18.05.05 QF-PCR Flashcards

1
Q

What is QF-PCR?

A

QF-PCR is a molecular method of determining the copy number of chromosome specific sequences by amplification of STRs on chromosomes of interest.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are STRs? How are they utilitsed in QF-PCR?

A

STRs (short tandem repeats or microsatellites) are a pattern of two or more nucleotides that are repeated directly adjacent to each other.

Repeats can vary in length from 2 bp to 6 bp per repeat.

STRs are amplified by PCR using fluorescently labelled primers and separated by capillary electrophoresis. Repeat size and quantification.

STRs known highly polymorphic markers; a patient is therefore likely to have different numbers of repeat units on each allele.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the stages of QF-PCR set-up?

A
  1. DNA is extracted from the AF/CVS/blood/tissue
  2. Extracted DNA is added to the primer multiplex and PCR amplified over 24 cycles. This can be increased to 26 cycles in cases with small amounts of DNA. A
    .
    PCR has 2 phases
    - Exponential phase when all rx constituents are in abundance and amount of amplified product is directly proportional to original amount of starting material
    - Plateau phase near the end of cycling, reagents are in short supply and some fragments may amplify and some may not. The results will be quantitative if PCR is stopped while in exponential phase i.e. after 26 cycles).
  3. PCR products analysed on a capillary based genetic analyser alongside a size marker.
    If there is a chance of overlap between the allele sizes of different markers these are labelled in different colours.
  4. QF-PCR multiplex assays permit the detection of major numerical chromosome disorders within 1-2 days. Cheap to perform.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How many makers should be used per chromosome?

A

4

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the composition of the majority of markers and PCR primers used in QF-PCR?

A

STRs are selected that exhibit high heterogeneity so that the copy number can be easily determined.

Majority of markers are usually 4bp repeats (tetra) but can also use tri/penta/hexanucleotide repeats. These have fewer stutter peaks (see below) than dinucleotide markers.

Dinucleotide repeat markers are acceptable if few suitable markers within the region of interest.

Markers should have high heterozygosity within the population to avoid uninformative tests.

Primers used must be >22bp in length and avoid homology to repetitive DNA and SNP’s.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is the AMEL marker used for?

A

AMEL is a non-polymorphic marker with identical peaks in all patients, with a 4bp difference between the X and Y alleles. This cannot be used to quantify the number of X chromosomes in a female fetus, but can detect XXY,XYY

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is the TAF9 marker used for?

A

TAF9 is used to compare the number of X chromosomes relative to the number of chromosome 3s.

TAF9 primers amplify similar sequences present on on 3p24.2 and Xq21, which produce products 2bp different in length.

A normal female would be expected to have a 1:1 ratio (2 Xs and 2 3s), where as a normal male would be expected to have a 2:1 ratio of 3s:Xs (2 chromosome threes and 1 X).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the range for normal allele ratios?

A

Normal range for allele ratios should not exceed 0.8-1.4.

For alleles separated by more than 24bp, an allele ratio of 1.5 is acceptable as the smaller allele will be preferentially amplified.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How many informative makers are required for a normal interpretation of that chromosome?

A

Best Practice: Need at least 2 informative markers on a single chromosome with a normal biallelic pattern to interpret as normal (all other markers for that chromosome can be uninformative).

Can report a normal pattern with 1 marker but this must be stressed in the report.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What allele ratio is consistent with an abnormal triallelic pattern? What is the acceptable range?

A

1: 1:1

0. 8-1.4

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What allele ratio is consistent with an abnormal biallelic pattern?

A

2: 1/1:2

0. 45 &0.65 and 1.8 &2.4.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How many informative makers are required for an abnormal interpretation of that chromosome?

A

Best Practice: Two informative abnormal markers required for trisomy (all other makers for that chromosome can be uninformative).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What can be interpreted about the mechanism of trisomy formation from the QF-PCR result?

A

The presence of 3 peaks for 1 or more marker is consistent with a meiosis I non-dysjunction event.

The absence of such a result (ie all abnormal markers have two allele with a 1:2/2:1 ratio) indicates a possible meiosis II or mitotic non-dysjunction event.
Where this pattern is seen in CVS the risk of CPM is increased (see Mosaicism).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What ratios indicate an uninformative result?

A

Ratios fall between normal and abnormal, i.e. 0.65-0.8 and 1.4/1.5-

Best Practice: Cannot report a sample as trisomic if any ratios are inconclusive or if any normal ratios for an otherwise trisomic chromosome are obtained.

Inconclusive ratios may be the result of preferential amplification of the smaller allele. This is more likely to occur if the distance between the alleles is increased, and may be more commonly observed for some markers than others.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How and why are trisomic results confirmed?

A

Confirm sample identity

In accordance with the current (2012) best practice guidelines, samples that give abnormal results should be confirmed before the result is issued (e.g. test maternal sample or second test on the original sample- according to individual lab policy).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What issues can make interpretation of QF-PCR difficult?

A

MCC

mosaicism

17
Q

How can MCC be indicated from the results of QF-PCR?

A

May show skewed allele pattern for all chromosomes.

Mixture of two related genotypes therefore will be three peaks if both genotypes are normal.

The two smaller peaks height should add up to the larger peak height i.e. skewed bialllelic results & triallelic results will arise where the sum of the peak areas of the mat specific and foetal specific alleles approximate to the area of the shared mat-foetal allele (a = b+c).

