Multi-mutational analysis of fifteen common mutations of the glucose 6-phosphate dehydrogenase gene in the Mediterrranean population

Abstract

Background Glucose-6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defence against oxidizing agents and in reductive biosynthetic reactions. Many variants of G6PD have been described, mostly produced from missense mutations, with wide ranging levels of enzyme activity and associated clinical symptoms. Method A single base extension assay is used, yielding a single base difference of the extended products. Primers are designed to amplify products of different sizes with distinct fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. Results We present the first application of a multiplex multicolour assay to detect 15 of the most frequent G6PD-related mutations in Spain, which are studied in three multiplex reactions. Capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high-resolution discrimination between wild-type and mutant alleles, even though various mutations may be present in the multiplex analysis. Conclusion The analytical method described herein offers greater diagnostic power in Spanish and Mediterranean populations and would facilitate automated genotyping in routine molecular diagnostics and large-scale genetic studies (e.g., newborn screening programs).