Abstract
Background: a-thalassemia, the most prevalent of all thalassemias, is an inherited single gene disorder of the hemoglobin synthesis characterized by the absence or the reduced expression of a-globin genes resulting in an imbalanced ratio between a- and b-gobin chain synthesis. While some DNA alterations lead to microcytic anemia, the most others cause severe anemia and intrauterine death if co-inherited in a homozygous constellation. The most common genetic defects leading to the a-thalassemia phenotype are deletions of one or more of the two highly homologous a-globin genes. Less frequently, a-thalassemia derives from nondeletional mutations located in parts of the a-globin gene that are critical for its normal expression. So far the identification of the genetic background of a-thalassemic patients has included specific amplification of each a-globin gene followed by laborious and expensive mutation analysis methods such as denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) or direct sequencing of the entire a-genes.
Method: We attempted to evaluate the performance of denaturing high performance liquid chromatography (dHPLC) technique to identify nondeletional mutations located in the a1- or a2-globin gene. Due to the high sequence homology of a1- and a2-globin genes, separate amplification of the two genes followed by nested PCR were performed. This generated four overlapping amplicons (a-dHPLC1 to 4) covering the whole a1/a2-globin locus including the 3′- and 5′-untranslated regions. The formation of DNA hetero-/homoduplexes was ensured by denaturing and slow reannealing of crude PCR products. Through a DNA separation column under partial-denaturing conditions, DNA homoduplexes were retained longer than their corresponding heteroduplexes generating distinguishable chromatographic profiles and hence allowing for the differentiation between wildtype and mutant DNA. Samples which differed from the wildtype in their elution profile were sequenced in both directions.
Results: 50 patients showing a presumptive a-thalassemic hematological profile not due to deletional mutations (excluded by gap PCR technique) were tested. 10 carriers of a-thalassemia nondeletional alleles (previously identified by endonuclease restriction analysis) and 10 healthy patients were also included in the evaluation.
Conclusion: Our results show that dHPLC is a reliable, sensitive, and specific screening method to detect pointmutations and small deletions located in the a-globin genes. Furthermore, the low costs of analysis and the rapidity of the screening (about 6 min/sample) make the dHPLC technique an appropriate alternative to technically demanding and time consuming mutation screening methods such as DGGE or SSCP analysis.
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