Prenatal diagnosis using free fetal DNA
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Prenatal diagnosis using free fetal DNA






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Prenatal diagnosis using free fetal DNA Prenatal diagnosis using free fetal DNA Presentation Transcript

  • 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 Aneuploidy Single gene disorders Haemoglobinopathies CVS CORDOCENTESIS AMNIOCENTESIS
  • Other sources of fetal tissue for PND Difficult to isolate and persist for years after pregnancy Cell free fetal DNA in the maternal circulation Detectable from 5 weeks’ Cleared from circulation within 30 minutes of delivery 3 – 6% of total circulating cell free DNA Originates from trophoblast Fetal cells in maternal circulation erythroblasts trophoblastic cells leucocytes View slide
  • Quantification and normal ranges Lo et al. 1998 Am J Hum Genet 25.4ge/ml 3.4% 292.2ge/ml 6.2% 0 50 100 150 200 250 300 11-17 weeks 37- 42 weeks fetal DNA conc % total DNA View slide
  • Current use for NIPD in the UK
    • Fetal sex determination
    • Fetal RhD status in high risk pregnancies
    • Some single gene disorders
  • Fetal sex determination Indications
    • 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 – 16 0 )
    • 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 2004-6
  • Effect on management 22 avoided CVS 4 declined CVS 2 25 29 X-linked 2 avoided amniocentesis 0 1 2 Ambiguous genitalia 5 avoided CVS 2 avoided steroids 4 stopped steroids <11w 1 3 6 CAH 46% reduction in invasive procedures 3 29 39 Total Effect on management Failed Female Male Indication 1 avoided amniocentesis 0 2 Discordant genotype / phenotype
    • 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
    P rospective R egister of O utcomes O f F ree-fetal DNA testing PROOF 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
  • Gestational age at testing
  • Results 88.5% outcomes obtained 91.2% accuracy overall in cases with outcomes 97.8% accuracy in results in tests > 7 weeks Sex on testing/USS/birth 25 71 66 161 total 4 5 2 11 no result 16 63 2 81 female 5 3 62 70 male n/k female male n ffDNA
  • Discordant results CAH. USS indicated female retested 16 weeks = male female male 5 CAH. USS indicated female Retested later in pregnancy = female female male 8 DMD. CVS showed female. Would have failed with new standards ?vanishing twins Retest at 12 weeks = female female male 9 Ectodermal dysplasia - CVS Retested at 12 wks = male male female 8 CAH. Too early repeat advised. Retested >7 wks = male male female 5 comment outcome ffDNA weeks
  • Reporting times Sample received in lab – report issued 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
  • Trends in use of ffDNA for fetal sexing 04.06 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 Potential in routine antenatal care Check fetal RHD group Potential for 44% reduction in AntiD Prevent exposure to blood products Save £1,000,000 pa AntiD 28 weeks AntiD 34 weeks AntiD 40 weeks +ve -ve No Anti-D
  • Study of high throughput analysis 1614 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 Birmingham 1440 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 Birmingham 1440 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 = 132bp Wild type = 132bp G380R G>A heterozygote = 132bp + 112bp + 20bp 100bp 150bp Courtesy of Gail Norbury, NE Thames Regional Genetics 50bp ladder Uncut PCR product 5ml of 400ml plasma sample 10ml of 400ml plasma sample 20ml of 400ml plasma sample 5ml of 800ml plasma sample 10ml of 800ml plasma sample 20ml of 800ml plasma sample Normal control G380R G>A positive control Water blank
    • 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 applications
    • 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 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 For 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 information
  • Non-invasive prenatal diagnosis Implications for antenatal diagnosis and the management of high risk pregnancies - Joint RCOG/SAFE Meeting Thursday 13 March 2008