Rapid, simultaneous genotyping of five common Southeast Asian beta-thalassemia mutations by multiplex minisequencing and denaturing HPLC.

Abstract

β-Thalassemias are inherited hemoglobin disorders characterized by reduced production of β-globin chain. The severe forms of β-thalassemia produce marked anemia starting a few months after birth, and survival relies on regular blood transfusion and the lifelong use of drugs to prevent iron accumulation (1). β-Thalassemias are among the commonest genetic disorders in the world, and more than 200 mutations have been described to date. However, each at-risk population has a few common mutations (usually four or five) and a variable number of rare alleles (1). Common Southeast Asian β-thalassemia mutations include −28 A>G, codon (CD) 26 G>A, CD 41/42 −CTTT, CD 71/72 +A, IVS2 + 654 C>T, and others. The mutation CD 26 G>A produces hemoglobin E, presents like thalassemia, and is particularly common in Thailand, Cambodia, Vietnam, and Malaysia (1). There are many methods for genotyping known β-thalassemia mutations (2). Here we describe a rapid method for simultaneous genotyping of these five common mutations. The study was approved by the Human Subject Ethics Subcommittee of the Hong Kong Polytechnic University. The method is based on multiplex minisequencing followed by analysis of the single-base-extended products by denaturing HPLC (DHPLC). In minisequencing, a primer anneals to a DNA template immediately upstream of a mutation site to be analyzed and is extended by a single base as a result of incorporating a dideoxyribonucleotide triphosphate (ddNTP) (3). The single-stranded extension products are then separated by ion-pair reversed-phase liquid chromatography under completely denaturing condition, and the retention time is a function of both size and base composition (4). The DNA template for minisequencing was first generated by amplifying the whole human β-globin gene using primers HBBpF1 and HBBpR1 (Table 1⇓ ). The 25-μL reaction mixture contained 0.1 μM each of the primers, 50 ng of genomic DNA, 2.0 mM MgCl2 …