Molecular characterization of porphyrias in Italy: a diagnostic flow-chart.

The porphyrias are disorders associated with inherited or acquired enzyme deficiencies in the heme biosynthetic pathway. The differential diagnosis is often difficult since the phenotype is very similar in some forms and the biochemical tests are not commonly available. Here we provide an update on the molecular diagnosis of porphyrias in Italy and a flow-chart to facilitate the identification of mutations in heme biosynthetic genes. The molecular analysis has allowed us to identify the molecular defect underlying the disease in 66 probands with different porphyrias [acute intermittent porphyria (AIP), variegate porphyria (VP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP)]. No Italian patients with defects in coproporphyrinogen oxidise (CPOX) gene, responsible for hereditary coproporphyria (HCP), have been detected. The molecular characterization has been extended to 115 relatives with the identification of 55 asymptomatic mutation carriers and 60 normal subjects. We have so far identified 50 different mutations among 4 genes associated with the most common porphyrias showing a high molecular heterogeneity: 22 in the hydroxymethylbilane synthase (HMBS) gene (AIP), 7 in the protoporphyrinogen oxidase (PPOX) gene (VP), 16 in the uroporphyrinogen decarboxylase (UROD) gene (PCT) and 5 in the ferrochelatase (FECH) gene (EPP). Among the 50 molecular defects, 29 seem to be restricted to the Italian population.     

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.     

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 (HFE) and transferrin receptor-1 (TFRC1) genes in sporadic porphyria cutanea tarda (sPCT).

Porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis, is caused by a decreased activity of the hepatic enzyme uroporphyrinogen decarboxylase (UROD). Two forms of PCT have been described: a familial one (fPCT) with an inherited decrease of UROD activity in all tissues and a sporadic one (sPCT) with a decreased UROD activity restricted to the liver. Iron overload and acquired factors including hepatic viral infections, alcohol, drugs contribute to the expression of PCT. In 65 French sPCT patients and 108 controls we have evaluated the respective role of iron and HCV status, the hemochromatosis (HFE) gene mutations frequencies (H63D. S65C, C282Y), and in a case control study we searched for an association between sPCT and the human transferrin receptor-1 (TFRC1) gene whose product is thought to be in functional association with the HFE protein: three single nucleotide polymorphisms (SNPs) previously characterized and 2 novel ones were studied. The iron-related parameters and transaminases were higher in sPCT patients than those of non-porphyric controls. Of the sPCT patients studied, 28% were HCV positive. In the HFE gene, 17% of sPCT patients carried C282Y mutation compared to 4% in controls, no significant differences were found with H63D and S65C variants. Compound heterozygous genotypes, C282Y/H63D or C282Y/S65C, were not significantly different in sPCT and control groups. Independently from HFE gene mutations, an association was found between the IVS4+198 T allele in the TFRC1 gene and sPCT patients. Analysis of HFE genotypes indicated that C282Y (but not H63D nor S65C) is a susceptibility factor for the development of sPCT in West European continental patients. However, analysis of TFRC1 genotypes suggest that sPCT should be considered as a multifactorial disorder in which other intracellular iron metabolism genes could be involved.     

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.     

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.     

Porphyria in Sweden

In a brief survey the work of Swedish porphyrinologists through time is presented, from the organic chemist Jakob Berzelius 1840 to the molecular biologists of today. The building up in Stockholm of a Swedish national competence centre for porphyria is touched upon and the emergence of a computerized national register on the porphyria gene carriers in the country described. Figures for the prevalences of the seven different forms of porphyria diagnosed in Sweden are given. The geographical distribution of gene mutation spectra is shown for the most frequent form, acute intermittent porphyria. The organisation at Porphyria Centre Sweden of its diagnostic and consultative services is described, as is the decentralized model for porphyria care applied in the form of a clinical network covering the long and sparsely populated country. The ideas and activities of the Swedish Porphyria Patients' Association are presented. Its focus on protection-by-information of the porphyria gene carrier against maltreatment in health service contacts, and against other exposures to environmental threats to his or her health, is discussed. The combined efforts of the national porphyria centre and the patients' association have resulted in early and accurate diagnosis of most of the porphyria gene carriers in the country. The information to the carriers and to the health service regarding the mechanisms of the diseases and the importance of avoiding exposure to disease triggering environmental factors have greatly reduced porphyric morbidity. In the case of the acute porphyrias, by this programme and after the introduction of heme arginate in the therapy, mortality in the acute phase has become extremely rare in Sweden. In contrast, probably due to greater awareness of the high risk for liver cancer in acute porphyrias the number of hepatoma cases diagnosed has increased. The current research activities at the Porphyria Centre which aim at finding ways to substitute the mutated gene in acute intermittent porphyria for an undamaged one, or to substitute the enzyme deficiency by administration of exogenously produced enzyme, are mentioned, as is the work to establish a reliable drug porphyrinogenicity prediction model for evidence based drug counselling.     

