Combine-ARMS: A Rapid and Cost-Effective Protocol for Molecular Characterization of β-Thalassemia in Malaysia

Abstract

Beta-thalassemia major patients have chronic anemia and are dependent on blood transfusions to sustain life. Molecular characterization and prenatal diagnosis of beta3-thalassemia is essential in Malaysia because about 4.5% of the population are heterozygous carriers for beta-thalassemia. The high percentage of compound heterozygosity (47.62%) found in beta-thalassemia major patients in the Thalassaemia Registry, University of Malaya Medical Centre (UMMC), Malaysia, also supports a need for rapid, economical, and sensitive protocols for the detection of beta-thalassemia mutations. Molecular characterization of beta-thalassemia mutations in Malaysia is currently carried out using ARMS, which detects a single beta-thalassemia mutation per PCR reaction. We developed and evaluated Combine amplification refractory mutation system (C-ARMS) techniques for efficient molecular detection of two to three beta-thalassemia mutations in a single PCR reaction. Three C-ARMS protocols were evaluated and established for molecular characterization of common beta-thalassemia mutations in the Malay and Chinese ethnic groups in Malaysia. Two C-ARMS protocols (cd 41-42/IVSII #654 and -29/cd 71-72) detected the beta-thalassemia mutations in 74.98% of the Chinese patients studied. The CARMS for cd 41-42/IVSII #654 detected beta-thalassemia mutations in 72% of the Chinese families. C-ARMS for cd 41-42/IVSI #5/cd 17 allowed detection of beta-thalassemia mutations in 36.53% of beta-thalassemia in the Malay patients. C-ARMS for cd 41-42/IVSI #5/cd 17 detected beta-thalassemia in 45.54% of the Chinese patients. We conclude that C-ARMS with the ability to detect two to three mutations in a single reaction provides more rapid and cost-effective protocols for beta-thalassemia prenatal diagnosis and molecular analysis programs in Malaysia.

Untitled

“…We were able to multiplex successfully nearly all common mutations of an ethnic population except for the b-thalassemia mutation IVSII-654, which is commonly found in Asians. This mutation was amplified in a separate reaction due to its lower annealing temperature [20].…”

“…In a recent report, two to three mutations were amplified in a single reaction in a Malaysian population [20]. However, Chang et al demonstrated the amplification of all the common Taiwanese/Chinese mutations in a single reaction [24].…”

Rapid Detection of Common Southeast Asian ß-Thalassemia Mutations by Nonisotopic Multiplex PCR-SSCP Analysis

“…One recent report described the detection of nine common Southeast Asian mutations (excluding IVS2 ϩ 654 C Ǟ T) by two separate PCRs and nine separate restriction digestion reactions (Pramoonjago et al, 1999). Another recent study employed three separate multiplex amplification refractory mutation systems (ARMS) to detect six common Southeast Asian ␤-thalassemia mutations (Tan et al, 2001). Finally, it is of interest to note that an expensive commercial kit (mDx™ BeTha Gene 2 from Bio-Rad) is available for the detection of eight common Southeast Asian mutations (Ϫ28, CD17, CD19, ␤ E , IVS1 ϩ 5, CD41-42, CD71-72, and IVS2 ϩ 654).…”

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

“…Minisequencing is a direct method that can be used to generate allele-specific DNA products, which is potentially useful for multiplex high-throughput genotyping (Syvanen 1999;Tan et al 2001). Relatively short primer extension products templated on experimental samples can be produced and analyzed rapidly.…”