Analysis of the non-deletion alpha-thalassemia mutations by PCR temperature gradient gel electrophoresis

Objective: To develop a PCR-temperature gradient gel electrophoresis(TGGE) method applied to screening for the point mutations causing the non-deletion alpha-thalassemia.

Methods: The entire alpha2-globin gene fragment (1085 bp) was selectively amplified from human genomic DNA with different genotypes of alpha-thalassemia and a 543 bp fragment spanning the exon 2 and exon 3 of the alpha2-globin gene was amplified with a pair of nested primers. The condition of perpendicular and parallel TGGE for the two different fragments in length was optimized and the candidate mutant was confirmed by DNA sequencing. In pilot study, 15 samples with suspected non-deletion alpha-thalassemia were screened for point mutations in alpha-globin gene.

Results: Hb Constant Spring (Hb CS) and Hb Quong Sze (Hb QS), two most commonly types of the non-deletion alpha-thalassemia distributed in Chinese, and a rare type of the Hb Westmead mutation, alpha2 CD122 CAC-->CAG(His-->Gln) could be detected by PCR-TGGE. Among those 15 samples screened for alpha-thalassemia mutation were 10 samples with Hb CS mutation, 2 samples with Hb QS mutation, 2 samples with Hb Westmead mutation, and a previously unreported one, alpha2 CD31 AGG-->AAG(Arg-->Lys), confirmed by DNA sequencing.

Conclusions: The present PCR-TGGE method could be a useful tool for the molecular screening for the point mutations causing alpha-thalassemia and alpha-globin gene polymorphism.