医学遗传学检测技术课件

1、医学遗传学检测技术在临床诊断的应用基因和基因组 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 基因与个体发育及疾病的关系基因组: 细胞核DNA成分和线粒体DNA分子的总和基因: 基因组内一个个具体的结构和功能单位功能：精确控制胚胎发育和分化的每一个步骤;决定了个体的所有生命特征; 决定了个体

2、患各种疾病的可能性.遗传病及特征遗传病：遗传物质发生突变所引起的疾病，生殖细胞或受精卵的突 变、体细胞突变。种类：确定的遗传疾病超过24000种。 单基因病- 涉及一对基因，AR、AD、XR、XD。 多基因病-多对基因和环境共同作用所导致的疾病。 染色体病- 数目异常及结构异常引起的疾病。 体细胞遗传病- 体细胞突变如肿瘤。 线粒体病- 线粒体及核基因异常。特征：垂直传递、终生性、发病率低、危害严重、家族性发病、多 无有效治疗。成为危害人类健康的主要疾病 基因组异常与基因病的诊断方法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的预防、诊断、治疗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技术明显的优势核型分析：带型复杂、个体差异，不可能全部机械化FISH技术：位点受限、需已知探针序列传统CGH ：人为因素的限制，需要一定的经验技术和劳动力支持，必需依靠经验丰富的细胞遗传学家。阵列CGH：高通量、高分辨，靶序列特定检测, 精确地定位

8、。其结果自动操纵控制，既快速又直观。 Array CGH 的技术限制1. aCGH 不能检测：平衡染色体重排某些多倍体DNA序列的碱基改变探针没有覆盖区域的异常2. aCGH检测结果正常不等于受检者遗传学检测完全正常3. 异常的aCGH检测结果可能临床意义不明CytoScan 750K适合临床对于低成本需求的芯片 适合临床对于低成本需求的细胞遗传学芯片 含有超过75万种探针，包括 20万种可以分型的SNP探针 55万个拷贝数探针 所有探针都是经过人工真实实验筛选 所有探针是从2400万个探针库中筛选而得到的，这个是独一无二的设计策略 以基因为中心进行芯片设计 全基因组骨架(backbone)设

9、计 覆盖15,400 RefSeq genes 领先的基因覆盖率指标 100%覆盖国际细胞遗传学学会认可的基因 100%覆盖癌症相关基因 93%覆盖X染色体基因 83%覆盖OMIM疾病相关基因 80%覆盖RefSeq genes 简化的实验流程 所有重要试剂打包提供 免费的分析软件ChAsCytoScan HD产前、产后、癌症、其它遗传疾病研究 密度最高的细胞遗传学芯片 含有超过270万种探针，包括 75万种可以分型的SNP探针 195万个拷贝数探针 所有探针都是经过人工真实实验筛选 所有探针是从2400万个探针库中筛选而得到的，这个是独一无二的设计策略 以基因为中心进行芯片设计 全基因组骨架

10、(backbone)设计 覆盖18,500 RefSeq genes 领先的基因覆盖率指标 100%覆盖国际细胞遗传学学会认可的基因 100%覆盖癌症相关基因 100%覆盖X染色体基因 98%覆盖OMIM疾病相关基因 96%覆盖RefSeq genes 简化的实验流程 所有重要试剂打包提供 免费的分析软件ChAs 在人类遗传性疾病中的应用1. ArrayCGH技术：与G显带和FISH一致；高分辨、大规模、高通量、自动化、更客观、省时。2.多发畸形儿、智力低下儿病因学：30% MCA，3.产前诊断中的应用：胎儿性别、特定染色体片段、胎儿全染色体组进行筛查、避免传统方法不足、POC、PGD.、非侵

11、入性PD。4.发现新的染色体异常：低频率的嵌合体、新的重复片段综合征、平衡易位的进一步分析、Veltman对20例已知细胞遗传学异常的特发性痴呆患者进行了双盲研究，检测出所有已知异常，几个新的基因拷贝数改变。5.剖析复杂的人类基因组多态性现象：揭示与疾病相关的多态性基因。6. 多基因病的病因分析：关联分析7. 儿童孤独症：10% vs 90%, 原发缺少和重复，8.发现新基因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.芯片检出9p和9q部分纯合缺失，导致乳糖血症，父母系近亲结婚。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反应条件1.DNA变性：98度加热5分钟。2.杂交：加入SALSA探针混合物和buffer于95度

25、孵育1分钟，然后于60度杂交16个小时。3.连接：加连接酶和buffer于54度孵育15分钟。再于98度加热5分钟使连接酶失活。4.加引物：dNTP，聚合酶，然后开始PCR扩增。5.毛细管电泳：输出片段长度和峰面积，软件分析结果。MLPA的优点1.MLPA技术能用于检测许多不同的疾病，差别只是探针不同而已。2.MLPA是多重反应，一个反应提供了将近50个靶位点。3.MLPA反应费用较低。4.MLPA重复性好，简单易操作，可以同时检测多个样本。5.MLPA敏感性高，仅需20 ng 人DNA，结果不受样品DNA量的影响。6.MLPA能区分单个碱基的差异，能检测少量的拷贝数改变，如2个，3个拷贝数改

26、变。7.MLPA可以检测单一外显子的缺失和插入突变。8.反应条件优化，所有试剂盒基本适用同一反应条件，试剂盒包含了所有必需试剂。9.高通量：24小时内得到结果。10.仪器要求：只需普通PCR仪和测序仪。11.检测范围：从点突变到大染色体缺失/插入均能检测MLPA目前的应用领域1.检测小的重排：BRCA1，BRCA2，MSH2，MLH1，DMD，APC，SMA，NF1，NF2，VHL，TSC1/2，MECP2，NSD1，LDLR，FBN1，CFTR，DPYD，COOL5A1，CACNA1A，PKHD1，BRIP1，SLC26A4，LNM1B，PRSS1，FRMD7，TPMT，FLCN，DNAI1

27、，EP300，DNAH5，UBE3A，PCCA，PCDH15等。2.检测大范围的染色体重排：williams syndrome，prader-willi/angelman syndrome，digeorge syndrome，cri du chat，pelizaeus-merzbacher，CMT1，HNPP等。3.检测亚端粒区的拷贝数改变。4.检测染色体非整倍体改变，（包括羊水样本）。5.肿瘤诊断：ALL，CLL，Oligodendrogliomas，melanomas，neuroblastomas等病的拷贝数改变。6.甲基化定量检测：Parader-willi、angelman syndr

28、ome，beckwith wiedemann，MGMT，MLH1，Fragile X，抑癌基因的失活。7.mRNA分析细胞凋亡和炎症反应。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 第一代测序 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新一代测序仪(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公司推出的个体化操作基因组测序仪是革命性的半导体芯片测序技术平台，通过密布于半导体芯片上的微反应孔和专有的大规模并行芯片感应器，组合独特的微流体机械和流体设计，实现2小时内，获得10Mb到1Gb以上的高精确度序列。技术优势配合半导体芯片技术，较市场上其他测序技术更简单、经济。 测序过程简单，人工操作时间短，样本处理快速。 经济的标准试剂，基本可通过试剂盒完成数据处理相对简单、快速，可将原始数据导入软件包中，就可以进行SNP分析，多序列比对，进化树分析。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)脊髓性肌萎缩症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