View
217
Download
1
Category
Tags:
Preview:
Citation preview
Great Ormond Street Hospital for ChildrenGreat Ormond Street Hospital for Children
North East London Regional Cytogenetics LaboratoryNorth East London Regional Cytogenetics Laboratory
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
From Robinson et al., 2002
The early embryo: late first trimester
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
mc
t
Direct preparation: 46,XX – trophoblast
LTC: 47,XX,+21 – mesenchymal core
Amniocentesis: 47,XX,+21 – mesoderm/ectoderm
Fetal tissue: 47,XX,+21 – mesoderm/ectoderm/endoderm
Trisomy 21 conceptus with trisomy rescue in trophoblast cells
Bianchi D
W et al., 1993
QF-PCR should assay both cell lineages
mc
t
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] – mesenchymal core
Amniocentesis: 47,XX,+21[30]
Postnatal blood: 47,XX,+21[30]
Bianchi D
W et al., 1993
UK data: PCR/Karyotype complete discrepancies as of 2006
Lab CVS Complete discrep-ancies
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)
25,000 12 2cVF+D(1)
PCR: X0; LTC: XY+21; XYY,+21,+21
+ 11 others
12:25,000 (0.05%)
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 responded9 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 assayBiallelic trisomy 18 case – Liverpool (case 2)
1.20
1.24
1.66
2.22
0.93
0.91
0.70
0.64
frond
mush
digest
culture
Six informative markers all suggesting mosaic trisomy 18 at PCR
All diallelic (post-zygotic non-disjunction)
Cultured cells
all showed +18
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
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 disomy 21 47,XX,+21[30]
biallelic
AF: trisomy 21 (PCR)
47,XX,+21[60]
Results of audit
trisomy 18 results no of markers routinely used
4 (2 labs), 5 (1 lab), 6 (2 labs), 7,8 or 9 (1 lab each), 11 (1 lab) 253 trisomy 18 samples 43 showed biallelic results: average: 17.0%
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 18PCR/Karyotype complete discrepancy
Case Extract
method
PCR LTC
Karyotype
Follow up
1
BWH
whole villi (2) disomy 18 47,XY,+18
PCR: trisomy 18
biallelic
2
BWH
enzyme 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 LTC
Karyotype
Follow up
1 NWP
enzyme digest
(5mg sample)
trisomy 13
biallelic
46,XY [30]
FISH: disomy 13[60] 2 cultures
TOP
not available
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
may help to ensure adequate representation of mesenchymal cells – usually a better predictor of fetal karyotype
use of glass beads should be evaluated biallelic PCR results
in some cases might now be reported differently with greater experience
marker number dependent – minimum number? report caveats
complete discrepancies complete discrepancies are rare events usually associated with biallelic trisomies
follow-up should be undertaken if at all possibleInform 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
Recommended