SMIM1 missense mutations exert their effect on wild type Vel expression late in erythroid differentiation

Background: Vel expression on erythrocytes is variable due to polymorphisms, complicating Vel typing. Weak Vel expression can be caused by mutations within SMIM1 in a heterozygous setting, suggesting a dominant negative effect of SMIM1 mutants on wild type (wt)SMIM1 expression. Here we report how SMIM1 expression is regulated during erythropoiesis, to understand its variable expression on erythrocytes. Study Design and Methods: Peripheral blood reticulocytes at different stages, cultured erythroid precursors and HEK293T cells were used to investigate expression and putative competition between wtSMIM1 and mutated SMIM1 VEL*01W.01, (c.152T>A (p.Met51Lys)), VEL*01W.02 (c.152T>G (p.Met51Arg)), and VEL*01W.03 (c.161T>C (p.Leu54Pro)). Results: Depending on the mutations in SMIM1 an effect on total and membrane expression of SMIM1 was observed in transfected HEK293T cells, but co-expression of wtSMIM1 and mutatedSMIM1 did not have an effect on wtSMIM1 membrane expression. During differentiation of donors expressing VEL*01W.01, VEL*01W.03, Vel-positive, Vel-negative (homozygote SMIM1*64_80del), and Vel-heterozygote SMIM1*64_80del primary human erythroblasts no overt defect was found in Vel expression dynamics or total SMIM1 expression levels when compared with wtSMIM1 erythroblasts. However, during enucleation, total Vel expression was significantly lower on reticulocytes of Vel-weak donors expressing heterozygote mutated SMIM1 compared to Vel-positive or Vel-heterozygote SMIM1*64_80del donors, while Vel expression on extruded nuclei was maintained. In addition, reticulocyte maturation in vivo showed further loss of Vel expression in these individuals and nearly absent on erythrocytes. Conclusion: These results suggest that SMIM1 mutations exert a dominant negative effect on wtSMIM1 probably by affecting SMIM1 multimerization and thereby Vel epitope presentation at the latest stages of erythroid differentiation.