5. QF-PCR Flashcards
What is QF-PCR routinely used to test for?
Common aneuploidies - trisomy 13, 18, 21
Triploidy
Sex chromosome aneuploidy (Turner, Klinefelter)
What is amplified in QF-PCR for aneuploidies?
STRs (highly polymorphic) - usually tri/tetra/penta/hexa nucleotide repeats
What it the advantage of QF-PCR in the prenatal setting?
Reliable even with small quantities of DNA, e.g. from AF, CVS, fetal blood, cultured and uncultured cells
Quick and inexpensive
What sex chromosome markers are used?
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
What does one peak for a marker mean?
Likely homozygous but could be monosomy –> therefore uninformative
What signifies a trisomy?
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)
How do the BPG define a normal QF-PCR result?
At least 2 informative markers per chromosome (others must be uninformative)
What does it mean if a result shows 3 peaks for at least 1 marker?
Trisomy
Meiosis I non-disjunction
Maternal and paternal homologs from one parent, one homolog from other parent
What does it mean if all abnormal markers show 2:1 ratio?
Meiosis II or mitotic non-disjunction
Both sister chromatids from one parent, one homolog from other parent
What risk is associated with all abnormal alleles showing 2:1 ratio?
Mitotic non-disjunction leading to mitotic CPM
What constitutes an inconclusive result?
Otherwise trisomic chromosome where
- Any of the ratios are inconclusive
- Any of the ratios are normal
What can cause inconclusive ratios?
Preferential amplification of the smaller allele - more likely if distance between alleles in increased
Or primer site polymorphism
What does a mosaic QF-PCR result look like?
Extra peaks and skewed allele ratios on a of chromosome-specific group of markers
Difficult to distinguish from MCC
How is an abnormal mosaic cell line generated meiotically?
Trisomy rescue generates normal cell line
How is an abnormal cell line generated mitotically?
Mitotic non-disjunction –> abnormal cell line will only have 2:1 or 1:2 ratios
When should a CVS QF-PCR result be reported with caution?
Mosaic trisomy with 2:1 peaks - suggestive of mitotic non-disjunction which could be due to CPM
Alternatively, could be meiosis II non-disjunction
What is the TAF9 marker?
Invariant (fixed size) present on chrX and chr3 - used to count the number of X chr relative to 3
When should the additional TAF9 marker be used?
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
How many markers are amplified per chromosome? Why?
4 markers per chromosome - eliminates false negative where parents share the same alleles
How many cycles are there in QF-PCR? Why?
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
What is the normal range for allele ratios?
0.8 - 1.4
What is the range of allele ratios for an abnormal biallelic pattern
2:1
1.8-2.4 and 0.45-0.65
Why are tissues tested by QF-PCR?
Pregnancy loss - determine if there’s a genetic cause
TOP - determine if there’s a genetic cause of abnormalities seen on scan
What might cause single inconsistent markers and how do they appear?
- Primer site polymorphism - skewed ratio of 2 alleles
- Somatic microsatellite mutation - 3 unequal peaks
- Submicroscopic duplication - if PSP is excluded
How is PSP excluded?
PCR of single marker with reduced annealing temp - less specificity
But don’t use marker to determine copy number
