Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

[Acute porphyrias viewed differently]. / Akute Porphyrien mal anders betrachtet.

The acute porphyrias are a group of four metabolic defects in which the heme synthesis in the liver is disrupted. They are characterized by massively painful acute attacks, which can be life-threatening if not diagnosed. To raise the awareness for these rare disorders, a heme molecule in cartoon style is introduced, which accurately explains the basic biochemical processes in the body and mediates important information on the acute hepatic porphyrias in a simplified and attractive way. The article is complemented by a case report.

Delta-aminolevulinic acid synthase 2 expression in combination with iron as modifiers of disease severity in erythropoietic protoporphyria.

Deficiency in ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway, leads to an accumulation of protoporphyrin IX (PPIX) that causes a severely painful phototoxic reaction of the skin in patients with erythropoietic protoporphyria (EPP). Besides phototoxicity of the skin, EPP patients often present with symptoms of iron deficiency in form of a microcytic and hypochromic anemia with low serum iron and ferritin. In addition, elevated aminolevulinic acid synthase 2 (ALAS2) both at the mRNA and protein levels have been observed among EPP patients. ALAS is the first enzyme in the pathway and exists in two isoforms, whereby the isoform 2 (ALAS2) is expressed exclusively in erythropoiesis. The mRNA of ALAS2 contains an iron response element (IRE) at its 5'UTR. When iron is limited, iron response element binding protein 2 (IRP2) binds to the IRE of ALAS2 mRNA and suppresses its translation. In this study, we demonstrated that iron deprivation increased the amount of ALAS2 mRNA as well as the ratio of ALAS2 to FECH mRNAs in cultured erythroleukemic K562 cells. At the protein level, however, iron deprivation in the cell line caused reductions in both enzymes as shown by the Western blot analysis. A comparable increase in the ratio of ALAS2 to FECH mRNAs was also found in EPP patients indicating an imbalance in heme biosynthesis. As iron cannot be completely missing from an organism, we assume that in EPP patients, a certain amount of ALAS2 mRNA is translated despite a partial deficiency of FECH. The increase in ALAS2 enzyme contributes to the accumulation in PPIX in the patients. Targeted inhibition of ALAS2 could therefore be a treatment option for EPP.

A next-generation-sequencing panel for mutational analysis of dominant acute hepatic porphyrias.

Molecular diagnosis of autosomal dominant acute hepatic porphyrias (AHPs) plays an important role in the management of these disorders. To introduce next generation sequencing (NGS) to the porphyria diagnosis, we designed a panel that contained four genes, ALAS1, HMBS, CPOX and PPOX for mutational analysis of acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). To validate the AHP panel, 30 samples with known pathogenic variants as determined by Sanger sequencing, were analyzed using the Ion PGM™. Among them, nine have so far not been reported. The pathogenic variants were identified and annotated manually in IGV by three individuals who were blinded to the Sanger results. The AHP panel consists of 95 amplicons that covers 92% of the coding region of the four genes. Of the 95 amplicons, 93 had an average read-depth of >500 reads. In 29 of the 30 tested samples, pathogenic variants were correctly identified and annotated. The number of reads from the mutated alleles were approximately 50% of the total. The annotation of a 22-bp duplication with NGS differed from that of Sanger by one nucleotide. NGS showed an advantage in allelic discrimination over Sanger sequencing and was also able to detect a known somatic variant in the HMBS gene. The AHP panel will be applied in the initial diagnosis of new patients. Any sequence variations with a frequency of ≥10% will be confirmed by Sanger sequencing. The cost-effectiveness of a NGS approach for AHP in a diagnostic laboratory needs to be further assessed.

[Porphyria - when to think about how to clarify and treat?] / Porphyrien ­ wann daran denken, wie abklären und behandeln?

Porphyria - when to think about how to clarify and treat? Abstract. Porphyrias are a group of metabolic disorders that are mostly hereditary. They manifest either as abdominal colic or as skin changes at light-exposed areas. During the symptomatic phase the diagnosis of porphyria can be made by cost-effective screening tests. If the screening gives a positive result, further testing is required to determine the exact type of porphyria and to establish the best therapeutic option for the patient in a specialized porphyria center.

Biallelic inactivation of protoporphyrinogen oxidase and hydroxymethylbilane synthase is associated with liver cancer in acute porphyrias.

