1、基因芯片技術(microarray) 的臨床應用基因芯片技術(microarray) 的臨床應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 x 30 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）染色體數量異常Trisomy 21 （唐氏綜合癥）Trisomy 18Trisomy 13Sex chromosomal aneuploidiesMosaic trisomies of other chromosomes染色體結構變化More th

2、an 200 known disordersMore than 1000 rare 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人類基因及基因組Dominant線粒體病腫瘤也是基因及基因組中國年出生1600萬，出生缺陷發生率在5.6%, 每年新增出生缺陷數約90萬例。(嬰兒在出生的一年內，體格上出現明顯的結構異常和需要手術矯正的畸形）智力低下遲發性疾病-Thompson & Thompson Genetics In Medicine. Eighth Edition中國年出生1600萬，出生缺陷發生率在5.6%, 每年

3、新增出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）非測序分子生物學技術（non-DNA techniques）核型分析（Karyotyping）基因/基因組 檢測基因芯片（Microarray）酶學檢測高效液相色譜-串聯質譜電感耦合等 離子體質譜氣相色譜-質譜超高效 液相色譜蛋白質及代 謝產物檢測遺傳病的實驗室診斷二代測序（NGS）原位熒光雜交一代測序非測Chromosome Microarray Analysis (CMA)Principles of CMACurrent Status of CMA Application for

4、 Clinical ServiceFuture Trends of CMA for Clinical ServiceChromosome Microarray AnalysisaCGH techniquesSNP microarray199220032005 Indicating the presence of uniparental disomy (UPD)Indicating the presence of consanguinityIndicating the presence of shared ancestryIdentify recessive gene mutationsConf

5、irm CNV calls by checking SNP allele patternsIncrease sensitivity for detection of mosaicismIdentify triploidy for which aCGH fails to detectDetermine parental origin of a de novo CNVImproves our understanding of genetic aberrationsEnhances the quality control in the diagnostic laboratory workflowId

6、entify genomic regions with LOH related to tumorigenesisPrinciples of CMAsaCGH techniquesSNP microarray1PathogenicLikely pathogenicUncertain clinical significanceLikely benignbenignClassification of Copy Number Variants identified by CMA based on their clinical significancesPathogenicClassification

7、of CoCMA applications for clinical service受孕胚胎植入前的基因及基因組檢測產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后判斷治療監控復發基因克隆檢出CMA applications for clinical Validations of CMA platforms for Clinical ServicesTechnical ValidationsClinical Valida

8、tionsValidation-Agilent aCGH-244KYu, S. Bittel, DC. Kibiryeva, N. Zwick, D L. Cooley, LD.Validation of the Agilent 244K oligonucleotide array-based comparative genomic hybridization platform for clinical cytogenetic diagnosis. Am J Clin Pathol 2009;132(3):349-60.Verification of aCGH findingsYu S, Ki

9、elt, M, Stegner A, Bittel, DC. Cooley, LD.Application of Quantitative Real-Time PCR Methods for the Verification of Genomic Imbalances Detected by Microarray-based Comparative Genomic Hybridization. Genet Test Mol Biomarkers 2009;13(6):751-60.Validations of CMA platforms faCGH for postnatal diagnosi

10、s (1)Identify Novel Genomic DisordersaCGH for postnatal diagnosis (Bellone RR et al. Differential gene expression of TRPM1, the potential cause of congenital stationary night blindness (先天性靜止 性 夜 盲 癥 ) and coat spotting patterns (LP) in the Appaloosa horse (Equus caballus). Genetics. 2008 Aug;179(4)

11、:1861-70.Lepichon 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 Bitt

12、el DC.Genome wide gene expression in a patient with 15q13.3 homozygous microdeletion syndrome Eur J Hum Genet. 2013, 1-7.15q13.3 homozygous microdeletionBellone RR et al. DifferentialAbdelmoity AT, Hall JJ, Bittel DC, Yu S.1.39 Mb inherited interstitial deletion in 12p13.33 associated with developme

13、ntal delay.Eur J Med Genet. 2011 Mar-Apr;54(2):198-203.12p13.33 deletionAbdelmoity AT, Hall JJ, BittelRamalingam 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.

14、11 duplicationRamalingam A, Zhou XG, FiedlerYu S and Graf WD.BRAF gene deletion broadens the clinical spectrum neuro-cardio-facial-cutaneous syndromes.J Child Neurol. 2011 Dec;26(12):1593-6.BRAF gene deletionYu S and Graf WD.BRAF gene delYu S, Shao L, Kilbride H, Zwick DL.Haploinsufficiencies of FOX

15、F1 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 microdeletionYu S, Shao L, Kilbride H, ZwicaCGH for postnatal diagnosis (2)Discover Novel Genetic MechanismsaCGH for postnatal diagnosis (Telomere cap

16、ture 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 deletion associated with inverted duplication.Cytogenet Genome Res. 2010;12

17、9(4):265-74.Telomere capture as a frequentGenomic profile of copy number variants on the short arm of human chromosome 8Yu S, Fiedler S, Stegner A, and Graf WD.Genomic profile of copy number variants on the short arm of human chromosome 8. Eur J Hum Genet.2010 Oct;18(10):1114-20.Genomic profile of c

