Non-invasive prenatal sex determination

1
Non-invasive prenatal sex determination single
gene disorder testing using cell-free DNA

• Cathy Meaney
• NE Thames Regional Molecular Genetics

Great Ormond Street Hospital for Children NHS
Trust
2
Prenatal Diagnosis

• Invasive techniques
• amnio, CVS, PGD
• risks, sampling time, costs
• Non invasive prenatal diagnosis
• 1989 fetal cells in maternal blood
• Low number (1-6 cells/ml blood)?
• Fetal cell enriching and isolation - time
  consuming
• Labor intensive or difficult on large scale
• Low sensitivity PND
• Cells can remain in circulation for many years
• gt confusion as to which pregnancy testing
• 1996 cell-free tumour DNA
• Found in plasma serum from cancer patients
• Monitor treatment without BMT for karyotyping

3
1997 cell-free fetal DNA

• Lo et. al. gt maternal plasma contains both fetal
  maternal DNA
• Fetal DNA is stable in high concs in maternal
  plasma
• Cleared rapidly from maternal circulation
  following birth
• (with a half-life of minutes)?
• NB. Genome equivalent obtained using conversion
  factor of 6.6pg of DNA/cell
• Likely that circulating fetal DNA released from
  fetal and/or
• placental cells undergoing apoptosis
• Syncytiotrophoblasts in the form of apoptotic
  fragments packaged into microvesicles
• Ties in with observed different sizes
• (fetal lt 300bp and maternal gt 350bp)?
• Detect fetal abnormality without invasive
  procedure
• ? problems with multiple pregnancies

4
Clinical Applications

• Blood typing
• Genotyping Rh-ve women
• Fetus could inherit RhD allele from father
• Fetal sexing (detecting Y chromosome sequences
  eg. SRY, DYS)?
• X-linked diseases
• CAH (pregnant women get dexamethasone for
• CAH female fetuses)?
• STRs for fetal alleles, trisomy 13, 18, 21
• Fetal aneuploidy detection (T21 gt2-fold increase
  in total
• fetal cfDNA)?
• Pregnancies at risk for pre-eclampsia
• gt increase in cell-free fetal DNA
• Single gene disorders
• Achondroplasia, myotonic dystrophy, prenatal
  exclusion in CF,
• beta-thalassaemia

5
Challenges

• Sensitivity
• Early detection when fetal DNA at low levels
  (1gE/PCR)?
• Specificity
• Low amount of fetal DNA (1-6) in high
  background of maternal DNA
• Dynamic Range
• 1-560gE/ml fetal DNA and 70gt4000gE/ml total DNA
• Lack of reliable fetal-specific markers
• Confirm presence of fetal DNA
• Mother fetus share, on average, half of their
  genomic DNA seqs
• Only detect uniquely fetal DNA sequences that
  are paternally
• inherited
• Technology / platforms
• Real time PCR, SABER MALDI-TOF MS

6
Extraction of cell-free DNA
PLASMA 6-10mls (cell-free maternal fetal
DNA)? 1 ml plasma aliquots gt store
-20?C Extract DNA using Qiagen QIAamp MinElute
Virus Kit (manual or EZ1 robot)? Elute into
50-100?l RNAse free water
16-20mls maternal EDTA blood from 7 weeks
gestation (pref within 24hrs)? Spin blood at
3000g
plasma
MBC
RBC
BUFFY COAT (maternal white blood
cells)? Extract DNA on EZ1 robot or manual
salting out method
7
Real-Time PCR

• Fetal sex determination
• Polymorphic markers
• Achondroplasia common mutation

8
Cell-Free fetal DNA analysisusing Real Time PCR
Theoretical

• Real-Time PCR allows us to see the
  exponential phase so can calculate how much DNA
  started with
• Closed system contamination
• Probes - specificity, sensitivity
• Melt curve analysis - primer
• dimer mis-priming
• Optimisation
• Reagent cost
• Multiplex

Log Target DNA
Real Life
Ct
Threshold Cycle, Ct The point at which
fluorescence rises appreciably above background
9
End Point Measurements
96 replicates of the identical reaction can have
very different final amounts of fluorescence
10
Taqman probes

• A hybridization probe is constructed with a
  fluorescent reporter
• at one end to a nearby quencher.
• The reporter is excited but its emitted
  fluorescence is captured
• by the nearby quencher.
• No reporter fluorescence is detected.

During the extension step, the polymerase
encounters the TaqMan probe and chews off the
end.

• Once freed from the quencher the reporter
  fluorescence
• can be detected indicating amplification has
  occurred

11
Fetal sex determination using Real Time PCR
12
Fetal sex determination on AB 7300

• Applied Biosystems Assay-on Demand kits
• 60C annealing temp
• (95C/10, 50 cycles of 95C/15 sec and
  60C/1min)?

