醫(yī)學(xué)遺傳學(xué)檢測技術(shù)課件

1、醫(yī)學(xué)遺傳學(xué)檢測技術(shù)在臨床診斷的應(yīng)用基因和基因組 46 chromosomes 2 x 3,000,000,000 DNA base pairs in each haploid encoding 21,000 genes Each mitochondrial -16,500 base pairs in each mitochondrial DNA molecule encoding 37 genes 基因與個體發(fā)育及疾病的關(guān)系基因組: 細(xì)胞核DNA成分和線粒體DNA分子的總和基因: 基因組內(nèi)一個個具體的結(jié)構(gòu)和功能單位功能：精確控制胚胎發(fā)育和分化的每一個步驟;決定了個體的所有生命特征; 決定了個體

2、患各種疾病的可能性.遺傳病及特征遺傳病：遺傳物質(zhì)發(fā)生突變所引起的疾病，生殖細(xì)胞或受精卵的突 變、體細(xì)胞突變。種類：確定的遺傳疾病超過24000種。 單基因病- 涉及一對基因，AR、AD、XR、XD。 多基因病-多對基因和環(huán)境共同作用所導(dǎo)致的疾病。 染色體病- 數(shù)目異常及結(jié)構(gòu)異常引起的疾病。 體細(xì)胞遺傳病- 體細(xì)胞突變?nèi)缒[瘤。 線粒體病- 線粒體及核基因異常。特征：垂直傳遞、終生性、發(fā)病率低、危害嚴(yán)重、家族性發(fā)病、多 無有效治療。成為危害人類健康的主要疾病 基因組異常與基因病的診斷方法Laboratory Diagnosis Genetic MethodsCytogeneticsMolecula

3、r GeneticsBiochemical Genetics Non-Genetic MethodsPathologyCytologyImmunologyNon-Laboratory Diagnosis RadiologyClinical Evaluation Carrier detection Preimplantation genetic diagnosis Prenatal diagnosis Newborn screening Postnatal diagnostic testing Germ line mutationsSomatic mutation Predictive, pre

4、symptomatic testing and Personalized Medicine基于aCGH&NGS的預(yù)防、診斷、治療Variability in preparing chromosomesKaryotyping variation as result of operator skillsResolution can range from 5 Mb to 10 Mb depending on preparationMicroarrays offer unbiased means of detecting chromosomal aberrations. The same chromo

5、some prepared 6 different times!16 TELOMERIC PROBES17DiGeorge Syndrome ( microdeletion on 22 ) 22q13 specific control signal 22q11.2 specific DiGeorge probeSRY locus specific probe. SRY/X probe was used. X chromosome centromer specific probe SRY probeLocus specific probes18Centromer specific probes

6、chromosome 7 centromer specific signal chromosome 8 centromer specific signal19Whole chromosome painting probesChromosome 8 painting probe20R x FISH=Cross-species colour FISH /./v1/n1/full/nprot.2006.41.html21Spectral Karyotyping (SKY)22Multiplex FISHMetaphase spread of a lung cancer cell line after

7、 hybridization of the 5-fluorochrome M-FISH mix shown in true-color (a) and classification-color representation (b). The karyotype is shown in (c).2008Nature Education ArrayCGH技術(shù)明顯的優(yōu)勢核型分析：帶型復(fù)雜、個體差異，不可能全部機械化FISH技術(shù)：位點受限、需已知探針序列傳統(tǒng)CGH ：人為因素的限制，需要一定的經(jīng)驗技術(shù)和勞動力支持，必需依靠經(jīng)驗豐富的細(xì)胞遺傳學(xué)家。陣列CGH：高通量、高分辨，靶序列特定檢測, 精確地定位

8、。其結(jié)果自動操縱控制，既快速又直觀。 Array CGH 的技術(shù)限制1. aCGH 不能檢測：平衡染色體重排某些多倍體DNA序列的堿基改變探針沒有覆蓋區(qū)域的異常2. aCGH檢測結(jié)果正常不等于受檢者遺傳學(xué)檢測完全正常3. 異常的aCGH檢測結(jié)果可能臨床意義不明CytoScan 750K適合臨床對于低成本需求的芯片 適合臨床對于低成本需求的細(xì)胞遺傳學(xué)芯片 含有超過75萬種探針，包括 20萬種可以分型的SNP探針 55萬個拷貝數(shù)探針 所有探針都是經(jīng)過人工真實實驗篩選 所有探針是從2400萬個探針庫中篩選而得到的，這個是獨一無二的設(shè)計策略 以基因為中心進行芯片設(shè)計 全基因組骨架(backbone)設(shè)

