20.05.05 QF PCR Flashcards
When is QF-PCR performed
- Rapid prenatal screening
- Testing for trisomy 13, 18, 21 routines
- Sex chromosome aneuploidy in a subset of referrals indicating a likely sex chromosome abnormality.
QF PCR TAT
3 working days
Benefits of QF PCR
Cheap, rapid, reliable with small quantities of DNA (often the case in prenatal samples)
Principle of QF PCR
- Determines copy number of chromosome-specific sequences by amplification of STRs on chromosomes of interest.
- Low number of PCR cycles (~24), stopped during exponential phase as it needs to be quantitative.
What are STRs
- Short tandem repeats
- Highly polymorphic markers
How many markers should be used for each chromosome
4 (eliminates false negatives in cases where parents share same allele)
What size are most STR markers
- Usually 4bp repeats (3-6bp also ok)
- Produce fewer stutter peaks than dinucleotide repeats.
What other characteristics must markers have
High heterozygosity in population (to avoid uninformative tests)
What sex chromosome markers are there
- AMEL: non-polymorphic (identical in all patients). Cannot quantify X but can differentiate between X and Y.
- DXYS218 (Xp) and X22 (Xq): map to pseudoautosomal regions of X and Y.
- HPRT: polymorphic X chromosome marker.
- SRY: non-polymorphic marker to confirm male fetus
- TAF9: Used to compare X chromosomes to chromosome 3. Amplifies a similar sequence on 3p24.2 and Xq21 (differs 2 bp in length) . Female= 1:1 (2 Xs, 2 3s), male= 2:1 (2 3s 1 X)
Interpreting a normal QF PCR result
- Normal 1:1 ratio is consistent with a normal heterozygote biallelic pattern.
- Ratio should not exceed 0.8-1.4 (unless they are separated by more than 24bp, where smaller allele will preferentially amplify)
- One peak could indicate monosomy or homozygous for the marker.
- Best practice: need at least 2 informative markers on a single chromosome with a normal biallelic pattern to interpret as normal
- If only 1 informative marker: need a caveat on report (should be confirmed by karyotype or FISH)
Interpreting a trisomy result
- Three alleles in the ratio 1:1:1 is consistent with triallelic pattern
- 2:1 or 1:2 ratio suggests abnormal biallelic pattern (2 of the 3 alleles have the same size microsatellite).
- If all three alleles have the same size marker= 1 peak. i.e. uninformative.
- Best practice: 2 informative abnormal markers required for trisomy. Can’t report if normal ratio is seen in an otherwise trisomic chromosome
What does a 1:1:1 trisomy indicate
-Meiosis I non-dysjunction event
What does a 2:1 or 1:2 trisomy indicate
- Meiosis II or mitotic non-dysjunction event.
- If this pattern is seen in CVS, risk of CPM is increased
What do 2018 best practice guidelines recommend to confirm a Trisomic result
-Confirm sample identity either by repeating test or genotype comparison with a maternal sample.
What issues can cause problems with QF PCR interpretation
- MCC
- Mosaicism
Normal and abnormal allele patterns for a single chromosome, due to:
- Somatic microsatellite mutations
- Submicroscopic microsatellite duplications (SMD)
- Partial chromosome imbalance
- CNVs
- Primer site polymorphisms
- Homozygosity
- Twin pregnancies
Other technical problems that may interfere with interpretation
- Stutter peaks. Usually 1bp shorter than true allele. Due to Taq polymerase slippage.
- Spikes. Similar height in all colours (analyser artefact). As long as it doesn’t overlap with an allele peak can be ignored. If overlaps then re-inject.
- Bleed through. Small peaks in the same position as high alleles in a different dye.
How does MCC cause issues
- QF PCR can detect ~10% MCC
- Can affect amnios (blood-stained) and CVS (maternal decidua)
- Mixture of two related genotypes to give three peaks if both genotypes are normal
- If MCC suspected then maternal blood must be run to compare alleles.
What 3 groups do BP guidelines use for blood stained prenatals
- Low level MCC: present but low (majority is fetal) and no inconclusive allele ratios= report. Maternal blood should be tested.
