Repurposing ciclopirox as a pharmacological chaperone in a model of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme's stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme's catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease.

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.

A molecular study of congenital erythropoietic porphyria in cattle.

Previous studies have shown that congenital erythropoietic porphyria (CEP) in cattle is caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS) encoded by the UROS gene. In this study, we have established the pedigree of an extended Holstein family in which the disease is segregating in a manner consistent with autosomal recessive inheritance. Biochemical analyses demonstrated accumulation of uroporphyrin, thus confirming that it is indeed insufficient activity of UROS which is the cause of the disease. We have therefore sequenced all nine exons of UROS in affected and non-affected individuals without detecting any potential causative mutations. However, a single nucleotide polymorphism (SNP) located within the spliceosome attachment region in intron 8 of UROS is shown to segregate with the disease allele. Our study supports the hypothesis that CEP in cattle is caused by a mutation affecting UROS; however, additional functional studies are needed to identify the causative mutation.

Mutational analysis of uroporphyrinogen III cosynthase gene in Iranian families with congenital erythropoietic porphyria.

Porphyrias are rare metabolic hereditary diseases originating from defects in specific enzymes involved in the heme biosynthesis pathway. Congenital erythropoietic porphyria (CEP) is the rarest autosomal recessive porphyria resulting from a deficiency of uroporphyrinogen III cosynthase (UROS), the fourth enzyme in heme biosynthesis. CEP leads to an excessive production and accumulation of type Ι porphyrins in bone marrow, skin and several other tissues. Clinical manifestations are presented in childhood with severe cutaneous photosensitivity, blistering, scarring and deformation of the hands and the loss of eyebrows and eyelashes. Less than 200 cases of CEP have been reported to date. Four CEP patients and their family members were studied for the first time in Iran. A missense mutation in the UROS gene was identified in this family. A, T to C change at nucleotide 34313, leading to a substitution of Leucine by Proline at codon 237, was observed in the homozygous state in these 4 patients and heterozygous state in their parents. Our data from the Iranian population emphasizes the importance of codon 237 alone, given the rarity of this disease. This fact can be taken into consideration in the mutational analysis of UROS. This work emphasizes the advantages of molecular genetic techniques as diagnostic tools for the detection of clinically asymptomatic heterozygous mutation carriers as well as CEP within families.

Late onset erythropoietic porphyria (Gunther's disease).

Congenital erythropoietic porphyria (CEP) or Gunther's disease is inherited disorder of porphyrin heme synthetic pathway that usually presents early in life. A very rare form of this disease has its onset in later years of life, called late onset erythropoietic porphyria (late onset EP). Fourteen cases of late onset EP have been reported to-date. We report another case of this rare entity in a 40 years old male with associated findings of haemolysis and thrombocytopenia.

Structural, thermodynamic, and mechanistical studies in uroporphyrinogen III synthase: molecular basis of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal disease ultimately related to deleterious mutations in uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the biosynthetic route of the heme group. UROIIIS catalyzes the cyclization of the linear tetrapyrrol hydroxymethylbilane (HMB), inverting the configuration in one of the aromatic rings. In the absence of the enzyme (or when ill-functioning), HMB spontaneously degrades to the by-product uroporphyrinogen I, which cannot lead to the heme group and accumulates in the body, producing some of the symptoms observed in CEP patients. In the present chapter, clinical, biochemical, and biophysical information has been compiled to provide an integrative view on the molecular basis of CEP. The high-resolution structure of UROIIIS sheds light on the enzyme reaction mechanism while thermodynamic analysis revealed that the protein is thermolabile. Pathogenic missense mutations are found throughout the primary sequence of the enzyme. All but one of these is rarely found in patients, whereas C73R is responsible for more than one-third of the reported cases. Most of the mutant proteins (C73R included) retain partial catalytic activity but the mutations often reduce the enzyme's stability. The stabilization of the protein in vivo is discussed in the context of a new line of intervention to complement existing treatments such as bone marrow transplantation and gene therapy.