9、計 覆蓋15,400 RefSeq genes 領(lǐng)先的基因覆蓋率指標(biāo) 100%覆蓋國際細(xì)胞遺傳學(xué)學(xué)會認(rèn)可的基因 100%覆蓋癌癥相關(guān)基因 93%覆蓋X染色體基因 83%覆蓋OMIM疾病相關(guān)基因 80%覆蓋RefSeq genes 簡化的實驗流程 所有重要試劑打包提供 免費的分析軟件ChAsCytoScan HD產(chǎn)前、產(chǎn)后、癌癥、其它遺傳疾病研究 密度最高的細(xì)胞遺傳學(xué)芯片 含有超過270萬種探針，包括 75萬種可以分型的SNP探針 195萬個拷貝數(shù)探針 所有探針都是經(jīng)過人工真實實驗篩選 所有探針是從2400萬個探針庫中篩選而得到的，這個是獨一無二的設(shè)計策略 以基因為中心進行芯片設(shè)計 全基因組骨架

10、(backbone)設(shè)計 覆蓋18,500 RefSeq genes 領(lǐng)先的基因覆蓋率指標(biāo) 100%覆蓋國際細(xì)胞遺傳學(xué)學(xué)會認(rèn)可的基因 100%覆蓋癌癥相關(guān)基因 100%覆蓋X染色體基因 98%覆蓋OMIM疾病相關(guān)基因 96%覆蓋RefSeq genes 簡化的實驗流程 所有重要試劑打包提供 免費的分析軟件ChAs 在人類遺傳性疾病中的應(yīng)用1. ArrayCGH技術(shù)：與G顯帶和FISH一致；高分辨、大規(guī)模、高通量、自動化、更客觀、省時。2.多發(fā)畸形兒、智力低下兒病因?qū)W：30% MCA，3.產(chǎn)前診斷中的應(yīng)用：胎兒性別、特定染色體片段、胎兒全染色體組進行篩查、避免傳統(tǒng)方法不足、POC、PGD.、非侵

11、入性PD。4.發(fā)現(xiàn)新的染色體異常：低頻率的嵌合體、新的重復(fù)片段綜合征、平衡易位的進一步分析、Veltman對20例已知細(xì)胞遺傳學(xué)異常的特發(fā)性癡呆患者進行了雙盲研究，檢測出所有已知異常，幾個新的基因拷貝數(shù)改變。5.剖析復(fù)雜的人類基因組多態(tài)性現(xiàn)象：揭示與疾病相關(guān)的多態(tài)性基因。6. 多基因病的病因分析：關(guān)聯(lián)分析7. 兒童孤獨癥：10% vs 90%, 原發(fā)缺少和重復(fù)，8.發(fā)現(xiàn)新基因Indications for Ordering Individuals with an unexplained abnormal phenotype,such as: Autism/autism spectrum dis

12、order (ASD)/pervasive developmental disorder (PDD) Developmental delay/intellectual disability, with or without dysmorphic features Multiple congenital anomalies Heart defects & Epilepsy/seizuresScreening for microdeletions and microduplications associated with known syndromes/clinical phenotypes Sc

13、reening for unique microdeletions and microduplications not associated with known syndromes Identification of LCSH that may be suggestive of UPD or increased risk of a recessive disorderFurther characterizationof a chromosomal abnormality,including marker chromosomes, ring chromosomes, apparent term

14、inal deletions, unbalanced translocations, or subsequent analysis of an apparently balanced de novo rearrangement seen in patients with abnormal phenotypes ARUPFigure 2.Detection of genomic disorders. Detection of 22q.11.2 microdeletion syndrome and reciprocal 22q11.2 microduplication syndrome by Ar

15、rayCGHwithFISHconfirmation. (a1) ArrayCGHshowing a loss in copy number of chromosome band 22q11.2 involving the 22q11.2 deletion syndrome region (red circle). (a2)FISHanalysis shows lack of signal (red oval) for target probe on one chromosome 22, confirming the deletion (green signal is control prob

