Heterogeneity of familial porphyria cutanea tarda.

The concentration of immunoreactive uroporphyrinogen decarboxylase has been measured in erythrocytes from 17 patients with porphyria cutanea tarda (PCT) from 10 families, from 74 of their relatives, and from 47 control subjects. The 10 families were divided into two groups according to their erythrocyte enzyme concentrations. Group A contained four families in which at least two subjects had overt PCT. All members of these families, including seven patients with overt PCT, had normal erythrocyte uroporphyrinogen decarboxylase concentrations and activities. Apart from their family history, patients in group A were clinically and biochemically indistinguishable from cases of type I (sporadic) PCT. Group B contained six families with the only previously described form of familial PCT (type II PCT) in which decreased erythrocyte uroporphyrinogen decarboxylase segregates as an autosomal dominant trait. These findings show that familial PCT is heterogeneous and suggest that inheritance contributes to the pathogenesis of at least some cases of type I PCT.     

Hepatoerythropoietic porphyria: clinical, biochemical, and enzymatic studies in a three-generation family lineage

Hepatoerythropoietic porphyria is caused by a marked deficiency in the activity of uroporphyrinogen decarboxylase, an enzyme that is essential for heme biosynthesis. It has been hypothesized that uroporphyrinogen decarboxylase deficiency is inherited as a homozygous defect in the disease. This suggestion has been supported by reports of a deficiency of the enzyme in parents of patients with the disorder. Further confirmation would be provided by demonstrating a similar uroporphyrinogen decarboxylase deficiency in the offspring of such patients. This study follows the enzymatic defect throughout three generations of a family in which a second-generation male was shown to have hepatoerythropoietic porphyria. Detailed biochemical and enzymatic analyses revealed a moderate deficiency of uroporphyrinogen decarboxylase in both the proband's parents and in his three children, all of whom were asymptomatic. The mildness of the clinical symptoms in the proband correlated with a higher level of residual enzyme activity than that in previously described patients. We conclude that clinically manifested hepatoerythropoietic porphyria results from the homozygous inheritance of a defect in the uroporphyrinogen decarboxylase gene, that the severity of clinical symptoms is probably related to the level of residual enzyme activity, and that the genetic defect of uroporphyrinogen decarboxylase in hepatoerythropoietic porphyria can be heterogeneous.     

Increased frequency of HLA-A3 in subjects with sporadic porphyria cutanea tarda

The HLA-A3 alloantigen, usually associated with the hemochromatosis gene, was detected in 12 of 18 patients (67%) with sporadic porphyria cutanea tarda but in only 23 per cent of 328 normal subjects (p = 0.0006). This difference remained significant after correcting for the number of HLA-A locus antigens tested (p = 0.025). These results suggest that an HLA-linked hemochromatosis allele is present and may account for the iron abnormalities in many patients with sporadic porphyria cutanea tarda.     

Molecular analysis of the UROD gene in 17 Argentinean patients with familial porphyria cutanea tarda: characterization of four novel mutations

Porphyria cutanea tarda (PCT) is caused by decreased activity of uroporphyrinogen decarboxylase (UROD) in the liver. The disease usually occurs in adulthood and is characterized by cutaneous photosensitivity, hyperpigmentation, skin fragility and hypertrichosis, due to the accumulation of porphyrins produced by oxidation of uroporphyrinogen and other highly carboxylated porphyrinogens overproduced as a result of the enzyme deficiency. PCT is generally sporadic, but about 20-30% of patients have familial-PCT (F-PCT) which is associated with heterozygosity of mutations in the UROD gene. In the present study we have found the molecular defect in seventeen unrelated Argentinean patients with F-PCT, identifying a total of eleven UROD gene mutations: four novel and seven previously described. The novel mutations were: a guanine insertion at the 5' splice junction of intron 2, a three nucleotide deletion causing the lost of valine 90, a deletion of 22 bp in exon 6 and a deletion of part of the polyadenylation signal. Prokaryotic expression studies showed that the novel amino acid deletion resulted in an inactive protein. Mutations c.10insA and p.M165R, previously found in Argentinean patients, were recurrent in this study; they are the most frequent in Argentina accounting for 40% of the mutant alleles characterized to date.     

