Post on 10-Feb-2020
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GenomicsequencingidentifiessecondaryfindingsinacohortofparentstudyparticipantsShortrunningtitle:SecondaryandIncidentalFindingsfromWES/WGSMichelleL.Thompson,PhD1§,CandiceR.Finnila,PhD1§,KevinM.Bowling,PhD1,KyleB.Brothers,MD,PhD2,MatthewB.Neu,BS1,3,MichelleD.Amaral,PhD1,SusanM.Hiatt,PhD1,KellyM.East,MS,CGC1,DavidE.Gray,MS1,JamesM.J.Lawlor,MS1,WhitleyV.Kelley,MS,CGC1,EdwardJ.Lose,MD3,CarlaA.Rich,MA2,ShirleySimmons,RN3,ShawnE.Levy,PhD1,RichardM.Myers,PhD1,GregoryS.Barsh,MD,PhD1,E.MartinaBebin,MD,MPA3,GregoryM.Cooper,PhD1*1HudsonAlphaInstituteforBiotechnology,Huntsville,AL,USA2UniversityofLouisville,Louisville,KY,USA3UniversityofAlabamaatBirmingham,Birmingham,AL,USA§Theseauthorscontributedequallytothiswork.*CorrespondingAuthor:601GenomeWay,Huntsville,AL35806;256-327-9490;gcooper@hudsonalpha.org
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ABSTRACTPURPOSE:Clinicallyrelevantsecondaryvariantswereidentifiedinparentsenrolledwithachild
withdevelopmentaldelayandintellectualdisability.METHODS:Exome/genomesequencing
andanalysisof789‘unaffected’parentswasperformed.RESULTS:Pathogenic/likelypathogenic
variantswereidentifiedin21geneswithin25individuals(3.2%),with11(1.4%)participants
harboringvariationinagenedefinedasclinicallyactionablebytheACMG.Ofthe25individuals,
fivecarriedavariantconsistentwithapreviousclinicaldiagnosis,thirteenwerenotpreviously
diagnosedbuthadsymptomsorfamilyhistorywithprobableassociationwiththedetected
variant,andsevenreportednosymptomsorfamilyhistoryofdisease.Alimitedcarrierscreen
wasperformedyielding15variantsin48(6.1%)parents.Parentswerealsoanalyzedasmate-
pairstoidentifycasesinwhichbothparentswerecarriersforthesamerecessivedisease;this
ledtoonefindinginATP7B.Fourparticipantshadtwofindings(onecarrierandonenon-carrier
variant).Intotal,71ofthe789enrolledparents(9.0%)receivedsecondaryfindings.
CONCLUSION:Weprovideanoverviewoftheratesandtypesofclinicallyrelevantsecondary
findings,whichmaybeusefulinthedesign,andimplementationofresearchandclinical
sequencingeffortstoidentifysuchfindings.
Keywords:Secondaryfindings;genomicsequencing;diseaserisk;CSER;ACMG
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INTRODUCTIONWholeexomeandgenomesequencing(WES/WGS)haveproventobepowerfultestsfor
identifyingclinicallyrelevantgeneticvariation.Theexistenceofsecondaryandincidental
findingshascatalyzeddebateregardingthetypesoffindingsthatshouldbesoughtby
sequencinglabs,thecircumstancesinwhichcertaintypesofvariantsshouldbereturned,and
thenecessaryextentofpatientconsent,education,andgeneticcounseling.TheAmerican
CollegeofMedicalGeneticsandGenomics(ACMG)releasedrecommendationsaboutthe
interpretationofvariantsingenesconsideredtobeclinicallyactionable,includingthosethat
posehighriskofcancerandheartdisease.TheACMGsuggeststhatthesebesoughtand
providedtopatientsthatconsenttoreceivesuchresults1,2.Recommendationsrelatedtouse
ofspecificgenelistsandapproachesforreturningsecondaryfindingswereintendedtobeused
inclinicalcontexts,althoughitisalsoimportanttoexaminethemintranslationalresearch
contexts.
ThroughastudythatwaspartoftheClinicalSequencingExploratoryResearch(CSER)
Consortium3,weassessedtheutilityofWES/WGStoidentifygeneticcausesofdevelopmental
delay,intellectualdisability(DD/ID),andrelatedcongenitalanomalies.Wehavesequenced
affectedprobandsfrom455families,andhaveidentifiedDD/ID-relatedpathogenic/likely
pathogenic(P/LP)variantsin29%ofcases4.AsourDD/IDstudyincludesproband-parenttrios,
wehavetheabilitytoassesssecondaryfindingsinasizablecohortofadults4.
Weusetheterm‘secondaryfindings’throughoutthemanuscripttodescribevariation
identifiedviaproactivesearching5andreportratesandtypesofsecondaryfindingsincontext
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ofreportedsymptomsorfamilyhistory.Ourexperiencesanddatasuggestthevalueofgenomic
sequencinginaclinicalsettingnotonlyfordiseasepatients,butalsoforthosenotcurrently
exhibitinganovertdiseasephenotype.Wedemonstratetheutilityofdisseminationofsuch
findingsinacohortofparentstudyparticipants,andhighlightthisthroughcasestudyanalyses.
