationis

found, all other exons are sequenced. Conversely, if the progranulin level is not suggestive for the

presenceofnullmutations (i.e.>61.55ng/ml), the clinicalphenotype is taken intoaccount.C9orf72

genetic screening should have the priority in patientswith FTLD andMotorNeuronDisease (FTD–

MND),behaviouralvariantfrontotemporaldementia(bvFTD)withpsychosis,orFTLDwithcerebellar

features,whileMAPTgene(exons1,9-13,asfirst)shouldbethefirstchoiceinpatientswithbvFTDand

semanticdementia,progressivesopranuclearpalsy(PSP),corticobasalsyndrome(CBS)oranearlyage

atonset(AAO<60years).WhenbothC9orf72andMAPTresultednegative,thenGRNshouldbetested,

givenpossiblefalsenegativeinprogranulinplasmascreening.

WhenCSF tau andAβ levels arenormal ornot available, the algorithm is basedon theprogranulin

dosage,asdescribedpreviously.Whenpatient'slevelofprogranulinisnormal,theclinicalphenotypeis

taken into account for guiding the analysis. If the clinical phenotype is FTD-like, then the priority

betweenC9orf72andMAPT isdefinedaspreviouslydescribed.Iftheclinicalphenotypeisconsistent

with AD, then, according to the age at onset, APP, PSEN1 and PSEN2 are screened as described

previously.Whennomutationisfound,theotherbranchinthediagramisfollowed(Figure2).

Lastly, if theDNAanalysis isdone inanat-risksubject,only themutation found in thesymptomatic

relativeissearchedfor.

4.DISCUSSION

Genetic testinghas ethical, social, legal andpsychological implications for patients and their family:

when handled by an experienced and qualifiedmultidisciplinary team following structured criteria,

these issues can be faced serenely and confidently by families. Here, we present a standard and

structured protocol for genetic testing and counselling for symptomatic and at-risk individuals

belonging to familieswithsuspected formsofautosomaldominantADorFTLD.Thisprotocol isnot

designed for genetic counseling of people who carry susceptibility genetic risk for AD, such as the

ApolipoproteinEe4allele,asthisisonlyariskfactorfordementiaandnodefinitivepredictioncanbe

made about dementia in these subjects. Moreover, based on the current knowledge, wewould not

15

recommendgeneticcounsellingtoallsporadicFTDcasesasthelikelihoodoffindingamutationincases

withanegativefamilyhistoryispresumablylow[49]andwouldthusunnecessarilyincreaseburdento

patientsandtheirfamilies.TheIT-DIAfNprotocolprovidesguidanceontheschedule,theprocedures,

thetestsandtheprofessionalfiguresinvolvedingeneticcounselling,withtheaimtosupportandtake

responsible care of these families. The tests for clinical, cognitive and personality assessmentwere

accuratelychosentoobtainaconcisebutaccurateprofileoftheindividual,helpinginpersonalizingand

adaptingthesupportwithinthecounsellingtohis/herpossiblereactions.

WehavetakenintoaccounttheguidelinesforthegenetictestingofHuntington'sdisease[27,29,30],

whichisconsideredthegoldstandardforgenetictestingforadultonsetdiseases.Asrecommendby

theseguidelines,weusedamultidisciplinaryapproachtofacilitatethesubject'sautonomousdecision

making: theGeneticistas theexpert ingeneticcounselling forADandFTLD, theSpecializedMedical

Doctorastheclinicalcontactpersonofthepatients;andthePsychologist/Psychiatristasthecontact

personforcounselling,psychologicalappraisalandsupport.Thescheduleandtimespanofthevisitsare

similartothosedefinedforHuntington'sdisease:severalsessionsandphases(pre-,post-testingand

follow-ups)arecrucialtoallowfreechoice,withoutrushingintogenetictestingbeforeallthenecessary

explanationsandinformationaregiven.OtherrecommendationsprescribedintheHuntington'sdisease

guidelineswereadoptedhere:i)disclosureofthegeneticstatusisprovidedbothorallyandthrougha

writtenreport; ii)availabilityof the testonly to individualswhohavecomeofage; iii)possibilityof

storingthebiologicalsamplesinabiobankforresearchstudiesaftersigningtheinformedconsent.

