Setup of a Protocol of Molecular Diagnosis of β‐Thalassemia Mutations in Tunisia using Denaturing High‐Performance Liquid Chromatography (DHPLC)

Backgrounds β-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the β-globin gene. In Tunisia, β-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole β-globin gene (HBB) is screened for mutations covering about 90% of the spectrum. Methods We have performed the validation of a DHPLC assay for direct genotyping of 11 known β-thalassemia mutations in the Tunisian population. Results DHPLC assay was established based on the analysis of 62 archival β-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study. Conclusion Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known β-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals.