1、基因芯片技基因芯片技术术(microarray)的临床应用的临床应用人类基因及基因组人类基因及基因组DominantRecessive线粒线粒体病体病多基多基因病因病Genes+Environments肿瘤也是基因及基因组病肿瘤也是基因及基因组病 23 对染色体对染色体-2 x 30 亿个碱基亿个碱基 编编码码 21,000 个基个基因因 -编码序列占整编码序列占整个个基因基因组组 的的1.5%基因及基因组基因及基因组病病 （遗传病）（遗传病）染色染色体数体数量量异常异常Trisomy 21（唐（唐氏氏综综合合症症）Trisomy 18Trisomy 13Sex chromosomal ane
2、uploidiesMosaic trisomies of other chromosomes染色染色体结体结构构变化变化More than 200 known disordersMore than 1000 rare abnormalities单基单基因病因病 (more than 8,000)人类人类有有60多种恶性肿瘤多种恶性肿瘤所有肿瘤都含有基因及基因所有肿瘤都含有基因及基因组组异常异常中国年出中国年出生生1600万万，出生出生缺缺陷发生陷发生率率在在5.6%,每年新增出生缺陷数每年新增出生缺陷数约约90万万例。例。(婴儿在出生的一年内婴儿在出生的一年内，体格体格上上出现明出现明显显的结
3、的结构构异常和异常和需需要手要手术术矫正矫正的的畸形）畸形）智力低下智力低下迟发性疾病迟发性疾病-Thompson&Thompson Genetics In Medicine.Eighth Edition遗传病的实验室诊断遗传病的实验室诊断二代测序（二代测序（NGS）原位荧光杂交原位荧光杂交（FISH）一代测序一代测序（Sanger Sequencing）非测序分子生物学技术非测序分子生物学技术（non-DNA techniques）核型分析核型分析（Karyotyping）基因基因/基因组基因组 检测检测基因芯片基因芯片（Microarray）酶学检测酶学检测高效液相色谱高效液相色谱-串联质
4、谱串联质谱电感耦合等电感耦合等 离子体质谱离子体质谱气相色谱气相色谱-质谱质谱超高效超高效 液相色谱液相色谱蛋白质及代蛋白质及代 谢产物检测谢产物检测Chromosome Microarray Analysis(CMA)Principles of CMA Current Status of CMA Application for Clinical Service Future Trends of CMA for Clinical ServiceaCGH techniquesSNP microarray199220032005 Indicating the presence of unipare
5、ntal disomy(UPD)Indicating the presence of consanguinity Indicating the presence of shared ancestry Identify recessive gene mutations Confirm CNV calls by checking SNP allele patterns Increase sensitivity for detection of mosaicism Identify triploidy for which aCGH fails to detect Determine parental
6、 origin of a de novo CNV Improves our understanding of genetic aberrations Enhances the quality control in the diagnostic laboratory workflow Identify genomic regions with LOH related to tumorigenesisPrinciples of CMAs Pathogenic Likely pathogenic Uncertain clinical significance Likely benign benign
7、Classification of Copy Number Variants identified by CMA based on their clinical significancesCMA applications for clinical service受孕受孕胚胎胚胎植植入前入前的的基因基因及及基因基因组组检测检测产产前前筛查筛查及及诊断诊断新生新生儿儿筛筛查查及诊断及诊断 遗传遗传病病病病人人（儿童及成（儿童及成人人）诊断）诊断健康健康人群人群隐隐性遗性遗传传病携病携带带者检出者检出健康健康及亚及亚健健康人康人群群疾病疾病易易感基感基因因检测检测遗传病的基因及基因组检遗传病的基因及
8、基因组检测测肿瘤的基因及基因组检测肿瘤的基因及基因组检测遗传遗传性肿性肿瘤瘤携带携带者者检出检出无症无症状早状早期期筛查筛查分子分子诊断诊断靶向靶向药物药物的的选择选择预后预后判断判断治疗治疗监控监控复发复发基因基因克克隆检出隆检出Validations of CMA platforms for Clinical Services Technical Validations Clinical ValidationsValidation-Agilent aCGH-244KYu,Yu,S.S.Bittel,Bittel,DC.DC.Kibiryeva,Kibiryeva,N.N.Zwick,Zwi
9、ck,D D L.L.Cooley,Cooley,LD.LD.Validation of Validation of the the Agilent Agilent 244K 244K oligonucleotide oligonucleotide array-based comparative genomic array-based comparative genomic hybridization hybridization platform platform for for clinical clinical cytogenetic cytogenetic diagnosis.diagn
10、osis.Am J Clin PatholAm J Clin Pathol 2009;132(3):349-60.2009;132(3):349-60.Verification of aCGH findingsYu S,Yu S,Kielt,Kielt,M,M,Stegner Stegner A,A,Bittel,Bittel,DC.DC.Cooley,Cooley,LD.LD.Application of Application of Quantitative Quantitative Real-TimeReal-Time PCR PCR Methods Methods for the fo
11、r the Verification of Genomic Imbalances Detected Verification of Genomic Imbalances Detected by by Microarray-based Microarray-based Comparative Comparative Genomic Genomic Hybridization.Hybridization.Genet Genet Test Test Mol BiomarkersMol Biomarkers 2009;13(6):751-60.2009;13(6):751-60.aCGH for po
