Barman-Aksoezen J, Schneider-Yin X, Minder EI. J Rare Dis Res Treat. (2017) 2(4): 1-5 Journal of www.rarediseasesjournal.com Rare Diseases Research & Treatment

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Iron in erythropoietic protoporphyrias: Dr. Jekyll or Mr. Hyde? Jasmin Barman-Aksoezen1, Xiaoye Schneider-Yin1, Elisabeth I. Minder2* 1Stadtspital Triemli, Institute of Laboratory medicine, Zurich, Switzerland 2Stadtspital Triemli, outpatient clinics, Zurich, Switzerland

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Article Notes Erythropoietic protoporphyria consists of two different genetic diseases, Received: April 16, 2017 erythropoietic protoporphyria (EPP) and X-linked erythropoietic protoporphyria Accepted: June 02, 2017 (XLEPP). Both of them are often accompanied by iron deficiency. Iron *Correspondence: supplementation appears to be beneficial in XLEPP, although the clinical Elisabeth I. Minder experience until to date is limited. In EPP, iron supplementation is discussed Stadtspital Triemli, Porphyria outpatient clinics, Zurich, ambiguously and may cause harm in the majority of cases. Switzerland; E-mail: [email protected] This minireview summarizes the limited knowledge on the connections © 2017 Elisabeth I. Minder. This article is distributed under the of iron to regulation of and synthesis and the terms of the Creative Commons Attribution 4.0 International influence these regulations may have on disease severity in the protoporphyrias. License. Further, we propose clinical guidelines, how to manage iron deficiency in both Keywords XLEPP and EPP. Erythropoietic protoporphyria Iron supplementation Heme biosynthesis Introduction Porphyrin synthesis Regulation Two inborn errors of heme biosynthesis, erythropoietic Liver disease protoporphyria (EPP, OMIM #177000) and X-linked erythropoietic protoporphyria (XLEPP, OMIM #300752) lead to accumulation of protoporphyrin IX (PPIX) during erythropoiesis1. EPP is caused by inactivating mutations in the for the last enzyme of heme biosynthesis, (FECH). FECH catalyzes the incorporation of ferrous iron into protoporphyrin IX (PPIX) to produce heme. XLEPP is caused by activating mutations in the aminolevulinate synthase 2 (ALAS2) gene2. ALAS2 catalyzes the conversion of glycine and succinyl-CoA to delta-aminolevulinic acid - the exclusive heme precursor in the initial and rate-limiting step of heme biosynthesis during erythropoiesis. In both disorders, PPIX accumulates in the body and leads to acute and severely painful phototoxic reactions

Excess PPIX is excreted through the liver and cause damages to hepatocytesof the skin upon and cholangioles.exposure to light, In 2-5% both of sunlightthe patients, and theartificial cholestatic light. liver damage is so severe that liver transplantation - preferentially together with bone marrow transplantation to prevent relapse by ongoing PPIX overproduction in the native erythropoietic tissue -is needed to save patients’ lives3,4.

in 19615 and its unique inheritance was however resolved 40 years laterThe by clinicalGouya etdescription al.6. In over of 95% EPP ofwas the first patients, made aby combination Magnus et al.of a loss-of-function mutation in the FECH gene with a splice altering single nucleotide polymorphism (SNP) c.315-48C in trans reduces

