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Çiğdem Yüce Kahraman

2
PRENATAL DIAGNOSIS OF GENETIC DISORDERS AND
CONGENITAL DEFECTS
 The principal aim of prenatal diagnosis is to supply at-risk
families with information so that they can make informed
choices during pregnancy.
 The potential benefits of prenatal testing include
¤ providing reassurance to at-risk families when the result is
normal;
¤ providing risk information to couples who, in the absence of such
information, would not choose to begin a pregnancy;
¤ allowing a couple to prepare psychologically for the birth of an
affected baby;
¤ helping the health care professional to plan delivery,
management, and care of the infant when a disease is diagnosed
in the fetus;
¤ and providing risk information to couples for whom pregnancy
termination is an option.
 Indications for prenatal diagnosis:
’ advanced maternal age
’ previous child with a chromosome abnormality
’ family history of a chromosome abnormality
’ family history of single gene disorder
’ family history of a neural tube defect
’ family history of other congenital structural abnormalities
’ exposure to specific chemical or radiation agents
’ certain ultrasound findings
’ positive maternal or genetic screening outcomes
’ other high risk factors (consanguinity, poor obstetry history,
maternal illnesses
 INDICATIONS FOR PRENATAL DIAGNOSIS
Advanced Maternal Age
’ This has been the most common indication for offering
prenatal diagnosis.
’ There is a well-recognized association of advanced maternal
age with increased risk of having a child with Down syndrome
and the other autosomal trisomy syndromes.
’ Most centers routinely offer amniocentesis or CVS to women
age 37 years or older, and the option is often discussed with
women from the age of 35 years onward.
Previous Child with a Chromosome Abnormality
Although there are a number of series with slightly different
recurrence risk figures, for couples who have had a child with
Down syndrome because of non-disjunction, or a de novo
unbalanced Robertsonian translocation, the risk in a subsequent
pregnancy is usually given as the mother’s age-related risk plus
approximately 1%.
’ If one of the parents has been found to carry a balanced
chromosomal rearrangement, such as a chromosomal
translocation or pericentric inversion, that has caused a previous
child to be born with serious problems due to an unbalanced
chromosome abnormality,
’ the recurrence risk is likely to be between 1% to 2% and 15% to 20%.
’ The precise risk will depend on the nature of the parental
rearrangement and the specific segments of the individual
chromosomes involved
Family History of a Single-Gene Disorder

’ If prospective parents have already had an affected child,

or if one of the parents is affected or has a positive family
history of a single-gene disorder that conveys a
significant risk to offspring, then the option of prenatal
diagnosis should be discussed with them.
’ Prenatal diagnosis is available for a large and ever-
increasing number of single-gene disorders by either
biochemical or DNA analysis.
Family History of a Neural Tube Defect

’ Careful evaluation of the pedigree is necessary to

determine the risk that applies to each pregnancy. Risks
can be determined based on empiric data.
’ Incidence of NTD 1 /1000 in a population
’ In high-risk situations, ultrasonographic examination of
the fetus, possibly in conjunction with assay of maternal
serum AFP, can be offered.
Family History of Other Congenital Structural Abnormalities

’ As with NTDs, evaluation of the family pedigree should enable the

provision of a risk derived from the results of empiric studies.
If the risk to a pregnancy is increased, detailed ultrasonographic
examination looking for the specific structural abnormality can be
offered at around 16 to 18 weeks’ gestation.
’ Mid-trimester ultrasonography will detect most serious cranial,
cardiac, renal and limb malformations.
’ Some couples request detailed ultrasonographic scanning,
’ not because they wish to pursue the option of termination of
pregnancy but because they wish to prepare themselves if the
baby is found to be affected.
Family History of Undiagnosed Learning Difficulty

’ An increasingly common scenario is the urgent referral of a

pregnant couple who already has a child, or close relative,
with an undiagnosed learning difficulty, with or without
dysmorphic features.
’ Whereas in the past a standard karyotype and fragile X
syndrome test might be carried out, today it is
recommended for geneticists to use the latest technique of
microarray-CGH.
Abnormalities Identified In Pregnancy

