genetic diagnosis-工具与遗传病诊断-王小竹

1、Chapter 4Tools of Human Molecular GeneticsWang Xiaozhu 2013-04-08We will discussv The Polymerase Chain Reactionv Molecular cloningv Nucleic Acid Hybridizationv DNA sequence analysisAnalysis of individualDNA and RNA sequencesv Polymerase Chain Reaction (PCR)v Molecular CloningThe Polymerase Chain Rea

2、ction聚合酶链式反应PCR: Enzymatic amplification of a fragment of DNA located between a pair of primers回 -Am咄知 tion of 沁哼 屯 q,Um）Ori9inoIll ar钾3宁如 ble七阮ond4 C NA- - -g勹 怡n 二-3f制阳m 可 2- 、3l- l 、IPrim15-闷5护_匕啊 Co.mplementary_ j3to primer 2尸primen-3-，,Com曲础en厮 yiSepa呻如 nd广 b primerls5#s。心ann的 I primeliSdI晏心pri

3、 mers -.,s|3，PCR_technique丘d pfj1imemMolecular Cloningv To isolate a particular gene or other DNA sequence in large quantities.v To transfer the DNA sequence into a single cell of a microorganism, then reproduces the DNA sequence by grown of microorganismTools of molecular cloningv Vector (Plasimd,V

4、irus, Bacterial artificial chromsome, PAC,YAC)v Restriction enzymev Target genev Hast celln Restriction EnzymesEcoR（palindrome）5-G A A T T C-35-GA A T T C-33-C T T A A G-53-C T T A AG-5n VectorsExample: plasmid(35 years old)v Positive prenatal screening.v High danger family.v Other well-defined risk

5、 factors.1.2:11.10； 仅 广 气书g.0B忌 ，07呈8貌芍苍岛，仁中二 I -岊口04. 03. . 也; i,01？o At 础 n沁c entesis AJ., birth， 飞匕丘鼠心1 堇. -o . 20,.2s iao. 年40必50 M啦 n氐1 Ellge. 声 团应 ur e l 8 - 1 叶 心I如皿I :a.g.心 J叩 皿虹，中 0f in0 den设 of tri虹呵2l 忒 birth, Rnd at 也 仇七 忒 叩 mb ccn志 氐 知如C佪佴扛 lO.h如 呴 J心心l 24女20开2038.）Ch叩 陨 0 咖 l 心 皿 皿 :il

6、i守 立-at 扣 mio如 郎 归 9ttd in h伈如 m,(1)囮fro.tn HO咄 阻 ，Cr岱 5 万Sch叫 心. 113Ch如 ; Dli 9B3The content of prenatal diagnosisv Noninvasive testing Maternal serum screening (MSS), Ultrasonographyv Invasive testingAmniocentesis,Chorionic villus sampling, Cordocentesisv Laboratoryservice Cytogenetic testing, Bio

7、chemical testing,Gene testingNoninvasive testingNoninvasive testingv Maternal serum screeningv UltrasonographyMaternal serum screening (MSS1)v MSS: also called triple screen measures three blood markers:Alphafetoprotein(MS-AFP),unconjugated estriol(uE3),human chorionic gonadotrophin (HCG).v These ma

8、rkers are produced by the fetus and/or placenta.v Performed between 15-20+6 weeks at gestational age.Maternal serum screening (MSS2)v These triples markers are used to screen three diseases: Down syndrome Trisomy 18 neural tube defect (NTD)Down syndromeTrisomy 18 syndromeNeural tube defect (NTD)Cond

9、itionMSAFPuE3HCGNeural tube defectNormalNormalTrisomy 21Trisomy 18t.壹古，尊二n ysS-Enph-pul石gP邓邸 hl Di嗯 吩 邓365,_ii i 1中；亭，护,重嘉 2十一仆5.0nl亡凸dO,5, 0 .8 1 5432Me.t仑咋 I se n.J订1 汗 F (mLJliple.of narmal rn祠 an;|1020压郡 芯 18 - 4. M啦 皿甘 叩 m 呴址 fetop邓 血 晒 ）颂 氓lm ri OJI，叫沉 函 扭 ml:llti产 df the m奾 lllt, i.n 沁 n吐 知il

10、S邸，函 U5邸 面 th open neum压 妇 如缸 沁 d fe出 5邸 wi, r:b. Dowl1 平 血 亡，戊忒均1心如ll ，固d NS C 匹 klc I-IS I98? R红心1tg动妇心 s in s氓却 ing for neuml mbe d吐虹 S 皿 d. Dovm s,;叫 心 血 m R咄 eek C七tlJ p如 飞血 l p 均萨 o啦B诅k如 Tm 忐u,严o.n，卯俨研项The caution of MSSv MSS is only screening test, not diagnostic test.v Further counseling and

11、 diagnostic testing should be offered to women whose MSS screening test result is positive.v Negative result of MSS The risk is zeroUltrasonography in prenatal diagnosisv It is important to detectfetal assessment: fetal age, multiple pregnancies, fetal viability.v It is important for the detection o

