M. Mazzoli, A. Ricci, A.E. Vaudano, M. Marcacci, S. Marchini, P. Bergonzini, E. Di Pierro, E. Pischik, L. Iughetti, A. Pietrangelo, S. Meletti, P. Ventura

European Review for Medical and Pharmacological Sciences 2024; 28: 3268-3274

BACKGROUND: We describe the first case of a pediatric patient with acute intermittent porphyria and severe chronic porphyric neuropathy treated with givosiran, a small-interfering RNA that drastically decreases delta-aminolevulinic acid production and reduces porphyric attacks’ recurrence. CASE REPORT: A 12-year-old male patient with refractory acute intermittent porphyria and severe porphyric neuropathy was followed prospectively for 12 months after givosiran initiation (subcutaneous, 2.5 mg/kg monthly). Serial neurological, structural, and resting-state functional magnetic resonance imaging (MRI) evaluations were performed, including clinical scales and neurophysiological tests. Delta-aminolevulinic acid urinary levels dropped drastically during treatment. In parallel, all the administered neurological rating scales and neurophysiological assessments showed improvement in all domains. Moreover, an improvement in central motor conduction parameters and resting-state functional connectivity in the sensory-motor network was noticed. At the end of the follow-up, the patient could walk unaided after using a wheelchair for 5 years. CONCLUSIONS: A clear beneficial effect of givosiran was demonstrated in our patient with both clinical and peripheral nerve neurophysiologic outcome measures. Moreover, we first reported a potential role of givosiran in recovering central motor network impairment in acute intermittent porphyria (AIP), which was previously unknown. This study provides Class IV evidence that givosiran improves chronic porphyric neuropathy.

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Mitochondrial DNA Copy Number Drives the Penetrance of Acute Intermittent Porphyria

Elena Di Pierro, Miriana Perrone, Milena Franco, Francesca Granata, Lorena Duca, Debora Lattuada, Giacomo De Luca and Giovanna Graziadei

Life 2023; 13: 1923

No published study has investigated the mitochondrial count in patients suffering from acute intermittent porphyria (AIP). In order to determine whether mitochondrial content can inﬂuence the pathogenesis of porphyria, we measured the mitochondrial DNA (mtDNA) copy number in the peripheral blood cells of 34 patients and 37 healthy individuals. We found that all AIP patients had a low number of mitochondria, likely as a result of a protective mechanism against an inherited heme synthesis deﬁciency. Furthermore, we identiﬁed a close correlation between disease penetrance and decreases in the mitochondrial content and serum levels of PERM1, a marker of mitochondrial biogenesis. In a healthy individual, mitochondrial count is usually modulated to ﬁt its ability to respond to various environmental stressors and bioenergetic demands. In AIP patients, coincidentally, the phenotype only manifests in response to endogenous and exogenous triggers factors. Therefore, these new ﬁndings suggest that a deﬁciency in mitochondrial proliferation could affect the individual responsiveness to stimuli, providing a new explanation for the variability in the clinical manifestations of porphyria. However, the metabolic and/or genetic factors responsible for this impairment remain to be identiﬁed. In conclusion, both mtDNA copy number per cell and mitochondrial biogenesis seem to play a role in either inhibiting or promoting disease expression. They could serve as two novel biomarkers for porphyria.

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Hyperhomocysteinemia in acute hepatic porphyria (AHP) and implications for treatment with givosiran

Paolo Ventura, Eliane Sardh, Nicola Longo, Manisha Balwani, Jorge Plutzky, Laurent Gouya, John Phillips, Sean Rhyee, Mary-Jean Fanelli, Marianne T. Sweetser & Petro E. Petrides

Expert Review of Gastroenterology & Hepatology 2022. Pubblicato online: 25 Aug 2022

Introduction: Homocysteine is a sulfur-containing amino acid formed in the intermediary metabolism of methionine. Amino acid metabolism and heme biosynthesis pathways are complexly intertwined. Plasma homocysteine elevation, hyperhomocysteinemia (HHcy), has been reported in patients with acute hepatic porphyria (AHP), a family of rare genetic disorders caused by defects in hepatic heme biosynthesis.
Areas covered: This article summarizes published case series in which givosiran, a subcutaneously administered small interfering RNA approved for AHP treatment, appeared to exacerbate dysregulated homocysteine metabolism in patients with AHP. A comprehensive exploratory analysis of ENVISION trial data demonstrated that on a population level, givosiran increased homocysteine but with wide interpatient variations, and there is no proof of correlations between HHcy and changes in efficacy or safety of givosiran.
Expert opinion: The strong correlation and co-increase of homocysteine and methionine suggest that HHcy associated with givosiran is likely attributable to the impaired trans-sulfuration pathway catalyzed by cystathionine β-synthase, which uses vitamin B6 as a cofactor. Data-based consensus supports monitoring total plasma homocysteine and vitamin B6, B12, and folate levels before and during givosiran treatment; supplementing with pyridoxine/vitamin B6 in patients with homocysteine levels >100 μmol/L; and involving patients with homocysteine levels >30 μmol/L in decisions to supplement.

