18 ??????? Prenatal Diagnosis of Disease

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18 ??????? Prenatal Diagnosis of Disease
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• 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.

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• Furthermore, 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.

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1. 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.

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• The general indications of prenatal diagnosis
  include maternal age and the results of
  noninvasive serum biochemical screening.

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• Specific indications include a positive family
  history and the birth of a previous child
  affected by a particular genetic disease.

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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.

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• A. Amniocentesis
• Amniocentesis 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.

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• Traditionally 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  to permit 20 to 30 ml to be withdrawn safely.

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• 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.

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Amniocentesis performed concurrently with
ultrasound scanning.
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• Safety of amniocentesis
• Any procedure that involves passing a
  device into an organ, especially the pregnant
  uterus, carries with it risks amniocentesis is
  no exception.

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• 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.

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• Fetal risks include spontaneous abortion,
  needle puncture injuries, placental abruption,
  chorioamnionitis, preterm labour, and amniotic
  band formation.

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• Several 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.

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• 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-resolution ultrasound
  utilised.

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• 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 gestation.  

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• There is evidence that early
  amniocentesis is associated with a higher fetal
  loss rate and a more frequent occurrence of
  certain congenital abnormalities.

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• B. 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.

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• 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.

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• The 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.

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• Safety of CVS
• Maternal 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.

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• 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
  therapeutic purposes.

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Indications for
fetal blood sampling Diagnostic
Rapid karyotyping Fetal anomaly on ultrasound Late attending patients who require fetal karyotyping
Alloimmunisation Rhesus Platelet antigens
Fetal infection Toxoplasmosis Cytomegalovirus infection
Genetic Haemoglobinopathies Metabolic disorders and enzyme deficiencies
Fetal well being Severe intrauterine growth retardation
Therapeutic
Transfusion Red cell alloimmunisation
Transplantation Stem cells
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• FBS 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.

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• Safety of FBS
• Maternal complications from FBS are
  uncommon but include amnionitis, infection,
  rhesus sensitisation and transplacental
  haemorrhage.

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• Fetal 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.

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• Other fetal complications include
  infection, premature rupture of membranes,
  haemorrhage, severe bradycardia and umbilical
  cord thrombosis.

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• D. Fetal biopsy
• Although 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.

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• 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.

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3. Analytical methods

• Following the acquisition of fetal
  tissues, these materials are then subjected to
  analysis using a variety of techniques.

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• A. Cell culture and conventional cytogenetics
• These are the most commonly used methods for
  the diagnosis of chromosomal aneuploidies such as
  Down syndrome.

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• B. 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.

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• Using 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.

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• 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
  obtained following amniocentesis, CVS or fetal
  blood sampling.

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• 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 .

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• PCR-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.

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• Genetic 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.

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• D. 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.

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• 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 established within the tissue of interest.

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• 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.

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• E. Microarray Analysis
• Much of the excitement today centers on
  gene expression profiling that uses a technology
  called microarrays.

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• 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.

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• A 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.

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4. New methods for prenatal diagnosis

• A. Preimplantation diagnosis
• Preimplantation diagnosis is the
  performance of prenatal genetic analysis on
  embryos or oocytes prior to implantation.

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• This technology has the advantage that it
  allows prenatal diagnosis to be carried out much
  earlier than existing methods such as
  amniocentesis and CVS.

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• 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.

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• In 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.

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• Access 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.

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• For 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.

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• For 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.

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• In both situations, only the embryos
  confirmed not to possess the full disease-causing
  genotype are then implanted back into the uterus.

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• Diagnostic methods
• Preimplantation diagnosis may be achieved
  using PCR, FISH or by measurement of embryonic
  secretory products such as certain enzymes.

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• This 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.

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• Worldwide, 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.

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• 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.

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• However, convincing proof of the
  existence of these cells have to await the
  development of molecular biological techniques,
  especially the PCR.

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• Using 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.

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• Fetal 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.

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• At 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.

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• Isolation 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.

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• Genetic 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.