• Separate assays
• SRY primers and probe (FAM) specific for
• Y chromosome sequences
• CCR5 primers and probe (FAM) as control
• for total DNA measurement
• 20ul PCR reaction in 96 well microtitre plate
• AB Universal Mastermix
• Store primers probes in small aliquots in
• freezer use once
• Accurate pipetting using filter tips
• Plastic seal, spin plate, no bubbles

13
Plate Setup
 
14
Amplification Plots of SRY CCR5
SRY
CCR5
DRn
S2
S3
S4
S1
ffDNA
S1
S4
S2
S3
ffDNA
Standard Curves
S4
S4
CCR5
SRY
S3
S3
S2
S2
S1
S1
15
RQ-PCR Results Validation

16

• Examples of X-linked diseases tested
• Metabolic (Fabry, Hunters)?
• X-linked immunodeficiencies (Wiskott Aldrich
  sydrome, XSCID, XL-CGD)?
• X-linked Adrenoleukodystrophy (ALD)?
• Haemophilia
• DMD/BMD
• X-linked icthyosis
• Menkes
• Retts
• Ambiguous genitalia
• CAH

17
PND for ALD cfDNA analysis - male
18
PolymorphicMarkers

• SRY negative result
• True female result or low circulating fetal DNA
  concentrations or fetal DNA loss during sampling
  processing?
• Panel of 8 biallelic markers tested by
• RQ-PCR
• Detect fetal markers not present in maternal
  DNA to confirm presence of fetal DNA

Maternal EDTA - neg for S03, S04, S05, S06
Plasma - positive for marker S05
19
RASSF1A universal marker

• RASSF1A promoter
• hypermethylated in placenta (gtfetus)?
• hypomethylated in maternal blood cells
• (Lo et. al. 2007)?
• Use BstU1 methylation sensitive restriction
  enzyme to digest unmethylated maternal DNA
• amplify with RASSF1A primers/probe
• detection of placental-derived hypermethylated
• RASSF1A sequences in maternal plasma

20
SAFE QC Workshop 2Showing the quality of
performance of non-invasive prenatal genotyping
in Europe

• 0.6 ml plasma sent out at RT
• 18 laboratories received 4-6 samples each
• Tested for RhD SRY
• Results on 105 RhD and 109 SRY
• Correct results 193 (90.2)?
• False results 10 (4.7)
• 6 false positive and 4 false negative
• Inconclusive results 11 (5.1)?
• 8 in positive samples and 3 in negative samples

21
Fetal sex results 2006-2007
22
False positives

• CAH - 5 6 weeks gestation
• ? Contamination
• strict validation criteria
• repeat test on a 2nd aliquot of plasma
• request 2nd EDTA sample
• Risk of FP due to sex discordant DZ vanishing
• twins is 1/70
• Early good sonography
• More wary in assisted pregnancies and mothers
• with higher incidence DZ twins

23
2007-2009

• Samples gt 7 weeks gestation
• About 190 patients tested
• Of those samples there were repeat samples
• or 2 samples tested at same time

24
(No Transcript)
25
Single Gene Disorders

• Skeletal Dysplasia
• Achondroplasia
• Craniosynostosis
• Apert syndrome
• Crouzon syndrome
• Torsion Dystonia

26
Achondroplasia

• Applied Biosystems Assay-by-Design for SNP
  genotyping assays
• Taqman MGB labelled probes (FAM VIC) using real
  time PCR
• Designed for genotyping specific SNPs
• Each assay enables scoring of both alleles in a
  single well
• Confidence levels are low for detecting fetal
  mutant DNA in maternal background
• enrichment of fetal DNA required
• fetal DNA lt350bp, maternal DNA gt350bp

27
Achondroplasia - G380R ggta

• Extracted DNA from 400ul 800ul plasma from
  mother with known ACH pregnancy
• Digested with BsrGI enzyme
• 400ul plasma 800ul plasma

Ladder Uncut 5ul DNA 10ul DNA 20ul DNA 5ul
DNA 10ul DNA 20ul DNA Normal control G380Rggta
control H20
132bp 112bp
28
Early-onset primary dystonia (DYT1)?

• Usually presents in childhood or early
  adolescence with a minority occurring as an adult
• Dystonic muscle contraction causing uncontrolled
  distortion
• of parts of the body
• Regardless of ethnicity, majority of DYT1
  patients have a
• 3bp GAG deletion (c.907_909delGAG / p.Glu303del)
  
• in exon 5 of TOR1A (encodes torsinA)?
• Autosomal dominant disease with reduced
  penetrance
• 30-40 of offspring from an affected or
  asymptomatic parent develop symptoms
• 70 of mutations are parental derived with
  remaining
• de novo