’ The widespread introduction of prenatal diagnostic

screening procedures, such as triple testing and fetal
anomaly scanning, has meant that many couples
unexpectedly present with diagnostic uncertainty during the
pregnancy that can be resolved only by an invasive procedure
such as amniocentesis or CVS.
’ Other factors, such as poor fetal growth, can also be an
indication for prenatal chromosome analysis, as
confirmation of a serious and non-viable chromosome
abnormality, such as trisomy 18 or triploidy, can influence
subsequent management of the pregnancy and mode of
delivery.
 Other High-Risk Factors
’ These factors include parental consanguinity, a poor obstetric
history, and certain maternal illnesses.
Parental consanguinity increases the risk that a child will have a
hereditary disorder or congenital abnormality. Consequently, if the
parents are concerned, it is appropriate to offer detailed
ultrasonography to try to exclude a serious structural abnormality.
’ A poor obstetric history, such as recurrent miscarriages or
a previous unexplained stillbirth, could indicate an
increased risk of problems in a future pregnancy and
detailed ultrasonographic monitoring.
A history of three or more unexplained miscarriages should
be investigated by parental chromosome studies to exclude a
chromosomal rearrangement such as a translocation or
inversion.
’ Maternal illnesses, such as poorly controlled diabetes
mellitus or epilepsy treated with anticonvulsant
medications such as sodium valproate, would also be
indications for detailed ultrasonography. Both of these
factors convey an increased risk of structural abnormality
in a fetus
What can be evaluated?
’ Chromosomal aberrations:
’ Trisomy,
’ Monosomy,
’ Polyploidy,
’ Marker chromosome,
’ Deletion, duplication, inversion, translocation, ring
chromosome .
’ Genetic aberrations (DNA)
’ Infectious disease
’ Biochemical markers (AFP)
’ Protein/enzyme levels
 Invasive techniques
• Fetal visualization
– Embryoscopy
– Fetoscopy
• Fetal tissue sampling
– Amniocentesis
– Chorionic villus sampling (CVS)
– Percutaneous umbilical blood sampling (PUBS)
– Percutaneous skin biopsy
– Other organ biopsies, including muscle and liver
biopsy
 Amniocentesis
’ Amniocentesis is traditionally performed at 15 to 17 weeks after a
pregnant woman's last menstrual period (LMP).
After real-time ultrasound imaging localizes the placenta and
determines the position of the fetus, a needle is inserted through
the abdominal wall into the amniotic sac. Between 20 and 30 mL of
amniotic fluid is withdrawn; this fluid contains living cells
(amniocytes) shed by the fetus.
’ The amniocytes are cultured to increase their number (a
procedure that requires up to 7 days), and standard cytogenetic
studies are carried out on the cultured amniocytes.
In addition, cells can be grown for biochemical assays or DNA-
based diagnosis of any genetic disease for which mutation testing
is available. The results of cytogenetic studies are typically available
in 10 to 12 days.
’ Because fluorescent in situ hybridization (FISH) can be carried
out on a small number of uncultured amniocytes, it can provide
an indication of fetal aneuploidy in just 1 to 2 days. If the FISH
result is positive, subsequent, confirmatory diagnosis by routine
cytogenetic methods is recommended.
Second-trimester amniocentesis