18、opy numberYu 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):620-5.8p23.1 genomic duplicationsYu S, Zhou XG, Fiedler SD, BraaCGH for postnatal diagnosis

19、 (3)Refine breakpoints of genomic disordersaCGH for postnatal diagnosis (Genomic 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):113-20.Yu S, Graf WD

20、, 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.Genomic Disorders onYu S, BittButler MG, Bittel

21、DC, Kibiryeva N, Cooley LD, Yu S. An interstitial 15q11-q14 deletion: expanded Prader-Willi syndrome phenotype. Am J Med Genet A. 2010 Feb;152A(2):404-8.expanded Prader-Willi syndromeButler MG, Bittel DC, KibiryevaCGH for postnatal diagnosis (4)Characterize origin of marker chromosomeaCGH for postna

22、tal diagnosis (A neocentric supernumerary marker chromosome originating from the Xp distal regionYu 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 G

23、enome Res 2007;116(1-2):141-5.A neocentric supernumerary marYu S, Fiedler 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 Chromosom

24、es (SMCs)Yu S, Fiedler DS, Brawner SJ, SNP Microarray for Clinical ApplicationsWhy should SNP microarray be used to replace aCGH ?What is a SNP?What is a SNP array?Advantages ofSNP arrays over aCGH?Applications of SNP MicroarraySNP Microarray for Clinical ApSingle Nucleotide Polymorphism (SNP)Defini

25、tion: a single nucleotide change in a DNA sequence that occurs in asignificant proportion ( 1%) in a large population.Single Nucleotide PolymorphismDifferent Levels of polymorphisms in human genomeinv(9)Different Levels of polymorphiSNP factsRepresent 90% of genomic variationsin human genome,There i

26、s 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 their response to medicines, environmental factors.SNP factsRepresent 90% of genoDNA Sequen

27、cingHybridizationMicroarraysTaqMan, Molecular BeaconsAllele-specific PCRFRETIntercalating DyesPrimer ExtensionMALDI-tofSNaPshotLigationPadlock ProbesRolling Circle AmplificationEndonuclease CleavageRFLPPIRA/RFLPMethods to discover novel SNPs and detect known SNPsDNA SequencingMethods to discoClinica

28、l SNP arraysNumbers of Markers (probes)Average Marker Spacing (base pairs)Genes covered (25 markers/100 kb)AgilentIlluminaAffymetrixThe CytoScan HD Array from AffymetrixClinical SNP arraysNumbers of Indicate the presence of uniparental disomy (UPD)Indicate the presence of consanguinityIndicate the p

29、resence of shared ancestry Identify recessive gene mutationsConfirm CNV calls by checking SNP allele patternsIncrease sensitivity for detection of mosaicismIdentify triploidy for which aCGH fails to detectDetermine parental origin of a de novo CNVImproves our understanding of genetic aberrationsEnha

30、nces the quality control in the diagnostic laboratory workflowIdentify genomic regions with LOH related to tumorigenesisSNP Microarray Analysis for Clinical Service Indicate the presence of unipSNP array for Clinical Service (5)Indicate the presence of uniparental disomy (UPD)SNP array for Clinical

31、ServiceUPD chromosomes associated with imprinting disordersUPD chromosomes associated witSNP array for Clinical Service (6)Indicate the presence of consanguinitySNP array for Clinical ServiceROH indicating consanguinityTwo siblings with high percentage ROHindicating consanguinity marriageROH indicat

32、ing consanguinityTGlobal Consanguinity RatesHamamy H. Consanguineous marriages: Preconception consultation in primary health care settings. J Community Genet. 2012 Jul;3(3):185-92.Global Consanguinity RatesHamaSNP array for Clinical Service (7)Identify recessive gene mutationsSNP array for Clinical

33、ServiceROH facilitating identification of recessive genesmonth-old boy with multiple endocrine &structural issues:congenital primary hypothyroidismhyperinsulinism in the face of hypoglycemiagrowth hormone deficiencycortisol deficiencyresolved direct and indirect hyperbilirubinemiacortical visual imp

34、airmentrespiratory issues, aspirationsignificant developmental delayhypotoniadysmorphic facial featureshirsutism with low anterior hairlineROH facilitating identificatiCMA testing for CNV is recommended as a first-line test in the initial postnatal evaluation of individuals with the following: Multi

35、ple 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 retardation, speechdelay, and other less well-studied indications is recommended, part

36、icularly 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 clinical genetic evaluation and counseling.Graf WD, Le Pichon JB, Bittel DC,

37、 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 Child Neurology Society. Neurology. 2010;74(13):1080-1.Ledbetter DH.

38、 Cytogenetic technology-genotype and phenotype. N Engl J Med. 2008;359(16):1728-30.Bejjani BA and Shaffer LG. Clinical utility of contemporary molecular cytogenetics. Annu Rev Genomics Hum Genet. 2008;9:71-86.CMA testing for CNV is recommeClinical indicationTesting不孕不育( about 1/3 due to genetic/geno

39、mic 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，WGSCMA for PGS/PGD (preimplantation genetic screening/diagnosis (8)Colls, P. et al. Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embr

40、yos. 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 embryos. 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 phasin