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Population Screening > Aneuploidy Screening By QF-PCR > Flashcards

Aneuploidy Screening By QF-PCR Flashcards

1
Q

QF-PCR results are usually confirmed by what method?

A

FISH

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2
Q

What are some of the benefits of QF-PCR in comparison to FISH?

A
  • Smaller amounts of material required
  • Suitable for high throughput
  • More likely to detect regional imbalances
  • Much cheaper cost
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3
Q

How does QF-PCR work?

A
  • Microsatellites = repetitive DNA sequences made up of rpt units 2-10bp in length located across genome
  • Amplify microsatellite regions by PCR using flanking primers
  • PCR products quantified to determine chromosome copy number
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4
Q

How many cycles are used for QF-PCR and why?

A
  • Number of cycles limited to exponential phase so product is proportional to the starting material (currently 28)
  • Additional cycles may deplete reagents and may not get accurate quantitation
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5
Q

How many microsatellite markers are used for aneuploidy by QF-PCR?

A
  • 9 microsatellite markers: 3 each from chromosomes 13, 18 and 21
  • 2 informative markers required for each chromosome
  • 6 additional microsatellite markers can be amplified if needed
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6
Q

What is an informative marker?

A
  • One that has either two or three peaks

- If only one peak then you are unsure if this is due to it being homozygous or if they have a deletion

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7
Q

How is the chromosome copy number calculated?

A
  • Peak height 1 divided by peak height 2 gives the dosage ratio
  • Normal values = 0.8-1.4
  • Peak area may also be used
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8
Q

What are the dosage ratios used for detection of a trisomy by QF-PCR?

A

2 or more markers on the same chromosome should show either:

  • 2:1 ratio (1.8-2.4 or 0.45-0.65)
  • 3 peaks of equal size
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9
Q

How would a triploidy be detected by QF-PCR?

A
  • All markers show either a 2:1 ratio or 1:1:1 (3 peaks)
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10
Q

What are some issues that arise through QF-PCR aneuploidy testing?

A
  • Bloodstained amnios (potential MCC)
  • Inconclusive markers (primer binding site polymorphisms)
  • Submicroscopic dups or other CNVs
  • Somatic microsatellite mutations
  • Mosaicism
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11
Q

What are some ways in which MCC can be spotted via QF-PCR aneuploidy testing?

A
  • Majority of markers show inconclusive ratios

- triallelic results not 1:1:1

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12
Q

A clinically significant regional imbalance may present as what rough QF-PCR results?

A
  • Discrepant results for the most proximal/distal QF-PCR markers
  • Abnormal markers flanked by normal markers
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13
Q

If microsatellite markers give inconclusive ratios via QF-PCR what may be cause and how can it be combatted?

A
  • Most likely due to primer binding site polymorphism (PSP)
  • Reduce the annealing temp
  • If persists could suggest mosaic imbalance at one marker
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14
Q

How can you distinguish between a PSP and a single abnormal marker/submicroscopic dup in QF-PCR analysis?

A

Reducing the annealing temp would combat a PSP but not the SMD

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15
Q

How may mosaicism present through QF-PCR?

A

Inconclusive ratios for one chromosome with or without trisomy

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16
Q

According to QF-PCR BPG, what constitutes a normal result?

A
  • At least two informative markers with a normal pattern are required
  • Acceptable to report on cases with a single informative marker but must add caveat for follow up with final karyotype/FISH
17
Q

According to QF-PCR BPG, what constitutes an abnormal result?

A
  • At least two informative markers consistent with a triallelic genotype
  • Confirmation of sample identity
18
Q

How is MCC interpreted and reported in context of QF-PCR aneuploidy results?

A
  • Low level MCC: can be ignored if all ratios are normal and consistent with foetal genotype
  • High level MCC: large number of inconclusive ratios - await karyotype
19
Q

QF-PCR testing using CVS has some issues - why?

A
  • Discrepant results found between CVS and karyotype when using previous CVS lysis method
  • Now changed to whole enzyme digest method which has helped to reduce this
20
Q

What do you need to be wary of when interpreting results from CVS QF-PCR?

A
  • Possibility of maternal cells affecting karyotyping from cultured CVS
  • Fully biallelic results (no triallelic markers found), which may represent mitotic nondysjunction events, would need caveat on report regarding risk of confined placental mosaicism

Population Screening (9 decks)

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