医学遗传学检测技术在临床诊断的应用

1、医学遗传学检测技术在临床诊断的应用金域检验遗传病诊断中心广州医科大学金域检验学院基因和基因组基因和基因组 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与个体发育及疾病的关系基因组基因组: 细胞核细胞核DNA成分和线粒体成分和线粒体DNA分子的总和分子的总和基因

2、基因: 基因组内一个个具体的结构和功能单位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; 决定了个体患各种疾病的可能性.遗传病及特征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。

3、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基因组异常与基因病的诊断方法Laboratory Diagnosis v Genetic MethodsCytogeneticsMolecular GeneticsBiochemical Geneticsv Non-Genetic MethodsPathologyCytologyImmunologyNon-Laboratory Diagnosis vRadiologyvClinical Evaluation Carrier detection Preimplantation genetic diagnosis Prenatal diagn

4、osis Newborn screening Postnatal diagnostic testing vGerm line mutationsvSomatic mutation Predictive, presymptomatic 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 d

5、epending on preparationMicroarrays offer unbiased means of detecting chromosomal aberrations. The same chromosome 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 pro

6、be was used. X chromosome centromer specific probe SRY probeLocus specific probes18Centromer specific probes chromosome 7 centromer specific signal chromosome 8 centromer specific signal19Whole chromosome painting probesChromosome 8 painting probe20R x FISH=Cross-species colour FISH 21Spectral Karyo

7、typing (SKY)22Multiplex FISHMetaphase spread of a lung cancer cell line after hybridization of the 5-fluorochrome M-FISH mix shown in true-color (a) and classification-color representation (b). The karyotype is shown in (c).2008 Nature Education ArrayCGHArrayCGH技术明显的优势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F

8、ISH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。其结果自动操纵控制，既快速又直观。 Array CGH 的技术限制的技术限制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

9、，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、其它遗传疾病研究产前、产后、癌症、其它遗传疾病

10、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(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在

11、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

12、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 disorder (ASD)/pervasive developmental disorder (PDD) Developmental delay/intellectual disability, with or without dysmorphic features Multiple congenital anomali

13、es Heart defects & Epilepsy/seizuresScreening for microdeletions and microduplications associated with known syndromes/clinical phenotypes Screening for unique microdeletions and microduplications not associated with known syndromes Identification of LCSH that may be suggestive of UPD or increas

14、ed risk of a recessive disorderFurther characterizationof a chromosomal abnormality,including marker chromosomes, ring chromosomes, apparent terminal deletions, unbalanced translocations, or subsequent analysis of an apparently balanced de novo rearrangement seen in patients with abnormal phenotypes

15、 ARUPFigure 2. Detection of genomic disorders. Detection of 22q.11.2 microdeletion syndrome and reciprocal 22q11.2 microduplication syndrome by Array CGH with FISH confirmation. (a1) Array CGH showing a loss in copy number of chromosome band 22q11.2 involving the 22q11.2 deletion syndrome region (re

16、d circle). (a2) FISH analysis shows lack of signal (red oval) for target probe on one chromosome 22, confirming the deletion (green signal is control probe, red signal is target probe). Insert-G-banded chromosome analysis showing the deletion on one chromosome 22 (black arrow). (b1) Array CGH showin

17、g a gain in copy number of chromosome band 22q11.2 involving the 22q11.2 duplication syndrome region (red circle). (b2) FISH analysis shows three signals for the target probe, confirming the duplication (green signal is control probe, red signal is target probe). Insert-G-banded chromosome analysis

18、showing the duplication on one chromosome 22 (black arrow).Figure 3. Detection of UPD and IBD by SNP Array analysis. SNP Array analysis showing evidence of uniparental disomy (UPD) and identity by descent (IBD). (a) UPD for the entire chromosome 15 is indicated by absence of heterozygosity (top pane

19、l-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 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.