16、e, red signal is target probe). Insert-G-banded chromosome analysis showing the deletion on one chromosome 22 (black arrow). (b1) ArrayCGHshowing a gain in copy number of chromosome band 22q11.2 involving the 22q11.2 duplication syndrome region (red circle). (b2)FISHanalysis shows three signals for

17、the target probe, confirming the duplication (green signal is control probe, red signal is target probe). Insert-G-banded chromosome analysis showing the duplication on one chromosome 22 (black arrow).Figure 3.Detection ofUPDand IBD bySNPArray analysis.SNPArray analysis showing evidence of uniparent

18、al disomy (UPD) and identity by descent (IBD). (a)UPDfor the entire chromosome 15 is indicated by absence of heterozygosity (top panel-lack of signal at 0.5 B allele frequency (BAF) which represents genotype A/B) and no change in copy number (bottom panel-all signals are at 0 Log ratio). (b) Blocks

19、of absence of heterozygosity (AOH) of the proximal regions of chromosome 9p and 9q as demonstrated by lack of signals at the 0.5 BAF. Within the block of AOH at 9p (red oval) is the gene for galactosaemia,GALT. The patient is affected with galactosaemia due to a homozygous mutation in theGALTgene. T

20、he parents are consanguineous, which is consistent with the multiple blocks of AOH.芯片檢出9p和9q部分純合缺失，導(dǎo)致乳糖血癥，父母系近親結(jié)婚。Comparative Genomic Hybridization in the Study of Human Disease, Sau Wai Cheung, 2011This SNP array karyotype from a colorectal carcinoma demonstrates allele-specific analysis (red and g

21、reen plot) generated using the Oscar algorithm (Yamamoto G, et al. Am J Hum Genet. 2007 Jul;81(1):114-26). The allele-specific plot provides the relative copy number of each allele separately. In a normal diploid state, there is one copy of the red allele and one copy of the green allele (one from e

22、ach parent), for an overall copy number of two (e.g., chromosome 2 in this tumor). When there is copy neutral LOH/acquired UPD, both copies are coming from the same parent, so there are two copies of the red allele and zero copies of the green allele, for an overall copy number of two (e.g., 5q in t

23、his tumor). In a trisomy, there are two copies of the red allele and one copy of the green allele, for an overall copy number of 3 (e.g., chromosome 7 in this tumor). A deletion is shown for 11q in this tumor. There is one copy of the red allele and zero copies of the green allele for an overall cop

24、y number of 1.Note: The tumor-initiating event in this case was copy neutral LOH of the APC gene on chromosome 5q. In otherwords, the 2nd hit for this patient was via acquired uniparental disomy rather than deletion.iKaryos and iKaryos DiagnosticsMLPA反應(yīng)條件1.DNA變性：98度加熱5分鐘。2.雜交：加入SALSA探針混合物和buffer于95度

25、孵育1分鐘，然后于60度雜交16個小時。3.連接：加連接酶和buffer于54度孵育15分鐘。再于98度加熱5分鐘使連接酶失活。4.加引物：dNTP，聚合酶，然后開始PCR擴增。5.毛細(xì)管電泳：輸出片段長度和峰面積，軟件分析結(jié)果。MLPA的優(yōu)點1.MLPA技術(shù)能用于檢測許多不同的疾病，差別只是探針不同而已。2.MLPA是多重反應(yīng)，一個反應(yīng)提供了將近50個靶位點。3.MLPA反應(yīng)費用較低。4.MLPA重復(fù)性好，簡單易操作，可以同時檢測多個樣本。5.MLPA敏感性高，僅需20 ng 人DNA，結(jié)果不受樣品DNA量的影響。6.MLPA能區(qū)分單個堿基的差異，能檢測少量的拷貝數(shù)改變，如2個，3個拷貝數(shù)改

