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5: Prenatal diagnosis > 5. QF-PCR > Flashcards

5. QF-PCR Flashcards

1
Q

What is QF-PCR routinely used to test for?

A

Common aneuploidies - trisomy 13, 18, 21
Triploidy
Sex chromosome aneuploidy (Turner, Klinefelter)

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2
Q

What is amplified in QF-PCR for aneuploidies?

A

STRs (highly polymorphic) - usually tri/tetra/penta/hexa nucleotide repeats

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3
Q

What it the advantage of QF-PCR in the prenatal setting?

A

Reliable even with small quantities of DNA, e.g. from AF, CVS, fetal blood, cultured and uncultured cells

Quick and inexpensive

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4
Q

What sex chromosome markers are used?

A

AMEL located in PAR - differentiates between X & Y due to 4bp difference

SRY - to confirm male fetus

TAF9 - X chromosome counting marker

Multiple STR markers

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5
Q

What does one peak for a marker mean?

A

Likely homozygous but could be monosomy –> therefore uninformative

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6
Q

What signifies a trisomy?

A

3 alleles in 1:1:1 ratio (marker different length in all 3 alleles)

2:1 ratio - suggest abnormal bilallelic pattern (2/3 alleles the same size)

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7
Q

How do the BPG define a normal QF-PCR result?

A

At least 2 informative markers per chromosome (others must be uninformative)

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8
Q

What does it mean if a result shows 3 peaks for at least 1 marker?

A

Trisomy

Meiosis I non-disjunction

Maternal and paternal homologs from one parent, one homolog from other parent

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9
Q

What does it mean if all abnormal markers show 2:1 ratio?

A

Meiosis II or mitotic non-disjunction

Both sister chromatids from one parent, one homolog from other parent

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10
Q

What risk is associated with all abnormal alleles showing 2:1 ratio?

A

Mitotic non-disjunction leading to mitotic CPM

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11
Q

What constitutes an inconclusive result?

A

Otherwise trisomic chromosome where

  1. Any of the ratios are inconclusive
  2. Any of the ratios are normal
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12
Q

What can cause inconclusive ratios?

A

Preferential amplification of the smaller allele - more likely if distance between alleles in increased

Or primer site polymorphism

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13
Q

What does a mosaic QF-PCR result look like?

A

Extra peaks and skewed allele ratios on a of chromosome-specific group of markers

Difficult to distinguish from MCC

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14
Q

How is an abnormal mosaic cell line generated meiotically?

A

Trisomy rescue generates normal cell line

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15
Q

How is an abnormal cell line generated mitotically?

A

Mitotic non-disjunction –> abnormal cell line will only have 2:1 or 1:2 ratios

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16
Q

When should a CVS QF-PCR result be reported with caution?

A

Mosaic trisomy with 2:1 peaks - suggestive of mitotic non-disjunction which could be due to CPM

Alternatively, could be meiosis II non-disjunction

17
Q

What is the TAF9 marker?

A

Invariant (fixed size) present on chrX and chr3 - used to count the number of X chr relative to 3

18
Q

When should the additional TAF9 marker be used?

A

Turner’s like referral, female fetus with uninformative sex markers

Compares no. of X chr to no. of chr 3

Confirms whether original result is due to homozygosity or monosomy

19
Q

How many markers are amplified per chromosome? Why?

A

4 markers per chromosome - eliminates false negative where parents share the same alleles

20
Q

How many cycles are there in QF-PCR? Why?

A

24 (26 if low DNA input)

PCR is stopped while in the exponential phase when the amount of product is doubling with every cycle & is proportional to the amount of starting material

21
Q

What is the normal range for allele ratios?

A

0.8 - 1.4

22
Q

What is the range of allele ratios for an abnormal biallelic pattern

A

2:1

1.8-2.4 and 0.45-0.65

23
Q

Why are tissues tested by QF-PCR?

A

Pregnancy loss - determine if there’s a genetic cause

TOP - determine if there’s a genetic cause of abnormalities seen on scan

24
Q

What might cause single inconsistent markers and how do they appear?

A
  1. Primer site polymorphism - skewed ratio of 2 alleles
  2. Somatic microsatellite mutation - 3 unequal peaks
  3. Submicroscopic duplication - if PSP is excluded
25
Q

How is PSP excluded?

A

PCR of single marker with reduced annealing temp - less specificity

But don’t use marker to determine copy number

5: Prenatal diagnosis (11 decks)

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