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Iron Metabolism in the Disorders of Heme Biosynthesis

Andrea Ricci, Giada Di Betto, Elisa Bergamini, Elena Buzzetti, Elena Corradini and Paolo Ventura

Metabolites 2022; 12: 819

Given its remarkable property to easily switch between different oxidative states, iron is essential in countless cellular functions which involve redox reactions. At the same time, uncontrolled interactions between iron and its surrounding milieu may be damaging to cells and tissues. Heme—the iron-chelated form of protoporphyrin IX—is a macrocyclic tetrapyrrole and a coordination complex for diatomic gases, accurately engineered by evolution to exploit the catalytic, oxygen-binding, and oxidoreductive properties of iron while minimizing its damaging effects on tissues. The majority of the body production of heme is ultimately incorporated into hemoglobin within mature erythrocytes; thus, regulation of heme biosynthesis by iron is central in erythropoiesis. Additionally, heme is a cofactor in several metabolic pathways, which can be modulated by iron-dependent signals as well. Impairment in some steps of the pathway of heme biosynthesis is the main pathogenetic mechanism of two groups of diseases collectively known as porphyrias and congenital sideroblastic anemias. In porphyrias, according to the speciﬁc enzyme involved, heme precursors accumulate up to the enzyme stop in disease-speciﬁc patterns and organs. Therefore, different porphyrias manifest themselves under strikingly different clinical pictures. In congenital sideroblastic anemias, instead, an altered utilization of mitochondrial iron by erythroid precursors leads to mitochondrial iron overload and an accumulation of ring sideroblasts in the bone marrow. In line with the complexity of the processes involved, the role of iron in these conditions is then multifarious. This review aims to summarize the most important lines of evidence concerning the interplay be-tween iron and heme metabolism, as well as the clinical and experimental aspects of the role of iron in inherited conditions of altered heme biosynthesis.

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Disease burden in patients with acute hepatic porphyria: experience from the phase 3 ENVISION study

Bruce Wang, Paolo Ventura, Kei‑ichiro Takase, Manish Thapar, David Cassiman, Ilja Kubisch, Shangbin Liu, Marianne T. Sweetser and Manisha Balwani

Orphanet Journal of Rare Diseases 2022; 17: 327

Background: Acute hepatic porphyria (AHP) is a family of four rare genetic diseases, each involving deficiency in a hepatic heme biosynthetic enzyme. Resultant overproduction of the neurotoxic intermediates δ‑aminolevulinic acid (ALA) and porphobilinogen (PBG) leads to disabling acute neurovisceral attacks and progressive neuropathy. We evaluated the AHP disease burden in patients aged ≥ 12 years in a post hoc analysis of the Phase 3, randomized, double‑blind, placebo‑controlled ENVISION trial of givosiran (NCT03338816), an RNA interference (RNAi) therapeutic that targets the enzyme ALAS1 to decrease ALA and PBG production. We analyzed baseline AHP severity via chronic symptoms between attacks, comorbidities, concomitant medications, hemin‑associated complications, and quality of life (QOL) and evaluated givosiran (2.5 mg/kg monthly) in patients with and without prior hemin prophylaxis on number and severity of attacks and pain scores during and between attacks.
Results: Participants (placebo, n = 46; givosiran, n = 48) included patients with low and high annualized attack rates (AARs; range 0–46). At baseline, patients reported chronic symptoms (52%), including nausea, fatigue, and pain; comorbidities, including neuropathy (38%) and psychiatric disorders (47%); concomitant medications, including chronic opioids (29%); hemin‑associated complications (eg, iron overload); and poor QOL (low SF‑12 and EuroQol visual analog scale scores). A linear relationship between time since diagnosis and AAR with placebo suggested worsening of disease over time without effective treatment. Givosiran reduced the number and severity of attacks, days with worst pain scores above baseline, and opioid use versus placebo.
Conclusions: Patients with AHP, regardless of annualized attack rates, have considerable disease burden that may partly be alleviated with givosiran.

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Endothelial Dysfunction in Acute Hepatic Porphyrias

Andrea Ricci, Gilda Sandri, Matteo Marcacci, Elena Di Pierro, Francesca Granata, Chiara Cuoghi, Stefano Marchini, Antonello Pietrangelo and Paolo Ventura

Diagnostics 2022; 12: 1303

Background: Acute hepatic porphyrias (AHPs) are a group of rare diseases caused by dysfunctions in the pathway of heme biosynthesis. Although acute neurovisceral attacks are the most dramatic manifestations, patients are at risk of developing long-term complications, several of which are of a vascular nature. The accumulation of non-porphyrin heme precursors is deemed to cause most clinical symptoms. Aim: We measured the serum levels of endothelin-1 (ET-1) and nitric oxide (NO) to assess the presence of endothelial dysfunction (ED) in patients with AHPs. Forty-six patients were classiﬁed, according to their clinical phenotype, as symptomatic (AP-SP), asymptomatic with biochemical alterations (AP-BA), and asymptomatic without biochemical alterations (AP-AC). Results: Even excluding those under hemin treatment, AP-SP patients had the lowest NO and highest ET-1 levels, whereas no signiﬁcant differences were found between AP-BA and AP-AC patients. AP-SP patients had signiﬁcantly more often abnormal levels of ED markers. Patients with the highest heme precursor urinary levels had the greatest alterations in ED markers, although no signiﬁcant correlation was detected. Conclusions: ED is more closely related to the clinical phenotype of AHPs than to their classical biochemical alterations. Some still undeﬁned disease modiﬁers may possibly determine the clinical picture of AHPs through an effect on endothelial functions.

