2. Diagnosis I.
Knowledge of the disease in question is a
prerequisite of genetic counseling
The use and interpretation of different diagnostic
procedures (e.g. sonography, karyotyping)
Obtaining old medical records, pedigree analysis,
careful history taking, clinical examinations of
relatives, special laboratory tests should be relevant
3. Diagnosis II.
Until the recent past, couples at high risk of
genetic disorder had the choice:
to take the risk
to consider other reproductive options
(long term contraception, sterilisation, TOP, or adoption)
In 1966 the relation of advanced maternal age and
increase risk of Down syndrome was noticed
prenatal diagnosis started to develope
4. Objectives of prenatal diagnosis
AIMS:
to detect abnormalities and allow termination
to allow couples at high risk to know that the disorder
could be confirmed or excluded by testing
to provide a range of informed choice to the couples at risk
of having a child with abnormality
to provide reassurance and reduce anxiety, especially
among high-risk groups
to allow the couples the option of appropriate management
(psychological, postnatal)
to enable prenatal treatment of the affected fetus
5. To make prenatal diagnosis more effective
Less invasive techniques
Earlier testing
Individual diagnosis
6. Methods of prenatal diagnosis
INVASIVE
Genetic amniocentesis
(GAC)
Chorionic villus sampling
(CVS)
Chordocentesis (percutan
umbilical blood sampling
– PUBS)
(Biopsy from fetal tissues)
(Coelocentesis)
NON-INVASIVE
Ultrasonography (US)
Magnetic resonance
imaging (MRI)
(Fetal cells and) cell-free
fetal DNA in maternal
blood (NIPT)
7. Indications of prenatal diagnosis
advanced maternal age (≥ 35 yrs)
positive family history for
chromosome aberration
a monogenic disorder
neural tube defect
other congenital structural abnormalities
abnormalities suspected in pregnancy
other high risk factors (consanguinity, obstetric history,
maternal diseases)
8. Maternal age
career or
motherhood?
socio-economic
problem
average age of
primiparous women
increases
increase of incidence
of fetal aneuploidies
increasing number
of obstetrical
complications
9. Positive family history of
chromosomal abnormality
usually no increase in risk compared to general
population since most chromosomal disorders
arise as a result of non-disjunction
however each situation should be confirmed by
nature of chromosome abnormality in affected
individual
10. Positive family history for a
monogenic disorder
previous affected child
affection of one or both parents
positive family history
recurrence risk 25% (AR) or 50% (AD)
prenatal diagnosis should be offered in case of
certain conditions as many can be diagnosed by
DNA analysis or biochemical testing (cystic
fibrosis, achondroplasia, Huntington disease,
neurofibromatosis, haemophilia, muscular
dystrophy, etc.)
11. Positive family history for neural
tube defects
Risk of recurrence: 3-5%
Screening: MSAFP 16th
week
Diagnosis: US
Small closed neural tube
defects can be missed even
with the most skilled person
12. Positive history for other
congenital malformations
Evaluation of the family history
Calculation of the risk of recurrence
In case of suspected disorder detailed ultrasound is
indicated (majority of the malformations can be
diagnosed by US)
Fetal heart malformation (fetal echocardiography)
13. Other risk factors
Parental consanguinity: increased risk of AR and
multifactorial disorders
Poor obstetric history (recurrent miscarriages and
stillbirths) increases the risk of future pregnancies
Maternal diseases (diabetes, etc.)