Page 1 of 5 Barman-Aksoezen J, Schneider-Yin X, Minder EI. J Rare Dis Res Treat. (2017) 2(4): 1-5 Journal of Rare Diseases Research & Treatment the enzyme activity to below a critical threshold of 35% In contrast, our study on longitudinal data of three and leads to overt disease. Homozygous or compound EPP-patients found that iron availability (as assessed by heterozygous mutations are rare7. hemoglobin levels) positively correlated with the amount of PPIX in the individual patient. In two patients, iron substitution increased liver damage as seen by an increase e.g. a mild microcytic, hypochromic anemia and low serum in liver transaminases, which led to discontinuation of the iron,EPP low patients transferrin frequently saturation show and signs ferritin of iron and deficiency a slightly treatment23,24. In the same direction points the observation elevated zinc-protoporphyrin8-13. Female patients, who are of lower PPIX levels in females than in males, the former 13. Additionally, the to have lower PPIX levels than male patients13, 14. physiologically more likely to develop iron deficiency, tend majority of EPP-patients under our care, who received iron In 2008, gain-of-function mutations affecting the beingsupplementation more prone at some to iron point deficiency in their life from their family responsible for a second form of protoporphyria, XLEPP2. informally reported worsening of photosensitivity after iron firstThe mutations enzyme of result heme in biosynthesis, an over activity ALAS2, of ALAS2 were and found in substitution.doctor or other How physicians can iron substitution for suspected in protoporphyria iron deficiency, turn, leads to accumulation of PPIX and to a similar extent, also zinc-protoporphyrin (ZnPP). XLEPP is suspected in to characters in the famous novel of Robert Louis Stevenson patients with both high PPIX and high ZnPP concentration “Strangebe benevolent Case ofand Dr. maleficent Jekyll and at Mr. the Hyde” same25 time,? comparable genetic testing. Patients with XLEPP also show signs of iron Beneficial effects of iron substitution in XLEPP in the erythrocytes,15. In XLEPP, and the the amount diagnosis of has PPIX to accumulationbe confirmed by is In XLEPP, the overproduction of PPIX is caused by the on the average more pronounced and liver damage is more overly active rate-limiting enzyme ALAS2, while the activity frequentdeficiency compared to EPP15. of the last enzyme, FECH, is normal. Human FECH is able The prevalence of protoporphyria is 1:100.000 – Overly active ALAS2 increases one of the substrates of 1:200.000 in Europe and the US, 2- 10% of all cases are to use iron and, with a lesser affinity, zinc, as substrates. XLEPP15. Before 2008, XLEPP was included in the statistic FECH reaction, PPIX. The other substrate, iron, might of EPP, as the two diseases are clinically indistinguishable. become depleted and the second best substrate, zinc, is used. Substitution of iron therefore might help to further Ambivalent experiences with iron substitution reduce the pool of metal free PPIX, thereby reducing As patients of both types of protoporphyria show signs phototoxic reactions, at the same time reducing the 8, iron substitution appears to be an obvious therapeutic approach for treatment of including anemia. In our experience with three XLEPP liver damage by PPIX and further correct iron-deficiency bothof an anemia iron deficient and possibly anemia even the photosensitivity. The cases from our Swiss porphyria center, iron substitution hypothesis in this approach is that substituted iron, as a co- indeed reduced PPIX and light sensitivity and improved substrate of FECH, may be able to deplete excess amount general health (unpublished results). A recent case report of PPIX to form heme. The clinical experience however from 128 µmol/l (reference <0,09) at the time of diagnosis is ambiguous: Iron substitution was shown to indeed todescribed between a 20boy to with 40 µmol/l confirmed under XLEPP: oral iron his substitutionPPIX dropped26. increase hemoglobin and decrease photosensitivity in The authors also raise the question, whether case reports some cases and was even able to reverse liver damage16,17. In the majority of published case reports however, discovery of XLEPP in 2008 might contained this subgroup iron substitution was accompanied by an increase in ofregarding protoporphyria. beneficial Theiron substitutioncurrent knowledge published is beforethat iron the photosensitivity and / or PPIX levels18-20. The ambiguity is illustrated by a peculiar case with severe 21,22, who prior to iron supplementation effectsis only of beneficial iron substitution, for patients such as with a possible XLEPP, iron however overload, the exhibited a PPIX concentration of 18 µmol/ L. Oral iron arepathophysiology still unknown. of the iron deficiency and the long-term substitutioniron deficiency improved the photosensitivity and had Adverse effects of iron substitution in EPP intermittent oral iron PPIX concentrations varied between As stated above, the role of iron supplementation is more 30-40initially µmol/L, no influence which again on his dropped PPIX levels, to 21.4 but comparable later under to ambivalent for EPP and our group was intrigued by our clinical observations that iron supplementation increased photosensitivity, the amount of PPIX and even adversely the initial value after intensified intravenousof protoporphyrin iron therapy. affected liver parameters in EPP patients. A potential The general well-being improved each time5 have by intensified not been irondiscussed therapy in this (seasonal publications). fluctuations expression of the rate-limiting enzyme in erythropoietic concentrations which may be significant explanation of this unexpected findings is the fact that the