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Psychological Aspect and Quality of Life in Porphyrias: A Review

Francesca Granata, Annamaria Nicolli, Alessia Colaiocco, Elena Di Pierro and Giovanna Graziadei

Diagnostics 2022; 12: 1193

The World Health Organization (WHO) describes “health” as a state of physical, mental, and social well-being and not merely the absence of disease or inﬁrmity. Therefore, a biopsychosocial approach should be considered as an integral part of patients’ management. In this review, we summarize the available data starting from 1986 on the biological, psychological, and social aspects of porphyrias in order to provide a useful tool for clinicians about the missing knowledge within this ﬁeld. Porphyrias are a group of rare metabolic disorders affecting the heme biosynthetic pathway and can be categorized into hepatic and erythropoietic. Here, a total of 20 articles reporting the psychological and the quality of life (QoL) data of porphyria patients affected by acute hepatic porphyrias (AHPs), Porphyria Cutanea Tarda (PCT), and Erythropoietic Protoporphyria (EPP) were analyzed. These 13 articles include reported quantitative methods using questionnaires, while the reaming articles employed qualitative descriptive approaches through direct interviews with patients by psychology professionals. We conclude that the use of questionnaires limits the complete description of all areas of a patient’s life compared to a direct interview with specialists. However, only a combined use of these methods could be the best approach for the correct disorder management.

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Givosiran for the treatment of acute hepatic porphyria

Andrea Ricci and Paolo Ventura

Expert Review of Clinical Pharmacology. Pubblicato on line 11 maggio 2022

Introduction: Acute hepatic porphyrias (AHPs) are a family of rare inherited disorders characterized by enzyme dysfunctions in the hepatic pathway of heme biosynthesis. In AHPs, accumulation of the neurotoxic porphyrin precursors delta-aminolevulinic acid and porphobilinogen, caused by enhanced activity of hepatic aminolevulinate synthase 1 (ALAS1), is associated with acute, potentially life- threatening neurovisceral attacks. Symptoms during and between attacks dramatically reduce patients’ quality of life (QoL). Givosiran is the first mRNA-targeted treatment for AHPs, silencing ALAS1 expression.
Areas covered: For givosiran, this review summarizes its chemistry, mechanism of action, pharmaco-kinetics, pharmacodynamics, safety, preclinical and clinical data in AHP, postmarketing surveillance, and regulatory status. A literature search of public and internal databases was performed, bibliographies of retrieved articles were manually searched to identify additional studies of relevance, and information was also provided by Alnylam Pharmaceuticals.
Expert opinion: Givosiran is a small interfering RNA (siRNA) therapeutic that reduces hepatic activity of ALAS1 and decreases accumulation of neurotoxic porphyrin precursors in patients with AHPs, ultimately reducing the number of acute attacks and improving symptoms and QoL between attacks. As AHPs are lifelong diseases, long-term safety data are needed for givosiran as an siRNA-based therapy.

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Challenges in diagnosis and management of acute hepatic porphyrias: from an uncommon pediatric onset to innovative treatments and perspectives

Matteo Marcacci, Andrea Ricci, Chiara Cuoghi, Stefano Marchini, Antonello Pietrangelo and Paolo Ventura

Orphanet Journal of Rare Diseases 2022; 17: 160

Acute hepatic porphyrias (AHPs) are a family of four rare genetic diseases resulting from a deficiency in one of the enzymes involved in heme biosynthesis. AHP patients can experience potentially life-threatening acute attacks, characterized by severe abdominal pain, along with other signs and symptoms including nausea, mental confusion, hyponatraemia, hypertension, tachycardia and muscle weakness. Some patients also experience chronic manifestations and long-term complications, such as chronic pain syndrome, neuropathy and porphyria-associated kidney disease. Most symptomatic patients have only a few attacks in their lifetime; nevertheless, some experience frequent attacks that result in ongoing symptoms and a significant negative impact on their quality of life (QoL). Initial diagnosis of AHP can be made with a test for urinary porphobilinogen, δ-aminolaevulinic acid and porphyrins using a single random (spot) sample. However, diagnosis is frequently missed or delayed, often for years, because the clinical symptoms of AHP are non-specific and mimic other more common disorders. Delayed diagnosis is of concern as some commonly used medications can trigger or exacerbate acute attacks, and untreated attacks can become severe, potentially leading to permanent neurological damage or fatality. Other attack triggers include hormonal fluctuations in women, stress, alcohol and low-calorie diets, which should be avoided in patients where possible. For the management of attacks, intravenous hemin is approved, whereas new therapeutic approaches are currently being investigated as a baseline therapy for prevention of attacks and improvement of QoL. Among these, a novel siRNA-based agent, givosiran, has shown very promising results in a recently concluded Phase III trial and has been approved for the management of AHPs. Here, we propose a challenging case study-with a very unusual pediatric onset of variegate porphyria-as a starting point to summarize the main clinical aspects (namely, clinical manifestations, diagnostic challenges, and therapeutic management) of AHPs, with a focus on the latest therapeutic innovations.

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Kidney Involvement in Acute Hepatic Porphyrias: Pathophysiology and Diagnostic Implications

Andrea Ricci, Claudio Carmine Guida, Paola Manzini, Chiara Cuoghi and Paolo Ventura

