Uroporphyrinogen decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria.

A deficiency in uroporphyrinogen decarboxylase (UROD) enzyme activity, the fifth enzyme of the heme biosynthetic pathway, is found in patients with sporadic porphyria cutanea tarda (s-PCT), familial porphyria cutanea tarda (f-PCT), and hepatoerythropoietic porphyria (HEP). Subnormal UROD activity is due to mutations of the UROD gene in both f-PCT and HEP, but no mutations have been found in s-PCT. Genetic analysis has determined that f-PCT is transmitted as an autosomal dominant trait. In contrast, HEP, a severe form of cutaneous porphyria, is transmitted as an autosomal recessive trait. HEP is characterized by a profound deficiency of UROD activity, and the disease is usually manifest in childhood. In this study, a strategy was designed to identify alleles responsible for the HEP phenotype in three unrelated families. Mutations of UROD were identified by direct sequencing of four amplified fragments that contained the entire coding sequence of the UROD gene. Two new missense mutations were observed at the homoallelic state: P62L (proline-to-leucine substitution at codon 62) in a Portuguese family and Y311C (tyrosine-to-cysteine substitution at codon 311) in an Italian family. A third mutation, G281E, was observed in a Spanish family. This mutation has been previously described in three families from Spain and one from Tunisia. In the Spanish family described in this report, a paternal uncle of the proband developed clinically overt PCT as an adult and proved to be heterozygous for the G281E mutation. Mutant cDNAs corresponding to the P62L and Y311C changes detected in these families were created by site-directed mutagenesis. Recombinant proteins proved to have subnormal enzyme activity, and the Y311C mutant was thermolabile.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

Characterization of a new mutation (R292G) and a deletion at the human uroporphyrinogen decarboxylase locus in two patients with hepatoerythropoietic porphyria

Summary A deficiency in the activity of uroporphyrinogen decarboxylase (UROD), the fifth enzyme of the haem biosynthetic pathway, is found in familial porphyria cutanea tarda (F-PCT) and hepatoerythropoietic porphyria (HEP). A new mutation (R292G) and a deletion have been found in a pedigree with two HEP patients (two sisters). The R292G mutation was not detected in 13 unrelated affected patients with F-PCT, so it appears to be uncommon. The possibility that the arginine 292 may participate at the active site of the enzyme is discussed. A summary of the 7 mutations/deletions found in the UROD gene with their frequency is presented.     

The role of iron in experimental porphyria and porphyria cutanea tarda

Porphyria cutanea tarda (PCT) and experimental porphyria are characterized by a decreased activity of the enzyme uroporphyrinogen decarboxylase, and accumulation of uroporphyrins and heptacarboxylporphyrins in the liver. Iron (Fe) plays an important role in PCT and experimental porphyria. Biochemically and electron microscopically, we examined the relationship between Fe and porphyrins in liver tissue of C57BL/10 mice made porphyric by administration of iron dextran as Imferon^® (IMF), and in liver biopsies of patients with symptomatic PCT. Accumulation of uroporphyrins and heptacarboxylporphyrins, and an increased amount of Fe were observed in livers of mice treated with IMF and in liver biopsies of patients with PCT. In mice treated with IMF, the activity of uroporphyrinogen decarboxylase was decreased. Both in livers of mice treated with IMF and in livers of patients with PCT, needle-like structures, representing uroporphyrin crystals, were observed by electron microscopy. Uroporphyrin crystals and Fe (as ferritin) were observed in the same hepatocyte. Moreover, there was a striking morphological correlation between uroporphyrin crystals and ferritin-Fe, suggesting a role for (ferritin-)Fe in the pathogenesis of porphyria.     

Familial and sporadic porphyria cutanea

Summary Uroporphyrinogen (URO) decarboxylase was measured in hemoglobin-free erythrocytes from subjects with familial porphyria cutanea: the mean activity was about 50% of that found in erythrocytes from normal subjects. Asymptomatic carriers were always found in the family. No enzyme deficiency was found in erythrocytes from subjects with sporadic porphyria cutanea. The measurement of URO decarboxylase in erythrocytes seems to allow an easy distinction between these two groups of porphyria cutanea.     

