疾病的產(chǎn)前診斷PrenatalDiagnosisofDisease共頁課件

1、18 疾病的產(chǎn)前診斷 Prenatal Diagnosis of Disease Genetic diseases and congenital malformations occur in approximately 2 to 5 % of all live births, account for up to 30% of paediatric admissions to hospital, and are an important cause of death under the age of 15 years. Furthermore, the psychological stress

2、on families with children with serious genetic disorders is incalculable. Until gene therapy becomes a practical reality, the only option available for the control of genetic disease is prenatal diagnosis. 1. Indications for prenatal diagnosisThe use of prenatal diagnosis is determined by balancing

3、the risk of the birth of an abnormal child against the risk of an investigative procedure. The general indications of prenatal diagnosis include maternal age and the results of noninvasive serum biochemical screening. Specific indications include a positive family history and the birth of a previous

4、 child affected by a particular genetic disease.2. Methods for obtaining fetal tissues for prenatal diagnosis To perform prenatal diagnosis, fetal-derived tissues must first be obtained. All of the commonly used methods that yield fetal tissues are invasive. A. Amniocentesis Amniocentesis is the wit

5、hdrawal of amniotic fluid from the amniotic sac surrounding the fetus. For over two decades this has been the primary technique utilised for the diagnosis of fetal genetic disorders. Traditionally amniocentesis has been performed around 15 to 16 weeks gestation. At this time the uterus is easily acc

6、essible to a transabdominal approach, and a sufficient volume of amniotic fluid (approximately 200 ml) exists to permit 20 to 30 ml to be withdrawn safely. Amniocentesis is normally performed as an outpatient facility. An ultrasound examination is normally done immediately before the procedure to ev

7、aluate fetal number and viability, perform fetal biometric measurements, establish placental location, and estimate amniotic fluid volume. Amniocentesis performed concurrently with ultrasound scanning. Safety of amniocentesis Any procedure that involves passing a device into an organ, especially the

8、 pregnant uterus, carries with it risks; amniocentesis is no exception. Amniocentesis involves potential danger to both mother and fetus. Serious maternal risks are quite low but include amnionitis which can lead to fetal loss, haemorrhage or injury to an intra-abdominal viscus and leakage of amniot

9、ic fluid. Fetal risks include spontaneous abortion, needle puncture injuries, placental abruption, chorioamnionitis, preterm labour, and amniotic band formation. Several controlled studies have been done to evaluate the risks of amniocentesis. The data indicate that the risk of pregnancy loss attrib

10、utable to amniocentesis may be as high as 0.5%. In general, risks will depend on (1) the experience of the obstetrician performing the procedure;(2) clinical characteristics of a particular case (e.g., presence or absence of biochemical markers of fetal abnormality); (3) the quality of the high-reso

11、lution ultrasound utilised. Early amniocentesis With development of higher resolution ultrasound equipment, some centres have begun offering amniocentesis before 15 weeks gestation, usually between 10 and 14 weeks. The majority of procedures have been performed during the 13th and 14th weeks of gest

12、ation. There is evidence that early amniocentesis is associated with a higher fetal loss rate and a more frequent occurrence of certain congenital abnormalities.B. Chorionic villus sampling Chorionic villus sampling (CVS) is the only tested method for first-trimester fetal genetic diagnosis that is

13、currently in clinical use and is usually performed between 9 and 11 weeks. The procedure involves the passing of a sampling instrument into the chorion (developing placenta). A good procedure yields from 10 to 25 mg of tissue which is adequate for cytogenetic, enzymatic or DNA analysis. The main adv

14、antage of CVS over amniocentesis is the applicability of CVS earlier in gestation. This results in considerably reduced social, emotional and psychological stress for the couple.Safety of CVS Maternal complications include bleeding and infection. Fetal loss following CVS has been reported to be arou

15、nd 2%. There are also reports of limb reduction defects in infants born to mothers who have had CVS between 56 and 66 days of gestation. C. Fetal blood sampling (FBS) Fetal blood can be safely and directly sampled from approximately 18 weeks gestation onwards. FBS can be used for both diagnostic and

16、 therapeutic purposes. Indications for fetal blood samplingDiagnosticRapid karyotypingFetal anomaly on ultrasoundLate attending patients who require fetal karyotypingAlloimmunisationRhesusPlatelet antigensFetal infectionToxoplasmosisCytomegalovirus infectionGeneticHaemoglobinopathiesMetabolic disord

17、ers and enzyme deficienciesFetal well beingSevere intrauterine growth retardationTherapeuticTransfusionRed cell alloimmunisationTransplantationStem cells FBS is contraindicated if the mother is suffering from infections that can be transmitted to the fetus by the procedure. Examples include human im

18、munodeficiency virus and hepatitis B virus infection. Safety of FBS Maternal complications from FBS are uncommon but include amnionitis, infection, rhesus sensitisation and transplacental haemorrhage. Fetal loss rates following FBS have been reported to be approximately 1% in several large series. T

19、he presence of structural abnormalities or severe growth retardation of the fetus is associated with a much increased fetal loss rate. Other fetal complications include infection, premature rupture of membranes, haemorrhage, severe bradycardia and umbilical cord thrombosis. D. Fetal biopsy Although

20、advances in molecular and biochemical genetics have made the diagnosis of many Mendelian disorders possible by analysis of amniotic fluid cells or chorionic villi, some conditions still require direct analysis of tissues in which the disorder is manifested. Tissues which have been successfully biops

21、ied include fetal skin, liver and muscle.Safety of fetal biopsy Due to the relatively small numbers performed in different centres, no precise figures for the safety of fetal biopsy is available.3. Analytical methods Following the acquisition of fetal tissues, these materials are then subjected to a

22、nalysis using a variety of techniques. A. Cell culture and conventional cytogenetics These are the most commonly used methods for the diagnosis of chromosomal aneuploidies such as Down syndrome. B. Molecular cytogenetics using FISH FISH involves the hybridization of DNA probes representing a specifi

23、c chromosome or chromosomal region to target DNA such as metaphase chromosomes or interphase nuclei, where the probe binds to homologous sequences in the cell. Using FISH, several groups have demonstrated that trisomies such as trisomy 21 and trisomy 18 can be detected in uncultured interphase nucle

24、i as three positive hybridisation signals rather than the normal two. The main advantage is speed: thus results are available in 24 to 48 hours compared with the 10 to 14 days more typical of standard culture-based cytogenetic analysis. This type of technology can be applied to fetal materials obtai

25、ned following amniocentesis, CVS or fetal blood sampling. C. DNA-based techniques The main advantage of DNA-based techniques is that any nucleated fetal cell can be used. Techniques which are used include the polymerase chain reaction (PCR) and Southern blotting . PCR-based techniques allow a rapid

26、diagnosis to be made in several hours. These methods can be used for direct mutation detection or linkage analysis. The latter type of analysis is needed when the exact mutation or gene causing the disease is not known. Genetic diagnosis is then carried out by analysing DNA sequences within the gene

27、 itself or DNA loci closely linked to it. An analysable difference or polymorphism must exist between the disease-carrying allele and the normal allele to distinguish them. D. Metabolic analysis of fetal tissues Fetal tissues or fluids can be subjected to analysis to detect the characteristic metabo

28、lic or cellular defects of an inherited metabolic disease. For this type of analysis to be carried out, the specific enzyme or metabolite of interest must be expressed in the fetal tissues sampled, and the range of normal values as well as the assay sensitivity and reproducibility must be establishe

29、d within the tissue of interest. Although an increasing number of inherited metabolic diseases are amenable to direct DNA-based diagnosis, enzyme-based techniques are still useful in situations where the disease-causing gene has not been identified or where the precise mutation is not known. E. Micr

30、oarray Analysis Much of the excitement today centers on gene expression profiling that uses a technology called microarrays. A DNA microarray is a thin-sized chip that has been spotted at fixed locations with thousands of single-stranded DNA fragments corresponding to various genes of interest. A si

31、ngle microarray may contain 10,000 or more spots, each containing pieces of DNA from a different gene. Fluorescent-labeled probe DNA fragments are added to ask if there are any places on the microarray where the probe strands can match and bind. Complete patterns of gene activity can be captured wit

32、h this technology. 4. New methods for prenatal diagnosisA. Preimplantation diagnosis Preimplantation diagnosis is the performance of prenatal genetic analysis on embryos or oocytes prior to implantation. This technology has the advantage that it allows prenatal diagnosis to be carried out much earli

33、er than existing methods such as amniocentesis and CVS. Furthermore, couples who are at exceptionally high genetic risk and those who have had previous terminations for genetic indications may find preimplantation diagnosis a more acceptable form of prenatal testing. In the future, preimplantation d

34、iagnosis may also be used in conjunction with gene therapy. At present, given the expense of the procedure and the small number of centres equipped to perform this form of diagnosis, preimplantation diagnosis is unlikely to become a standard procedure in the foreseeable future.Access to oocyte and e

35、mbryonic cells Preimplantation genetic analysis can be carried on either embryonic cells or oocytes. In the later situation, diagnosis is carried out even prior to fertilisation. Individual oocytes are aspirated and their polar body biopsied. For preimplantation diagnosis carried out on embryonic ce

36、lls, the embryo may be fertilised in vitro and then individual blastomeres biopsied. Alternatively, the embryo may be fertilised in vivo and then the embryos are obtained by uterine lavage followed by biopsy and genetic analysis. For heterozygous women carrying one mutant and one normal allele of a

37、disease-causing gene, in the absence of chromosomal crossing-over, the aspirated polar body containing a mutant allele would indicate that the primary oocyte pronucleus is carrying the normal allele. In both situations, only the embryos confirmed not to possess the full disease-causing genotype are

38、then implanted back into the uterus.Diagnostic methods Preimplantation diagnosis may be achieved using PCR, FISH or by measurement of embryonic secretory products such as certain enzymes. This type of analysis has been carried out successfully for the determination of fetal sex for the avoidance of

39、sex-linked disorders such as Duchenne muscular dystrophy and haemophilia A, and for the diagnosis of single gene disorders such as cystic fibrosis, alpha-1-antitrypsin deficiency, Tay-Sachs disease, fragile X and sickle cell anaemia. Worldwide, preimplantation diagnosis of embryos has been attempted

40、 on over 1,200 in vitro fertilisation cycles in 2019, with clinical pregnancy resulted in 20%. No increase in the occurrence of abnormalities has been observed in the liveborns.B. Noninvasive prenatal diagnosis using fetal cells isolated from maternal blood Circumstantial evidence that fetal nucleated cells exist in maternal peripheral blood can be traced back to 1969. However, convincing proof of the existence of these cells have to await the development of molecular biological techniques, especially the PCR. Using the PCR, investigators are able to demonstrate the presence of cel