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

1
Proof of Principle for the Non-Invasive Prenatal
Diagnosis of Fetal Trisomy 21

• Sarah Fielding
• 12-04-2010

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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
• lt10 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
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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
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Dosage of chromosome 21(RNA-SNP allelic ratio
method)

• Quantitative analysis of SNPs in PLAC4 mRNA

Trisomy 21 pregnancy
Euploid pregnancy
T
T
T
C
C
T C 2 1
T C 1 1
Allele Ratio
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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
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Testing Procedure

• Stored in Trizol
• RNeasy Kit (Qiagen)
• 1.6mL plasma
• 60 µ L of cfRNA (15ng/ µL)
• 81 ? 93 weeks gestation

• ABI Allelic Discrimination Assays

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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

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PLAC4 mRNA Quantification - Results
5 replicates of cfRNA sample
Plasma cfRNA Sample
Quantity of PLAC4 mRNA 21 copies/µL
RT -ve

• 85 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

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Results84 week gestation pregnancy rs8130833
Fetal Genotype T/T
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Results84 week gestation pregnancy rs7844
Fetal Genotype C/C
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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 21 or 12 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

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T21 simulation experiments
rs8130833 Ratio of DNA genotype controls in synthetic T21 sample Ratio of DNA genotype controls in synthetic T21 sample Ratio of DNA genotype controls in synthetic T21 sample
rs8130833 T/T T/C C/C
Synthetic TTC 0.5 1
Synthetic TCC 1 0.5
VIC/FAM ratio Mean for T/C Controls 0.376 Mean
for synthetic TTC 0.722 (p2.8x10-4) Mean for
synthetic CCT 0.210 (p1.48x10-9)
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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 G/C GCC or GGC
rs8130833 T/C T/T TTT or TTC
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T21 sample rs8130833
16
T21 sample rs8130833
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T21 sample
Paternal DNA Maternal DNA Fetal cfRNA
rs7844 G/C G/C GCC or GGC
rs8130833 T/C T/T TTT or TTC

• 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
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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 84 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

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Acknowledgements

• Gail Norbury
• Lucy Jenkins
• Lighta Godinho
• Bhaneeta Mistry
• all staff
• Lyn Chitty
• Darryl Wang

Guys Hospital
North East Thames Regional Genetics Laboratory
Institute of Child Health