Erythropoietic Protoporphyria Research Articles

Effects of iron supplements in individuals with erythropoietic protoporphyria

BackgroundProtoporphyrin IX (PPIX) is the final precursor of heme, forming heme when iron is inserted. Individuals with erythropoietic protoporphyrias (EPP) have accumulation of PPIX, causing photosensitivity and increased liver disease risk. Many also have iron deficiency and anemia. We investigated outcomes of oral iron supplements in individuals with EPP. MethodsA systematic review identified literature on oral iron supplements in EPP patients. Subsequently, we administered iron supplements to EPP patients with iron deficiency. The primary outcome was impact on PPIX level. Secondary outcomes were adverse events and relative differences in hemoglobin and iron parameters. ResultsThe systematic review found 13 case reports and one uncontrolled clinical trial with uncertain results. From our department 10 patients with EPP and iron deficiency took daily dosages of 330 mg of ferrous fumarate for two months. Five of our patients had anemia at baseline. After 2 months of supplementation seven patients had increased PPIX level compared to baseline, two had decrease, one remained unchanged. The administration of iron led to a rise in ferritin, and in four of the anemic patients also to an improvement in blood hemoglobin. A small transiently elevation in plasma alanine transaminase concentration was observed during supplementation. ConclusionsOverall, iron supplementation in EPP patients replenished iron stores and elevated erythrocyte PPIX and plasma alanine transaminase. For anemic patients, there was some degree of normalization of the hemoglobin level. If iron therapy is needed for EPP patients, monitoring of photosensitivity, PPIX, hemoglobin, and plasma liver enzymes is advisable.

The effects of cholecalciferol and afamelanotide on vitamin D levels in erythropoietic protoporphyria; a multicentre cohort study.

Erythropoietic protoporphyria (EPP) patients experience lifelong painful photosensitivity resulting in a lack of sunlight exposure. Previous studies have shown that 47-63% of EPP patients suffer from vitamin D deficiency and a high prevalence of osteoporosis. As of 2016 an effective treatment for EPP is available: the alpha-MSH analogue afamelanotide. So far studies on vitamin D levels in EPP have only investigated patients who were not treated with afamelanotide. To investigate the effects of afamelanotide treatment on vitamin D levels in EPP. A multi-centre observational cohort study, in adult patients with EPP from the Erasmus Medical Centre, the Netherlands and the University Hospital Düsseldorf, Germany. Routinely-collected vitamin D levels between 2005 and 2021 were used for analysis. Patient exposure to cholecalciferol or afamelanotide was categorized into four treatment groups; untreated, cholecalciferol, afamelanotide, and combined treatment. A linear mixed model for longitudinal data was applied to measure the effect of the treatment groups, compared to the untreated, on vitamin D levels. A total of 230 patients and 1774 vitamin D measurements were included. Prevalence of vitamin D deficiency remained high despite afamelanotide treatment: <50 nmol/l in 71.8% of patients, and severe deficiency <30 nmol/l in 48.1%. Afamelanotide treatment alone did not lead to a significant average increase in vitamin D levels (β:0.5, 95% Confidence Interval [CI]: -3.2 - 4.2). In contrast, cholecalciferol and combined therapy with afamelanotide, led to a significant increase in vitamin D levels (β:11.6, CI: 7.2-15.9 and β:15.2, CI: 12.3-18.1). Cholecalciferol remains essential for treatment of vitamin D deficiency in EPP, irrespective of new treatment options like afamelanotide. Afamelanotide treatment did not affect vitamin D levels. We suggest that future guidelines include continuous monitoring of vitamin D and prescription of cholecalciferol in all patients with EPP, including those treated with afamelanotide.

Observational pilot study of multi-wavelength wearable light dosimetry for erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) causes painful light sensitivity, limiting quality of life. Our objective was to develop and validate a wearable light exposure device and correlate measurements with light sensitivity in EPP to predict and prevent symptoms. A wearable light dosimeter was developed to capture light doses of UVA, blue, and red wavelengths. A prospective observational pilot study was performed in which five EPP patients wore two light dosimeters for 3 weeks, one as a watch, and one as a shirt clip. Standard deviation (SD) increases from the mean in the daily blue light dose increased the odds ratio (OR) for symptom risk more than the self-reported outdoor time (OR 2.76 vs. 2.38) or other wavelengths, and a one SD increase from the mean in the daily blue light wristband device dose increased the OR for symptom risk more than the daily blue light shirt clip (OR 2.45 vs. 1.62). The area under the receiver operator curve for the blue light wristband dose was 0.78, suggesting 78% predictive accuracy. These data demonstrate that wearable blue light dosimetry worn as a wristband is a promising method for measuring light exposure and predicting and preventing symptoms in EPP.

Liver transplantation and primary liver cancer in porphyria.

The porphyrias are a heterogeneous group of metabolic disorders that result from defects in heme synthesis. The metabolic defects are present in all cells, but symptoms are mainly cutaneous or related to neuropathy. The porphyrias are highly relevant to hepatologists since patients can present with symptoms and complications that require liver transplantation (LT), and some porphyrias are associated with a high risk for primary liver cancer (PLC). Among the cutaneous porphyrias, erythropoietic protoporphyria (EPP) can lead to cholestatic liver failure where LT cures the liver disease but not the porphyria. In acute porphyria (AP), neurotoxic porphyrin precursors are produced in the liver and LT is a curative treatment option in patients with recurrent severe neuropathic attacks. Patients with AP, mainly acute intermittent porphyria, have a significantly increased risk for PLC that warrants surveillance and adequate follow-up of high-risk groups. LT is well established in both EPP with liver failure and AP with recurrent attacks, but most transplant centres have little porphyria experience and cooperation between transplant hepatologists, and porphyria experts is important in the often-difficult decisions on timing and management of comorbid conditions.

Treatment strategy for erythropoietic protoporphyria accompanied by severe abdominal pain and liver injury

Treatment strategy for erythropoietic protoporphyria accompanied by severe abdominal pain and liver injury

Development and Content Validation of Novel Patient-Reported Outcome Measures to Assess Disease Severity and Change in Patients with Erythropoietic Protoporphyria: The EPP Impact Questionnaire (EPIQ).

Erythropoietic protoporphyria (EPP), a rare inherited disorder, presents in early childhood with severe, painful phototoxicity, with significant impacts on health-related quality of life (HRQoL). Previous studies have not captured all concepts important to patients. Therefore, this study sought to develop a novel, comprehensive, and content valid patient-reported outcome (PRO) measure to assess the efficacy of new therapies. Qualitative interviews were conducted with EPP participants and clinical experts to obtain views on concepts relevant to patients. Results informed the development of novel PROs, which were debriefed during subsequent combined concept elicitation and cognitive debriefing interviews. Twenty-three interviews were conducted with 17 adults and 6 adolescents with EPP. Concept elicitation revealed that participants experienced many symptoms with significant variability. The most common were burning, pain, swelling, and tingling. Tingling was the most common prodromal symptom, while burning was the most bothersome, and pain was the worst full reaction symptom. Participants reported being negatively impacted in their ability to do daily activities, and social and emotional functioning. Many reported impacted ability to work and be productive at their job. Participants reviewed and completed the newly developed PRO measures assessing full reactions and ability to do activities, as well as items to assess severity and change in severity of prodromal symptoms, full reactions, and EPP overall. All measures were found to be comprehensive, clear, and relevant. PRO measures are needed to assess important aspects of HRQoL and evaluate therapeutic response. These PRO measures are unique in assessing overall severity and change in EPP.

Diagnosis and management of protoporphyria-related liver dysfunction in erythropoietic protoporphyria and x-linked protoporphyria: A patient-friendly summary of the 2023 evidence-based consensus guidelines.

Diagnosis and management of protoporphyria-related liver dysfunction in erythropoietic protoporphyria and x-linked protoporphyria: A patient-friendly summary of the 2023 evidence-based consensus guidelines.

Illuminating Dersimelagon: A Novel Agent in the Treatment of Erythropoietic Protoporphyria and X-Linked Protoporphyria.

Erythropoietic protoporphyria (EPP) is a genetic disorder stemming from reduced ferrochelatase expression, the final enzyme in the pathway of heme biosynthesis. A closely related condition, X-linked protoporphyria (XLP), bears similar clinical features although it arises from the heightened activity of δ-aminolevulinic acid synthase 2 (ALAS2), the first and normally rate-controlling enzyme in heme biosynthesis in developing red blood cells. Both of these abnormalities result in the buildup of protoporphyrin IX, leading to excruciating light sensitivity and, in a minority of cases, potentially fatal liver complications. Traditionally, managing EPP and XLP involved sun avoidance. However, the emergence of innovative therapies, such as dersimelagon, is reshaping the therapeutic landscape for these conditions. In this review, we summarize salient features of the properties of dersimelagon, shedding light on its potential role in advancing our understanding of treatment options for EPP and XLP.

Cimetidine Does Not Inhibit 5-Aminolevulinic Acid Synthase or Heme Oxygenase Activity: Implications for Treatment of Acute Intermittent Porphyria and Erythropoietic Protoporphyria.

Acute intermittent porphyria (AIP) is characterized by acute neurovisceral attacks that are precipitated by the induction of hepatic 5-aminolevulinic acid synthase 1 (ALAS1). In erythropoietic protoporphyria (EPP), sun exposure leads to skin photosensitivity due to the overproduction of photoreactive porphyrins in bone marrow erythroid cells, where heme synthesis is primarily driven by the ALAS2 isozyme. Cimetidine has been suggested to be effective for the treatment of both AIP and EPP based on limited case reports. It has been proposed that cimetidine acts by inhibiting ALAS activity in liver and bone marrow for AIP and EPP, respectively, while it may also inhibit the hepatic activity of the heme catabolism enzyme, heme oxygenase (HO). Here, we show that cimetidine did not significantly modulate the activity or expression of endogenous ALAS or HO in wildtype mouse livers or bone marrow. Further, cimetidine did not effectively decrease hepatic ALAS activity or expression or plasma concentrations of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which were all markedly elevated during an induced acute attack in an AIP mouse model. These results show that cimetidine is not an efficacious treatment for acute attacks and suggest that its potential clinical benefit for EPP is not via ALAS inhibition.

Application of a Validated Method for Quantifying Circulating Protoporphyrin IX to the Beacon Trial of Bitopertin in Erythropoietic Protoporphyria

Introduction: Accumulation of the photoreactive heme precursor protoporphyrin IX (PPIX) is a signature of the disease pathophysiology for both erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP), and PPIX levels correlate with disease severity and light tolerance. Additionally, an elevated ratio of circulating metal-free PPIX vs. metal-bound PPIX is observed in patients with protoporphyrias, and the extent of metal-free elevation is distinguishable between EPP (85-100%) and XLP (50-85%). Therefore, the accurate determination of metal-free PPIX and metal-bound PPIX is of particular interest in patients with EPP or XLP. Methods: Novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed and validated to quantify circulating metal-free and zinc (Zn)-complexed PPIX in whole blood and plasma. Each of the four assays were developed and validated according to applicable regulatory guidance for bioanalytical assay validation with regard to accuracy, precision, sensitivity, and selectivity. The minimum required sample volume for each assay was 200 µL, compared to &amp;gt;1 mL for most currently available protoporphyria-related assays. During bioanalytical method development, rapid degradation of metal-free PPIX (≥90%) and Zn-PPIX (up to 96%) was observed in plasma samples within 24 hours under ambient light conditions. In contrast, whole blood was less susceptible to photodegradation (i.e., ≤20% for both metal-free PPIX and Zn-PPIX) under the same conditions. The most effective method for maintaining sample integrity was reduction of light exposure during sample collection, handling, and processing. Implementation of light-reduction controls was used during method validation and facilitated improved stability of metal-free PPIX and Zn-PPIX in plasma and whole-blood samples. For the metal-free PPIX plasma assay validation, the effect of hemolysis was evaluated due to the apparent sequestration of metal-free PPIX in red blood cells. The bioanalytical work informed specific sample-collection procedures to mitigate instability and hemolysis, such as the use of opaque consumables for sample collection, controls for plasma aliquoting, and operating under low-light conditions for all sample handling and processing. BEACON is a Phase 2, randomized, open-label, parallel-arm trial (ACTRN12622000799752) of approximately 22 participants who will receive oral, once-daily administration of 20 or 60 mg of bitopertin for 6 months. Baseline light tolerance was assessed using 2 patient-reported outcomes measures during a 2- to 4-week screening period. Participants were asked to conduct weekly sun exposure challenges and time to first prodromal symptom (itching, burning, tingling, etc.) was averaged for the screening period. Results: More than 600 clinical samples from 17 participants in the BEACON study have been collected as of 06 June 2023 and analyzed using the validated porphyrin assays. Measured metal-free PPIX baseline values ranged from 1440 - 34,100 ng/mL (2.6 - 61 µM) in whole blood and 177 - 2560 ng/mL (0.31 - 4.5 µM) in plasma ( n=16). Baseline percentage of intra-patient plasma/whole-blood concentrations ranged from 2.5 - 16%. Each participant's baseline values were converted to erythrocyte metal-free PPIX concentrations using measured hematocrit levels and ranged from 3590 - 88400 ng/mL (6.4 - 157 µM). These values were comparable to data previously reported in the literature (6-139 µM 1). Additionally, higher baseline whole-blood metal-free PPIX concentrations were associated with decreased averages for time to prodrome (Table 1). Conclusion: Fully-validated LC-MS/MS assays to quantify metal-free PPIX and Zn-PPIX in plasma and whole blood were used to analyze clinical samples from the BEACON study evaluating bitopertin in EPP. PPIX values were consistent with previously reported values for patients with EPP. The methods also provided improved accuracy and specificity for metal-free vs. metal-complexed PPIX and reduced sample volume requirements. In addition, higher baseline whole-blood PPIX values were associated with decreased light tolerance within the trial population, supporting its use as a measure of disease severity in EPP.

Interim Analyses from the Beacon Trial: A Phase 2, Randomized, Open-Label Trial of Bitopertin in Erythropoietic Protoporphyria

Introduction: Erythropoietic protoporphyria (EPP) is associated with accumulation of photoreactive protoporphyrin IX (PPIX) in the skin and other organs, causing debilitating phototoxic skin reactions following exposure to sunlight, and potentially life-threatening protoporphyric hepatopathy in some patients. Reduction of PPIX is associated with amelioration of disease in the settings of hematopoietic stem cell transplant, pregnancy, and extracorporeal photoinactivation. Glycine transporter 1 (GlyT1) supplies extracellular glycine for the initial step of heme biosynthesis in erythroid cells. Bitopertin is an investigational, orally-administered inhibitor of GlyT1. It is hypothesized that GlyT1 inhibition leads to a decrease in heme pathway intermediates, including PPIX, and can improve light tolerance. Methods: BEACON is a Phase 2, randomized, open-label, parallel-arm trial (ACTRN12622000799752) of 22 participants who will receive oral, once-daily administration of 20 mg or 60 mg of bitopertin for 24 weeks. The trial is being conducted at 2 sites in Australia and includes participants ≥18 years of age with a confirmed diagnosis of EPP. The primary efficacy endpoint is percent change in whole-blood metal-free PPIX. Additional endpoints include daily patient-reported outcomes (PRO) of light tolerance and quality of life, as well as safety and tolerability. Results: As of data cutoff (05 July 2023), a total of 17 subjects had been enrolled. Treatment with bitopertin resulted in mean (SD) decreases in PPIX of -39% ± 17% by Day 43 (n=12), with more pronounced decreases observed with 60 mg compared to 20 mg (Figure 1). Bitopertin also improved multiple measures of light tolerance. A participant randomized to 20 mg bitopertin reported a &amp;gt;80-fold increase in sunlight tolerance on Day 88 of treatment, increasing from 4.5 minutes at baseline to over 6 hours; no prodromes were reported during any sunlight challenge after Day 20. A participant randomized to 60 mg bitopertin reported a &amp;gt;200-fold increase in sunlight tolerance on Day 74 of treatment, increasing from 1.3 minutes at baseline to over 4 hours, and did not report a prodrome during any sunlight challenge after Day 120. Aggregate measures of light tolerance also improved over time, including time to prodrome averaged over a 2-week period and weekly averages of total time in sunlight. The proportion of prodrome-free sunlight challenges increased from 2% during screening to 49% while receiving bitopertin (n=17), and the proportion of days without symptoms (with sun exposure) increased from 26% during screening to 74% while receiving bitopertin (n=17). Improvements in weekly averages of total time in sunlight were consistent and observed in 15/15 participants with post-baseline PPIX values. The greatest increases in weekly averages of total time in sunlight were observed in participants who experienced reductions in PPIX of at least 30% (Figure 2), for whichtotal time in sunlight increased from a weekly average of 339 minutes at baseline to a maximum of 999 minutes with bitopertin (n=11; mean duration of treatment: 133 days [range: 76 to ≥169 days]). Patient-reported phototoxic reactions decreased by 93% while on treatment compared to baseline (n=17). By Day 43, 15/16 participants reported in the Patient Global Impression of Change (PGIC) their EPP was much better or a little better, and 15/16 reported in the Patient Global Impression of Severity (PGIS) their EPP was mild or not at all severe. A majority (13/16) of participants responded in a novel PRO, the EPP Impact Questionnaire (EPIQ), that EPP had little or no impact on their quality of life while receiving bitopertin. No serious adverse events, discontinuations due to adverse events, or dose reductions have been reported. A total of 12 (71%) participants reported treatment-emergent adverse events; only dizziness and headache were reported in more than one subject. No meaningful changes in hemoglobin have been observed. Conclusion: By reducing whole-blood PPIX levels, bitopertin targets the underlying pathophysiology of EPP, resulting in consistent improvements in multiple measures of light tolerance and quality of life. Bitopertin has been well tolerated to date with no changes in hemoglobin, and its safety profile in EPP is consistent with prior studies that enrolled more than 4000 participants. Updated results will be presented at the meeting.

The Relationship between Protoporphyrin IX and Platelet Count in the Protoporphyrias

Introduction: Erythropoietic protoporphyria and X-linked protoporphyria, collectively known as the protoporphyrias, are inherited disorders of heme biosynthesis characterized by the accumulation of protoporphyrin IX in the skin and other organs. Clinical manifestations include severe pain with exposure to sunlight, as well as liver disease in a subset of patients. Approximately half of patients with protoporphyria have mild microcytic anemia, which is not well understood but thought to be related either to decreased iron availability or to decreased heme biosynthesis. Several reports have described mild thrombocytopenia in patients with protoporphyria (Delaby et al., 2009, Wensink et al., 2022), although thrombocytosis would be expected with iron deficiency as low iron biases megakaryocyte-erythroid progenitors to the megakaryocyte lineage (Xavier-Ferrucio et al., 2019). We sought to further characterize the relationship between erythrocyte metal-free protoporphyrin IX (PPIX) levels, platelet count, and iron status in patients with protoporphyria. Methods: We conducted a single-center cohort study of adults with protoporphyria treated at the Massachusetts General Hospital Porphyria Center from March 2021 through June 2023. Patient-level data across multiple time points were collected, including platelet count, ferritin, and alanine transaminase (ALT). Generalized Estimating Equation (GEE) models with an identity link and gaussian family distribution were performed to determine the association, accounting for within-participant associations. Results: A total of 38 patients with protoporphyria were included in the analysis. The median PPIX level was 1169.5ug/dl (interquartile range [IQR] 729.50 - 2069.75, reference range &amp;lt;20). The median platelet count was 208 x10 9/L (IQR, 171-243, reference range 150-400). Only 3 patients had a platelet count &amp;lt;150 x10 9/L and none &amp;lt;100 x10 9/L. A 1ug/dl increase in protoporphyrin level was associated with an 0.02 x10 9/L decrease in platelet count on average (p= &amp;lt; 0.00001, Figure). We next sought to evaluate whether the relationship between PPIX and platelet count was affected by iron status (assessed by ferritin) or liver disease (assessed by ALT). When adjusting for ferritin and ALT, the relationship between PPIX and platelet count remained statistically significant (p= &amp;lt; 0.00001). When adjusting for PPIX and ferritin, an increase in platelets was associated with an increase in ALT (p=0.002), but there was no significant relationship between ferritin and PPIX when adjusting for the other variables in the model. Conclusion: In this study of 38 patients with protoporphyria, higher levels of PPIX were associated with lower platelet counts. This association was independent of markers of iron status or hepatic dysfunction. Only 3 patients had thrombocytopenia, which was mild in all cases. Although increased PPIX was not associated with clinically significant thrombocytopenia, the reason for the relationship between PPIX and platelets in this patient population remains unclear. Considering the well-described risk of anemia in protoporphyria, there may exist some unknown effect on hematologic cells and their precursors, either due to the accumulation of PPIX or variants in heme biosynthetic enzymes. Understanding this fundamental biology may pave the way for further treatment targets in protoporphyria and other hematologic conditions.

A Phase I/II, Intra-Patient Dose-Escalation Study of Bitopertin, a Selective Inhibitor of GlyT1 and Heme Synthesis, for Steroid-Refractory Diamond-Blackfan Anemia

Background: Diamond-Blackfan anemia (DBA) is a heritable bone marrow failure disorder characterized by macrocytic anemia with reticulocytopenia and bone marrow erythroid hypoplasia, usually presenting within the first two years of life. Treatment consists of systemic corticosteroids. Treatment failure or steroid intolerance occurs in approximately half of all cases, necessitating chronic transfusions, which carry the risks of iron overload and infections. Hematopoietic cell transplant remains the sole curative therapy for DBA. In cases where a causative mutation can be identified, with rare exceptions, the affected genes encode ribosomal proteins (RPs), defining DBA as prototypic haploinsufficient ribosomopathy. Erythrocyte hemoglobinization requires balanced heme synthesis and globin translation. Various in vitro and animal models suggest that RP insufficiency results in reduced ribosomal biogenesis, disrupting this balance and leading to excess of heme, resulting in toxicity to developing erythroid cells and premature termination of erythroid differentiation. Bitopertin is an investigational, orally administered, inhibitor of the GlyT1 glycine transporter. Outside the central nervous system (CNS), GlyT1 is expressed almost exclusively on developing erythrocytes. By reducing intracellular glycine, a rate-limiting substrate for heme synthesis, bitopertin slows heme synthesis and thus we hypothesize rebalances hemoglobin synthesis. In preclinical DBA models, bitopertin rescues erythropoiesis. The drug was originally developed for CNS indications, and has been shown to exhibit a favorable safety profile in prior studies with cumulative enrollment of over 4,000 patients and healthy volunteers. It is currently under clinical investigation for the treatment of erythropoietic protoporphyria. Study Design and Methods: This is a phase I/II, intra-patient dose-escalation study of bitopertin for steroid-refractory DBA, conducted at the National Institutes of Health (NIH) Clinical Center in Bethesda, Maryland. Subjects visit the NIH for initial evaluation and eligibility assessment and at the primary endpoint, with interim follow-up conducted via telehealth and local laboratory assessments. Subjects must be 18 years of age or and older, diagnosed with DBA and with clinically significant anemia (hemoglobin &amp;lt; 9/dL and/or requiring 2 units PRBC per 8 weeks). They must have relapsed and/or steroid refractory disease or be intolerant of systemic steroids, and be without significant comorbidities. Ongoing transfusions and chelation therapy are allowed, but other DBA-directed therapy, most notably steroids and growth factors, are not. Each subject will take oral bitopertin daily, escalating the dose monthly (5 mg, 10 mg, 20 mg, 40mg, and 60 mg) until reaching either the study maximum dose of 60 mg, the minimum effective dose (MED), or the maximum tolerated dose (MTD). Subjects will be followed for 8 months (32 weeks), including a minimum of 4 months (16 weeks) at the final dose (MED, MTD or study maximum). Response will be assessed on an ongoing basis and will be defined as an increase in hemoglobin ≥by 1.5 g/dL and/or a reduction in the units of PRBC transfusions by ≥50% from baseline. Furthermore, a robust response will be defined as a hemoglobin ≥10 g/dL and transfusion independence for 8 weeks. Patients with response (robust or non) by the 8-month primary endpoint will be allowed to continue extended therapy for 3 additional years. The primary endpoint is erythroid response. Secondary endpoints include safety as defined by the toxicity profile over 8 months of therapy and on extended therapy, relapse, survival, clonal evolution, health-related quality of life, and ancillary laboratory studies including the impact of bitopertin upon erythroid cell dynamics. The study employs a minmax design. A minimum of 15 patients will be enrolled. If the minimum efficacy threshold is met or surpassed (≥13% response rate), an additional 10 patients will be enrolled. Status: This study has been granted an FDA IND (165778) and is open for enrollment. It is monitored under the NIH Institutional Review Board (001528-H) and is registered with the U.S. National Library of Medicine (NCT05828108). Funding: This study is supported by the NHLBI Division of Intramural Research and in part through a Cooperative Research and Development Agreement (CRADA) with Disc Medicine, Inc. (Watertown, MA).

The Clpx G298D Mutation in Clpx-EPP Reveals That Clpx Regulates the Mitochondria Enzymes in Erythroid Heme Synthesis Via Distinct Mechanisms

Terminally differentiating red cells synthesize large quantities of heme for hemoglobin production, requiring tight coordination between mitochondrial iron import and synthesis of photosensitive protoporphyrin IX (PPIX). PPIX causes erythropoietic porphyria from loss of function mutations in FECH, or gain of function mutations in ALAS2. We previously reported that a dominant heterozygous point mutation in human CLPX (G298D) was a novel cause of erythropoietic protoporphyria (Yien et al. 2017, PNAS). CLPX encodes a ubiquitous ATPase-dependent mitochondrial protein unfoldase and is required for tight regulation of the mitochondrial heme synthesis enzymes, ALAS 1 and 2, FECH and PPOX, as well as mitochondrial iron utilization. CLPX facilitates degradation of the ALAS enzymes, but paradoxically, is also required for activity of the PPOX and FECH enzymes. CLPX function is required for porphyrin synthesis and for the incorporation of iron into heme (Rondelli et al. 2021, JBC). Because the role of CLPX in heme regulation is complex and cell-specific, we sought to interrogate the mechanism by which the CLPX G298D mutant protein caused erythroid protoporphyria. To determine the requirement for CLPX for maximal heme synthesis, we labeled wild-type and Clpx -/- 3T3 murine fibroblasts and G1E-ER4 erythroid cells with 14C-aminolevulinate (ALA) and measured heme synthesis by liquid scintillation to ensure that equal amounts of labelled ALA were entering the heme synthesis pathway, uncoupling the effects of CLPX on the first and terminal steps of heme synthesis. We found that Clpx -/- cells synthesized significantly less 14C-ALA -labelled heme, indicating that CLPX is required for activation of the terminal heme synthesis enzymes in both erythroid and non-erythroid cells. Mass spectrometric quantification of FECH and PPOX protein and enzyme activity assays indicated that their protein levels and activities were decreased in Clpx -/- cells. Since CLPX ubiquitously regulates the mitochondrial steps of heme synthesis, we sought to determine why the porphyrin accumulation in CLPX -EPP predominantly occurred in erythroid cells and to interrogate the pathogenic mechanism of CLPX-EPP. To address this question, we knocked in the human G298D/mouse G299D mutation into murine erythroleukemia cells. The heterozygous mutant cells, denoted as WT/G299D, synthesized normal levels of heme. Further, differentiating WT/G299D cells synthesized significantly more porphyrin than wild-type cells, phenocopying the patient. In contrast, non-differentiating WT/G299D cells produced wild-type levels of porphyrin. ALAS2 was stabilized in the mutant expressing cells, likely accounting for the increase in porphyrin during erythroid differentiation. Iron supplementation was effective in decreasing photosensitivity and porphyrin levels in the patient (Ducamp et al., Hematologica 2021) as was predicted for porphyrias caused by increases in ALAS2 activity (Landefeld et al., Br. J. Hematol. 2016). Similarly, supplemental iron was also able to decrease porphyrin levels in WT/G299D cells to wild-type levels (Figure 1). In contrast to the WT/G299D cells, wild-type cells did not experience a decrease in porphyrin synthesis. We also did not observe increased heme synthesis with iron supplementation, indicating that iron, rather than promoting heme synthesis, is inhibiting an upstream step of porphyrin synthesis in WT/G299D cells. Secondly, co-immunoprecipitation assays indicated ATPase activity or ATP binding was dispensable for the interaction between CLPX and PPOX/FECH, likely accounting for the normal heme synthesis observed in CLPX G299D expressing cells. Lastly, the G299D mutant protein has decreased ALAS degradation activity, while retaining its ability to activate PPOX/FECH. This indicates that the CLPX protein is multifunctional in heme regulation. The control of heme synthesis by CLPX unveils a novel cause of protoporphyria and the erythroid specificity of the disease indicates that CLPX has erythroid-specific functions in heme regulation. Further, while iron promotes ALAS2 translation, iron supplementation also suppresses porphyrin synthesis in CLPX-EPP. We reveal an important erythroid-specific regulatory node where the mitochondrial protein homeostasis intersects with heme synthesis, offering important insights on the pathogenesis and treatment of porphyrias.

Erythropoietic protoporphyria: case reports for clinical and therapeutic hints

BackgroundErythropoietic protoporphyria is a rare disorder which represents an important health problem in children, causing painful photosensitivity. Little is known on the correlation between genetic profile and clinical manifestations. The standard of care for Erythropoietic protoporphyria is based on avoiding sun and using sun protections, but recent literature has suggested that cimetidine may have a role in improving sun sensitivity. Herein we report our case series describing the successful use of cimetidine and analyzing potential phenotype-genotype correlations.Case presentationThis case series describes five patients presented to our Rheumatology Service complaining sun sensitivity. Blood exams and genetic analysis were consistent with the diagnosis of erythropoietic protoporphyria. Four of 5 patients received cimetidine in addition to standard therapies and the effect of treatment was evaluated by Erythropoietic Protoporphyria - Quality of Life questionnaire.ConclusionsErythropoietic protoporphyria usually manifests in early childhood after a short sun exposure. Skin manifestations are the main reason for investigations, although sometimes they can be more subtle, leading to a significant diagnostic delay. Skin diseases in children can have profound effects on their family and social relationships. A treatment with cimetidine appears to be an excellent therapeutic option in children with Erythropoietic protoporphyria.

Development and content validation of a sunlight exposure diary in patients with erythropoietic protoporphyria

BackgroundErythropoietic protoporphyria is a rare, inherited disorder presenting in early childhood with severe, painful phototoxicity. EPP has significant impacts on health-related quality of life, though there is variable disease severity. Accurately capturing how much time individuals with EPP can spend outdoors before they develop symptoms is critical to understanding HRQoL and measuring therapeutic response. Therefore, the goal of this study was to develop a comprehensive and content valid sun exposure diary to assess the efficacy of new therapies in individuals with EPP.MethodsQualitative interviews were conducted with adult and adolescent EPP participants, as well as five clinical experts, to obtain their input on the content of an existing sun exposure diary. Revisions to the diary were made based on evidence generated in cognitive debriefing interviews analyzed in eight consecutive groups of EPP participant.ResultsInterviews were conducted with 17 adults and 6 adolescents with EPP. The average age of adults was 40 years and of adolescents was 14 years. Clinical experts thought the original diary needed clarification on the description of symptoms, how time outdoors was captured, and the distinction between direct vs. indirect sunlight. Participants with EPP also noted these items needed revision, and that the distinction between prodromal symptoms and full reaction symptoms should be clarified. In the final diary version, participants with EPP found most items to be clear and easy to complete/think about. Seventy-six percent of participants (13/17) asked thought the diary was easy to complete. The remainder thought the majority of the diary was easy to complete with the exception of select questions.ConclusionsEvaluating a new treatment for EPP requires accurately capturing time in sunlight and symptoms in this unique disorder. The newly developed sun exposure diary is content valid and can be used to assess important aspects of symptoms and daily life and therefore evaluate clinically meaningful therapeutic response.

Current trials in erythropoietic protoporphyria: are placebo controls ethical?

A new active substance called “dersimelagon” (MT-7117) is being tested as an alternative treatment option for Erythropoietic protoporphyria (EPP). At the moment, dersimelagon is being tested both in the US and in Europe in a phase III placebo-controlled RCT. However, given the availability of an already approved treatment option for EPP the use of a placebo arm is questionable from an ethics point of view. We analyze the issue and suggest that a noninferiority active-control trial without placebo is an ethically and scientifically more valid design to test the efficacy of dersimelagon as well as other EPP treatments.

Erythropoietic protoporphyria and afamelanotide: apatient's perspective.

The patient explores the profound impact of erythropoietic protoporphyria (EPP) on her life to date. The clinician’s comment reviews the symptoms and diagnostic criteria for EPP, as well as the novel treatment afamelanotide for EPP.

Hepatic farnesoid X receptor is necessary to facilitate ductular reaction and expression of heme biosynthetic genes.

Bile, which contains bile acids, the natural ligands for farnesoid x receptor (FXR), moves from the liver to the intestine through bile ducts. Ductular reaction often occurs during biliary obstruction. A subset of patients with erythropoietic protoporphyria, an inherited genetic mutation in heme biosynthetic enzyme ferrochelatase, accumulate porphyrin-containing bile plugs, leading to cholestasis. Here, we examined the link between FXR, bile plug formation, and how heme biosynthesis relates to this connection. We treated female and male wild-type and global and tissue-specific Fxr knockout mice with a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine, an inhibitor of ferrochelatase, and examined the expression of heme biosynthetic genes. We mined FXR mouse ChIP-Seq data, performed biochemical and histological analysis, and tested HepG2 and primary human hepatocytes after treatment with obeticholic acid, an FXR agonist. We observed that hepatic but not intestinal Fxr loss resulted in reduced bile plugs and ductular reaction in the liver. Then, we examined if FXR plays a regulatory role in heme biosynthesis and found significantly lower porphyrin accumulation in 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-fed Fxr knockout mice. Gene expression and FXR mouse ChIP-Seq atlas analysis revealed that FXR orchestrates the expression of multiple heme biosynthetic enzymes. Finally, human HepG2 cells and primary human hepatocytes treated with obeticholic acid, showed increased expression of several heme biosynthetic genes. Overall, our data show that hepatic Fxr is necessary to maintain ductular reaction and accumulation of bile plugs. FXR can direct the expression of multiple heme biosynthetic genes. Thus, modulating FXR activity in EPP patients may help alleviate its associated liver disease.

When the diagnosis is written in the DNA: a case of erythropoietic protoporphyria in a patient with a chromosome 18 deletion

We present a case of erythropoietic protoporphyria (EPP) in a 21-year-old man who sought medical attention in April 2022 due to diffuse edema and erythema of the hands, which he had been experiencing since childhood and occurring shortly after sun exposure. The patient's medical history revealed a partial deletion of the long arm of chromosome 18. Based on the patient's medical background and clinical symptoms, we conducted various tests, including measuring total erythrocyte protoporphyrin levels and evaluating the fluorometric emission peak of plasma porphyrins using a spectrofluorometer. Additionally, a genetic analysis revealed a complete deletion of the FECH gene on one allele and the presence of an intronic variant on the other allele, identified as c.315–48T&gt;C (IVS3–48T&gt;C) and classified as a susceptibility polymorphism. In June 2022, the patient underwent an Afamelanotide implant, which resulted in an improvement in his clinical condition.