A new mutation responsible for severe G6PD deficiency in two ethnic Chinese with different clinical presentations:...

To test the hypothesis that clinical manifestation in G6PD deficiency correlates with a molecular lesion, we investigated the G6PD gene of two Chinese Americans both of whom had G6PD deficiency, but who manifested different clinical presentations. In this study, we have developed a direct PCR sequencing protocol to examine the human G6PD gene. By using optimized PCR conditions with internal primers, we were able to amplify a 4.2 kb DNA fragment (covering exon 3 through 13 of the G6PD gene) of consistently high quality. From this we were then able to generate high quality single-stranded DNA templates by asymmetric PCR for subsequent sequencing. We also overcame the crossband problem by using internal primers, high temperature reaction with Taq I DNA polymerase, and/or sequencing with gene 32 protein. We could consistently amplify exons 1 and 2 despite their high G/C content by substituting 75% of dGTP with deoxy-7-deaza-guanosine triphosphate. By using this novel approach, we have identified a new mutation at cDNA position 1376 from G to T, which causes substitution of Leu for Arg at amino acid position 459. This mutation has not been reported in other ethnic groups. It is the only genetic defect in the coding regions of the G6PD gene of these two G6PD deficient individuals. We speculate that in addition to a defect in the G6PD gene, other factors also play a role in the clinical manifestation of G6PD deficiency.