The first validated criteria for effective screening and a new simplified method for α-globin gene sequencing for diagnosis of uncommon α-globin mutations

Abstract

No well-defined phenotypes that distinguish between unknown α- and β-globin mutations have been reported to date. Direct DNA sequencing of α-globin genes can be technically challenging, as α1- and α2-globin genes are nearly indistinguishable. To detect hemoglobin variants (HbXs) on Hb analysis, the entire β- and α-globin genes were directly sequenced using a newly developed sequencing protocol for α-globin genes. An algorithm to distinguish between α- and β-HbXs was constructed and subsequently validated in the independent validation group. Distinctive characteristics that can distinguish 39 α-HbXs from 24 β-HbXs were the presence of unidentifiable variants of HbA₂ and/or HbX of <37% on isoelectric focusing and <31% on high-performance liquid chromatography. Another set of 67 HbXs was employed to validate our algorithm. This accurately predicted 33 α-HbXs with 100% sensitivity and 97.1% specificity. Our sequencing protocol for α-globin genes was able to identify 11 rare mutations among all exons of both α-globin genes from 72 subjects. Six of these variants were first discovered in Thais. This is the first well-characterized algorithm for distinguishing unknown Hb variants in a large cohort. Our validated criteria and DNA sequencing procedure are highly efficient for molecular characterization of rare Hb mutations.

Keywords: DNA sequencing; Screening algorithm; Validation; α-Hemoglobin variants.