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