session 6: prenatal Flashcards
where does cffDNA originate from?
placenta - shed highly fragmented DNA into the maternal circulation during normal apoptosis
what is the fetal fraction and what amount is there in maternal blood? from what stage can it be detected? how do we know it is specific to the current pregnancy? how is fetal DNA distinguished from mums DNA?
- the total cell free DNA in maternal plasma that comes from the placenta (up to 20%)
- reliably detected from 7 weeks
- increases with increasing gestation
- rapidly cleared from circulation within an hour after delivery
- fetal DNA is shorter -approximately 200bp for fetal fragments and larger for maternal fragments. in silico size selection in data analysis enriches for fetal DNA
what are the technical challenges of isolating cffDNA?
- low abundance - outnumbered by cell free mat DNA
- need specialist STRECK tube to stabilise maternal blood and prevent release of maternal DNA
- DNA is indistinguishable from mat DNA (apart from size)
what advantage do intact fetal cells in maternal circulation have over cff-DNA?
stays in blood for 27 years whereas fetal DNA cleared rapidly after delivery
how does NIPD work?
- uses informative SNPS where mum homozygous to quantify level of paternal SNP present
- can also look at methylation in fetal epigenetic markers but some methods such as bisulphite conversion degrade DNA
why is an invasive procedure needed for NIPT but not NIPD?
- Confined placental mosaicism has not been reported for single gene diseases. USS recommended to rule out vanishing twin however that might interfere with genotyping
Describe uses of NIPD for Fetal sex determination. How does the testing work? why might there be a false negative result (no Y material in a male?) how can this be mitigated? what is the accuracy?
- sex determination to rule out X linked diseases eg. DMD/CAH. DMD -If Y chromosome material detected > do invasive sampling or stop if female. CAH - administer dexamethasone in female fetus to prevent virilisation (abnormal genitalia). not required for males. RT-PCR quantified amount of SRY on Y chromosome present.
- undetectable levels of cff-DNA - overcome by having cut-off limit, have control locus eg. CCR5 to check for presence of fetal DNA, replicate testing of each sample
- accuracy = 95.5%
- RAPID project still recommends scan to confirm fetal sex
Describe uses of NIPD for AD/de novo mutation detection?
- cffDNA will contain altered alleles that are not present in the high background of maternal cfDNA
- may use targeted NGS panel eg. FGFR3 or bespoke RT-PCR assay for relevant variant
- can only detect SVs (expansion, insertion or duplication ) <300 bp due to shorter fetal DNA length
- workup from affected proband or carrier parent required beforehand
how does NIPD work for AR or XL disorders? paternal exclusion testing and RMD/RHDO
PATERNAL EXCLUSION TESTING
- test fetal DNA for paternal variant, if absent the fetus is at most a carrier
- invasive testing offered if paternal variant identified
- eg. cystic fibrosis offered by targeted NGS at GOSH
relative mutation dosage (RMD)
- difficult to test for maternally-inherited variants due to background of maternal cfDNA
- In relative mutation dosage (RMD) the relative amounts of mutant and normal alleles in the maternal plasma are determined.
- • Equal amounts of wild type and mutant alleles would be expected in a female carrier with a carrier fetus.
- more mutant than expected = fetus homozygous for mutation
- more wildtype than expected = normal fetus
- need high enough fetal fraction for accurate calculation
advantage = doesnt require proband or dad
disadvantage = technically challenging , targeted to one mutation
Relative haplotype dosage
- multiple SNPs around region of interest are sequenced with targeted NGS
- o Haplotyping (linkage analysis) is used to determine whether the fetus is likely to have inherited a high risk or low risk haplotype
- o In diagnostic use for SMA, DMD and CF
advantages: statistically robust as uses several SNPs, can be used for mutation types or genes that are not amenable to NGS - disadvantages: risk of recombination, requires proband and paternal sample, expensive, consaguinity may be an issue with SNPS
what might the future hold for NIPD?
whole fetal genome present in mat blood so may be able to do WGS
non-invasive exome screening in USS abnormalities for paternally inherited alleles and de novo pathogenic variants
many more disorders added for RHDO and ‘proband free’ direct parental haplotyping from cell-free DNA
what was the RAPID project?
Project - 5 year UK national programme funded by NIHR. Aims: (1) to improve the quality of NHS prenatal diagnostic services by evaluating early non-invasive options based on cffDNA and RNA in plasma, (2) to develop standards and recommendations for use of NIPD/NIPT in NHS practice
what are the benefits of NIPD?