Variegate porphyria (VP) and acute intermittent porphyria (AIP), the two most common types of acute porphyrias (AHPs), result from a partial deficiency of protoporphyrinogen oxidase (PPOX) and hydroxymethylbilane synthase (HMBS), respectively. A rare but serious complication in the AHPs is hepatocellular carcinoma (HCC). However, the underlying pathomechanisms are yet unknown. We performed DNA sequence analysis in cancerous and non-cancerous liver tissue of a VP and an AIP patient, both with HCC. In samples of both cancerous and non-cancerous liver tissues from the patients, we identified the underlying PPOX and HMBS germline mutations, c.1082dupC and p.G111R, respectively. Additionally, we detected a second somatic mutation, only in the cancer tissue i.e., p.L416X in the PPOX gene of the VP patient and p.L220X in the HMBS gene of the AIP patient, both located in trans to the respective germline mutations. Both somatic mutations were not detected in 10 non-porphyria-associated HCCs. Our data demonstrate that in the hepatic cancer tissue of AHP patients, somatic second-hit mutations result in nearly complete inactivation of the enzymes catalyzing major steps in the heme biosynthetic pathway. Both PPOX and HMBS, which might act as tumor suppressors, play a crucial role in the development of HCC in these individuals.

In ferrochelatase-deficient protoporphyria patients, ALAS2 expression is enhanced and erythrocytic protoporphyrin concentration correlates with iron availability.

The activity of the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase (ALAS2), the first and rate-limiting enzyme in heme biosynthesis, is down-regulated during iron-deficiency. Ferrochelatase (FECH), the last enzyme of this pathway, inserts iron into protoporphyrin IX (PPIX) to form heme. Patients with erythropoietic protoporphyria (EPP), an inherited deficiency in FECH, often show signs of iron deficiency in addition to phototoxicity which is caused by PPIX accumulation. However, iron supplementation often leads to exacerbation of phototoxicity. We report three EPP patients who had reduced erythrocytic PPIX concentrations when they were iron-deficient and their microcytic and hypochromic anemia deteriorated. Additionally, we found a significant increase in the amount of ALAS2 mRNA and protein among EPP patients. To verify the connection between FECH deficiency and ALAS2 over-expression, we inhibited FECH in cultured cells and found a subsequent increase in ALAS2 mRNA. We conclude that the primary deficiency in ferrochelatase leads to a secondary increase in ALAS2 expression. The combined action of these two enzymes within the heme biosynthetic pathway contributes to the accumulation of PPIX. Furthermore, we hypothesize that EPP patients may benefit from a mild iron deficiency since it would limit PPIX production by restricting ALAS2 over-expression.

Evaluation of the immunogenicity of the synthetic α-melanocyte-stimulating hormone (α-MSH) analogue afamelanotide ([Nle4-D-Phe7]-α-MSH, Scenesse®) in erythropoietic protoporphyria patients by ELISA detecting both anti-afamelanotide and anti-α-MSH antibodies.

UNLABELLED: Afamelanotide is an α-melanocyte-stimulating hormone (α-MSH) agonist with proven efficacy in photodermatoses such as erythropoietic protoporphyria (EPP). This peptide drug, repeatedly administered over prolonged time, may induce anti-drug antibodies (ADA). Here, we describe a new ELISA method developed to monitor the occurrence of ADA against afamelanotide as well as against α-MSH. Covalent binding instead of absorption of antigen onto the microtitre wells prevented antigen leakage and enabled extensive washings followed by lower background. The cut-off between antibody-negative and -positive sera was determined. Inhibition of the antigen-antibody reaction by excess soluble antigen tested for specificity. The sensitivity of the ELISA was 608 and 1,390 ng/ml of specific ADA against afamelanotide and α-MSH, respectively. This ELISA method enabled us to investigate the occurrence of ADA during long-term administration of afamelanotide. No immunoreactivity was found in 23 of the 26 EPP patients exposed to the drug for up to 6 years. Pre-existing immunoreactivity against afamelanotide as well as α-MSH was found in 3 patients, whose titres did not change during afamelanotide administration. CONCLUSION: The new ELISA is suitable to determine ADA against afamelanotide and α-MSH. Afamelanotide did not elicit ADA during long-term administration in patients with EPP.