Page 2 of 5 Barman-Aksoezen J, Schneider-Yin X, Minder EI. J Rare Dis Res Treat. (2017) 2(4): 1-5 Journal of Rare Diseases Research & Treatment heme synthesis, ALAS2, connects iron to heme metabolism 27: The mRNA of ALAS2 carries an iron-responsive element hepatic peptide hepcidin is considered to be the master (IRE) at its 5`untranslated region (UTR). Under low iron regulatorabsorption of or iron an unidentifiedmetabolism mechanismand controls of intestinal iron loss. ironThe condition, iron-responsive 1 or 2 (IRP1/ IRP2) absorption by degradation of the iron transporter protein bind to these IREs and thereby prevent translation of ferroportin thereby preventing dietary iron uptake35. ALAS2 mRNA into active enzyme. This process adapts Hepcidin is upregulated in iron overload, infection and heme precursor synthesis to iron availability. chronic disease. In addition, hepcidin hinders the export of iron from its stores such as macrophages to the circulation. Our investigations support an effect of enhanced ALAS2 and XLEPP is due to an inadequately elevated hepcidin, PPIX synthesis in EPP, as we found elevated ALAS2 similarThe question to other arose chronic whether diseases. the iron Bossi deficiency et al. investigated in both EPP mRNAexpression and onprotein the rate concentrations of heme precursor in peripheral and specifically blood iron uptake in a group of EPP patients with normal iron samples of patients. Moreover, in-vitro FECH inhibition status and found no block of intestinal absorption and was accompanied by induced ALAS2 mRNA expression in normal hepcidin expression in urine and serum12. However, cultures of erythroleukemic K562 cells24. And, thirdly, we the patients selected by Bossi et al. had a normal iron expect that the regulation of the porphyrin biosynthesis status without prior supplementation and therefore in our in EPP behaves similarly to the pathophysiologically opinion, do not qualify to investigate the pathophysiology related congenital (CEP, OMIM #263700). In these condition, elevated ALAS2 expression of 67 EPP patients, which comprised both anemic and not- exacerbates porphyrin intermediates production and of iron deficiency in EPP. In our own investigation, a cohort disease severity, which can be improved by iatrogenic- hepcidin concentration compared to healthy volunteers13. 28,29. Therefore, our hypothesis anemic individuals, exhibited a significantly decreased is that the translation of increased expressed ALAS2 elevated hepcidin to be responsible for the observed iron inducedmRNA into iron enzyme deficiency protein is stimulated by an unlimited The results of both studies finally excluded an inadequately iron availability and that this mechanism accounts for the increase in PPIX after iron supplementation that we deficiencyBossi et in al. EPP further patients. investigated in the same iron-replete observed in the EPP patients. EPP-subgroup the expression of erythropoietin and of the serum cytokines G-CSF, IFN, IL-8, MCP-1, MIP-1b or TNF-a, which all did not differ from the control group. However, protects against exacerbated phototoxic symptoms. On the same limitation as mentioned above applies. the Onother the hand, one iron hand, is slightnecessary iron for deficiency the expression apparently and stability of FECH: The enzyme carries a FeS-Cluster, which stabilizes the newly translated protein and is necessary for The main conundrum about the observed iron deficiency the enzymatic function30,31. Under iron depleted condition, this respect it is interesting to mention that inactivating remains, why do EPP patients show iron deficiency? In the half-life of FECH was shown to be reduced in pulse mutations of ALAS2 in sideroachrestic anemia (OMIM chase and cell culture experiments32,33. In addition, iron #300751) lead to iron accumulation, whereas activating mutations in XLEPP or an increased expression of ALAS2 acceptor site in FECH intron 3 (c.315-63), reducing the amountdeficiency of correctly increases spliced the usageFECH mRNA of the and aberrant protein 34 splice. This facts point to a direct physiological link between ALAS2 in EPP lead to iron deficiency. We wonder whether this aberrant acceptor splice site also happens to be activated 6 feedback regulation. . Indeed, expression and iron metabolism by a hitherto unidentified Proposed clinical guidelines for the management serumby the ferritin,disease definingshow normal SNP ashemoglobin discussed concentrationsearlier 13 of iron deficiency in the protoporphyrias indicatingEPP-patients that with iron replete availability, iron stores rather as definedthan FECH by a activity, normal Iron is an essential factor for human life, but toxic in is the limiting factor in the heme synthesis of EPP patients. excess. In both types of protoporphyria, all studies describe EPP patients with normal hemoglobin also exhibit normal iron stores as accessed by ferritin13. Therefore, the question remains unsolved, why are many EPP patients patientsan increased with XLEPP frequency and our of ironguideline deficiency. includes The a generous current ironknowledge substitution is that based iron supplementation on general health, is beneficialanemia and for ironThe deficient?fate of iron in EPP ZnPP-concentrations. As no controlled mechanism to excrete iron is In EPP, we prefer to apply iron supplementation only known, iron homoeostasis is thought to be regulated suffering from severe fatigue, low ferritin and microcytic protoporphyrias is either caused by a block in intestinal andto patients hypochromic with anemia severe ironwith a deficiency, hemoglobin especially concentration those by its absorption only. The observed iron deficiency in

Page 3 of 5 Barman-Aksoezen J, Schneider-Yin X, Minder EI. J Rare Dis Res Treat. (2017) 2(4): 1-5 Journal of Rare Diseases Research & Treatment of below 10g/dL. During iron supplementation, preferably on small doses and applied during seasons with short 26-35. and a high prevalence of X-linked protoporphyria. Mol Med. 2013; 19: daylight, the patients are monitored closely for their PPIX 16. Kniffen J. Protoporphyrin removal in intrahepatic porphyrastasis. concentrations and signs of liver damage. Current trials on 36 iron supplementation in both XLEPP and EPP will help to 17. Gastroenterology. 1970; 58: 1027. hepatic dysfunction in erythropoietic protoporphyria. Ann Intern Gordeuk VR, Brittenham GM, Hawkins CW, et al. Iron therapy for and the effect of iron substitution on symptoms, PPIX- concentrationfurther clarify andthe hemoglobinpathophysiology synthesis. of the iron deficiency 18. Med.Baker 1986; H. Erythropoietic 105(1): 27-31. protoporphyria provoked by iron therapy.

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290(2): G199-G203. MontgomeryCalifornia and D otherBissell. locations. University study of started California December San Francisco 1, 2016; iron metabolism. Am J Physiol Gastrointest Liver Physiol. 2006; estimated completion: August 1, 2019; principal investigator: 36. Bisell M.D.(principal investigator). Oral Iron for Erythropoietic NCT02979249, Last accessed April 8th, 2017 Protoporphyrias: a study on Erythropoietic Protoporphyria X-Linked (UCSF); clinical trials webpage: http://clinicaltrials.ucsf.edu/trial/

Protoporphyria; for people ages 18 years and up at San Francisco,

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