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METHODS
Studyparticipantpopulation
Therewasnopublicrecruitmentforthisstudy.Parentandchildren(n=455families)
participantswereenrolledatNorthAlabamaChildren’sSpecialistsinHuntsville,AL.Consent
wasobtainedforstudyparticipationandpublicationofdatageneratedbythisstudy.Review
boardsatWesternInstitutionalReviewBoard(20130675)andtheUniversityofAlabamaat
Birmingham(X130201001)approvedandmonitoredthisstudy.
Patientpreferencesandconsent
WedevelopedthePreferencesInstrumentforGenomicSecondaryResults(PIGSR)6toelicit
parents’preferencesforreceivingcategoriesofsecondaryresults.Thisinstrumentdivides
secondaryfindingsinto13distinctdiseasecategories(Figure1).Resultswerereturnedto
parentparticipantsonlywhentheyoptedtoreceivesecondaryfindings.Decisionsregard
disclosureofsecondaryfindingssolelyintheprobandwerebasedonacombinationofparent
preferencesforthemselvesandmedicalrelevancetotheprobandduringchildhood.Inthecase
ofadoptedprobands,preferencesweresolicitedfromtheadoptiveparentsonbehalfofthe
proband.
Phenotyping
Atenrollment,ageneticcounselorgeneratedathree-generationpedigreebasedonfamily
historyreportedbytheparents/guardiansoftheproband.Parents’healthrecordswerenot
availabletothestudynorwasaphysicalexamperformed.Thegeneticcounselorasked
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questionsrelatedtofamilyhistoryofcancerandsudden/unusualdeathsofadults(e.g.cardiac
arrestoraneurysm).Cascadesequencingwasnotconductedaspartofthisstudy.Wehave(1)
retainedthelanguageusedbytheparticipantand(2)includedanyreportedfamilyhistorythat
isplausiblyrelatedtothephenotypeofconcern.
Returnofresults
Parentparticipantsthatreceivedsecondaryfindingswerescheduledforprivatedisclosurewith
amedicalgeneticistandgeneticcounselor.Theclinicalsignificanceoffindingswasaddressed
anddocumentsdetailingvariantinformationandrelevantresourceswereprovided.Secondary
findingswerenotbydefaultplacedintheparticipant’smedicalrecordandnoformalreferrals
torelevantspecialistsweremade.Iftheparticipantschosetoshareresultswiththeir
healthcareprovider,formalreferralscouldbecoordinated.
Sequencingandvariantinformation
FurtherdetailsregardingWES/WGS,readalignment,variantcalling,filtering,classification,and
validationcanbefoundinourpreviousreport4andinSupplementalMethods.Briefly,we
identifiedsecondaryvariationinACMGgenes1,2;P/LPvariationinClinVar(notinACMGgenes);
recessivevariationinindividualswhoharboredtwoormoreP/LPvariantsinthesamegene;
variationinOMIMgenesinwhichbothparentsofparentalpairharboredP/LPvariationforthe
samerecessivedisorder;andcarrierstatusinformationinCFTR,HEXA,andHBB.OnlyP/LP
variantswerereturned.
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DatasharingIdentifiedvariantsinparentparticipantshavebeensharedthroughClinVaranddbGaP,with
consent.AdditionalinformationisprovidedinSupplementalMethods.
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RESULTS
Demographicsofstudypopulation
Of455enrolledfamilies,424includedatleastoneparent,andbothparentswereavailablefor
365families.Demographicsforthe789parentparticipantsarereportedinTable1.Thestudy
populationhadameanageof41yearsandincluded422femalesand367males.80.5%self-
reportedtobeofEuropeanancestry(“White”),8.5%asAfrican-American(“Black”),and8.2%as
“OtherorMultiracial”.Over25%hadahighschooldiplomaorless,while34.5%reportedsome
collegeeducation(Table1).
PatientPreferences
Onegoalofourstudywastounderstandpatientpreferencesastheyrelatetoreceiptof
secondaryfindingsacrossvariousdiseasecategories6.85%ofparentsrequestedallsecondary
findings,while1.6%declinedtoreceiveallfindings.Themostfrequentlyrequestedcategory
wasriskforgender-specificcancers(breast,ovarian,testicularandprostate;n=584,96.1%).The
leastfrequentlyrequestedresultwasriskfordevelopingobesity(n=542,89.2%)(Figure1).
Carrierstatusfindings
Weconductedalimitedcarrierscreenforvariantsrelevanttocysticfibrosis(CFTR,MIM:
219700),beta-thalassemia(HBB,MIM:613985),sicklecelldisease(HBB,MIM:603903),and
Tay-Sachsdisease(HEXA,MIM:272800),whichareamongthemostcommonMendelian
diseases(averagecarrierriskis1/40)7-9.WeobservedeightP/LPvariantsinCFTRacross35
individuals(4.4%ofparentcohort),fourHEXAvariantsacrossfiveindividuals(0.6%),andthree
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HBBvariantsacrosseightindividuals(1%)(Table2;TableS2).Additionally,wesearchedfor
casesinwhichparental“matepairs”werebothcarriersforvariantsinageneassociatedwitha
recessivedisorder.Thisanalysisledtoreturnablefindingsforoneofthe365parentalpairs;
bothparentsharboredvariationinATP7BassociatedwithWilsondisease(MIM:277900)(Table
S2).
