| α-Thalassemia is the most common inherited disorder in South China. The molecular lesions can be categorized into α+ (a-thalassemia-2) or α0 (α-thalassemia-1) defects, depending on whether they partially or completely abolish a-globin chain production, respectively. There are four clinical classifications according to their phenotypes, silent carrier, a-thalassemia trait, Hemoglobin (Hb) H disease, and Hb Bart's hydrops fetalis. The a-thalassemia-2 (α+) deletions are the most common forms of a-thalassemia, with two major forms predominating (-α3.7 and -α4.2), which are found throughout the world. And the -α4 2 deletion is subdivided into two types of defects, the common -α4.2 and -α4.2HbQ (-α4.2 allele linked with HbQ sequence). At present, the sequence characterizations of the -α3.7 and -α4.2 deletions are uncertain. Southern blot analysis is the traditional method to detect gene deletions. But it is time-consuming and labor-intensive, and is not feasible for application to large-scale population screening. In addition, the inherent problems of gap-PCR amplification of the GC-rich and sequence-homology a-globin gene cluster are difficult to overcome. The purpose of this study is to obtain the specific single-nucleotide variations and haplotypes linked with the -α3.7 and -α4.2 gene deletions by sequencing of multi-sample, and then to establish a novel gene diagnosis method for rapid deletion detection using the sequence characterization. In addition, the study results can provide some useful information for elucidating the molecular mechanism of the -α3.7 and -α4.2 deletions.Design and MethodsWe designed the PCR system for amplification of the -a3'7, -a4'2, -a4'2HbQ allele, and the normal homology segments including the X boxes and Z boxes in a-globin locus. All of the PCR products were sequenced directly. The sequencing results were analyzed by homology comparison. Based on the new finding of DNA sequences, we developed the PCR-DHPLC (Denaturing-5-High Performance Liquid Chromatography) technology for genotyping the deletional α-thalassemia containing the -α42 allele using point mutation analysis strategy instead of traditional gap-PCR for deletion detection.Results and InterpretationWe obtained the sequences of the Z homology boxes and the X homology boxes of the a-globin locus in 25 DNA samples from Chinese and Israel individuals including 8 with -a gene, 10 with -α42 gene (including one Israel sample), 3 with -α4 2HbQ gene and 4 with wild-type allele as normal control. The results of homology sequencing analysis: (1) These sequences are highly conservative. And only one polymorphism site of A/G was found in the Z2 box of the normal and -α37 allele. (2) 5 single-nucleotide sequence markers linked with the -α3 7 deletion were identified according to the sequencing results of the Z boxes of the -α3.7 allele. But it is pity that we got no more information than previous observation by Okin et al. (3) The sequencing results of the -α2 allele indicated that there are 13 single-nucleotide markers linked with the -α42deletion from X2 box and X1 box, respectively. We can infer from these sites that the X box of the -α4 2 and-α4.2HbQ was a hybrid of X2 box and XI box, with 6 single-nucleotide variations discovered in these two subtypes of the -α42 deletion. The 6 single-nucleotide variations are the particular genetic markers linked with the -α42 deletion, and constructed their specific haplotypes. Using these single-nucleotide variations, we established a new method to detect deletionalα-thalassemia by using PCR combined with DHPLC technology. Firstly, PCR primers were designed to amplify the specific segments containing four single-nucleotide variations. And then the PCR amplification products were analyzed by DHPLC to detect the heteroduplexes in PCR products. The experimental results indicated that the four types of common genotypes,-6--α4-2/aa, -α4.2HbQ /act, -α42/--SEA and aa/aa, can be clearly clarified by analysis of distinctive DHPLC profile...
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