Editorial on the Special Issue "Heme Metabolism and Porphyria".

Porphyria denotes a heterogeneous group of metabolic disorders caused by anomalies in the biosynthesis of heme, a crucial component of hemoglobin and other vital hemoproteins [...].

Fair Funding Decisions: Consistency of the Time Horizons Used in the Calculation of Quality-Adjusted Life Years for Therapies for Very Rare Diseases by the National Institute for Health and Care Excellence in England.

The National Institute for Health and Care Excellence (NICE) in England uses quality-adjusted life years (QALYs) to assess the cost-effectiveness of treatments. A QALY is a measure that combines the size of the clinical benefit of a treatment with the time the patient benefits from it, i.e., the time horizon. We wanted to know how consistently QALY gains are calculated at NICE. Therefore, we have analysed information on the time horizons used for the QALY calculations of the concluded evaluations conducted under the Highly Specialised Technologies programme for treatments of very rare diseases at NICE. For treatments with final guidance published by December 2023 (n = 29), a time horizon of median 97.5 years (range: 35 to 125 years) was used to calculate the QALY gains. For most QALY calculations, the accepted time horizon was longer than either the expected treatment duration or the estimated life expectancy. In contrast, for the only technology with a final negative funding decision, i.e., afamelanotide for treating the lifelong chronic disease erythropoietic protoporphyria, a time horizon that was shorter than the expected treatment duration was used. The fairness and consistency of the evaluation process of treatments for very rare diseases at NICE should be reviewed.

Iron availability modulates aberrant splicing of ferrochelatase through the iron- and 2-oxoglutarate dependent dioxygenase Jmjd6 and U2AF(65.).

Erythropoietic protoporphyria (EPP) results from partial deficiency of ferrochelatase (FECH). Genetically, EPP patients differ from asymptomatic mutation carriers at the unmutated FECH allele, the expression of which is modulated by single nucleotide polymorphism IVS3-48C/T. The IVS3-48C genotype, which is present among patients, leads to correct splicing of 60% of the pre-mRNA and to alternative splicing of 40%, the latter mRNA-product being destroyed by nonsense-mediated decay. An IVS3-48T genotype generates 80% correct and 20% aberrant products. Our study demonstrated that under iron deficient conditions, the aberrant splice product was increased to 56% and 50% of total FECH mRNA in erythroleukemic K562 and lymphoblastoid cell lines, respectively, both being homozygous for IVS3-48T. Concomitantly, FECH protein was decreased. Iron deficiency had less effect on the FECH splice ratio in an IVS3-48C/C lymphoblastoid cell line. Effects similar to iron deficiency were generated by siRNA knockdown of either splicing factor U2AF(65) or Fe(II)- and 2-oxoglutarate-dependent dioxygenase Jumonji domain-containing protein 6 (Jmjd6), which interacts with U2AF(65) by lysyl-hydroxylation. Based on these results, we propose that the availability of iron, a co-factor of Jmjd6, modulates U2AF(65)-lysyl-hydroxylation. This in turn, influences the relative amounts of correct and aberrant FECH mRNA splice products and thus, regulates the FECH enzyme activity.

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.

Quality-Adjusted Life Years in Erythropoietic Protoporphyria and Other Rare Diseases: A Patient-Initiated EQ-5D Feasibility Study.

Erythropoietic protoporphyria (EPP) is an ultra-rare inborn error of metabolism characterised by painful phototoxic burn injuries after short exposure times to visible light. Patients with EPP are highly adapted to their condition which makes the quantification of their health-related quality of life (QoL) challenging. In the presented patient-initiated feasibility study, we describe a new approach to assess treatment benefits in EPP by measuring QoL with the generic EQ-5D instrument in five patients under long-term (≥two years) treatment with afamelanotide, the first approved therapy for EPP. For the study, we selected patients with EPP who in addition were affected by an involuntary treatment interruption (caused by a temporary reimbursement suspension) because we hypothesized that individuals who had previously unlearned their adaptation are better able to assess their life without treatment than treatment-naïve patients. QoL under treatment was comparable to the age-matched population norm, and retrospective results for a treatment interruption and phototoxic reaction time point were comparable to the QoL of patients with chronic neuropathic pain and acute burn injuries, respectively. The results were accepted by the National Institute for Health and Care Excellence in England for their evaluation of the cost-effectiveness of afamelanotide, i.e., the calculation of quality-adjusted life years.