26、變。7.MLPA可以檢測單一外顯子的缺失和插入突變。8.反應(yīng)條件優(yōu)化，所有試劑盒基本適用同一反應(yīng)條件，試劑盒包含了所有必需試劑。9.高通量：24小時內(nèi)得到結(jié)果。10.儀器要求：只需普通PCR儀和測序儀。11.檢測范圍：從點突變到大染色體缺失/插入均能檢測MLPA目前的應(yīng)用領(lǐng)域1.檢測小的重排：BRCA1，BRCA2，MSH2，MLH1，DMD，APC，SMA，NF1，NF2，VHL，TSC1/2，MECP2，NSD1，LDLR，F(xiàn)BN1，CFTR，DPYD，COOL5A1，CACNA1A，PKHD1，BRIP1，SLC26A4，LNM1B，PRSS1，F(xiàn)RMD7，TPMT，F(xiàn)LCN，DNAI1

27、，EP300，DNAH5，UBE3A，PCCA，PCDH15等。2.檢測大范圍的染色體重排：williams syndrome，prader-willi/angelman syndrome，digeorge syndrome，cri du chat，pelizaeus-merzbacher，CMT1，HNPP等。3.檢測亞端粒區(qū)的拷貝數(shù)改變。4.檢測染色體非整倍體改變，（包括羊水樣本）。5.腫瘤診斷：ALL，CLL，Oligodendrogliomas，melanomas，neuroblastomas等病的拷貝數(shù)改變。6.甲基化定量檢測：Parader-willi、angelman syndr

28、ome，beckwith wiedemann，MGMT，MLH1，F(xiàn)ragile X，抑癌基因的失活。7.mRNA分析細(xì)胞凋亡和炎癥反應(yīng)。DNA SequencingSequencing DNAAllows us to determine the nucleotide sequence of a DNA fragment.The process which is used to sequence DNA is known as chain termination sequencing.The replicated section of DNA is made from a series of

29、small fragments instead of a whole strand.A radioactive or fluorescent marker is placed on the nucleotide which ends each fragment, a procedure called tagging.The fragments are run on a gel electrophoresis to properly identify the fragments and determine the nucleotide sequence of the original DNA s

30、trand 第一代測序 HGP: $3 Billions, 14 years, five nations (USA, UK, France, Japan and China) Celera: $300 million and hundreds of machines working 24 hours a day for 9 months.Sequencing centers producing the Sanger sequence data for mammalian genome projects are factory like outfits with a large number o

31、f personnel.Sanger sequencingDNA is fragmentedCloned to a plasmid vectorCyclic sequencing reactionSeparation by electrophoresisReadout with fluorescent tags新一代測序儀(NGS)In 2004, NIH initiated a project Technology development for the $1,000 genome to develop novel genome sequencing technologies that wo

32、uld allow extremely low-cost DNA sequencing with a de-novo sequence assembledwith 99.9% accuracy and with essentially no gaps. Sequencing of a 3 Gb genome for $100,000 (achievable in five years-2009) Sequencing of a 3 Gb genome for around $1000 (achievable in ten years-2014) Third generationNanopore

33、 sequencingNucleic acids driven through a nanopore.Differences in conductance of pore provide readout.Real-time monitoring of PCR activityRead-out by fluorescence resonance energy transfer between polymerase and nucleotides orWaveguides allow direct observation of polymerase and fluorescently labele

34、d nucleotidesPGM(Personal Genome Machine)由Ion torrent公司推出的個體化操作基因組測序儀是革命性的半導(dǎo)體芯片測序技術(shù)平臺，通過密布于半導(dǎo)體芯片上的微反應(yīng)孔和專有的大規(guī)模并行芯片感應(yīng)器，組合獨特的微流體機械和流體設(shè)計，實現(xiàn)2小時內(nèi)，獲得10Mb到1Gb以上的高精確度序列。技術(shù)優(yōu)勢配合半導(dǎo)體芯片技術(shù)，較市場上其他測序技術(shù)更簡單、經(jīng)濟。 測序過程簡單，人工操作時間短，樣本處理快速。 經(jīng)濟的標(biāo)準(zhǔn)試劑，基本可通過試劑盒完成數(shù)據(jù)處理相對簡單、快速，可將原始數(shù)據(jù)導(dǎo)入軟件包中，就可以進行SNP分析，多序列比對，進化樹分析。Case report Chromos

35、omal 16p microdeletion in Rubinstein-Taybi syndrome detected by oligonucleotide-based array comparative genomic hybridization: a case reportMd A Mohd Fadley13*, Azli Ismail1, Thong Meow Keong2, Narazah Mohd Yusoff3 and Zubaidah Zakaria1* Journal of Medical Case Reports 2012, 6:30 doi:10.1186/1752-19

