基因芯片技術(shù)的臨床應(yīng)用

基因芯片技術(shù)(microarray)的臨床應(yīng)用人類基因及基因組DominantRecessive線粒體病多基因病Genes+

Environments腫瘤也是基因及基因組病23

對染色體---

2

x30

億個堿基編碼

21,000

個基因

---編碼序列占整個基因組

的1.5%基因及基因組病

（遺傳病）染色體數(shù)量異常Trisomy

21

（唐氏綜合癥）Trisomy

18Trisomy

13Sexchromosomal

aneuploidiesMosaictrisomiesofother

chromosomes染色體結(jié)構(gòu)變化Morethan200known

disordersMorethan1000rare

abnormalities單基因病

(more

than

8,000)人類有60多種惡性腫瘤所有腫瘤都含有基因及基因組異常中國年出生1600萬，出生缺陷發(fā)生率在5.6%,

每年新增出生缺陷數(shù)約90萬例。(嬰兒在出生的一年內(nèi)，體格上出現(xiàn)明顯的結(jié)構(gòu)異常和需要手術(shù)矯正的畸形）智力低下遲發(fā)性疾病-------Thompson&ThompsonGeneticsInMedicine.Eighth

Edition遺傳病的實驗室診斷二代測序（NGS）原位熒光雜交（FISH）一代測序（Sanger

Sequencing）非測序分子生物學(xué)技術(shù)（non-DNA

techniques）核型分析（Karyotyping）基因/基因組檢測基因芯片（Microarray）酶學(xué)檢測高效液相色譜-串聯(lián)質(zhì)譜電感耦合等離子體質(zhì)譜氣相色譜-質(zhì)譜超高效液相色譜蛋白質(zhì)及代謝產(chǎn)物檢測ChromosomeMicroarrayAnalysis

(CMA)Principlesof

CMACurrentStatusofCMAApplicationforClinical

ServiceFutureTrendsofCMAforClinical

ServiceaCGH

techniquesSNP

microarray199220032005

Indicatingthepresenceofuniparentaldisomy

(UPD)Indicatingthepresenceof

consanguinityIndicatingthepresenceofshared

ancestryIdentifyrecessivegene

mutationsConfirmCNVcallsbycheckingSNPallele

patternsIncreasesensitivityfordetectionof

mosaicismIdentifytriploidyforwhichaCGHfailsto

detectDetermineparentaloriginofadenovo

CNVImprovesourunderstandingofgenetic

aberrationsEnhancesthequalitycontrolinthediagnosticlaboratory

workflowIdentifygenomicregionswithLOHrelatedto

tumorigenesisPrinciplesof

CMAsPathogenicLikely

pathogenicUncertainclinicalsignificanceLikely

benignbenignClassificationofCopyNumberVariantsidentifiedbyCMAbasedontheirclinical

significancesCMAapplicationsforclinical

service受孕胚胎植入前的基因及基因組檢測產(chǎn)前篩查及診斷新生兒篩查及診斷遺傳病病人（兒童及成人）診斷健康人群隱性遺傳病攜帶者檢出健康及亞健康人群疾病易感基因檢測遺傳病的基因及基因組檢測

腫瘤的基因及基因組檢測遺傳性腫瘤攜帶者檢出無癥狀早期篩查分子診斷靶向藥物的選擇預(yù)后判斷治療監(jiān)控復(fù)發(fā)基因克隆檢出ValidationsofCMAplatformsforClinical

ServicesTechnical

ValidationsClinical

ValidationsValidation-Agilent

aCGH-244KYu,

S.

Bittel,

DC.

Kibiryeva,

N.

Zwick,

DL.

Cooley,

LD.ValidationoftheAgilent244Koligonucleotidearray-basedcomparativegenomichybridizationplatformforclinicalcytogeneticdiagnosis.AmJClinPathol

2009;132(3):349-60.VerificationofaCGH

findingsYuS,

Kielt,M,StegnerA,Bittel,DC.Cooley,

LD.ApplicationofQuantitativeReal-Time

PCRMethodsfortheVerificationofGenomicImbalancesDetectedbyMicroarray-basedComparativeGenomicHybridization.GenetTestMolBiomarkers

2009;13(6):751-60.aCGHforpostnataldiagnosis

(1)IdentifyNovelGenomic

DisordersBelloneRRetal.DifferentialgeneexpressionofTRPM1,thepotential

cause

of

congenital

stationary

night

blindness

(先天性靜止性夜盲癥)andcoatspottingpatterns(LP)intheAppaloosahorse(Equuscaballus).Genetics.2008

Aug;179(4):1861-70.LepichonJB,BittelDC,GrafWD,Yu

S.A15q13.3homozygousmicrodeletionassociatedwithasevereneurodevelopmentaldisordersuggestsputativefunctionsoftheTRPM1,CHRNA7,andotherhomozygouslydeletedgenes.AmJMedGenetA.2010

