Rapid detection of multiple beta-globin gene mutations by a real-time polymerase chain reaction in beta-thalassemia carriers

Background beta-Thalassemia is a heterogeneous disorder caused by mutations that reduce or abolish the synthesis of the beta-globin chain. The clinical severity of thalassemia major makes it a priority genetic disease for prevention programs involving population screening of heterozygotes and an optional prenatal diagnosis for carrier couples. Aim of the study This study aimed to determine the most common beta-globin gene mutations in Egypt using a real-time PCR and fluorescently labeled hybridization probes specific for each mutation and to assess the feasibility of introducing this technique in an overall thalassemia prevention program. Participants and methods The study was carried out on 45 individuals: 37 beta-thalassemia carriers [including five amniotic fluid (AF) samples], seven beta-thalassemia major cases (including two AF samples), and one normal AF sample. The most common beta-thalassemia mutations were characterized by real-time PCR with fluorescently labeled hybridization probes specific for IVSI-110, IVSI-1, IVSI-6, codon 37, and codon 39 in 28/37 (75.7%) carriers. Results The most common mutation encountered was IVSI-110 (46%), followed by IVSI-1 (16.2%) and then IVSI-6 (13.5%). Codon 37 and codon 39 were not characterized in any sample. The genotype of the uncharacterized carriers was determined using a less sensitive method (reverse hybridization technique) and a relatively less common set of mutation was characterized as follows: IVSII-1(10.8%), codon 5 (5.4%), IVSII-745 (5.4%), and IVSI-116 (2.7%). The overall number of alleles detected using both techniques was calculated to be 51. The real-time PCR alone, with its assigned probes, detected 38/51(74.5%). Thirteen mutations (13/51=25.5%) remained uncharacterized by this technique (because of the unavailability of the corresponding probes). However, the reverse hybridization technique detected 48/51 alleles (94.1%). However, comparison between both techniques in terms of the shared mutations showed that the real-time PCR detected 38/38 (100%) of these mutations, whereas the reverse hybridization technique detected only 36/38 (94.7%). Conclusion Real-time PCR is a very rapid and accurate method for the detection of the beta-thalassemia mutation, which may be valuable in cases for which a rapid decision has to be taken. Impediments to prenatal diagnosis as encountered in this study were attributed to refusal of termination of pregnancy by the family for religious/reasons, abortion following amniocentesis, and failure to determine the correct genotype of the AF analyzed. (C) 2012 The Egyptian Society of Haematology.