Molecular assay of -alpha(3.7) and -alpha(4.2) deletions causing alpha-thalassemia by denaturing high-performance liquid chromatography

alpha-Thalassemia, the most common single gene disorder in humans, is due to the absence of one (-alpha/alphaalpha) or both (--/alphaalpha) of the two functional alpha-globin genes (alpha1 and alpha2). The -alpha(3.7) and -alpha(4.2) single gene deletions are common in Southeast Asian populations. Southern blotting analysis and gap PCR assay are commonly used for the detection of such alpha-thalassemia genotypes. The two genes are located on chromosome 16, with high homology (>96%). 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 -alpha(3.7) and -alpha(4.2) alleles. To demonstrate the utility of this approach, 40 DNA samples with known genotypes were analyzed, including -alpha(3.7)/alphaalpha (7 cases), -alpha(4.2)/alphaalpha (4 cases), -alpha(3.7)/--(SEA) (6 cases), -alpha(4.2)/--(SEA) (3 cases), and 20 unaffected subjects (alphaalpha/alphaalpha). We successfully distinguished all of the alpha-thalassemia genotypes through their characteristic chromatograms of alpha1 and alpha2 genes. The accuracy of this technique for our sample was 100% sensitivity and specificity. This novel and alternative DHPLC-based alpha-thalassemia genotype assay is easy, rapid, and highly accurate. This technique enables the diagnosis of silent alpha+ thalassemia and hemoglobin H disease for large scale population screening.