Haem biosynthesis and human porphyria cutanea tarda: effects of alcohol intake

The review describes the structural and biochemical properties of the haem biosynthetic enzyme, uroporphyrinogen decarboxylase (UROD), which sequentially catalyzes the removal of the four carboxyl groups from the acetate side chains of octacarboxylic uroporphyrinogen to form coproporphyrinogen, and the possible biochemical mechanism of the genesis of porphyria cutanea tarda (PCT). The disease is caused when the activity of UROD is significantly reduced. PCT is a multifactorial disease where both inherent and environmental factors such as alcohol, estrogens, halogenated aromatic hydrocarbons and viral infection (mainly hepatitis C) are involved in biochemical and clinical expression. In PCT, hepatic iron plays a key role. Alcohol intake could induce mobilization of iron from protein-bound ferritin. PCT should be managed by avoidance of these toxins and removal of iron by vigorous phlebotomy. Such iron-reduction therapy would provide additional benefit for hepatitis C patients by interferon therapy.     

Volatile anaesthetics induce biochemical alterations in the heme pathway in a B-lymphocyte cell line established from hepatoerythropoietic porphyria patients (LBHEP) and in mice inoculated with LBHEP cells

Hepatoerythropoietic porphyria (HEP) is the homozygous form of Porphyria Cutanea Tarda (PCT), characterized by an accumulation of porphyrins due to uroporphyrinogen decarboxylase deficiency.Fluorinated volatile anaesthetics are often used to produce general anaesthesia. Anaesthesia has certainly been implicated in the triggering of acute porphyria crisis.The effects of volatile anaesthetics in a B-lymphocyte cell line established from HEP patients (LBHEP) on heme metabolism have been investigated.LBHEP cells were exposed to sodium phosphate buffer containing dissolved Enflurane, Isoflurane or Sevoflurane (10mM) during 20min.Aminolevulinate synthase (ALA-S) activity, the regulatory enzyme of heme synthesis, was 300% induced by the anaesthetics. A 25-30% diminution of porphobilinogenase (PBG-ase) activity was found when Isoflurane or Sevoflurane were added to the cells, while no significant changes were detected after Enflurane treatment.Although some oxidative stress has been induced by the anaesthetics, reflected by the 35% diminution of glutathione (GSH), no alteration in heme oxygenase (HO) activity, the enzyme involved in heme breakdown and frequently induced as a response to stress stimuli, was observed.Studies using animals inoculated with LBHEP cells were also performed. Findings here described mimic biochemical alterations in the heme pathway, which are characteristic of another hepatic porphyria, similar to those previously reported when these anaesthetics were administered to animals, and they also advertise about the possible unsafe use of these drugs in the case of hepatic non-acute porphyrias.     

Defective human erythrocyte uroporphyrinogen decarboxylase in familial porphyria cutanea tarda: the metabolic lesion or the result of endogenous porphyrinemia?

We have demonstrated that oral charcoal therapy is as effective as therapeutic phlebotomy in reducing porphyrinemia in porphyria cutanea tarda. The effects of immediate and sustained reduction of porphyrinemia on the catalytic properties of partially purified (approximately 200-fold) preparations of red cell uroporphyrinogen decarboxylase of a patient with familial porphyria cutanea tarda were studied. All populations of the patient's red cells exhibited defective enzyme activity, and the apparent Michaelis constants (Km) determined with penta-, hepta-, and octa-carboxylic I porphyrinogen substrates were approximately 3-4 times higher as compared to the normal controls. Mixing experiments (normal and defective enzyme), and preincubation of the normal enzyme with porphyric plasma prior to purification, yielded data supporting the concept that the catalytic defects of red cell uroporphyrinogen decarboxylase in familial porphyria cutanea tarda are independent of interactions between circulating endogenous porphyrins and the enzyme.     

Levels of uroporphyrinogen decarboxylase (URO-D) in erythrocytes of Italian porphyria cutanea tarda patients.

Porphyria cutanea tarda (PCT) is a human metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen decarboxylase (URO-D) activity, the fifth enzyme of the heme biosynthetic pathway. A classification of inherited and non-inherited forms is based on the enzyme activity levels in red blood cells (RBC). Clinical manifestations of PCT are often precipitated by triggering factors such as alcohol, drug abuse, estrogens, virus infections, hepatotoxic chemicals and hepatic siderosis. We measured URO-D activity in RBC from a large sample of Italian PCT patients in order to define the enzyme activity distribution and to attempt a correlation among activity, risk factors and clinical outcome. Three classes of patients with low, normal and over-normal URO-D activity were defined according to control values. Low URO-D levels were present in 25.8% of patients, suggesting the familial form of PCT (type II). In this group, the outcome of PCT seems to be less influenced by risk factors. Patients with over-normal URO-D activity in RBC deserve further investigation.     

Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.     

Precipitating/aggravating factors of porphyria cutanea tarda in Spanish patients.

Erythrocyte uroporphyrinogen decarboxylase (UROD) activity was measured to classify 118 Spanish patients with porphyria cutanea tarda (PCT) into three subtypes: sporadic-, familial- and type III-PCT. Seventy-four patients (63%) had eythrocyte UROD activity within the normal range (74% to 126% of the mean activity of 43 healthy controls) and were classified as sporadic-PCT (47%) or as type III-PCT (16%) whenever a family history of PCT was documented. Forty-four patients (37%) had decreased UROD activity and were classified as familial-PCT. The frequency of both familial-PCT and type III-PCT was higher than reported in other countries. The clinical expression of PCT was associated with the coexistence of two or more risk factors in 80% of the sporadic-PCT patients and in 89% of the familial-PCT patients. Hepatitis C virus and alcohol abuse were risk factors frequently found in these patients, being unrelated to age of onset of skin lesions. A heavy alcohol intake was the main risk factor for type III-PCT. Estrogens appeared as a precipitating factor for women with familial-PCT. The H63D mutation in the hemochromatosis type 1 gene was more frequently found than the C282Y mutation. Both mutations appeared to play a role as precipitating factors in sporadic-PCT when associated with hepatitis C virus infection and alcohol abuse.     

Development of chronic hepatic porphyria (porphyria cutanea tarda) with inherited uroporphyrinogen decarboxylase deficiency under exposure to dioxin

• 1.1. Exposure to dioxin triggered a clinically manifest chronic hepatic porphyria (porphyria cutanea tarda) in two patients (brother and sister) with hereditary uroporphyrinogen decarboxylase deficiency.
• 2.2. The patients showed a decrease of erythrocyte uroporphyrinogen decarboxylase activity to ~ 50% of controls even in reinvestigations after three years, whereas clinical symptoms and porphyrinuria had improved considerably. Only a subclinical phase of chronic hepatic porphyria persisted. Subnormal uroporphyrinogen decarboxylase activity could be determined in altogether nine family members.
• 3.3. The remission of porphyria cutanea tarda into a subclinical phase occurred after chloroquine therapy. Subclinical phases of chronic hepatic porphyria (type A) in other family members remitted without special therapy.
• 4.4. Among the 60 persons dioxin-exposed by the Seveso accident, a secondary coproporphyrinuria was found in 22% of examined patients with transition to a subclinical chronic hepatic porphyria in 5 cases. The changes had subsided completely after one year. A persistence of the transition state in 3 cases is probably due to alcohol influence. None of these cases developed a porphyria cutanea tarda.
• 5.5. The investigations showed that a hereditary disposition is necessary for biochemical and clinical expression of chronic hepatic porphyria after a unique dioxin exposure. This is not given in the sporadic cases: after a unique dioxin exposure they indeed develop a symptomatic disturbance of porphyrin metabolism but not a clinically relevant chronic hepatic porphyria.
• 6.6. We conclude that a unique acute exposure to dioxin can trigger the chronic hepatic porphyria disease process in persons with an underlying genetic abnormality of uroporphyrinogen decarboxylase.

     

Immunoreactive uroporphyrinogen decarboxylase is unchanged in porphyria caused by TCDD and hexachlorobenzene

A method has been developed for the immuno-titration of rodent liver uroporphyrinogen decarboxylase (porphyrinogen carboxy-lyase, EC 4.1.1.37) and used to show that two porphyrogenic polyhalogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin and hexachlorobenzene, cause porphyria in rodents by decreasing the catalytic activity of uroporphyrinogen decarboxylase without altering the amount of immunoreactive enzyme protein. Investigation of the nature of the inactive form of uroporphyrinogen decarboxylase produced by these compounds should provide new information about the mechanism of their toxicity.     

Study of the origin of urinary porphyrins in experimental hexachlorobenzene-induced porphyria

The activity of the enzyme uroporphyrinogen decarboxylase was determined in the liver and the kidneys of C57BL/6 mice and Wistar albino rats with chronic hexachlorobenzene intoxication and the amount of the deposited uroporphyrin was measured in the both organs. In the control animals the activity of hepatic uroporphyrinogen decarboxylase was several times higher than the renal one. The administration of hexachlorobenzene led to an inhibition of the enzyme activity, which was equally expressed (about 2.5 times) in the liver and kidneys of the both species. The accumulation of uroporphyrin was more pronounced in the hepatic tissue than in the kidneys (about 9 times in mice and 5 times in rats on average). Taking into consideration the much higher uroporphyrin accumulation in the liver, the more active haem biosynthesis in this organ, as well as its larger size, one could accept that the predominant part of the urinary porphyrins in hexachlorobenzene porphyria has a hepatic and not a renal origin.     