Diagnostics 2021; 11: 2324

Porphyrias are a group of rare disorders originating from an enzyme dysfunction in the pathway of heme biosynthesis. Depending on the speciﬁc enzyme involved, porphyrias manifest under drastically different clinical pictures. The most dramatic presentation of the four congenital acute hepatic porphyrias (AHPs: acute intermittent porphyria—AIP, ALAD deﬁciency, hereditary coproporphyria—HCP, and porphyria variegata—VP) consists of potentially life-threatening neuro- visceral attacks, for which givosiran, a novel and effective siRNA-based therapeutic, has recently been licensed. Nonetheless, the clinical manifestations of acute porphyrias are multifaceted and do not limit themselves to acute attacks. In particular, porphyria-associated kidney disease (PAKD) is a distinct, long-term degenerating condition with speciﬁc pathological and clinical features, for which a satisfactory treatment is not available yet. In PAKD, chronic tubule-interstitial damage has been most commonly reported, though other pathologic features (e.g., chronic ﬁbrous intimal hyperplasia) are consistent ﬁndings. Given the relevant role of the kidney in porphyrin metabolism, the mechanisms possibly intervening in causing renal damage in AHPs are different: among others, δ-aminolevulinic acid (ALA)-induced oxidative damage on mitochondria, intracellular toxic aggregation of porphyrins in proximal tubular cells, and derangements in the delicate microcirculatory balances of the kidney might be implicated. The presence of a variant of the human peptide transporter 2 (PEPT2), with a greater afﬁnity to its substrates (including ALA), might confer a greater susceptibility to kidney damage in patients with AHPs. Furthermore, a possible effect of givosiran in worsening kidney function has been observed. In sum, the diagnostic workup of AHPs should always include a baseline evaluation of renal function, and periodic monitoring of the progression of kidney disease in patients with AHPs is strongly recommended. This review outlines the role of the kidney in porphyrin metabolism, the available evidence in support of the current etiologic and pathogenetic hypotheses, and the known clinical features of renal involvement in acute hepatic porphyrias.

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Efficacy and safety of givosiran for acute hepatic porphyria: 24-month interim analysis of the randomized phase 3 ENVISION study

Paolo Ventura, Herbert L. Bonkovsky, Laurent Gouya, Paula Aguilera-Peiró, D. Montgomery Bissell, Penelope E. Stein, Manisha Balwani, D. Karl E. Anderson, Charles Parker, David J. Kuter, Susana Monroy, Jeeyoung Oh, Bruce Ritchie, John J. Ko, Zhaowei Hua, Marianne T. Sweetser, Eliane Sardh, for the ENVISION Investigators

Liver Int. 2021; 00: 1–12

Background & Aims: Upregulation of hepatic delta-aminolevulinic acid synthase 1 with accumulation of potentially toxic heme precursors delta-aminolevulinic acid and porphobilinogen is fundamental to the pathogenesis of acute hepatic porphyria.
Aims: evaluate long-term efficacy and safety of givosiran in acute hepatic porphyria.
Methods: Interim analysis of ongoing ENVISION study (NCT03338816), after all ac- tive patients completed their Month 24 visit. Patients with acute hepatic porphyria (≥12 years) with recurrent attacks received givosiran (2.5 mg/kg monthly) (n = 48) or placebo (n = 46) for 6 months (double-blind period); 93 received givosiran (2.5 mg or 1.25 mg/kg monthly) in the open-label extension (continuous givosiran, n = 47/48; placebo crossover, n = 46/46). Endpoints included annualized attack rate, urinary delta-aminolevulinic acid and porphobilinogen levels, hemin use, daily worst pain, quality of life, and adverse events.
Results: Patients receiving continuous givosiran had sustained annualized attack rate reduction (median 1.0 in double- blind period, 0.0 in open- label extension); in pla- cebo crossover patients, median annualized attack rate decreased from 10.7 to 1.4. Median annualized days of hemin use were 0.0 (double- blind period) and 0.0 (open- label extension) for continuous givosiran patients and reduced from 14.98 to 0.71 for placebo crossover patients. Long-term givosiran led to sustained lowering of delta- aminolevulinic acid and porphobilinogen and improvements in daily worst pain and quality of life. Safety findings were consistent with the double- blind period.
Conclusions: Long-term givosiran has an acceptable safety profile and significantly benefits acute hepatic porphyria patients with recurrent attacks by reducing attack frequency, hemin use, and severity of daily worst pain while improving quality of life.

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Nutrients and Porphyria: An Intriguing Crosstalk

Elena Di Pierro and Francesca Granata

International Journal of Molecular Sciences (2020); 21: 3462

Porphyria refers to a group of fascinating diseases from a metabolic and nutritional standpoint as it provides an example of how metabolic manipulation can be used for therapeutic purposes. It is characterized by defects in heme synthesis, particularly in the erythrocytes and liver. Speciﬁc enzymes involved in heme biosynthesis directly depend on adequate levels of vitamins and minerals in the tissues. Moreover, micronutrients that are required for producing succinyl CoA and other intermediates in the Krebs (TCA) cycle are indirectly necessary for heme metabolism. This review summarizes articles that describe the nutritional status, supplements intake, and dietary practices of patients aﬀected by porphyria, paying special attention to the therapeutic use of nutrients that may help or hinder this group of diseases.

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Laboratory Diagnosis of Porphyria

Elena Di Pierro, Michele De Canio, Rosa Mercadante, Maria Savino, Francesca Granata,
Dario Tavazzi, Anna Maria Nicolli, Andrea Trevisan, Stefano Marchini and Silvia Fustinoni

Diagnostics (2021); vol. 11; pag. 1343

Porphyrias are a group of diseases that are clinically and genetically heterogeneous and originate mostly from inherited dysfunctions of speciﬁc enzymes involved in heme biosynthesis. Such dysfunctions result in the excessive production and excretion of the intermediates of the heme biosynthesis pathway in the blood, urine, or feces, and these intermediates are responsible for speciﬁc clinical presentations. Porphyrias continue to be underdiagnosed, although laboratory diagnosis based on the measurement of metabolites could be utilized to support clinical suspicion in all symptomatic patients. Moreover, the measurement of enzymatic activities along with a molecular analysis may conﬁrm the diagnosis and are, therefore, crucial for identifying pre-symptomatic carriers. The present review provides an overview of the laboratory assays used most commonly for establishing the diagnosis of porphyria. This would assist the clinicians in prescribing appropriate diagnostic testing and interpreting the testing results.

