1、技術(shù)(microarray) 的臨床應(yīng)用基因于MD, PhD, FACMG金域檢驗(yàn)（KingMed Diagnostics)&University of Washington School of Medicine人類基因及基因組- 2 x 30 億個(gè)堿基Ø 23 對(duì)Ø 編碼 21,000 個(gè)基因 -編碼序列占整個(gè)基因組的1.5%基因及基因組病 （遺傳?。?#167;數(shù)量異常Trisomy 21 （唐氏綜合癥）Trisomy 18Trisomy 13Sex chromosomal aneuploidiesMosaic trisomies of other chromo

2、somes結(jié)構(gòu)變化More than 200 known disordersMore than 1000 rare abnormalitiesØØØØاØا單基因病 (more than 8,000)ØØDominant Recessive腫瘤也是基因及基因組病§§線粒體病多基因病Ø 人類有60多種Ø所有腫瘤都含有基因及基因組異常ØGenes + EnvironmentsThompson & Thompson Genetic

3、s In Medicine. Eighth Edition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）Ø 智力低下Ø性疾病Ø -遺傳病的基因（Microarray）一代測(cè)序（Sanger Sequencing）原位熒光雜交（FISH）氣相色譜-質(zhì)譜超高效液相色譜酶學(xué)檢測(cè)基因/基因組檢測(cè)蛋白質(zhì)及代謝產(chǎn)物檢測(cè)核型分析（Karyoty非測(cè)序分子生物學(xué)技術(shù)（non-DNA techniques）二代測(cè)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

4、 Microarray Analysis (CMA)Ø Principles of CMAØ Current Status of CMA Application for Clinical ServiceØ Future Trends of CMA for Clinical ServicePrinciples of CMAsaCGH techniquesSNP microarray199220032005Ø Indicating the presence of uniparental disomy (UPD)Ø Indicating the pr

5、esence of consanguiØ Indicating the presence of shared ancestryØ Identify recessive gene mutationsØ Confirm CNV calls by checking SNP allele patternsØ Increase sensitivity for detection of mosaicismØ Identify triploidy for which aCGH fails to detectØ Determine parental

6、origin of a de novo CNVØ Improves our understanding of genetic aberrationsØ Enhances the quality control in the diagnostic laboratory workflowØ Identify genomic regions with LOH related to tumorigenesisClassification of Copy Number Variants identified by CMA based on their clinical si

7、gnificancesØØØØØPathogenicLikely pathogenicUncertain clinical significance Likely benignbenignCMA applications for clinical service遺傳病的基因及基因組檢測(cè)腫瘤的基因及基因組檢測(cè)vvv受孕胚胎植入前的基因及基因組檢測(cè)ØØØ遺傳性腫瘤攜帶者檢出無癥狀早期篩查分子產(chǎn)前篩查及新生兒篩查及Ø靶向的選擇vvv遺傳?。▋和俺扇耍?#216;ØØ預(yù)后治療復(fù)發(fā)基因克隆檢出

8、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Validations of CMA platforms for Clinical ServicesØ Technical ValidationsØ Clinical ValidationsValidation-Agilent aCGH-244KYu, S. Bittel, DC. Kibiryeva, N. Zwick, D L. Cooley, LD.Validation of the Agilent 244K oligonucleotide array-based comparative genomic hybrid

9、ization 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 Qutative Real-Time PCR Methods for the Verification of Genomic Imbalances Detected by Microarray-based ComparativeGen

10、omic Hybridization. Genet Test Mol Biomarkers 2009;13(6):751-60.aCGH for postnatal diagnosis (1)ØIdentify Novel Genomic Disorders15q13.3 homozygousmicretionBellone 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

11、 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 micretion associated with a severe neurodevelopmental disorder suggests putative functions of the TRPM1,CHRNA7, and other homozygously deleted

12、 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 micr Eur J Hum Genet. 2013, 1-7.etion syndrome12p13.33 deletionAbdelmoity AT, Hall JJ, Bittel DC, Yu S.1.39 Mb inherited interstitial deletion

13、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 spectrum of neuropsychiatric disorders. J Hum Genet. 2011 Jul;56(7):5

14、41-4BRAF 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 micretionYu S, Shao L, Kilbride H, Zwick DL.Haploinsufficiencies of FOXF1 and FOXC2 genes associated with lethal alveolar ca

