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 %
Amniotic fluid %

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:
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
Large deletions
Four regional groups:
Mediterranean, Indian, Chinese, African
Number per ethnic group
Common mutations
Rare mutations

7. b-Thalassaemia mutations diagnosed in the UK population
Total number of different alleles diagnosed for PND: 42
Mediterranean
Asian Indian
Southeast Asian
African
British
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:

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 (
Mutations: Ithanet base is a database containing up to date information for all known thalassaemia and Hb variant mutations.
Frequencies: Ithanet base lists the geographical distribution of all the b-thalassaemia mutations, and their allele frequencies in each country.
Genotype / Phenotype: Ithanet base will 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 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) genotypesAA AS SS

14. Pyrosequencing results to date
66 fetal samples tested: all pyrosequencing results agreed with the first diagnosis result. 1 fetal sample not reported due to maternal DNA contamination.
Sickle cell alleles
Hb S
Hb C
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)
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. Acknowledgements
Adele Timbs Michelle Rugless Alice Gallienne Anna Haywood Shirley Henderson