ORIGINAL ARTICLE

Rapid, accurate genotyping of the common -^4.2 thalassaemia deletion based on the use of denaturing HPLC

H Ou-Yang, L Hua, Q H Mo, X M Xu

Department of Medical Genetics, First Military Medical University, Guangzhou 510515, Guangdong, PR China

Correspondence to:
Correspondence to:
Dr X-M Xu
Department of Medical Genetics, First Military Medical University, Guangzhou 510515, Guangdong, PR China; gzxuxm{at}pub.guangzhou.gd.cn

Aims: To develop an alternative assay for specific genotyping of the -^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 -^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 -^4.2 breakpoint region was found in all of the 10 Chinese -^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 -^4.2 allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the 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 (-4.2/, -^4.2/--^SEA, and /) 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 ⁺ thalassaemia and Hb H disease.

Abbreviations: DHPLC, denaturing high performance liquid chromatography; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; TEAA, triethylammonium acetate

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