Rapid prenatal diagnosis of sickle cell disease and thalassaemia by pyrosequencing Dr. John Old...

Rapid prenatal diagnosis of sickle cell disease and thalassaemia by pyrosequencing

Dr. John Old

National Haemoglobinopathy Reference LaboratoryMolecular 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 85%• Amniotic fluid 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 230• Large deletions 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 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

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.

http://www.ithananet.eu/

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

C: 98%G: 1%T: 1%

A: 0%C: 100%T: 0%

EE SS TT AA GG TT CC

5

AA TT GG CC GG

10

TT GG CC AA0

50

100

150

A2: AC/ G/ TCAAC/A/TCTGCCCAGGGCCTCACCACCAACTTCA

C: 50%G: 49%T: 1%

A: 0%C: 100%T: 0%


5

AA TT GG CC GG

10

TT GG CC AA0

50

100

150

200


C: 0%G: 100%T: 0%

A: 0%C: 100%T: 0%


5

AA TT GG CC GG

10

TT GG CC AA0

50

100

150


βA/βA

βA/IVS1-5 G→C

IVS1-5 G→C/IVS1-5 G→C

*

*

*

Pyrosequecing – detection of sickle cell (Cd 6 A→T) genotypes

A: 1%T: 99%

EE SS GG AA TT CC AA

5

GG AA GG TT0

25

50

75

100

A2: WCAGGAGTCAGGTGCACCATGGTGTCT

A: 44%T: 56%


5

GG AA GG TT0

25

50

75

100


A: 98%T: 2%


5

GG AA GG TT0

25

50

75


AA

AS

SS

66 fetal samples tested: all pyrosequencing results agreed with the first diagnosis result.

1 fetal sample not reported due to maternal DNA contamination.

-thalassaemia allelesHb ECodon 8/9 +GCD 30 GAIVSI-1 GTIVSI-5 GCCD 41/42 (-TCTT)

Pyrosequencing results to date

-thalassaemia allelesHb Adana Hb Constant Spring

Sickle cell allelesHb SHb C

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

Adele TimbsMichelle RuglessAlice GallienneAnna HaywoodShirley Henderson

Acknowledgements

Rapid prenatal diagnosis of sickle cell disease and thalassaemia by pyrosequencing Dr. John Old...

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