QF-PCR

Question 1

Outline the QF-PCR service in Nottingham.

Answer 1

• Rapid aneuploidy testing by QF-PCR for chromosomes 13, 18, 21.
• Performed on all amniotic fluid samples (not yet CVS).
• Service since November 2006.
• Total 2270 amnios to date.
• Average report time = 1.6 days.
• All positive results confirmed by FISH.
• 0.6% failures (attempted by FISH).
• 2.8% unreportable due to MCC.
• Follow up karyotyping on all analysable samples.

Question 2

Outline the development and validation of prenatal QF-PCR in Nottingham.

Answer 2

• Tested >200 frozen amnio samples blind - results previously reported by FISH and karyotype.
• 100% concordance with cytogenetic results.
• May 2006-October 2006 - dual FISH and QF-PCR service.
• 100% concordance with cytogenetic results.
• November 2006 - full service.

Question 3

Compare FISH and QF-PCR.

Answer 3

FISH:

• Amnio and CVS
• Result 24-48 hrs
• Specificity = 100% (HTA)
• Sensitivity = 99% (HTA)
• Failure rate = 1-3%
• Larger amounts of material required
• Suitable for a small throughput
• Equipment set up and maintenance = minimal
• May miss regional imbalances
• Cost = £55 per sample

QF-PCR:

• Amnio and CVS
• Result 24-48 hrs
• Specificity = 100% (HTA)
• Sensitivity = 97% (HTA)
• Failure rate = 1.4%
• Smaller amounts of material required
• Suitable for high throughput
• Large initial cost and high maintenance of F.Analyser
• More likely to detect regional imbalances
• Cost = £5 per sample

Question 4

How is DNA extracted from amnios in Nottingham?

Answer 4

• Amnio DNA extraction using the Qiagen EX1 system.
• Allows automated purification of genomic DNA from amniocytes.
• Use 1.5ml amniotic fluid but can be scaled up or down (smallest = 0.5ml).
• Pellet cells before extraction.
• Wash with PBS.
• Water wash for bloodstained amnios.
• Can process 1-6 samples.
• Uses the Qiagen EZ1 tissue kit.
• Elute in 100ul.

Question 5

Describe the basis of the QF-PCR technique.

Answer 5

• Fluorescent PCR used to amplify microsatellite markers on chromosomes 13, 18 and 21.
• Microsatellites - repetitive DNA sequences made up of repeat units 2-10bp in size.
• Located throughout the genome.
• Repeat sizes vary in human population.
• Amplify microsatellite regions by PCR using flanking primers.
• PCR products quantitated to determine chromosome copy number.
• Quantitative PCR is so called because it is quantitative. The reason it remains quantitative is because we limit the number of cycles in the PCR to the exponential phase of the PCR and so the total amount of PCR product is proportional to the starting material. After the exponential phase additional cycles may deplete reagents and therefor you may not get accurate quantitation.

Question 6

Describe the specific QF-PCR method that is currently used in prenatal testing.

Answer 6

• Modified method published by Kathy Mann et al. in 2004.

1) . MULTIPLEX A2 - Single tube QF-PCR
- Amplify 9 microsatellite markers - 3 from each chr 13, 18 and 21. Different size ranges and fluorescent labelled with either blue, green, or black.
- Show 96-74% heterozygosity in general population.
- 2 informative markers required for each chromosome.

2). MULTIPLEX B - 6 additional microsatellite markers amplified in single tube.

Question 7

How does the QF-PCR determine the chromosome copy number?

Answer 7

• The QF-PCR determines the chromosome copy number by comparing the heights of the peaks.
• Peak height 1 / Peak height 2 = Dosage Ratio
• Normal vaulues = 0.8 - 1.4 (2 alleles)
• Peak area may also be used.

Question 8

What QF-PCR dosage ratio will you get in trisomic cases?

Answer 8

• You either get 3 peaks of approximately equal size, or 2 peaks showing a 2:1 dosage ratio.
• 2:1 dosage ratio will fall in range 1.8-2.4 or 0.45-0.65

Question 9

What QF-PCR dosage ratio will you get in triploidy cases?

Answer 9

• Shown by all markers.

- Either get 2:1 ratio or triallelic 1:1:1.

