Mass spectrometry-based detection of hemoglobin E mutation by allele-specific base extension reaction.

Background: The specific detection of a minor population of mutant DNA molecules requires methods of high specificity and sensitivity. While the single-allele base extension reaction (SABER) was shown to be useful for the detection of certain beta-thalassemia mutations, we encountered problems with false positivity during development of SABER for the noninvasive prenatal diagnosis of the hemoglobin E (HbE) disease. Systematic optimization resulted in an alternative protocol, the allele-specific base extension reaction (ASBER).

Methods: An artificial model was established by mixing genomic DNA of HbE carriers and normal individuals. Effects of terminator concentration and annealing temperature on the nonspecificity of SABER were then studied. The use of a single relevant terminator and the other 3 types of dideoxynucleotide as competing terminators were also compared in the development of the ASBER protocol. Thirteen cases of HbE-susceptible pregnancies were tested to compare the SABER and the ASBER protocols.

Results: Decreasing the single relevant terminator concentration and increasing the annealing temperature in SABER were found to improve specificity. The use of the other 3 types of dideoxynucleotide as competing terminators was shown to offer better detection sensitivity than a single terminator in ASBER. Genotyping results were all correctly determined by ASBER, except one false-negative detection (sensitivity: 80%, specificity: 100%).

Conclusions: An alternative mass spectrometry-based protocol for noninvasive prenatal diagnosis, ASBER, has been successfully developed to allow the detection of a minor DNA population with a point mutation.