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

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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