Role of phlebotomy in the treatment of liver damage related to erythropoietic porphyria.

Liver damage affects the prognosis of patients with erythropoietic protoporphyria (EPP). However, there is no radical cure for EPP patients with severe liver damage. This study aims to investigate the effectiveness of phlebotomy in patients with severe liver damage. We examined seven patients diagnosed with EPP and liver damage between 2010 and 2020. Of the 7 cases, phlebotomy was performed in 3 cases with severe hepatic disorder, and the improvement effect of hepatic disorder was observed in all cases. In addition, as an additional study, we also investigated the mechanism by which liver damage becomes more severe. Liver biopsy samples were stained with hematoxylin and eosin and immunohistochemistry was used to examine the expression of adenosine triphosphate-binding transporter G2 (ABCG2). Liver biopsies were performed in 3 of 7 patients with EPP. Of these three patients, ABCG2 expression was low in two patients, especially in the protoporphyrin (PP) deposition area. Two patients with reduced ABCG2 expression subsequently developed severe liver damage. However, the causal relationship between the decreased expression of ABCG2 and the exacerbation of liver damage has not been directly proved, and further investigation is required in the future. This study demonstrated the effectiveness of phlebotomy in EPP patients with severe liver damage.

Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models.

Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.

Genetic background influences hepcidin response to iron imbalance in a mouse model of hemolytic anemia (Congenital erythropoietic porphyria).

Clinical severity is heterogeneous among patients suffering from congenital erythropoietic porphyria (CEP) suggesting a modulation of the disease (UROS deficiency) by environmental factors and modifier genes. A KI model of CEP due to a missense mutation of UROS gene present in human has been developed on 3 congenic mouse strains (BALB/c, C57BL/6, and 129/Sv) in order to study the impact of genetic background on disease severity. To detect putative modifiers of disease expression in congenic mice, hematologic data, iron parameters, porphyrin content and tissue samples were collected. Regenerative hemolytic anemia, a consequence of porphyrin excess in RBCs, had various expressions: 129/Sv mice were more hemolytic, BALB/c had more regenerative response to anemia, C57BL/6 were less affected. Iron status and hemolysis level were directly related: C57BL/6 and BALB/c had moderate hemolysis and active erythropoiesis able to reduce iron overload in the liver, while, 129/Sv showed an imbalance between iron release due to hemolysis and erythroid use. The negative control of hepcidin on the ferroportin iron exporter appeared strain specific in the CEP mice models tested. Full repression of hepcidin was observed in BALB/c and 129/Sv mice, favoring parenchymal iron overload in the liver. Unchanged hepcidin levels in C57BL/6 resulted in retention of iron predominantly in reticuloendothelial tissues. These findings open the field for potential therapeutic applications in the human disease, of hepcidin agonists and iron depletion in chronic hemolytic anemia.

Congenital Erythropoietic Porphyria: A Rare Case of Photosensitivity with Hemolytic Anaemia and Mental Retardation.

Congenital erythropoietic porphyria, also called Gunther's disease, is a very rare genetic autosomal recessive diseaseaffecting less than 1 per 1,000,000 children. Pathogenesis involves genetic mutation encoding uroporphyrinogen-III cosynthase which leads to accumulation of porphyrin in many tissues, leading to extreme skin photosensitivity, red cell lysis, splenomegaly and reduced life expectancy. Herein, we report a 12-year mentally challenged girl with multiple blisters and scars on sun exposed sites since birth. She had hepatomegaly, erythrodontia, severe anaemia with haemolytic blood picture and mildly elevated liver enzymes. Skin biopsy showed deposition of amorphous eosinophilic porphyrins in the dermis, thus confirming a diagnosis of congenital erythropoietic porphyria.

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

Effects of the Usage of l-Cysteine (l-Cys) on Human Health.

This review summarizes recent knowledge about the use of the amino acid l-Cysteine (l-Cys) through diet, nutritional supplements or drugs with the aim to improve human health or treat certain diseases. Three databases (PubMed, Scopus, and Web of Science) and different keywords have been used to create a database of documents published between 1950 and 2017 in scientific journals in English or Spanish. A total of 60,885 primary publications were ultimately selected to compile accurate information about the use of l-Cys in medicine and nutritional therapies and to identify the reported benefits of l-Cys on human health. The number of publications about the use of l-Cys for these purposes has increased significantly during the last two decades. This increase seems to be closely related to the rise of nutraceutical industries and personalized medicine. The main evidence reporting benefits of l-Cys usage is summarized. However, the lack of accurate information and studies based on clinical trials hampers consensus among authors. Thus, the debate about the role and effectiveness of supplements/drugs containing l-Cys is still open.

Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Missense UROS mutations causing congenital erythropoietic porphyria reduce UROS homeostasis that can be rescued by proteasome inhibition.

Congenital erythropoietic porphyria (CEP) is an inborn error of heme biosynthesis characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in deleterious porphyrin accumulation in blood cells responsible for hemolytic anemia and cutaneous photosensitivity. We analyzed here the molecular basis of UROS impairment associated with twenty nine UROS missense mutations actually described in CEP patients. Using a computational and biophysical joint approach we predicted that most disease-causing mutations would affect UROS folding and stability. Through the analysis of enhanced green fluorescent protein-tagged versions of UROS enzyme we experimentally confirmed these data and showed that thermodynamic instability and premature protein degradation is a major mechanism accounting for the enzymatic deficiency associated with twenty UROS mutants in human cells. Since the intracellular loss in protein homeostasis is in excellent agreement with the in vitro destabilization, we used molecular dynamic simulation to rely structural 3D modification with UROS disability. We found that destabilizing mutations could be clustered within three types of mechanism according to side chain rearrangements or contact alterations within the pathogenic UROS enzyme so that the severity degree correlated with cellular protein instability. Furthermore, proteasome inhibition using bortezomib, a clinically available drug, significantly enhanced proteostasis of each unstable UROS mutant. Finally, we show evidence that abnormal protein homeostasis is a prevalent mechanism responsible for UROS deficiency and that modulators of UROS proteolysis such as proteasome inhibitors or chemical chaperones may represent an attractive therapeutic option to reduce porphyrin accumulation and prevent skin photosensitivity in CEP patients when the genotype includes a missense variant.

Acute hepatic and erythropoietic porphyrias: from ALA synthases 1 and 2 to new molecular bases and treatments.

PURPOSE OF REVIEW: Many studies over the past decade have together identified new genes including modifier genes and new regulation and pathophysiological mechanisms in inherited inborn diseases of the heme biosynthetic pathway. A new porphyria has been characterized: X-linked protoporphyria and the perspective to have innovative treatment at very short-term became a reality. We will summarize how recent data on both ALAS1 and ALAS2 have informed our understanding of disease pathogenesis with an emphasis on how this information may contribute to new therapeutic strategies. RECENT FINDINGS: The development of clinical and biological porphyria networks improved the long-term follow up of cohorts. The ageing of patients have allowed for the identification of novel recurrently mutated genes, and highlighted long-term complications in acute hepatic porphyrias. The treatment of hepatic porphyrias by an RNAi-targeting hepatic ALAS1 is actually tested and may lead to improve the management of acute attacks.In erythropoietic porphyrias, the key role of ALAS2 as a gate keeper of the heme and subsequently hemoglobin synthesis has been demonstrated. Its implication as a modifier gene in over erythroid disorders has also been documented. SUMMARY: The knowledge of both the genetic abnormalities and the regulation of heme biosynthesis has increased over the last 5 years and open new avenues in the management of erythropoietic and acute hepatic porphyrias.

Advances in understanding the pathogenesis of congenital erythropoietic porphyria.

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.

The emerging role of GATA transcription factors in development and disease.

The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.

Inducing iron deficiency improves erythropoiesis and photosensitivity in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of heme synthesis characterized by reduced activity of uroporphyrinogen III synthase and the accumulation of nonphysiologic isomer I porphyrin metabolites, resulting in ineffective erythropoiesis and devastating skin photosensitivity. Management of the disease primarily consists of supportive measures. Increased activity of 5-aminolevulinate synthase 2 (ALAS2) has been shown to adversely modify the disease phenotype. Herein, we present a patient with CEP who demonstrated a remarkable improvement in disease manifestations in the setting of iron deficiency. Hypothesizing that iron restriction improved her symptoms by decreasing ALAS2 activity and subsequent porphyrin production, we treated the patient with off-label use of deferasirox to maintain iron deficiency, with successful results. We confirmed the physiology of her response with marrow culture studies.

Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

Bullous skin lesions in a jaundiced infant after phototherapy: a case of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria is a rare autosomal recessive disorder of porphyrin metabolism in which the genetic defect is the deficiency of uroporphyrinogen III cosynthase (UIIIC). Deficiency of this enzyme results in an accumulation of high amounts of uroporphyrin I in all tissues, leading to hemolytic anemia, splenomegaly, erythrodontia, bone fragility, exquisite photosensitivity, and mutilating skin lesions. We discuss a female infantile case who was admitted for jaundice; bullous lesions appeared on her trunk during phototherapy in the neonatal period. The skin biopsy findings were consistent with epidermolysis bullosa. Due to persistent hepatosplenomegaly and cholestasis, metabolic tests and liver biopsy were performed. During the follow-up, hemolytic anemia and red urine were detected. The levels of porphyrin metabolites were determined at high concentrations in plasma, stool and urine analysis, which were suggestive of congenital erythropoietic porphyria.

In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

Phototolerance induced by narrow-band UVB phototherapy in severe erythropoietic protoporphyria.

Erythropoietic protoporphyria arises from an inherited disorder of porphyrin metabolism which leads to an accumulation of protoporphyrin IX in the erythropoietic system and other tissues. It is characterized by cutaneous photosensitivity, usually difficult to keep under control. Among the scant therapeutic options proposed to reduce photosensitivity in erythropoietic protoporphyria, narrow-band UVB phototherapy has occasionally been used to induce sunlight tolerance. We report an adult case of erythropoietic protoporphyria with a severe photosensitivity treated with narrow-band UVB that developed an appropriate sunlight phototolerance, without adverse events during phototherapy.

An explorative study of non-invasive ultra-weak photon emission and the anti-oxidative influence of oral zinc sulphate in light-sensitive patients with erythropoietic protoporphyria.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare, inherited disorder of haem biosynthesis owing to deficient ferrochelatase (FECH) and accumulation of protoporphyrin IX (PPIX). This results in acute cutaneous photosensitivity upon light exposure with production of reactive oxygen species (ROS) and ultra-weak photon emission (UPE) as a by-product. We investigated if UPE evaluated the light sensitivity in EPP patients and influence of zinc treatment. METHODS: Fourteen EPP patients took zinc sulphate (3 × 200 mg/day) during spring and summer. Using a photomultiplier (PM), UPE was measured from the buttock skin and dorsal hand before and after solar-simulated light (SUN) exposure. Blood samples were analysed routinely for plasma zinc, iron, ferritin, transferrin, haemoglobin, erythrocyte PPIX and Zn-PPIX. RESULTS: UPE in EPP patients resembled that seen in healthy individuals. Without treatment, a seasonal decrease was seen from spring to summer in four control patients. However, oral zinc treatment reduced ROS formation significantly regardless of SUN exposure. After SUN exposure, the initial burst was correlated to plasma iron and erythrocyte PPIX. During treatment, an inverse correlation was found between plasma zinc concentration and the initial burst. CONCLUSION: Measurements of UPE can be used for monitoring UVA-induced oxidative processes in vivo in the skin of EPP patients.

Protoporphyrin retention in hepatocytes and Kupffer cells prevents sclerosing cholangitis in erythropoietic protoporphyria mouse model.

BACKGROUND & AIMS: Chronic, progressive hepatobiliary disease is the most severe complication of erythropoietic protoporphyria (EPP) and can require liver transplantation, although the mechanisms that lead to liver failure are unknown. We characterized protoporphyrin-IX (PPIX)-linked hepatobiliary disease in BALB/c and C57BL/6 (Fechm1Pas) mice with mutations in ferrochelatase as models for EPP. METHODS: Fechm1Pas and wild-type (control) mice were studied at 12-14 weeks of age. PPIX was quantified; its distribution in the liver, serum levels of lipoprotein-X, liver histology, contents of bile salt and cholesterol phospholipids, and expression of genes were compared in mice of the BALB/c and C57BL/6 backgrounds. The in vitro binding affinity of PPIX for bile components was determined. RESULTS: Compared with mice of the C57BL/6 background, BALB/c Fechm1Pas mice had a more severe pattern of cholestasis, fibrosis with portoportal bridging, bile acid regurgitation, sclerosing cholangitis, and hepatolithiasis. In C57BL/6 Fechm1Pas mice, PPIX was sequestrated mainly in the cytosol of hepatocytes and Kupffer cells, whereas, in BALB/c Fechm1Pas mice, PPIX was localized within enlarged bile canaliculi. Livers of C57BL/6 Fechm1Pas mice were protected through a combination of lower efflux of PPIX and reduced synthesis and export of bile acid. CONCLUSIONS: PPIX binds to bile components and disrupts the physiologic equilibrium of phospholipids, bile acids, and cholesterol in bile. This process might be involved in pathogenesis of sclerosing cholangitis from EPP; a better understanding might improve diagnosis and development of reagents to treat or prevent liver failure in patients with EPP.

ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria.

Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.