Non-invasive molecular prenatal diagnosis

1. Non-invasive molecular prenatal diagnosis Oxford BRC molecular diagnostic laboratory Dr Shirley Henderson Consultant Clinical Scientist

2. Conventional approach to Prenatal Diagnosis Fetal sampling Ultrasound Chorionic villi Amniotic fluid DNA diagnostic test

3. Prenatal Diagnosis • 1982:- The first ever prenatal diagnoses by fetal DNA analysis in the UK (β-thalassaemia and sickle cell disease). John Old et.al • Carried out in the National Haemoglobinopathy Reference Laboratory (NHRL), John Radcliffe Hospital • This was performed on DNA extracted from an amniotic fluid (AF) sample. • Constituted a major breakthrough for prenatal diagnosis. • Now the standard approach – 30 years

4. Another safer source of fetal DNA for diagnosis? • 1997 :- Discovered in John Radcliffe Hospital, Oxford by Jim Wainscoat & Dennis Lo et. al. • Most cell free DNA in plasma is maternal (from haematopoietic system) • Free fetal DNA is from placenta (trophoblasts) • 5-15% of plasma DNA is from the fetus

5. Plasma DNA approach Cell free DNA Double spin plasma to remove any remaining white cells and platelets DNA extraction

6. Main problems with cff-DNA • Concentration of cff-DNA is low and it is degraded • Once a blood sample is taken the fetal DNA deteriorates very rapidly, background maternal DNA will increase as white blood cells in the sample breakdown. Plasma needs to be separated within 6 hours of the sample being taken. • Cell free fetal DNA molecules substantially out-numbered by cell free maternal molecules • Currently no way to separate fetal DNA from the maternal plasma DNA. Fetus inherits 50% of it’s genetic sequence maternally- making much of the cffDNA indistinguishable from the mother

7. Some success stories – all when allele not present in the mother Non-invasive fetal sex determination Maternal plasma with male fetus Maternal plasma with female fetus Female plasma xx xy xx xx xx xx xx xx xx xx xx xx xx xx xy xx xx xx xx xx xx xx xx xx xx Method used to detect the y chromosome, usually RT-PCR

8. Other examples:- • Fetal Rh status • Achondroplasia In development for other conditions but not straightforward:- • Need sensitive technique to assay low levels • DNA is degraded and can be difficult to work with - assays can need a lot of optimisation • Negative depends on “no result”

9. Down’s syndrome (Trisomy 21) • Main focus for NIPND research so far has been Down’s syndrome • Next generation sequencing (NGS), whole genome approach :-involves the random sequencing of millions of DNA molecules in maternal plasma. • Individual sequence tags are aligned to the human genome to determine the chromosome of origin of a particular sequence tag. • An increase in representation of sequence tags aligned to a particular chromosome indicating a potential trisomy. • Effective but very expensive and requires access to high-throughput NGS platforms. • World-wide there are currently only limited clinical services offering this service, none in the UK

10. What about the classic single gene disorders where the mother is a carrier for the condition? • Haemophilia • Cystic fibrosis • Sickle cell disease • Thalassaemia X-linked inheritance Autosomal recessive inheritance

11. Sickle cell disease - first molecular disease DNA-Sickle mutation Protein Pathophysiology Inheritance Prevalence Immigration

12. Sickle Cell Disease in the UK • Birth Incidence:- Nationally 1:2000 (higher than cystic fibrosis) S.E. London 3:1000 • Most common indication for invasive PND in the UK • Approximately 440 PNDs carried out per year in UK

13. NHS National Antenatal screening programme • Main purpose to identify couples at risk of having a child with a major haemoglobinopathy (sickle cell and thalassaemia) so prenatal diagnosis can be offered. • UK divided into 2 types of area:- • High prevalence areas - non-selective screening • Low prevalence areas - selective screening based on family origins

14. ?NIPND for sickle cell disease • Recent development has seen the launch of bench top sequencers which are scaled down cost-effective NGS platforms, driven by the need for faster and more cost-effective sequencing both in research and for the determination of genetic variants in patients:- • Life Technologies Ion torrent, Roche Junior, Illumina MiSeq • Targeted approach cheaper than whole genome approach.