May;152A(5):1300-4.LepichonJB,YuS,GrafWD,andBittelDC.Genomewidegeneexpressioninapatientwith15q13.3homozygousmicrodeletionsyndromeEurJHumGenet.2013,

1-7.15q13.3

homozygousmicrodeletionAbdelmoityAT,HallJJ,BittelDC,Yu

S.1.39Mbinheritedinterstitialdeletionin12p13.33associatedwithdevelopmentaldelay.EurJMedGenet.2011

Mar-Apr;54(2):198-203.12p13.33

deletionRamalingamA,ZhouXG,FiedlerSD,BrawnerSJ,JoyceJM,LiuHY,Yu

S.16p13.11duplicationisariskfactorforawidespectrumofneuropsychiatricdisorders.JHumGenet.2011

Jul;56(7):541-416p13.11

duplicationYuSandGraf

WD.BRAFgenedeletionbroadenstheclinicalspectrumneuro-cardio-facial-cutaneoussyndromes.JChildNeurol.2011

Dec;26(12):1593-6.BRAFgene

deletionYuS,ShaoL,KilbrideH,Zwick

DL.HaploinsufficienciesofFOXF1andFOXC2genesassociatedwithlethalalveolarcapillary

dysplasiaandcongenitalheart

disease.AmJMedGenetA.2010

May;152A(5):1257-62.16q24.1

microdeletionaCGHforpostnataldiagnosis

(2)DiscoverNovelGenetic

MechanismsTelomerecaptureasafrequentmechanismforstabilizationoftheterminalchromosomaldeletionassociatedwithinverted

duplication.YuS

andGraf

WD.Telomerecaptureasafrequentmechanismforstabilizationoftheterminalchromosomaldeletionassociatedwithinverted

duplication.CytogenetGenomeRes.

2010;129(4):265-74.Genomicprofileofcopynumbervariantson

theshortarmofhumanchromosome

8YuS,

FiedlerS,StegnerA,andGraf

WD.Genomicprofileofcopynumbervariantsontheshortarmofhumanchromosome8.EurJHum

Genet.2010Oct;18(10):1114-20.YuS,ZhouXG,FiedlerSD,BrawnerSJ,JoyceJM,Liu

HY.Cardiac

defects

are

infrequent

findings

in

individuals

with

8p23.1

genomic

duplications

containing

GATA4.CircCardiovascGenet.2011

Dec;4(6):620-5.8p23.1genomic

duplicationsaCGHforpostnataldiagnosis

(3)Refinebreakpointsofgenomic

disordersGenomicDisorders

onchromosome

22YuS,BittelDC,YuS,NewkirkH,KibiryevaN,ButlerMG,CooleyLD.Refiningthe22q11.2deletionbreakpointsinDiGeorgesyndromebyaCGH.CytogenetGenomeRes

2009;124(2):113-20.YuS,GrafWD,RamalingamA,BrawnerSJ,JoyceJM,FiedlerDS,ZhouXG,andLiuHY(2001)Identificationofcopynumber

variants

(CNV)

on

human

chromosome

22

in

patients

with

a

variety

of

clinical

findings.

Cytogenet

GenomeRes.2011;134(4):260-8.Epub2011Aug

17.ButlerMG,BittelDC,KibiryevaN,CooleyLD,YuS.

Aninterstitial15q11-q14deletion:expandedPrader-Willisyndromephenotype.AmJMedGenetA.2010Feb;152A(2):404-8.expandedPrader-WillisyndromeaCGHforpostnataldiagnosis

(4)Characterizeoriginofmarker

chromosomeAneocentricsupernumerarymarkerchromosomeoriginatingfromtheXpdistal

regionYuS,BarbouthD,BenkePJ,WarburtonPE,Fan

YS.CharacterizationofaneocentricsupernumerarymarkerchromosomeoriginatingfromtheXpdistalregionbyFISH,CENP-Cstaining,andarray

CGH.CytogenetGenomeRes

2007;116(1-2):141-5.YuS,FiedlerDS,BrawnerSJ,JoyceJM,ZhouXG,andLiu

HY.CharacterizingSupernumeraryMarkerChromosomes(SMCs)withCombinationofMultiple

Techniques.CytogenetGenomeRes.

2012;136(1):6-14.Characterizing

SupernumeraryMarkerChromosomes

(SMCs)SNPMicroarrayforClinical

ApplicationsWhyshouldSNPmicroarraybeusedtoreplaceaCGH

?Whatisa

SNP?WhatisaSNP

array?Advantages

of SNParraysover

aCGH?ApplicationsofSNPMicroarraySingleNucleotidePolymorphism

(SNP)Definition:asinglenucleotidechangeinaDNAsequencethatoccursin

asignificantproportion(≥1%)inalarge

population.DifferentLevelsofpolymorphismsinhuman

genomeinv(9)SNP

factsRepresent90%of

genomic

variations inhuman

genome,ThereisaSNPper100-300bpinhuman

genome,SNPscanoccurincoding(gene)andnoncodingregionsofthe

genome,ManySNPshavenoeffectoncellfunction,butotherscouldpredisposepeopletodiseaseorinfluencetheirresponsetomedicines,environmental