12、f morphological anomalies, such as, anencephaly, cystic hygrome(囊性水瘤).Ultrasonography in genetic disorderv chromosome aneuploidy.v single-gene disorders (Holt-Oram syndrome).v multifactorial disorders.v Determination of fetal sex.Detectingchromosome aneuploidyThe aortic isthmus(主动脉峡) is significantl

13、y narrower in trisomy 21,18,13,andTurner syndrome than in normal fetuses. Measurment of nuchal translucency thickness (NTT) at 10-14 weeks may prove to be a useful method for screening chromosomal defects.Nuchal translucency thickness (NTT)v Nuchal translucency thickness: between the skin and the so

14、ft tissue overlying the cervical spine. 0.12cm in a normal 11-week fetus.The accumulation of fluid behind the fetal neckNT Ultrasound for single-gene disorderNormal fetusHolt-Oram syndrome AD diseaseTBX5 transcription factor gene mutationInvasive testingThe principalindications for Invasive testingA

15、dvanced maternal age.Previous child with a de novo chromosome abnormality.Presence of structural chromosome abnormality in one of the parents.Family history of a genetic disorder that may be diagnosed or ruled out by biochemical or DNA analysis.Family history of an X-linked disorder for which there

16、is no specific prenataldiagnostic testRisk of a neural tube defect（such as, high MSAFP ofMSS)The positive result of MSS and ultrasoundThe methods of invasive testingv Amniocentesisv Chorionic villus samplingv CordocentsisAmniocentesis(羊膜腔穿刺）v Amniocentesis refers to the procedure of removing a sampl

17、e of amniotic fluid transabdominally by syringe.time: 15-16th week after the first day of the last menstrual period.Method: transabdominally , located in the amniotic cavity by ultrasound.How to avoid polluting with mothers blood.Culturing amniotic fluid cellsThe purpose of amniocentesisv The concen

18、tration of AFP(AFAFP):NTD and Anencephaly(无脑畸形）.v Chromosome analysis-Karyotype and FISHv Biochemical detection-enzyme analysisv DNA analysisClinical example-prenatal diagnosis of NTDRisk of a neural tube defect pregnancyMSSDetecting MSAFPIf MSAFP is at normal levelIt should be detected by ultrasoun

19、d to exclude NTDIf MSAFP is higher than normal level It should be given Amniocentesis to detect AFAFP.If AFAFP is higher than normal level, it should be excluded other causes of elevated amniotic fluid AFAFP, such as fetal blood contamination, Fetal death, twin pregnancy, etc.The complication of amn

20、iocentesisv miscarriage in the midtrimester.v talipes equinovarus （马蹄型内翻足）v Leakage of amniotic fluidv Infectionv Injury to the fetus by needle puncture.Chorionic Villus SamplingThe villi are derived from the trophoblast, the extra-embryonic part of the blastocystThe time of cvs: 10-12th weeks The v

21、illi sampled: tertiary villiThe purposeofCVSv Chromosome analysis-Karyotype and FISHv Biochemical detection-enzyme analysisv DNA analysisThe features of CVSv The advantages of CVS: the stage of samplingis earlier than amniotensis.v The disadvantages of CVS: AFP can not be assayed at this stage. The

22、rate of fetal loss increases approximately 1% about 2% of CVS samplings yield ambiguous results due to chromosomalmosaicism (true mosaicism and pseudomosaicism).Cordocentensis（脐血穿刺）v Cordocentensis isa procedure used to obtain a sample of fetal blood directly from the umbilical cord with ultrasonogr

23、aphic guidance.v Time: 19-21th week of pregnancy.cordocentensisLaboratorytestv Cytogeneticsv Biochemical testv DNA analysisPrenatal diagnosis by cytogeneticSummary Clinical diagnsisGenetic counselingLabortary testing Karyotype-G bandingGenetic counseling CVS or amniocentesis Karyotype-G bandingPrena

24、tal diagnosis by PCR-sequencingproband？mother Female fetusFeatures of laboratory testingProblems in chromosome analysis for prenatal diagnosisv Mosaicism: refers to the presence of two or more cell lines in an individual or tissue sample.v Culture failurev Unexpected adverse findings.无法预测表型的染色体畸形， 如

25、染色体数目正常但为常见变异体，罕见的重排或标记染色体,应确定双亲的核型，判断是遗传的还是新发的，才能评估这种变异对胎儿的影响。Mosaicismv True mosaicism：is detected in multiple colonies from several different primary cultures.Mosaicism is truly present in the fetus，v PseudomosaicismAn artifact occurring in tissue cultureMaternal cell contamination, especially CV

26、S.v Confined placental mosaicism, in CVSstudies, 2% of pregnancies.Mosaicism is present in the placental but not in the fetus.The features of Biochemical tests for prenatal diagnosisv Advantages:Biochemical assays can be detecteddirectly the gene product.Is not easy to be contaminated in the biochem

27、ical assays.v Disadvantage: Some proteins only express inspecial cell. For example, PAH only expresses in liver cell.The features of DNA analysisv DNA analysis can directly detect the mutant allele gene.v Sensitivelyv compensate for the inadequacies of biochemical analysis. such as, PAHv Combining DNA analysis with biochemical testing.Emerging technologies for prenatal diagnosisv Preimplanta