15. Principle of allelic imbalance in maternal plasma Sickle carrier plasma with disease (SS) fetus Sickle carrier plasma with carrier (AS)fetus Sickle carrier plasma with normal (AA) fetus Sickle carrier plasma 10 12 8 8 Sickle copies 8 12 10 8 Normal copies

16. Plan Amplify the sickle cell gene in maternal plasma – will result in millions of copies of the sickle cell gene Sequence all these reads using NGS technology Count how many of the reads have the sickle mutation and how many have the normal sequence. >50% sickle cell reads <50% sickle cell reads 50% sickle cell reads Sickle Disease Fetus Normal Fetus Sickle Carrier Fetus

17. Challenges • Obtaining fresh enough plasma • Measuring the proportion of fetal DNA in the sample • Efficient amplification of low copy number poor quality DNA • Need all fetal molecules to be amplified • Preservation of the starting ratio of sickle and normal molecules • PCR amplification can change ratios (cold PCR effects) • Development of NGS technology for this application

18. Process Plasma separation DNA extraction QIAamp Circulating Nucleic Acid Kit Plasma DNA concentration and fetal fraction determination RASSF1 assay / Qubit amplicon based Library preparation Nested PCR x3 amplicons Purification MinElute PCR Purification kit Quantification and Normalisation Bioanalyser Miseq Paired-end sequencing (84|6|84)

19. Targets • Design 3 overlapping small amplicons • Amplicon1 • chr11:5248185-5248276 • TCACTAGCAACCTCAAACAGACACCATggtgcatctgactcctgaggagaagtctgccgttactgccCTGTGGGGCAAGGTGAACGTGGATG • Amplicon 2 • chr11:5248192-5248274 • ACTAGCAACCTCAAACAGACACCATGgtgcatctgactcctgaggagaagtctgccgtTACTGCCCTGTGGGGCAAGGTGAAC • Amplicon 3 • chr11:5248202-5248270 • GCAACCTCAAACAGACACCATggtgcatctgactcctgaggagaagtctgcCGTTACTGCCCTGTGGGG • bp targets are in red: chr11:5,248,243; chr11:5,248,233; chr11:5,248,232

20. Reads data 5% AC Affected (SS) fetus 8% AC Normalised data for runs 1, 2 and 3

21. Normalised data – amplicons 1, 2, & 3 5% AC 8% AC Affected (SS) fetus

22. Normalised data - Amplicon 3, S mutation

23. Conclusions Successful proof of principle, spiking experiments and real samples – it works! Cost effective and rapid:- New bench top NGS analyser Targeted sequencing approach rather than whole genome. Simple amplicon based library prep – low cost and quick Next stage – proper clinical evaluation, validation etc.

24. Summary • cff-DNA is a potential “safe” source of fetal DNA for PND • NIVPND in routine use for a small number of conditions where allele in fetus is not present in the mother • NIVPND currently possible for aneuploidy – but expensive and not widely available • In development for single gene disorders – new lower cost NGS platforms hold great promise, NIVPND could become a cost effective reality in routine molecular diagnostic laboratories.

25. Acknowledgements

26. Acknowledgements Illumina David Bentley Mark Ross Jennifer Becq Sean Humphray David McBride Oxford Hematology/Oncology Chris Hatton Tim Littlewood Paresh Vyas Bass Hassan Mark Middleton ORB Tissue Banking Maite Cabes Research Nurse Christopher Levett UK NCRN CLL Subgroup Peter Hillmen Andy Pettitt Oxford BRC Genomics Jenny Taylor Sam Knight Chris Yau Chris Holmes Oxford BRC/NHS SIHMDS Anna Schuh John Old Adam Burns Ruth Clifford Adele Timbs Our Patients Clinical (Bio-)Informatics Jim Davies Joe Wood Minji Ding Jean Baptiste Cazier Michalis Titsias EUROSARC Oxford Pathology Runjan Chetty Liz Soilleux

28. Normalised data for run 7 25% AC 15% AC 8% AC 5% AC

29. Spiking experiments 8% AC 25% AC 15% AC 5% AC AS (50:50) control

30. Reproducibility comparison histograms of run 7 25% AC 15% AC 8% AC 5% AC

31. SS AS AA