Identification of mutations in the uroporphyrinogen III cosynthase gene in German patients with congenital erythropoietic porphyria

The porphyrias are heterogeneous disorders arising from predominantly inherited catalytic deficiencies of specific enzymes along the heme biosynthetic pathway. Congenital erythropoietic porphyria is a very rare disease that is inherited as an autosomal recessive trait and results from a profound deficiency of uroporphyrinogen III cosynthase, the fourth enzyme in heme biosynthesis. The degree of severity of clinical symptoms mainly depends on the amount of residual uroporphyrinogen III cosynthase activity. In this study, we sought to characterize the molecular basis of congenital erythropoietic porphyria in Germany by studying four patients with congenital erythropoietic porphyria and their families. Using PCR-based techniques, we identified four different mutations: C73R, a well-known hotspot mutation, the promoter mutation -86A that was also described previously, and two novel missense mutations, designated G236V and L237P, the latter one encountered in the homozygous state in one of the patients. Our data from the German population further emphasize the molecular heterogeneity of congenital erythropoietic porphyria as well as the advantages of molecular genetic techniques as a diagnostic tool and for the detection of clinically asymptomatic heterozygous mutation carriers within families.     

Heterogeneity of mutations in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria

Summary Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. We have previously described two missense mutations in the UROIIIS gene, confirming that the primary defect responsible for CEP is a structural alteration of this gene. We have extended our work to 5 additional unrelated families. Two new point mutations, a deletion and an insertion have been found in the messenger RNA. Our study shows that a molecular heterogeneity of the mutations exists in Günther's disease. One mutation (C73R), however, appears to be more frequent than the others. Finally, the different normal and mutated proteins have been expressed in Escherichia coli to determine the consequence of the mutations on the enzyme activity.     

Successful gene therapy of mice with congenital erythropoietic porphyria

Porphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by a deficiency in uroporphyrinogen III synthase (UROS) activity. Bone marrow transplantation represents a curative treatment for patients, as long as human leucocyte antigen-compatible donor is available. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut 248) mice resulted in a complete and long-term enzymatic, metabolic and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate for the first time that the cure of this mouse model of CEP at moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified HSCs.     

Uroporphyrinogen III cosynthetase in asymptomatic carriers of congenital erythropoietic porphyria

Mean activity of the enzyme uroporphyrinogen III cosynthetase in hemolysates from five asymptomatic carriers of bovine erythropoietic porphyria was intermediate to the means of the activities in normal controls and affected animals. Similarly, mean cosynthetase activity in hemolysates from eight presumed carriers of human congenital erythropoietic porphyria was lower than the mean for 38 nonporphyric individuals and higher than the activity in hemolysates from patients. These results are consistent with the known autosomal recessive mode of inheritance of the disorder in cattle and the presumed autosomal recessive inheritance in man. The activity of cosynthetase is higher in young red cells than in older ones, which explains the relatively high enzyme activities in blood from human patients with nonporphyric hemolytic disorders accompanied by reticulocytosis. It also explains why cosynthetase activity is higher in newborn porphyric calves than in older porphyric animals, because the former have a period of accelerated erythropoiesis and a high percentage of very young red cells in the circulation.Supported in part by U.S. Public Health Service Grants NB-05367 and AM-10858.     

Mapping the uroporphyrinogen III cosynthase locus in Bacillus subtilis.

Summary The gene hemD taking part in the formation of uroporphyrinogen III from porphobilinogen was mapped by two-and three-factor transduction crosses in Bacillus subtilis. This gene codes uroporphyrinogen III cosynthase. The gene hemD is linked to the hemA locus and is located between the hemA and pheA loci.     

Uroporphyrinogen III cosynthetase activity in fibroblasts from patients with congenital erythropoietic porphyria

The activity of uroporphyrinogen III cosynthetase is lower in extracts of fibroblasts from patients with congenital erythropoietic porphyria than in extracts of fibroblasts from control subjects. The porphyric extracts do not inhibit the cosynthetase activity of control extracts. The genetically determined enzymatic defect in this disease can thus occur in other tissues besides bone marrow.Supported in part by a National Institutes of Health Grant (NB-05367).     

Congenital erythropoietic porphyria with two mutations of the uroporphyrinogen III synthase gene (Cys73Arg, Thr228Met)

Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of uroporphyrinogen III synthase (UROS). We describe a 14-year-old girl with red urine since infancy, progressive blistering and scarring of the skin, and moderate hemolytic anemia. After years of skin damage, her face is mutilated; she has a bald patch on the scalp, hypertrichosis of the neck, areas of skin darkening, and limited joint movements of the hands. Total urine excretion and fecal total porphyrin were both markedly raised above normal levels. Sequencing of the UROS gene identified two mutations causing CEP (Cys73Arg, Thr228Met). The patient lesions are progressing. Bone marrow transplantation and/or gene therapy are proposed as the next steps in her treatment. In brief, we describe a CEP with confirmed two pathogenic mutations, severe phenotype and discuss the various treatment options available.     

