Single Nucleotide Variations In Spectrin-1β Accentuate The Red Blood Cell Storage Lesion

Abstract

IntroductionDuring routine storage packed red blood cells (PRBC) undergo biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. Donor-to-donor variability in the severity of the storage lesion has been reported. The extent to which donor-associated differences in blood component storage affect blood product quality and post-transfusion outcome remains unknown. Murine models with single nucleotide variants (SNV) in gene encoding spectrin-1β were used to investigate the impact of mutations on the RBC storage lesion. MethodsTwo murine lineages with N-ethyl-N-nitrosourea (ENU) generated single SNV in Spnb1, encoding spectrin-1β (Table 1), were selected from the Australian Phenomics Facility library (http://databases.apf.edu.au/mutations). Using genetic selection, homozygous (HOM), heterozygous (HET) and unaffected (WT) mice from each strain were generated (C57BL/6 background strain). Murine blood was leucoreduced, prepared in SAGM (0.4 HCT) and stored at 4°C for time course assessment of RBC characteristics. At day (D), D2, D7, D14 and D21 of storage, RBC integrity and evidence of storage-related changes were investigated using RBC osmotic fragility and flow cytometric analysis of CD44, CD47, TER119 and phosphatidylserine (PS). Data were generated from analysis of blood from Spnb1 (pedigree spectrin-1β a) homozygous (HOM, n=3), heterozygous (HET, n=3) and unaffected (WT, n=2 ); Spnb1 (pedigree spectrin-1β b) HOM (n=4), HET( n=4); C57BL/6 (n=4). The Mann-Whitney Test and ANOVA were utilised for statistical analyses (95% CI).Table 1Pedigrees selected for mutations in RBC structural genes (Build 38)Gene (Protein)ChromosomeBase changeAmino acid changeAmino acid positionMutation genomic position (bp)DomainPolyPhen scoreSpnb1 (Spectrin-1β) (a)12A - GS - P85776,620,753Spectrin binding0.24 benignSpnb1 (Spectrin-1β) (b)12T - CD - G66476,621,331Spectrin binding0.2 benign ResultsAt D2 of storage SNV in Spnb1 did not alter RBC characteristics, with all mice studied demonstrating a similar resistance to osmotic lysis and levels of CD44, CD47, TER119 and PS. By D7 of storage, clear pedigree-related differences in RBC characteristics were evident. At D7, RBC from spectrin-1β(a) HOM mice had significantly increased osmotic fragility and exposure of PS as well as significantly reduced CD44 and TER119 expression compared to unaffected siblings and background strain. Of note, these changes were not evident in the spectrin-1β(b) HOM mice at D7. For both strains at D7, heterozygous SNV did not exhibit altered storage parameters. By D14 both HOM and HET spectrin-1β(a) mice demonstrated a phenotype consistent with an exacerbated RBC storage lesion, characterised by significantly increased osmotic fragility and exposure of PS, and reduced CD44 and CD47 compared to background strain. At D14 there was also evidence of exacerbation of the storage lesion in stored RBC from HOM spectrin-1β(b) mice (significantly increased PS), though this was not to the extent observed in the spectrin-1β(a) mice. By D21 all murine RBC were substantially degraded under these storage conditions. ConclusionsSNV in Spnb1,encoding RBC structural protein spectrin-1β, resulted in both early onset and exacerbation of the RBC storage lesion. Further, the degree of storage lesion and the point at which RBC degradation was observed was not only dependent on the homozygous or heterozygous status, but the mutation itself. These data demonstrate that minor genetic variation in genes encoding important RBC proteins contribute to donor related differences in PRBC storage. Disclosures:No relevant conflicts of interest to declare.