prenatal diagnosis using free fetal dna
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Prenatal diagnosis using free fetal
DNA
Lyn Chitty
Reader in Genetics and Fetal Medicine
Clinical and Molecular Genetics, Institute of Child Health, London&
Fetal Medicine Unit, University College Hospital
1% risk of miscarriage
Not possible before 11 weeks’
Current prenatal diagnosis requires invasive procedures
CVS
CORDOCENTESIS
AMNIOCENTESIS
Aneuploidy
Single gene
disorders
Haemoglobinopathi
es
Other sources of fetal tissue for PND
Difficult to isolate and persist for years after pregnancy
Fetal cellsFetal cells in maternal in maternal circulationcirculation
erythroblaststrophoblastic cellsleucocytes
Cell free fetal DNACell free fetal DNA in the maternal circulationin the maternal circulation
Detectable from 5 weeks’ Cleared from circulation within 30 minutes of delivery3 – 6% of total circulating cell free DNAOriginates from trophoblast
Quantification and normal ranges
25.4ge/ml3.4%
292.2ge/ml
6.2%
0
50
100
150
200
250
300
11-17 weeks 37- 42 weeks
fetal DNA conc
% total DNA
Lo et al. 1998 Am J Hum Genet
Current use for NIPD in the UK
• Fetal sex determination
• Fetal RhD status in high risk pregnancies
• Some single gene disorders
Fetal sex determinationIndications
• Risk of X-linked disorders
• Risk of congenital adrenal hyperplasia
• Genital ambiguity detected on ultrasound
• As an aid to prenatal diagnosis in some renal anomalies or genetic syndromes
• 74 cases tested at 10 weeks (6+5 – 160)
• Sex determination using ffDNA
– Sex determined by invasive testing or at birth in 66
– Repeat testing needed in 7 (9%)
– Failed completely in 3 (4%)
– 2 miscarriages
– 3 lost to follow-up
• No discordant cases
UCLH experience 2004-6UCLH experience 2004-6
Effect on management
X-linked 29 25 2 22 avoided CVS4 declined CVS
Ambiguous genitalia
2 1 0 2 avoided amniocentesis
CAH 6 3 1 5 avoided CVS2 avoided steroids4 stopped steroids <11w
Total 39 29 346% reduction in invasive procedures
Indication Male Female Failed
Effect on management
Discordant genotype / phenotype
2 0 1 avoided amniocentesis
PProspective rospective RRegister of egister of OOutcomes utcomes OOf f FFree-fetal ree-fetal DNA testingDNA testing
PROOFPROOF
• Audit of all NIPD tests done for fetal sexing in the UK for clinical indications from 1.4.2006 – 31.3.2007
• Two labs offering the test. One using DYS14 and the other SRY
• Tests performed for units in UK, Ireland, Canada, Germany
• Samples sent in by post to arrive within 48 hours
• Ethical approval from ULCH REC
Finning et al in preparation
Results
• 160 women with 202 ffDNA tests performed
• 28 repeats due to no result issued
• 14 repeats for marker testing
55%
22%
11%
2%4%
5%1%
x linked
haemophilia
CAH
Other (renal)
ultrasound
discordant amnio
no indication given
Gestational age at testing
Gestation at First Sample
0102030405060
Results
Sex on testing/USS/birth
ffDNA n male female n/k
male 70 62 3 5
female 81 2 63 16
no result 11 2 5 4
total 161 66 71 25
88.5% outcomes obtained
91.2% accuracy overall in cases with outcomes 97.8% accuracy in results in tests > 7 weeks
Discordant results
weeks ffDNA outcome
comment
5 female male CAH. Too early repeat advised. Retested >7 wks = male
8 female male Ectodermal dysplasia - CVSRetested at 12 wks = male
9 male female DMD. CVS showed female. Would have failed with new standards ?vanishing twinsRetest at 12 weeks = female
8 male female CAH. USS indicated femaleRetested later in pregnancy = female
5 male female CAH. USS indicated femaleretested 16 weeks = male
Reporting timesSample received in lab – report issued
0
10
20
30
40
50
Number of Days from Receipt to Report
NR
F
M
72.0% within 3 days
98.7% within 7 days
Effect on invasive testing
There was no invasive test in: 45% all pregnancies
18% of those with male fetuses
66% of pregnancies with female fetuses
Invasive Procedures for X-Linked Conditions
0102030405060
Female Male NR
ffDNA Result
Unknown
Lost to FU
No Test
Invasive Test
Trends in use of ffDNA for fetal sexing
0
5
10
15
20
25
30
35
Month04.06 08.07
Manchester
Conclusions
• Fetal sexing using ffDNA is 98% accurate when performed >7 weeks.
