title: Screening for clinically significant non-deletional alpha thalassaemia mutations by pyrosequencing.
abstract: Non-deletional α(+)-thalassaemia is associated with a higher degree of morbidity and mortality than deletional forms of α(+)-thalassaemia. Screening for the common deletional forms of α-thalassaemia by Gap-PCR is widely practiced; however, the detection of non-deletional α-thalassaemia mutations is technically more labour-intensive and expensive, as it requires DNA sequencing. In addition, the presence of four very closely homologous alpha globin genes and the frequent co-existence of deletional forms of α-thalassaemia present another layer of complexity in the detection of these mutations. With growing evidence that non-deletional α-thalassaemia is relatively common in the UK, there is a demand for technologies which can quickly and accurately screen for these mutations. We describe a method utilising pyrosequencing for detecting the ten most common clinically significant non-deletional α-thalassaemia mutations in the UK. We tested 105 patients with non-deletional α-thalassaemia and found 100% concordance with known genotype as identified by Sanger sequencing. We found pyrosequencing to be simpler, more robust, quicker, and cheaper than conventional sequencing, making it a good choice for rapid and cost-effective diagnosis of patients with suspected non-deletional α-thalassaemia. The technique is also likely to help expedite prenatal diagnosis of pregnancies at risk of α-thalassaemia major.
authors.affiliation: National Haemoglobinopathy Reference Laboratory, Molecular Haematology, Haemophilia Centre, Churchill Hospital, Oxford Radcliffe NHS Trust, Oxford, UK OX3 7LJ. anna.haywood@orh.nhs.uk
authors.forename: Anna
authors.initials: A
authors.lastname: Haywood
authors.forename: Helene
authors.initials: H
authors.lastname: Dreau
authors.forename: Adele
authors.initials: A
authors.lastname: Timbs
authors.forename: Anna
authors.initials: A
authors.lastname: Schuh
authors.forename: John
authors.initials: J
authors.lastname: Old
authors.forename: Shirley
authors.initials: S
authors.lastname: Henderson
mesh_terms: D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D009154:Mutation; D011061:Poly A / Q000235:genetics; D026723:Polyadenylation / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055542:alpha-Globins / Q000235:genetics*; D017085:alpha-Thalassemia / Q000175:diagnosis / Q000235:genetics*
chemical_list: D018388:Codon, Terminator; D055542:alpha-Globins; D011061:Poly A