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When awareness makes the difference: diagnosing and treating the acute hepatic porphyrias

Paolo Ventura

Internal and Emergency Medicine (2021) vol. 16; pag. 25–27

Abstract non disponibile

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Liver Transplantation for Acute Intermittent Porphyria

Mattias Lissing, Greg Nowak, René Adam, Vincent Karam, Alexander Boyd, Laurent Gouya,
Wouter Meersseman, Espen Melum, Urszula Ołdakowska-Jedynak, Florian P. Reiter,
Jordi Colmenero, Rosario Sanchez, Uta Herden, Janneke Langendonk, Paolo Ventura,
Helena Isoniemi, Olivier Boillot, Felix Braun, Stéphanie Perrodin , Elizabeth Mowlem, Staffan Wahlin

Liver Transplantation vol. 27; pag. 491‒501 April 2021

Recurrent attacks of acute intermittent porphyria (AIP) result in poor quality of life and significant risks of morbidity and mortality. Liver transplantation (LT) offers a cure, but published data on outcomes after LT are limited. We assessed the pretransplant characteristics, complications, and outcomes for patients with AIP who received a transplant. Data were collected retrospectively from the European Liver Transplant Registry and from questionnaires sent to identified transplant and porphyria centers. We studied 38 patients who received transplants in 12 countries from 2002 to 2019. Median age at LT was 37 years (range, 18-58), and 34 (89%) of the patients were women. A total of 9 patients died during follow-up, and 2 patients were retransplanted. The 1-year and 5-year overall survival rates were 92% and 82%, which are comparable with other metabolic diseases transplanted during the same period. Advanced pretransplant neurological impairment was associated with increased mortality. The 5-year survival rate was 94% among 19 patients with moderate or no neuropathy at LT and 83% among 10 patients with severe neuropathy (P = 0.04). Pretransplant renal impairment was common. A total of 19 (51%) patients had a GFR < 60 mL/minute. Although few patients improved their renal function after LT, neurological impairments improved, and no worsening of neurological symptoms was recorded. No patient had AIP attacks after LT, except for a patient who received an auxiliary graft. LT is a curative treatment option for patients with recurrent attacks of AIP. Severe neuropathy and impaired renal function are common and increase the risk for poor outcomes. If other treatment options fail, an evaluation for LT should be performed early.

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Clinical and molecular epidemiology of erythropoietic protoporphyria in Italy

Paolo Ventura, Valentina Brancaleoni, Elena Di Pierro, Giovanna Graziadei, Annelise Macrì, Claudio Carmine Guida, Annamaria Nicolli, Maria Teresa Rossi, Francesca Granata, Valeria Fiorentino, Silvia Fustinoni, Raffella Sala, Piergiacomo Calzavara Pinton, Andrea Trevisan, Stefano Marchini, Chiara Cuoghi, Matteo Marcacci, Elena Corradini, Fiammetta Sorge, Caterina Aurizi, Maria Grazia Savino, Maria Domenica Cappellini, Antonello Pietrangelo (Gruppo Italiano Porﬁria)

European Journal of Dermatology, 2020 Oct 1; vol. 30(5): pag. 532-540

Background: Erythropoietic protoporphyria (EPP) is a rare inherited disease associated with heme metabolism, characterized by severe life-long photosensitivity and liver involvement.
Objectives: To provide epidemiological data of EPP in Italy.
Materials and methods: Prospective/retrospective data of EPP patients were collected by an Italian network of porphyria specialist centres (Gruppo Italiano Porfiria, GrIP) over a 20-year period (1996-2017).
Results: In total, 179 patients (79 females) with a clinical and biochemical diagnosis of EPP were assessed, revealing a prevalence of 3.15 cases per million persons and an incidence of 0.13 cases per million persons/year. Incidence significantly increased after 2009 (due to the availability of alfa-melanotide, which effectively limits skin photosensitivity). Mean age at diagnosis was 28 years, with only 22 patients (12.2%) diagnosed ≤10 years old. Gene mutations were assessed in 173 (96.6%) patients; most (164; 91.3%) were FECH mutations on one allele in association with the hypomorphic variant, c.315-48C, on the other (classic EPP), and nine (5.2%) were ALAS2 mutations (X-linked EPP). Only one case of autosomal recessive EPP was observed. Of the 42 different FECH mutations, 15 are novel, three mutations collectively accounted for 45.9% (75/164) of the mutations (c.215dupT [27.2%], c.901_902delTG [11.5%] and c.67 + 5G > A [7.2%]), and frameshift mutations were prevalent (33.3%). A form of light protection was used by 109/179 (60.8%) patients, and 100 (56%) had at least one α-melanotide implant. Three cases of severe acute liver involvement, requiring OLT, were observed.
Conclusions: These data define, for the first time, the clinical and molecular epidemiology of EPP in Italy.

