Rapid, accurate genotyping of β‐thalassaemia mutations using a novel multiplex primer extension/denaturing high‐performance liquid chromatography assay

Abstract

Summary. b-thalassaemia is a common inherited disorder of haemoglobin synthesis worldwide, with an estimated 3-10% frequency in certain regions. Rapid, accurate genotyping methodologies for specific, causative mutations of the b-globin gene are needed for pre-and postnatal screening and diagnosis of this disease in different ethnic populations. In this study, we performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) for simultaneously detecting and genotyping the five most common molecular lesions in the b-globin gene [codons (CDs) 41-42 (-TCTT), IVS-2-654 (C fi T), ) 28 (A fi G), CD17 (A fi T) and CD71-72 (+ A)] in Chinese populations. This method involved the amplification of b-globin target sequence followed by a purification step, a multiplex PE reaction that did not require labelled oligonucleotides, and a fully-denaturing DHPLC analysis on the Transgenomic Wave DNA fragment analysis system. In a blinded study, this technique accurately genotyped 100% (120/120) of samples previously characterized by reverse-dot blot and direct sequencing, and was used successfully for prenatal diagnosis of b-globin mutations in six Chinese families. This study validated the combined PE/DHPLC approach as simple, rapid, highly accurate and cost-effective for use in genotyping common disease-causing mutations, including substitutions, insertions and deletions in b-thalassaemia, and strongly suggests that this technique can be used successfully in other genetic diseases.

A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations

“…We previously developed and evaluated a denaturing high-performance liquid chromatography-based mutation screening method; however, it did not achieve widespread use because of its cost, tedious manipulations, and narrow detection of the mutation spectrum. 15 This experience taught us that a method for routine ␤-thalassemia carrier screening must be inexpensive, easy to use, rapid, accurate, and robust.…”

Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for β-thalassemia mutations

“…Minisequencing has been widely applied for multiplex genotyping of common b-thalassemia mutations. These assays utilize several different methods to characterize the primer extension products, including denaturing HPLC (Su et al 2003;Wu et al 2003;Yip et al 2003), solid-phase array (Kurg et al 2000), or fluorescence-based gel capillary electrophoresis .…”

Rapid, Simultaneous Genotyping of 10 Southeast Asian Glucose-6-Phosphate Dehydrogenase Deficiency–Causing Mutations and a Silent Polymorphism by Multiplex Primer Extension/Denaturing HPLC Assay

“…Our results, along with the relatively low reagent costs (approximately US $0.50/genotype) and short processing time (1 day for testing for 11 mutations in 65 samples) for our assay, suggest a potential use of this technology for screening programs. This approach has been applied to the detection of G6PD deficiency (present study) and ␤-thalassemia (14 ), and may be expanded to other common diseases, such as nondeletional ␣-thalassemia, Wilson disease, or cystic fibrosis. Great interindividual variability in drug response leads to variation in drug safety and efficacy.…”

“…1. In this technique, there are 3 possible DHPLC profiles for each potential mutation, which have been described previously (14 ), except that a primer peak plus a mutant peak (M in Fig. 1) indicates a hemizygous mutation in men, because the G6PD gene is X-linked, or a homozygous mutation in women.…”

“…The basic principle for identification of point mutations by this method is PE-based specific detection (13 ). Because the separation of oligonucleotides by DHPLC is both size and sequence dependent under fully denaturing conditions, modification of some of the oligonucleotides was necessary to provide an easily interpretable genotype pattern (14 ). The multiplex PCR was performed in a total volume of 25 L containing 12.5 L of 2ϫ PCR Master Mix [including deoxynucleotide triphosphates (dNTPs), buffer, MgCl 2 , and Taq DNA polymerase (Promega)], 0.2 g of genomic DNA, 0.2 M each of the primers for fragment 1 (5Ј-GACCCCAGAGGAACTCTCAA-3Ј and 5Ј-CAGGTA-GAGCCGGGATGAT-3Ј), 0.6 M each of the primers for fragment 2 (5Ј-ACGATGATGCAGCCTCCTAC-3Ј and 5Ј-GTTCTGCACCATCTCCTTGC-3Ј), and 0.8 M each of the primers for fragment 3 (5Ј-CCTGAGGGCTGCA-CATCT-3Ј and 5Ј-CCTTTCCTCACCTGCCATAA-3Ј).…”