QF-PCR

Question 1

What are the advantages of QF-PCR over FISH?

Answer 1

High throughput
Cheaper
Smaller amount of starting material
More likely to detect regional imbalance

Question 2

What is the basic concept of QF-PCR?

Answer 2

• It utilises repeat sequences in our DNA e.g. tetranucleotide repeats which are polymorphic between individuals.
• Fluorescently labelled primers amplify these repeats during PCR (fluoro tag is specific to particular repeat)
• Capillary gel electrophoresis is performed to quantify how much of each product is there and of what size.
• The ratio of product sizes are compared to indicate chromosome copy number e.g. 3 peaks = trisomy, 2 peaks with skewed ratio = trisomy, 2 peaks of same size = normal.

Question 3

How many cycles of PCR are used in QF-PCR?

Answer 3

PCR must stay in the exponential phase - the amount of product is directly proportional to the amount of target sequence present in the initial template.
Quantification is unreliable once reagents begin to use up.
BPG recommend 24-26 cycles, but more can be used if it is validated using known trisomies etc.

Question 4

How does the polymerase know when to stop?

Answer 4

• for the first round of replication it doesn’t- the end of the extension cycle will determine when the polymerase drops off so it could extend further than you want…
• but for subsequent rounds he extension of the fragment is DNA has an end point of where the primer initially annealed, there are no further nucleotides beyond this point.
• although some fragments in the DNA will be these longer start fragments they are in such a small number compared to all the other fragments that they are undetectable by QF-PCR.

Question 5

What is preferential amplification?

Answer 5

Where 2 alleles differ in size and the smaller size allele is favoured during the PCR reaction.
Reaction is optimised to try and prevent this however alleles that differ in size by >24bp have a higher ‘normal’ ratio cut off of 1.5.

Question 6

What is a primer site polymorphism? What can be done to confirm it?

Answer 6

A polymorphism that sits under the binding site of a primer. This reduces the efficiency of the PCR and may cause the allele to ‘drop out’.
This can be investigated by running the PCR at a lower annealing temperature to make the annealing less specific/less fussy. The annealing temperature is lowered to 52’C.

Question 7

What are the acceptable ranges for QF-PCR?

Answer 7

Normal dosage - 0.8-1.4
Normal dosage but >24bp apart - up to 1.5
Trisomy 2:1 - 1.8-2.4 or 0.45 - 0.65
Trisomy 1:1:1 - 3 equal peak areas

Question 8

What qualifies as an informative marker?

Answer 8

A marker with 2 or more peaks.

A single peak could be either homozygous for that marker or could represent a deletion.

Question 9

How many markers are used for our QF-PCR assay? What mixes are available?

Answer 9

Trisomy mix: 13, 18, 21 and AMEL.
XY mix: X and Y specific markers, pseudoautosomal markers, AMEL and TAF9 which sits on the X and chr3.
These are used as standard.
We also have multiplex kits which can be used to increase the number of markers on a particular chromosome e.g. if standard analysis is non-informative.
Single markers can also be run e.g. if a PSP is suspected.

Question 10

How can you tell the difference between a homozygous X female and a Turners female?

Answer 10

Using the TAF9 allele. This is present on the X and on the 3, therefore the ratio of these 2 alleles will ‘count’ the number of X’s in the sample.

Question 11

What is the sensitivity of QF-PCR?

Answer 11

Stated down to 20%. In practice my depend on abnormality e.g. full blown or mosaic.
A triallelic trisomy (e.g a 3rd peak) may be detectable at quite a low level.

Question 12

How would triploidy be detected?

Answer 12

All informative markers either 2:1 or 1:1:1.

Question 13

What is the minimum for an abnormal trisomy?

Answer 13

At least 2 informative markers consistent with trisomy.
All other markers uninformative.
Cannot class something as abnormal based on a single marker.

Question 14

What does a trisomy with at least one triallelic marker tell you?

Answer 14

That the trisomy has occurred due to a meiotic non-disjunction.

Question 15

Why would a trisomy without any triallelic markers be of concern? What sample type in particular does this relate to?

Answer 15

It suggests that the trisomy is of mitotic origin.
If the sample type is CVS then the concern here is for CPM. The trisomy may not be found in the fetus.
Particular caution should be taken in the absence of any scan findings.

Question 16

What findings might suggest MCC?

Answer 16

A ratio which is skewed into the inconclusive range
A small additional peak that can’t be attributable to bleed through
Triallelic peaks of varying heights if the contamination is significant.

Question 17

How can you check whether skewing is due to MCC?

Answer 17

A + B = C
The 2 smallest peaks e.g. the fetal peak and the contaminating maternal peak are added together (A + B) and they should be roughly equal to the larger peak C (as this peak represents the allele that fetus and mum share). When the new ratio is calculated it should be normal.

Question 18

What would you think if the A + B = C calculation for MCC is falling into the inconclusive/abnormal range for a particular set of markers?
What could you do about this?

Answer 18

There could be an underlying trisomy which is being masked by the MCC.
We could use FISH to check this out, especially if fetus is male.

Question 19

What must happen before an abnormal prenatal is reported?

Answer 19

The identity of the sample must be confirmed. Either by a repeat or by using a maternal sample (BPG).

The only time this doesn’t need to be done is if it is to confirm a positive NIPT.

Question 20

What would you think if a single marker was skewed?

Answer 20

If it was proximal or distal I would be concerned that this may be clinically significant. Further testing should be carried out. This could for example represent a rearrangement.

If it is central and flanked by normal marker it could be a CNV. A list of reported CNV’s can be obtained via email address in BPG. We could run parents and see if they have it also.

Could be a PSP - rerun marker at lower temp to check.

Question 21

Can you report a sample with MCC?

Answer 21

Yes, if it doesn’t skew the alleles into the inconclusive or abnormal ranges.

Question 22

What must we be careful of in an abnormal QF-PCR report?

Answer 22

State that markers are triallelic, we mustn’t say it is full trisomy as we have only tested certain markers.
Be careful to say ‘suggestive of’ or ‘consistent with’ trisomy XX.
Likewise, we must be careful to say ‘fetus is predicted to be affected with XX’.

Question 23

What is the minimum for a normal QF-PCR report?

Answer 23

2 informative markers for each chromosome with all other markers non-informative.

Question 24

What is a Somatic microsatellite mutation (SMM)?

Answer 24

Can cause a third smaller peak. Total of 2 smaller peaks should equal the bigger peak (give 1:1 ratio).
Error causes different size allele at single locus. Usually mosaic.
Associated with CVS samples. However, now we digest CVS to enrich the mesoderm cells which reduces chance of seeing SMM.
Historically could repeat on a different frond to confirm SMM.

Question 25

What is a submicroscopic duplication and deletion?

Answer 25

Duplication:
Appears as 2:1 skew or 1:1:1
Firstly would want to exclude PSP - rerun at lower annealing temp.
If still there could represent CNV or could be significant
Test parents to see if inherited?
?array if parents normal

Deletion:
In a normal sample - indistinguishable from homozygous marker
In a trisomy - could present as single 1:1 marker.
As above, test parents to see if it is inherited (- actually would show as homo marker so no point looking at parents)
?array to investigate further.

Question 26

What is the TAT for prenatal QF-PCR?

Answer 26

3 days.