1. Great Ormond Street Hospital for Children North East London Regional Cytogenetics Laboratory UK audit of biallelic abnormal PCR results in CVS Jonathan Waters 1, Kathy Mann 2 and Caroline Ogilvie 2 Gt Ormond St Hospital NHS Trust 1 and Guy’s Hospital Foundation Trust 2 ACC Spring Conference 2008 31st March to 2nd April Liverpool

2. From Robinson et al., 2002 The early embryo: late first trimester

3. Possible types of mosaicism within the fetal-placental unit From: Gardner RJM and Sutherland GR, 2004 oversimplification - cell-lineage specific mosaicism not considered

4. 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 DW et al., 1993

5. 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 DW et al., 1993

6. UK data: PCR/Karyotype complete discrepancies as of 2006 LabCVSComplete discrep- ancies Method (no of assays) DetailsFrequency BWH23212cVf (2)PCR: normal ; LTC: +181:232 (0.43% ) Guy’s (GOS) 3,70032cVf (2)PCR: trisomy 21; LTC: 46,XY PCR: normal; LTC: +21 3:3700 (0.08%) Glasgow36013cVf (1)PCR: normal ; LTC: +181:360 (0.28%) Bristol34202cVf (2) Liverpool53012cVf (2)PCR: normal ; LTC: +211:530 (0.19%) TDL (GOS) 25,000122cVF+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

7. 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

8. 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

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

10. 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

11. Evidence for value of enzyme digestion method for PCR assay Biallelic 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

12. 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

13. Results of audit – trisomy 21 PCR/Karyotype complete discrepancy CaseExtract method PCRLTC Karyotype Follow up 1 GOS whole villi (2)trisomy 21 biallelic 46,XYconsistent with disomy 21 2 GOS whole villi (2)disomy 2147,XY+21 biallelic Placental tissue showed trisomy 21 >> disomy 21 3 GOS whole villi (2)disomy 2146,XX,der (21q;21q) not available 4 LP whole villi (2)disomy 2147,+21[41] triallelic Postnatal blood: 47,+21[50] 5 TDL glass beaddisomy 2147,XX,+21[30] biallelic AF: trisomy 21 (PCR) 47,XX,+21[60]

14. 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

15. Results of audit – trisomy 18 PCR/Karyotype complete discrepancy CaseExtract method PCRLTC Karyotype Follow up 1 BWH whole villi (2)disomy 1847,XY,+18 PCR: trisomy 18 biallelic 2 BWH enzyme digest disomy 1847,XY,+18 PCR: trisomy 18 biallelic 3 TDL glass beaddisomy 1847,XY,+18 [53] PCR: trisomy 18 biallelic AF: 46,XY [60] PCR: disomy 18 4 GLW whole villi (3)disomy 1847,+18 [26] PCR: trisomy 18 biallelic AF: 47,+18 PCR: trisomy 18 POC: 47,+18 PCR: trisomy 18

16. 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

17. Results of audit – trisomy 13 PCR/Karyotype complete discrepancy CaseExtract method PCRLTC Karyotype Follow up 1 NWP enzyme digest (5mg sample) trisomy 13 biallelic 46,XY [30] FISH: disomy 13[60] 2 cultures TOP not available

18. 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)

19. 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)

20. 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 possible Inform next ACC/CMGS QF-PCR rapid aneuploidy testing Best Practice meeting

21. 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