Coupled-enzyme and direct assays for uroporphyrinogen III synthase activity in human erythrocytes and cultured lymphoblasts: Enzymatic diagnosis of heterozygotes and homozygotes with congenital erythropoietic porphyria

Rapid and reproducible assays for uroporphyrinogen III synthase (URO-S; EC 4.2.1.75) have been developed and used to determine the enzymatic activity in human erythrocytes and cultured lymphoid cells. In the coupled-enzyme assay, porphobilinogen was first converted to hydroxymethylbilane, the natural substrate for URO-S, by hydroxymethylbilane synthase which was conveniently obtained from heat-treated erythrocyte lysates. In the direct assay, synthetic hydroxymethylbilane was used as substrate. In both assays, the uroporphyrinogen reaction products were oxidized to their respective uroporphyrin isomers, which were then resolved and quantitated by reversed-phase high-pressure liquid chromatography. Both assays were optimized for pH, substrate concentration, and linearity with time and protein concentration. The mean URO-S activities in normal human erythrocyte lysates determined by the coupled-enzyme and direct assays were 7.41 ± 1.35 and 7.64 ± 1.73 units/mg protein, respectively. In normal human cultured lymphoid cells, the mean activities were 13.7 ± 1.39 and 17.6 ± 1.15 units/mg protein for the coupled-enzyme and direct assays, respectively. In four families with congenital erythropoietic porphyria, both assays reliably identified the markedly decreased URO-S activities in erythrocytes and cultured lymphoid cells from affected homozygotes and the half-normal activities in these sources from obligate heterozygotes. The coupled-enzyme assay was easier to perform and was suited for clinical diagnostic assays and for monitoring enzyme purification procedures, while the direct assay, which required substrate preparation and technical dexterity, was best for kinetic studies of URO-S.     

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.     

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.     

Mutational analysis of gene families in human cancer

The completion of the human genome project has marked a new beginning in biomedical sciences. Human cancer is a genetic disease and, accordingly, the field of oncology has been one of the first to be impacted by this historic revolution. Knowledge of the sequence and organization of the human genome facilitates the systematic analysis of the genetic alterations underlying the origin and evolution of tumors. Recent mutational analyses in colorectal and other cancers have focused on examination of gene families involved in signal transduction, such as kinases and phosphatases. This approach has been successful in identifying mutations in a variety of different genes, including the identification of PI3KCA as one of the most commonly mutated oncogenes in human cancer. Such genomic analyses have already demonstrated their utility in basic and clinical cancer research, and are expected to have an important impact on future diagnostic and therapeutic strategies.     

Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.     

Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene

The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther''s disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref {"type":"entrez-protein","attrs":{"text":"NP_000366.1","term_id":"4557873","term_text":"NP_000366.1"}}NP_000366.1, {"type":"entrez-nucleotide","attrs":{"text":"NM_000375.2","term_id":"171543867","term_text":"NM_000375.2"}}NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14^th Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.     

Decarboxylation of uroporphyrinogen I and III in 2,3,7,8-tetrachlorodibenzo-p-dioxin induced porphyria in mice

• 1.1. The decarboxylation of uroporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in mouse liver supernatant was compared in relation to substrate concentrations.
• 2.2. In this species uroporphyrinogen III was the best substrate judging by the criteria of K_m/V_max (estimated for total porphyrinogens) and was converted into coproporphyrinogen faster than its series I isomer.
• 3.3. The difference between the two isomers was mainly due to the first decarboxylation.
• 4.4. This difference was confirmed by calculation of the Hill coefficient and of Lineweaver-Burk plot which suggested that isomer I induced negative cooperativity in the active centre of the enzyme.
• 5.5. After treatment with a porphyrogenic dose of TCDD (25 μg/kg/week for 9 weeks) differences between uroporphyrinogen I and III as substrate were maintained.
• 6.6. In addition treatment reduced V_max and K_m (estimated for total porphyrinogens) of liver porphyrinogen carboxy-lyase to about half control values for both isomers.
• 7.7. V_max was reduced mainly because of the formation of smaller amounts of all products of decarboxylation, and K_m because more heptaporphyrinogen was formed than coproporphyrinogen.
• 8.8. Values of the Hill coefficient and Lineweaver-Burk plots suggested TCDD induced altered substrate affinity for isomer III too.
• 9.9. Treatment with TCDD did not affect the decarboxylation of uroporphyrinogen III by RBC porphyrinogen carboxy-lyase, estimated from K_m and V_max for total porphyrinogens formed.

     

Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. 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 used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros^mut248 mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.     

Identification of meso-hydroxyuroporphyrin I in the urine of a patient with congenital erythropoietic porphyria.

A new porphyrin, meso-hydroxyuroporphyrin I, was isolated from the urine of a patient with congenital erythropoietic porphyria. The structure was characterized as the methyl ester, ethyl ester and acetoxy derivatives by fast-atom-bombardment m.s., by conversion into uroporphyrin I and by chemical synthesis.     

A novel point mutation in congenital erythropoietic porphyria in two members of Japanese family

The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in two members of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Günther’s disease. The first patient was homoallelic for a novel missense mutation: a T to C transition of nucleotide 634 that predicted a serine to proline substitution at residue 212 (S212P). The second patient appeared heteroallelic, carrying the same missense mutation and a nonsense mutation: a C to T change at nucleotide 745, resulting in a premature stop at codon 249, instead of a glutamine (Q249X). The corresponding mutated proteins were expressed in Escherichia coli and no residual activity was observed. A family study was also performed to determine the carrier status. Received: 18 July 1995