Novel technique for rapid detection of α-globin gene mutations and deletions

Populations in Southeast Asia and South China have high frequencies of α-thalassemia caused by α-globin gene mutations and/or deletions. This study was designed to find an efficient and simple diagnostic test for the mutations and deletions. A duplex polymerase chain reaction (PCR)/denaturing high-pressure liquid chromatography (DHPLC) was used to detect the mutations and deletions. A blinded study of 110 samples, which included 92 α-thalassemia samples with various genotypes and 18 normal DNA samples, was carried out by the methods. The duplex PCR products of the sample with known Constand spring mutation (CS)/αα, Quonsze mutation (QS)/αα, and Weastmead mutation (WS)/αα DNA showed significantly different profiles, which suggests that DHPLC analysis at 63.8°C can detect potential mutations directly. The DHPLC at 50°C analysis can distinguish the --SEA and nondeletional alleles. The new assay is 100% concordant with the original genotype. In conclusion, the technique including the duplex PCR assay followed by DHPLC analysis can be used to diagnose α-thalassemia; this methodology is simple, rapid, accurate, semiautomatic, and high output, and thus, it is suitable for large-scale screening.

Abbreviations: CS, Constand spring mutation, DC, dissociation curve analysis, DHPLC, denaturing high-performance liquid chromatography, Duplex PCR, duplex polymerase chain reaction, GC, guanine-cytosine, QS, Quonsze mutation, RDB, reverse dot-blot, TEAA, triethylammonium acetate, WS, Weastmead mutation