Secondaryvariationinindividualswithapreviousclinicaldiagnosis
P/LPvariantswerefoundinfiveindividualswithaself-reportedpreviousclinicaldiagnosisbut
inwhomaspecificgeneticcausewasunknown.A35-year-oldfemaleindividualwasfoundto
harboraheterozygousmissensevariantinSLC4A1(spherocytosis,MIM:612653),andhad
familyhistoryofrelateddisease(Table3;TableS1).Weidentifiedthreemissensevariants(two
likelyincis)inSLC22A5ina37-year-oldfemalewithrecessivesystemicprimarycarnitine
deficiency(MIM:212140).Finally,acanonicalsplicedonorsite(D1)variantaffectingPKD2was
identifiedina36-year-oldfemalewithpolycystickidneydisease(MIM:613095).Thisindividual
alsoreportedafamilyhistoryofdisease(Table3;TableS1).
Secondarygeneticvariationaffectingcardiacgeneswasidentifiedintwoindividualswitha
previousclinicaldiagnosisandhadafamilyhistoryofcardiovascularphenotypes.One30-year-
oldfemalereportedtohaveexperiencedcardiomyopathypostpartum,hadapaternalfamily
historyofarrhythmia,andherpaternalunclesufferedtwo“heartattacks”priortoage40.She
wasfoundtoharboraframeshiftvariantinDSG2,ageneassociatedwitharrhythmogenicright
ventriculardysplasiaanddilatedcardiomyopathy(MIM:610193,MIM:612877).AlthoughDSG2
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hasnotpersebeenassociatedwithperipartumcardiomyopathy(PPCM),wefinditprobable
thatthevariantexplainsherdiseasehistory.TheclinicalsymptomsofPPCMaresimilartothat
ofdilatedcardiomyopathy10andothergeneticvariantsassociatedwithdilatedcardiomyopathy
mayrepresentsusceptibilityfactorsforPPCM11.Ina52-year-oldmalewithhypertrophic
cardiomyopathyandarrhythmia,weidentifiedmissensevariationinANK2,ageneassociated
withankyrin-B-relatedcardiacarrhythmiaandlongQTsyndrome(MIM:600919).Itisunknown
whetherthisindividualpresentswithlongQTintervals.Additionally,althoughnotclearly
relatedtoANK2variation,thisindividualalsoreportedhisfatherhadischemicheartdisease.
Finally,sixoftheeightparentscarryingP/LPvariationinHBBreportedhavingsicklecellor
thalassemiatraitattimeofenrollment(Table2;TableS2).
Secondarygeneticvariationinsymptomaticindividualsand/orthosethathavefamilyhistory
ofdisease
Weidentifiedsecondaryvariantsin13individualswithnopreviousdiagnosisorgenetictesting
despitethemanifestationofdiseaseand/orfamilyhistory(Table3;TableS1).Given
informationprovidedattimeofenrollment,sixofthesecases(CLCN1,MFN2,BRCA1,BRCA2,
BARD1,PMS2;Table3)wouldhavemetcriteriatojustifygeneticconsultationandtestingvia
standardclinicalrecommendations12,13.Givenadditionalphenotypicinformationacquiredat
returnofresults,twoadditionalcases(SCN4A,HARS;Table3)wouldhavemetsuchcriteria14,15.
Theseeightcasesaredescribedbelow.
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AheterozygousmissensevariantinCLCN1wasidentifiedina29year-oldfemalewhoreported
legcrampsandrestlesslegsbeginninginchildhood.VariationinCLCN1associateswith
myotoniacongenita(MIM:160800)characterizedbymusclestiffnessandinabilitytorelax
musclesaftervoluntarycontraction,symptomsthatareexacerbatedbycoldertemperatures.
Hermotherwasdiagnosedwithmyotoniacongenitawhenshewas10yearsoldandher
maternalgrandfatherhadamusclebiopsyperformedinhis30sduetopresentationof
symptoms,including“stiffness”thatoccurred“especiallyincold[temperatures]”.Inaseparate
case,aheterozygousmissensevariantinMFN2(Charcot-Marie-Tooth(CMT)Diseasetype
2A2A,MIM:609260)wasidentifiedina35-year-oldfemalewhoreportedbalancedifficulties
andweaknesssincechildhoodthathasprogressedtoseverecramping,myalgia,andnumbness
mostprominentlyinlowerextremities,andisexacerbatedbyexercise.Herfamilyhistoryis
notableforneuromusculardisorder,withsimilarsymptomspresentinherbrother,father,
paternalgrandmother,andpaternalaunt.Thoughaclinicianhasnotformallyevaluatedher,
shereportedthatherbrotherwasdiagnosedwithCMT.
Wealsoidentifiedcancerriskvariantsinanumberofindividualswhoreportfamilyhistoryof
cancer.WeidentifiedaframeshiftvariantinBRCA1(familialbreast/ovariancancer,MIM:
604370)ina40-year-oldmalewhosemotherwasdiagnosedwithbreastcancerinherthirties.