- no invasive sampling & therefore no miscarriage risk
- less expertise required for blood sample
- earlier testing to allow for decisions
- better targeting of anti-D therapy, which is currently given to all RhD negative women without testing to prevent a potentially fatal maternal immune response against a RhD positive fetus)
what are the limitations of NIPD?
- • In multiple pregnancies it is not possible to differentiate between the foetuses
- • The fetal fraction cffDNA in women with a high BMI is lower
- • The potential for incorrect results: false negatives can be the result of failure to extract or detect sufficient material, due to the low amount of total cell-free DNA and the small proportion of fetal versus maternal cell-free DNA - mitigated by fetal fraction quantification. False positives - result of either technical issues, such as contamination, or clinical abnormalities such as the presence of a non-identical vanishing twin
- wider issues include consent, equity of access, false negatives and positives, paternity testing, sex selection, , available DTC - should this be regulated?
what is aneuploidy?
a cell, having one or more chromosomes extra or missing from the normal full euploid set
how does QF-PCR work? what are the advantages?
amplification of STRs on chromosomes of interest used to determine copy number
● STRs (short tandem repeats or microsatellites) are a pattern of 2 - 6 bp that are repeated directly adjacent to each other.
● STRs are known highly polymorphic markers; a patient is therefore likely to have different numbers of repeat units on each allele.
- 4 markers for each chromosome of interest and sex chromosome markers if referral indicates sex chromosome abnormality eg. AMEL, SRY and DXYS218(Xp) and X22
- quantitative pcr - extracted DNA added to fluorescent primer multiplex and pcr. The reaction must be quantitative to detect copy number, therefore the PCR is stopped while in exponential phase to detect copy number -“ During the exponential phase of the reaction, the amount of product is directly proportional to amount of template “
- rapid, cheap, small quantities of DNA needed
what does a normal QF-PCR result ratio look like? what is the ratio range? how many informative markers are needed on one chromosome to interpret as normal?
1:1 if heterozygous or 1:0 if homozygous (uninformative as can be monosomic)
ratio is 0.8-1.4
need at least 2 informative markers per chromosome to interpret as normal
what does a trisomic QF-PCR result ratio look like? what is the ratio range? how many informative markers are needed on one chromosome to interpret as trisomy?
2:1 (0.45 &0.65 and 1.8 &2.4.) or 1:1:1 (0.8-1.4)
2 abnormal markers required for trisomy
if all alleles have same sized marker it is uninformative
● Best Practice: Cannot report a sample as trisomic if any ratios are inconclusive (between normal and abnormal) or if any normal ratios for an otherwise trisomic chromosome are obtained.
1:1:1 or 2:1
which is associated with M1 event? what might the other possibility be associated with and what could this mean if seen in a CVS sample?
1:1:1
M2 or mitotic non-disjunction
Where this pattern is seen in CVS the risk of CPM is increased
Best practice: The villi are now chopped/dissociated together and tested to give better sample representation (inner mesenchyme core and the outer cytotrophoblast layer
what is an inconclusive QF-PCR result? how can this be resolved?
result between normal and abnormal
● May be resolved by using single chromosome kits.
● Inconclusive ratios may be the result of preferential amplification of the smaller allele. This is more likely to occur if the distance between the alleles is increased,
do you need to confirm a positive QF-PCR result? do you need to confirm a negative QF-PCR result? In what circumstance should it be confirmed and how?
no - but sample identity must be confirmed prior to reporting (N.B. by a repeat test of the original sample or genotype comparison with a maternal blood sample).
- Normal results do not need repeating (N.B. there is a requirement for rigorous checking of sample transfers), unless the result is only based on a single marker result – this should be stated in the report and these results should be confirmed by a second method, e.g. karyotype or FISH.
what level of MCC can QF-PCR detect? how can this be identified from the trace? what should be done if MCC is suspected? when can a result be reported if there is MCC? If there is MCC, what alternative testing should be done?
10% - May show skewed allele pattern for all chromosomes.
if foetus is normal will have 3 peaks and the sum of the two smaller will equal the larger peak
run mums sample to compare genotypes
– if a low level of maternal genotype present but majority is foetal & has no inconclusive allele ratios
- QF-PCR on cultured cells or karyotype
how is mosaicism identified on QF-PCR?
skewed allele ratios and extra peaks
in diploid/triploid mosaic - all markers will be affected
what might normal and abnormal allele patterns obtained for a single chromosome with QF-PCR mean?
