UK audit of biallelic abnormal PCR results in
CVS
Jonathan Waters1, Kathy Mann2
and Caroline Ogilvie2
Gt Ormond St Hospital NHS Trust1 and Guy’s Hospital Foundation Trust2
ACC Spring Conference 2008
31st March to 2nd April
Liverpool
Great Ormond Street Hospital for Children
North East London Regional Cytogenetics Laboratory
The early embryo:
late first trimester
From Robinson et al., 2002
Possible types of mosaicism within the fetal-placental unit
From: Gardner RJM and Sutherland GR, 2004
oversimplification - cell-lineage specific mosaicism not considered
chromomosomal mosaicism in CVS may be
cell-lineage specific
Bianchi DW et al., 1993
t
mc
Direct preparation: 46,XX
– trophoblast
LTC: 47,XX,+21
–
Amniocentesis: 47,XX,+21
– mesoderm/ectoderm
Fetal tissue: 47,XX,+21
– mesoderm/ectoderm/endoderm
Trisomy 21 conceptus with trisomy rescue in trophoblast cells
mesenchymal core
QF-PCR should assay both cell lineages
Bianchi DW et al., 1993
t
mc
Cardiff case: P07-2481
QF-PCR: disomy 21 (50%) / trisomy 21 (50%)
–
trophoblast + mesenchymal core
Direct preparation: 46,XX[14]
–
trophoblast
LTC: 47,XX,+21[44]/46,XX[1]
–
Amniocentesis: 47,XX,+21[30]
Postnatal blood: 47,XX,+21[30]
mesenchymal core
UK data: PCR/Karyotype complete discrepancies as of 2006
Lab
CVS
Complete
discrepancies
Method
(no of
assays)
Details
Frequency
BWH
232
1
2cVf (2)
PCR: normal ; LTC: +18
1:232
(0.43% )
Guy’s
(GOS)
3,700
3
2cVf (2)
PCR: trisomy 21; LTC: 46,XY
PCR: normal; LTC: +21
PCR: normal; LTC: +21
3:3700
(0.08%)
Glasgow
360
1
3cVf (1)
PCR: normal ; LTC: +18
1:360
(0.28%)
Bristol
342
0
2cVf (2)
Liverpool 530
1
2cVf (2)
PCR: normal ; LTC: +21
1:530
(0.19%)
TDL
(GOS)
12
2cVF+D
(1)
PCR: X0; LTC: XY+21;
XYY,+21,+21
12:25,000
(0.05%)
25,000
+ 11 others
Table (with modifications) from data supplied by Val Davison, Birmingham
CVS double testing: QF-PCR and Karyotyping
 test selectivity

different populations of cells are assayed

PCR – cytotrophoblast (ct) + mesenchymal (mc) cells
 biological constraint: cytotrophoblast cells > or >> mc cells

LTC karyotype – in vitro selection for subset of mc cells
 mosaicism within the biopsy sample


1-2% CVS karyotypes are mosaic
>80% of this mosaicism is confined to the placenta
 test sensitivity
 QF-PCR abnormal results

Biallelic (AAB, AAC, etc) trisomies may represent mitotic
errors and may rarely lead to discrepant results
PCR/Karyotype complete discrepancy
- practical steps
 QF-PCR - sample representation
 from whole fronds to use of minced,
enzyme - digested whole sample mix
 QF-PCR (biallelic) abnormal results
 reported with a caveat that the result may
represent post-zygotic non-disjunctional events
and may not be representative of the fetus
 suggested a UK audit might be helpful

Waters et al., 2007. Prenat Diagn 37: 332-9
Results of audit – outline
10 laboratories providing a service responded
9 provided comprehensive data
 Method of villus preparation for PCR assay
 Complete discrepancies



trisomy 21
trisomy 18
trisomy 13
 biallelic results
 conclusions - recommendations to Best Practice
committee
Results of audit
 villus preparation for PCR assay




three fronds in one assay: 1 lab
three fronds in one assay and/or cellular aggregate: 1 lab
cellular aggregate (enzymatic digestion +/- finely chopped
pretreatment): 7 labs
glass bead preparation: : 1 lab
6 labs have significantly changed their sample preparation
approach from whole villi to cell aggregate
2 labs (Guy’s, TDL) provided overall discrepancy incidence
data before and after change in sample preparation
Evidence for value of enzyme digestion method for PCR assay
Biallelic trisomy 18 case – Liverpool (case 2)
1.20
frond
0.93
1.24
mush
0.91
1.66
digest
0.70
2.22
culture
0.64
Data courtesy of Julie Sibbring, Regional Molecular
Genetics Laboratory, Liverpool women’s Hospital
See also Mann K et al 2007. Prenat Diagn 27:285-9
 Six informative
markers all
suggesting mosaic
trisomy 18 at PCR
 All diallelic (postzygotic nondisjunction)
 Cultured cells
all showed +18
Results of audit
 trisomy 21 results

no of markers routinely used



4 (2 labs), 5 (2 labs), 7 (1 lab), 8 (3 labs), 10 (1 lab)
688 trisomy 21 samples
71 showed biallelic results; average: 10.3%
with 10 markers: 6.7%
 With 4/5 markers: 13.9%


