Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A^- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (> 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery.

Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H₂O_2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion.

Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery.