Jing You, Nara L. Sobreira, Dustin L. Gable, Julie Jurgens, · PDF fileRichholt, Stephanie E. Vallee, Mary Beth P. Dinulos, David Valle, Mary Armanios, and Julie Hoover-Fong. Supplemental

The American Journal of Human Genetics, Volume 98

Supplemental Data

A Syndromic Intellectual Disability Disorder

Caused by Variants in TELO2, a Gene Encoding

a Component of the TTT Complex

Jing You, Nara L. Sobreira, Dustin L. Gable, Julie Jurgens, Dorothy K. Grange, NewellBelnap, Ashley Siniard, Szabolcs Szelinger, Isabelle Schrauwen, Ryan F.Richholt, Stephanie E. Vallee, Mary Beth P. Dinulos, David Valle, MaryArmanios, and Julie Hoover-Fong

SupplementalMaterials

CaseReports

Family1:IndividualsII-2andII-3(17yearold,maleandfemale,respectively)are

fraternaltwins(seeFigure1)borntoa21yearoldmotherbyvaginaldeliveryfollowing

a35weekpregnancycomplicatedbypreeclampsia.Againstnormativebirthparameters

fortwingestation,birthweightswere1.9kg(10-25%)and1.7kg(5-10%),respectively,

andbirthlengthswere43.8cm(25%)and38.7cm(<3%).IndividualII-2had‘low’Apgar

scoresandremainedhospitalizedfor2-1/2weeksduetopoorfeedingandapnea.

IndividualII-3hadpoorrespiratoryeffortatbirthandrequiredresuscitationfor15

minuteswith‘low’Apgarscores.Acleftpalateandshrillcrywerenotedafterher

resuscitationandsheremainedhospitalizedfor1.5monthswithnasogastrictube

feeding.IndividualII-4wasbornattermwithbirthweightof2.8kg(10-25%)andbirth

length47.0cm(25%)anddischargedat1weekwithdifficultyfeedingandhypotonia.

Hehad10hypospadiasandfailedhisnewbornhearingscreen.

Frominfancy,individualII-2hadsymmetricalpoorgrowthanddelayeddevelopment.He

satindependentlyat2years,crawledat3yearsandstoodat4years,butwas

wheelchair-dependentbymid-childhoodduetoimpairedbalancethathasbecome

morepronouncedwithage.At17years,hecanholdapencilorutensilbutisunableto

writeorfeedhimselfindependently.Heoccasionallyusessinglewords,rarelymakes

eyecontact,andisdescribedas‘distantandwithdrawn’exceptintheevenings,when

hisbehaviorchangesdramaticallytouncontrollablelaughingandhugging.Whileawake,

hisbodyisconstantlymovingwithoutpurposebutwithoutoverttremororchorea.

Therehasbeenslowacquisitionbutnoregressionofskills.Hewasbornwithapatent

ductusarteriosus(PDA)andaventricularseptaldefect(VSD).Bothclosed

spontaneously,buthealsowasfoundtohaveadoubleaorticarchwithvascularring

andcleftmitralvalvewithmitralregurgitation.Thusfar,nocardiacsurgeryhasbeen

required.Hehasbilateralhearinglossandcorticalvisualimpairment(CVI).A

comprehensive,dilatedophthalmologicexamat17yearsofagerevealedgrosslynormal

eyestructure,butvisualacuitycouldnotbeassessedduetopoortracking/jerkypursuits

andnocompliance.Hewasdiagnosedwithautisminlatechildhood,basedonno

languageorcommunicationandrepetitive,self-stimulatorymovements.

Hypothyroidismwasdiagnosedat15years.Uponexaminationatage17years,weight

was28.7kg(-7.6SD),height130.4cm(-5.6SD)andOFC48.0cm(<<3%;50%for1.5

years).Hehasbrittlehair,bluesclera,darkcirclesunderhiseyes,anintactpalate,

pectuscarinatum,decreasedelbowextensionbutotherwisejointlaxity,broadgreat

toeswith4/5toesyndactyly,eczema,andgeneralizedhypotoniawithspasticity.Hehas

sparsefacialandpubichair.PriortoenrollmentforresearchWES,hehadseveral

normaltestsandstudiesincluding:brainMRI(2yearsofage),comparativegenomic

hybridization(10years),EEG(12years),serumuricacid,andmoleculartestingfor

fragileX.

