Accurate detection of unknown deletions in beta-Globin gene cluster in beta thalassemia carriers using Real-time PCR and MLPA

Background and objectivesAlthough beta thalassemia is mainly caused by mutations involving single base substitutions and small deletions, there have been reports showing deletions of large regions of beta- globin genes play a similar causing role. The strategy to identify beta thalassemia carriers with known deletions is based on PCR techniques such as Gap PCR. There are however some unknown deletions that can not be detected by the above methods. To overcome this limitation, Real-time PCR and MLPA were developed as two quantitative assays for analysis of beta-globin gene cluster. Materials and MethodsThe subjects were evaluated in a case-control study. Among individuals referred to genetic laboratories of Pasteur Institute of Iran and Kawsar Genetic Research Center, 40 were suspected of having a large deletion in β-globin gene cluster. The including criteria were hematological findings such as low blood indices (MCV <80 fl and MCH <27 pg), normal HbA2 and raised or normal HbF. Genomic DNA was extracted from peripheral blood. A Real-time PCR assay was developed using comparative threshold cycle (Ct) method for analysis of gene copy number. In addition, gene dosage was analyzed using MLPA method. ResultsReal-time PCR results for quantitative analysis of Beta, Delta, G-gamma genes showed the ratio (2-ΔΔCt) of 0.96 ± 0.18 for normal individuals and 0.58 ± 0.04 for carriers of deletions in beta globin gene cluster. MLPA results showed nearly 50% reduction in the height of the peaks corresponding to regions of deletions. ConclusionsMLPA results confirmed the presence of the same deletions detected by Real-time PCR in all of the carrier individuals. It would be ideal to combine these quantitative assays to confirm corresponding results for accurate diagnosis of known and unknown deletions in beta thalassemia carriers.