2Genetic 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.
3Furthermore, the psychological stress 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.
41. Indications for prenatal diagnosis The use of prenatal diagnosis is determined by balancing the risk of the birth of an abnormal child against the risk of an investigative procedure.
5 The general indications of prenatal diagnosis include maternal age and the results of noninvasive serum biochemical screening.
6 Specific indications include a positive family history and the birth of a previous child affected by a particular genetic disease.
72. 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.
8A. AmniocentesisAmniocentesis is the withdrawal 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.
9Traditionally amniocentesis has been performed around 15 to 16 weeks gestation. At this time the uterus is easily accessible 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approach, and a sufficient volume of amniotic fluid (approximately 200 ml) exists to permit 20 to 30 ml to be withdrawn safely.
10Amniocentesis is normally performed as an outpatient facility Amniocentesis is normally performed as an outpatient facility. An ultrasound examination is normally done immediately before the procedure to evaluate fetal number and viability, perform fetal biometric measurements, establish placental location, and estimate amniotic fluid volume.
11Amniocentesis performed concurrently with ultrasound scanning.
12Safety of amniocentesis Any procedure that involves passing a device into an organ, especially the pregnant uterus, carries with it risks; amniocentesis is no exception.
13Amniocentesis involves potential danger to both mother and fetus 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 amniotic fluid.
14Fetal risks include spontaneous abortion, needle puncture injuries, placental abruption, chorioamnionitis, preterm labour, and amniotic band formation.
15Several controlled studies have been done to evaluate the risks of amniocentesis. The data indicate that the risk of pregnancy loss attributable to amniocentesis may be as high as 0.5%.
16In 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-resolution ultrasound utilised.
17Early amniocentesisWith 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 gestation.
18There is evidence that early amniocentesis is associated with a higher fetal loss rate and a more frequent occurrence of certain congenital abnormalities.
19B. Chorionic villus sampling Chorionic villus sampling (CVS) is the only tested method for first-trimester fetal genetic diagnosis that is currently in clinical use and is usually performed between 9 and 11 weeks.
20The 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.
21The main advantage 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.
22Safety of CVSMaternal complications include bleeding and infection. Fetal loss following CVS has been reported to be around 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.
23C. 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 therapeutic purposes.
24Diagnostic Therapeutic Rapid karyotyping Fetal anomaly on ultrasound Indications for fetal blood samplingDiagnosticRapid karyotypingFetal anomaly on ultrasoundLate attending patients who require fetal karyotypingAlloimmunisationRhesusPlatelet antigensFetal infectionToxoplasmosisCytomegalovirus infectionGeneticHaemoglobinopathiesMetabolic disorders and enzyme deficienciesFetal well beingSevere intrauterine growth retardationTherapeuticTransfusionRed cell alloimmunisationTransplantationStem cells
25FBS is contraindicated if the mother is suffering from infections that can be transmitted to the fetus by the procedure. Examples include human immunodeficiency virus and hepatitis B virus infection.
26Safety of FBSMaternal complications from FBS are uncommon but include amnionitis, infection, rhesus sensitisation and transplacental haemorrhage.
27Fetal loss rates following FBS have been reported to be approximately 1% in several large series. The presence of structural abnormalities or severe growth retardation of the fetus is associated with a much increased fetal loss rate.
28Other fetal complications include infection, premature rupture of membranes, haemorrhage, severe bradycardia and umbilical cord thrombosis.
29D. Fetal biopsyAlthough 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 biopsied include fetal skin, liver and muscle.
30Safety of fetal biopsyDue to the relatively small numbers performed in different centres, no precise figures for the safety of fetal biopsy is available.
313. Analytical methodsFollowing the acquisition of fetal tissues, these materials are then subjected to analysis using a variety of techniques.
32A. Cell culture and conventional cytogenetics These are the most commonly used methods for the diagnosis of chromosomal aneuploidies such as Down syndrome.
33B. Molecular cytogenetics using FISH FISH involves the hybridization of DNA probes representing a specific chromosome or chromosomal region to target DNA such as metaphase chromosomes or interphase nuclei, where the probe binds to homologous sequences in the cell.
34Using FISH, several groups have demonstrated that trisomies such as trisomy 21 and trisomy 18 can be detected in uncultured interphase nuclei as three positive hybridisation signals rather than the normal two.
35The 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 obtained following amniocentesis, CVS or fetal blood sampling.
36C. 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 .
37PCR-based techniques allow a rapid 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.
39Genetic diagnosis is then carried out by analysing DNA sequences within the gene 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.
42D. Metabolic analysis of fetal tissues Fetal tissues or fluids can be subjected to analysis to detect the characteristic metabolic or cellular defects of an inherited metabolic disease.
43For 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 established within the tissue of interest.
44Although 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.
45E. Microarray AnalysisMuch of the excitement today centers on gene expression profiling that uses a technology called microarrays.
46A 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.
47A single 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 with this technology.
494. New methods for prenatal diagnosis A. Preimplantation diagnosisPreimplantation diagnosis is the performance of prenatal genetic analysis on embryos or oocytes prior to implantation.
50This technology has the advantage that it allows prenatal diagnosis to be carried out much earlier than existing methods such as amniocentesis and CVS.
51Furthermore, 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.
53In the future, preimplantation diagnosis 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.
54Access to oocyte and embryonic 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.
55For preimplantation diagnosis carried out on embryonic cells, 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.
56For heterozygous women carrying one mutant and one normal allele of a 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.
57In both situations, only the embryos confirmed not to possess the full disease-causing genotype are then implanted back into the uterus.
59Diagnostic methodsPreimplantation diagnosis may be achieved using PCR, FISH or by measurement of embryonic secretory products such as certain enzymes.
60This type of analysis has been carried out successfully for the determination of fetal sex for the avoidance of 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-Sach's disease, fragile X and sickle cell anaemia.
61Worldwide, preimplantation diagnosis of embryos has been attempted on over 1,200 in vitro fertilisation cycles in 1997, with clinical pregnancy resulted in 20%. No increase in the occurrence of abnormalities has been observed in the liveborns.
62B. 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.
63However, convincing proof of the existence of these cells have to await the development of molecular biological techniques, especially the PCR.
64Using the PCR, investigators are able to demonstrate the presence of cells possessing fetal genetic markers circulating in the peripheral blood of pregnant women. The isolation of these cells offer the possibility of a noninvasive and safe method for prenatal diagnosis.
65Fetal nucleated cell types in maternal blood Three populations of fetal nucleated cells are currently known to be present in maternal peripheral blood: fetal lymphocytes, trophoblasts and fetal nucleated red cells.
66At present, the isolation of fetal nucleated red cells is receiving the most attention from investigators in the field due to the availability of relatively specific monoclonal antibodies against these cells.
67Isolation of fetal cells and genetic analysis A combination of physical and immunological methods are used to isolate fetal nucleated cells from maternal blood. Physical methods include density gradient centrifugation and micromanipulation techniques while immunological methods include the use of monoclonal antibodies.
68Genetic analysis of these isolated fetal cells can be performed using PCR or FISH. Fetal cells in maternal blood have been successfully used on a research level to diagnose trisomy 21, trisomy 18, beta-thalassaemia and sickle cell anaemia. Actual clinical use will have to await further technological development to improve its reliability and clinical trials to assess the sensitivity and specificity of this approach.