Su Yi-Ning, Lee Chien-Nan, Hung Chia-Cheng, Chen Chi-An, Cheng Wen-Fang, Tsao Po-Nien, Yu Chia-Li, Hsieh Fon-Jou
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
Hum Mutat. 2003 Oct;22(4):326-36. doi: 10.1002/humu.10265.
Beta-thalassemia is a common inherited disease, resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (HBB). Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at-risk populations based upon a molecular diagnosis. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by coupling heteroduplex and primer-extension analysis based on the denaturing high performance liquid chromatography (DHPLC) system. A total of 161 healthy heterozygous Taiwanese carriers featuring 10 different HBB mutations and 30 patients exhibiting 12 different compound heterozygous or homozygous HBB mutations were subjected to DHPLC. The elution profile for the heteroduplex analysis of DHPLC could be successfully used to identify the common disease-causing mutations of HBB. To further confirm the sequence variants, we developed a technique combining multiplex primer-extension analysis coupled with DHPLC for the genotyping of eight common disease-causing mutations in the HBB gene. Overall, by coupling heteroduplex and primer-extension analysis based upon DHPLC, we were able to unambiguously identify the most-common beta-thalassemia mutations corresponding to more than 99% of HBB alleles among the Taiwanese population. In conclusion, compared to classic approaches to mutation screening for this malady, we suggest that DHPLC is an excellent technique to be applied to the genetic screening of prenatal and postnatal individuals as a part of a diagnosis program for beta-thalassemia and provides a more-efficient, economic, and sensitive means to undertake such a screening program.
β地中海贫血是一种常见的遗传性疾病，由β珠蛋白基因（HBB）中总共200多种不同突变中的一种或多种引起。为了基于分子诊断为高危人群制定适当的预防计划，高效可靠的突变筛查方法至关重要。我们开发了一种快速且高度特异性的突变筛查测试，通过基于变性高效液相色谱（DHPLC）系统结合异源双链分析和引物延伸分析来诊断β地中海贫血。共有161名具有10种不同HBB突变的健康台湾杂合子携带者和30名表现出12种不同复合杂合或纯合HBB突变的患者接受了DHPLC检测。DHPLC异源双链分析的洗脱图谱可成功用于鉴定HBB常见的致病突变。为了进一步确认序列变异，我们开发了一种技术，将多重引物延伸分析与DHPLC相结合，用于对HBB基因中8种常见致病突变进行基因分型。总体而言，通过基于DHPLC结合异源双链和引物延伸分析，我们能够明确鉴定出台湾人群中超过99%的HBB等位基因对应的最常见β地中海贫血突变。总之，与针对这种疾病的经典突变筛查方法相比，我们认为DHPLC是一种优秀的技术，可作为β地中海贫血诊断计划的一部分应用于产前和产后个体的基因筛查，并提供了一种更高效、经济且灵敏的手段来开展此类筛查计划。
