1、基因芯片技術(shù)(microarray) 的臨床應(yīng)用于世輝 MD, PhD, FACMG金域檢驗（KingMed Diagnostics) &University of Washington School of Medicine人類基因及基因組 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?。┤旧w數(shù)量異常Trisomy 21 （唐氏綜合癥） Trisomy 18Trisomy 13Sex chromosomal aneuploidiesMosaic trisomies of other chromosomes染色體結(jié)構(gòu)變

2、化More than 200 known disorders More than 1000 rare abnormalities單基因病 (more than 8,000)Dominant Recessive腫瘤也是基因及基因組病線粒體病多基因病 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Genes + EnvironmentsThompson & Thompson Genetics In Medicine. Eighth Edition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）

3、 智力低下 遲發(fā)性疾病 -遺傳病的實驗室診斷基因芯片（Microarray）一代測序（Sanger Sequencing）原位熒光雜交（FISH）氣相色譜-質(zhì)譜超高效液相色譜基因/基因組檢測酶學(xué)檢測蛋白質(zhì)及代謝產(chǎn)物檢測非測序分子生物學(xué)技術(shù)（non-DNA techniques）核型分析（Karyotyping）二代測序（NGS）高效液相色譜-串聯(lián)質(zhì)譜電感耦合等離子體質(zhì)譜Chromosome Microarray Analysis (CMA) Principles of CMA Current Status of CMA Application for Clinical Service Futu

4、re Trends of CMA for Clinical ServicePrinciples of CMAsaCGH techniquesSNP microarray199220032005 Indicating the presence of uniparental disomy (UPD) Indicating the presence of consanguinityIndicating the presence of shared ancestry Identify recessive gene mutationsConfirm CNV calls by checking SNP a

5、llele 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 genetic aberrationsEnhances the quality control in the diagnostic laboratory workflow Identify genomic regions wit

6、h LOH related to tumorigenesisClassification of Copy Number Variants identified by CMA based on their clinical significancesPathogenicLikely pathogenicUncertain clinical significance Likely benignbenignCMA applications for clinical service遺傳病的基因及基因組檢測腫瘤的基因及基因組檢測vvv受孕胚胎植入前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

7、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 產(chǎn)前篩查及診斷新生兒篩查及診斷vvv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Validations of CMA platforms for Clinical Services Technical Validations Clinical ValidationsValidation-Agilent aCGH-244KYu, S. Bittel, DC. Kibiryeva, N. Zwick, D L. Cooley, LD.Validation of the Agilent 244K o

8、ligonucleotide array-based comparative genomic hybridization platform for clinical cytogenetic diagnosis. Am J Clin Pathol 2009;132(3):349-60.Verification of aCGH findingsYu S, Kielt, M, Stegner A, Bittel, DC. Cooley, LD.Application of Quantitative Real-Time PCR Methods for the Verification of Genom

9、ic Imbalances Detected by Microarray-based Comparative Genomic Hybridization. Genet Test Mol Biomarkers 2009;13(6):751-60.aCGH for postnatal diagnosis (1)Identify Novel Genomic Disorders15q13.3 homozygous microdeletionBellone RR et al. Differential gene expression of TRPM1, the potential cause of co

10、ngenital 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):1861-70.Lepichon JB, Bittel DC, Graf WD, Yu S.A 15q13.3 homozygous microdeletion associated with a severe neurodevelopmental disorder suggests putative f

11、unctions 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 expression in a patient with 15q13.3 homozygous microdeletion syndrome Eur J Hum Genet. 2013, 1-7.12p13.33 deletionAbdelmoity AT, H

12、all 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):198-203.16p13.11 duplicationRamalingam A, Zhou XG, Fiedler SD, Brawner SJ, Joyce JM, Liu HY, Yu S.16p13.11 duplication is a risk factor for a wide spect

13、rum of neuropsychiatric disorders. J Hum Genet. 2011 Jul;56(7):541-4BRAF gene deletionYu S and Graf WD.BRAF gene deletion broadens the clinical spectrum neuro-cardio-facial-cutaneous syndromes.J Child Neurol. 2011 Dec;26(12):1593-6.16q24.1 microdeletionYu S, Shao L, Kilbride H, Zwick DL.Haploinsuffi

14、ciencies of FOXF1 and FOXC2 genes associated with lethal alveolar capillary dysplasia and congenital heart disease.Am J Med Genet A. 2010 May;152A(5):1257-62.aCGH for postnatal diagnosis (2)Discover Novel Genetic MechanismsTelomere capture as a frequent mechanism for stabilization of the terminal ch