IndividualII-3alsoexperiencedsymmetricpooroverallgrowthafterbirthassociated

withdelayeddevelopmentwithoutregression.Shesatindependentlyat5yearsofage

andhasneverwalked,butcanstandwithsupport.Shehasnowordsbutmakessounds

tocommunicate.Sheisthemostattentivetoherenvironmentofthethreeaffected

childreninFamily1.Sheoftenexhibitshandflappingwhensheisfrustrated,throws

herselfbackintoherwheelchairwhensheisangry,andhasanaffinityforwater.Her

cleftpalatewasrepairedat14monthsandsherequiredafrenulectomyfor

ankyloglossiaat3yearsofage.Sherequiredmultiplesetsoftypanostomytubesfor

otitismedia.Inearlychildhood,shewasfoundtohaveadoubleaorticarchwithan

atreticleftarchcreatinganincompletevascularringimpingingontheupperesophagus

butnotthetrachea.Hermedicalcoursewascomplicatedbyseveregastrointestinal

refluxdiseaseandrecurrentepisodesofaspirationpneumoniathatdiminished

followingG-tubeplacementatage12years.ShehasbilateralhearinglossandCVI.On

examat12and1/3years,herheightwas108.5cm(-6.1SD),weight18.1kg(-6.6SD),

andOFC47.0cm(<<3%,50%for1.5years).Shehadsynophrys,bluesclera,up-slanting

palpebralfissures,downturnedmouth,largetonguewithtetheredsublingualfrenulum,

torticollis,pectusexcavatum,anteriorlyplacedrectum,brachydactyly(middledigit<<

3%ile),4/5toesyndactyly,jointlaxity,andgeneralizedhypotonia.She,too,isin

constantmotionwhileawakewithspasticityinherlowerextremities.Shehasrotatory

nystagmus.Priortoenrollmentinthisresearch,shehadmultiplenormalclinicalstudies

including:karyotype,subtelomereand22q11FISH,7dehydrocholesterollevels,and

BACarray(4,000probes).

Followingbirth,individualII-4remainedinhospitalfordifficultyfeedingandhypotonia

untilage1week.Hisexamwasnotableforhypospadias.Hesatat15monthsbuthas

neverambulated.Hefailedhisnewbornhearingscreenandallsubsequenthearing

tests,andhasnospeech.Basedonhisbehavior,itisthoughtthathealsohasCVI.He

wasnotedtohaveanaccessoryoralfrenuliandcoarctationoftheaorta.Onexamat

age9years,hewassymmetricallysmallwithheight100.4cm(-5.9SD),weight15.1kg(-

6.3SD)andOFC46.5cm(<<3%for1year).Hehadbluesclera,alargemouthwiththin

upperlip,loosejoints,hypotonia,brachydactylyand5thfingerclinodactyly.Heexhibited

self-injuriousbehaviorduringtheexamwithconstantmotormovementandnowords.

Hehad3+patellarreflexesbutnospasticity.

Family2:

IndividualII-2isa5yearoldwhitefemalewhowasbornattermafteranuncomplicated

pregnancy.Shewasdeliveredvaginallytoa30yearold,gravida2,para1woman.Her

birthweightwas3.71kg(50%);length50.8cm(60%);andOFC35cm(60%).Concerns

regardingdelayeddevelopmentbeganat2months.Sherolledoverat5months,sat

aloneat8months,andwalkedat14months.At20monthssheknewabout20words,

butherparentsfeltherverbalskillsregressed.Ahearingtestat2yearsofagewas

normal.Atage30months,shehadformaltestingusingtheDevelopmentalAssessment

ofYoungChildren(DAYC-2)testwithadevelopmentalquotient(DQ)of55inexpressive

andlanguageskills,40infineandgrossmotorskills,40insocialskillsand37in

cognition.Shewasre-testedatage60monthsandscored<50inalldomains.

ShehadanormalcranialMRIatage2andstrabismussurgeryatage3.5yearsfor

intermittentesotropia.Shehashadachronicsleepdisturbancewithdifficultyfalling

asleepandoftenstayedawakescreamingduringthenight.AnEEGatage3and10/12

showedslightlyslowbackgroundactivitywith2briefburstsofbifrontalepileptiform

activity.Therehavebeennoclinicallydetectableseizures.

Physicalexamatage4and9/12revealedaheightof106cm(50%),aweightof16.8kg

(45%),andaheadcircumferenceof47.5cm(<3%,50%for18months).Shewasnon-

dysmorphicandambulatorywithintermittentabnormaltruncalmovements.