14. Methods of prenatal diagnosis
INVASIVE
Genetic amniocentesis
(GAC)
Chorionic villus sampling
(CVS)
Chordocentesis (percutan
umbilical blood sampling
– PUBS)
(Biopsy from fetal tissues)
(Coelocentesis)
NON-INVASIVE
Ultrasonography (US)
Magnetic resonance
imaging (MRI)
(Fetal cells and) cell-free
fetal DNA in maternal
blood (NIPT)
15. Genetic amniocentesis
Ultrasound-guided intervention between 16-20
gestational week
Fetal karyotype analysis: from fetal cells (cell
culture). Result of karyotyping from cell culture
takes ~3 weeks
Ability of QF-PCR for most common trisomies
Material for diagnosing monogenic disorders
Measurement of the amniotic fluid alpha-
fetoprotein (AFAFP)
16. Ultrasound prior to amniocentesis: to determine
fetal cardiac activity, estimated gestational age,
location of placenta, amniotic fluid volume,
number and position of fetuses
Avoidance of the placenta is recommended
Sterile technique
Volume of amniotic fluid removed: 8-15 ml
After the examination: 2 days bed rest
Genetic amniocentesis II.
17. Complications:
Fetal loss: 0.5-1%
Fetal injury: very rare because of ultrasound
guidance
Leakage of amniotic fluid, bleeding, uterine
irritability
Genetic amniocentesis III.
18. Chorionic villus sampling (CVS)
The most common first trimester invasive prenatal
diagnosis technique: chromosomal abnormalities,
monogenic disorders
Ultrasound-guided procedure between the 10-12th
gestational weeks
Initially transcervical, nowadays transabdominal
CVS obtains chorionic tissue from the developing
placenta
19. Ultrasound is performed prior to CVS: fetal cardiac
activity, gestational age, number of fetuses, uterine fibroid
Sterile technique
Ultrasound-guided intervention
Pregnancy loss: 2-3%
Placental mosaicism can cause diagnostic difficulties
Chorionic villus sampling (CVS) II.
20. Amniocentesis vs. CVS
AMNIOCENTESIS CVS
Procedure
AF removed by needle
CV removed by catheter
(TC) or needle (TA)
Timing 16-20th week 10-12th week
Fetal malform. risk
-
1:3000 vascular limb
malformation
Pregnancy loss
0.5-1% 2-3%
Time required for
cytogenetic dg.
2-3 weeks 1 week
Accuracy Highly accurate Highly accurate
Risk of placental
mosaicism
21. Chordocentesis (PUBS)
Ultrasound guided puncture of the umbilical cord
„pure” fetal blood can be sampled
The sample is rich in cells
Can be used in cases of Rh isoimmunisation:
diagnosis, fetal therapy (tranfusion)
22. Ultrasound
Screening (chromosome aberrations) and
diagnostics (e.g. spina bifida) in one
Five US scan during pregnancy
0. US: to diagnose pregnancy (embryonal heart
function)
1st US scan: 12th week (NT<3 mm!)
2nd US scan: 17-21st week (detailed examination:
diagnosis of congenital malformations)
23. 3rd US scan: 28-32nd week (IUGR, flowmetry:
placental circulation)
4th US scan: 36-38th week: fetal position,
estimated weight, placental position, width of the
scar)
Fetal echocardiography: 18-22nd week: fetal heart
anatomy and function
Ultrasound II.
24. Fetal MRI I.
Fetal MRI: an adjunct tool to US in fetal screening
Images to be obtained from any directions
Excellent soft tissue contrast
Substituting US in case of oligohydramnios or
obesity
25. INDICATIONS
Central nervous system malformations (NTD,
holoprosencephaly, hydranencephaly, AV
malformations, intracranial haemorrhage)
Cervical teratoma (relationship to vessels and
airways)
Chest masses (diaphragmatic hernia, CAM)
Adrenal neuroblastoma
Fetal MRI II.
