1. Proof of Principle for the Non-Invasive Prenatal Diagnosis of Fetal Trisomy 21 Sarah Fielding 12-04-2010 NE Thames Regional Genetics Service

2. Non-Invasive Aneuploidy Detection  Cell free fetal DNA and RNA detectable in maternal plasma  Several diagnostic applications:  RhD typing in rhesus-negative mothers  Fetal sex determination  Inheritance of paternal mutations  Potential for detection of aneuploidies  <10% total cell-free DNA is fetal DNA  Sufficient for detection but…  INSUFFICIENT for quantitative analysis  Proposed solution –  Target sub-fraction of plasma nucleic acids that are completely fetal specific  Target fetal RNA of placental origin  Use fetal specific RNA markers to determine chromosome copy number QUALITATIVE DETECTION OF FETAL SPECIFIC SEQUENCE QUANTITATIVE ASSESSMENT

3. PLAC4  21q22.3  Expressed exclusively by placenta PLAC4 mRNA can be detected in all three trimesters of pregnancy but NOT in plasma of non-pregnant individuals Clearance of PLAC4 mRNA within 24hrs after delivery – specificity to pregnancy Lo et al., 2007. Nature Medicine 13(2): 218-223

4. Dosage of chromosome 21 (RNA-SNP allelic ratio method)  Quantitative analysis of SNPs in PLAC4 mRNA Trisomy 21 pregnancyEuploid pregnancy T T C T C T : C 2 : 1 T : C 1 : 1 Allele Ratio

5. RNA-SNP allelic ratio method  Lo et al (2007) – determined allelic ratio of PLAC4 SNP rs8130833  Correctly identified 90% trisomy 21 cases in +ve controls  Excluded trisomy 21 in 96% normal controls  RNA-SNP allelic ratio method chosen for evaluation Lo et al., 2007. Nature Medicine 13(2): 218-223

6. Testing Procedure ► Stored in Trizol ► RNeasy Kit (Qiagen) 1.6mL plasma 60 μ L of cfRNA (~15ng/ μ L) ► 8+1  9+3 weeks gestation ► ABI Allelic Discrimination Assays

7. PLAC4 mRNA Quantification  Real-time quantitative PCR  ABI7300 real-time PCR machine  Two steps (single reaction):  1-step reverse transcription PCR  TaqMan real-time PCR  TaqMan MGB Probe  Detects PLAC4 PCR product Reverse transcription -ve control  Standard curve used to determine quantity of PLAC4 mRNA  7 standards with known quantity of PLAC4 amplicon

8. PLAC4 mRNA Quantification - Results Plasma cfRNA Sample 5 replicates of cfRNA sample RT -ve Quantity of PLAC4 mRNA ~ 21 copies/ μ L ► 8+5 week gestation pregnancy ► In line with average 1 st trimester PLAC4 mRNA conc. reported in literature

9. PLAC4 SNP Genotyping  ABI SNP genotyping assays for 2 PLAC4 SNPs:  rs8130833 (T 0.67/C 0.33)  rs7844 (G 0.63/C 0.37)  Each Assay:  2 primers  2 allele-specific TaqMan probes  Optimised to work directly with cfRNA  Two steps (single reaction)  1-step reverse transcription PCR  Allelic Discrimination PCR  60cycles  Whole reaction ~ 3.5 hours  Requires total 30 μ L cfRNA

10. Results 8+4 week gestation pregnancy – rs8130833 Fetal Genotype: T/T

11. Results 8+4 week gestation pregnancy – rs7844 Fetal Genotype: C/C

12. Allele Dosage – VIC/FAM ratio  Need to determine allelic ratio of SNPs in het fetuses VIC FAM  Heterozygous Genotype Controls  rs8130833 (Mean VIC/FAM = 0.387; Std Dev = 0.012)  rs7844 (Mean VIC/FAM = 0.327; Std Dev = 0.009)  Hypothesis:  Het T21 sample with 2:1 or 1:2 allele ratio –  VIC/FAM ratio would deviate from het controls  Potential to discriminate between euploid and trisomy 21 cases consistent between samples with same genotype

13. T21 simulation experiments rs8130833 Ratio of DNA genotype controls in synthetic T21 sample T/TT/CC/C Synthetic TTC 0.51 Synthetic TCC 10.5 VIC/FAM ratio: Mean for T/C Controls: 0.376 Mean for synthetic TTC: 0.722 (p=2.8x10 -4 ) Mean for synthetic CCT: 0.210 (p=1.48x10 -9 )

14. T21 sample  Maternal plasma aliquots from genuine T21 pregnancy  Parental samples genotyped  No result for rs7844  Instrument error  Result for rs8130833 consistent with T21 & TTC fetal genotype Paternal DNA Maternal DNA Fetal cfRNA rs7844 G/C GCC or GGC rs8130833 T/CT/TTTT or TTC

15. T21 sample – rs8130833

17. T21 sample  Maternal plasma aliquots from genuine T21 pregnancy  Parental samples genotyped  Result for rs8130833 consistent with T21 & TTC fetal genotype  VIC/FAM ratio  Heterozygous T/C controls – 0.408  T21 cfRNA sample – 0.866  At rs8130833 – proof of principle demonstrated Paternal DNA Maternal DNA Fetal cfRNA rs7844 G/C GCC or GGC rs8130833 T/CT/TTTT or TTC T-Test: p = 1.85 x 10 -4

18. Summary  Presence of fetal PLAC4 mRNA in maternal plasma successfully demonstrated and quantified  Method developed to determine allelic ratio of 2 PLAC4 SNPs using ABI allelic discrimination assays  Fetal PLAC4 mRNA from 8+4 week gestation pregnancy successfully genotyped at both loci  Analysis of cfRNA extracted from genuine T21 pregnancy supports results from T21 simulation experiments  Presented method can distinguish a T21 result from euploid result at ch21 PLAC4 SNP rs8130833

19. Acknowledgements Gail Norbury Lucy Jenkins Lighta Godinho Bhaneeta Mistry & all staff Lyn Chitty Darryl Wang North East Thames Regional Genetics Laboratory Institute of Child Health Guy’s Hospital