Non-invasive Prenatal Diagnosis (NIPD)

1. Non-invasive Prenatal Diagnosis (NIPD) Chris Wragg 07.01.2011 Exceptional healthcare, personally delivered

2. Prenatal testing • Prenatal testing falls into two categories: • Prenatal screening is offered to all pregnant women as part of routine antenatal care to determine if their fetus is at significant risk of having a particular disorder e.g. Down’s Syndrome, sickle cell anaemia. • In cases deemed to be high risk, prenatal diagnosis is offered, which aims to accurately determine whether the fetus has a particular disorder or characteristic Exceptional healthcare, personally delivered

3. Invasive Prenatal Diagnosis • Chorionic villus sampling (CVS) • a small sample of the placenta is taken for testing, either by inserting a needle through the abdominal wall or by passing a fine tube or forceps through the cervix • in around 8% of women, not enough tissue can be removed and the needle must be reinserted • This procedure is usually carried out between 11-14 weeks gestation • CVS causes miscarriage in around 1-2% of cases • Amniocentesis • a small amount of the amniotic fluid surrounding the fetus in the womb is removed for testing by passing a needle through the abdominal wall • in around 2% of women, not enough fluid can be removed and the needle must be reinserted. • This procedure is usually carried out after 15 weeks gestation. • Amniocentesis causes miscarriage in around 1% of cases Exceptional healthcare, personally delivered

4. Invasive Prenatal Diagnosis • The invasive approach to obtaining fetal tissue and DNA is currently the gold standard for prenatal diagnosis • Many women are reluctant to undergo invasive testing, either due to the risk to the pregnancy, or because they would not terminate the pregnancy irrespective of the results. • A reliable and convenient method for non-invasive prenatal diagnosis (NIPD) would reduce this risk of miscarriage and allow earlier diagnosis. Exceptional healthcare, personally delivered

5. Circulating Fetal Material • Placenta not an impenetrable barrier – bidirectional traffic between mother and foetus • Fetal cells (erythroblasts, trophoblasts, leukocytes) • Difficult to isolate • If isolated could, in theory culture and karyotype, however have proven intractable to this approach • Persist for years after pregnancy (up to 27 years) • Cell free fetal nucleic acid (cffNA i.e. DNA and RNA) Exceptional healthcare, personally delivered

6. cffNA • Cell-free fetal DNA (cffDNA) from the Y chromosome of male fetuses was first discovered in maternal blood • This DNA originates from placental cells, which expel their DNA into the maternal circulation during normal cell death, breaking up the chromosomes into short fragments (apoptosis). • Cell-free fetal RNA (cffRNA) derived from placentally active genes was also detected in the maternal circulation e.g. PLAC4 on chr.21 • (Unlike fetal cells) cffNA are rapidly cleared from the maternal circulation • Although all fetal elements in the maternal blood are diluted by maternal elements, the amount of cffDNA increases with gestation, comprising ~5-10% of the total cell-free DNA in maternal blood during the first and second trimesters Exceptional healthcare, personally delivered

7. cffDNA • Distinguishing (or ideally isolating), fetally derived cell-free DNA is a significant technical challenge: • The concentration of all cell-free DNA in blood is relatively low • Cell-free fetal DNA molecules are substantially outnumbered by cell-free maternal DNA molecules • The fetus inherits half its genetic sequence from the mother, making much of the cffDNA indistinguishable from maternal cell-free DNA. • A number of methods have been developed to address these problems: • Ensure no cellular contamination and maximise DNA extraction yield • Application of highly sensitive techniques e.g. digital PCR, massively parallel sequencing applications (Shotgun sequencing, padlock probes etc.), mass spectrometry (particularly RNA-SNP allelic ratio methods) • Selective enrichment of cffDNA e.g. size fractionation (fetal cffDNA fragments are generally smaller than maternal fragments), chemical enrichment also attempted (not reproducible method) Exceptional healthcare, personally delivered

8. Universal fetal specific markers • Universal fetal specific markers • Provide +ve control for other cffDNA based assays (highlight false –ve) • Act as diagnostic test in their own right • Detection of paternally inherited SNPs/STRs: • Issues with paternity and similarity/homozygosity between pat and mat polymorphisms (more common in certain populations) • Ideally want an independent marker • Detection of epigenetic differences between mother and fetus, specifically methylation • E.g. the promoter region of two TSGs – maspin and RASSF1A – are differentially methylated in the placenta relative to maternal cells • Detection of mRNA uniquely derived from active fetal DNA: • Numerous RNA species have been shown to be of placental origin and detectable in maternal plasma during pregnancy • Since the expression of these genes is unique to pregnancy, complete isolation from background maternal RNA is possible e.g. PLAC4 (chr. 21) only expressed from the placenta (i.e. fetus) Exceptional healthcare, personally delivered

9. Clinical applications • High genetic risk families e.g. family history of a known heritable genetic disorder: • fetal sex determination (in the case of sex-linked disorders and certain endocrine disorders) • certain autosomal single gene disorders (particularly where the paternal disease causing allele differs from the maternal one) • Routine antenatal screening for all pregnancies at a population level e.g. testing for Down’s Syndrome and other aneuploidies. • Management of pregnancy for pregnancies at risk of specific complications e.g. fetal RHD testing in RhD negative women (currently receive anti-D prophylaxis without testing). Exceptional healthcare, personally delivered

10. ELSI • Ethical, Legal and Social Issues • Improved safety (possible increase in uptake) • Earlier diagnosis (possible increase in terminations of pregnancies that may otherwise miscarry) • Easier testing (may mean that fully informed decisions are not facilitated) • Screening vs. diagnosis (especially for aneuploidy testing) • Direct to consumer testing; voluntary code of conduct • IP rights held by private companies e.g. Sequenom • Non-clinical applications e.g. social sex selection Exceptional healthcare, personally delivered

11. High risk genetic families • Fetal sex determination via detection of Y sequence (SRY or DYS14) in maternal plasma thereby preventing unnecessary invasive test • Presence of a female fetus inferred from negative result therefore inherently prone to false negative results • Sensitivity 99.9%, specificity 94.9% (PHG preliminary meta-analysis) • Clinical applications: • Sex-linked disorders e.g. haemophilia • Certain endocrine disorders e.g. CAH • Autosomal single gene disorders (particularly where the paternal disease causing allele differs from the maternal one) • Detection of point mutations challenging (especially homozygosity) and enrichment of cffDNA generally required • Paternally inherited AD conditions e.g. Huntington’s disease • Exclusion of AR disease via compound heterozygosity where paternal allele has not been inherited e.g. CF, β-thalassaemia (often compound heterozygosity and therefore may be amendable to this approach) • Reduction in # invasive tests, and as cffDNA testing ~half cost of invasive test may offer a slight reduction in cost of antenatal care • Each test would require UKGTN gene dossier Exceptional healthcare, personally delivered

12. High risk genetic families • Reduction in # invasive tests, and as cffDNA testing ~half cost of invasive test may offer a slight reduction in cost of antenatal care • Need to maintain expertise in invasive techniques • Relatively low throughput therefore managed by current infrastructure (Regional Genetics Labs) • Each test would require UKGTN gene dossier • ?Local labs offer all approved tests • Different specialist labs offering tests nationally for specific diseases Exceptional healthcare, personally delivered

13. Routine Antenatal Screening • Aneuploidy; current practice all women receive antenatal screening (serum screening, U/S) with invasive testing for at risk pregnancies • NIPD may: • increase the accuracy of the screening risk assessment, or if it sufficiently robust • reduce or replace the need for invasive testing • Techniques required to detect the relative amount of specific chromosomal material e.g. chr.21 • Broadly 2 current methods Exceptional healthcare, personally delivered

14. Routine Antenatal Screening • Exploits presence of heterozygous SNPs to calculate a ratio between the SNPs • e.g. Sequenom announced 5000 patient trial of mass spec. method to detect multiple SNPs across several placentally derived genes reported to offer 95% detection rate for US Caucasian population • Detection rate will vary with ethnicity Exceptional healthcare, personally delivered

