Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common α-thalassemia point mutations and deletions

Abstract

Background: α-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of α-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations. Methods: We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 α-globin mutations: two single gene deletions (-α3.7; -α4.2), five double gene deletions [ -MED; -SEA; -THAI; -FIL; -(α)20.5], αααanti-3.7gene triplication, two point mutations in the α1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the α2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2). Results: Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods. Conclusions: The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of α-globin mutations in endemic areas worldwide. ©2007 by Walter de Gruyter.