Development of a quantitative real-time PCR assay for detection of unknown α-globin gene deletions

BackgroundAlpha-Thalassemia is the most common inherited disorder of hemoglobin (Hb) synthesis in the world. Unlike beta-thalassemia, in which non-deletional mutations predominate, most of recognized α-thalassemia mutations include deletion of one or both α-globin genes. The importance of α-thalassemia detection is mainly due to its shared blood parameters with beta-thalassemia and its impact on discrimination between unknown α-thalassemia and normal HbA2 beta-thalassemia during thalassemia prevention program.Materials and methodsCases with hematologic profile of low MCV, MCH, and normal HbA2 were enrolled in this study. Common α-globin deletional mutations including α3.7 kb, α4.2 kb, α20.5 kb, and αMED and point mutation including 5nt, Constant Spring (CS), and C19 were checked using either GAP-PCR or ARMS-PCR. Cases with unknown molecular defects were investigated further by direct gene sequencing. Finally, further study was done for probable unknown deletions by gene dosage analysis using real-time PCR. For this, five pairs of primers were used spanning from θ-globin gene up to the 3′ upstream of α2 gene.ResultsAfter validation of primers specificity and performing serial dilution analysis in order to calculate PCR efficiency, the assay was performed on normal samples and cases with known alpha-globin gene deletions as positive and negative controls, respectively. The assay was able to diagnose the control groups successfully. In 21 out of 29 unknown cases (72.4%), the assay showed various patterns of deletions in at 2 to 5 screened regions (θ gene up to the upstream of alpha2 gene). In 8 (27.6%) cases, deletions were seen in all regions.ConclusionGene dosage study by quantitative real-time PCR can be suggested as a rapid and reliable assay to screen probable carrier of α-thalassemia for unknown alpha-globin gene deletions.