20、 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 the GALT gene. The parents are consanguineous, which is consistent with the multiple blocks of AOH.芯片检芯片检出出9p和和9q部分纯合缺失，导致乳糖血症，父母系近亲结婚。部分纯合缺失，导致乳糖

21、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 green plot) generated using the Oscar algorithm (Yamamoto G, et al. Am J Hum Genet. 2007 Jul;81(1):114-2

22、6). 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 each parent), for an overall copy number of two (e.g., chromosome 2 in this tumor). When there is copy n

23、eutral 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 this tumor). In a trisomy, there are two copies of the red allele and one copy of the green allele, for

24、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 copy number of 1.Note: The tumor-initiating event in this case was copy neutral LOH of the APC gene on chr

25、omosome 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

26、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。1

27、1.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，EP300，DNAH5，UBE3A，PCCA，PCDH15等。2.检测大范围的染色体重排：williams syndrome，prader-willi/angelman syndrome，digeorg

28、e 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 syndrome，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 DNAAllow

29、s 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 small fragments instead of a whole strand.A radioactive or fluorescent marker is placed on the nucleot

30、ide 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 strand 第一代测序第一代测序 HGP: $3 Billions, 14 years, five nations (USA, UK, France, Japan and China) Celera: $

31、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 of personnel.Sanger sequencing新一代测序仪新一代测序仪(NGS)In 2004, NIH initiated a project Technology develop

32、ment for the $1,000 genome to develop novel genome sequencing technologies that would allow extremely low-cost DNA sequencing with a de-novo sequence assembledwith 99.9% accuracy and with essentially no gaps.v Sequencing of a 3 Gb genome for $100,000 (achievable in five years-2009)v Sequencing of a

33、3 Gb genome for around $1000 (achievable in ten years-2014) Third generationPGM(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

34、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 Chromosomal 16p microdeletion in Rubinstein-Taybi syndrome detected by oligonucleotide-based array comparative genomic hybridization: a case report Md A Mohd Fadley13*, Azli Ismail1, Thong Meow Keong2, Narazah Mohd Yusoff3 and Zubaidah

35、 Zakaria1* Journal of Medical Case Reports 2012, 6:30 doi:10.1186/1752-1947-6-30AbstractIntroductionChromosomal 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 Rubinste

36、in-Taybi syndrome patients, leaving the diagnosis in 45% of patients to rest 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 eviden

37、ce of a microdeletion of 16 p13.3 with contiguous gene deletion, comprising 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

38、a three-year-old Malaysian Chinese girl with a de novo microdeletion on the 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 to

39、es, a moderate size atrial septal defect, hypotonia and feeding difficulties. 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 ba

40、nd 16 p13.3. ConclusionA mutation or abnormality in the cyclic adenosine 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 protei

41、n and tumor necrosis factor receptor-associated protein 1 genes is a rare 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 s

42、yndromic conditions of young children. This case report demonstrates the 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 m

43、ore accurate genetic counseling and provide further insight into the syndrome.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 imbalance

44、s may reveal complex or uncommon inheritance mechanisms. MethodsA familial, 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 pre

45、natally detected 16p13.11 interstitial microduplication disclosed a balanced 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 o

46、bserved in the mother probably occurred as a consequence of interchromosomal 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

47、Medical Genetics 2014, 15:116 Spinal Muscular Atrophy (SMA)脊髓性肌萎缩症1. Muscular 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 4:631634SMN1SMN2 1 Copies of SMN

48、1 on One Chromosome 5 with a Deletion (Carrier)SMN Gene: in #5 Chromosome(5q11.2-13.3) 1 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 convers

49、ionMFSMN1 & SMN2 TestingSMN1. SMN1 (survival motor neuron 1) is the primary SMA-related gene.Approximately 95%-98% of individuals with a clinical diagnosis of SMA are homozygous for a deletion or gene conversion of SMN1 Typically determined by lack ofexon 7 in both copies of SMN1 (i.e., they are homozy