Presentation on theme: "Dr. John Old National Haemoglobinopathy Reference Laboratory"— Presentation transcript:
1 Rapid prenatal diagnosis of sickle cell disease and thalassaemia by pyrosequencing Dr. John OldNational Haemoglobinopathy Reference LaboratoryMolecular HaematologyJohn Radcliffe Hospital, Oxford, UK
2 Prenatal diagnosisNHRL carries out approx 180 PNDs per year for UK patients:56% sickle cell disease42% -thalassaemia2% -thalassaemiaSources of fetal DNA :Chorionic villi %Amniotic fluid %
3 Current best practice PND procedure 1. Use fresh parental blood samples for control DNAs2. Use cleaned & sorted chorionic villi3. Test for mutations in more than one fetal DNA aliquot with appropriate controlsConfirm result by a different diagnostic methodCheck for maternal DNA contaminationReport result with error risk3 working days turnaround time target
4 The two DNA methods for PND’s Sickle cell:ARMS-PCRRE-PCR using Dde 1b-thalassaemia:Sanger sequencinga-thalassaemia:Hb Bart’s hydrops: Gap-PCR + MLPAHb H hydrops: Sanger sequencing
5 Problems with current approach Second method can be time consumingRE-PCR for Hb S requires O/N digestionSanger sequencing takes 2 days.Sanger sequencing is expensive as confirmatory methodFor very rare b-thalassaemia mutations, only have one approach – Sanger sequencing.
6 Molecular basis of b-thalassaemia Total number mutationsPoint mutationsLarge deletionsFour regional groups:Mediterranean, Indian, Chinese, AfricanNumber per ethnic groupCommon mutationsRare mutations
7 b-Thalassaemia mutations diagnosed in the UK population Total number of different alleles diagnosed for PND: 42MediterraneanAsian IndianSoutheast AsianAfricanBritishTotal number diagnosed for all patients: 68Total number diagnosed in indigenous Britons: 9Reference: 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 (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 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-PCRCVS DNA 50 casesAF DNA casesUsed pyrosequencing as the 2nd confirmatory test instead ofRE-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
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 allelesHb SHb Cb-thalassaemia allelesHb ECodon 8/9 +GCD 30 GAIVSI-1 GTIVSI-5 GCCD 41/42 (-TCTT)a-thalassaemia allelesHb AdanaHb Constant Spring
15 Pyrosequencing – conclusions 1 Cheaper, simpler and quicker than conventional Sanger sequencingApprox 1/5 the cost of Sanger sequencingHigher degree of accuracy than ARMSMutation is detected in the context of its surround sequenceFewer pitfalls than gel based methodscan test for several mutations at oncedon’t need positive control for every mutationMuch quicker than RE-PCR methodology
16 Pyrosequencing – conclusions 2 More robust – lower failure rate than Sanger sequencingSanger sequencing takes 2 days (5 steps)Pyrosequencing takes 0.5 days (2 steps)More sensitive than Sanger sequencing and RE-PCRWorks with much lower quantities of DNAResults are quantitative – results reflect any allelic imbalance: mosaicism, vanishing twin or maternal contaminationSet up costs are cheaper than Sanger sequencing – more suitable for PND in developing countries
17 AcknowledgementsAdele Timbs Michelle Rugless Alice Gallienne Anna Haywood Shirley Henderson