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

Sarah Fielding

12-04-2010

NE Thames Regional Genetics Service

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

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

pregnancyLo et al., 2007. Nature Medicine 13(2): 218-223

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

Quantitative analysis of SNPs in PLAC4 mRNA

Trisomy 21 pregnancyEuploid pregnancy

TTC

TC

T : C 2 : 1

T : C 1 : 1 Allele Ratio

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

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

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

PLAC4 mRNA Quantification - Results

Plasma cfRNA Sample

5 replicates of cfRNA sample

RT -ve

Quantity of PLAC4 mRNA ~ 21 copies/μL

► 8+5 week gestation pregnancy

► In line with average 1st trimester PLAC4 mRNA conc. reported in literature

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

Results8+4 week gestation pregnancy – rs8130833

Fetal Genotype: T/T

Results8+4 week gestation pregnancy – rs7844

Fetal Genotype: C/C

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

T21 simulation experiments

rs8130833Ratio of DNA genotype controls in synthetic T21 sample

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

(p=2.8x10-4)

Mean for synthetic CCT: 0.210

(p=1.48x10-9)

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

T21 sample – rs8130833

T21 sample – rs8130833

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 G/C GCC or GGC

rs8130833 T/C T/T TTT or TTC

T-Test: p = 1.85 x 10-4

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

AcknowledgementsGail Norbury Lucy JenkinsLighta GodinhoBhaneeta Mistry& all staff

Lyn ChittyDarryl Wang

North East Thames Regional Genetics Laboratory

Institute of Child Health

Guy’s Hospital