Congenital erythropoietic porphyria: characterization of murine models of the severe common (C73R/C73R) and later-onset genotypes.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder due to the deficient activity of uroporphyrinogen III synthase (UROS). Knock-in mouse models were generated for the common, hematologically severe human genotype, C73R/C73R, and milder genotypes (C73R/V99L and V99L/V99L). The specific activities of the UROS enzyme in the livers and erythrocytes of these mice averaged approximately 1.2%, 11% and 19% of normal, respectively. C73R/C73R mice that survived fetal life to weaning age (~12%) had a severe microcytic hypochromic anemia (hemoglobin 7.9 g/dL, mean cellular volume 26.6 fL, mean cellular hemoglobin content 27.4 g/dL, red cell distribution width 37.7%, reticulocytes 19%) and massively accumulated isomer I porphyrins (95, 183 and 44 µmol/L in erythrocytes, spleen and liver, respectively), but a nearly normal lifespan. In adult C73R/C73R mice, spleen and liver weights were 8.2- and 1.5-fold increased, respectively. C73R/V99L mice were mildly anemic (hemoglobin was 14.0 g/dL and mean cellular hemoglobin was 13.3), with minimally accumulated porphyrins (0.10, 5.54 and 0.58 µmol/L in erythrocytes, spleen and liver, respectively), whereas adult V99L/V99L mice were normal. Of note, even the mildest genotype, V99L/V99L, exhibited porphyria in utero, which disappeared by 2 months of age. These severe and mild mouse models inform therapeutic interventions and permit further investigation of the porphyrin-induced hematopathology, which leads to photo-induced cutaneous lesions. Of significance for therapeutic intervention, these mouse models suggest that only 11% of wild-type activity might be needed to reverse the pathology in CEP patients.

Intracellular rescue of the uroporphyrinogen III synthase activity in enzymes carrying the hotspot mutation C73R.

A single mutation (C73R) in the enzyme uroporphyrinogen III synthase (UROIIIS) is responsible for more than one-third of all of the reported cases of the rare autosomal disease congenital erythropoietic porphyria (CEP). CEP patients carrying this hotspot mutation develop a severe phenotype of the disease, including reduced life expectancy. Here, we have investigated the molecular basis for the functional deficit in the mutant enzyme both in vitro and in cellular systems. We show that a Cys in position 73 is not essential for the catalytic activity of the enzyme but its mutation to Arg speeds up the process of irreversible unfolding and aggregation. In the mammalian cell milieu, the mutant protein levels decrease to below the detection limit, whereas wild type UROIIIS can be detected easily. The disparate response is not produced by differences at the level of transcription, and the results with cultured cells and in vitro are consistent with a model where the protein becomes very unstable upon mutation and triggers a degradation mechanism via the proteasome. Mutant protein levels can be restored upon cell treatment with the proteasome inhibitor MG132. The intracellularly recovered C73R-UROIIIS protein shows enzymatic activity, paving the way for a new line of therapeutic intervention in CEP patients.

Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluation.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients. METHODS: RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose. RESULTS: We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34(+) cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo. CONCLUSIONS: The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation.

Feline congenital erythropoietic porphyria: two homozygous UROS missense mutations cause the enzyme deficiency and porphyrin accumulation.

