Galehdari Hamid, Bijanzadeh Mehdi, Azarshin Seyedeh Zohreh, Shafee Mohammad, Heydaran Sogand
Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Department of Genetics, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Indian J Hematol Blood Transfus. 2021 Jul;37(3):436-441. doi: 10.1007/s12288-020-01358-w. Epub 2021 Mar 20.
Beta-thalassemia is the most frequent hemoglobin disorder in Iran resulting from disrupting mutations in the beta globin () gene that causes decreased or complete absent of beta-globin chains. The screening of beta-thalassemia minor and major individuals and prenatal diagnosis is important for familial planning. Therefore, it is essential, depending on the ethnicity and local frequency of changes, to develop a rapid and accurate method for molecular diagnosis of beta-thalassemia. Here, we developed reverse slot blot (RSB) assay for the simultaneous detection of six common pathogenic changes in the gene (-88, -28, IVSII-745, IVSII-848, Codon 6 [G → A] for HbC, Codon 6 [A → T] for HbS) in the Khuzestan Province of Iran. We designed normal and mutant oligonucleotide probes for each selected mutation and fixed them on positively charged nylon membrane. In the next step, a multiplex-polymerase chain reaction (PCR) performed for the amplification of the entire gene using labelled 5'-biotinylated primers. The PCR products were hybridized to immobilized oligonucleotide probes on the membrane at the appropriate temperature. Finally, we developed the membrane by chemically colorimetric reaction using nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate. For the best probe concentration, we made a serial dilution of probe pairs for each mutation. The optimal probe concentration for each mutation varied from 25 to 50 pmol. In the next step, DNA samples from homozygous affecting individuals were subjected for multiple PCR. Hybridization of each PCR products on the nylon membrane with probe pairs revealed specific bands with expected signal intensity without any background. Our designed RSB test is a rapid, sensitive and cost-effective method for screening of regional specific beta-thalassemia mutations in the Khuzestan population of Iran, which might be extended for the detection of any desired pathogenic changes.
β地中海贫血是伊朗最常见的血红蛋白疾病，由β珠蛋白（）基因突变引起，导致β珠蛋白链减少或完全缺失。对轻型和重型β地中海贫血患者进行筛查以及产前诊断对于计划生育至关重要。因此，根据种族和当地变异频率，开发一种快速准确的β地中海贫血分子诊断方法至关重要。在此，我们开发了反向斑点杂交（RSB）检测法，用于同时检测伊朗胡齐斯坦省基因中的六种常见致病突变（-88、-28、IVSII-745、IVSII-848、HbC的密码子6 [G→A]、HbS的密码子6 [A→T]）。我们为每个选定的突变设计了正常和突变寡核苷酸探针，并将它们固定在带正电荷的尼龙膜上。下一步，使用标记的5'-生物素化引物进行多重聚合酶链反应（PCR），以扩增整个基因。PCR产物在适当温度下与膜上固定的寡核苷酸探针杂交。最后，我们使用硝基蓝四唑-5-溴-4-氯-3-吲哚磷酸通过化学比色反应使膜显色。为了确定最佳探针浓度，我们对每个突变的探针进行了系列稀释。每个突变的最佳探针浓度在25至50 pmol之间。下一步，对纯合受累个体的DNA样本进行多重PCR。每个PCR产物与尼龙膜上的探针杂交后，显示出具有预期信号强度且无任何背景的特异性条带。我们设计的RSB检测法是一种快速、灵敏且经济高效的方法，用于筛查伊朗胡齐斯坦人群中区域特异性的β地中海贫血突变，该方法可能会扩展用于检测任何所需的致病突变。
