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2467 Uncoupling Alas2 from Iron-Regulatory Proteins Demonstrates the Protective Role of Alas2 Iron-Responsive Element in Protoporphyrias

Program: Oral and Poster Abstracts
Session: 102. Iron Homeostasis and Biology: Poster II
Hematology Disease Topics & Pathways:
Research, Genetic Disorders, Diseases, Biological Processes, Pathogenesis
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Sarah Ducamp, PhD1, Dean R. Campagna2*, Paul J. Schmidt, PhD2 and Mark D. Fleming, MD, DPhil1

1Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA
2Department of Pathology, Boston Children's Hospital, Boston, MA

Introduction. Many genes relevant to iron metabolism include a transcribed cis-regulatory element called iron-responsive element (IRE) that couples their expression to the availability of intracellular iron. Canonical IREs consist of a conserved stem-loop structure in the 5’ or 3’ untranslated region (UTR) of these mRNAs. IREs are bound by two iron-regulatory proteins (IRP1 & IRP2) to either limit the translation of the targeted transcript or to stabilize it. Among the 5’UTR IRE- genes, ALAS2, the erythroid-specific aminolevulinate synthase, catalyzes the first step of heme biosynthesis. Loss-of-function and gain-of-function mutations cause X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP). A deficiency in the last enzyme of the pathway, ferrochelatase (FECH), causes the most common Erythropoietic Protoporphyria (EPP). A single-family with protoporphyria has been described with a mutation in the mitochondrial unfoldase CLPX. In the mouse, targeted deletion of IRP2 also results in protoporphyria.

Several lines of evidence suggest a role for Alas2 in modifying the severity of erythroid porphyrias. In 4 patients with congenital erythropoietic porphyria due to the same UROS genotype, an XLPP mutation was also found in the most severely affected individual. A mutation in the Alas2-IRE has been described as a modifier in the CLPX-family. Furthermore, increased levels of Alas2 mRNA have been reported in EPP patients. Lastly, other reports suggest that iron deficiency may be protective in protoporphyrias.

Methods. We generated a mouse line lacking the loop of Alas2-IRE (Alas2ΔLOOP), which should uncouple ALAS2 expression from IRP-dependent regulation. We phenotyped hemizygous male Alas2ΔLOOP/Y mice from 1 to 8 weeks of age. We compared their phenotype to established mouse models of protoporphyria (EPP FECHm1Pas/m1Pas, XLPP Alas2Q548X/Y, and IRP2-/-) at baseline. We also challenged animals with iron deficiency and iron overload. Lastly, we investigated the severity of EPP animals in the presence or absence of Alas2-IRE.

Results.

(a) Phenotype. Alas2ΔLOOP/Y mice have a transitory protoporphyria that resolves nearly completely by 8 weeks of age. The RBC protoporphyric phenotype is similar to XLPP mice, but substantially less severe than the EPP, XLPP, and IRP2 models.

(b) Iron status. EPP, XLPP, and Alas2ΔLOOP male animals were challenged with either iron deficiency or iron overload at weaning. In iron-poor conditions, all three models have an increase in erythroid PPIX, with the Alas2ΔLOOPanimals having by far the most dramatic change after 5 weeks (mean MFI PPIX [SD] for Alas2ΔLOOPiron-replete: 12.8[6.0] v. iron poor 752[374]). However, only the Alas2 mutants (XLPP, Alas2ΔLOOP) see a modest but prolonged increase in erythroid PPIX when injected once with iron dextran.

(c) Disease modifier. The double mutant Alas2ΔLOOP/Y FECHm1Pas/m1Pas (EPP-ΔLOOP) accumulate more erythroid PPIX than the EPP animals at 8 weeks of age (MFI PPIX mean [SD] for Alas2ΔLOOP 18.8[8.9], EPP 128.6 [19.1], EPP-ΔLOOP 659.6[384.7]). The fraction of fluorocytes (erythroid cells in peripheral blood abnormally containing PPIX) is also increased (%mean [SD] for EPP 57.8[8.9], EPP-ΔLOOP 97.6[2.6]). XLPP animals lacking one allele of IRP2 (Alas2Q548X/Y IRP2wt/-) present a phenotype similar to the EPP-ΔLOOP animals while Alas2Q548X/Y IRP2-/- are not viable.

Conclusion. Altogether, our results confirm that iron status strongly modifies the erythroid PPIX accumulation, particularly when Alas2 expression is uncoupled from the IRPs control. We also demonstrated that the IRE-IRP system plays a critical role in mitigating the severity of protoporphyria in the rodents, providing evidence that targeting the Alas2-IRE may have pharmacological benefits.

Disclosures: Schmidt: DISC medicine: Research Funding. Fleming: Dyne Therapeutics: Consultancy; Affyimmune: Consultancy; DISC medicine: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Evolve Immune: Consultancy.

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