Inanothercase,variationinacanonicalacceptorsplicesiteofBRCA2(familialbreast/ovarian
cancer,MIM:612555)wasidentifiedina38-year-oldfemalewhohadahistoryofbreastcancer
onbothsidesofthefamily-paternalgrandmother(diagnosisatunknownage)andmaternal
grandfather(age60).AframeshiftvariantinBARD1(MIM:114480)wasidentifiedina33-year-
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oldfemalewhosematernalgrandmotherhadbladder,lung,andperitonealcanceraswellasa
great-grandmotherdiagnosedwithbreastcancerinherfifties.Additionally,aframeshiftvariant
inPMS2(hereditarynonpolyposiscolorectalcancer;MIM:614337)wasidentifiedina43-year-
oldmalewithfamilyhistoryofcoloncancer-father(diagnosedinhissixties)andpaternalaunt
(forties).Thisindividualalsohadapaternalauntandgrandmotherwhowerediagnosedwith
breastcancerintheirsixtiesandfifties,respectively.Afterreceiptofthisfinding,thestudy
participantfollowed-upwithacolonoscopy,foundtobenegative.Hereportsthathewill
continueperiodicassessment.
Secondaryvariantswerealsoidentifiedintwosymptomaticindividualswhowerenotaware
thattheirsymptomswereunusualandthusneverhadclinicalorgeneticevaluation(Table3).
Atenrollment,neitherindividualreportedrelevantphenotypestothevariantsidentified.In
onecase,a28-year-oldfemalewasfoundtoharborapathogenicmissensevariantinSCN4A,
implicatedinhyperkalemicperiodicparalysisandparamyotoniacongenita(MIMs:170500;
168300),neuromusculardisorderscharacterizedbyintermittentmuscleweaknessand/or
myotonia.Atresultsreturn,shereportedahistoryofpainfulstiffnessduringexercisethat
beganatapproximatelyagefiveandthatherthroat“locksup”afterdrinkingcoldliquids.
Additionally,shereportedthathereyelids“stick”and“becomeheavy”throughouttheday.She
notedthathermotherdisplayssimilarphenotypes.Thisindividualhasplanstofollow-upwitha
neurologist.Inasecondcase,a41-year-oldmalewasfoundtoharborpathogenicvariationin
HARS,associatedwithCharcot-Marie-Toothdisease(MIM:616625)characterizedbygait
difficultiesandsensoryimpairmentcausedbyperipheralneuropathy.Atreturnofresultsthis
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individualindicatedthathewas“clumsy”,dischargedfrommilitarybootcampduetohis
inabilitytomarchinformation,andoftenwearsoutshoesbecauseoffeetshuffling.
Identificationofgeneticriskfactorsinindividualswhoarecurrentlyasymptomaticandhave
nofamilyhistoryofdisease
WealsoidentifiedP/LPvariantsinindividualsthatarecurrentlyasymptomaticandnofamily
historyofdisease(Table3).Twounrelatedindividuals,a52-year-oldfemaleanda50-year-old
male,werefoundtoharborvariationinSCN5A(LongQTsyndrome,MIM:603830)andDSG2
(dilatedcardiomyopathy,MIM:618277),respectively.A31-year-oldmalestudyparticipantwas
foundtoharboramissensevariantinACTN1,associatedwithableedingdisorder(MIM:
615193).Finally,P/LPcancer-associatedvariantswereidentifiedinfourparticipantswithno
personalorfamilyhistory,includingoneineachofMSH2,BARD1,BRCA2,andRET(Table3;
TableS1).Notably,apathogenicmissensevariant(C609Y)inRET,associatedwithmultiple
endocrineneoplasiatype2A(MEN;MIM:171400),medullarythyroidcarcinoma(MTC;MIM:
155240),and/orHirschsprung'sdisease(MIM:142623),wasidentifiedina52-year-oldmale
participantwhoreportednohistoryofRET-associatedcancer.C609Yhasbeenobservedin
manyMTC-affectedindividualsandhasbeendesignatedaslevelBriskfromtheAmerican
ThyroidAssociation(levelDishighestrisk),withexpectedageofonsetoflessthan30years
16,17.RecommendationsforC609Ycarriersvarybutoftenincludeprophylacticthyroidectomyat
ayoungage18,19.However,morerecentstudiesindicateRETC609Ymayhavelowerpenetrance
orlateronsetofMTCthanpreviouslynoted20,21,whichareconsistentwiththeobservationof
nopersonalorfamilyhistoryofcancerinthisfamily.Interestingly,whileC609Ywasnot
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transmittedtotheenrolled,developmentallydelayedproband,thefamilyreportedthatthey
haveanotherdaughter(notenrolled)whohasHirschsprung’sdiseaseandisthereforelikelyto
haveinheritedC609Y.Thefamilywasreferredforgeneticcounselingtotestforthevariantin
theHirschsprung’s-affecteddaughterandthatboththefatheranddaughterfollowupwith
oncologists.