15、romosomal 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;129(4):265-74.Genomic profile of copy number variants on the short

16、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):1114-20.8p23.1 genomic duplicationsYu S, Zhou XG, Fiedler SD, Brawner SJ, Joyce JM, Liu HY.Cardiac defects are infrequent f

17、indings in individuals with 8p23.1 genomic duplications containing GATA4. Circ Cardiovasc Genet. 2011 Dec;4(6):620-5.aCGH for postnatal diagnosis (3)Refine breakpoints of genomic disordersGenomic Disorders on chromosome 22Yu S, Bittel DC, Yu S, Newkirk H, Kibiryeva N, Butler MG, Cooley LD.Refining t

18、he 22q11.2 deletionbreakpoints in DiGeorge syndrome by aCGH. Cytogenet Genome Res 2009;124(2):113-20.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 fi

19、ndings. Cytogenet Genome Res. 2011;134(4):260-8. Epub 2011 Aug 17.expanded Prader-Willi syndromeButler MG, Bittel 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.aCGH for postnatal diagnosis (4)

20、Characterize origin of marker chromosomeA 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,

21、and array CGH.Cytogenet Genome Res 2007;116(1-2):141-5.Characterizing Supernumerary Marker Chromosomes (SMCs)Yu 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

22、-14.SNP Microarray for Clinical ApplicationsWhy should SNP microarray be used to replace aCGH ?v What is a SNP?v What is a SNP array?v Advantages of SNP arrays over aCGH?v Applications of SNP MicroarraySingle Nucleotide Polymorphism (SNP)Definition: a single nucleotide change in a DNA sequence that

23、occurs in a significant proportion ( 1%) in a large population.Different Levels of polymorphisms in human genomeinv(9)SNP factsv Represent 90% of genomic variationsin human genome,v There is a SNP per 100-300 bp in human genome,v SNPs can occur in coding (gene) and noncoding regions of the genome,v

24、Many SNPs have no effect on cell function, but others could predispose people to disease or influence their response to medicines, environmental factors.Methods to discover novel SNPs and detect known SNPsDNA Sequencing HybridizationMicroarraysTaqMan, Molecular Beacons Allele-specific PCRFRETInterca

25、lating Dyes Primer ExtensionMALDI-tof SNaPshotLigationPadlock ProbesRolling Circle Amplification Endonuclease CleavageRFLP PIRA/RFLPClinical SNP arraysAgilent Illumina AffymetrixThe CytoScan HD Array from AffymetrixNumbers of Markers (probes)Average Marker Spacing (base pairs)Genes covered (25 marke

26、rs/100 kb)SNP Microarray Analysis for Clinical Service Indicate the presence of uniparental disomy (UPD) Indicate the presence of consanguinity Indicate the presence of shared ancestry Identify recessive gene mutations Confirm CNV calls by checking SNP allele patterns Increase sensitivity for detect

27、ion of mosaicism Identify triploidy for which aCGH fails to detect Determine parental 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 tumorigenesisSNP array for C

28、linical Service (5) Indicate the presence of uniparental disomy (UPD)UPD chromosomes associated with imprinting disordersSNP array for Clinical Service (6) Indicate the presence of consanguinityROH indicating consanguinityTwo siblings with high percentage ROH indicating consanguinity marriageGlobal

29、Consanguinity RatesHamamy H. Consanguineous marriages: Preconception consultation in primary health care settings. J Community Genet. 2012 Jul;3(3):185-92.SNP array for Clinical Service (7)Identify recessive gene mutationsROH facilitatingidentificationofrecessive genes16-month-old boy with multiple

30、endocrine & structural issues:vvvvvvvvvvvcongenital primary hypothyroidism hyperinsulinism in the face of hypoglycemia growth hormone deficiencycortisol deficiencyresolved direct and indirect hyperbilirubinemia cortical visual impairmentrespiratory issues, aspiration significant developmental delay

31、hypotoniadysmorphic facial features hirsutism with low anterior hairline1.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 Neuro

32、logy and the Practice Committee of the Child Neurology Society. Neurology. 2010;74(13):1080-1.Ledbetter DH. 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 Gene

33、t. 2008;9:71-86.2.3.CMA testing for CNV is recommended as a first-line 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 t

34、he use of CMA testing for the evaluation of the child with growth retardation, 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, t

35、o include cytogenetic/FISHstudies of the patient, parental evaluation, and clinical genetic evaluation and counseling.CMA for PGS/PGD (preimplantation genetic screening/diagnosis (8)Colls, P. et al. Validation of array comparative genome hybridization for diagnosis of translocations in preimplantati

36、on 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 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 phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequen