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.     

Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda.

The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins. Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation. Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type. The excreted porphyrins comprised mainly dehydroisocoproporphyrin. Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme. A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant. The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda.     

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.     

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.     

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.

     

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.     

Oral contraceptives and porphyria cutanea tarda

The clinical and biochemical findings in two cases of porphyria cutanea tarda (PCT) are presented. The cases are unique in their early age of onset (24 and 22 years) and their apparent precipitation by low-dosage oral contraceptives.Review of the literature reveals these to be the two youngest patients with estrogen-induced PCT. Only one other case of PCT induced by low-dosage oral contraceptives has been reported. In most cases of PCT induced by estrogens, high dosages have been taken by patients over age 35.     

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.     

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.     

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.     

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.     

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.     

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.     

Autoimmunity and HCV infection in porphyria cutanea tarda: a controlled study.

Autoimmunity and high rates of autoantibodies have been implicated in the pathogenesis of porphyria cutanea tarda. These abnormalities could be in part virus-induced, since porphyria cutanea tarda in most geographical regions is highly associated with hepatitis C virus infection. We analyzed the link of autoantibodies, autoimmune hepatitis and systemic lupus erythematosus in 111 patients with porphyria cutanea tarda and sex- and age-matched controls (mean age 58+/-13 years) in Germany, a region with a low prevalence of hepatitis C virus infection. Patients with porphyria cutanea tarda displayed lower rates of anti-nuclear antibodies (16/111, 14% vs 28/111, 25%, p<0,05) and of antibodies against smooth muscle (25/111, 23% vs 48/111, 43%, p<0,01), than controls. The percentage of patients with porphyria cutanea tarda with positive anti-HCV was low but significantly higher than in our controls (9/111, 8% vs 0/111, 0%, respectively), (p<0,05). Two patients with porphyria cutanea tarda (2/111, 2%) fulfilled the criteria for systemic lupus erythematosus and not one of 65 patients was found to have clinical autoimmune hepatitis. In the first controlled study of a large cohort of patients with porphyria cutanea tarda no increased prevalence of selected autoantibodies and autoimmune hepatitis was found. However, a higher prevalence of HCV infection and systemic lupus erythematosus in patients with porphyria cutanea tarda was confirmed.