12、stnatal diagnosis(1)Identify Novel Genomic DisordersBellone RR et al.Differential gene expression of TRPM1,the potential cause of congenital stationary night blindness(先先天性静天性静止止 性性 夜夜 盲盲 症症 )and coat spotting patterns(LP)in the Appaloosa horse(Equus caballus).Genetics.2008 Aug;179(4):1861-70.Lepich
13、on JB,Bittel DC,Graf WD,Yu S.A 15q13.3 homozygous microdeletion associated with a severe neurodevelopmental disorder suggests putative functions of the TRPM1,CHRNA7,and other homozygously deleted genes.Am J Med Genet A.2010 May;152A(5):1300-4.Lepichon JB,Yu S,Graf WD,and Bittel DC.Genome wide gene e
14、xpression in a patient with 15q13.3 homozygous microdeletion syndrome Eur J Hum Genet.2013,1-7.15q13.3 homozygous microdeletionAbdelmoity AT,Hall JJ,Bittel DC,Yu S.1.39 Mb inherited interstitial deletion in 12p13.33 associated with developmental delay.Eur J Med Genet.2011 Mar-Apr;54(2):198-203.12p13
15、.33 deletionRamalingam A,Zhou XG,Fiedler SD,Brawner SJ,Joyce JM,Liu HY,Yu S.16p13.11 duplication is a risk factor for a wide spectrum of neuropsychiatric disorders.J Hum Genet.2011 Jul;56(7):541-416p13.11 duplicationYu S and Graf WD.BRAF gene deletion broadens the clinical spectrum neuro-cardio-faci
16、al-cutaneous syndromes.J Child Neurol.2011 Dec;26(12):1593-6.BRAF gene deletionYu S,Shao L,Kilbride H,Zwick DL.Haploinsufficiencies of FOXF1 and FOXC2 genes associated with lethal alveolar capillary dysplasiaand congenital heart disease.Am J Med Genet A.2010 May;152A(5):1257-62.16q24.1 microdeletion
17、aCGH for postnatal diagnosis(2)Discover Novel Genetic MechanismsTelomere capture as a frequent mechanism for stabilization of the terminal chromosomal deletion associated with inverted duplication.Yu S and Graf WD.Telomere capture as a frequent mechanism for stabilization of the terminal chromosomal
18、 deletion associated with inverted duplication.Cytogenet Genome Res.2010;129(4):265-74.Genomic Genomic profile profile of of copy copy number variants onnumber variants on the the short short arm arm of human chromosomeof human chromosome 8 8Yu Yu S,S,Fiedler S,Stegner A,and Graf WD.Genomic profile
19、of copy number variants on the short arm of human chromosome 8.Eur J Hum Genet.2010 Oct;18(10):1114-20.Yu S,Zhou XG,Fiedler SD,Brawner SJ,Joyce JM,Liu HY.Cardiac defects are infrequent findings in individuals with 8p23.1 genomic duplications containing GATA4.Circ Cardiovasc Genet.2011 Dec;4(6):620-5
20、.8p23.1 genomic duplicationsaCGH for postnatal diagnosis(3)Refine breakpoints of genomic disordersGenomic Disorders onchromosome 22Yu S,Bittel DC,Yu S,Newkirk H,Kibiryeva N,Butler MG,Cooley LD.Refining the 22q11.2 deletion breakpoints in DiGeorge syndrome by aCGH.Cytogenet Genome Res 2009;124(2):113
21、-20.Yu S,Graf WD,Ramalingam A,Brawner SJ,Joyce JM,Fiedler DS,Zhou XG,and Liu HY(2001)Identification of copy number variants(CNV)on human chromosome 22 in patients with a variety of clinical findings.Cytogenet Genome Res.2011;134(4):260-8.Epub 2011 Aug 17.Butler MG,Bittel DC,Kibiryeva N,Cooley LD,Yu
22、S.An interstitial 15q11-q14 deletion:expanded Prader-Willi syndrome phenotype.Am Am J J Med Med Genet Genet A.A.2010 Feb;152A(2):404-8.expanded Prader-Willi syndromeaCGH for postnatal diagnosis(4)Characterize origin of marker chromosomeA neocentric supernumerary marker chromosome originating from th
