Molecular abnormalities of a human glucose-6-phosphate dehydrogenase variant associated with undetectable enzyme...

Among a large number of glucose-6-phosphate dehydrogenase (G6PD) variants associated with different severity of clinical manifestations, enzyme deficiency, and kinetic abnormalities found in humans, only one variant exhibits no measurable activity and lacks an immunologically cross-reacting material in blood cells and other tissues. The mRNA content of the patient's lymphoblastoid cells was found to be normal, and the size of mRNA was also normal (i.e., approximately 2.4 kb). Western blot hybridization indicated that the patient's cells did not produce cross-reacting material. The variant mRNA was reverse transcribed and amplified by PCR. Nucleotide sequencing of the variant cDNA showed the existence of three nucleotide base changes, i.e., a C----G at nucleotide 317 (counting from adenine of the initiation codon), which should cause Ser----Cys substitution at the 106th position (counting from the initiation Met); a C----T at nucleotide 544, which induces the Arg----Trp at the 182d position; and a C----T at nucleotide 592, which induces Arg----Cys at the 198th position of the protein. The existence of three mutation sites was confirmed by sequencing of selected regions of the variant gene. No base deletion or frameshift mutation was found in the variant cDNA. No nucleotide change was detected in the extended 5' region, which included the most distal cap site. When the variant cDNA was expressed in Escherichia coli, the G6PD activity was approximately 2% of that expressed by the normal cDNA, and cross-reacting material was undetectable. However, when the variant mRNA was expressed in the in vitro translation system of rabbit reticulocytes, the variant protein was produced. These results suggest that extremely rapid in vivo degradation or precipitation of the variant enzyme induced by the three amino acid substitutions could be the major cause of the molecular deficiency.