Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthon 4 Rd., Phuttamonthon, Nakornpathom 73170, Thailand.
J Mol Diagn. 2010 May;12(3):354-8. doi: 10.2353/jmoldx.2010.090136. Epub 2010 Feb 26.
alpha-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of alpha-globin protein. Couples who both carry the alpha-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the alpha-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for alpha-thalassemia diagnosis was developed by genotyping alpha-thalassemia-1, the Southeast Asian type (--(SEA)) and Thai type (--(THAI)) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the --(SEA) and --(THAI) deletions and two normal fragments, psizeta- and alpha2-globin gene. The primers were capable of distinguishing alpha-thalassemia 1 heterozygotes from alpha-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the --(THAI), --(SEA), psizeta-globin, and alpha2-globin gene fragments were 79.9 +/- 0.2, 89.4 +/- 0.5, 92.8 +/- 0.2, and 85.0 +/- 0.2 degrees C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia.
α-地中海贫血是一种遗传性血红蛋白疾病，由α-珠蛋白合成缺陷引起。夫妇双方均携带α-地中海贫血-1 基因时，胎儿有患 Bart 胎儿水肿综合征的风险。快速准确地筛查携带α-地中海贫血-1 基因的个体是预防和控制这种严重地中海贫血的最有效策略。在这项研究中，通过多重 PCR 结合熔解曲线分析，建立了一种新的、准确的α-地中海贫血-1 基因（东南亚型[--(SEA)]和泰国型[--(THAI)])缺失基因分型方法。设计了引物特异性扩增两个缺失片段，即--(SEA)和--(THAI)缺失片段和两个正常片段，psiζ和α2-珠蛋白基因。引物能够区分α-地中海贫血 1 杂合子和α-地中海贫血 2 纯合子，而标准血液学数据和血红蛋白分型无法诊断后者。--(THAI)、--(SEA)、psiζ-珠蛋白和α2-珠蛋白基因片段的熔解温度分别为 79.9±0.2°C、89.4±0.5°C、92.8±0.2°C 和 85.0±0.2°C。在 130 例患者中与常规缺口-PCR 分析平行进行了熔解曲线分析，结果显示 100%一致。该方法消除了费力的 PCR 后电泳过程，允许高通量筛选，适合用于地中海贫血的预防和控制的大规模人群筛查。