23、e Xp distal regionYu Yu S S,Barbouth D,Benke PJ,Warburton PE,Fan YS.Characterization of a neocentric supernumerary marker chromosome originating from the Xp distal region by FISH,CENP-C staining,and array CGH.Cytogenet Cytogenet Genome Genome ResRes 2007;116(1-2):141-52007;116(1-2):141-5.Yu S,Fiedle
24、r DS,Brawner SJ,Joyce JM,Zhou XG,and Liu HY.Characterizing Supernumerary Marker Chromosomes(SMCs)with Combination of Multiple Techniques.Cytogenet Genome Res.2012;136(1):6-14.Characterizing Supernumerary Marker Chromosomes(SMCs)SNP Microarray for Clinical ApplicationsWhy should SNP microarray be use
25、d to replace aCGH?What is a SNP?What is a SNP array?Advantages of SNP arrays over aCGH?Applications of SNP MicroarraySingle Nucleotide Polymorphism(SNP)Definition:a single nucleotide change in a DNA sequence that occurs in asignificant proportion(1%)in a large population.Different Levels of polymorp
26、hisms in human genomeinv(9)SNP facts Represent 90%of genomic variations in human genome,There is a SNP per 100-300 bp in human genome,SNPs can occur in coding(gene)and noncoding regions of the genome,Many SNPs have no effect on cell function,but others could predispose people to disease or influence
27、 their response to medicines,environmental factors.DNA Sequencing Hybridization Microarrays TaqMan,Molecular Beacons Allele-specific PCR FRET Intercalating Dyes Primer Extension MALDI-tof SNaPshot Ligation Padlock Probes Rolling Circle Amplification Endonuclease Cleavage RFLP PIRA/RFLPMethods to dis
28、cover novel SNPs and detect known SNPsClinical SNP arraysNumbers of Markers(probes)Average Marker Spacing(base pairs)Genes covered(25 markers/100 kb)AgilentIlluminaAffymetrixThe CytoScan HD Array from Affymetrix Indicate the presence of uniparental disomy(UPD)Indicate the presence of consanguinity I
29、ndicate the presence of shared ancestry Identify recessive gene mutations Confirm CNV calls by checking SNP allele patterns Increase sensitivity for detection of mosaicism Identify triploidy for which aCGH fails to detect Determine parental origin of a de novo CNV Improves our understanding of genet
30、ic aberrations Enhances the quality control in the diagnostic laboratory workflow Identify genomic regions with LOH related to tumorigenesisSNP Microarray Analysis for Clinical ServiceSNP array for Clinical Service(5)Indicate the presence of uniparental disomy(UPD)UPD chromosomes associated with imp
31、rinting disordersSNP array for Clinical Service(6)Indicate the presence of consanguinityROH indicating consanguinityTwo siblings with high percentage ROHindicating consanguinity marriageGlobal Consanguinity RatesHamamy H.Consanguineous marriages:Preconception consultation in primary health care sett
32、ings.J Community Genet.2012 Jul;3(3):185-92.SNP array for Clinical Service(7)Identify recessive gene mutationsROH facilitating identification of recessive genes16month-old boy with multiple endocrine&structural issues:congenital primary hypothyroidism hyperinsulinism in the face of hypoglycemia grow
33、th hormone deficiency cortisol deficiency resolved direct and indirect hyperbilirubinemia cortical visual impairment respiratory issues,aspiration significant developmental delay hypotonia dysmorphic facial features hirsutism with low anterior hairlineCMA testing for CNV is recommended as a first-li