28. Cell-free fetal DNA in maternal blood
Both the mother and fetus produce cell-free DNA from
apoptotic cells
– maternal DNA originates in bone marrow
– fetal DNA originates in placenta
• 150-200bp
• Adequate amounts for clinical testing after 10 weeks (detected after
32 days gestation)
• Can also be used in the third trimester
• Undetectable after 2 hours postpartum
An average maternal plasma sample contains
– ~90% maternal cell-free DNA
– ~10% fetal cell-free DNA
isolate cell-free fetal DNANot to
29. Euploid fetus
Fetus with Down
syndrome
Maternal plasma
Maternal cell-free DNA
from chromosome 21
Fetal cell-free DNA
from chromosome 21
10 11
Fetus with trisomy 21 releases an an extra amount of cell-free fetal
DNA representing chromosome 21 into the maternal circulation
30. Improvements in DNA sequencing
gel-based systems
capillary sequencing
massively parallel
sequencing
kb/day/machine
1 000 000 000
100 000 000
10 000 000
1 000 000
100 000
10 000
1 000
100
10
1980 1990 2000 2010
year
31. Massively parallel sequencing
Both maternal and fetal DNA are examined from a
peripherial maternal blood sample
– Fetal DNA is NOT isolated from maternal blood, BOTH maternal and
fetal DNA are sequenced
• library preparation
• cluster generation
• sequencing
MPS is used as a molecular counting system rather than as a
sequencer
– 25-36 base pair reads
Alignment of reads
– Measure counts relative to a reference value (from a normal genome)
32. Overall performance of NIPT for
aneuploidy
Sensitivity for detection of trisomy 21 is >99%
False positive rates are extremely low (~0,5%)
– May have a biological basis
False negative rates are even lower (but not 0%)
– Mostly due to low fetal cell-free DNA fraction
33. Screening for a genetic disease
Screening program’s aim:
the prevention of a certain disease
the early diagnosis of a certain disease
Effective screening programs need general
agreement, in the society or population
There may be opportunities for prenatal prevention:
primary (e.g. folic acid supplementation vs. NTD),
secondary (induced abortion)
35. Biochemical screening tests
II. trimester
Triple test:
AFP, hCG, unconjugated estriol
Quadruple test:
AFP, hCG, unconjugated estriol, inhibin-A
I. trimester
Serum screening alone:
free beta-hCG, PAPP-A
Combined test:
free beta-hCG, PAPP-A + NT
Nondisclosure sequential
screening (results not
reported until all are
complete)
Integrated serum test
Integrated test
Stepwise sequential
screening (intermediate
results reported as they are
ready)
Contingent screening (only
those with borderline risks
have second-trimester tests)
36. Detection rate
– Maternal age 30%
– Quadruple test (FPR 5%) 60-65%
– NT alone 70-79%
– Combined test (NT + free beta-hCG and PAPP-A)
(FPR 5%) 87%
– NT + nasal bone
(FPR 1-2%) 90%
– Combined test + nasal bone
(FPR 1%) 95%
– Integrated test, stepwise sequential screening
(FPR 4-5%) 95%
Biochemical screening tests II.
37. Decision making, parental rights and
responsibilities
The couple decide whether or not to undertake
pregnancy (before 12th week)
The couple decide whether or not to accept the offer of
prenatal diagnosis
Genetic counselor: gives the adequate information
Parents: make the decision
Factors influencing the decision: optimistic/pessimistic
attitude, ethical or religious principles etc.
38. Termination of pregnancy (TOP) I.
TOP may be permitted at any time when serious disease
threatens the mother’s life (e.g. heart failure, obstetric
complication)
TOP may be permitted at any time when fetal disease is
incompatible with postpartum life (e.g. anencephaly)
TOP up to the 12th gestational week is permitted, when
the risk of genetic disorder or teratogenic damage to the
fetus exceeds 10%
TOP is possible until the 24th gestational week, when the
risk of a severe, difficultly curable or incurable fetal
disease is 50-100% (e.g. Down syndrome)
39. Fetal indications, e.g.
The probability of a severe AD disorder is 50%
(Huntington disease)
Mother carrying an XR-gene is pregnant with a male
fetus
Severe CNS malformation
Severe bilateral kidney disease
Severe chromosome aberration
Termination of pregnancy (TOP) II.
40. Malformations with better prognosis
E.g. Cleft lip, cleft palate, mild ventriculomegaly,
multicystic kidney etc.
Follow-up during the pregnany (US)
Exclude associating malformation, chromosome
aberrations
The optimal hospital or clinic for the delivery
Caesarean section vs. vaginal delivery?
Detailed neonatological examinations
Postnatal medical treatment
Postnatal surgery