The first feline model of human congenital erythropoietic porphyria (CEP) due to deficient uroporphyrinogen III synthase (URO-synthase) activity was identified by its characteristic clinical phenotype, and confirmed by biochemical and molecular genetic studies. The proband, an adult domestic shorthair cat, had dark-red urine and brownish discolored teeth with red fluorescence under ultraviolet light. Biochemical studies demonstrated markedly increased uroporphyrinogen I in urine and plasma (2,650- and 10,700-fold greater than wild type, respectively), whereas urinary 5-aminolevulinic acid and porphobilinogen were lower than normal. Erythrocytic URO-synthase activity was <1% of mean wild-type activity, confirming the diagnosis and distinguishing it from feline phenocopies having acute intermittent porphyria. Sequencing of the affected cat's UROS gene revealed two missense mutations, c.140C>T (p.S47F) in exon 3 and c.331G>A (p.G111S) in exon 6, both of which were homozygous, presumably owing to parental consanguinity. Neither was present in 100 normal cat alleles. Prokaryotic expression and thermostability studies of the purified monomeric wild-type, p.S47F, p.G111S, and p.S47F/G111S enzymes showed that the p.S47F enzyme had 100% of wild-type specific activity but ~50% decreased thermostability, whereas the p.G111S and p.S47F/G111S enzymes had about 60% and 20% of wild-type specific activity, respectively, and both were markedly thermolabile. Molecular modeling results indicated that the less active/less stable p.G111S enzyme was further functionally impaired by a structural interaction induced by the presence of the S47F substitution. Thus, the synergistic interaction of two rare amino acid substitutions in the URO-synthase polypeptide caused the feline model of human CEP.

Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively spliced transcripts.

Splicing mutations account for approximately 10% of lesions causing genetic diseases, but few branchpoint sequence (BPS) lesions have been reported. In 3 families with autosomal recessive congenital erythropoietic porphyria (CEP) resulting from uroporphyrinogen III synthase (URO-synthase) deficiency, sequencing the promoter, all 10 exons and the intron/exon boundaries did not detect a mutation. Northern analyses of lymphoblast mRNAs from 2 patients and reverse-transcribed polymerase chain reaction (RT-PCR) of lymphoblast mRNAs from all 3 patients revealed multiple longer transcripts involving intron 9 and low levels of wild-type message. Sequencing intron 9 RT-PCR products and genomic DNA in each case revealed homozygosity for a novel BPS mutation (c.661-31T-->G) and alternatively spliced transcripts containing 81, 246, 358, and 523 nucleotides from intron 9. RT-PCR revealed aberrant transcripts in both wild-type and CEP lymphoblasts, whereas BPS mutation reduced the wild-type transcript and enzyme activity in CEP lymphoblasts to approximately 10% and 15% of normal, respectively. Although the +81-nucleotide alternative transcript was in-frame, it only contributed approximately 0.2% of the lymphoblast URO-synthase activity. Thus, the BPS mutation markedly reduced the wild-type transcript and enzyme activity, thereby causing the disease. This is the first BPS mutation in the last intron, presumably accounting for the observed 100% intron retention without exon skipping.

Feline acute intermittent porphyria: a phenocopy masquerading as an erythropoietic porphyria due to dominant and recessive hydroxymethylbilane synthase mutations.

Human acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn error of heme biosynthesis due to the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Here, we describe the first naturally occurring animal model of AIP in four unrelated cat lines who presented phenotypically as congenital erythropoietic porphyria (CEP). Affected cats had erythrodontia, brownish urine, fluorescent bones, and markedly elevated urinary uroporphyrin (URO) and coproporphyrin (COPRO) consistent with CEP. However, their uroporphyrinogen-III-synthase (URO-synthase) activities (deficient in CEP) were normal. Notably, affected cats had half-normal HMB-synthase activities and elevated urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), the deficient enzyme and accumulated metabolites in human AIP. Sequencing the feline HMB-synthase gene revealed different mutations in each line: a duplication (c.189dupT), an in-frame 3 bp deletion (c.842_844delGAG) identical to that causing human AIP and two missense mutations, c.250G>A (p.A84T) and c.445C>T (p.R149W). Prokaryotic expression of mutations c.842_844delGAG and c.445C>T resulted in mutant enzymes with <1% wild-type activity, whereas c.250G>A expressed a stable enzyme with approximately 35% of wild-type activity. The discolored teeth from the affected cats contained markedly elevated URO I and III, accounting for the CEP-like phenocopy. In three lines, the phenotype was an autosomal dominant trait, while affected cats with the c.250G>A (p.A84T) mutation were homozygous, a unique recessive form of AIP. These animal models may permit further investigation of the pathogenesis of the acute, life-threatening neurological attacks in human AIP and the evaluation of therapeutic strategies. GenBank Accession Numbers: GQ850461-GQ850464.

Congenital erythropoietic porphyria: mutation update and correlations between genotype and phenotype.

High quality genotype/phenotype analysis is a difficult issue in rare genetic diseases such as congenital erythropoietic porphyria (CEP) or Günther's disease, a heme biosynthesis defect due to uroporphyrinogen III synthase deficiency. The historical background and the main phenotypic features of the disease are depicted together with an update of published mutants and genotype/phenotype correlations. General rules concerning the prediction of disease severity are drawn as a guide for patient management and therapeutic choices. The phenotypic heterogeneity of the disease is presented in relation with a likely influence of modifying factors, either genetic or acquired.

Uroporphyrinogen III synthase mutations related to congenital erythropoietic porphyria identify a key helix for protein stability.

In the present study we have investigated deleterious mutants in the uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutants found in CEP patients have been cloned, expressed, and purified. Their enzymatic activities have been measured relative to wild-type UROIIIS activity. All mutants retain measurable activity, consistent with the recessive character of the disease. Most of the mutants with a significant decrease in activity involve residues likely associated in binding. However, other mutants are fully active, indicating that different mechanisms may contribute to enzyme missfunction. UROIIIS is a thermolabile enzyme undergoing irreversible denaturation. The unfolding kinetics of wild-type UROIIIS and the suite of mutants have been monitored by circular dichroism. This analysis allowed the identification of a helical region in the molecule, essential to retain the kinetic stability of the folded conformation. C73R is found in one-third of CEP patients, and Cys73 is part of this helix. The integrated analysis of the enzymatic activity and kinetic stability data is used to gain insight in the relationship between defects in UROIIIS sequence and CEP.

Biochemical and molecular diagnosis of erythropoietic protoporphyria in an Ashkenazi Jewish family.

Erythropoietic protoporphyria (EPP) is a rare hereditary disorder due to a partial deficiency of ferrochelatase (FECH). The genotype of EPP patients features a mutation on one allele of the FECH gene and a common hypomorphic FECH IVS3-48c on the other allele (M/c). The resulting enzyme activity in patients is ∼35% of that in normal individuals. Ferrochelatase deficiency results in the accumulation of protoporphyrin in the skin, which is responsible for the clinical symptom of cutaneous photosensitivity in patients. In this study, we report the identification of a novel FECH mutation delT23 in an 11-member EPP family of Jewish origin. Two EPP siblings shared an identical genotype of delT23/IVS3-48c (M/c). They were both photosensitive and showed highly increased erythrocyte protoporphyrin. The genotype of the patients' mother, who did not present with any EPP clinical symptoms, was delT23/IVS3-48t (M/t). The patients' father, an offspring of consanguineous parents, was homozygous IVS3-48 c/c. He exhibited a mild photosensitivity, and an increase of 4-fold in erythrocyte protoporphyrin. His FECH mRNA amount was 71% of that of genotype t/t. It is the first reported case of an individual with c/c genotype who exhibits both biochemical and clinical indications of EPP. These results suggest that IVS3-48c is a functional variant of ferrochelatase. The clinical symptoms and biochemical abnormalities in the patients' father could be the result of an interaction between genetic and environmental factors. In addition, the frequency of IVS3-48c in the Ashkenazi Jewish population was estimated at 8%, which is similar to that in the European populations.

Uroporphyrinogen III synthase knock-in mice have the human congenital erythropoietic porphyria phenotype, including the characteristic light-induced cutaneous lesions.

Congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error, results from the deficient but not absent activity of uroporphyrinogen III synthase (URO-synthase), the fourth enzyme in the heme biosynthetic pathway. The major clinical manifestations include severe anemia, erythrodontia, and disfiguring cutaneous involvement due to the accumulation of phototoxic porphyrin I isomers. Murine models of CEP could facilitate studies of disease pathogenesis and the evaluation of therapeutic endeavors. However, URO-synthase null mice were early embryonic lethals. Therefore, knock-in mice were generated with three missense mutations, C73R, V99A, and V99L, which had in vitro-expressed activities of 0.24%, 5.9%, and 14.8% of expressed wild-type activity, respectively. Homozygous mice for all three mutations were fetal lethals, except for mice homozygous for a spontaneous recombinant allele, V99A(T)/V99A(T), a head-to-tail concatemer of three V99A targeting constructs. Although V99A(T)/V99A(T) and C73R/V99A(T) mice had approximately 2% hepatic URO-synthase activity and normal hepatic microsomal heme and hemoprotein levels, they had 20% and 13% of wild-type activity in erythrocytes, respectively, which indicates that sufficient erythroid URO-synthase was present for fetal development and survival. Both murine genotypes showed marked porphyrin I isomer accumulation in erythrocytes, bone, tissues, and excreta and had fluorescent erythrodontia, hemolytic anemia with reticulocytosis and extramedullary erythropoiesis, and, notably, the characteristic light-induced cutaneous involvement. These mice provide insight into why CEP is an erythroid porphyria, and they should facilitate studies of the disease pathogenesis and therapeutic endeavors for CEP.

A knock-in mouse model of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a recessive autosomal disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. The severity of the disease, the lack of specific treatment except for allogeneic bone marrow transplantation, and the knowledge of the molecular lesions are strong arguments for gene therapy. An animal model of CEP has been designed to evaluate the feasibility of retroviral gene transfer in hematopoietic stem cells. We have previously demonstrated that the knockout of the Uros gene is lethal in mice (Uros(del) model). This work describes the achievement of a knock-in model, which reproduces a mutation of the UROS gene responsible for a severe UROS deficiency in humans (P248Q missense mutant). Homozygous mice display erythrodontia, moderate photosensitivity, hepatosplenomegaly, and hemolytic anemia. Uroporphyrin (99% type I isomer) accumulates in urine. Total porphyrins are increased in erythrocytes and feces, while Uros enzymatic activity is below 1% of the normal level in the different tissues analyzed. These pathological findings closely mimic the CEP disease in humans and demonstrate that the Uros(mut248) mouse represents a suitable model of the human disease for pathophysiological, pharmaceutical, and therapeutic purposes.

Two brothers with mild congenital erythropoietic porphyria due to a novel genotype.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease caused by the deficient activity of the heme biosynthetic enzyme, uroporphyrinogen III synthase (URO-synthase), and the accumulation of the nonphysiologic and phototoxic porphyrin I isomers. Clinical manifestations range from severe mutilation to mild erosions and blisters on sun-exposed areas. Evaluation of the URO-synthase mutation and residual enzyme activity has been correlated with the phenotypic expression of the disease. OBSERVATIONS: We describe 16- and 4-year-old brothers with CEP with a mild phenotype due to a novel genotype, one allele having a promoter mutation (-76G-->A) and the other having an exonic missense mutation (G225S). The father and a 4-year-old fraternal twin brother were carriers of the -76G-->A mutation, whereas the mother and a 15-year-old brother were carriers of the G225S mutation. Previous in vitro expression studies demonstrated that the G225S mutation severely decreased URO-synthase activity to 1.2% of normal, whereas the promoter mutation decreased the activity to approximately 50% of wild type, accounting for the mild clinical phenotype. CONCLUSION: The mild disease phenotype in these patients is a further example of the clinical heterogeneity seen in CEP and is additional proof that in vitro enzyme expression studies provide dependable genotype-phenotype correlations.