34、ne test in the initial postnatal evaluation of individuals with the following:Multiple anomalies not specific to a well-delineated genetic syndrome.Apparently nonsyndromic DD/ID.Autism spectrum disorders.Further determination of the use of CMA testing for the evaluation of the child with growth reta
35、rdation,speechdelay,and other less well-studied indications is recommended,particularly by prospective studies and aftermarket analysis.Appropriate follow-up is recommended in cases of chromosome imbalance identified by CMA,to include cytogenetic/FISH studies of the patient,parental evaluation,and c
36、linical genetic evaluation and counseling.1.Graf WD,Le Pichon JB,Bittel DC,Abdelmoity AT,and Yu S.Practice parameter:evaluation of the child with microcephaly(an evidence-based review):report of the quality standards subcommittee of the American Academy of Neurology and the Practice Committee of the
37、 Child Neurology Society.Neurology.2010;74(13):1080-1.2.Ledbetter DH.Cytogenetic technology-genotype and phenotype.N Engl J Med.2008;359(16):1728-30.3.Bejjani BA and Shaffer LG.Clinical utility of contemporary molecular cytogenetics.Annu Rev Genomics Hum Genet.2008;9:71-86.Clinical indicationTesting
38、不孕不不孕不育育(about 1/3 due to genetic/genomic defects)已知基已知基因因基因组异常基因组异常 核型分析核型分析 原位荧光杂交原位荧光杂交 基因芯基因芯片片(BlueGnome)MES,WES,WGS胚胎植胚胎植入入前前检检测测(PGS&PGD)已知基已知基因因基因组异常基因组异常 原位荧光杂交原位荧光杂交 基因芯片基因芯片 单基因检测单基因检测，WGSCMA for PGS/PGD(preimplantation genetic screening/diagnosis(8)Colls,P.et al.Validation of array compa
39、rative genome hybridization for diagnosis of translocations in preimplantation human embryos.Reprod Biomed Online.2012;24:621629.Treff,N.R.et al.Single nucleotide polymorphism microarray-based concurrent screening of 24-chromosome aneuploidy and unbalanced translocations in preimplantation human emb
40、ryos.Fertil Steril.2011;95:16061612.Johnson,D.S.et al.Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol.Hum Reprod.2010;25:10661075.Peters BA et al.Detection and phasing of single base de novo mutations in biopsies from human in vitro fert
41、ilized embryos by advanced whole-genome sequencing.Genome Res.2015 Mar;25(3):426-34.CMA for Prenatal Screening/Diagnosis(9)Norton ME,et al.Cell-free DNA analysis for noninvasive examination of trisomy.N Engl J Med.2015 Apr 23;372(17):1589-97.CMA for Molecular characterization of Tumors(10)SNP microa
42、rray for health individuals(11)祖先和家谱信息 疾病携带者信息 FDA于2015年10月批准了其包含35项检测的个人基因 组服务，对于基因检测的合法市场化具有里程碑式意义遗传特征信息药物代谢信息Future of CMA for Clinical serviceCMA for Genetic/Genomic Disorders受孕胚胎植入前的基因受孕胚胎植入前的基因及及基因基因组组检测检测产前筛查及诊断产前筛查及诊断遗传病病人（儿童及成遗传病病人（儿童及成人人）诊断）诊断CMA for Characterization of TumorsCMADeletion/D
43、uplication+GenotypingWhole Genome Sequencing(WGS)Point mutations-GenotypingAll kinds of genomic abnormalitiesCMAs function in clinical service will be covered gradually by WGS Cost AccuracyGenomic MedicineUnderstanding the structure of genomesUnderstanding the biology of genomesAdvancing the science of MedicineImproving the effectiveness of healthcareImproving the effectiveness of healthcarefrom Base Pairs to BedsideGreen ED et al.Charting a course for genomic medicine from base pairs to bedside.Nature.2011;470(73 33):204-13.