SecondaryfindingsinDD/ID-affectedchildren
Forthreeenrolledchildren,weidentifiedsecondaryvariationnotinheritedfromaparent.Two
individualswhosebiologicalparentswerenotavailableharboredpathogenicvariationinCFTR
(Phe508del)andBRCA2(Leu579*),respectively.Also,asix-year-oldfemaleharboreda
pathogenicdenovovariantinFBN1(Asn2144Ser).Attimeofanalysis,thisprobanddidnot
exhibitMarfanphenotypes(MIM:154700),withexceptionofcrowdedteethandscoliosis.In
threeadditionalprobands,compoundheterozygousvariationassociatedwithrecessivedisease
wasidentified.TwoP/LPvariants,oneinheritedfromeachparent,inOCA2(oculocutaneous
albinismtypeII,MIM:203200)wereidentifiedinaneleven-year-oldmaleandhissix-year-old
brother;bothpresentedwithalbinism.Inathirdcase,anine-year-oldfemalewithcataracts
wasfoundtoinheritaP/LPvariantfromeachparentinFYCO1,ageneassociatedwithcataract
18(MIM:610019).
DISCUSSION
TheACMGestimatedthatsecondaryfindingsingenesrelevanttoadefinedlistofactionable
phenotypes(e.g.cardiacarrhythmias,cancers)wouldbefoundin~1%ofsequencedindividuals
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1,2.WeobservedvariationinACMGgenesin1.4%ofparentparticipants,consistentwiththat
estimateandthe1%-5.6%reportedbyotherresearchandclinicallaboratories22-25.
Ourstudyassessedcarrierstatusinallparticipantsforonlythreegenes,HBB,HEXA,andCFTR,
leadingtotheidentificationofP/LPvariationin~6.1%ofparentparticipants.Thesegeneswere
selectedbasedontheiranticipatedfrequenciesinthepopulationsampledandourdesireto
balanceyieldwithanalyticalandcostburden.Hadweassessedallgenesknowntoassociate
withrecessivedisease26,theburdenofanalysiswouldhaveincreasedsubstantially27,28.
Further,expandedcarrierscreeninganddiscoveryeffortswouldhaveincreasedSanger
validationcostsandthetimerequiredfromgeneticcounselorsandmedicalgeneticistsfor
returnofresults.Thus,whileourchoiceofgenesastargetsforcarrieranalysiswassemi-
arbitrary,therestrictiontoonlythreegenesimposedminimalanalyticalburdenandledtoa
substantialbutmanageableyield.
Oneadditionalmorecomprehensivecarrierstatusstrategyweusedwastosearchwithinboth
parentsofaparentalpairforP/LPvariantsinthesamegene(expandingbeyondCFTR,HBBand
HEXA).Ofthe365parentalpairsenrolled,recessivediseaserisk(i.e.,25%forfuturechildren)
wasidentifiedinone.Thissmallnumberwaslikelyduetoourrelativelystringentcriteriafor
classifyingvariantsofthistypeaspathogenicorlikelypathogenic,butthesenumbersarelikely
togrowinthefutureasevidenceaccruesonthepathogenicityofvariantsingenescausing
Mendeliandisorders22.Thetreatmentofparentalpairsasunitsofanalysisforcarrierstatusis
aneffectivewaytominimizeanalyticalandcostburdenandyeteffectivelycapturethose
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carrierresultslikelytohavethegreatestpotentialimpact.
Copy-numbervariation(CNV)wasnotexploredinparentsasasourceofsecondaryfindings.
Thisdecisionwasdrivenbytheconsiderablemanualscrutinythatisrequiredtoevaluatethe
technicalqualityofCNVs,thecostsandchallengesofCNVvalidation,andtheabsenceofrobust
CNVpopulationfrequencydata,particularlyforsmallerevents.AnalysesofsecondaryP/LP
CNVsmaybeofinteresttofutureeffortstoincreasetheoverallyieldofmedicallyrelevant
variationwithinsequencingdata.
Patientpreferences
Thequestionofwhetherpatientsandresearchparticipantsneedtobeofferedchoicesabout
receivingsecondaryfindingshasbeendebated,especiallyafterthereleaseofACMG’soriginal
secondaryfindingsrecommendationsin20131.Multiplestudieshavedocumentedthatmost
participantswantmost,andusuallyall,possiblesecondaryfindings.Thistrendisconsistent
betweenstudiesaskingthisquestionasahypothetical29-33ortoinformactualreturnofresults
34-37.Consistentwiththesepreviousstudies,thevastmajority(84.8%)ofparentsparticipating
inourstudychosetoreceiveall13categoriesofpotentialsecondaryresults.However,aminor
butsubstantialfractionofparticipants(15.2%)declinedatleastonecategoryofsecondary
results;1.6%declinedallsuchresults.OneofthesecondaryfindingslistedinTable3wasnot
returnedbecausetheparenthaddeclinedtherelevantcategory.
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Challengesassociatedwithvariantinterpretation
Oneofthemostchallengingtaskswhenanalyzingsecondaryfindingsisinterpretationof
geneticvariation,particularlyforvariantsthathavenotbeenpreviouslydescribedinscientific
literatureorinclinicalgeneticdatabases.Eventhosevariantslabeledaspathogenicinvariant
databasesareoftensupportedbyonlyweakunderlyingevidenceorareevenassociatedwith
strongevidenceforbeingbenign38.Interpretationismadeevenmorechallengingwhenan
individualharborspotentialdisease-associatedvariationbutdoesnotpresentwiththe
associatedphenotypeorhaveafamilyhistoryofdisease.Thatsaid,inthisstudy,ACMG
evidencecodeswereassignedandvariantsthatweredeemedtobeP/LPwereofferedfor
returnregardlessofthepresenceorabsenceofanyparticularphenotypeorfamilyhistory.
