FETAL DNA IN MATERNAL SERUM

DR KAPIL DEV

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

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.

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 - 1-

2%

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%

• Complications associated with invasive prenatal diagnostic tests:

• Pregnancy loss (0.15%-1.0%)• PROM• Vaginal bleeding• Worsening sensitization of alloimmunization• Infection• Placental mosaicism

• 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.

• 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)

Background:Fetal Nucleic Acids in Maternal Plasma

Library preparation

Cluster Generation

Massively Parallel Sequencing

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

Trisomy Detection – Fetal fraction matters

• Trisomy 21• Disomic Chr

• Total: 100

• (Maternal: 90)• (Fetal: 10)

• 10% fetal DNA in circulation

• Total: 105

• (Maternal: 90)• (Fetal: 15)

20% fetal DNA in circulation

Total: 100(Maternal: 80)(Fetal: 20)

Trisomy 21

Total: 110(Maternal: 80)(Fetal: 30)

Disomic Chr

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

Some Limitations

• Diagnostic testing is recommended following all positive results .Chance pregnancy affected ≥ 50%

• Negative result does not ensure unaffected pregnancy .

• Cost efficacy

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