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

Abstract

Background: Alpha-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 alpha-thalassemia mutations include deletion of one or both alpha-globin genes. The importance of alpha-thalassemia detection is mainly due to its shared blood parameters with beta-thalassemia and its impact on discrimination between unknown alpha-thalassemia and normal HbA2 beta-thalassemia during thalassemia prevention program.

Materials and methods: Cases with hematologic profile of low MCV, MCH, and normal HbA2 were enrolled in this study. Common alpha-globin deletional mutations including alpha(3.7)kb, alpha(4.2)kb, alpha(20.5)kb, and alpha(MED) and point mutation including 5 nt, 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 theta-globin gene up to the 3' upstream of alpha(2) gene.

Results: After 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 (theta gene up to the upstream of alpha2 gene). In 8 (27.6%) cases, deletions were seen in all regions.

Conclusion: Gene dosage study by quantitative real-time PCR can be suggested as a rapid and reliable assay to screen probable carrier of alpha-thalassemia for unknown alpha-globin gene deletions.