Evenforthosewithindicationsofdisease,theparticularphenotypesreported(Table3)arenot
necessarilydirectlyrelatedtothepresenceofthegivenvariant.Imprecisionand
incompletenessofself-reporteddiseasesandfamilyhistoriesandlimitationstoknowledgeof
penetranceandexpressivityforanygivengene,andespeciallyanygivenvariant,canallmake
interpretationmorechallenging.
Utilityofsecondaryfindings
Thesecondarygeneticfindingsthatweidentifiedmaybeofconsiderableutilitytotheparent
participants.Forfiveindividuals,wewereabletoconfirm,andgeneticallyexplain,aprevious
clinicaldiagnosis(Table3).Suchinformationmayproveusefulforfutureclinicalmanagement
andindiscussionswithfamilymembersthatmaycarrythesamevariant.Secondarygenetic
findingswerealsoidentifiedin13individualswhoreportedfamilyhistoryorsymptomsthatare
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likelytoassociatedwiththedetectedvariant.Asdescribedindetailintheresultssection,itis
clearthatgeneticcounselingandtestingcould/shouldhavebeenperformedoneightcases
basedonpresentationofsymptomsand/orfamilyhistory.Additionally,weidentifiedsecondary
geneticvariantsinfourindividualswhohaveanincreasedriskofdiseasewithmodestbutnon-
trivialevidencefordisease(twocasesofKCNQ1;onecaseeachofMYBPC3andDDX41).
Throughparticipationinourstudy,theseindividualsnowhaveabetterunderstandingoftheir
causeorriskofdiseaseandareinpositiontobettermanagethatdiseaseorriskofdisease.
Wealsoidentifiedsecondarygeneticvariationinsevenindividualswhoreportneither
symptomsnorfamilyhistoryofdisease(MSH2,RET,BARD1,BRCA2,ACTN1,SCN5A,DSG2).
Thesestudyparticipantsappeartobeatincreasedriskofdiseaseandithasbeensuggested
thattheytofollow-upwithanappropriatespecialist(Table3)inthehopesthatactionscanbe
takentoscreenfor,prevent,ormitigateunobserveddiseaseintheseindividuals.
Finally,wealsoidentifiedsecondaryvariationinDD/IDaffectedprobandsthatwerenot
identifiedinparents,eitherduetounavailabilityofparents,(n=2)orasaresultofthevariant
arisingdenovo(n=1).Further,threechildrenexhibitedrecessivediseaseunrelatedtoDD/ID
andwerefoundtoharborcompoundheterozygousvariationthatexplainedtheirdisease(i.e.
albinismandcataracts).
Challengesofreturningunexpectedvariantstofamilies
Manyparentsinthisstudyhaveexperiencedadiagnosticodysseyinhopesofidentifyingthe
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causeoftheirchild’sdevelopmentaldisabilities.IndividualswhocarriedP/LPsecondary
variantsthereforerequiredcounselingandrecommendationsforclinicalfollow-upregarding
theirsecondaryfindings,inadditiontoinformationregardingthecareandwell-beingoftheir
affectedchildren.Returninggeneticinformationrelevanttoaneworunexpecteddiseaserisk
maybeparticularlyproblematicwhennoresultsarefoundrelevanttotheprimaryindication
fortesting.Inourstudy,51%ofthesecondaryfindingsidentifiedintheparentswere
transmittedtotheDD/ID-affectedproband,and56%ofthe71parentsthatharboreda
secondaryfindingdidnotreceiveaprimaryresultfortheirenrolledDD/ID-affectedchild.The
lackofaprimaryresultmayincreasetheshockvalueofasecondaryfinding.Aparentmay
expecttheconversationtorevolvearoundtheirchild’shealthbutinsteadspendstime
discussingthemeaningoftheirowndiseaseriskand/oranadditionaldiseaseriskrelevantto
theiralreadyaffectedchild.Thisfacthighlightsthepotentialfinancial,emotional,andclinical
implicationsofsecondaryfindingsthatshouldbeclearlyaddressedintheinformedconsent
discussionpriortosequencingsothatfamiliesareawareofallthepossibleoutcomesofthis
typeoftesting.
Conclusions
Ourstudydescribestheidentificationandreturnofsecondaryvariationtoparentswhowere
subjecttogenomicsequencinginhopesofreceivingageneticdiagnosisforadevelopmentally
delayedchild.Althoughthereturnofsecondarygeneticvariationhasbeendebated39,40,alarge
majorityofparentparticipantsinthisstudyoptedtoreceiveallidentifiedsecondaryfindings,
regardlessofdiseasecategory,suggestingthatparticipantsaregenerallyopentoreceiving
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geneticinformationthatmayberelevanttotheirhealth.Thisstudydemonstratestheutilityof
returningsecondaryvariants,asitmayfacilitatepreventativescreeningforindividualswhoare
geneticallypredisposedtoseriousdiseases.Thisinformationcanalsobeusefultoindividuals
whohavebeenclinicallydiagnosedwithaspecificconditionbutforwhomthecausalgenetic
varianthasbeenunknown.Wehavealsoshownthatsecondarygeneticinformationmaylead
toclinicaldiagnosisinindividualswhohaveexperiencedsymptomsrelatedtoadisordernot
previouslydiagnosed.Someindividualsalsodescribedsignificantfamilyhistorythatwouldhave
justified,butdidnotleadto,geneticevaluationindependentoftheirparticipationinthisstudy.
