Rapid, accurate genotyping of the common -alpha(4.2) thalassaemia deletion based on the use of denaturing HPLC.
OBJECTIVE
To develop an alternative assay for specific genotyping of the -alpha(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 -alpha(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 -alpha(4.2) breakpoint region was found in all of the 10 Chinese -alpha(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 -alpha(4.2) allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the alpha 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 (-alpha4.2/alphaalpha, -alpha(4.2)/--SEA, and alphaalpha/alphaalpha) 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 alpha+ thalassaemia and Hb H disease.
Ou-Yang
H
H
Department of Medical Genetics, First Military Medical University, Guangzhou 510515, Guangdong, PR China.
Hua
L
L
Mo
Q H
QH
Xu
X M
XM
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D017353:Gene Deletion; D006580:Genetic Carrier Screening / Q000379:methods*; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D017085:alpha-Thalassemia / Q000235:genetics*
D005914:Globins