IBMS Congress

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Advances in haemoglobinopathy diagnostics

Session 3

Haematology

Dr John Old

Learning outcomes

• the different types of abnormal haemoglobins and thalassaemias, with reference to those types found in the UK population
• the methods of haematological screening and carrier diagnosis for the UK screening programme
• the methods used for the molecular analysis and prenatal diagnosis of the haemoglobinopathies

Recent advances in haemoglobinopathy diagnostics include application of capillary electrophoresis for carrier screening, the characterisation of the spectrum of mutations in the UK population for the rare Hb variants, silent β-thalassaemia, δ-thalassaemia and βδ-thalassaemia, and the development of pyrosequencing for fetal genotyping.

Abnormal haemoglobins have traditionally been detected by acid/alkali gel electrophoresis methods, HPLC and isoelectric focussing. Capillary electrophoresis gives better separation of haemoglobins than acid/alkali gels and also measures the % Hb levels similar to HPLC. It offers very clean separation patterns and the advantage of separating Hb E from HbA2. The best resolution of Hb variants is obtained with IEF, but even this method does not give an accurate presumptive diagnosis for the rare variants. We have now characterised 225 different Hb variants in the UK population by DNA analysis, HPLC and IEF, and found that many have either the same IEF position or HPLC retention time. However we have shown that by combining the HPLC retention time with the IEF position to make a presumptive diagnosis, the accuracy of the prediction of a rare variant can be greatly improved.

Beta thalassaemia carriers are identified by a Hb A₂ level above 3.5% and reduced red cell indices. However a small number of β-thalassaemia carriers have an atypical silent phenotype, with borderline-normal Hb A₂ levels of 3.0-3.5%, and in some cases, normal red cell indices.  A study of UK patients with this phenotype showed that silent/normal HbA₂ β-thalassaemia is caused by 8 different mild β⁺-thalassaemia mutations, including a novel Poly A mutation found only in the indigenous British population.  The Hb A₂ level in carriers may also be lowered to normal levels by the co-inheritance of δ-thalassaemia or a δ-chain variant, and a study of the UK population has revealed types of 3 δ-thalassaemia and 8 δ-chain variants, including 3 novel ones.

The molecular diagnosis of β-thalassaemia mutations in our laboratory is achieved by the techniques of ARMS-PCR, DNA sequencing and MLPA. The latter technique has been developed recently for the detection of large DNA deletion mutations in the globin genes, and a study of UK patients with a raised Hb F level has revealed 17 different δβ-thalassaemia, εγδβ-thalassaemia and HPFH deletions. DNA sequencing is a time consuming procedure when used for prenatal diagnosis and we have developed the new technique of pyrosequencing as its replacement for mutation analysis of fetal DNA. The results over the last year for 173 prenatal diagnoses of sickle cell disease and β-thalassaemia have shown that pyrosequencing is an accurate, robust and much quicker approach than DNA sequencing and it has now been adopted routinely by our laboratory for the prenatal diagnosis of the haemoglobinopathies.