15、pillary dysplasiaand congenital heart disease.Am J Med Genet A. 2010 May;152A(5):1257-62.aCGH for postnatal diagnosis (2)Ø Discover Novel Genetic MechanismsTelomere capture as a frequent mechanism for stabilization of the terminal chromosomal deletion associated with inverted duplication.Yu S a

16、nd 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 arm of human chromosome 8Yu S, Fiedler S, Stegner A, and Graf

17、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 findings in individuals with 8p23.1 genomic duplications contai

18、ning GATA4. Circ Cardiovasc Genet. 2011 Dec;4(6):620-5.aCGH for postnatal diagnosis (3)Refine breakpoints of genomic disordersØGenomic 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

19、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 findings. Cytogenet Genome Res. 2011;134(4):260-8. Epub 2

20、011 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)Characterize origin of marker chromosomeØA neocentr

21、ic 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 Genome Res 2007;116(1-2):1

22、41-5.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):6-14.SNP Microarray for Clinical ApplicationsWhy sh

23、ould 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 occurs in asignificant proportion ( 1%) in a large

24、 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 regionsof tome,v Many SNPs have no effect on cell function, but others coul

25、d predispose people to disease or influence their response to medicines, environmental factors.Methods to discover novel SNPs and detect known SNPsv DNA Sequencingv HybridizationØ MicroarraysØ TaqMan, Molecular Beaconsv Allele-specific PCRØ FRETØ Intercalating Dyesv Primer Extens

26、ionØ MALDI-tofØ SNaPshotv LigationØ Padlock ProbesØ Rolling Circle Amplificationv Endonuclease CleavageØ RFLPØ PIRA/RFLPClinical SNP arraysv Agilentv Illuminav AffymetrixThe CytoScan HD Array from AffymetrixNumbers of Markers (probes)Average Marker Spacing (base pairs)G

27、enes covered (25 markers/100 kb)SNP Microarray Analysis for Clinical ServiceØ Indicate the presence of uniparental disomy (UPD)Ø Indicate the presence of consanguiØ Indicate the presence of shared ancestryØ Identify recessive gene mutationsØ Confirm CNV calls by checking SNP

28、 allele patternsØ Increase sensitivity for detection of mosaicismØ Identify triploidy for which aCGH fails to detectØ Determine parental origin of a de novo CNVØ Improves our understanding of genetic aberrationsØ Enhances the quality control in the diagnostic laboratory work

29、flowØ Identify genomic regions with LOH related to tumorigenesisSNP array for Clinical Service (5)Ø Indicate the presence of uniparental disomy (UPD)UPD chromosomes associated with imprinting disordersSNP array for Clinical Service (6)Ø Indicate the presence of consanguiROH indicating

30、 consanguiTwo siblings with high percentage ROHindicating consanguimarriageGlobal ConsanguiRatesHamamy H. Consanguineous marriages: Preconception consultation inprimary health care settings. J CommuGenet. 2012 Jul;3(3):185-92.SNP array for Clinical Service (7)ØIdentify recessive gene mutationsR

31、OH facilitating identification of recessive genes16-month-old boy with multiple endocrine &structural issues:vvvvvvvvvvvcongenital primary hypothyroidism hyperinsulinism in the face of hypoglycemia growth hormone deficiencycortisol deficiencyresolved direct and indirect hyperbilirubinemia cortic

32、al visual impairmentrespiratory issues, aspiration significant developmental delay hypotoniadysmorphic facial featureshirsutism 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 revi

33、ew): 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. Cytogenetic technology-genotype and phenotype. N Engl J Med. 2008;359(16):1728-30.Bejjani BA and Shaffer LG. C

34、linical utility of contemporary molecular cytogenetics. Annu Rev Genomics Hum Genet. 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.A

35、pparently 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, particularly by prospective studies and aftermarket analysis. Appropri

36、ate 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.CMA for PGS/PGD (preimplantation genetic screening/diagnosis (8)ØColls, P. et al. Validation o

37、f array comparative 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 prei

38、mplantation human embryos. Fertil Steril. 2011; 95: 16061612.ØØJohnson, D.S. et al. Preclinical validation of a microarray method for full molecular karyoty blastomeres in a 24-h protocol. Hum Reprod. 2010; 25: 10661075.ofPeters BA et al. Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome seq