Functional Overlap between the SEC23 Paralogs Suggests a Novel Treatment Paradigm for Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemia type II (CDAII), an autosomal recessive disease characterized by ineffective erythropoiesis and increased percentage of bi-nucleated erythroid precursors in the bone marrow (BM), results from loss of function mutations in SEC23B, which encodes a core component of COPII vesicles. Approximately 8,000 secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII vesicles, suggesting that a defect in this pathway would result in a profound systemic phenotype. However, CDAII patients exhibit a specific erythroid phenotype, with no other defects described. Mammals have 2 paralogs for SEC23, SEC23A and SEC23B. In contrast to SEC23B mutations, bi-allelic SEC23A loss of function mutations in humans result in cranio-lenticulo-sutural dysplasia, a disease characterized by skeletal defect but normal erythropoiesis.

We previously demonstrated that a SEC23B-A chimeric protein composed of the first 122 amino acids of SEC23B followed by amino acids 123-765 of SEC23A overlaps in function with SEC23B, suggesting that the 2 SEC23 paralogs are functionally interchangeable. However, to rule out the possibility that the functional overlap was due to the first 122 amino acids of SEC23B, we generated a bacterial artificial chromosome (BAC) transgene that expresses the full Sec23a coding sequence from the endogenous genomic locus of Sec23b (Sec23b-a BAC). We crossed the Sec23b-a BAC to the Sec23b null allele (Sec23b^-) and demonstrated that this BAC rescues the phenotype of mice deficient in Sec23b (Sec23b^-/-). Therefore, we now conclusively demonstrate that the SEC23A protein functionally replaces SEC23B when expressed from the endogenous regulatory elements of Sec23b.

We have previously shown that mice with erythroid-specific and pan-hematopoietic SEC23B deficiency exhibit a normal erythroid phenotype. In light of the functional overlap between SEC23A and SEC23B, we hypothesized that mice with erythroid-specific deficiency for SEC23A, alone or in combination with SEC23B, might exhibit an erythroid phenotype. First, we generated mice with erythroid-specific (EpoR-Cre) SEC23A deficiency. These mice were observed at the expected Mendelian ratios at weaning. Complete (or near complete) excision of the Sec23a floxed (Sec23a^fl) allele was confirmed in the erythroid cells. Peripheral blood counts, BM cellularity and morphology, and percent and distribution of BM erythroid cells among the 5 stages of maturation were indistinguishable between mice with erythroid SEC23A deficiency and wildtype littermate controls. Additionally, the percentage of bi-nucleated erythroid precursors were not increased in Sec23a^fl/flEpoR-Cre+ mice. Thus, mice with erythroid-specific SEC23A deficiency do not exhibit an erythroid phenotype. Similarly, mice with pan-hematopoietic SEC23A deficiency (Vav1-Cre) do not exhibit a hematologic phenotype.

Next, we generated mice with Sec23a deletion and Sec23b haploinsufficiency in the erythroid compartments. These mice exhibited normal survival, a mild reduction in hemoglobin levels (p = 0.014), and a block in late erythroid maturation (Stage V erythroid cells were reduced to 22.6% compared to 30.3% in control mice; p=0.08). In contrast, mice with erythroid-specific deletion for all 4 Sec23 alleles (combined SEC23A/B deficiency) died at mid-embryogenesis exhibiting reduced size and appearing pale compared to wildtype littermate controls, with histologic evidence of dyserythropoiesis reminiscent of human CDAII. Overall, these results suggest a requirement for a threshold level of total SEC23 (combined SEC23A/B) expression in the erythroid compartment. These results also suggest that the defect in CDAII is intrinsic to the RBC.

Finally, we generated K562 cells with either SEC23B or SEC23A deletion using CRISPR/Cas9 genome editing. SEC23B or SEC23A deletion alone was tolerated in the K562 cells. However, combined deletion of SEC23A and SEC23B was not tolerated.

Taken together, the results summarized above demonstrate that SEC23A and SEC23B appear to compensate for one another's function in murine and human erythroid cells. This finding suggests a potential therapeutic role for increasing expression of SEC23A to compensate for SEC23B deficiency in CDAII. This work is currently ongoing.