文稿diagnosis遺傳病診斷

1、v vClinical diagnosisvPedigree analysisvLaboratory testsPedigree analysisTo confirm whether it is genetic diseaseTo analyze its inherited mannersTo estimate the risk of the disease in this familyvDiagnosis: for chromosomes diseases or/and cancer（research).vTechnique: Karyotype-G banding Fluorescence

2、 In Situ Hybridization( FISH) Comparison Genetic Hybridization (CGH）vMaterial: Peripheral Blood- lymphocyte Amniotic fluid Tissue Clinical example(1)- Karyotype18-trisomysyndrome Clinical diagnosis confirming clinical diagnosis genetic counselingG-banding KARYOTYPEmalformation of many Systems.Develo

3、pment retardationMental retardationspecial fist posture etcLab testing- KaryotypevCollecting material: Peripheral Blood, Amniotic fluid, vHarvesting cellsvCulturing cells 1-2 daysvArresting cells in metaphase with colchicine, treating with 0.075M Potassium Chloride and fixed with fixative solution.v

4、Spreading cells on slide, which can be used for FISH or Karyotype.vstaining with Giemasa.vCounting chromosomes in 20 representative cells vCapturing image of 5 “best” cells and construct karyotypes for eachFluorescence in situ hybridization( FISH)2pterPreparing slides for hybridization.Preparing pro

5、bes labeled with biotin/dig according to BAC/YAC etc.Making hybridization at 37 overnight.Washing slides with washing buffer.Dying with anti-biotin or anti dig with fluorescence.Painting with DIPA. vDiagnosis: for metabolic diseasesvDiagnosed by enzyme assayvMaterial: Peripheral Blood- lymphocyte Am

6、niotic fluid Chorionic villusBiochemical tests for metabolic diseasesUseful for metabolic diseasesDiagnosed by enzyme assayGene test vMeans of closely linked markers- to linkage analysis. PCR-STR(PCR) RFLP（Southern blotting) vDirect detection of the mutation. PCR-sequencing to detect point mutation

7、PCR-SSCP to detect point mutation MLPA (Multiplex Ligation Probes Assay) to detect duplication/deletion Southern blotting to detect duplication/deletion5.7kb3.4kb2.4kbRFLP-Linkage analysisClinical Example（1）-DMD/BMD (XR)probandmotherDuplication mutation detected by MLPASummaryClinical diagnosisPredi

8、gree analysisgene testingConfirming clinical diagnosisGenetic counselingvNew tech (since2002)vHigh throughputvLow costvHigh specificityv40 mutations in 1 tubevApplications:TrisomyDeletion/DuplicationMethylation mRNAMultiplex ligation-dependent Probe amplification MLPAPCR-Sequencing to detect point m

9、utationFatherprobandMotherClinical Example（2） - Methylmalonicacidemia (AR) Development retardationMental retardationVomiting, etc.Vitb12 diagnosis therapyvConcept: make a diagnosis for genetic diseases before appearing clinical symptom early diagnosis, early therapy.vExample: wilsons disease(肝豆狀核變性）

10、，once diagnosed, the patient can take Penicillamine. Prenatal diagnosisPrenatal diagnosisvPrenatal diagnosis determines the health and condition of an unborn fetus. The history of prenatal diagnosisvIn 1966, Steele and Breg detected Down syndrome. Cell cultured Karyotype analysisThe requires of pren

11、atal diagnosisvObstetricsvClinical genetic (including genetic counseling)vUltrasonographyvLaboratory serviceThe goals of prenatal diagnosisTo provide a range of informed choice. To provide reassurance and to reduce anxiety. especially among high-risk groups.have a health one.the option of appropriat

12、e management.To enable prenatal treatment of the affected fetus.Indication for prenatal diagnosisvThe age of pregnant woman(35 years old)vPositive prenatal screening.vHigh danger family.vOther well-defined risk factors.The content of prenatal diagnosisvNoninvasive testing Maternal serum screening (M

13、SS), UltrasonographyvInvasive testing for laboratory service Amniocentesis, Chorionic villus sampling, CordocentesisvLaboratory service Cytogenetic testing, Biochemical testing, Gene testing Noninvasive testingNoninvasive testingvMaternal serum screeningvUltrasonographyMaternal serum screening (MSS1

14、)vMSS: also called triple screen measures three blood markers: Alphafetoprotein(MS-AFP), unconjugated estriol(uE3), human chorionic gonadotrophin (HCG). vThese markers are produced by the fetus and/or placenta.vPerformed between 15-20+6 weeks at gestational age.Maternal serum screening (MSS2)vThese

