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
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
85%
15%
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
4.
Confirm result by a different diagnostic method
5.
Check for maternal DNA contamination
6.
Report result with error risk
7.
3 working days turnaround time target
The two DNA methods for PND’s
1. Sickle cell:
•
•
ARMS-PCR
RE-PCR using Dde 1
2. -thalassaemia:
•
•
ARMS-PCR
Sanger sequencing
3. -thalassaemia:
•
•
Hb Bart’s hydrops: Gap-PCR + MLPA
Hb H hydrops:
Sanger sequencing
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 -thalassaemia mutations, only
have one approach – Sanger sequencing.
Molecular basis of -thalassaemia
Total number mutations
• Point mutations
• Large deletions
230
18
Four regional groups:
• Mediterranean, Indian, Chinese, African
Number per ethnic group
• Common mutations 3 - 10
• Rare mutations
10 - 30
-Thalassaemia mutations diagnosed in
the UK population
Total number of different alleles diagnosed for PND: 42
•
•
•
•
•
Mediterranean
Asian Indian
Southeast Asian
African
British
15
16
8
2
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
PND for -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 -thalassaemia, especially for
cases involving combinations of rare mutations.
it is an interactive database on the Ithanet Portal (www.ithananet.eu)
•
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 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.
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 -thalassaemias (20)
• Sanger sequencing for -thalassaemias (3)
Pyrosequencing
•Technology is now more robust
• Instrumentation costs have gone down
Example: detection of the -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
Pyrosequecing – detection of -thalassaemia
mutation IVSI-5 G→C genotypes
A2 : AC/ G/ TCAAC/ A/ TCTGCCCAGGGCCTCACCACCAACTTCA
C : 98%
A: 0%
A A
G: 1%
C : 100%
T: 1%
T: 0%
β /β
150
100
50
0
E
S
T
A
G
T
C
A
T
G
C
5
A3 : AC/ G/ TCAAC/ A/ TCTGCCCAGGGCCTCACCACCAACTTCA
C : 50%
A: 0%
G: 49%
C : 100%
T: 1%
T: 0%
G
T
G
C
A
10
*
βA/IVS1-5 G→C
200
150
100
50
E
S
T
A
G
*
0
T
C
A
T
G
C
G
5
T
G
C
A
10
A4 : AC/ G/ TCAAC/ A/ TCTGCCCAGGGCCTCACCACCAACTTCA
C : 0%
A: 0%
G: 100%
C : 100%
T: 0%
T: 0%
IVS1-5 G→C/IVS1-5 G→C
150
100
50
0
E
S
T
A
G
*
T
C
5
A
T
G
C
G
10
T
G
C
A
Pyrosequecing – detection of sickle cell
(Cd 6 A→T) genotypes
A2: WCAGGAGTCAGGTGCACCATGGTGTCT
A: 1%
T: 99%
AA
100
75
50
25
0
E
S
G
A
T
C
A
G
A
G
T
5
A3: WCAGGAGTCAGGTGCACCATGGTGTCT
A: 44%
T: 56%
AS
100
75
50
25
0
E
S
G
A
T
C
A
G
A
G
T
5
A5: WCAGGAGTCAGGTGCACCATGGTGTCT
A: 98%
T: 2%
SS
75
50
25
0
E
S
G
A
T
C
A
5
G
A
G
T
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.
-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
-thalassaemia alleles
Hb Adana
Hb Constant Spring
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
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
Acknowledgements
Adele Timbs
Michelle Rugless
Alice Gallienne
Anna Haywood
Shirley Henderson