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Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria

M. Balwani, E. Sardh, P. Ventura, P.A. Peiró, D.C. Rees, U. Stölzel, D.M. Bissell, H.L. Bonkovsky, J. Windyga, K.E. Anderson, C. Parker, S.M. Silver, S.B. Keel, J.-D. Wang, P.E. Stein, P. Harper, D. Vassiliou, B. Wang, J. Phillips, A. Ivanova, J.G. Langendonk, R. Kauppinen, E. Minder, Y. Horie, C. Penz, J. Chen, S. Liu, J.J. Ko, M.T. Sweetser, P. Garg, A. Vaishnaw, J.B. Kim, A.R. Simon, and L. Gouya

New England Journal of Medicine, vol. 382, n. 24, pag. 2289-2301

BACKGROUND: Up-regulation of hepatic delta-aminolevulinic acid synthase 1 (ALAS1), with resul-tant accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen, is cen-tral to the pathogenesis of acute attacks and chronic symptoms in acute hepatic porphyria. Givosiran, an RNA interference therapy, inhibits ALAS1 expression.
METHODS: In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned symptomatic patients with acute hepatic porphyria to receive either subcutaneous givosiran (2.5 mg per kilogram of body weight) or placebo monthly for 6 months. The primary end point was the annualized rate of composite porphyria attacks among patients with acute intermittent porphyria, the most common subtype of acute hepatic porphyria. (Composite porphyria attacks resulted in hospitalization, an urgent health care visit, or intravenous administration of hemin at home.) Key secondary end points were levels of ALA and porphobilinogen and the annualized attack rate among patients with acute hepatic porphyria, along with hemin use and daily worst pain scores in patients with acute intermittent porphyria.
RESULTS: A total of 94 patients underwent randomization (48 in the givosiran group and 46 in the placebo group). Among the 89 patients with acute intermittent porphyria, the mean annualized attack rate was 3.2 in the givosiran group and 12.5 in the placebo group, representing a 74% lower rate in the givosiran group (P<0.001); the results were similar among the 94 patients with acute hepatic porphyria. Among the pa-tients with acute intermittent porphyria, givosiran led to lower levels of urinary ALA and porphobilinogen, fewer days of hemin use, and better daily scores for pain than placebo. Key adverse events that were observed more frequently in the givosiran group were elevations in serum aminotransferase levels, changes in serum creati-nine levels and the estimated glomerular filtration rate, and injection-site reactions.
CONCLUSIONS: Among patients with acute intermittent porphyria, those who received givosiran had a significantly lower rate of porphyria attacks and better results for multiple other disease manifestations than those who received placebo. The increased ef-ficacy was accompanied by a higher frequency of hepatic and renal adverse events. (Funded by Alnylam Pharmaceuticals; ENVISION ClinicalTrials.gov number, NCT03338816.)

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Hyperhomocysteinemia in patients with acute porphyrias: A potentially
dangerous metabolic crossroad?

Paolo Ventura, Elena Corradini, Elena Di Pierro, Stefano Marchini, Matteo Marcacci, Chiara Cuoghi, Elena Buzzetti, Antonello Pietrangelo

European Journal of Internal Medicine (2020); vol. 79: pag. 101-107

Background: Acute porphyrias (AP) are characterized by heme deficiency and induction of hepatic 5-aminolevulinate synthase (ALAS1). Hyperhomocysteinemia (HHcy) is associated with endothelial damage, neurotoxicity and increased risk for vascular diseases. Interestingly, both heme biosynthesis and sulphur amino acid metabolism require vitamin B6, (Pyridoxal-phosphate, PLP) an important cofactor of ALAS1 and of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) enzymes that catabolize homocysteine (Hcy). Moreover, heme itself is an important cofactor for CBS.
Aim: to assess plasma Hcy status and HHcy main determinants in patients with AP.
Materials and methods: A total of 46 patients with AP (31 with Acute Intermittent Porphyria,15 with Variegate Porphyria) were assessed for clinical status (symptomatic vs. asymptomatic), serum Hcy, Cysteine (Cys), Vit.B6, Vit.B12, red blood cell folates and urinary delta-aminolevulinic acid (ALA) and porphobilinogen(PBG) levels (mean of six measurements).
Results: Symptomatic AP patients had significantly higher urinary ALA and PBG levels, plasma Hcy, HHcy prevalence and Hcy/Cys ratio when compared to asymptomatic carriers of AP. Even though no significant correlation was observed between ALA/PBG urinary levels and serum Hcy levels, patients with higher levels of ALA and PBG had significantly higher levels of Hcy, a higher prevalence of moderate-to severe HHcy and serum PLP levels below the 25 th percentile of a reference assessment with 300 healthy Italian subjects(<45nmol/L).
Conclusions: Most patients with symptomatic AP present HHcy resulting from alterations in sulphur amino acid metabolism. HHcy may represent an indirect marker of ALAS1 induction and its prevalence may be suggestive of a role of HHcy in the pathogenesis and/or comorbidities of AP.

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Inflammatory involvement into phototoxic reaction in erythropoietic protoporphyria (EPP) patients

Francesca Granata, Lorena Duca, Giovanna Graziadei, Valentina Brancaleoni, Pasquale Missineo, Giacomo De Luca, Silvia Fustinoni and Elena Di Pierro

Immunologic Research (2019); vol. 67(4-5): pag. 382-389

Phototoxic reaction is a known feature of EPP at least in part triggered by the oxidative status, complement system activation, and mast cell response. The aim of this study was to verify some aspects involved in phototoxic reaction during a season. The complement system was evaluated by C3 assay, alternative pathway by factor-B, and classical pathway by C1q; oxidative status was tested with malondialdehyde (MDA) and mast cell by IL-10 assay. The serum samples were collected in winter and summer from 19 EPP patients and 13 controls. The reaction to sun exposure within each group was monitored without any invasive treatment. In summer, C3 and factor B were higher in patients than in controls ( p = 0.002 and < .0001 respectively), while no change was detected for C1q. The oxidative stress was increased in summer in comparison with the control group ( p = 0.04), and IL-10 an assay was normal in both seasons. The correlation between the C3 and factor-B in summer was significant. This study shows that the phototoxic reaction is not limited to the dermis but can also exert a systemic response, which could affect the general health of a patient. The knowledge of the pathophysiology of phototoxic reaction is essential for identifying new disease markers useful for improving clinical studies of known and future drugs.

