Microarray analysis unmasked two siblings with pure hereditary spastic paraplegia shared a run of(3)

2.3.Exomesequencing

Paired-endsequencingwasperformedonIlluminaGAIIx/HiSeq2000instruments(Illumina,SanDiego,CA,USA)availableatShanghaiBiotechnologyCo.,Ltd.ExoncapturewasconductedusingAgilentSureSelectTechnology(Agilent,SantaClara,CA,USA).Forsequencealignment,variantcallingandannotation,thesequencesweremappedtotheirlocationwiththehumangenomereferencesequence(hg19build)usingaBurrows-WheelerAligner.Localrealignmentofthepotentialinsertion/deletionsiteswascarriedoutwithaGenomeanalysistool(GATK).SNPandindelvariantswereperformedagainstreferencedbSNP138.Allvariantswereannotatedwithreferencetocon-sensuscodingsequences(CCDS)(NCBIrelease20090902)andRefSeq(UCSCdumped20101004).ThenovelvariantswerecheckedwiththeIntegrativeGenomicsViewer(IGV),andthenfurtherinvestigatedinthefamilyandnormalChinesepopulation.3.Results3.1.Clinicaldata

ThepatientsaretwosiblingsfromaHanChinesefamily.Theeldersister(III-1)iscurrently18yearsold.Shewasbornattermwithanuneventfuldelivery.Sheexperiencedprogressiveweaknessofthelegsandtumbledseveraltimessincenine.Overthenextfewyears,gaitim-pairmentprogressedcontinuously;footdropandfootinversionwerepresentinherrightside.Atthetimeofadmissiontoourhospital,shewasbedriddenwithverylittlemobilityinherlegs.Theyoungerbrother(III-2)iscurrentlytenyearsold.Hewasnoticedslowlyprogressiveweaknessoflegsandgaitdisturbanceat7yearsold.Withthe

passage

2.2.Homozygositymapping

TotalgenomicDNAofthetwosiblingsandtheirmotherwereisolat-edandpuri edfromperipheralbloodusingtheQIAampDNAbloodminikit(Qiagen,Valencia,CA,USA).Then,theDNAsampleswereana-lyzedbyCytoScanHDarraysaccordingtothemanufacturer’sprotocol(Affymetrix,SantaClara,CA,USA).Brie y,250ngDNAwasdigestedwithNspIandligatedtoadaptersforsubsequentPCRampli cation.Theampli cationproductswerepuri edusingpuri cationbeads,fragmented,labeled,andhybridizedfor16–18hintheGeneChipHybridizationOven,ThearrayswerethenwashedwithaGeneChipFluidicsStation450,andscannedwithaGeneChipScanner30007G(Affymetrix).CopynumberandROHanalysiswereperformedusingtheAffymetrixChromosomeAnalysisSuitesoftware.

Fifteenmicrosatellitemarkerson3q28–q29includedD3S1580,D3S3530,D3S1294,D3S1288,D3S2747,D3S3043,D3S1601,D3S3663,D3S3669,D3S1523,D3S2305,D3S1305,D3S1272,D3S3707andD3S3550,nineofwhichwereconsistentwithG.Vazzaetal.[9]previ-ouslyreported.GenomicDNAoftheprobands,theirmotheranduncleswereextractedandusedforampli cations.FragmentswereanalyzedusinganABIPrism3730GeneticAnalyzer(AppliedBiosystems,ForsterCity,CA,USA)withthedenaturingPOP7polymer.ElectropherogramswereanalyzedusingGeneMappersoftware.