Seven novel point mutations in the uroporphyrinogen decarboxylase (UROD) gene in patients with familial porphyria cutanea tarda (f-PCT)

In this work, we describe seven novel molecular defects in the uroporphyrinogen decarboxylase gene responsible for familial porphyria cutanea tarda in Italian subjects with reduced erythrocyte URO-D activity. Four of these molecular abnormalities (R142Q, L161Q, S219F, P235S) are missense mutations, one (Q206X) is a nonsense mutation, one (IVS8-1 G>C) is a splicing defect causing the exon 9 deletion and one (1107 G>A) is located in the 3' untranslated region of UROD gene. All the amino acid substitutions fall in conserved regions in several organisms suggesting an important role in catalysis or in the protein structure stabilization. Three of these mutations have been detected in more than one subject. These results suggest a molecular heterogeneity at the UROD locus in Italian PCT patients although recurrent mutations have been identified.     

Dual porphyria in double heterozygotes with porphobilinogen deaminase and uroporphyrinogen decarboxylase deficiencies

A coexistent dual deficiency of porphobilinogen deaminase (PBG-D; EC 4.3.1.8) and uroporphyrinogen decarboxylase (EC 4.1.1.37) in erythrocytes was recognized in five individuals, four males and one female. Clinically, the female and one male were diagnosed as suffering from acute intermittent porphyria (AIP), and the other two males were diagnosed as having porphyria cutanea tarda (PCT). Biochemically, the excretion pattern of urinary and fecal heme precursors exhibited a complex constellation with signs characteristic for both AIP and PCT. A coexistent dual enzyme deficiency of PBG-D and URO-D could be confirmed by repeated studies over 10 years. Clinical courses of both disease manifestations were observed. Family investigations have shown that the two disorders do not consistently segregate together. The findings suggest that the dual porphyria reflects a double heterozygous condition of coexistent AIP and PCT genes in the same individual.     

C282Y and H63D mutation of the hemochromatosis gene in German porphyria cutanea tarda patients

BACKGROUND AND AIMS: Patients with porphyria cutanea tarda (PCT) have a susceptibility to reversible inactivation of hepatocyte uroporphyrinogen decarboxylase, which can be triggered by alcohol, hepatitis C virus, and other agents. Inherited factors that may predispose to PCT include the C282Y mutation in the hemochromatosis (HFE) gene. METHODS: We analyzed the hemochromatosis mutations C282Y and H63D in liver biopsies and serum samples of 190 German patients (mean age 48+/-12.5 years) with sporadic PCT. The hepatic iron concentration was determined within the liver tissue. Age-matched healthy blood donors (115 donors) served as controls. RESULTS: The C282Y and H63D mutations were found in 75 (39%) and 85 (45%) of 190 patients with PCT, respectively. Twenty-two patients (12%) were homozygous for the C282Y mutation, and eighteen patients (9%) were compound heterozygotes, displaying both the C282Y and the H63D mutation. Within the control group, 3 of 115 patients were heterozygous for C282Y (3%) and 12 for H63D (10%). Serum and hepatic iron, ferritin, transferrin saturation, or liver enzymes did not differ significantly between patients with or without HFE mutations. CONCLUSIONS: The high frequency of homo- and heterozygosity for the C282Y and H63D alleles strongly suggests that these mutations are important predisposing factors for PCT in German patients.     

Pathobiochemical transition of secondary coproporphyrinuria to chronic hepatic porphyria in humans

Summary Secondary coproporphyrinurias can be observed in one third of all patients affected by liver damage, especially by alcohol liver syndromes. Potentially, every secondary coproporphyrinuria can turn into a chronic hepatic porphyria, which progresses along several biochemically detectable latent phases and which indicates clinical manifestation by cutaneous symptoms.Two requirements are necessary for the transition of a secondary coproporphyrinuria to a chronic hepatic porphyria: 1. Genetic disposition of an autosomal dominantly inherited defect in uroporphyrinogen decarboxylase, which only becomes subclinically or clinically relevant through alcohol or estrogens (also oral contraceptives) in connection with liver damage. 2. A toxic inhibition of uroporphyrinogen decarboxylase in the liver caused by polyhalogenated hydrocarbons, such as hexachlorobenzene, polychlorinated and polybrominated biphenyls, dioxins and probably also methyl and vinyl chloride in combination with a present liver damage or caused by one of these agents. Experimental results, further, prove a direct negative effect of alcohol on the activity of uroporphyrinogen decarboxylase.In all liver conditions one should look out for porphyrinuria, because it represent an important criterium for toxic liver damage and early diagnosis of a chronic hepatic porphyria is assessed by differentiating hepatic porphyrinurias.     

Multiple nodular foci in the liver associated with chronic hepatic porphyria after previous treatment of breast cancer

Summary More than 7 years after the diagnosis and treatment of breast cancer (T1N1aM0), multiple nodular foci were observed in the liver of a 40-year-old woman at ultrasonographic examination. The lesions were confirmed by CT scan, but CT-guided liver biopsy revealed only non-specific alterations. At subsequent peritoneoscopic examination, bluish-brown foci were indeed visible on the liver surface, but guided liver biopsies again failed to corroborate the suspected metastases. Instead, histology showed mild portal fibrosis, moderate steatosis and siderosis of hepatocytes, as before. Only the intense red fluorescence of part of the biopsy material under Wood's light suggested the diagnosis of chronic hepatic porphyria or porphyria cutanea tarda, here presumably as a consequence of prolonged alcohol consumption. Subsequent porphyrin studies in urine, faeces and plasma yielded the typical constellation of latent porphyria cutanea tarda (chronic hepatic porphyria type C). The activity of erythrocyte uroporphyrinogen decarboxylase was normal, which argued against a genetic predisposition. After 1 year of strict alcohol abstinence and low-dose chloroquine treatment the nodular foci in the liver were no longer visible on ultrasonogram and CT scan; only proton-weighted NMR imaging (SE 1500/30) still showed ill-defined areas of higher signal intensity. The renal excretion of porphyrins had decreased considerably. The levels are now consistent with the diagnosis of subclinical chronic hepatic porphyria type A. Modern non-invasive imaging techniques are tremendously useful, but they have their pitfalls. Focal liver lesions may present serious diagnostic problems, especially when they are found in a patient with a history of carcinoma at an extrahepatic primary site. A rare example is described.Abbreviations gamma-GT gamma glutamyl transpeptidase - CEA carcinoembryonic antigen - CA-15-3 breast cancer associated antigen - TPA tissue polypeptide antigen - CHP chronic hepatic porphyria - PCT porphyria cutanea tarda - delta-ALA delta-amino-laevulinic acid - CT computerised axial tomography - NMR nuclear magnetic resonance - T1, T2 relaxation time - TE echo time - TR repetition time - SE spin echo     

Ultrastructural study of the liver in hepatic porphyria

In intermittent acute porphyria there are no specific alterations in the ultrastructure of the liver. In contrast, the constant characteristic of porphyria cutanea tarda is siderosis of the hepatocytes and Kupffer cells. Therefore, between the two types of hepatic porphyria there are no similar ultrastructural lesions. It is still doubtful whether the presence of hepatosiderosis in porphyria cutanea tarda is connected with the hepatopathy or is secondary to an alteration in the porphyrinic metabolism. The role played by lysosomes is discussed.     

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterization of six novel mutations associated with familial PCT

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M01T, F229L, and M324T), two splice mutations (IVS3‐2A→T and IVS5‐2A→G) leading to exon skipping, and a 2‐bp deletion (415‐416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a considerable increase in thermolability. The six new mutations bring to a total of 29 the number of disease‐related mutations in the UROD gene. The DGGE assay presented greatly improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases. Hum Mutat 14:222–232, 1999. © 1999 Wiley‐Liss, Inc.     

Hepatic fluorescence in porphyria cutanea tarda studied in fine needle aspiration biopsy smears

Fine needle aspiration biopsy smears from 19 patients with porphyria cutanea tarda were studied by fluorescence microscopy. Fluorescence was arbitrarily graded from 0 to 4. All patients-those with clinically manifest as well as those with latent disease-showed bright red fluorescence of porphyrin type in the liver cells, though in latent cases the fluorescence commonly was of a low grade. In patients with manifest disease fluorescence of grade 3 or 4 was present.In patients with fluorescence of grades 1 or 2 the uroporphyrin excretion in urine was normal or slightly increased. At higher grades all levels of porphyrin excretion were encountered.Of 22 control subjects, only one showed hepatic fluorescence of a porphyrin character. The control patient with a positive finding was found to have definitely increased urinary uroporphyrin excretion and probably has latent porphyria cutanea tarda.The procedure described should be useful as a screening test for hepatic cutaneous porphyria.     

Hepatic porphyrins and urinary prophyrins and prophyrin precursors in liver cirrhosis

Summary In 21 liver cirrhotics (19 men, 2 women) the urinary excretion of-aminolevulinic acid, porphobilinogen, uro- and coproporphyrin, and hepta-, hexa-, and pentacarboxylic porphyrin was studied. Porphyrins in liver biopsy tissue were also analyzed in 17 of the 21 cases. Only a single patient had slight clinical symptoms of porphyria cutanea tarda.Porphyrin excretion was normal in six of the patients, and six showed moderate to severe coproporphyrinuria. Excretion of-aminolevulinic acid and porphobilinogen was normal in all cases. Under 366 nm UV light the liver tissue from three patients, including the one with porphyria cutanea tarda, exhibited the red fluorescence characteristic of porphyrin. On the basis of the constellation of urinary porphyrins and the storage of uro- and heptacarboxylic porphyrin in the liver, chronic hepatic porphyria Type A was diagnosed in two patients, and chronic hepatic porphyria Type B, Type C, and porphyria cutanea tarda in one patient each. In eight of the fourteen non-red-fluorescent hepatic tissue specimens the content of uroporphyrin, and in some of these cases of heptacarboxylic porphyrin as well, was usually slightly elevated, as observed both in four patients with normal and in four with abnormal coproporphyrin excretion. The increased levels of porphyrin in three patients were possibly due to certain drugs, the porphyrinogenic effect of which is known from experiments.The present study includes all stages of biochemical manifestation of chronic disturbance of hepatic porphyrin synthesis, i.e., four patients with clinically inapparent chronic hepatic porphyria.Types A, B, and C, in the order of degree of porphyrin accumulation in the liver and the order in which the individual urinary porphyrins increase. This supports the contention that hepatic cirrhosis in itself provides the conditions triggering the development of chronic hepatic porphyria. Induction of-aminolevulinic acid synthase by sexual hormones and their metabolites due to impaired steroid conjugation may be one factor in the pathogenesis, which is of complex nature; just why liver cirrhosis should produce secondary coproporphyrinuria in some cases and chronic hepatic porphyria and porphyria cutanea tarda in others still remains an open question.No correlation was found between the concentrations of porphyrins in liver and urine and clinical-chemical data from serum analysis and the bromosulfophthalein test.     

Porphyrinanalyse im Lebercylinder bei Porphyria cutanea tarda

Zusammenfassung Es wird eine Methode zur dünnschichtchromatographischen Analyse der Porphyrine aus dem Biopsiematerial der Leber mitgeteilt. Das Lebergewebe enthält bei physiologischer Porphyrinsynthese, Proto-, Kopro- und Uroporphyrin in niedrigen Konzentrationen, wobei Protoporphyrin überwiegt. Charakteristisch für die Leber bei Porphyria cutanea tarda sind hohe Konzentrationen von Uround Heptacarboxyporphyrin.

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Summary A method is described for the analysis of porphyrins in liver needle biopsy material by thin-layer chromatography. In normal liver tissue proto-, copro-, and uroporphyrin were found in small concentrations. Protoporphyrin was the main component. High concentrations of uroporphyrin and heptacarboxylic porphyrin are characteristic for the liver of patients with porphyria cutanea tarda.

     

Chronic hepatic prophyria type C

Summary In four patients with liver disease (fatty liver, fibrosis, and cirrhosis) the concentrations of uroporphyrin and heptacarboxylic porphyrin in the liver were found to be considerably elevated (up to 90 µg/g). The patients also excreted increased amounts of porphyrin with the urine (0.5–1 mg/l), although cutaneous symptoms were not present. The constellation of urinary porphyrins differed from that in chronic hepatic porphyria Types A and B, but was similar to that characteristic ofporphyria cutanea tarda. The relative proportion of heptacarboxylic porphyrin in the liver is, however, 50% lower than that inporphyria cutanea tarda. The biochemical abnormality found in these four patients is given the name of chronic hepatic porphyria type C. Fasting, glycine loading, alcohol, and high fat diet bring about an increase in porphyrin excretion.

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Zusammenfassung Chronische hepatische Porphyrie Typ C. Vier Patienten mit Leberschäden (Fettleber, Fibrose, Cirrhose) wiesen einen stark erhöhten Uro- und Heptacarboxyporphyringehalt in der Leber (bis 90 µg/g) und eine erhöhte Porphyrinausscheidung im Urin (0,5–1 mg/l) auf, ohne daß cutane Symptome bestanden. Die Konstellation der Urinporphyrine unterschied sich von denjenigen bei chronischer hepatischer Porphyrie Typ A und Typ B, korrelierte jedoch mit derjenigen, wie sie bei Porphyria cutanea tarda vorkommt. Dagegen war der relative Anteil des Heptacarboxyporphyrins in der Leber um 50% geringer als bei Porphyria cutanea tarda. Die bei den vier Patienten gefundene biochemische Abnormität wird als chronische hepatische Porphyrie Typ C bezeichnet. Hunger, Glycinbelastung, Alkohol und fettreiche Diät verursachen eine Erhöhung der Porphyrinausscheidung.

     

Fermentbestimmungen in Erythrocyten von Kranken mit Porphyria cutanea tarda

Zusammenfassung In Erythrocyten von 12 Kranken mit Porphyria cutanea tarda konnte eine Aktivitätssteigerung der Enzyme G-6-PDH, 6-PGDH sowie der NADH- und der NADPH-abhängigen Glutathionreduktion festgestellt werden, deren Ursache diskutiert wird.

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Summary In erythrocytes of 12 patients with porphyria cutanea tarda an increase in activity of the enzymes G-6-PDH, 6-PGDH and also the NADH-, and the NADPH-dependent glutathione reduction could be detected. These findings are discussed.

     

Vancomycin-resistant Aureobacterium species cellulitis and bacteremia in a patient with acute myelogenous leukemia.

A 39-year-old male with acute myelogenous leukemia and concomitant porphyria cutanea tarda was admitted to the hospital for consolidation chemotherapy of his leukemia. During his hospitalization, he developed cellulitis of the left hand and persistent bacteremia with a yellow-pigmented, nonfermenting coryneform bacterium that was identified as Aureobacterium sp. The portal of entry for the Aureobacterium infection was probably through the skin lesions due to porphyria cutanea tarda. The infection developed while the patient was receiving vancomycin prophylaxis, and the vancomycin MIC for the isolate was 32 micrograms/ml.     

Demonstration of needle-shaped hepatic inclusions in porphyria cutanea tarda using the ferric ferricyanide reduction test

Summary In 18 of 19 biopsy and 2 of 3 autopsy samples of hepatic tissue from cases of porphyria cutanea tarda needle-shaped cytoplasmic inclusions are capable of reducing ferric ions in the ferric ferricyanide reduction test. Thanks to the resulting Turnbull's Blue the inclusions are clearly visible, which facilitates their histological demonstration. In 20 biopsy and 20 autopsy samples of hepatic tissue from cases other than porphyria cutanea tarda the inclusions are not present. These needle-shaped inclusions are thus considered to be a specific histological feature. The ferric ferricyanide reduction test represents a simple method for their visualization, which can be used in routine diagnostic practice.     

Vinyl chloride-induced hepatic coproporphyrinuria with transition to chronic hepatic porphyria

Summary A chronic hepatic disorder of prophyrin metabolism was found in 36 workers with vinyl chloride (VC)-induced hepatic injury following long-time industrial exposure. Pathologic porphyrinuria, especially secondary coproporphyrinuria with transition to subclinical chronic hepatic porphyria, is a consistent pathobiochemical parameter for the recognition of VC hepatic lesions. The porphyrinuria is of diagnostic value for the incipient toxic phase. Erythrocyte uroporphyrinogen decarboxylase activity studied in six cases with initial chronic hepatic porphyria was normal, suggesting that VC affects only this enzyme in the liver.This study is dedicated to Hans Popper, M.D., Ph.D., Dr. (hon.) mult., Gustave L. Levy Distinguished Service Professor, Mount Sinai School of Medicine, New York, on the occasion of his 80th birthday on Nov. 24th, 1983.This investigation has been supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Grant Do 134)     

Erythrocytapheresis

Summary We report on the progress of a new modified method of phlebotomy, erythrocytapheresis, as a means of fast therapeutic removal of erythrocytes from the circulation. We performed erythrocytapheresis in 65 patients. In 18 patients with central venous thrombosis of the eye, the beneficial effect of this procedure proved superior to the traditional approach. In 29 patients with primary or secondary polycythemia, hemoglobin, hematocrit, and blood viscosity could be lowered drastically for up to 11 months by a single erythrocytapheresis. We performed erythrocytapheresis in an effort to deplete the iron stores in patients with hemochromatosis (14 cases) and in patients with porphyria cutanea tarda (4 cases). Several consecutive erythrocytaphereses were necessary, however, to even slightly lower the amounts of stored iron as measured by serum iron, iron-binding capacity, and serum ferritin in these patients. The intervals between treatment were 2 to 11 months, thus much longer than the intervals between blood-lettings. We did not observe any adverse side effects. There was no significant influence on the clotting system, and no reactive thrombocytosis as described after phlebotomies.Abbreviations EA Erythrocytapheresis - CVT Central venous thrombosis of the eye - PPV Primary polycythemia vera - SPV Secondary polycythemia vera - ACD Anticoagulant solution - HC Hemochromatosis - PCT Porphyria cutanea tarda Dedicated to Professor N. Zöllner on the occasion of his 65th birthday