Rapid prenatal diagnosis of sickle cell disease and thalassaemia by pyrosequencing

1. Rapid prenatal diagnosis of sickle cell disease and thalassaemia by pyrosequencing Dr. John Old National Haemoglobinopathy Reference Laboratory Molecular Haematology John Radcliffe Hospital, Oxford, UK

2. Prenatal diagnosis NHRL carries out approx 180 PNDs per year for UK patients: • 56% sickle cell disease • 42% -thalassaemia • 2% -thalassaemia Sources of fetal DNA : • Chorionic villi 85% • Amniotic fluid 15%

3. Current best practice PND procedure 1. Use fresh parental blood samples for control DNAs 2. Use cleaned & sorted chorionic villi 3. Test for mutations in more than one fetal DNA aliquot with appropriate controls • Confirm result by a different diagnostic method • Check for maternal DNA contamination • Report result with error risk • 3 working days turnaround time target

4. The two DNA methods for PND’s • Sickle cell: • ARMS-PCR • RE-PCR using Dde 1 • b-thalassaemia: • ARMS-PCR • Sanger sequencing • a-thalassaemia: • Hb Bart’s hydrops: Gap-PCR + MLPA • Hb H hydrops: Sanger sequencing

5. Problems with current approach • Second method can be time consuming • RE-PCR for Hb S requires O/N digestion • Sanger sequencing takes 2 days. • Sanger sequencing is expensive as confirmatory method • For very rare b-thalassaemia mutations, only have one approach – Sanger sequencing.

6. Molecular basis of b-thalassaemia Total number mutations Point mutations 230 Large deletions 18 Four regional groups: Mediterranean, Indian, Chinese, African Number per ethnic group Common mutations 3 - 10 Rare mutations 10 - 30

7. b-Thalassaemia mutations diagnosed in the UK population Total number of different alleles diagnosed for PND:42 • Mediterranean 15 • Asian Indian 16 • Southeast Asian 8 • African 2 • British 1 Total number diagnosed for all patients:68 Total number diagnosed in indigenous Britons: 9 Reference: Incidence of haemoglobinopathies in various populations - The impact of immigration. Henderson S, Timbs A, McCarthy J, Gallienne A, Van Mourik M, Masters G, May A, Khalil M, Schuh A, Old J. Clinical Biochemistry (2009); 42:1745-1756

8. PND for b-thalassaemia: Ithanet base Ithanet base is being developed as a unique database resource of genotype / phenotype information and thus will become a valuable tool to aid the prenatal diagnosis of b-thalassaemia, especially for cases involving combinations of rare mutations. it is an interactive database on the Ithanet Portal (www.ithananet.eu) • Mutations: Ithanet baseis a database containing up to date information for all known thalassaemia and Hb variant mutations. • Frequencies: Ithanet baselists the geographical distribution of all the b-thalassaemia mutations, and their allele frequencies in each country. • Genotype / Phenotype: Ithanet basewill be designed to collect haematological data for each mutation and mutation combination. The Ithanet base is part of the Ithanet Portal project, funded by the Research Promotion Foundation of Cyprus through structural funds.

9. Pyrosequencing Pilot Project • Looked at 67 fetal samples which had been tested first by ARMS-PCR • CVS DNA 50 cases • AF DNA 17 cases • Used pyrosequencing as the 2nd confirmatory test instead of • RE-PCR for Hb SS and Hb SC disease (44) • Sanger sequencing for b-thalassaemias (20) • Sanger sequencing for a-thalassaemias (3)

10. Pyrosequencing • Technology is now more robust • Instrumentation costs have gone down

11. Example: detection of the a-gene mutation: Cd 68 AAG→AAC * * Normal DNA: Cd 68 AAG→AAC. Results read 100%C * * Heterozygous DNA; Cd 68 AAG→AAC. Results show 50% C and 50% G

12. Pyrosequecing – detection of b-thalassaemia mutation IVSI-5 G→C genotypes βA/βA * βA/IVS1-5 G→C * IVS1-5 G→C/IVS1-5 G→C *

13. Pyrosequecing – detection of sickle cell (Cd 6 A→T) genotypes AA AS SS

14. 66 fetal samples tested: all pyrosequencing results agreed with the first diagnosis result.1 fetal sample not reported due to maternalDNA contamination. Pyrosequencing results to date b-thalassaemia alleles Hb E Codon 8/9 +G CD 30 GA IVSI-1 GT IVSI-5 GC CD 41/42 (-TCTT) Sickle cell alleles Hb S Hb C a-thalassaemia alleles Hb Adana Hb Constant Spring

15. Pyrosequencing – conclusions 1 • Cheaper, simpler and quicker than conventional Sanger sequencing • Approx 1/5 the cost of Sanger sequencing • Higher degree of accuracy than ARMS • Mutation is detected in the context of its surround sequence • Fewer pitfalls than gel based methods • can test for several mutations at once • don’t need positive control for every mutation • Much quicker than RE-PCR methodology

16. Pyrosequencing – conclusions 2 • More robust – lower failure rate than Sanger sequencing • Sanger sequencing takes 2 days (5 steps) • Pyrosequencing takes 0.5 days (2 steps) • More sensitive than Sanger sequencing and RE-PCR • Works with much lower quantities of DNA • Results are quantitative – results reflect any allelic imbalance: mosaicism, vanishing twin or maternal contamination • Set up costs are cheaper than Sanger sequencing – more suitable for PND in developing countries

17. Adele TimbsMichelle RuglessAlice GallienneAnna HaywoodShirley Henderson Acknowledgements