Genetic studies of human apolipoproteins. VII. Population distribution of polymorphisms of apolipoproteins A-I, A-II, A-IV, C-II, E, and H in Nigeria.

Familial porphyria cutanea tarda (PCT) results from a generalized deficiency of uroporphyrinogen decarboxylase (URO-D) activity. The molecular defect responsible for this disorder has not been characterized. To determine whether decreased levels of URO-D mRNA are responsible for subnormal URO-D activity, steady-state levels of URO-D mRNA in lymphoblastoid cells were determined. Northern blots were hybridized with a URO-D cDNA probe and quantified by densitometry. No difference in the levels of URO-D mRNA was detected between affected individuals and their normal relatives. Thus, the deficiency of URO-D activity in two familial PCT pedigrees characterized here does not arise from a deficiency of URO-D mRNA.     

CYP1A2*1F and GSTM1 alleles are associated with susceptibility to porphyria cutanea tarda

Porphyria cutanea tarda (PCT) is a cutaneous porphyria with sporadic (type 1) and familial (type 2) subtypes, both resulting from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. Environmental and genetic factors are involved in the development of PCT, and genetic variants in the cytochrome P450 (CYP ) genes, CYP1A1 and CYP1A2, have been implicated. We investigated the association between PCT and variants in CYP1A1, CYP1A2 and CYP2E1, and the glutathione-S-transferase (GST ) genes, GSTM1 and GSTT1. PCT diagnosis was based on urinary or plasma porphyrin profiles. Patients were classified as type 1 or 2 PCT based on UROD mutation analysis. The CYP1A2*1F promoter A allele frequency was significantly higher (P < 0.022) and the A/A genotype frequency marginally higher in PCT patients overall (P < 0.057), with the A/A genotype significantly more common in type 1 PCT (P < 0.043). The presence of the wild-type GSTM1 allele also was associated significantly with PCT (P < 0.019). Neither hemochromatosis (HFE) mutations, tobacco smoking, hepatitis C and HIV infection, ethanol consumption, nor estrogen use were associated with these allelic variants. Age at onset was significantly lower in type 2 PCT patients (P < 0.001), as observed previously. Thus, positive associations between PCT and the CYP1A2*1F promoter A allele and A/A genotype and the wild-type GSTM1 allele indicates that these functional hepatic biotransformation enzymes are risk factors for the development of this disease.     

Hemochromatosis gene sequence deviations in German patients with porphyria cutanea tarda

Patients with porphyria cutanea tarda (PCT) reveal a susceptibility to reversible inactivation of hepatic uroporphyrinogen decarboxylase, which might be triggered by alcohol, hepatitis C virus infection, and iron overload. Inherited factors that may predispose to clinically overt PCT also include sequence deviations in the HFE gene that is mutated in classical hemochromatosis. Here, we studied the prevalence of both common and rare hemochromatosis gene variations in 51 PCT patients and 54 healthy controls of German origin. The frequency of the common HFE gene mutation C282Y was 15.7 % in PCT patients and 2.8 % in healthy control individuals (P < 0.001). By contrast, the frequencies of the common H63D mutation did not differ, and the allele frequencies of the less frequently observed sequence deviations as substitution S65C in the HFE gene and mutation Y250X in the TFR2 gene underlying hemochromatosis type 3 (HFE3) were < 0.02 both in PCT patients and controls. Our results comprise the first molecular studies of both common and rare hemochromatosis gene variants in German PCT patients, indicating a significant role of the C282Y mutation in the pathogenesis of PCT.     

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.     

Genetic heterogeneity in acute intermittent porphyria: characterisation and frequency of porphobilinogen deaminase mutations in Finland.

The occurrence of different porphobilinogen deaminase mutant types in 68 patients with acute intermittent porphyria from 33 unrelated families in Finland was studied with biochemical and immunological techniques. In this fairly homogenous population four different porphobilinogen deaminase mutant types were identified and their frequencies determined. Most (about 80%) of the mutations were cross reacting immunological material (CRIM) negative, including a large kindred with normal erythrocyte porphobilinogen deaminase activities. The remainder of the families had CRIM positive mutations, including an unusual type (type 2) that had an immunoreactive, non-catalytic porphobilinogen deaminase level considerably greater than the maximal theoretical ratio of CRIM to activity of 2.0 for a single mutant allele. Correlations of the amount of residual porphobilinogen deaminase activity and the occurrence of acute clinical manifestations in each mutant type suggested that CRIM positive type 2 patients may have fewer acute symptoms.     

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker''s yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.