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Primers A and B were used with MARMS, and primers C and D were used with the single ARMS. A pair of primers was used as an internal control for each reaction. Primers to detect IVS 1–1 (G–A), Cd 43 (G–T), Cd 19 (A–G) and Initiation Cd (ATG–AGG) mutations were designed during the course of the project. Most of the primer sequences were obtained from earlier publications (14–17). Another mutation of IVS 2–654 (C–T) was detected in a single ARMS. In MARMS-E, we screened for the –88 (C–T), Initiation Cd (ATG–AGG), Cd 15 (G–A), and –29 (A–G) mutations. In MARMS-D, we screened for the Cd 19 (A–G) and cap +1 (A–C) mutations. In MARMS-C, we screened for the IVS 1–1 (G–A), Cd 43 (G–T), Cd 16 (–C), and Poly A (A–G) mutations. Multiplex Amplification Refractory Mutation System– Polymerase Chain Reaction (MARMS–PCR)įive MARMS and one ARMS were used to detect 20 mutations: In MARMS-A, we screened for the IVS 1–5 (G–C), Cd 41/42 (–TTCT), Cd 17 (A–T), and Cd 26 (G–A) HbE mutations. Direct sequencing and reverse dot blot hybridization analysis, using β-Globin StripAssay SEA™ (ViennaLab Diagnostics GmbH, Vienna, Austria) were used for mutation confirmation. Five sets of MARMS and one single ARMS were used to detect 20 different mutations of the β-globin gene. Based on earlier reported studies of β-thalassaemia heterogeneity among Malaysians ( 11, 13, 14), we modified the MARMS developed by Bhardwaj et al., ( 15) by adding primers, suitable for the local mutation heterogeneity. The aims of this study are to design and use a rapid, inexpensive, and simple PCR approach to update, and characterise the spectrum of β-globin gene mutations among Malaysians. A molecular diagnostic approach to the Malaysian population has been reported previously ( 6, 10) using ARMS, and MARMS to detect most of the common mutations, and with either PCR or genomic sequencing for the rarer mutations ( 9, 11, 12). It is common among the Malaysian Malays and Chinese ( 8), with a structural haemoglobin variant, HbE found very commonly among the Malays ( 8, 9). Blood transfusions and iron-chelator treatment for patients with β-thalassaemia major are expensive, and beyond the economic reach of many families, and therefore, the provision of genetic counselling even before marriage is important.Ībout 4.5% of the Malaysian population are carriers for β-thalassaemia ( 6, 7). The majority of the β-thalassaemia mutations are nucleotide substitutions, frameshifts or minor deletions ( 4), and rarely, large deletions are also reported ( 5). To date, the database of haemoglobin variants and thalassaemia mutations, HbVar, has recorded more than 800 mutation entries involving the β-globin gene ( –bin/hbvar/query_vars3). Generally, a defective β-globin gene leads to a reduction (β +) or absence (β°) of the gene production. β-globin chain deficit leads to the intracellular precipitation of excess α-globin chains, causing ineffective erythropoiesis ( 2, 3). Β-thalassaemia is an autosomal haematological disorder resulting in a genetically deficient synthesis of the β-globin chain in haemoglobin ( 1).