Question 10

What problems may you encounter with QF-PCR in prenatal screening?

Answer 10

• Bloodstained amnios.
• Inconclusive markers - primer binding site polymorphism.
• Submicroscopic duplications / other CNVs/
• Somatic microsatellite mutations.
• Mosaicism.

Question 11

Describe what we do with regards to bloodstained amnios and MCC in prenatal QF-PCR testing.

Answer 11

• All bloodstained amnios are tested.
• 22% of amnios showed some degree of blood staining (10-100% of amnio pellet).
• 8% showed heavy blood staining (>90% of pellet size).
• Possible that majority genotype may be maternal. Use 2 different approaches to determine this.
  1) . Test sample using amelogenin PCR which distinguishes between X and Y chromosomes and only report is male (if no maternal sample avaliable).
  2) . If mat sample available then can compare results with maternal genotype. Will process the mat blood if the amnio is visibly bloodstained.
• If two genotypes are present then there is a characteristic pattern involving extra alleles / inconclusive dosage ratios. Majority of markers show inconclusive ratios or triallelic results not at 1:1:1.
• Not all blood stained amnios show MCC - foetal blood.
• 2nd genotype present with no blood staining may be due to a maternal tissue plug or a vanishing twin.

Question 12

Describe what effect primer site polymorphisms can have in prenatal QF-PCR.

Answer 12

• Some markers may give inconclusive ratios (allele ratios 0.65-0.80 or 1.4-1.8).
• Most likely due to PSP.
• Reduce the annealing temperature.
• If persists could suggest mosaic imbalance at 1 marker.

Question 13

What may cause single abnormal markers in QF-PCR results?

Answer 13

1) . Primer Site Polymorphisms (PSP).
- Some markers may give inconclusive ratios (allele ratios 0.65-0.80 or 1.4-1.8).
- Most likely due to PSP.
- Reduce the annealing temperature.
- If persists could suggest mosaic imbalance at 1 marker.
2) . Submicroscopic Duplications (SMD).
- Discrepant results obtained for most proximal/distal markers may represent clinically significant regional imbalance.

• If the abnormal markers flanked by normal markers more likely to represent a CNV.
  E.g. Single triallelic or 2:1/1:2 diallelic marker. All other markers normal.
• Single marker assay at lower annealing temp rules out primer site polymorphism.
• Very small regional copy number variant may not be detected cytogenetically.
• Request prenatal blood samples - if present in one parent = polymorphic variant - if de novo = clinical significance?
• Rare = 1/790 amnio samples.
  3) . Somatic microsatellite mutations (SMM).
• A single marker showing triallelic results not at 1:1:1.
• Caused by defective proofreading during DNA replication in early embryogenesis/
• SMM reported in about 0.1% of amnios and 4.2% of CVS samples.
• Can identify SMMs by testing different cell populations e.g. cultured cells.

Question 14

Describe what effect submicroscopic duplications may have on prenatal QF-PCR results.

Answer 14

• Discrepant results obtained for most proximal/distal markers may represent clinically significant regional imbalance.
• If the abnormal markers flanked by normal markers more likely to represent a CNV.
  E.g. Single triallelic or 2:1/1:2 diallelic marker. All other markers normal.
• Single marker assay at lower annealing temp rules out primer site polymorphism.
• Very small regional copy number variant may not be detected cytogenetically.
• Request prenatal blood samples - if present in one parent = polymorphic variant - if de novo = clinical significance?
• Rare = 1/790 amnio samples.

Question 15

Describe what effect Somatic microsatellite mutations (SMMs) may have on prenatal QF-PCR results.

Answer 15

Somatic microsatellite mutations (SMM).

• A single marker showing triallelic results not at 1:1:1.
• Caused by defective proofreading during DNA replication in early embryogenesis/
• SMM reported in about 0.1% of amnios and 4.2% of CVS samples.
• Can identify SMMs by testing different cell populations e.g. cultured cells.

Question 16

What would the QF-PCR results for prenatal testing look like in cases where mosaicism is present?

Answer 16

• Inconclusive ratios for one chromosome with or without trisomy.
• Don’t say that we can detect mosaicism with QF-PCR - difficult to detect.