15、triples markers are used to screen three diseases: Down syndrome Trisomy 18 neural tube defect (NTD) Down syndromeTrisomy 18 syndromeNeural tube defect (NTD)Condition MSAFP uE3 HCG Neural tube defect Normal Normal Trisomy 21 Trisomy 18 The caution of MSSvMSS is only screening test, not diagnostic te

16、st.vFurther counseling and diagnostic testing should be offered to women whose MSS screening test result is positive.vNegative result of MSSThe risk is zeroUltrasonography in prenatal diagnosisvIt is important to detect fetal assessment: fetal age, multiple pregnancies, fetal viability. vIt is impor

17、tant for the detection of morphological anomalies, such as, anencephaly, cystic hygrome(囊性水瘤).Ultrasonography in genetic disordervchromosome aneuploidy.vsingle-gene disorders (Holt-Oram syndrome).vmultifactorial disorders.vDetermination of fetal sex.Detecting chromosome aneuploidy The aortic isthmus

18、(主動脈峽) is significantly narrower in trisomy 21,18,13,and Turner 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)vNuchal translucency thickness: betwe

19、en the skin and the soft 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 syndromeAD diseaseTBX5 transcription factor gene mutationInvasive testingThe principal indication

20、s for Invasive testingAdvanced 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 d

21、isorder for which there is no specific prenatal diagnostic testRisk of a neural tube defect（such as, high MSAFP of MSS)The positive result of MSS and ultrasoundThe methods of invasive testingvAmniocentesisvChorionic villus samplingvCordocentsisAmniocentesis(羊膜腔穿刺）vAmniocentesis refers to the procedu

22、re of removing a sample 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 am

23、niocentesisvThe concentration of AFP(AFAFP):NTD and Anencephaly(無腦畸形）.vChromosome analysis-Karyotype and FISH vBiochemical detection-enzyme analysisvGene testClinical example-prenatal diagnosis of NTDRisk of a neural tube defect pregnancy MSS Detecting MSAFPIf MSAFP is at normal levelIt should be de

24、tected by ultrasound toexclude NTDIf MSAFP is higher than normal levelIt should be given Amniocentesis to detect AFAFP.If AFAFP is higher than normal level, it should be excluded other causes of elevated amnioticfluid AFAFP, such as fetal blood contamination,Fetal death, twin pregnancy, etc.The comp

25、lication of amniocentesisvmiscarriage in the midtrimester.vtalipes equinovarus （馬蹄型內(nèi)翻足）vLeakage of amniotic fluidvInfectionvInjury 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 w

26、eeksThe villi sampled: tertiary villiThe purpose of CVSvChromosome analysis-Karyotype and FISH vBiochemical detection-enzyme analysisvDNA analysisThe features of CVSvThe advantages of CVS: the stage of sampling is earlier than amniotensis. vThe disadvantages of CVS: AFP can not be assayed at this st

27、age. The rate of fetal loss increases approximately 1% about 2% of CVS samplings yield ambiguous results due to chromosomal mosaicism (true mosaicism and pseudomosaicism).Cordocentensis（臍血穿刺）vCordocentensis is a procedure used to obtain a sample of fetal blood directly from the umbilical cord with u

28、ltrasonographic guidance.vTime: 19-21th week of pregnancy.Laboratory testvCytogeneticsvBiochemical testvDNA analysisPrenatal diagnosis by cytogeneticSummaryClinical diagnsisGenetic counselingLabortary testingKaryotype-G bandingGenetic counselingCVS or amniocentesisKaryotype-G bandingPrenatal diagnos

29、is by PCR-sequencingProband CarriersisterAffected fetus？Prenatal diagnosis by MLPA？ProbandCarrier motherNormal fetusFeatures of laboratory testingProblems in chromosome analysisfor prenatal diagnosisvMosaicism: refers to the presence of two or more cell lines in an individual or tissue sample.vCultu

30、re failurevUnexpected adverse findings.無法預(yù)測表型的染色體畸形，如染色體數(shù)目正常但為常見變異體，罕見的重排或標記染色體,應(yīng)確定雙親的核型，判斷是遺傳的還是新發(fā)的，才能評估這種變異對胎兒的影響。MosaicismvTrue mosaicism：is detected in multiple colonies from several different primary cultures.Mosaicism is truly present in the fetus，vPseudomosaicismAn artifact occurring in tissu

31、e/cell culturingMaternal cell contamination, especially CVS.vConfined placental mosaicism, in CVS studies, 2% of pregnancies.Mosaicism is present in the placental but not in the fetus.The features of Biochemical testsfor prenatal diagnosisvBiochemical assays can be detected directly the gene product, although heter