2 discrepant results from this data set (3 from
previous data set)
4 based on PCR analysis of individual villi
 1 based on glass bead sample

Results of audit – trisomy 21
PCR/Karyotype complete discrepancy
Case Extract
method
PCR
LTC
Karyotype
Follow up
1
GOS
whole villi (2) trisomy 21
biallelic
46,XY
consistent with
disomy 21
2
GOS
whole villi (2) disomy 21
47,XY+21
biallelic
Placental tissue
showed trisomy 21 >>
disomy 21
3
GOS
whole villi (2) disomy 21
46,XX,der
(21q;21q)
not available
4
LP
whole villi (2) disomy 21
47,+21[41]
triallelic
Postnatal blood:
47,+21[50]
5
TDL
glass bead
47,XX,+21[30] AF: trisomy 21 (PCR)
biallelic
47,XX,+21[60]
disomy 21
Results of audit
 trisomy 18 results

no of markers routinely used



253 trisomy 18 samples
43 showed biallelic results: average: 17.0%



4 (2 labs), 5 (1 lab), 6 (2 labs), 7,8 or 9 (1 lab each), 11 (1 lab)
with 11 markers: 14.1%
with 4/5 markers: 25%
4 discrepant results



2 based on PCR analysis of individual villi (x1 or x2)
1 based on enzyme digest
1 based on glass bead sample
Results of audit – trisomy 18
PCR/Karyotype complete discrepancy
Case Extract
method
PCR
LTC
Karyotype
Follow up
1
whole villi (2) disomy 18
BWH
47,XY,+18
PCR: trisomy 18
biallelic
2
enzyme
BWH digest
disomy 18
47,XY,+18
PCR: trisomy 18
biallelic
3
TDL
glass bead
disomy 18
47,XY,+18 [53]
PCR: trisomy 18
biallelic
AF: 46,XY [60]
PCR: disomy 18
4
GLW
whole villi (3) disomy 18
47,+18 [26]
PCR: trisomy 18
biallelic
AF: 47,+18
PCR: trisomy 18
POC: 47,+18
PCR: trisomy 18
Results of audit
 trisomy 13 results

no of markers routinely used

4 (2 labs), 5 (3 labs), 6 (2 labs), 7 or 8 (2 labs)

122 trisomy 13 samples

43 showed biallelic results: average: 7.4%

1 discrepant result

1 based on enzyme digest
Results of audit – trisomy 13
PCR/Karyotype complete discrepancy
Case
Extract
method
PCR
1
NWP
enzyme digest trisomy 13
(5mg sample) biallelic
LTC
Karyotype
Follow up
46,XY [30]
TOP
not available
FISH: disomy 13[60]
2 cultures
Results – sample preparation
 use of mince/enzymatic digestion



experimental evidence from two laboratories – Liverpool
and Guy’s (data not shown) suggests that this method
enhances peak ratios for accurate analysis
two cases reported with complete discrepancies using
this approach
use of glass bead approach should be monitored
Relevant CVS data – incidence of complete discrepancy
Guy’s : 3/4,025 (whole villi x 2) ¼,167 (mince, enzyme digest)
TDL : 12/25,000 (whole villi X 1) 2/3,500 (glass bead)
Results – biallelic trisomies
 biallelic trisomic results

9/10 discrepancies associated with biallelic trisomy

3 labs distinguish between biallelic and triallelic results in
QF-PCR report

proportion of biallelic trisomic results is as follows:
trisomy 18: 17.0% > trisomy 21: 10.3% > trisomy 13: 7.4%
-chromosome 18 markers less informative
biallelic percentage dependent on number of markers
used in assay

For trisomy 21 with 10 markers: biallelic incidence is 6.7%
close to 4.5% for mitotic error rate previously reported in
Down syndrome (Antonarakis SE et al., 1993)
Summary

method of sample preparation



biallelic PCR results




in some cases might now be reported differently with greater
experience
marker number dependent – minimum number?
report caveats
complete discrepancies



may help to ensure adequate representation of mesenchymal
cells – usually a better predictor of fetal karyotype
use of glass beads should be evaluated
complete discrepancies are rare events
usually associated with biallelic trisomies
follow-up should be undertaken if at all possible
Inform next ACC/CMGS QF-PCR rapid aneuploidy testing
Best Practice meeting
Acknowlegements
 Sue Hamilton, QF-PCR User Group, Manchester
 participating laboratories










Aberdeen
Birmingham
Bristol
Cardiff
Glasgow
Guy’s (with GOS, NWP, St George’s)
Liverpool
Oxford
Manchester
TDL