36、47-6-30AbstractIntroductionChromosomal aberrations of chromosome 16 are uncommon and submicroscopic deletions have rarely been reported. At present, a cytogenetic or molecular abnormality can only be detected in 55% of Rubinstein-Taybi syndrome patients, leaving the diagnosis in 45% of patients to r

37、est on clinical features only. Interestingly, this microdeletion of 16 p13.3 was found in a young child with an unexplained syndromic condition due to an indistinct etiological diagnosis. To the best of our knowledge, no evidence of a microdeletion of 16 p13.3 with contiguous gene deletion, comprisi

38、ng cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes, has been described in typical Rubinstein-Taybi syndrome. Case presentationWe present the case of a three-year-old Malaysian Chinese girl with a de novo microdeletion on t

39、he short arm of chromosome 16, identified by oligonucleotide array-based comparative genomic hybridization. Our patient showed mild to moderate global developmental delay, facial dysmorphism, bilateral broad thumbs and great toes, a moderate size atrial septal defect, hypotonia and feeding difficult

40、ies. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XX karyotype. Further investigation by high resolution array-based comparative genomic hybridization revealed a 120 kb microdeletion on chromosomal band 16 p13.3. ConclusionA mutation or abnormality in the cyclic adenosine

41、monophosphate-response element-binding protein has previously been determined as a cause of Rubinstein-Taybi syndrome. However, microdeletion of 16 p13.3 comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes is a rare

42、 scenario in the pathogenesis of Rubinstein-Taybi syndrome. Additionally, due to insufficient coverage of the human genome by conventional techniques, clinically significant genomic imbalances may be undetected in unexplained syndromic conditions of young children. This case report demonstrates the

43、ability of array-based comparative genomic hybridization to offer a genome-wide analysis at high resolution and provide information directly linked to the physical and genetic maps of the human genome. This will contribute to more accurate genetic counseling and provide further insight into the synd

44、rome.Familial imbalance in 16p13.11 leads to a dosage compensation rearrangement in an unaffected carrierBackgroundWe and others have previously reported that familial cytogenetic studies in apparently de novo genomic imbalances may reveal complex or uncommon inheritance mechanisms. MethodsA familia

45、l, combined genomic and cytogenetic approach was systematically applied to the parents of all patients with unbalanced genome copy number changes. ResultsDiscordant array-CGH and FISH results in the mother of a child with a prenatally detected 16p13.11 interstitial microduplication disclosed a balan

46、ced uncommon rearrangement in this chromosomal region. Further dosage and haplotype familial studies revealed that both the maternal grandfather and uncle had also the same 16p duplication as the proband. Genomic compensation observed in the mother probably occurred as a consequence of interchromoso

47、mal postzygotic nonallelic homologous recombination. ConclusionsWe emphasize that such a dualistic strategy is essential for the full characterization of genomic rearrangements as well as for appropriate genetic counseling.BMC Medical Genetics 2014, 15:116Spinal Muscular Atrophy (SMA)脊髓性肌萎縮癥1. Muscu

48、lar Disease2. Autosomal Recessive Inheritance3. Second most common genetic disease after Thalassemia4. Carrier rate as high as 1/301/50 around the world。Rodrigues NR, et al (1995) Hum Mol Genet 4:631634SMN1 and SMN2 GeneSMN1SMN2 1 Copies of SMN1 on One Chromosome 5 with a Deletion (Carrier)SMN Gene:

49、 in #5 Chromosome(5q11.2-13.3) SMN1: has 90function, translation Full stable and functional ProteinSMN2: has 10function, translation Non-stable proteins1 Copy of SMN1 on Each Chromosome 5 (Not a Carrier)1 Copies of SMN1 on One Chromosome 5 with a Conversion (Carrier)Type I MajorType IIILightType IIIntermediateHomozygousdeletionSMN1 deletion/conversionSMN1 conversionMFSMN1 & SMN2 TestingSMN1.SMN1(survival motor neuron 1) is the primary SMA-relatedgene.Approximately 95%-98% of individuals with a clinical diagnosis of SMA are homozygous for adeletionorgene convers