Finally,ourstudydescribesaframeworkforidentifyingsecondarygeneticvariationinabroad
yetmanageablemanner,includingalimitedbutproductivecarrierscreenononlyafew
commonrecessivediseasesalongwithamorecomprehensivescreenthattreatsparentsas
matepairs.Themethodsandresultsrelatedtosecondaryvariationidentificationmaybeofuse
tootherresearchandclinicallaboratoriesthatareconductinggenomicsequencing.
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Acknowledgements
Funding:ThisworkwassupportedbygrantsfromtheUSNationalHumanGenomeResearch
Institute(NHGRI;UM1HG007301)andtheNationalCancerInstitute(NCI;R01CA197139).
Conflictofinterest.Theauthorsdeclarenoconflictsofinterests.
Wearegratefultothefamilieswhocontributedtothisstudy.WethankthestaffatNorth
AlabamaChildren’sSpecialists(Children’sofAlabamainHuntsville,AL),theHudsonAlpha
SoftwareDevelopmentandInformaticsteam,theGenomicServicesLaboratoryat
HudsonAlpha,andtheHudsonAlphaClinicalServicesLab,LLC.whocontributedtodata
acquisitionandanalysis.
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Figure1.Participantpreferencesforreceiptofsecondarygeneticfindings.Participantpreferenceswereassessedforreturnofgeneticvariationacrossanumberofdifferentdiseasecategories.Anoverwhelminglylargemajority(85%)ofstudyparticipantschosetoreceiveanyidentifiedsecondaryvariant,regardlessofdiseaseassociation(n=789).
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Table1.DemographicsofparentparticipantsenrolledintheHudsonAlphaCSERproject. Total,mean(SD)
(n=789)Males,mean(SD)
(n=367)Females,mean(SD)
(n=422)Age 40.95(9.4) 42.63(9.67) 39.49(8.94)Race* Total(%oftotal) Total(%ofmales) Total(%offemales)
White 635(80.5%) 295(80.4%) 340(80.6%)
BlackorAfrican-American
67(8.5%) 28(7.6%) 39(9.2%)
AmericanIndian/Alaska
Native
7(0.9%) 6(1.6%) 1(0.2%)
Other/Multiracial 65(8.2%) 30(8.2%) 35(8.3%)
NoAnswer 15(1.9%) 8(2.2%) 7(1.7%)
Ethnicity* Total(%oftotal) Total(%ofmales) Total(%offemales)HispanicorLatino 32(4.0%) 16(4.4%) 16(3.8%)
NotHispanicorLatino
750(95.1%) 349(95.1%) 401(95.0%)
NoAnswer 7(0.9%) 2(0.5%) 5(1.2%)
Education* Total(%oftotal) Total(%ofmales) Total(%offemales)LessthanHighSchoolDiploma
79(10.0%) 44(12.0%) 35(8.3%)
HighSchoolDiploma/GED
122(15.4%) 67(18.2%) 55(13.0%)
SomeCollege 272(34.5%) 113(30.8%) 159(37.7%)
Bachelor’sDegree 197(25.0%) 84(22.9%) 113(26.8%)
GraduateDegree 118(15.0%) 58(15.8%) 60(14.2%)
NoAnswer 1(0.1%) 1(0.3%) 0(0.0%)*Self-reported
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Table2.UniquevariantsofcarrierstatusinCFTR,HEXA,andHBBUniqueVariantInfo No.ofindividualsCFTR(MIM:219700) 4.4%oftotalpopulation
F508del 22
G685fs 3
D1152H 2
G551D 2
G542* 2
R117H 2
c.489+1G>T 1
F342Hfs 1
HEXA(MIM:272800) 0.6%oftotalpopulationY427Ifs 2
c.986+3A>G 1
c.459+5G>A 1
c.1073+1G>A 1
HBB(MIM:603903;613985) 1%oftotalpopulation
E7V 6
E27K 1
G40* 1
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Table3.Secondaryfindingsofenrolledparentssegregatedinto“Clinicallydiagnosed”,“Notablefamilyhistoryand/orsymptomatic”,and“Currentlyasymptomaticwithnofamilyhistory”. Age
(Male/Female)
Gene VariantInfo AssociatedPhenotype(MIM) Phenotypesorfamilyhistoryreportedbyparentparticipants*
Clinicallydiagnosed(0.6%totalpopulation)
35-F SLC4A1 V488M Spherocytosis,type4(612653)
Clinicallydiagnosedwithspherocytosis;Twodaughtersandfatherwithspherocytosis
37-F SLC22A5 A142S;T440M,R488H
Carnitinedeficiency,systemicprimary(212140) Clinicallydiagnosedwithcarnitinedeficiency
36-F PKD2 c.1319+1G>A Polycystickidneydisease2(613095)
Clinicallydiagnosedwithpolycystickidneydisease(PKD);mother,brother,2nieces,maternalaunt,uncleandgrandmotherwithPKD
30-F DSG2 V986fs
Cardiomyopathy,dilated,1BB;Arrhythmogenicrightventriculardysplasia10