- somatic microsatellite mutation. usually increase or decrease of 1-2bp. sum of mutation and original peaks should give normal ratio when compared to unaffected allele
- Polymorphic Submicroscopic Duplications/Submicroscopic microsatellite duplications: a single marker consistent with trisomy and all others are normal . usually parents have same pattern for that marker.
- CNV: abnormal markers flanked by normal markers
- primer binding site polymorphism - primer binding less efficient and results in skewed ratios. lower annealing temperature.
what advantages does QF-PCR have over FISH?
● Less sample volume required (0.5-1ml amniotic fluid for PCR vs 3-4ml for FISH).
● Greater range of gestation (12-34wks PCR vs 15-21wks FISH).
● Less intense labour and higher through-put possible.
● Cheaper cost.
● Can detect MCC in all samples rather than just male conceptions.
● Can be used to infer UPD.
● Can detect certain unbalanced rearrangements (distal 13q, 18q, 21q).
what is the UK screen-positive cut-off rate for Down Syndrome?
> 1:150
what NT thickness is associated with increased aneuploidy risk?
> 3.5mm
Increased NT reflects foetal heart failure and is strongly associated with a chromosomal abnormality
(+21,18,13, 45,X and triploidy) also structural anomolies including cardiac if normal karyotype
which screening test only includes down syndrome and when does this occur?
quadruple
2nd trimester
Trisomies 13 and 18 can be picked up on the ultrasound scan from 18 weeks
when is the more detailed USS performed? what % of malformations does this detect?
from 18 weeks
identified 40% of malformation
what is the miscarriage rate for CVS 12 weeks and amnio 16 weeks and fetal blood >18 weeks (usually following abnormal USS?
- <2%
- <1%
<2.5%
what is the sensitivity of cff-DNA for Trisomy 21? what happens when a positive result is found?
sensitivity of 99% and false positive rate of <0.1%. It is not as reliable for other aneuploidies such as trisomy 18 and 13 but can detect both. PPV for turners is only 40%
• All positive NIPT results need to be confirmed using an invasive diagnostic test such as CVS or amniocentesis.
what was the result of the RAPID study?
UKNSC recommended that NIPT should be implemented into the NHS as part of the Fetal Anomaly Screening Programme.
when is cff-DNA testing offered for NIPT in the uk?
screening risk >1:150
what does the combined test take into account?
maternal age, biochemical markers – free beta human chorionic gonadotropin (bhCG) and pregnancy associated plasma protein-A (PAPP-A) ultrasound measurements – nuchal translucency (NT) and crown rump length
what does the quadruple test take into account?
AFP, β-hCG, uE3+ Inhibin A
screens for T21 only, when NT measurement cannot be obtained
can FISH detect mosaicism?
Yes and No (depends on if 18/X/Y used and if fetus is male)
can FISH detect triploidy
can qf-pcr detect triploidy
both yes
when should a prenatal array result be reported
where there is clinical significance i.e. the finding explains the observed (ultrasound) abnormality, or a clinically actionable result has been obtained.
It is important that the laboratory informs the clinician when a pathogenic CNV is not consistent with the ultrasound findings or when the origin of the CNV may have a contributing effect.
The BSGM has reported a list of incidental findings not to be reported.
when is prenatal array most likely to be used?
to pregnancies with abnormal ultrasound scan (USS) results and a normal QF-PCR result for the common aneuploidies
can arrays be performed on uncultured samples?
yes
if DNA extraction on uncultured material is too poor for a quality microarray result, cultured cells can be grown for repeat DNA extraction
what are the Advantages of Microarrays in prenatal diagnosis
- don’t need to culture cells > faster TAT and no artefacts
- high resolution and pick-up rate - Increases detection of chromosome abnormalities by up to 6% compared to karyotype for USS findings
- carrier status for balanced translocations not revealed
what are the disadvantages of Microarrays in prenatal diagnosis
- difficult to interpret & limited TAT
- VUS - whether to report requires MDT
- incidental findings
- requires high quality and quantity of DNA
- doesn’t detect balanced rearrangements (may have phenotypic consequence if gene disrupted)
- misses low level mosaicism <10%
- doesn’t detect triploidy
- may need to culture for follow-up FISH
- cost for array + follow-up more expensive than karyotype
- balancing coverage and optimal resolution
- counselling for variable penetrance/phenotype
why might a pathogenic CNV be inherited from a normal parent?
- may have SNV in AR gene
- may be imprinted region and only show effect when inherited from certain gender
- incomplete penetrance
