DHPLC for screening of B-thalassaemia gene mutations

Abstract

ẞ thalassaemia is a heterogeneous group of disorder resulted from more than 200 mutations to date with most of the defects involve a few base pair changes. Denaturing high performance liquid chromatography (DHPLC) is a relatively new technique for mutation screening, which have been reported to have a high sensitivity rate but its application for ẞ thalassaemia mutation screening is still very limited. DHPLC was used in this study to screen for ẞ thalassaemia gene mutations in 25 participants; 19 known B thalassaemias (16 patients and 3 carriers) and 6 normal controls. Five sets of primers were designed to cover the ẞ gene with consequential 45 amplicons have been analysed. DHPLC was only able to detect 6 of 28 mutation-containing amplicons resulting in its sensitivity of 21.4%. Twelve of the ẞ thalassaemia patients were homozygous for the common ẞ thalassaemia mutations causing these to be undetected. Only one compound heterozygous patient was successfully detected. The mutations in the 3 carriers also failed to be detected, most likely due to their locations at the end of the DA sequence and possibly due to the stabilized the heteroduplex molecules. The pre-DHPLC preparation steps were labour intensive, analysis was lengthy, optimization and standardization were difficult. The cost, on the other hand could potentially be advantageous in the long run. In conclusion, this study showed that DHPLC was labour intensive, was technically difficult in terms of optimization and standardization and was also insensitive for mutation screening in ẞ thalassaemia but had an advantage of being relatively cheap for long term use.