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EXPLORE: A Prospective, Multinational, Natural History Study of Patients with Acute Hepatic Porphyria with Recurrent Attacks

Laurent Gouya, Paolo Ventura, Manisha Balwani , D. Montgomery Bissell, David C. Rees, Ulrich Stölzel, John D. Phillips, Raili Kauppinen, Janneke G. Langendonk, Robert J. Desnick, Jean-Charles Deybach, Herbert L. Bonkovsky , Charles Parker, Hetanshi Naik, Michael Badminton, Penelope E. Stein, Elisabeth Minder, Jerzy Windyga, Radan Bruha, Maria Domenica Cappellini, Eliane Sardh, Pauline Harper, Sverre Sandberg, Aasne K. Aarsand, Janice Andersen, Félix Alegre, Aneta Ivanova, Neila Talbi, Amy Chan, William Querbes, John Ko, Craig Penz, Shangbin Liu, Tim Lin, Amy Simon and Karl E. Anderson

Hepatology (2020); vol. 71(5): pag. 1546-1558

Background and Aims: Acute hepatic porphyria comprises a group of rare genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long-term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients.
Approach and Results: EXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months before the study, patients reported a median (range) of 6 (0-52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a health care facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0-37.0]). Elevated levels of hepatic δ-aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ-aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased health care utilization.
Conclusions: Patients experienced attacks often requiring treatment in a health care facility and/or with hemin, as well as chronic symptoms that adversely influenced day-to-day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies.

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Brain perfusion defects by SPET/CT and neurostat semi-quantitative analysis in two patients with congenital erythropoietic porphyria

Vincenzo Frusciante, Cristina Ferrari, Manuela Totaro, Guido Valle, Claudio Carmine Guida, Filippo Aucella, Paola Caputo, Giuseppe Rubini

Hell J Nucl Med 2018; 21(1): 43-47

BACKGROUND: Congenital erythropoietic porphyria (CEP) is a rare autosomal recessively inherited disorder with chronic and relatively stable presentation. Till now brain blood flow derangements have been described only in acute hepatic porphyrias. We describe the first findings of brain perfusion defects, studied by single photon emission tomography/computed tomography (SPET/CT), in two patients affected by CEP, by using a semi-quantification anatomic-standardized voxel-based program compared with magnetic resonance imaging (MRI) results.
SUBJECTS AND METHODS: Two Pakistanis brothers were investigated for CEP confirmed by a genetic test. The disease was severe with: skin burning, mood depression and haemolytic anemia. Considering depression, patients underwent brain SPET/CT and MRI. Single photon emission tomography/CT images were processed by neurostat semi-quantitative software. Data obtained were compared to a normal database and z-score images were generated.
RESULTS: In both patients we found several perfusion defects evident in transaxial slices and in z-score images obtained by neurostat processing. Magnetic resonance imaging was negative in both patients. Biochemical mechanisms inducing localized brain hypoperfusion are uncertain. However, mismatch between SPET/CT data and MRI was probably due to absence of necrosis.
CONCLUSION: In our opinion, SPET/CT could have a key role in this setting of patients due to its high sensitivity and reliability in mild-to-moderate brain perfusion defects detection. Moreover, the quantitative analysis by using neurostat may allow to recognize even mild brain perfusion alterations, difficult to detect only visually.

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The assessment of noncoding variant of PPOX gene in variegate porphyria reveals post-transcriptional role of the 5′ untranslated exon 1.

Fiorentino V, Brancaleoni V, Granata F, Graziadei G, Di Pierro E.

Blood Cells Mol Dis. 2016 Oct;61:48-53

The PPOX gene encodes for the protoporphyrinogen oxidase, which is involved in heme production. The partial deficiency of protoporphyrinogen oxidase causes variegate porphyria. The tissue-specific regulation of other heme biosynthetic enzymes is extensively studied, but the information concerning transcriptional and post-transcriptional regulation of PPOX gene expression is scarcely available. In this study, we characterized functions of three variants identified in the regulatory regions of the PPOX gene, which show a novel role for the 5′ untranslated exon 1. Using luciferase assays and RNA analysis, we demonstrated that only c.1-883G>C promoter variant causes a significant loss in the transcriptional activity of PPOX gene whereas c.1-413G>T 5′ UTR variant inhibits translation of PPOX mRNA and c.1-176G>A splicing variant causes 4bp deletion in 5′ UTR of PPOX mRNA variant 2. These observations indicate that the regulation of PPOX gene expression can also occur through a post-transcriptional modulation of the amount of gene product and that this modulation can be mediated by 5′ untranslated exon 1. Moreover this study confirms that these regulatory regions represent an important molecular target for the pathogenesis of variegate porphyria.

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Advances in understanding the pathogenesis of congenital erythropoietic porphyria.

Di Pierro E, Brancaleoni V, Granata F.

Br J Haematol. 2016 May;173(3):365-79.