18
Q

What should be done is MCC is suspected? What are the three groups of MCC?

A

Maternal blood must be run to compare alleles.

  1. Low level MCC – if a low level of maternal genotype present but majority is foetal & has no inconclusive allele ratios: report.
  2. Single foetal genotype present therefore no MCC: report.
  3. Inconclusive allele ratios: the foetal genotype should not be interpreted.

In cases 1 and 2, the foetal genotype can be determined by analysis of a maternal DNA.

19
Q

According to BPG, what should happen if a QF-PCR result cannot be issued due to MCC?

A

In cases where a QF-PCR result is not available due to MCC of the sample, karyotype analysis of the cultured cells should be carried out. Note that maternal blood cells would not be expected to grow in the culture, but if the contamination results from maternal tissue then this may grow; care is required when undertaking analysis in these cases.

20
Q

How can mosaicism be indicated from the results of QF-PCR?

A

Mosaicism for trisomy and normal cell lines distinguished by extra peaks & skewed allele ratios on a chromosome specific group of markers.

Results may be subtle – skewed allele ratios representing the mosaic chromosome may be in the normal or abnormal range.

Subtle or consistent skewing or small and/or extra peaks should be investigated further.

Diploid/triploid mosaic: all markers affected. Difficult to distinguish from MCC. The two main alleles in a triallelic marker should give 1:1 ratio

21
Q

How may mosaicism arise?

A

Meiotic generation of abnormal cell line: Mitotic rescue event generating a normal cell line may result in mosaicism. Detectable when the trisomy cell line contributes at least 15%.

Mitotic generation of an abnormal cell line in a normal conception. Mitotic non-disjunction event generates the abnormal cell line.

Abnormal cell will only have a 2:1/1:2 biallelic ratio.
Detectable when abnormal cell line contributes at least 20%.

22
Q

Occasionally both normal and abnormal allele patterns can be obtained for a single chromosome. What may thie represent?

A
  1. SMM
  2. SMD
  3. Partial chromosome imbalance
  4. CNV
  5. Primer binding site polymorphism
23
Q

What is a SMM?

A

Somatic Microsatellite Mutations (SMMs)

Mosaicism for a de novo allele.
3 alleles present with unequal peak heights. T
he two peaks with lower peak area represent one allele which underwent a somatic mutation. The foetus is therefore mosaic for this one marker.

Mutation usually involves an increase or decrease of 1-2 repeats.

All the cells should contain one copy of either the original or mutated allele – the sum of the area/heights for these alleles should give a ratio in the normal range when compared with the unaffected allele.

SMM’s may be identified by testing a different cell population e.g. cultured cells.

24
Q

What is a SMD?

A

Characterised by a single marker consistent with trisomy (either 1:1:1 or 2:1 or 1:2). All other markers are normal. i.e. 2 copies of marker are present in tandem.

Distinguished by looking at parental genotypes as they usually have the same pattern for that marker. Does not need to be reported.

In rare cases the finding may reflect a real imbalance particularly if the discrepant abnormal/normal results are obtained for the most distal or proximal markers.

Findings should be detailed in the report and further tests with extra flanking markers recommended

25
Q

How may a partial chromosome imbalance manifest in QF-PCR?

A

Distinguished by normal and abnormal results on one chromosome.

Two or more consecutive markers (most distal/proximal) showing the same result can be reported.

Single discrepant distal/proximal markers may indicate partial chromosome imbalance or a polymorphism. Follow up required.

?PSP, SMD or SMM: run parental bloods, karyotype.

26
Q

How may a CNV manifest in QF-PCR? What follow up is required?

A

Abnormal markers flanked by normal may represent a CNV.

If marker has previously been reported to represent a CNV inherited from a normal parent, no requirement to report the abnormal marker.

For markers not previously reported as representing an inherited CNV it is recommended that the abnormal & normal results are detailed in the report.

Run parental samples to provide info on significance of the result.

Karyotype or array CGH (if referred for abnormal scan) may give further information/refinement.

27
Q

How may a PBS poly manifest in QF-PCR? What follow up is required?

A

A polymorphism on the template DNA strand where a PCR primer anneals.

Results in a reduced primer annealing due to sequence mismatch at primer binding site.

Primer binding is less efficient & results in skewed alleles, ratios lie in the range 1.4-1.8 & 0.65-0.8.

Re-testing using a lower PCR annealing temperature should correlate with greater amplification of the PSP allele & a change in allele ratio

28
Q

What causes stutter peaks?

A

Peaks detected primarily 1 repeat shorter than the true allele (can also be 1 repeat larger).

Due to Taq polymerase slippage during PCR amplification of repeated sequences by one repeat unit.

29
Q

What are the advantages of using QF-PCR over FISH/Anuescreen/?

A

Less sample volume required (0.5-1ml amniotic fluid for PCR vs 3-4ml for FISH)

Greater range of gestation (12-34wks PCR vs 15-21wks FISH)

Less intense labour and higher through-put possible

Cheaper cost

Can detect maternal cell contamination in all samples rather than just male conceptions

Can be used to infer UPD

Can detect certain unbalanced rearrangements (distal 13q, 18q, 21q)

30
Q

When should trisomy results be reported with caution?

A

Trisomy results in CVS samples with no evidence of a meiotic non-disjunction event (ie 3 peaks) should be reported more cautiously, since this could reflect placental mosaicism