15. Routine Antenatal Screening • Chromosome dosage measured directly by using highly sensitive techniques to compare the ratio of the chromosome of interest to a reference chromosome; • Does not rely upon polymorphisms • Techniques include: • Digital PCR, in which enriched single template DNA or RNA molecules are isolated by dilution and then amplified (labour intensive) • Direct high-throughput shotgun sequencing, in which millions of sequence tags are detected across the genome Exceptional healthcare, personally delivered

16. Routine Antenatal Screening • Logistics of a population level service: • Most NHS genetic labs would not be able to offer at present • Higher throughput and more specialist technologies would need to be implemented • Commissioning • Cost of cffNA testing currently unclear • Would significantly reduce the number of invasive tests • Unlikely would replace US • However uptake of cffNA much higher (~80-90% of pregnancies) • Current cost of biochemical screening (£15-25) significantly lower than predicted cost of cffNA • Accurate economic assessment required before implementation • However new screening programme would need to meet requirements of NSC Exceptional healthcare, personally delivered

17. Routine Antenatal Screening • Any new test would need to fulfil the formal screening criteria (see www.nsc.nhs.uk/pdfs/criteria.pdf ) and a large evidence base would be required • In the UK a number of national bodies would be involved in assessing the technology prior to offering it at a population level: • The National Screening Committee (NSC), which would ultimately be responsible for approving and implementing any national screening programmes. Using research evidence, pilot programmes and economic evaluation, it assesses the evidence for programmes against a set of internationally recognised criteria • The National Institute of Clinical Excellence (NICE) • The Health Technology Assessment (HTA) Programme (run by the UK National Institute for Health Research (NIHR) • NIHR funds RAPID project (Reliable Accurate Prenatal non-Invasive Diagnosis http://www.rapid.nhs.uk/ ) • The Human Genetics Commission (HGC) is the government’s advisory body on new developments in human genetics Exceptional healthcare, personally delivered

18. SAFE and RAPID • The Special non-invasive Advances in Fetal and neonatal Evaluation (SAFE) Network of Excellence (NoE) – EU funded 2004-2009 • Reliable Accurate Prenatal non-Invasive Diagnosis (RAPID) is a 5 year UK national programme funded by the National Institute for Health Research: • Hosted by NGRL (Wessex) • Develop NIPD for DS testing in collaboration with ICH / GOSH using • targeted new generation sequencing • (MALDI-TOF mass spectrometry) • digital PCR • Define Down Syndrome (DS) test analytical sensitivity and specificity • Develop prototype reference materials in collaboration with NIBSC & NGRL (M) • Produce standardised protocols in collaboration with GOSH & NGRL (M) • Participate in a model-based economic evaluation to assess incremental cost effectiveness of NIPD versus current testing methodology Exceptional healthcare, personally delivered

19. UK National Screening Committee (NSC) • The remit and terms of reference of the UK NSC are: • Advise Ministers and the NHS in all four UK countries about:: • the case for implementing new population screening programmes not presently provided by the NHS within each of the countries in the UK; • screening technologies of proven effectiveness but which require controlled and well-managed introduction; • the case for continuing, modifying or withdrawing existing population screening programmes. In particular, programmes inadequately evaluated or of doubtful effectiveness, quality, or value; • Generic issues relating to screening programmes and policy. • Call on sound evidence to inform its advice and recommendations. In particular: • HTA • NIHR (Chief Scientist's Office and NHS Quality Improvement Scotland) • calling on other and appropriate sources of sound evidence • Agree standards for the new programmes • The UK NSC will be informed by reports from the Advisory Groups or committees for specific programmes in each country on the performance of those programmes and issues that arise which would have relevance to general screening policy. Exceptional healthcare, personally delivered

20. References • Reliable Accurate Prenatal non-Invasive Diagnosis (RAPID) http://www.rapid.nhs.uk/ • Cell-free fetal nucleic acids for non-invasive prenatal diagnosis. Report of the UK expert working gtoup. Caroline Wright January 2009 (www.phgfoundation.org) • Wright C. The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis. Human Reproduction Update 2009; 15(1):139-151 • Lo Y. Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art. BJOG 2009;116:152-157 • UK National Screening Committee ( www.scrrening.nhs.uk ) Exceptional healthcare, personally delivered