11/05 Non-Invasive Analysis of the Fetal Genome

Question 1

Isolation of nucleated fetal cells from maternal circulation

Answer 1

• The presence of fetal cells in maternal tissues including blood observed in 1893.
• Number of fetal cells present in maternal blood is extremely low, with estimates ranging from 1 to 6 cells per mL of maternal blood.
• Emphasis has been placed on cell selection using cell surface antigens as markers but no markers are uniquely indicative of fetal origin
• No published method has had the sensitivity or specificity to be of clinical utility
• Approach has largely failed to deliver. Some recent revival.

Question 2

Current testing for aneuploidy

Answer 2

• Definitive prenatal diagnosis requires direct collection of fetal material through invasive procedures (amnio, CVS)
• Risk of miscarriage 0.1-1.0% higher
• First trimester screen has detection rate for trisomy 21 of 82-87% between 10-13 weeks gestation with a false positive rate of 5%.
• Possibly 300,000 pregnant women offered invasive tests due to false positive screening results
• 50% uptake results in significant number of potentially avoidable miscarriages

Question 3

Detection of Paternally inherited alleles in maternal plasma

Answer 3

1) Isolate plasma
2) Extract “cell free” DNA
3) Detect “a” (Mass spec, TaqMan)

Question 4

What was the struggle in detecting alleles in maternal plasma?

Answer 4

• Figuring out which alleles were maternally inherited or paternal alleles that are identical to mom’s

Question 5

Analysis of Cell-Free Fetal DNA in Maternal Plasma

Answer 5

• Presence of circulating nucleic acids in plasma and serum (1948; Mandel and Metais)
• Demonstrated in 1997 that Y chromosome DNA derived from a male fetus can be detected by PCR in maternal plasma and serum
• Fetal DNA constitutes approximately 1-20% of the total cell-free DNA in maternal plasma at the end of the first trimester
• Fetal Rhesus D blood group status is determined routinely in UK (etc) by non-invasive plasma based methods

Question 6

Aneuploidy detection by via Methylation-Specific PCR

Answer 6

Trying to use physical techniques (methylation in this case) to try and identify fetal DNA

Question 7

RNA-Based Purification of Fetal Alleles

Answer 7

a) Transcribed SNP locus on chromosome 21
b) RNA expressed in placenta
c) Circulating placental RNA in maternal plasma

Question 8

Illumina Sequencing

Answer 8

• Sequence data is gathered via high resolution imaging of DNA extension products
• DNA tag lengths vary between 50 and 150 bp
• Billions of tag reads are possible
• 10 million are required to detect trisomy 21

Question 9

2 Methods of testing fetal DNA

Answer 9

1) Take all of DNA and shotgun sequence it

2) Or take part of it and target areas of interest

Question 10

NIPT for Microdeletions: Challenges

Answer 10

Important variables that impact sensitivity and specificity include:

i) sequencing read depth
ii) the size of the deleted or duplicated region
III) the frequency of fetal genome equivalents in the maternal plasma DNA sample
iv) the GC content of the region of interest

Question 11

By targeting:

Answer 11

you increase the number of reads that you can see

Question 12

NIPT Future

Answer 12

• All pregnancies irrespective of “risk”
• Lower cost, higher resolution, greater sensitivity
• Targeting (reduces cost and increases resolution…also reduces output)
• Eliminate sequencing? (microarray? Open array? Microfluidic?)
• Cell Capture??? - so much better to have pure sample; probs where everything is heading eventually.- Capture needs to be done on genome not antibodies