Congenital dyserythropoietic anemia type-II (CDAII) is an autosomal recessive disease characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from mutations in SEC23B, one of two closely related mammalian SEC23 paralogs. SEC23 is a core component of COPII coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum (ER) to the Golgi apparatus. Bone marrow transplantation cures CDAII, suggesting that the pathologic defect in this disease is restricted to the hematopoietic compartment. However, the mechanism by which SEC23B-deficiency results in CDAII remains unknown. We previously reported that mice homozygous deficient for SEC23B (Sec23bgt/gt) exhibit massive pancreatic degeneration. The latter resulted in perinatal mortality precluding evaluation of the adult hematopoietic compartment. To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, fetal liver cells (FLC) were harvested from Sec23bgt/gt or wildtype (WT) control E17.5 embryos and transplanted into lethally irradiated C57BL/6J mice. Recipients of Sec23bgt/gt FLC had normal peripheral blood counts and were indistinguishable from recipients of WT FLC. To test for a more subtle hematopoietic defect, Sec23bgt/gt FLCs were tested directly against WT FLC for their ability to reconstitute hematopoiesis in a competitive repopulation assay. SEC23B deficient FLC exhibited no competitive disadvantage at reconstituting erythropoiesis relative to WT FLC over 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. We also generated a second, conditional Sec23b allele, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 with Cre-recombinase results in a frame shift leading to a stop codon in exon 7. Mice with erythroid-specific SEC23B deficiency were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice maintained normal erythropoiesis indistinguishable from their WT littermates. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to p48-Cre or Pdx1-Cre transgenes generated phenotypes indistinguishable from complete SEC23B deficiency, demonstrating that loss of pancreatic Sec23b expression alone is sufficient to explain the perinatal lethality observed in Sec23bgt/gt and Sec23b-/- mice. Our results conclusively demonstrate that in contrast to humans, SEC23B-deficiency results in massive pancreatic degeneration in mice, but no CDAII in these animals.

To investigate the cause of the disparate human and mouse SEC23B-deficient phenotypes, SEC23B/SEC23A expression ratios were examined in endogenous tissues from both species. This ratio (normalized to the ratio in liver mRNA as 1.0) was higher in mouse pancreas (12.7) compared to BM (2.6), with the reverse pattern observed in human BM (7.8) relative to pancreas (5.5). These data, taken together with the high degree of identity between SEC23A and SEC23B (∼ 85% amino acid identity), suggest that the tissue-specific functions of SEC23A and SEC23B may have shifted during evolution between humans and mice. To test the role of SEC23A, we generated a mouse with a conditional Sec23a allele, in which exon 3 is flanked by loxP sites (Sec23afl). Cre-recombinase mediated deletion of exon 3 results in a frame shift leading to a stop codon in exon 7 (Sec23a-). Mice with erythroid-specific SEC23A-deficiency (Sec23afl/-/EpoR-CreTg+) maintained normal red blood cell counts indistinguishable from their WT littermates.

In summary, we have shown that SEC23B-deficient humans and mice exhibit disparate phenotypes. We have also demonstrated variations in the gene expression programs for SEC23A and SEC23B potentially explaining the pancreatic phenotype of SEC23B-deficiency in mice and the erythroid phenotype in humans. These results suggest that the two SEC23 paralogs have overlapping functions and that therapeutic strategies that increase the expression of either SEC23A or SEC23B in erythroid cells might be effective in CDAII. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII.

Disclosures:

No relevant conflicts of interest to declare.

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