Microarray analysis unmasked two siblings with pure heredita(3)

Microarray analysis unmasked two siblings with pure hereditary spastic paraplegia shared a run of homozygosity region onchromosome 3q28–q29

W.Yuetal./JournaloftheNeurologicalSciences359(2015)351–355353

oftime,heshowedslurredspeechandmildfootdrop.Hecanwalkwiththehelpofwalkingaidswhenarrivedatourhospital.Neurologicexam-inationofthetwopatientsrevealedweaknessandspasticityinthelowerlimbswithpositivepyramidaltractsigns(hypertonicityofthelowerlimbs,markedbilateralankleclonusandbilateralBabinskisigns),pescavusandplantarhypermyotonia.Noneofthemdisplayedabnormalitiesonelectroencephalography(EEG).Theirbrainswerealsonormalbasedonmagnetic-resonanceimaging(MRI).Atthetimeofrecentvisit,theeldersisterwascon nedtobedwithmalnutritionandamyotrophy,whichmightbepartiallyascribedtodisuseatrophy.Nystagmuswasobservedinbothpatients.ThedetailedinformationissummarizedinTable1.Bothofthemwereclassi edaspureHSPbytwoexperiencedneurologists(Dr.Q-LSongandDr.JSu).ExceptforIII-1andIII-2,otherindividualsfromthisfamilyarenormalinclinicalmanifestationsandauxiliaryexaminations.

3.3.Mutationanalysis

Whole-exomesequencingwasperformedontheIII-1,III-2andII-6.Theresultscovered93%ofthetargetregionwiththemedianreaddepth(102×,100×and81×),26,033SNPsand258indelswereidenti ed.SinceHSPwasassumedtobeaveryrareconditionandnoclinicalsig-ni cantmutationinknownHSPgeneswasdetected,weexcludedheterozygouschanges,changesnotsharedbytheaffectedsiblingsandhomozygouschangesconsistentwiththeirmother.Tenhomozygousvariationswithafrequencyofb1%indbSNP138wereidenti ed,twoofwhichwerein5′UTRregionsandonewasintheintronicregion.Thefrequencyofthesethreevariantsrangedfrom0.1to0.4%referencedinthedbSNP138(rs185421189,rs147536472andrs191327348).NoneofthemwaslikelypathogenicsincetheydidnotaffectsplicesitesthroughpredictionbytheHumanSplicingFinder(HSF).Thesevenre-mainingvariantswerelocatedinexoncodingsequences,threeofthemweremissensevariantsinC3orf43,CRLF1andKIR3DL1genespreviouslyreportedinthedbSNP138,HapMap,and1,000GenomesDatabase(rs118152136,rs117193413andrs150084719respectively),theywerepredictedasbenignbySIFTandPolyphen2predictionpro-grams.Theremainingweresynonymousmutations,twoofwhichhadfrequency0.5%indbSNP138(rs35480846,rs139525246),andwerepredictedtobenon-pathologicbyHSF.Theothertwonovelsynony-mousvariant,oneofwhichlocatedatthe rstexonoftheMUC4gene(c.G10605C),theotherwasavariantintheFBXO45genec.33CNT.However,theywerebothpredictednottoaffectadjacentsplicesitesoractivatecrypticsplicesites.

Furthermore,Sangersequencingwasperformedontheexonsandpromoterregionsofcandidateeightgenes(IL1RAP,GEMC1,SNAR-I,OSTN,UTS2D,CCDC50,PYDC2andFGF12).27variantswereidenti ed,ninelocatedinexonoruntranslatedregions(UTR)regions.TheSangersequencingresultsoftheninevariantswereconsistentwiththoseofexomesequencing(SupplementTable2).Asthephenotypeofourpa-tientsappearedtobeanautosomalrecessivemodel,wefocusedonho-mozygousmutationsintheaffectedindividualsthatwereheterozygousintheirmother.Fourhomozygousvariationswithafrequencyofb1%indbSNP138wereidenti ed(markedwithboldfont).However,noneof

3.2.Theresultsofhomozygositymapping

Thecytogeneticsrevealednormalkaryotypes(46,XXand46,XY)inthetwosiblingswithnoabnormalitydetected.Microarrayanalysisofthetwosiblingsandtheirmotherdidn'tidentifyanyclinicalsigni canceregionsofcopynumbervariation.Assmallerstretchesofhomozygosity(b3Mb)spreadthroughoutthegenomearecommoneveninoutbredpopulations[10],wesetasizethreshold(N3Mb).Thesegmentsbelow3Mbwerenotconsideredtobesigni cant.AnROHonthechro-mosome3q28–q29(Fig.2)wasdetectedinthetwosiblings,whichwasabout7.7Mbandcoveredthebasepairpositionsfrom190172058to197851260(hg19)including68genes.Moreover,thisregionpartiallyoverlappedwiththeSPG14locusandtheoverlappingregionincluded8genes(IL1RAP,GEMC1,SNAR-I,OSTN,UTS2D,CCDC50,PYDC2andFGF12)(SupplementTable1).

Tocon rmthehomozygositysharedbythetwosiblings,wegenotypedpolymorphismsusing15single-nucleotidemicrosatellitemarkersinfamilymembers.Theresultscon rmedtheROHregionofthetwosiblingsonthetelomereofchromosome3betweenmarkersD3S1288andD3S3707(Fig.1).

Table1

Clinicalfeaturesoffamilieswith3q28–q29ROH

PresentfamilyIII-1

ROHregionEthnicitySex

Ageatexamination,yAgeatonset,ySymptomatonsetUpperlimbsSpasticityWeaknessHyperre exia

LowerlimbsSpasticityAnkleclonusAmyotrophyWeaknessHyperre exiaDystoniaBabinskisignPescavus

ExtensorplantarresponsesWalkingabilityCognitivedeclineNystagmusEEGMRI

a

G.Vazzaetal

III-2

Chinese-HanMale107

Weaknessoflowerlimb

II-2

II-6

II-8ItalianFemale52

Spasticgait

Chr3:190172058–197851260Chinese-HanFemale189

Weaknessoflowerlimb;spasticgaitChr3:188542793–192502314ItalianItalianMaleFemale6254

Average30

SpasticgaitSpasticgaitNRaNRNR

––––––NRNRNRNRNRNR

+++++++++

Walker–

+(mild)NormalNormal++–++++++

Walker–

+(mild)NormalNormal+NRNR+++NR++

Unaided+NR

NormalNormal+NRNR+++NR++

Unaided+NR

NormalNR+NRNR+++NR++

Unaided+NR

NormalNR

NRindicates“NotReported”.