factors.DNA

SequencingHybridizationMicroarraysTaqMan,Molecular

BeaconsAllele-specific

PCRFRETIntercalating

DyesPrimer

ExtensionMALDI-tofSNaPshotLigationPadlock

ProbesRollingCircle

AmplificationEndonuclease

CleavageRFLPPIRA/RFLPMethodstodiscovernovelSNPsanddetectknown

SNPsClinicalSNP

arraysNumbersofMarkers

(probes)AverageMarkerSpacing(base

pairs)Genescovered(25markers/100

kb)AgilentIlluminaAffymetrixTheCytoScan?HDArrayfrom

Affymetrix

Indicatethepresenceofuniparentaldisomy

(UPD)Indicatethepresenceof

consanguinityIndicatethepresenceofshared

ancestry

Identifyrecessivegene

mutationsConfirmCNVcallsbycheckingSNPallele

patternsIncreasesensitivityfordetectionof

mosaicismIdentifytriploidyforwhichaCGHfailsto

detectDetermineparentaloriginofadenovo

CNVImprovesourunderstandingofgenetic

aberrationsEnhancesthequalitycontrolinthediagnosticlaboratory

workflowIdentifygenomicregionswithLOHrelatedto

tumorigenesisSNPMicroarrayAnalysisforClinical

ServiceSNParrayforClinicalService

(5)Indicatethepresenceofuniparentaldisomy

(UPD)UPDchromosomesassociatedwithimprinting

disordersSNParrayforClinicalService

(6)Indicatethepresenceof

consanguinityROH

indicatingconsanguinityTwosiblingswithhighpercentage

ROHindicatingconsanguinity

marriageGlobalConsanguinity

RatesHamamyH.Consanguineousmarriages:Preconceptionconsultationinprimaryhealthcaresettings.JCommunityGenet.2012

Jul;3(3):185-92.SNParrayforClinicalService

(7)Identifyrecessivegene

mutationsROHfacilitatingidentificationofrecessive

genesmonth-oldboywithmultipleendocrine

&structural

issues:congenitalprimary

hypothyroidismhyperinsulinisminthefaceof

hypoglycemiagrowthhormone

deficiencycortisol

deficiencyresolveddirectandindirect

hyperbilirubinemiacorticalvisual

impairmentrespiratoryissues,

aspirationsignificantdevelopmental

delayhypotoniadysmorphicfacial

featureshirsutismwithlowanterior

hairlineCMAtestingforCNVisrecommendedasafirst-linetestintheinitialpostnatalevaluationofindividualswiththefollowing:Multipleanomaliesnotspecifictoawell-delineatedgenetic

syndrome.Apparentlynonsyndromic

DD/ID.Autismspectrum

disorders.FurtherdeterminationoftheuseofCMAtestingfortheevaluationofthechildwithgrowthretardation,

speechdelay,andotherlesswell-studiedindicationsisrecommended,particularlybyprospectivestudiesandaftermarketanalysis.Appropriatefollow-upisrecommendedincasesofchromosomeimbalanceidentifiedbyCMA,toincludecytogenetic/FISHstudiesofthepatient,parentalevaluation,andclinicalgeneticevaluationand

counseling.GrafWD,LePichonJB,BittelDC,AbdelmoityAT,andYuS.Practiceparameter:evaluationofthechildwithmicrocephaly(anevidence-basedreview):reportofthequalitystandardssubcommitteeoftheAmericanAcademyofNeurologyandthePracticeCommitteeoftheChildNeurologySociety.Neurology.

2010;74(13):1080-1.LedbetterDH.Cytogenetictechnology--genotypeandphenotype.NEnglJMed.

2008;359(16):1728-30.BejjaniBAandShafferLG.Clinicalutilityofcontemporarymolecularcytogenetics.AnnuRevGenomicsHumGenet.

2008;9:71-86.Clinical

indication Testing不孕不育(

about

1/3duetogenetic/genomic

defects)已知基因\基因組異常核型分析原位熒光雜交基因芯片(BlueGnome)MES,WES,

WGS胚胎植入前檢測(PGS

&

PGD)已知基因\基因組異常原位熒光雜交基因芯片單基因檢測，WGSCMAforPGS/PGD(preimplantationgeneticscreening/diagnosis

(8)Colls,P.etal.Validationofarraycomparativegenomehybridizationfordiagnosisoftranslocationsinpreimplantationhumanembryos.ReprodBiomedOnline.2012;24:

621–629.Treff,N.R.etal.Singlenucleotidepolymorphismmicroarray-basedconcurrentscreeningof24-chromosomeaneuploidyandunbalancedtranslocationsinpreimplantationhumanembryos.FertilSteril.2011;95:

1606–1612.Johnson,D.S.etal.Preclinicalvalidationofamicroarraymethodforfullmolecularkaryotypingofblastomeresina24-hprotocol.HumReprod.2010;25:1066–1075.PetersBAetal.