AsdescribedinotherexistentcounsellingprotocolsforADandFTLD[21,22],werecommendedthati)

the legal guardian of the symptomatic patient is present during the counselling to help her/him in

understandingitspurposeandpossibleresultsofthegenetictestandinexpressingachoice,andthat

ii)asymptomaticfamilymembershouldbetestedbeforeanat-riskindividual.

TheguidelinesforgeneticcounsellingforADdefinedbytheAmericanCollegeofMedicalGeneticsand

theNational SocietyofGeneticCounsellors [21] specified thatgenetic counselling couldbedone in-

personorthroughvideoconferences.TheHuntington'sdiseaseguidelinesclearlystatedthattheresults

ofthetestshouldberevealedin-person,andnotbyphoneormail[27,30],whilepsychosocialsupport

16

shouldbeavailableclosetotheperson’scommunity,byphoneortelemedicinewherenecessary.Afew

pilotprojectsdemonstratedthattelemedicinecanbeusedtooffermedicalgeneticsserviceonaremote

basis [53]. Preliminary evidence suggests that genetic counselling can be effectively delivered in

underserved areas, also in late-onset genetic disorders such as familial cancer [54]. The advent of

telegenetics could allow the implementation of this genetic counselling protocol in clinical research

centreswithoutloweringtheclinicalstandard;itmayalsofacilitatetheenrolmentinclinicaltrialsof

individualswhocouldnotaccesstotertiarycaregeneticscentres.Nevertheless,itisrecommendedthat

thePsychologist/Psychiatristshouldbealwayspresentinpersonduringthevisits.

We developed two specific paths, one for symptomatic patients and one for asymptomatic at-risk

subjects,beingawareofthedifferenceofthetwoscenarios. Infact, insymptomaticsubjects,genetic

testingisaimedatconfirmingtheclinicaldiagnosisandthegeneticcauseofthedisease.Apositiveresult

canprovokehopelessnessandsenseofguiltyforthepossibilityoftransmissionofthemutationtothe

offspring.Inthecaseofasymptomaticsubjectspsychological,socialandethicalissuesareevenmore

critical,especiallyintheabsenceofpreventionortreatmentoptions:acorrectandcarefulcounselling

willofferthemtheoptionofknowingtheircondition,copepositivelywiththeirfutureandeventually

theymightbeofferedthepossibilitytoenteranovelpreventionclinicaltrial.Itmustbeunderlinedthat

a negative result has differentmeanings according to the twodifferent scenarios: in a symptomatic

subject,thiscouldnotexcludedefinitelythatthediseasehasageneticcause(thusitissporadic),because

an unknown/newmutation could be present but not yet been discovered; in an at-risk relative, a

negative result confirms almostwith absolute certainty that he/she has not inherited the causative

mutationfoundintheaffectedrelativeandthushe/shehasthesameriskofthegeneralpopulationto

developthediseaseinthefuture.

Considering the results reported in Binetti et al., 2006 [20], Italian families with familial dementia

showedpoorknowledgeof thediseaseandpoorawarenessofpersonalriskofdevelopingdementia

duringlifetime.Thismeansthatthefirstconsultationshouldaddresstheseaspectsindetails,guiding

familiestoafreeandinformedchoice.

17

Wedevelopedastep-by-stepalgorithmforguidinggeneticscreeningonthebasisofbiomarkerresults

andclinicaldata,toreducetimeandcostofthelaboratoryanalysis.Specifically,thedecisiontreewas

definedaccordingto(i)theobservedfrequencyofgenesinspecificclinicalphenotypesintheliterature

[49,55-58],andspecificallyinItalianclinicalseries[52,59-65],(ii)thedirectexperienceoftheItalian

centres,and(iii)practicalconsiderations,e.g.thehighspeedandlowcostofagivenscreeningprocedure

(i.e. plasma progranulin dosage). In the future, other biomarkers can be taken into account and

implementedwithintheflowchart,suchastheamyloidPET.

