Molecular assay of -α3.7 and -α4.2 deletions causing α-thalassemia by denaturing high-performance liquid chromatography

Objectives: α-Thalassemia, the most common single gene disorder in humans, is due to the absence of one (-α/αα) or both (--/αα) of the two functional α-globin genes (α1 and α2). The -α3.7 and -α4.2 single gene deletions are common in Southeast Asian populations. Southern blotting analysis and gap PCR assay are commonly used for the detection of such α-thalassemia genotypes. The two genes are located on chromosome 16, with high homology (> 96%). Design and methods: Based on the sequence variation within the two Z boxes, a denaturing high-performance liquid chromatography (DHPLC)-based assay was developed for rapid genotyping of the -α3.7 and -α4.2 alleles. To demonstrate the utility of this approach, 40 DNA samples with known genotypes were analyzed, including -α3.7/αα (7 cases), -α4.2/αα (4 cases), -α3.7/--SEA (6 cases), -α4.2/--SEA (3 cases), and 20 unaffected subjects (αα/αα). Results: We successfully distinguished all of the α-thalassemia genotypes through their characteristic chromatograms of α1 and α2 genes. The accuracy of this technique for our sample was 100% sensitivity and specificity. Conclusion: This novel and alternative DHPLC-based α-thalassemia genotype assay is easy, rapid, and highly accurate. This technique enables the diagnosis of silent α+ thalassemia and hemoglobin H disease for large scale population screening. ? 2007 The Canadian Society of Clinical Chemists.