Abstract

Aims: To develop an alternative assay for specific genotyping of the −α^4.2 thalassaemia deletion based on the DNA sequence features surrounding the breakpoint.

Methods: The 5′ and 3′ ends of the breakpoint regions of the −α^4.2 allele and the normal homologous segments were sequenced in Chinese individuals. A sequence haplotype composed of four single nucleotide variations within the X2/X1 box of the −α^4.2 breakpoint region was found in all of the 10 Chinese −α^4.2 thalassaemia alleles studied. Based on these findings, a novel polymerase chain reaction (PCR)/denaturing high performance liquid chromatography (DHPLC) assay was developed for rapid genotyping of the −α^4.2 allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the α globin target sequence encompassing these four polymorphic sites, followed by a partially denaturing HPLC analysis using the transgenomic WAVE DNA fragment analysis system.

Results: The three major genotypes (−α4.2/αα, −α^4.2/--^SEA, and αα/αα) could be distinguished through the characteristic chromatograms generated by the WAVE system. The accuracy of this technique was evaluated blindly, and the results were 100% (40 of 40) concordant with the genotypes previously characterised by Southern blotting or Gap-PCR.

Conclusions: This study validates the PCR/DHPLC approach as a simple, rapid, highly accurate, and cost effective method, potentially adaptable for use in epidemiological surveys, genetic screening, and diagnosis of silent α⁺ thalassaemia and Hb H disease.