Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment for XLSA is mainly supportive, except in pyridoxine-responsive patients. Female XLSA often represents a late onset of severe anemia, mostly due to the acquired skewing of X-chromosome inactivation. Here, we successfully generated active wild-type and mutant ALAS2 induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and two daughters in a family with pyridoxine-resistant XLSA due to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared to that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. Additionally, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared to that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent wild-type ALAS2 allele in active mutant HPCs and ameliorated erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Pyridoxine Response in Mouse Alas2 Knock-in Models of X-Linked Sideroblastic Anemia and X-Linked Protoporphyria

Introduction. Pyridoxal 5' Phosphate (PLP) is the cofactor form of vitamin B6 in ~60 human enzymes. The first enzyme of the heme biosynthesis pathway, delta-aminolevulinic acid synthase (ALAS), that catalyzes the condensation of glycine and succinyl-CoA to form 5-ALA the sole precursor of porphyrins and heme, is PLP dependent. The erythroid specific isoform of ALAS is ALAS2. Inherited ALAS2 mutations cause two rare diseases: X-linked Sideroblastic Anemia (XLSA), due to loss-of-function mutations located throughout the gene, and X-linked Protoporphyria (XLPP), due to gain-of-function mutations specifically located in the C-terminal domain. Male XLSA patients have a microcytic hypochromic anemia of variable severity characterized by abnormal erythroid mitochondrial iron deposits, in nucleated and enucleate cells (ring sideroblasts and siderocytes). XLPP patients develop acute photosensitivity, due to an abnormal erythroid accumulation of free protoporphyrin IX (PPIX), the substrate of ferrochelatase, the last enzyme in the pathway. In two-thirds of XLSA patients, the anemia is responsive to oral pyridoxine supplementation. Isoniazid, an antituberculosis agent, that can cause sideroblastic anemia by limiting PLP availability to ALAS2, may limit free PPIX accumulation in protoporphyric patients. While being well tolerated, isoniazid treatment of protoporphyric patients did not reduce erythroid free PPIX accumulation. Given these clinical findings, we sought to explore the effects of dietary supplementation and restriction of vitamin B6 in animal models of the diseases. Methodology. Using CRISPR-CAS9 editing technology, we generated C57BL/6N mouse models of p.R170H, p.R452H and p.R411H found in XLSA patients with B6-sensitive or -refractory disease, and p. Q548X, a XLPP allele. Fed our normal chow containing 8ppm of pyridoxine, XLSA mouse males develop phenotypes ranging from severe (p.R411H) to trivial (p.R170H) anemia; XLPP animals have the expected protoporphyric phenotype. At weaning, we fed XLSA, XLPP and control male littermates with diets with defined amounts of B6 (Envigo: 0ppm, 2ppm or 10ppm). We performed complete blood counts (CBC) and quantified erythroid free PPIX by flow cytometry after 2, 5 and 8 weeks on diet and evaluated steady state and stress erythropoiesis by flow cytometry at 8 weeks. Results. B6-depleted animals have a growth delay that is more severe in the XLSA animals. Similarly treated control and XLPP animals develop a mild microcytic anemia with siderocytes only after 8 weeks. XLPP depleted animals accumulate less free PPIX compared to normal diet, while a 2 ppm B6 diet did not affect free PPIX accumulation. On 0 ppm B6, all XLSA depleted animals developed a very severe anemia characterized by profound reticulocytopenia and massive splenomegaly. Blood smears revealed many fragmented red blood cells and siderocytes. Flow cytometry analyses reveal a blockage of erythropoiesis, at early stages of differentiation, in both the marrow and the spleen. Feeding with 2ppm B6, demonstrated variable responses in each of the three mutants, with the p.R411H being the most severe and the R170H being the least. Conclusion. All XLSA mutations are sensitive to B6 depletion. Thus, the tendency to develop B6 deficiency with age may account for later clinical presentations in patients with pyridoxine-sensitive mutations. The limited PPIX response and development of siderocytic anemia in B6 deficient XLPP animals may suggest why the B6 inhibitor isoniazid had limited clinical efficacy. Thus our novel XLSA and XLPP mice model the each disease accurately and have demonstrated their potential for evaluating experimental treatments. Disclosures Schmidt: Disc Medicine, Inc.: Research Funding. Fleming: Disc Medicine: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Biallelic mutations in the SARS2 gene presenting as congenital sideroblastic anemia

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