Fetal DNA in maternal serum

1. FETAL DNA IN MATERNAL SERUM
DR KAPIL DEV

2. Trisomies
• Trisomy 21 is due to an extra chromosome 21 also called
Down Syndrome and is the most common trisomy at the time
of birth.
• Trisomy 18 is due to an extra chromosome 18. also called
Edwards Syndrome, is associated with a high rate of
miscarriage.
• Trisomy 13 is due to an extra chromosome 13. also called
Patau Syndrome, is associated with a high rate of miscarriage

4. How are trisomies diagnosed?
• Current methods of fetal genetic testing typically
involve obtaining samples of amniotic
fluid, placenta, fetal blood, or rarely, other fetal
tissues or fluids.
• The invasive techniques required for obtaining
fetal samples (eg, amniocentesis, chorionic villus
biopsy, fetal venipuncture, fetoscopy-guided
biopsy) place the fetus at risk of injury or death.

5. Chorionic villi sampling (CVS)
• A small amount of tissue from the developing
placenta.
• tested chromosomes.
• CVS is typically performed between 11 and 14 weeks
of pregnancy.
• CVS is associated with a small risk of miscarriage - 12%

6. Amniocentesis
• withdraws a small amount of fluid that
surrounds the fetus.
• tested for chromosomes.
• An amniocentesis is usually performed
around or after the 15 weeks of pregnancy.
• Amniocentesis is associated with a small risk
of miscarriage- 1%

7. • Complications associated with invasive prenatal
diagnostic tests:
•
•
•
•
•
•
Pregnancy loss (0.15%-1.0%)
PROM
Vaginal bleeding
Worsening sensitization of alloimmunization
Infection
Placental mosaicism

8. • Fetal DNA in maternal plasma or serum was first detected by PCR amplification
of a Y-specific fetal sequence, followed by the analysis with agarose-gel
electrophoresis and ethidium-bromide staining.
• In a subsequent study Lo et al
• Developed a real-time quantitative PCR assay to measure the concentration of
fetal DNA in maternal plasma and serum.
• This system is sensitive enough to detect the DNA equivalent of a single target
cell.
• Other advantages include a large dynamic range of over five orders of
magnitude, a high through put, and the use of a homogeneous amplification
and detection system that minimizes the risk of carry-over contamination.
• The main limitation of PCR analysis of Y-chromosomal sequences in maternal
plasma is that this approach can be used only in pregnancies involving male
fetuses.

9. Background:
Fetal Nucleic Acids in Maternal Plasma
•First report of free fetal DNA in maternal circulation. (Lo YMD et
al. Lancet 1997;350:485-7)
•Fetal DNA clears rapidly from maternal circulation after the
baby is delivered. (Lo YMD et al. Am J Hum Genet 1999;64:218-24)
•First report of free fetal RNA in maternal circulation. (Poon LLM
et al. Clin Chem 2000;46:1832-4)
•Prenatal diagnosis of fetal RHD status by molecular analysis of
maternal plasma. (Lo YMD et al. N Engl J Med 1998;339:1734-8)

14. LOW RISK PATIENTS
(HIGH RISK SHOULD BE SCHEDULED FOR
COUNSELING AT 10 WEEKS)
Blood draw for PAPP-A and
HCG at 9-12 weeks.
High Risk
> 1/65 risk for Down syndrome
Counseling- tests offered
·Invasive testing
·Maternal serum fetal
DNA test
·Complete Sequential
Ultrasound scheduled per usual
11 4/7 weeks to 14 weeks
(12 weeks optimal)
Intermediate Risk
1/66 to 1/2000 risk for Down
syndrome
Brief Counseling
·Sequential screen
> 1/270 risk -counseling
< 1/270 riskCounseling based on results
Low Risk
< 1/2000 risk for Down
syndrome
No further testing

18. Library preparation

19. Cluster Generation

20. Massively Parallel Sequencing

23. Data Analysis
• Alignment (chromosome matching) using human genome
database
• One matching error per 36 bases allowed
• Interpretation of results:
% of matches on chromosome 21
Z score for each sample

26. Trisomy Detection – Fetal fraction matters
 Trisomy detection via cell-free DNA analysis depends on the fetal fraction (proportion of
fetal to maternal cell-free DNA within the sample)
 The higher the fetal fraction the easier it becomes to detect aneuploidy
10% fetal DNA in circulation
Disomic Chr
Trisomy 21
Total: 100
Total: 105
(Maternal: 90)
(Fetal:
10)
(Maternal: 90)
(Fetal:
15)
20% fetal DNA in circulation
Disomic Chr
Trisomy 21
Total: 100
Total: 110
(Maternal: 80)
(Fetal:
20)
(Maternal: 80)
(Fetal:
30)

29. Some Limitations
• Diagnostic testing is recommended following
all positive results .
Chance pregnancy affected ≥ 50%
• Negative result does not ensure unaffected
pregnancy .
• Cost efficacy

31. THANK YOU