Examinationofherchest,spine,heartandabdomenwasnormalaswereherdeep

tendonreflexes.

Pertinentlaboratorytestingincludednormalchemistries;normal15qmethylation

testingforPraderWillisyndromeandAngelmansyndrome;normalMECP2sequencing

anddeletion/duplicationtesting;normaltransferrinisoelectricfocusingfor

carbohydratedeficienttransferrin;normalcreatinedisorderspanel;andnormalurine

purinesandpyrimidines.

Family3:

IndividualII-1isa17yearoldfemalewhowasbornat37.5weeksofgestationafteran

uncomplicatedpregnancy.Shewasdeliveredwithvacuumandforcepsassistancedue

tounspecifieddystocia.BirthweightisunknownandApgarscoreswerereportedas

“low”.ShehadpoorrespiratoryeffortandwasobservedintheNICUforseveralhours

butdischargedat2daysofage.Herparentsnotedslowacquisitionofmilestones.She

satindependentlyat2years,neverdevelopedwordstocommunicate,canassistwith

transfersfromchairtofloorbutcannotstandorwalkindependently.Shehasnothad

formalpsychometricsbutcurrently(age17)sheisnon-ambulatory,non-verbal,and

incontinentandperformsnoactivitiesofdailyliving.Formalaudiologyassessmentin

earlychildhoodwasnormal.Sheisinspecialschool4hoursadayandhasanirregular

sleeppattern.Onoccasions,shedoesnotsleepatnightforseveraldays.Shehas

nighttimeoutburstsoflaughingifsheisawakeforprolongedperiods.

Seizuresbeganinearlychildhoodand,currently,sheexperiencesabsence,partial

complexandgeneralizedtonicclonicseizures.Attimesthefrequencyhasbeenashigh

as20or30perday,butcurrentlyshehas~6permonthonTrileptalandclonazepam.

Shehaskyphoscoliosiswithprogressivevertebraldeformation,whichbeganinmid-

childhood.Herkyphoscoliosisworsenedinpubertyinassociationwithalineargrowth

spurt.

Atage15,shehadaroutineechocardiogramandlabworkinpreparationforpossible

spinesurgery.Herparentsweretoldhercardiacfunctionwassufficienttotoleratethe

surgerybutareunawareifanystructuralanomalieswerenoted.Therewerenospecific

pre-operativelaboratoryabnormalitiesotherthanlowvitaminDandanemia.The

formerhasbeentreatedwithsupplementaloralvitaminD(50,000IUweekly)andthe

latterwithoraliron,thoughtheanemiawasreportedtobeminimallyresponsive.

Onexamatage17yearsofage,herheadcircumstanceis51cm(<3%,50%for7years

old).Herfaceisexpressionlesswithslightlyelongatedpalpebralfissuresandsheholds

hertongueoutsidehermouth.Herneckandthoraxaregrosslynormalwithnormal

breastdevelopment.Shehassmallhandswithwastingofherperipheralmuscles.Her

hearingisgrosslynormal.Shetrackspeoplemovingaroundherandcan‘spot’smalltoys

orobjects.Sheavoidseyecontactandhasdifficultyreachingobjectswithashaky,poor

grasp.

Family4:

IndividualII-1isan8yearoldwhitemalewhowasthe45.7cm(<3%)and3.09kg(25%)

productofa38week,uncomplicatedpregnancyanddelivery.Apgarscoreswere9at1

and5minutes.Herequiredphototherapyforhyperbilirubinemiabutwasdischargedat

age5days.Developmentwasslow:hesatat12months,crawledat3years,andwalked

at4years.Therehasbeennoregression.Hehasdysphagiawithdifficultyswallowing

thinliquidsandchronicrespiratorycongestion.Herequiredafrenulectomy,multiple

typanostomytubesforchronicotitismediaandunilateralnasolacrimalductprobingfor

anobstructedtearduct.Hedevelopedkyphoscoliosisinchildhoodandisnon-verbal.

HehasCVIandamovementdisorderdiagnosedasataxiabyconsultingneurologists.

MultiplecranialMRIshavebeenreadasnormalexceptforsmallbrainsize.Hehasnot

hadseizuresandanEEGatage7.5wasnegative.Hisphysicalexamatage7years9

monthswasremarkableforshortstature(height113.5cm,1%),smallsize(weight

20.6kg,8%)andOFC47cm(<<3%,50%of1year).HEENTexamwasremarkablefor

downslantingpalpebralfissures;ascaronhisrightuppereyelidfromsurgicalrepairof

trauma;short,smoothphiltrum;broadnasalbridge;andachronicallyopenmouth.