Congenital erythropoietic porphyria (CEP) is a rare genetic disease resulting from the remarkable deficient activity of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthetic pathway. This enzyme defect results in overproduction of the non-physiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I. The predominant clinical characteristics of CEP include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. The severity of clinical manifestations is markedly heterogeneous among patients; and interdependence between disease severity and porphyrin amount in the tissues has been pointed out. A more pronounced endogenous production of porphyrins concomitant to activation of ALAS2, the first and rate-limiting of the haem synthesis enzymes in erythroid cells, has also been reported. CEP is inherited as autosomal recessive or X-linked trait due to mutations in UROS or GATA1 genes; however an involvement of other causative or modifier genes cannot be ruled out.

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X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria.

Brancaleoni V, Balwani M, Granata F, Graziadei G, Missineo P, Fiorentino V, Fustinoni S, Cappellini MD, Naik H, Desnick RJ, Di Pierro E.

Clin Genet. 2016 Jan;89(1):20-6.

X-linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain-of-function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X-linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X-chromosomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc-finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X-chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild-type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin-containing peripheral blood fluorocytes. When the wild-type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP.

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Congenital erythropoietic porphyria linked to GATA1-R216W mutation: challenges for diagnosis.

Di Pierro E, Russo R, Karakas Z, Brancaleoni V, Gambale A, Kurt I, Winter SS, Granata F, Czuchlewski DR, Langella C, Iolascon A, Cappellini MD.

Eur J Haematol. 2015 Jun;94(6):491-7.

Congenital erythropoietic porphyria (CEP) is a rare genetic disease that is characterized by a severe cutaneous photosensitivity causing unrecoverable deformities, chronic hemolytic anemia requiring blood transfusion program, and by fatal systemic complications. A correct and early diagnosis is required to develop a management plan that is appropriate to the patient’s needs. Recently only one case of X-linked CEP had been reported, describing the trans-acting GATA1-R216W mutation. Here, we have characterized two novel X-linked CEP patients, both with misleading hematological phenotypes that include dyserythropoietic anemia, thrombocytopenia, and hereditary persistence of fetal hemoglobin. We compare the previously reported case to ours and propose a diagnostic paradigm for this variant of CEP. Finally, a correlation between phenotype variability and the presence of modifier mutations in loci related to disease-causing gene is described.

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A challenging diagnosis for potential fatal diseases: recommendations for diagnosing acute porphyrias.

Ventura P, Cappellini MD, Biolcati G, Guida CC, Rocchi E; Gruppo Italiano Porfiria (GrIP).

Eur J Intern Med. 2014 Jul;25(6):497-505.

Acute porphyrias are a heterogeneous group of metabolic disorders resulting from a variable catalytic defect of four enzymes out of the eight involved in the haem biosynthesis pathway; they are rare and mostly inherited diseases, but in some circumstances, the metabolic disturbance may be acquired. Many different environmental factors or pathological conditions (such as drugs, calorie restriction, hormones, infections, or alcohol abuse) often play a key role in triggering the clinical exacerbation (acute porphyric attack) of these diseases that may often mimic many other more common acute medical and neuropsychiatric conditions and whose delayed diagnosis and treatment may be fatal. In order to obtain an accurate diagnosis of acute porphyria, the knowledge and the use of appropriate diagnostic tools are mandatory, even in order to provide as soon as possible the more effective treatment and to prevent the use of potentially unsafe drugs, which can severely precipitate these diseases, especially in the presence of life-threatening symptoms. In this paper, we provide some recommendations for the diagnostic steps of acute porphyrias by reviewing literature and referring to clinical experience of the board members of the Gruppo Italiano Porfiria (GrIP).

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The acute porphyrias: a diagnostic and therapeutic challenge in internal and emergency medicine.

Ventura P, Cappellini MD, Rocchi E.

Intern Emerg Med. 2009 Aug;4(4):297-308.

The porphyrias are a heterogeneous group of metabolic diseases resulting from a variable catalytic defect of one of the eight enzymes involved in the heme biosynthesis pathway; they are mostly inherited diseases, but in some circumstances the metabolic disturbance may be acquired. The specific patterns of tissue overproduction (and hence accumulation and excretion) of toxic heme precursors, associated with each enzymatic deficiency, are responsible for the characteristic biochemical and clinical features of each of these diseases. Moreover, even in the presence of a specific inherited enzymatic defect, many different environmental factors (such as drugs, calorie restriction, hormones, sunlight exposition, infections, etc.) often play a key role in triggering the clinical expression of the various forms of porphyrias. The porphyrias are often misdiagnosed diseases, due their multiform clinical manifestations, able to mimic many other more common diseases. For this reason, many different specialists, such as surgeons, psychiatrists, gastroenterologists, neurologists, emergency physicians and dermatologists may be variably involved in the diagnostic process, especially for the forms presenting with acute and life-threatening clinical features. According to the clinical features, the porphyrias can be classified into neuropsychiatric (characterized by neurovisceral crises involving autonomic and central nervous system but also the liver and the kidney with possible consequences in terms of neurological, psychic, cardiac, respiratory, liver and kidney functions), dermatological (mostly presenting with cutaneous lesions due to photosensitivity), and mixed forms. From a strictly clinical point of view, porphyrias presenting with neurovisceral attacks are also referred as acute porphyrias: they are the object of the present review. An accurate diagnosis of acute porphyria requires knowledge and the use of correct diagnostic tools, and it is mandatory to provide a more appropriate therapeutic approach and prevent the use of potentially unsafe drugs, able to severely precipitate these diseases, especially in the presence of life-threatening symptoms. To date, availability of a relatively stable haem preparation (haem arginate) has significantly improved the treatment outcome of acute porphyric attacks, so the knowledge about the diagnosis and the management of these diseases may be relevant for physicians working in internal medicine, neurology and emergency units.