(612877;610193)
Clinicallydiagnosedwithpostpartumcardiomyopathy;Paternalfamilyhistoryofarrhythmia;paternalunclewithtwo“heartattacks”priortoage40
52-M ANK2 E1458G Cardiacarrhythmia,ankryin-B-related,LongQTsyndrome4
(600919)
Clinicallydiagnosedwithhypertrophiccardiomyopathyandarrhythmia;fatherdiedwithischemicheartdisease
Notablefamilyhistoryand/orsymptomatic(1.6%oftotalpopulation)
29-F CLCN1 F413C Myotoniacongenita,dominant(160800)
Legcrampsandrestlesslegsinchildhood,stilloccasionallyhascramps;Motherdiagnosedwithmyotoniacongenita,10years;Maternalgrandfatherwithamusclebiopsyperformedin30sand“stiffness”especiallyincold,30s
35-F MFN2 W740S Charcot-Marie-Toothdisease,axonal,type2A2A(609260)
Historyofmusclewastinginback,lowerextremities;brotherclinicallydiagnosedwithCMT,30s;multiplefamilymembersaffectedwith“unspecifiedmuscledisorder”
40-M BRCA1 G1756fs Breast-ovariancancer,familial1(604370)
Motherwithbreastcancer,30s
38-F BRCA2 c.8488-1G>A Breast-ovariancancer,familial2(612555)
Maternalgrandfatherwithbilateralbreastcancer,60s;Paternalgrandmotherwithbreastcancer,ageunknown
33-F BARD1 E652fs Breastcancersusceptibility(114480)
Maternalgreat-grandmotherwithbreastcancer,50s;Maternalgrandmotherhadbladder,lung,andperitonealcancer,ageunknown
43-M PMS2 P246fsHereditarynonpolyposiscolorectalcancer,type4
(614337)
Father(60s)andpaternalaunt(40s)hadcoloncancer;Paternalaunt(60s)andgrandmother(50s)withbreastcancer
28-F SCN4A T1313M Paramyotoniacongenita(168300)
Atenrollment,noreportofneuromuscularphenotypes.Atreturnofresults,indicatedthatshehadmusclestiffnessbutalwaysthoughtshewas“easilyfatigued”andhad“lowstamina”;Motherdisplayssimilarsymptoms
41-M HARS R137Q Charcot-Marie-Tooth,axonal,type2W(616625)
Atenrollment,noreportofneuromuscularphenotypes.Atreturnofresults,indicatedthathehadCMT-associatedphenotypes.Alwaysthoughthewas“justclumsy”
32-F KCNQ1 R366W LongQTsyndrome1(192500) Fatherwithcoronaryarterydiseasewithtripleby-pass,early50s,paternalauntwithearly-onsetstroke,late30s
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47-M KCNQ1 P7S LongQTsyndrome1
(192500))
Mother“fainted”and“hitthefloor”-wastoldthisimpactpreventedcardiacarrest
39-M MYBPC3 E542Q
Hypertrophiccardiomyopathy4;Dilatedcardiomyopathy1MM(115197;615396)
“Leakyheartvalve”;Dadhaspacemakerandmomhas“leakyheartvalve”,60s
30-M DDX41 D140fs
Susceptibilitytofamilialmyeloproliferative/lymphoproliferativeneoplasms(616871)
Paternalcousinwithlymphoma“unspecified”,ageunknown
37-F MC4R C271Y Obesity,autosomaldominant(601665) Obese(BMI:41)
Currentlyasymptomaticwithnofamilyhistory(0.9%oftotalpopulation)
52-F SCN5A T1303M LongQTsyndrome-3(603830)
Recommendedtohavecardiovascularevaluation
50-M DSG2 E1020fs
Cardiomyopathy,dilated,1BB;Arrhythmogenicrightventriculardysplasia10
(612877;610193)
Recommendedtohavecardiovascularevaluation
31-M ACTN1 V105I
Bleedingdisorder,platelettype,15(615193)
Recommendedtohaveacompletebloodcountandfunctionalplateletstudy
33-M MSH2 Y570fs
Hereditarynonpolyposiscolorectalcancer,type1
(120435)
Recommendedtofollow-upandhavecolonoscopy
36-F BARD1 Y404* Breastcancersusceptibility(114480)
Recommendedtodiscusswithphysicianandcancergeneticcounselor
47-M BRCA2 V220fs Breast-ovariancancer,familial2(612555)
Recommendedtohaveself-andclinical-breastexams;Discusswithcancergeneticcounselor
52-M RET C609Y
Medullarythyroidcarcinoma(155240);SusceptibilitytoHirschsprungdisease1
(142623)
Recommendedtofollow-upandtestdaughterwithHirschsprung’sdisease
*Wehave(1)retainedthelanguageusedbytheparticipantand(2)includedanyreportedfamilyhistorythatisplausiblyrelatedtothephenotypeofconcern.
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