Use of denaturing gradient gel electrophoresis in screening unknown β-thalassemia mutations in Egyptian patients

Abstract

The molecular defects resulting in a β-thalassemia phenotype, in the Egyptian population show a clear heterogenic pattern. Many studies have embarked on the molecular detection and characterization of these mutations, using a wide array of the available techniques with successful detection of both known and unknown mutations. PCR based techniques, as well as direct DNA sequencing are effective with some limitations as regards the time, effort and high cost to reach a final diagnosis. Intermediary screening techniques have proved to be effective tools to overcome these drawbacks. This study aims to assess the use of the denaturing gradient gel electrophoresis (DGGE)¹ to detect b-thalassemia mutations prior to the performance of direct sequencing to minimize the cost and workload involved in the process. In this study, forty-two previously genotyped patients in a study by El-Gawhary et al. in 2007, have been analyzed by DGGE for fragment 2 then 1. These are the β-globin gene fragments showing the majority of the β-thalassemia mutations. Sixty-eight alleles out of 79 mutant alleles in total were detected within these two fragments. The 11 undetected alleles comprise 9 alleles that require further examination using other DGGE fragments (0, 4 and 5) and correspond to -87(C > G), intervening sequence (IVS)II-1(G >A), IVS II-745, and IVS II-848(C > A). The remaining two that failed detection correspond to codon (CD) 37(G> A). Although, IVS-II 745(C >G) is undetectable within these two fragments, its 100% linkage polymorphism (+20 C> T) was detected in fragment 1 gel. DGGE is a sensitive technique to screen for β-thalassemia mutations. For simultaneous analysis of multiple samples with unknown mutations, it is recommended that direct DNA sequencing be coupled with DGGE whenever available to reduce time, effort and cost.

Keywords: β-Thalassemia; DGGE; PCR; Mutations; Polymorphism; Screening