• 14 - 20 wk
• Aspirate initial 1-2 ml then 20 ml of fluid
• Chromosome , DNA analysis, AFP
• Complication
– Transient vaginal spotting or Amniotic fluid leakage
1-2%
– Chorioamnionitis 0.1%
– Injury to fetus - rare
– Fetal loss 0.5%
– Rh sensitization
 Amniocentesis is also used to measure AFP, a fetal protein
that is produced initially by the yolk sac and subsequently by
the fetal liver.
 The AFP level normally increases in amniotic fluid until
about 10 to 14 weeks' gestation and then decreases steadily.
 Amniotic fluid AFP is significantly higher in pregnancies in
which the fetus has an NTD. When an amniotic fluid AFP
assay is used with ultrasonography in the second trimester,
more than 98% of fetuses with an open spina bifida and
virtually all of those with anencephaly can be recognized.
Amniocentesis, the withdrawal of amniotic fluid during
pregnancy, is performed at about 16 weeks post-LMP and
is used to diagnose many genetic diseases. The amniotic
α-fetoprotein level is elevated when the fetus has a
neural tube defect and provides a reliable prenatal test
for this condition. The rate of fetal loss attributable to
this procedure is approximately 1/200 above the
background risk level. Amniocentesis can also be
performed earlier in the pregnancy; some studies
indicate an elevated rate of fetal loss after early
amniocentesis.
 Chorionic Villus Sampling
 Chorionic villus sampling (CVS) is performed by aspirating
fetal trophoblastic tissue (chorionic villi) by either a
transcervical or transabdominal approach.
 Because it is usually performed at 10 to 11 weeks post-LMP, CVS
has the advantage of providing a diagnosis much earlier in
pregnancy than second-trimester amniocentesis. This may be
important for couples who consider pregnancy termination an
option.
 Cell culture (as in amniocentesis) and direct analysis from
rapidly dividing trophoblasts can provide material for
cytogenetic analysis. Confined placental mosaicism
(mosaicism in the placenta but not in the fetus itself) is seen in
about 1% to 2% of cases in which direct analysis of villus
material is performed. This can confuse the diagnosis, because
the mosaicism observed in placental (villus) material is not
actually present in the fetus. This problem can usually be
resolved by a follow-up amniocentesis.
CVS
DNA studies
 CVS, like amniocentesis, is generally a safe procedure.
 Several collaborative studies revealed a post-CVS fetal loss
rate of approximately 1% to 1.5% above the background
rate, compared with 0.5% above background for
amniocentesis.
 Cordocentesis
’ Cordocentesis, or percutaneous umbilical blood
sampling (PUBS), has become the preferred method to
access fetal blood.
’ PUBS is usually carried out after the 16th week of gestation
and is accomplished by ultrasound-guided puncture of the
umbilical cord and withdrawal of fetal blood.
’ The fetal loss rate attributable to PUBS is low, but it is slightly
higher than that of amniocentesis or CVS.
 There are three primary applications of PUBS.
¤ It is used for cytogenetic analysis of fetuses with structural
anomalies detected by ultrasound when rapid diagnosis is
required. Cytogenetic analysis from fetal blood sampling is
completed in 2 to 3 days, whereas diagnosis after
amniocentesis can require 10 to 12 days if amniocytes must
be cultured. This time difference can be critical in the later
stages of a pregnancy.
¤ A second application is diagnosis of hematological diseases
that are analyzed most effectively in blood samples or
diagnosis of immunologic disorders such as chronic
granulomatous disease.
¤ PUBS is also used to make a rapid distinction between true
fetal mosaicism and false mosaicism caused by maternal
contamination of an amniotic fluid sample.
 Percutaneous umbilical blood sampling (PUBS, or
cordocentesis) is a method of direct sampling of fetal
blood and is used to obtain a sample for rapid
cytogenetic or hematological analysis or for
confirmation of mosaicism.
 Noninvasive techniques
• Fetal visualization
– Ultrasound
– Fetal echocardiography
– Magnetic resonance imaging (MRI)
– Radiography
• Serum marker
– Measuring Maternal Serum AFP
– Measuring maternal unconjugated estriol
– Measuring maternal serum beta-human
chorionic gonadotropin (HCG)
– Others
 Ultrasonography
’ A transducer placed on the mother's abdomen sends
pulsed sound waves through the fetus. The fetal tissue
reflects the waves in patterns corresponding to tissue
density. The reflected waves are displayed on a monitor,
allowing real-time visualization of the fetus.
’ Ultrasonography can help to detect many fetal
malformations, and it enhances the effectiveness of
amniocentesis, CVS, and PUBS.
Risk for trisomy 21 (Down syndrome) by maternal age, for different absolute values of
nuchal translucency (NT) at 12 weeks’ gestation.
 Ultrasonography is sometimes used to test for a specific
condition in an at-risk fetus (e.g., a short-limb skeletal
dysplasia).
 More often, fetal anomalies are detected during the
evaluation of obstetrical indicators such as uncertain
gestational age, poor fetal growth, or amniotic fluid
abnormalities.
 Second-trimester ultrasound screening has become
routine in developed countries. Studies of ultrasound
screening suggest that sensitivity for the detection of most
major congenital malformations ranges from 30% to 50%.
Specificity, however, approaches 99%.
The sensitivity of ultrasonography is higher for some
congenital malformations.
 In particular, ultrasound can detect virtually all fetuses
with anencephaly and 85% to 90% of those with spina
bifida.
It also sometimes identifies a fetus with a chromosome
abnormality by detecting a congenital malformation,
intrauterine growth retardation, hydrops (abnormal
accumulation of fluid in the fetus), or an alteration of the
amniotic fluid volume.
 Prenatal diagnosis includes invasive techniques
designed to analyze fetal tissue (CVS, amniocentesis,
PUBS) and noninvasive procedures that visualize the
fetus (ultrasonography, MRI).
’ Ultrasonography is the technique used most commonly
for fetal visualization, but other techniques are also used.
’ Radiography is still used occasionally, for example, to
evaluate a fetus for skeletal defects.
’ Magnetic resonance imaging (MRI) offers much greater
resolution than ultrasonography and is becoming more
widely available for prenatal screening.
Maternal serum screening
’ At 11+0 to 13+6 gestational weeks, first-trimester screening using
ultrasound is done to scan the fetal neck for enlarged nuchal
translucency (NT). Increased NT is associated with not only
trisomy 21 but also other chromosomal abnormalities. First-
trimester aneuploidy screening considers a combination of
maternal age, NT, and maternal serum (i.e., pregnancy
associated plasma protein A (PAPP-A) and free β-human
chorionic gonadotropin (free β-hCG)).
’ This method can identify about 90% of fetuses with Down
syndrome with a false positive rate of 5% .
’ The second trimester maternal serum triple test is applied
between 14 and 21 gestational weeks and determine the risk for
fetal chromosomal abnormalities combining maternal serum
levels of AFP, E3, human chorionic gonadotropin (hCG) and
maternal age.
Condition MSAFP uE3 HCG InhibinA PAPP-A USG

