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Proof of Principle for the Non-Invasive Prenatal Diagnosis of Fetal Trisomy 21

NE Thames Regional Genetics Service. Proof of Principle for the Non-Invasive Prenatal Diagnosis of Fetal Trisomy 21. Sarah Fielding 12-04-2010. Non-Invasive Aneuploidy Detection. Cell free fetal DNA and RNA detectable in maternal plasma Several diagnostic applications:

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Proof of Principle for the Non-Invasive Prenatal Diagnosis of Fetal Trisomy 21

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

  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 QUALITATIVEDETECTION OF FETAL SPECIFIC SEQUENCE QUANTITATIVEASSESSMENT

  3. PLAC4 • 21q22.3 • Expressed exclusively by placenta Clearance of PLAC4 mRNA within 24hrs after delivery – specificity to pregnancy PLAC4 mRNA can be detected in all three trimesters of pregnancy but NOT in plasma of non-pregnant individuals 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 Euploid pregnancy Trisomy 21 pregnancy T T T C 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/ μL) • 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 5 replicates of cfRNA sample Plasma cfRNA Sample Quantity of PLAC4 mRNA ~ 21 copies/μL RT -ve • 8+5 week gestation pregnancy • In line with average 1st 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 30μL cfRNA

  10. Results8+4 week gestation pregnancy – rs8130833 Fetal Genotype: T/T

  11. Results8+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 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

  15. T21 sample – rs8130833

  16. 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 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 Guy’s Hospital North East Thames Regional Genetics Laboratory Institute of Child Health

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