A versatile denaturing HPLC approach for human β‐globin gene mutation screening

Abstract

Hemoglobinopathies represent the most common genetic disorder worldwide, with a higher prevalence among populations with a history of malaria endemicity. More than 690 mutations in the human beta-globin gene are usually the cause of beta-type hemoglobinopathies. Here, we report a rapid and highly sensitive beta-globin gene mutation screening approach based on denaturing high-performance liquid chromatography (DHPLC), which contrary to the previously described ones can be used in every HPLC apparatus. The sensitivity and specificity of the method were tested in 120 healthy Greek subjects and 25 beta-thalassemia heterozygotes and homozygotes, in which 11 different beta-globin sequence variations had been previously characterized by denaturing gradient gel electrophoresis. Using this method, we were able to rapidly identify the commonest beta-globin gene mutations, accounting for more than 90% of the mutant beta-globin alleles reported for the Hellenic population. Compared to classical mutation screening approaches, our DHPLC approach provides the means for rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.