29
DYT1 family

• Family
• Father known to carry the common DYT1mutation
  c.907_909delGAG (p.Glu302del)
• Mother not tested
• Invasive PND on CVS DNA for first pregnancy
• PND requested for subsequent pregnancies
• Maternal plasma samples taken for non-invasive
  testing
• 2nd pregnancy (75 weeks and 85 weeks
  gestation)?
• 3rd pregnancy (73, 83 and 9 weeks gestation)?
• Spin 16-20 mls EDTA blood (within 24 hrs) ?
  plasma
• extract cfDNA using manual Qiagen Virus
  MinElute
• spin column kit

30
Confirm mutation status in parental genomic DNA

• Tested maternal gDNA to confirm mother did not
  carry mutation (only detect uniquely fetal DNA
  sequences that are paternally inherited or de
  novo)?
• Tested paternal gDNA to verify previous 3bp
  deletion heterozygote result
• Confirm presence of fetal DNA in extract
• Fetal sexing on cell-free DNA extract by
  real-time PCR
• SRY not present ? consistent female fetus
• presence of fetal DNA could not be confirmed in
  
• cfDNA extract

31
DYT1 mutation detection c.907_909delGAG
(p.Glu303del) in cfDNA

• Designed primers for short PCR product
• Run on 12 ATTO gel

cfDNA Mother gDNA Pos cont Blank H20 cont
Pos cont Wild type cont Blank H20
cont cfDNA cfDNA Mother gDNA Father gDNA
80bp 77bp
75 weeks
85 weeks
32
Confirmation on fetal tissue

• Sample from fetal tissue
• confirmed the heterozygous c.907_909delGAG
• (p.Glu303del) mutation result from testing
• maternal plasma

Ladder Fetal tissue Fetal tissue Maternal
gDNA Pos cont (het)? Wildtype Blank H20 control
Heteroduplex 80 bp 77 bp
33
Single gene disorders tested
34
SGD testing conclusion

• Analysis using PCR/polyacrylamide gel does not
  have sensitivity compared to RTqPCR or MS
• high specificity due to the detection of what
  can only
• be a paternally derived fetal mutant allele
  in the
• maternal plasma
• Primers designed ? short PCR fragments lt 200bp
• amplification of smaller-sized fetal cfDNA more
• efficient
• Advantages of using this method are simplicity,
  low costs and rapid results

35
SUMMARY

• Fetal sexing
• NIPD for fetal sexing using cfDNA successfully
  introduced as a service from 7 weeks gestation
  for XL diseases CAH
• Quality control
• Quality controls in place, SAFE QC workshops
• Minimised false /-ves by testing 2 samples a
  weeks gestation apart if lt 9 weeks 2 samples
  simultaneously if gt 9 weeks
• Maximise amount of cfDNA yield
• Confirmation of presence of fetal DNA in cfDNA
  extract
• Polymorphic markers used for female predicted
  (SRY negative) results were laborious not
  always informative
• ? Fetus specific (? RASSF1A) disease specific
  markers
• Single gene disorders
• NIPD testing set up for some skeletal
  dysplasias/cranio DYT1

36
Future

• Paternally derived or de novo mutation detection
• gt enrichment of fetal DNA and higher cfDNA
  yield (as high background of maternal DNA
  interferes with detection)?
• Develop fetal and disease specific markers for
  confirming presence of fetal DNA in extract

37
Future (cont)?

• Detect maternally or paternally derived mutations
  with new technologies such as digital PCR and
  whole gen sequencing
• Digital PCR (Lo et al, Fan)?
• NA sample is extremely diluted parallel PCRs
  undertaken so most reactions contain either a
  single or no target molecule
• Digital readout from microfluidic chip with 9180
  (12x765) 6nl reaction wells where PCR is either
  -ve or ve
• Correlates to absence or presence of target
  molecule
• Statistical analysis of proportion of ve
  reactions among the total number of PCRs analysed
  allows measurement of number of target molecules
  in input sample
• Detection of Downs b-thalassaemia in fetus and
• LOH in tumour samples and plasma of cancer
  patients

38

• High through-put shotgun sequencing Lo et al
  Fan
• (massively parallel whole genome sequencing)?
• Sequence cfDNA
• on average, 5 million sequence tags per patient
  sample
• Measure number of sequence tags mapped to each
  chromosome
• over or underrepresentation of any chromosome in
  maternal
• plasma DNA contributed by an aneuploid fetus
• Polymorphism-independent therefore universally
  applicable for NIPD of fetal aneuploidy
• said to be more sensitive that digital PCR
• Fan et al., successfully identi?ed all 9 x T21, 2
  x T18 1 x T13 in a cohort of 18 normal
  aneuploid pregnancies
• 14th week gestation earliest

39
Acknowledgements Laboratory (Regional Molecular
Genetics)? Gail Norbury Bhaneeta Mistry Lighta
Godinho Clinical (Fetal Medicine Unit, UCH)? Lyn
Chitty Melissa Whitten Taita Stojilkovic Louise
Thomasson Technical (Bristol)? Kirsten Finning,
IBGRL