Neural tube
Increased Normal Normal - - √
defect

Trisomy 21 Low Low Increased Increased Low √

Trisomy
18,13
Low Low - - Low √

Molar
Low Low Very High - - √
pregnancy

Multiple
Increased Normal Increased - - √
gestation

Fetal death
(stillbirth)
Increased Low Low - - √
 10 - 13 weeks Chorionic villus sampling (CVS)

10 - 14 weeks Early amniocentesis

Standard amniocentesis
16-18 weeks

Multiple marker screening

(MSAFP + additional markers)
Optimum** 16-18 weeks

18 weeks - term Targeted ultrasound

Optimum* 17 1/2 - 24 weeks

18 weeks- term Fetal echocardiography

Optimum* 18 - 24 weeks

Percutaneous umbilical blood

18 weeks - term sampling
(PUBS)

*Optimum times for procedures are dependent on maternal habitus.

**Optimum times for serum tests are based on best times for neural tube
defect detection.
 Preimplantation Genetic Diagnosis
 Several new approaches to prenatal diagnosis are now in the testing or
early application stages. These include preimplantation genetic
diagnosis (PGD) at three different stages: polar body, blastomere, and
blastocyst.
 The most common type of PGD is carried out on a blastomere obtained
in the course of in vitro fertilization.
 Diagnosis is begun 3 days after fertilization, when the embryo contains
six or eight cells. One or two cells are removed from the embryo for
diagnosis (this does not harm it). FISH analysis can be used to diagnose
aneuploidy. Also, DNA from the cell can be amplified using PCR,
permitting the diagnosis of single-gene diseases.
 If the embryo is morphologically normal and neither the disease-
causing mutation nor aneuploidy is detected, the embryo is implanted
into the mother's uterus. Testing protocols have been developed for
dozens of genetic diseases (e.g., cystic fibrosis, Tay-Sachs disease, β-
thalassemia, myotonic dystrophy, Huntington disease, Duchenne
muscular dystrophy).
Preimplantation genetic diagnosis can be carried out on
polar bodies, blastomeres, or blastocyst cells, on which
PCR analysis and/or FISH is performed. Diagnosis of
genetic conditions permits implantation of only
unaffected embryos and avoids the issue of pregnancy
termination.
Analysis of Fetal DNA in Maternal Circulation
During pregnancy, a small number of fetal cells cross the
placental barrier to enter the mother's circulation. (only
about one fetal cell is present in 1 ml of maternal blood)
 testing cells for surface proteins specific to the fetus.
 FISH analysis and pcr analysis.
Cell-free fetal DNA is also present in the mother's
circulation and has been used to identify the sex of the
fetus and its Rh blood type.
NIPT(noninvasive prenatal testing) is a screening test; it is
not diagnostic. Results should be confirmed by diagnostic
testing prior to making any pregnancy management
decisions.(chromosome 13,18,21,X,Y)
 Prenatal Treatment
’ A possible model for successful prenatal treatment is provided by
the autosomal recessive disorder congenital adrenal hyperplasia
(CAH).
’ Affected female infants are born with virilization of the external
genitalia. There is evidence that in a proportion of cases the
virilization can be prevented if the mother takes a powerful
steroid known as dexamethasone in a very small dose from 4 to 5
weeks’ gestation onward. Specific prenatal diagnosis of CAH can
be achieved by DNA analysis of CV tissue. If this procedure
confirms that the fetus is both female and affected, the mother
continues to take low doses of dexamethasone throughout
pregnancy, which suppresses the fetal pituitary–adrenal axis and
can prevent virilization of the female fetus. If the fetus is male and
either affected or unaffected, the mother ceases to take
dexamethasone and the pregnancy can proceed uneventfully.
 Role of Genetic Counseling:
Prior to Testing

Causes of prenatal loss and congenital abnormalities

Cause Prenatal loss Congenital abnormality
Unknown 40-50% 50-60%

Chromosome 50-60% 6-7%

abnormalities
Monogenic 7-8%

Polygenic 20-25%

Environmental 7-10%
’ Detailed review of family & medical history
’ Comprehensive pedigree analysis
’ Genetic risk assessment & interpretation
’ Genetic testing options, including risks, benefits &
limitations
’ Provide educational materials
’ Facilitate patient informed consent