Rapid, Simultaneous Genotyping of 10 Southeast Asian Glucose-6-Phosphate Dehydrogenase Deficiency–Causing Mutations and a Silent Polymorphism by Multiplex Primer Extension/Denaturing HPLC Assay

Abstract

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the most common inherited metabolic enzyme disorder in the world, with a very high incidence throughout the tropics and subtropics as a result of malarial selection (1)(2)(3). More than 140 mutations or combinations of mutations of the X-linked gene for G6PD have been characterized at the DNA level from different ethnic populations worldwide (4)(5). In areas of Southeast Asia, including southern China, at least 25 deficiency-causing point mutations have been identified in the human G6PD gene. Ten mutations (95A→G, 392G→T, 487G→A, 493A→G, 592C→T, 871G→A, 1024C→T, 1360C→T, 1376G→T, and 1388G→A) account for >80% of all known mutations in Southeast Asian countries (4)(5)(6)(7)(8). The current molecular approaches to identifying G6PD-deficiency–causing mutations rely on various methods for allele differentiation (7)(8)(9)(10). Each of these approaches has advantages and limitations, but the technical aspects and/or cost have limited their routine use in most laboratories. Rapid and accurate genotyping of G6PD-deficiency–causing mutations is necessary to meet the requirements for genetic counseling, genetic epidemiology, and clinical molecular diagnosis of this disorder. We have developed a multiplex primer extension/fully denaturing HPLC (PE/DHPLC) assay capable of simultaneously detecting the above 10 G6PD mutations and 1 common polymorphism (1311C→T) (11). Eleven genomic DNA samples with different known genotypes, including the above 11 G6PD mutations and the silent polymorphism as previously determined by direct sequencing, were used to validate this application. A total of 209 blood samples with various G6PD-deficiency–causing mutations, the silent polymorphism, or the wild-type G6PD gene sequence identified by direct sequencing were obtained to test the specificity of this assay by blind analysis. The genotypes of these DNA samples are shown in Table 1 of the Data Supplement that accompanies the …