Glucose-6-phosphate dehydrogenase Aveiro: a de novo mutation associated with chronic nonspherocytic hemolytic anemia

The patient is a 5-year-old boy from Aveiro, Portugal, born to healthy, nonconsanguineous parents with no known family history of anemia. He had chronic hemolytic anemia since birth (hemoglobin between 7.1 and 9.8 g/dL) and underwent exchange blood transfusion in the immediate neonatal period. His clinical history includes 6 episodes of severe acute intravascular hemolysis that required hospitalization and erythrocyte transfusion. On physical examination, his spleen was palpable 6 cm below the left costal margin. Except for anemia, he has had normal health and development.

G6PD activity was assayed according to the recommendations of the World Health Organization.6 To assay the enzyme activity on granulocytes, these cells were purified after centrifugation on a double-density gradient (Histopaque 1119 and 1077; Sigma, St Louis, MO). The cytochemical test was performed according to the procedures described by Dacie and Lewis.7 

Polymerase chain reaction amplification and single strand conformational polymorphism (SSCP) analysis were performed as previously described.8,9 Sequencing was carried out on newly generated amplicons of fragments that had revealed aberrant conformers. The respective forward and reverse primers were used in independent sequencing reactions. Chromosome X-linked microsatellite markers DXS548,10 STR44, STR45, STR49, and STR50¹¹ were analyzed by capillary electrophoresis using fluorochrome-labeled primers.

The diagnosis of G6PD deficiency was based on an undetectable G6PD activity in spite of reticulocytosis (30%). The patient's mother showed a normal enzymatic activity (105%) and no evidence of deficient cells in the cytochemical test (methemoglobin elution test). Biochemical characterization of the mutant G6PD was not feasible because of the total absence of enzymatic activity. The same was true when granulocyte cells were used as the patient enzyme source.

After screening for known mutations by differential endonuclease restriction, SSCP analysis of the patient's G6PD gene showed a mobility shift in exon 8 (Figure 1). Sequencing revealed that this was a G to A transition at nucleotide 806 (Figure 2), resulting in a cysteine-to-tyrosine change at amino acid 269. The entire coding region was later sequenced, but no further alteration was detected. Close analysis of the sequencing histogram revealed a small but visible G peak at the mutation site. This mosaicism was later confirmed by sequencing and SSCP analysis of the patient's buccal cells, which again revealed the presence of 2 cell populations (Figures 1 and 2).

The C269Y mutation was not found in the patient's mother and sister, in whom both hematopoietic and buccal cell samples were analyzed. Haplotyping using X-linked markers confirmed the mother-child relationships. The G6PD intragenic markers were not informative, but the patient and his sister were found to have inherited the same maternal allele for the FRAXA marker DXS548, which maps nearby. This, together with the evidence of mosaicism in the patient, suggests a de novo mutational event that must have occurred during a very early stage of embryogenesis.12 

Most class I mutations are located in exon 10, in an area believed to be involved in dimerization.5 The variant described in this report, G6PD Aveiro, results from a mutation in exon 8, close to a minor cluster of known CNSHA mutations.2 It is difficult to predict the conformational changes brought about by this C269Y mutation. However, based on the preliminary model proposed by Naylor et al,5 it may be speculated that this variant behaves in a manner analogous to G6PD Corum (E274K). The class I phenotype of the latter is thought to result from the disruption of a dimer-stabilizing salt bridge normally formed between Glu274 and Arg348. Because Glu274 is at the end of the alpha helix αi, where Cys269 is located, and considering the general rule that there are 3.6 amino acids per alphahelix turn, these 2 residues are expected to be spatially close. As such, the constraints on the orientation of the alpha helix imposed by the substitution of the small side chain of Cys by the large one of Tyr in G6PD Aveiro could be expected to change the spatial position of Glu274, thereby impairing the formation of the above-mentioned salt bridge. The resultant dimer instability could then explain the severe clinical picture observed in our patient. A recent report by Au et al13 reports further advances in the elucidation of the G6PD crystal structure. However, the reported data are so far insufficient to evaluate our model. Therefore, validation of this proposed mechanism requires further experimental data on the 3-dimensional structure of human G6PD.