Question 17

Outline the reporting of prenatal QF-PCR results.

Answer 17

1) . NORMAL RESULTS:
- At least two informative markers with a normal pattern are required to interpret as NORMAL.
- Acceptable to report on cases with a single informative marker but must add caveat for follow up with final karyotype or FISH.

2) . ABNORMAL RESULT:
- At least two informative markers consistent with a triallelic genotype.
- Confirmations of sample identity - FISH/QF-PCR on pour off sample - or by analysis of maternal sample.

Question 18

Outline the interpretation and reporting of single abnormal markers in prenatal QF-PCR.

Answer 18

SINGLE ABNORMAL MARKERS:

• Somatic microsatellite mutations (SMMs) not reported.
• Submicroscopic Duplications (SMDs) not reported if previously reported to be inherited. Otherwise request parental blood to provide information on significance.
• Persistent inconclusive marker - no further action if flanked either side by a normal marker and previously reported as inherited SMD. Otherwise, not reported buy alert cyto to analyse closely at the marker location on karyotype.

Question 19

Outline the interpretation and reporting of MCC in prenatal QF-PCR.

Answer 19

1) . Low level maternal genotype:
- Can be ignored if all ratios are normal / consistent with foetal genotype.

2) . Single genotype:
- If maternal then await karyotype results.
- If foetal report as normal.

3) . High level maternal genotype:
- large number of inconclusive ratios.
- Await karyotype.

Question 20

Outline sex chromosome aneuploidy testing by QF-PCR.

Answer 20

SEX CHROMOSOME ANEUPLOIDY:

• Testing for sex chromosome aneuploidy may occur in a subset of referrals with appropriate indications.
• XY QF-PCR is a highly stringent screen for monosomy X but not a diagnostic test.
• Abnormal ranges for autosomes may not apply and may instead represent mosaicism for monosomy X.
• Sex chromosome markers may give ratios >2.4 or <0.45, e.g. with rarer sex chromosome aneuploidies.
• A result consistent with monosomy X may represent a normal female homozygous for all markers tested.
• Abnormal results suggesting monosomy X should be confirmed by another technique ( or reported from QF-PCR with appropriate caveat).

Question 21

Outline the problems what have been documented when using QF-PCR to test CVS samples.

Answer 21

• Discrepant results reported for QF-PCR and karyotype on CVS - 2006 Audit 18 ‘incorrect’ results from 5 labs.
• These incorrect results were put down the to method of CVS lysis before DNA extraction - Previously CVS lysis method on fronds - Resulted in only outer trophoblast cells analysed.
• Since then BPGs have changed to whole enzyme digest method on at least 2 fronds. This results in trophoblast and mesodermal tissue being assayed.
• New method has reduced discrepancies but not 100%.
• Preparation quality of CVS is important - removal of all maternal decidua.
• Be wary of possibility of maternal cells affecting karyotyping cultured CVS.
• Beware of fully biallelic results (no triallelic results found) which may represent mitotic disjunction events (caveats regarding risk of confined placental mosaicism on report).

Question 22

Is stand alone QF-PCR a good idea?

Answer 22

• Some centres have decided not to karyotype following QF-PCR.
• Caroline Ogilvie - 2008 audit of T21 referrals found less than 0.5% had sex chromosome abnormalities and 0.6% had other chromosome abnormalities. 0.4% uncertain prognosis, 0.07% poor prognosis.
• A paper published in 2010 showed that by only karyotyping if there is an abnormal USS, raised NT measurement, T21 soft markers, family history, or abnormal by QF-PCR resulted in karyotyping only 25% of 9737 samples. Some additional samples (9%) were karyotyped either as a result of commissioning differences or private tests and it was found that QF-PCR detected 85% of all prenatally detected abnormalities and 99.9% of all clinically significant abnormalities in samples referred with no clinical indication for karyotyping.

Question 23

Summarise QF-PCR.

Answer 23

• QF-PCR analysis is an excellent technique for targeted trisomy detection.
• Provides additional useful information - sample identity / MCC / mosaicism / triploidy.
• Most results are very straightforward.
• Both molecular and cytogenetic expertise is needed for interpretation of results.
• Some centres use as a stand alone test for pregnancies presenting without foetal structural abnormalities.