20.05.09 NIPD/NIPT - ffDNA and RNA in maternal serum & applications Flashcards
1
Q
What is NIPD and NIPT
A
- Non invasive prenatal testing
- NIPD= diagnosis (diagnostic)
- NIPT= testing (screening)
2
Q
What is the basis for NIPD/T
A
-Cell free fetal DNA (CffDNA) in maternal blood during pregnancy (discovered in 1997)
3
Q
Why are intact fetal cells not used for non-invasive testing
A
- Present in low numbers
- lack distinct cell markers for easy enrichment
- Testing has low sensitivity, specificity and reproducibility
- Fetal cells persist in mother blood for years after pregnancy, so could give false positive results
4
Q
Review of cffDNA
A
- 10-20% of total cell free DNA in maternal plasma comes from placenta (cffDNA)
- Detectable after 4-5 weeks, reliably from 7 weeks
- Rapidly cleared from circulation after delivery (mean half life= 16-17 mins)
- Originates from placental cells, which shed DNA into maternal circulation.
- Average length of fragments= 166bp
5
Q
Review of stable fetal mRNA (cffmRNA)
A
- RNA is usually unstable as susceptible to nuclease attack
- cffmRNA is stable in peripheral blood due to association with trophoblast derived microparticles, which protect it.
- Total cffmRNA levels don’t increase during gestation, although relative abundance of transcripts changes.
- Detectable in maternal plasma by 4th week gestation
- Rapidly cleared from maternal blood after delivery. Half life= 14 mins.
6
Q
Technical challenges of isolating cffDNA
A
- Concentration is relatively low
- Nucleated blood cells must be stabilised to prevent release of maternal gDNA, which would reduce fetal fraction. Blood collected in Streck tubes and processed within 5 days.
- Often low fetal fraction
- Fetus inherits half of genetic information from mother, so much of the cffDNA is indistinguishable from maternal cell-free DNA.
7
Q
Main clinical applications of NIPD
A
- Fetal sex determination in pregnancies at risk of sex-linked disease
- Diagnosis of certain single gene disorders, detection of paternally inherited pathogenic variant.
- Detection of fetal aneuploidy (Down syndrome)
- Diagnosis of fetal blood type in pregnancies at risk of incompatibility (e.g. RhD)
- Detection of high risk allele using RHDO (relative haplotype dosage) analysis
8
Q
Review of NIPD for use in fetal sex determination
A
- Detection of male Y chromosome DNA
- Offered after 7th week pregnancy
- Used in pregnancies at risk of serious x-linked conditions (DMD, adrenoleukodystrophy)
- If male fetus is detected by NIPD, then invasive testing is offered for definitive molecular diagnosis.
- If fetus is female then invasive testing not required.
9
Q
Method of fetal sex determination NIPD
A
- Maternal blood is spun to isolate maternal plasma
- Circulating nucleic acids are isolated from plasma (maternal and fetal)
- RT-PCR used to detect and quantify SRY (sex determining region Y)
- Repeated in a number of replicates over two different extraction methods.
- Female fetuses inferred from negative result.
- 99.5% accurate
10
Q
How can false positives be overcome in sex determination NIPD
A
- A false positive= negative result due to very low levels of cffDNA
- Avoided by:
a) having a cut-off limit for the minimum amount of cffDNA required
b) Testing for universal fetal markers (paternal SNPs or CCR5), as a positive control for the presence of cffDNA
c) carrying out replicate testing of each sample
11
Q
What method does the RAPID project recommend to confirm fetal sex
A
- RAPID= Reliable accurate prenatal non invasive diagnostic project
- Recommend the use of ultrasound to confirm fetal sex.
12
Q
Review of NIPD to diagnose single gene disorders
A
- AD disorders from father or de novo (i.e. different from maternal genotype)
- Restricted to mutations <300bp, due to fragmented nature of cffDNA.
- Highly accurate, reducing the need for invasive confirmatory tests.
- Assay work up done before pregnancy
- Compound het disorders can be tested for paternal allele, reduce number of invasive tests by 50%
- if recessive (mother and father carry same variant) then RMD (Relative mutation dosage) could detect relative amounts of mutant and normal alleles (ddPCR), still in research.
13
Q
What fetal-specific markers can be used to confirm the presence of cffDNA
A
- Paternal-specific SNPs, limited to the SNP being het
- cffDNA is more fragmented and so shorter compared to maternal DNA. Size fragmentation can enrich for fetal sequences.
- Fetal epigenetic markers. Methylation of genes, specifically expressed in placenta. e.g.
1. RASSF1 (hypermethylated in fetal tissue, hypomethylated in maternal tissue can be removed by methylation-sensitive restriction enzymes).
2. SERPINB5 (hypomethylated in placental tissue, hypermethylated in maternal tissue). Could use methylation-specific PCR to detect. However, bisulfite conversion degrades 95% of input DNA.
14
Q
Review of fetal aneuploidy NIPT
A
- Not diagnostic yet
- Gold standard is karyotyping.
- NIPT requires detection and quantification of DNA derived from a specific chromosome.
15
Q
How can aneuploidy be calculated in NIPT
A
- Targeting fetal specific nucleic acids (DNA/RNA) to differentiate from background maternal DNA. Relative amounts of alleles are then calculated by het SNPs. If there is aneuploidy, one allele will be over represented. Multiple markers needed as homozygous SNPs are not informative.
- Direct measurement of chromosome dosage. Compares ratio of chr of interest to a reference chr. 1:1= normal, 2:3= aneuploidy. Advantages= does not rely on SNPs, therefore universally applicable to aneuploidies in all populations.
- Digital PCR. Sample partitioned into microreactions, so each contains 1 copy of of DNA
- NGS. Sequencing of specific sequences from each chromosome and comparing number of sequences to reference. Useful when screening for multiple pathogenic variants, can be focused on area of interest. However, kits need to be bespoke, requires 4-5% cffDNA, usually over 10 weeks gestation.
