Influence of iron metabolism on gene expression in erythropoietic protoporphyria

Abstract

Erythropoietic protoporphyria (EPP, OMIM 177000) is a hereditary disease characterized by extremely painful photosensitivity. The underlying defect is a partial deficiency of the enzyme ferrochelatase (FECH) which catalyzes the last step of heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX (PPIX). As a result of the enzyme deficiency, a large amount of the phototoxic substance PPIX accumulates in precursors of erythrocytes. In addition, up to 60% of EPP patients show disturbances of iron metabolism, e.g. microcytic, hypochromic anemia, low transferrin saturation and low ferritin. However, administration of iron is observed to worsen the photosensitivity. Genetically, over 97% of EPP patients carry, in addition to a loss of function mutation in trans, the identical SNP FECH IVS3-48C which enhances aberrant splicing. Since iron and heme metabolism as well as splicing are highly regulated we hypothesized that a mechanism exists interconnecting the three processes. An indepth characterization of FECH intron 3 was performed to identify possible sequence features that may be related to the disease causing splice defect or respond directly or indirectly to iron, heme or other products and substrates of heme metabolism. We could show that two homopolymeric tracts in FECH intron 3 differ in length between individuals, and that the low expression allele IVS3-48C is associated with longer poly-C tracts. Although homopolymeric tracts are conserved and significantly over-represented in the genome than simply by chance, we could not find a correlation between disease features and the length of these sequences yet. However, we could demonstrate that cultured cells derived from patients and healthy controls show enhanced aberrant splicing of FECH intron 3 and a subsequent decrease of the amount of FECH protein under iron depletion. The effect is more pronounced in the more frequent SNP IVS3-48T. In the low expression allele (IVS3-48C), a higher baseline level of aberrant splicing is seen under iron saturated condition, which is less enhanced by iron depletion. The consequence of an IVS3-48C genotype is therefore equivalent to the effect iron depletion exerts on cells from individuals with the more common genotype. The observed effect is mediated by the iron and 2-oxoglutarate-dependent dioxygenase Jmjd6, acting as a link between iron availability and splice regulation. To elucidate systemic aspects of iron metabolism in EPP, we further tested our hypothesis that the rate limiting enzyme of erythroid heme biosynthesis, 5-aminolevulinic acid synthase 2 (ALAS2), is involved in the adverse reaction on iron supplementation observed in patients. ALAS2 mRNA harbors an iron-responsive element (IRE) which prevents translation of the enzyme in case iron is scarce. Since iron is deficient in most EPP patients, the translation of ALAS2 mRNA might be partly repressed. Consequently, administration of iron could lead to an increase in ALAS2 protein due to a de-repression of the translation block and subsequently stimulate synthesis of PPIX, worsening the photosensitivity. In a longitudinal study, a positive correlation between hemoglobin levels and PPIX was seen in the three patients investigated – demonstrating the dependence of both PPIX and heme synthesis on iron availability in EPP.