Steap3 Is a Novel Candidate Gene for Regulating the RBC Blood Storage Lesion By Mitigating Peroxidation of Membrane Lipids in a Mouse Model

Background: There is substantial donor-to-donor variability in the post-transfusion survival of stored human RBCs. RBCs from different strains of inbred mice also store differently; RBCs from C57BL/6 (B6) mice store well, whereas RBCs from FVB/NJ (FVB) mice store poorly, as defined as 24-hr post-transfusion RBC recoveries (24hr-recoveries). We hypothesized that observed differences in RBC storage between inbred mouse strains are heritable and can be mapped using mouse genetic tools.

Methods: B6 and FVB mice were crossed to generate F1 mice, which were then intercrossed to generate 156 F2 animals. RBCs from single donor mice were stored for 7 days, followed by transfusion into B6xFVB F1 recipients that were transgenic for GFP, which is expressed in essentially 100% of F1 RBCs. 24hr-recoveries were measured by bleeding recipients 24 hours post-transfusion and enumerating non-fluorescent donor RBCs. The use of F1 recipients avoided crossing allo-antigenic barriers. Prior to transfusion, a sample of each donor RBC unit was frozen at -80^oC; all frozen samples were subjected to LC-MS/MS to generate an untargeted metabolomics profile. DNA from each mouse was applied to a 1,414 SNP Illumina BeadChip. Quantitative Trait Loci (QTL) analysis was performed for 24hr-recoveries and also for each LC-MS/MS analyte identified, by means of fitting a linear model at each SNP, and adjusting for the number of tests using a false discovery rate (FDR) procedure. For each LC-MS/MS analyte, correlation coefficients were calculated to 24-hr recoveries. Correlations of LC-MS/MS metabolites to 24hr-recoveries were combined with QTL mapping and referenced to known metabolic pathways to generate a blood storage metabolomics profiles associated with an RBC storage phenotype and linked to genotype. Additional genetic mapping resolution was obtained by backcrossing F2 mice with poor storage to B6 parents, and selecting poor-storing progeny to breed for each next generation.

Results: 24hr-recoveries exhibited a Gaussian distribution in F2 mice. LC-MS/MS quantified 554 analytes in each stored RBC sample. QTL analysis of the 24hr-recoveries using 813 informative SNPs identified a significant QTL (maximum peak p=2.09x10^-31), that we have termed Rbcstor1, spanning a ~149 Mb interval on chromosome 1 (rs13475827 to rs13476300). Fine mapping using backcrossed populations refined Rbcstor1 to a 9.5 Mb interval containing 64 genes. Filtering for coding genes harboring nonsynonymous B6-FVB SNPs identified 5 genes (Gli2, Steap3, Ccdc93, Rab3gap1, Tli). Steap3 is a functional enzyme in erythroid cells, in which it is the primary ferrireductase converting Fe³⁺ to Fe²⁺, both mitigating oxidative stress as well as allowing transferrin-dependent iron uptake. Steap3 harbors two FVB-B6 non-synonymous SNPs (p.A350V and p.N455S). Metabolomics analysis revealed that oxidized products of lipid metabolism strongly correlated with post-transfusion RBC survival, including the bioactive lipids Leukotriene B4 (r=0.71, p=1.7x10^-25) and Prostaglandin E2 (r=0.81, p=2.6x10^-37). In addition, a wide variety of dicarboxylic fatty acids (e.g. dodecanedioate (r=-0.81, p=1.1x10^-44) and octanedioate (r=-0.85, p=3.4x10^-53)) strongly correlated with RBC storage. Based upon additional QTL analysis of products of lipid peroxidation, a significant QTL was identified, which we have termed Rbcstormet1. Rbcstormet1 overlaps extensively with Rbcstor1.

Conclusion: We have identified Rbcstor1 on chromosome 1 as a QTL strongly associated with 24hr-recoveries. Within this region, Steap3 is a strong candidate gene. Steap3 has been previously implicated in erythroid phenotypes: mice lacking Steap3 are profoundly anemic, and a human family carrying a STEAP3 nonsense mutation has been reported to exhibit a congenital hypochromic anemia. However, to the best of our knowledge, Steap3 has no known function in mature RBC biology or RBC storage. We hypothesize that the FVB Steap3 allele is a hypomorphic variant, which adversely impacts RBC storage biology by decreasing the ability of RBCs to handle oxidative stress, leading to lipid peroxidation that generates inflammatory lipids, lysolipids, and dicarboxylic acids. In addition to identifying a novel genetic locus associated with 24hr-recoveries of stored RBCs, these studies suggest that polymorphisms in Steap3 or in related proteins could contribute to human blood donor variability.