First Report of a Low-Frequency Mosaic Mutation in the Hydroxymethylbilane Synthase Gene Causing Acute Intermittent Porphyria.

Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient's blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics.

Quantifying the impact of symptomatic acute hepatic porphyria on well-being via patient-reported outcomes: Results from the Porphyria Worldwide Patient Experience Research (POWER) study.

Acute hepatic porphyria (AHP) is a group of rare genetic diseases of heme biosynthesis resulting in severe neurovisceral attacks and chronic complications that negatively impact patients' well-being. This study evaluated the impacts of AHP on patients' physical and emotional health from a global perspective. Adult patients from the United States, Italy, Spain, Australia, Mexico, and Brazil with AHP with >1 porphyria attack within the past 2 years or receiving intravenous hemin and/or glucose for attack prevention completed an online survey assessing demographics, health characteristics, and patient-reported outcomes. Results were analyzed collectively and by patient subgroups. Ninety-two patients with AHP across the six countries completed the survey. More than 70% of patients reported that their physical, emotional, and financial health was fair or poor. Among patients who reported pain, fatigue, and muscle weakness, 94.3%, 95.6%, and 91.4%, respectively, reported that these symptoms limited daily activities. Moderate to severe depression was present in 58.7% of patients, and moderate to severe anxiety in 48.9% of patients. Of the 47% of patients who were employed, 36.8% reported loss in productivity while at work. Among patients, 85.9% reported that they had to change or modify goals that were important to them because of AHP. Aside from differences in healthcare utilization and pain severity, scores did not significantly vary with attack rate or use of hemin or glucose prophylactic treatments. AHP substantially impacts patients' physical and emotional well-being, regardless of hemin or glucose prophylactic treatment or frequency of attacks. This multinational study demonstrates that there is substantial disease burden for patients with AHP, even among those experiencing sporadic attacks or using prophylactic treatment.

Heme Biosynthetic Gene Expression Analysis With dPCR in Erythropoietic Protoporphyria Patients.

Background: The heme biosynthesis (HB) involves eight subsequent enzymatic steps. Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in the ferrochelatase (FECH) gene, which in the last HB step inserts ferrous iron into protoporphyrin IX (PPIX) to form heme. Aim and method: The aim of this work was to for the first time analyze the mRNA expression of all HB genes in peripheral blood samples of patients with EPP having the same genotype FECH c.[215dupT]; [315-48T > C] as compared to healthy controls by highly sensitive and specific digital PCR assays (dPCR). Results: We confirmed a decreased FECH mRNA expression in patients with EPP. Further, we found increased ALAS2 and decreased ALAS1, CPOX, PPOX and HMBS mRNA expression in patients with EPP compared to healthy controls. ALAS2 correlated with FECH mRNA expression (EPP: r = 0.63, p = 0.03 and controls: r = 0.68, p = 0.02) and blood parameters like PPIX (EPP: r = 0.58 p = 0.06). Conclusion: Our method is the first that accurately quantifies HB mRNA from blood samples with potential applications in the monitoring of treatment effects of mRNA modifying therapies in vivo, or investigation of the HB pathway and its regulation. However, our findings should be studied in separated blood cell fractions and on the enzymatic level.

Repurposing of glycine transport inhibitors for the treatment of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare disease in which patients experience severe light sensitivity. It is caused by a deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis (HBS). The lack of FECH causes accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in patients' erythrocytes. Here, we explored an approach for the treatment of EPP by decreasing PPIX synthesis using small-molecule inhibitors directed to factors in the HBS pathway. We generated a FECH-knockout clone from K562 erythroleukemia cells, which accumulates PPIX and undergoes oxidative stress upon light exposure. We used these matched cell lines to screen a set of publicly available inhibitors of factors in the HBS pathway. Inhibitors of the glycine transporters GlyT1 and GlyT2 lowered levels of PPIX and markers of oxidative stress selectively in K56211B4 cells, and in primary erythroid cultures from an EPP patient. Our findings open the door to investigation of glycine transport inhibitors for HBS disorders.