• It reduces invasive testing by around 45%
• Criteria for reporting sex must be very stringent
• Further investigation of the aetiology of false positive males is needed.
• Development of sex-independent fetal markers is needed.
• Early ultrasound (in an FMU) can be offered to confirm sex from 12 weeks’.
Male Female
Opinion
• Women should be informed of risks and benefits of ffDNA testing and allowed to make informed choices.
• There appears to be a trend towards offering sexing using ffDNA rather than USS is some conditions, eg haemophilia where invasive testing would not usually be offered. This may have significant service and economic implications.
• The audit must be continued to review change in laboratory practice and inform patient counselling
NIPD and RHD
• D+ / D- phenotype is usually due to the presence or absence of the RHD gene, respectively
• If RHD gene sequences are detected in the plasma of a D- woman, the fetus is predicted to be D+
• If no RHD is detected, the fetus is predicted to be D-
NIPD and RHD
RHD RHCE
D+
RHD RHCE
or
D-
Potential for 44% reduction in AntiD
Prevent exposure to blood products
Save £1,000,000 pa
NIPD and RHD Potential in routine antenatal care
AntiD 28 weeks
AntiD 34 weeks
AntiD 40 weeks
+ve
Check fetal RHD group -ve
No Anti-D
Study of high throughput analysis1614 samples at 28 weeks
• High-throughput screening for fetal RHD blood group is possible and is highly accurate – 97% with 2.4% inconclusive results
• If current programmes remain unchanged, knowledge of fetal RHD status will be needed BEFORE 28 weeks
• The amount of fetal DNA in maternal plasma increases throughout the pregnancy
• Next step is to test samples earlier in pregnancy– at booking – with DSS screening – At 20 week anomaly scan
Finning et al BMJ n press
RfPB study at ULCH, Bristol and RfPB study at ULCH, Bristol and BirminghamBirmingham
1440 RHD- women1440 RHD- women
All RhD- women - NIPD for fetal RhD status at booking
Check RhD on all cord bloods
RhD-
No anti-D
RhD+
Anti-D
RhD+
Anti-D
RhD-
Repeat NIPD testing at 28 weeks
RfPB study at ULCH, Bristol and RfPB study at ULCH, Bristol and BirminghamBirmingham
1440 RHD- women1440 RHD- women
All RhD- women - NIPD for fetal RhD status at booking
Check RhD on all cord bloods
RhD-
No anti-D
RhD+
Anti-D
RhD+
Anti-D
RhD-
Repeat NIPD testing at 28 weeks
Consent
• Myotonic dystrophy
• Achondroplasia
• Cystic fibrosis
• B-thalassaemia
• Congenital adrenal hyperplasia
• Huntingtons disease
Detection or exclusion of paternal or de-novo mutation
Single gene disorders
NIPD of achondroplasia
PCR product = 132bpWild type = 132bpG380R G>A heterozygote = 132bp + 112bp + 20bp
50
bp ladder
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150bp
Courtesy of Gail Norbury, NE Thames Regional Genetics
• ffDNA is a reliable way of determining fetal sex from around 7 weeks gestation
– It can reduce the need for invasive procedures in high risk women by up to 50%
– In CAH it can result in early cessation of steroid treatment
– NIPD using ffDNA should be offered as an alternative to invasive testing or fetal ultrasound for early determination of fetal sex.
• ffDNA will be useful in screening for fetal RHD in RHD- women
• ffDNA can be useful in diagnosis of genetic disorders occurring de novo or to look for paternal mutations
Summary of current Summary of current applicationsapplications
• Improved diagnosis of single gene disorders– Improved extraction of cffDNA– Better fetal markers
• Role in early determination of fetal RHD status to target immunoprophylaxis
• Diagnosis of aneuploidy– RNA ratios– Epigenetics– Proteomics
• Role of circulating fetal nucleic acids and proteins in management of obstetric complications
The futureThe future
• Development of laboratory protocols
• Careful evaluation of laboratory and clinical
utility
• Health professional and public education
• Careful consideration of the ethical issues
The future The future For implementationFor implementation
Acknowledgements
• IBGRL – in Bristol for the data on RHD typing
• ICH/GOSH Science development fund
• SAFE – funding personnel performing laboratory
development and the audit
For more informationFor more information
www.safenoe.org
Non-invasive prenatal diagnosis
Implications for antenatal diagnosis and the management of high risk pregnancies - Joint RCOG/SAFE Meeting
Thursday 13 March 2008
www.rcog.org.uk/meetings
www.safenoe.org