TheresearchprotocolreportedhereinreflectsthecurrentpracticeofItaliancentrestakingparttothe

IT-DIAfN initiative. Should important innovationsbe introduced in the clinicalpracticeaswell as in

laboratoryanalyses,theprotocolwillbeamended.Followingthewideavailabilityofnextgeneration

sequencing (NGS)panels for themolecular genetics analysis, thedecision tree is subject to changes

accordingly.NGSisnotcurrentlyappliedinallcentersparticipatingintotheIT-DIAfNframework,but

appearsastheupcomingtechnologyforthemutationanalysisinheterogeneousgeneticdisorderssuch

asADandFTLD.Todate,theNGSapproachprovidesthesimultaneousanalysisofapanelof17genes

(PRNP, PSEN1, PSEN2, APP, GRN, MAPT, TREM2, CHMP2B, CSF1R, FUS, ITM2B, NOTCH3, SERPINI1,

TARDBP, TYROBP, VCP, SQSTM1). Other experimental panels of additional genes and SNP

polymorphisms are currently under development within the IT-DIAfN group. The mutation search

shouldbecompletedwithtwoPCRanalysesoftheC9orf72hexanucleotiderepeatexpansionandthe

octapeptiderepeatregionofPRNP[66].

Thisprotocol,developedbyexpertsinthefield,providesItaliancentreswithalineofsharedpractice

forofferinggeneticcounsellingtoeligibleindividualswhomaybenefitfromgenetictestingforfamilial

AD and FTLD, and recruit them through a procedure which is compliant with International

recommendationsandgoodpractices.

Future steps will be the validation of the harmonized protocol of genetic counselling. This will be

performedintwosteps:i)afterapprovalbylocalethicscommittees,theprotocolwillbeimplemented

inallcentresparticipatingintotheIT-DIAfNproject;ii)datafromeachcentrewillbecollectedandthe

resultsfromthepilotstagewillbecriticallyreviewedbytheworkinggroupwhodevelopedtheprotocol.

18

Thegeneticcounsellingprotocolwillalsobedisseminatedthroughmedicalsocietiesandproposalsfor

amendmentswillbeacknowledged.

Weare aware that theentireprocedure, including counselling, repeatedassessments and testing, is

highly demanding, especially in terms of human resources. The procedure is expected to be

accomplished within a research environment by a multidisciplinary team in centres with specific

expertise–assuchitwasnotdesignedtobeappliedinroutineclinicalpracticeinitscurrentversion.In

thelightofthesuggestionraisedduringthevalidationphase,anoptimizedprotocolcouldbeeventually

transferred to clinics, provided that evidence of clinical utility has been produced and a health

technologyassessmenthassupporteditsimplementationinclinicalpractice.

5.FUNDING

Thisstudywasfundedthrough"ItalianDominantlyInheritedAlzheimer’sandFrontotemporalNetwork

(IT-DIAfN)", a project funded by the Italian Ministry of Health (RF-2010-2319722, Bando Ricerca

Finalizzata2010)andcoordinatedbyGiovanniB.FrisoniattheIRCCSIstitutoCentroSanGiovannidi

DioFatebenefratelli,Brescia,Italy.

19

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28

Table1.Surveyofthegeneticcounselingprotocolsinuseatthelocalcentres.

IRCCSIstitutoCentroSan

GiovannidiDioFatebenefratelli

Brescia

IRCCSFondazioneIstituto

NeurologicoCarloBestaMilano

SpedaliCivili/UniversitàdiBrescia

UniversitàdiFirenze

CentroRegionaleNeurogeneticaLameziaTerme

FondazioneIRCCS

CàGrandaOspedaleMaggiorePoliclinicoMilano

Schedulingandphases

3Consultations(+followup)

3Consultations(+followup)

4Consultations(+followup)

4Consultations(+followup)

4Consultations(+followup)

3Consultations(+followup)

1-Informationalmeeting,Familyhealthhistorycollection,psychosocialassessment,neurologicalexamination,

1-Informationalmeeting,Familyhealthhistorycollection,psychologicalquestionnaires,neurologicalexamination