- Single fetal genotype present, i.e no MCC= report. Maternal blood should be tested.
- Inconclusive allele ratios= fetal genotype should not be interpreted.
Ways to get around MCC
- Cultured cells as maternal blood cells would not grow in culture.
- However if there is maternal tissue this could contaminate culture.
How can mosaicism affect QF PCR analysis
- Mosaicism for trisomy can be distinguished by extra peaks or skewed ratios on a chromosome specific group of markers.
- Subtle or consistent skewing and extra peaks should be investigated
- Diploid/triploid mosaic is hard to distinguish from MCC.
2 ways mosaicism can arise
- Meiotic generation of abnormal cell line. Mitotic rescue event generating a normal cell line.
- Mitotic generation of an abnormal cell line in a normal conception. Mitotic non-disjunction event generates the abnormal cell line. In CVS- could be confined placental mosaicism.
What has CVS testing changed to account for confined placental mosaicism
- Used to test and compare 2 villi separately
- Now villi are chopped/mixed and tested together to give a better sample representation
What is somatic microsatellite mutations (SMMs)
- Mosaicism for a de novo allele. 3 alleles with unequal peak heights. Two peaks with lower area represent one allele that has undergone a somatic mutation.
- Often involves an increase or decrease of 1-2 repeats.
- If SMM is seen in a single marker, does not need to be reported.
- Could be investigated further by looking at culture cells
What are submicroscopic microsatellite duplications (SMDs)
- Where a single marker is consistent with trisomy (1:1:1 or 1:2 or 2:1). Other markers are normal
- Distinguished by looking at parental genotype.
- Does not need to be reported
- In rare cases may reflect a real imbalance, particularly at distal or proximal markers.
- Further tests with extra flanking markers recommended. Should be put in report.
Partial chromosome imbalance
- Normal and abnormal result on one chromosome
- Two or more consecutive markers (most distal/proximal) showing the same result can be reported.
- Follow up required if just a single discrepant distal/proximal marker. Could be a polymorphism or partial imbalance. Test parental bloods or karyotype
CNVs on QF-PCR
- Abnormal markers flanked by normal
- If previously reported to represent a CNV inherited from a normal parent, not required to be reported.
- Markers not previously reported as representing an inherited CNV should be reported.
- Karyotype, arrayCGH, test parents.
Primer binding site polymorphisms in QF PCR
- Where a polymorphism occurs on the DNA where a primer anneals. Causes reduced primer annealing due to mismatch, so skewed alleles.
- Re-testing using a lower PCR annealing temp should correlate with greater amplification of PSP allele and a change in allele ratio.
How can homozygosity impact QF PCR interpretation
- If all markers for a single chromosome are uninformative, extra markers need to be used.
- E.g. a female fetus (in Turner-like referrals), with uninformative sex chromosome markers, monosomy X must be confirmed. E.g. using TAF9
- If enough sex chromosome markers are used then FISH not required.
How do twin pregnancies impact QF PCR analysis
- Can be dizygotic or monozygotic.
- Sampling of the same twin twice cannot be excluded.
Advantages of QF PCR over FISH Aneuscreen
- Less sample volume required
- Greater range of gestation (12-34 weeks compared to 15-21 wks)
- Less intense labor and high throughput possible
- Cheaper
- Can detect MCC
- Can be used to infer UPD
- Can detect certain unbalanced rearrangements (distal 13q, 18q, 21q)
What should be included on prenatal QF PCR reports
- ASsumption that fetal material is tested
- Limitations that mosaicism and small segment imbalances may not be detected
- “consistent with trisomy 13/18/21” as only specific sequences from each chromosome are tested
- “Consistent with” e.g. downs syndrome
Reporting of trisomy in prenatal QF PCR reports
- Trimosy in CVS samples with no evidence of meiotic non-disjunction event (i.e. 3 peaks) should be reported cautiously as could reflect placental mosaicism.
- Recommend waiting for karyotype result, especially in absence of ultrasound abnormalities.
Recommendations when reporting of abnormal results
-Abnormal results may indicate a familial chromosome rearrangement. Should be followed by karyotype and testing parents to assess recurrence risk.
