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Path 19.05 > 20.05.05 QF PCR > Flashcards

20.05.05 QF PCR Flashcards

1
Q

When is QF-PCR performed

A
  • Rapid prenatal screening
  • Testing for trisomy 13, 18, 21 routines
  • Sex chromosome aneuploidy in a subset of referrals indicating a likely sex chromosome abnormality.
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2
Q

QF PCR TAT

A

3 working days

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3
Q

Benefits of QF PCR

A

Cheap, rapid, reliable with small quantities of DNA (often the case in prenatal samples)

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4
Q

Principle of QF PCR

A
  • 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.
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5
Q

What are STRs

A
  • Short tandem repeats

- Highly polymorphic markers

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6
Q

How many markers should be used for each chromosome

A

4 (eliminates false negatives in cases where parents share same allele)

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7
Q

What size are most STR markers

A
  • Usually 4bp repeats (3-6bp also ok)

- Produce fewer stutter peaks than dinucleotide repeats.

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8
Q

What other characteristics must markers have

A

High heterozygosity in population (to avoid uninformative tests)

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9
Q

What sex chromosome markers are there

A
  1. AMEL: non-polymorphic (identical in all patients). Cannot quantify X but can differentiate between X and Y.
  2. DXYS218 (Xp) and X22 (Xq): map to pseudoautosomal regions of X and Y.
  3. 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)
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10
Q

Interpreting a normal QF PCR result

A
  • 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)
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11
Q

Interpreting a trisomy result

A
  • 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
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12
Q

What does a 1:1:1 trisomy indicate

A

-Meiosis I non-dysjunction event

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13
Q

What does a 2:1 or 1:2 trisomy indicate

A
  • Meiosis II or mitotic non-dysjunction event.

- If this pattern is seen in CVS, risk of CPM is increased

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14
Q

What do 2018 best practice guidelines recommend to confirm a Trisomic result

A

-Confirm sample identity either by repeating test or genotype comparison with a maternal sample.

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15
Q

What issues can cause problems with QF PCR interpretation

A
  • 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
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16
Q

Other technical problems that may interfere with interpretation

A
  • 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.
17
Q

How does MCC cause issues

A
  • 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.
18
Q

What 3 groups do BP guidelines use for blood stained prenatals

A
  1. Low level MCC: present but low (majority is fetal) and no inconclusive allele ratios= report. Maternal blood should be tested.
  2. Single fetal genotype present, i.e no MCC= report. Maternal blood should be tested.
  3. Inconclusive allele ratios= fetal genotype should not be interpreted.
19
Q

Ways to get around MCC

A
  • Cultured cells as maternal blood cells would not grow in culture.
  • However if there is maternal tissue this could contaminate culture.
20
Q

How can mosaicism affect QF PCR analysis

A
  • 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.
21
Q

2 ways mosaicism can arise

A
  1. Meiotic generation of abnormal cell line. Mitotic rescue event generating a normal cell line.
  2. 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.
22
Q

What has CVS testing changed to account for confined placental mosaicism

A
  • Used to test and compare 2 villi separately

- Now villi are chopped/mixed and tested together to give a better sample representation

23
Q

What is somatic microsatellite mutations (SMMs)

A
  • 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
24
Q

What are submicroscopic microsatellite duplications (SMDs)

A
  • 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.
25
Q

Partial chromosome imbalance

A
  • 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
26
Q

CNVs on QF-PCR

A
  • 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.
27
Q

Primer binding site polymorphisms in QF PCR

A
  • 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.
28
Q

How can homozygosity impact QF PCR interpretation

A
  • 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.
29
Q

How do twin pregnancies impact QF PCR analysis

A
  • Can be dizygotic or monozygotic.

- Sampling of the same twin twice cannot be excluded.

30
Q

Advantages of QF PCR over FISH Aneuscreen

A
  • 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)
31
Q

What should be included on prenatal QF PCR reports

A
  • 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
32
Q

Reporting of trisomy in prenatal QF PCR reports

A
  • 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.
33
Q

Recommendations when reporting of abnormal results

A

-Abnormal results may indicate a familial chromosome rearrangement. Should be followed by karyotype and testing parents to assess recurrence risk.

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