Chest,cardiovascularandabdominalexamswerenormal.Hehadasmallpeniswith

mildchordee.Histonewasmildlyincreasedinhislowerextremitieswithnormaldeep

tendonreflexesandabnormalcoordinationwithataxia.Anechocardiogramat8years

ofagewasnormalwithnoabnormalitiesofthegreatvessels.

Pertinentlaboratorytestingincludednormalchemistriesandplasmaaminoacids;

normal15qmethylationtestingforPraderWillisyndromeandUBE3Asequencingfor

Angelmansyndrome;normalNIPBLsequencingforCorneliadeLangesyndrome1;

normalMECP2sequencing;normal7-dehydrocholesterolanduricacidlevels;normal

transferrinisoelectricfocusingforcarbohydratedeficienttransferrin;andnormalFragile

Xtesting.Hiskaryotypewas46,XYandachromosomalmicroarrayrevealeda

heterozygous40kbdeletionat22q12.3thatremovedsegmentsofC22orf42andRFLP2.

Hisasymptomaticmotherhasthesamecopynumbervariant.

Family 1: c.2159A>T

Supplemental Data Figure S1

Family 1: c.1100G>T

Family 2:c.2296G>A

Family 3: c.779C>T

Family 2: c.1100G>T

Family 3: c.1826G>A

Figure S1. Sequence of TELO2 variants in Family 1 (II-3, II-4 and II-5), Family 2 (II-2) and Family 3 (II-1). Family.1: TELO2, p.Cys367Phe (c.1100G>T) in exon 8 and p.Asp720Val (c.2159A>T) in exon 18. Family.2: TELO2, p.Cys367Phe (c.1100G>T) in exon 8 and p.Val766Met (c.2296G>A) in exon 20. Family.3: TELO2, p.Pro260Leu (c.779C>T) in exon 5 and p.Arg609His (c.1826G>A) in exon 15.

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C.1 C.2 C.3 II-2 II-3 II-4

Family 1 Controls

Figure S2

Figure S2. Comparison of TELO2 mRNA expression in affected individuals from Family 1 and controls Levels of TELO2 mRNA are within normal range in RNA extracted from primary affected individual-derived fibroblast cell lines in Family 1 (II-2, II-3 and II-4) (n=3 experiments, performed in triplicate; error bars indicate 1 SEM, Student’s t-test)

C.1 C.2 C.3 II-2 II-3 II-4

TELO2

TTI1

TTI2

β-Actin

Family 1

Figure S3

Figure S3. Repeated western blot of steady state expression of TTT complex in fibroblasts extracts in the affected individuals of Family 1 (II-2, II-3 and II-4) and three normal controls (C.1, C.2 and C.3).

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Colony survival assay (8 days) Controlmean(n=3)

I-1

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II-4

AtaxiaTelangiectasia

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eexpression

controls(n=3)heterozygous(n=2)C367F/D720V(n=3)

Figure S4

Figure S5

Figure S5. Colony survival assay following exposure to ionizing irradiation. LCLs from affected individuals in Family 1 (II-2, II-3 and II-4) are labeled in red and the mean of three controls is labeled in green. LCLs from an affected individual with molecularly confirmed ataxia-telangiectasia (ATM nonsense variant) was used as a positive control.

Figure S4. Steady state expression of PIKKs in LCL extracts in the affected individuals of Family 1 (II-2, II-3 and II-4), their heterozygous parents (I-1 and I-2) and three normal controls (C.1, C.2 and C.3). There was no significant difference between control, affected individuals and heterozygous parents (Student’s t-test).

FigureS7

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MMC(MitomycinC)survivalassay(12hrs)

controls(n=3)

C367F/D720V(n=3)

Figure S6

Figure S7. Telomere length measurement in affected individuals’ primary lymphocytes and granulocytes. The percentile lines are derived from 200 normal controls. Affected individuals and heterozygous parents from Families 1 and 2 were measured.

Figure S6. Mitomycin C (MMC) survival assay Cultured skin fibroblasts from affected individuals in Family 1 (II-2, II-3 and II-4) and controls were used to measure the survival fraction following MMC treatment. The assay was performed in triplicate, and the plot shows the means of the 3 affected individuals and the 3 controls. There was no significant difference between controls and affected individuals in survival fraction to MMC treatment (Student’s t-test).