1-Informationalmeeting,Familyhealthhistorycollection

1-Familyhealthhistorycollection,neuropsychologicalassessment,neurologicalexaminationandinformationalmeetingonthedisease

1-Familyhealthhistorycollection,neuropsychologicalandpersonalityassessment,neurologicalexamination

1-Informationalmeeting,Familyhealthhistorycollection,psychologicalquestionnaires,neurologicalexamination

2-Psychologicalassessment,neuropsychologicalassessment,neurologicalexamination

2-Informationalmeetingonthegeneticriskandthegenetictest

2-Informationalmeeting

2-Bloodsamplecollection

2-Bloodsamplecollection

3-Bloodsamplecollection 3-Bloodsamplecollection

3-Bloodsamplecollection

2-Bloodsamplecollection

3-Disclosureofgenetictestresult

3-Disclosureofgenetictestresult

4-Disclosureofgenetictestresult

4-Disclosureofgenetictestresult

4-Disclosureofgenetictestresult

3-Disclosureofgenetictestresult

29

ProfessionalsintheTeam

Neurologist,Geneticist,Psychiatrist,Psychologist

Neurologist,Geneticist,Psychiatrist,Psychologist

Neurologist,Geneticist,Psychiatrist,Psychologist

Neurologist,Geneticist,Psychiatrist,Psychologist

Neurologist,Geneticist,Psychiatrist,Psychologist

Neurologist,Geneticist,Psychiatrist,Psychologist

Timetable60daysrequiredforthegenetic

testNotdefined Notdefined

About1monthbetween

firstandsecondconsultation;about1monthbetweenthirdandforthconsultation

Bloodsampleistakenwithin4weeksfromthe

secondconsultationNotdefined

FollowupAt12and24months

(irrespectiveoftestresult)

At6/12months(notmandatory)

At15and30days(incaseofpositivityatthe

test)

At1,3,6and12months(irrespective

oftestresult)

At1,3,6and12months

(irrespectiveoftestresult)

At6/12months(notmandatory)

Clinical/Psychological,CognitiveandPersonality

Test

BDI,STAI-Y,BFQ,GHQ,ISS,HLC,SPQ,SLEQ,WHOQOL-100,

SDABE

Psychologicalself-

administeredQuestionnaires

Semi-structuredinterview,MMPI-2,STAI,ZUNG,

Neuropsychologicaltests

Neuropsychologicalassessment

MMPI,MentalDeterioration

Battery

Psychologicalself-

administeredQuestionnaires

ProtocolsDiagnosticandPredictiveprotocol

Predictiveprotocol

DiagnosticandPredictiveprotocol

Predictiveprotocol Predictiveprotocol Predictiveprotocol

Abbreviations:BDI,BeckDepressionInventory[38];STAI-Y,StateTraitAnxietyInventory[37];BFQ,BigFiveQuestionnaire[46,47];GHQ,General

HealthQuestionnaire[67];ISS,InformationSeekingStyle[68];HLC,HealthLocusofControl[42];SPQ,SocialProblemQuestionnaire[69];SLEQ,

StressfulLifeEventsQuestionnaire[709];WHOQOL-100,TheWorldHealthOrganizationQualityofLifeassessment[71];SDABE,DementiaAttitudes

30

BeliefsandExperiences[20,72,73];MMPI-2,MinnesotaMultiphasicPersonalityInventory-2[74];STAI,StateTraitAnxietyInventory[75];ZUNG,Zung

self-ratingdepressionandanxietyscales[76,77];MinnesotaMultiphasicPersonalityInventory[78];MentalDeteriorationBattery[79].

31

Figure1.GeneticCounsellingprotocol.Flowchartforsymptomaticsubjects(A)andforat-riskrelatives

(B).

A)

32

B)

33

Figure2.GeneticTestingFlowchart.Step-by-stepalgorithmtoassistinthesearchofgeneticmutations,forcaseswithabnormalAβandtaulevelsin

thecerebrospinalfluid(CSF)(leftpanel)andwithnormalCSFvaluesordatanotavailable(rightpanel).

34