A trisomy is a chromosomal condition that occurs when there are three copies of a particular chromosome instead of the expected two.risk of having a baby with a trisomy increases with maternal age21 - intellectual disabilities and digestive disease and congenital heart defects. present in 1 out of every 700 newborns.18- congenital heart defects- present in approximately 1 out of every 5,000 newborns.13 - present in approximately 1 out of every 16,000 newborns
development of an accurate, safe, rapid, noninvasive test for prenatal diagnosis is an area of active investigation.
PM is detected in approximately 1-2% of ongoing pregnancies that are studied by chorionic villus sampling (CVS) at 10 to 12 weeks of pregnancy
Sample requirements: Whole blood drawn into 2 x 10ml blood tubesSamples received within 5 days of blood collection
FPR FALSE POSITIVE REPORT
single baby @after 10 completed weeks of pregnancy
Robertsonian translocation (ROB) is a common form of chromosomal rearrangement that in humans occurs in the five acrocentric chromosome pairs, namely 13, 14, 15, 21, and 22. Other translocations occur but do not lead to a viable fetus. They are named after the American biologist William Rees Brebner Robertson Ph.D. (1881–1941), who first described a Robertsonian translocation in grasshoppers in 1916. They are also called whole-arm translocations or centric-fusion translocations.the participating chromosomes break at their centromeres and the long arms fuse to form a single chromosome with a single centromere. The short arms also join to form a reciprocal product, which typically contains nonessential genes and is usually lost within a few cell divisions.
Where do fetal cell-free nucleic acids come from?DNA — Both the mother and the fetus produce cell free DNA. The primary source of fetal cell-free DNA in the maternal circulation is thought to be apoptosis of placental cells (syncytiotrophoblast), while maternal hematopoietic cells are the source of most maternal cell-free DNA .There is some evidence that apoptosis of fetal erythroblasts also generates fetal cell-free DNA, which can cross the placenta and enter the maternal circulation.RNA — Both fetal and placental cells contribute cell-free mRNA to the maternal
Samples consisting of longer fragments are first sheared into a random library of 100-300 base-pair long fragments.ends of the obtained DNA-fragments are repaired and anA-overhang is added at the 3'-end of each strandadaptors which are necessary for amplification and sequencing are ligated to both ends of the DNA-fragments. These fragments are then size selected and purified.
Single DNA-fragments are attached to the flow cell by hybridizing to oligos on its surface that are complementary to the ligated adaptors. The DNA-molecules are then amplified by a so called bridge amplification which results in a hundred of millions of unique clusters. Finally, the reverse strands are cleaved and washed away and the sequencing primer is hybridized to the DNA-templates.Illumina (Solexa) sequencingDNA molecules and primers are first attached on a slide and amplified with polymerase so that local clonal DNA colonies, later coined "DNA clusters", are formed. To determine the sequence, four types of reversible terminator bases (RT-bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides, then the dye, along with the terminal 3' blocker, is chemically removed from the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.Sequencing is the process of determining the order of nucleotide bases (A,C,T, and G) within a stretch of DNA. Sequencing the entire complement of DNA, or genome, of many animal, plant, and microbial species is indispensable for basic biological and medical researchcBot is the next generation of workflow improvements for Illuminasequencing.automated system that creates clonal clusters from single molecule DNA templates, preparing them for sequencing by synthesis on the Genome Analyzer.Balasubramanian and Klennerman from Cambridge University's chemistry departmentuse of clonal arrays and massively parallel sequencing of short reads using solid phase sequencing by reversible terminators (subsequently referred to as SBS sequencing) as the basis of a new DNA sequencing approach.
huge amount of generated clusters are sequenced simultaneously. The DNA-templates are copied base by base using the four nucleotides (ACGT) which are fluorescently-labeled and reversibly terminated. After each synthesis step, the clusters are excited by a laser which causes fluorescence of the last incorporated base. After that, the fluorescence label and the blocking group are removed allowing the addition of the next base. The flourescence signal after each incorporation step is captured by a built-in camera, producing images of the flow cel
, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugatedestriol (uE3) in the maternal blood
1. FETAL DNA IN MATERNAL SERUM
DR KAPIL DEV
• Trisomy 21 is due to an extra chromosome 21 also called
Down Syndrome and is the most common trisomy at the time
• Trisomy 18 is due to an extra chromosome 18. also called
Edwards Syndrome, is associated with a high rate of
• Trisomy 13 is due to an extra chromosome 13. also called
Patau Syndrome, is associated with a high rate of miscarriage
3. 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.
4. Chorionic villi sampling (CVS)
• A small amount of tissue from the developing
• tested chromosomes.
• CVS is typically performed between 11 and 14 weeks
• CVS is associated with a small risk of miscarriage - 12%
• 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%
6. • Complications associated with invasive prenatal
Pregnancy loss (0.15%-1.0%)
Worsening sensitization of alloimmunization
7. • 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
• 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
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)
9. LOW RISK PATIENTS
(HIGH RISK SHOULD BE SCHEDULED FOR
COUNSELING AT 10 WEEKS)
Blood draw for PAPP-A and
HCG at 9-12 weeks.
> 1/65 risk for Down syndrome
Counseling- tests offered
·Maternal serum fetal
Ultrasound scheduled per usual
11 4/7 weeks to 14 weeks
(12 weeks optimal)
1/66 to 1/2000 risk for Down
> 1/270 risk -counseling
< 1/270 riskCounseling based on results
< 1/2000 risk for Down
No further testing
10. Library preparation
11. Cluster Generation
12. Massively Parallel Sequencing
13. Data Analysis
• Alignment (chromosome matching) using human genome
• One matching error per 36 bases allowed
• Interpretation of results:
% of matches on chromosome 21
Z score for each sample
14. 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
20% fetal DNA in circulation
15. Some Limitations
• Diagnostic testing is recommended following
all positive results .
Chance pregnancy affected ≥ 50%
• Negative result does not ensure unaffected
• Cost efficacy