Diet and alcohol effects on the manifestation of hepatic porphyrias

Porphyria cutanea tarda (PCT) is the most frequently reported type of porphyria. The average patient is male more than 40 years old with a history of alcohol consumption. In women the incidence of PCT has increased with use of estrogens for birth control. The cutaneous features are those of chronic porphyrin photosensitivity on the light-exposed area of the skin: pigmentation, hirsuitism and fragility, and vesiculobullae, which has prompted the expression bullosa actinica et mechanica. One-third of the patients have glucose intolerance. PCT has been reported frequently among the Bantu people in South Africa as resulting from combinations of alcohol and cooking in ironware. The average patient has a higher than normal hematocrit, which is used as a guide to treatment by phlebotomy ranging from 8 to 14 units removed every 2-4 wk. Chemically induced PCT has been reported with chlorinated hydrocarbons, the best-known of which is hexachlorobenzene (HCB). Porphyria was noted in more than 3,000 patients in southeast Turkey between 1955 and 1961, because of consumption of seed wheat treated with HCB. In addition, more than 1,000 children under the age of 1 year died because HCB was transferred from the mother, either via the placenta or through breast milk.     

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.     

A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize.

The maize lesion mimic gene Les22 is defined by dominant mutations and characterized by the production of minute necrotic spots on leaves in a developmentally specified and light-dependent manner. Phenotypically, Les22 lesions resemble those that are triggered during a hypersensitive disease resistance response of plants to pathogens. We have cloned Les22 by using a Mutator-tagging technique. It encodes uroporphyrinogen decarboxylase (UROD), a key enzyme in the biosynthetic pathway of chlorophyll and heme in plants. Urod mutations in humans are also dominant and cause the metabolic disorder porphyria, which manifests itself as light-induced skin morbidity resulting from an excessive accumulation of photoexcitable uroporphyrin. The phenotypic and genetic similarities between porphyria and Les22 along with our observation that Les22 is also associated with an accumulation of uroporphyrin revealed what appears to be a case of natural porphyria in plants.     

Computational Identification of Pathogenic Associated nsSNPs and its Structural Impact in UROD Gene: A Molecular Dynamics Approach

Uroporphyrinogen decarboxylase is a cytosolic enzyme involved in the biosynthetic pathway of heme production. Decreased activity of this enzyme results in porphyria cutanea tarda and hepato erythropoietic porphyria. Nonsynonymous single nucleotide polymorphisms (nsSNPs) alter protein sequence and can cause disease. Identifying the deleterious nsSNPs that contribute to disease is an important task. We used five different in silico tools namely SIFT, PANTHER, PolyPhen2, SNPs&GO, and I-mutant3 to identify deleterious nsSNPs in UROD gene. Further, we used molecular dynamic (MD) approach to evaluate the impact of deleterious mutations on UROD protein structure. By comparing the results of all the five prediction results, we screened 35 (51.47 %) nsSNPs as highly deleterious. MD analysis results show that all the three L161Q, L282R, and I334T deleterious variants were affecting the UROD protein structural stability and flexibility. Our findings provide strong evidence on the effect of deleterious nsSNPs in UROD gene. A detailed MD study provides a new insight in the conformational changes occurred in the mutant structures of UROD protein.     

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.     

Porphyria cutanea tarda: the etiological importance of mutations in the HFE gene and viral infection is population-dependent.

A number of factors, including increased iron stores and alcohol consumption, are known to be associated with the development of porphyria cutanea tarda (PCT) in susceptible individuals. Recent reports have described a significant association between inheritance of the C282Y and H63D mutations in the HFE gene, associated with genetic hemochromatosis (GH) and PCT. A strong association between hepatitis C virus infection and PCT has also been demonstrated, while case reports record a link between human immunodeficiency virus (HIV) and PCT. We have investigated the frequency of these factors in a racially-mixed population of patients with PCT in Cape Town, South Africa. 57 patients with PCT drawn from three ethnic groups were screened for the presence of the C282Y and H63D mutations linked to GH, and the prevalences were compared with corresponding healthy control populations. The seroprevalence of markers for HCV, hepatitis B (HBV) and HIV infection were examined in 28 of these. In the control populations, we found that both the C282Y and H63D mutations are highly prevalent in South Africans of European origin. In a population of mixed or Asian origin, the C282Y mutation is very rare whereas the H63D mutation is common. Neither mutation was encountered in any African subject. Both mutations are associated with PCT, but the association is dependent on the ethnic origins of the population to which the patient belongs. In contrast to other studies, HCV infection is numerically unimportant in PCT in our patients. HIV infection is increasingly encountered in our patients with PCT, but the strength of the association cannot be determined in view of the high background prevalence of HIV infection in some sectors of the South African population. The contribution of specific risk factors may be heavily dependent on the population from which patients are drawn, and care should be taken in extrapolating from observations in one racial or geographic population to any other.     

Demystification of Chester porphyria: a nonsense mutation in the Porphobilinogen Deaminase gene

The porphyrias arise from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings, and specific enzyme assays. In some cases however, these diagnostic tools reveal overlapping findings, indicating the existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultaneously. Recently, it was reported that the so-called Chester porphyria shows features of both variegate porphyria and acute intermittent porphyria. Linkage analysis revealed a novel chromosomal locus on chromosome 11 for the underlying genetic defect in this disease, suggesting that a gene that does not encode one of the enzymes of heme biosynthesis might be involved in the pathogenesis of the porphyrias. After excluding candidate genes within the linkage interval, we identified a nonsense mutation in the porphobilinogen deaminase gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria. However, we could not detect a mutation in the coding or the promotor region of the protoporphyrinogen oxidase gene that is mutated in variegate porphyria. Our results indicate that Chester porphyria is neither a dual porphyria, nor a separate type of porphyria, but rather a variant of acute intermittent porphyria. Further, our findings largely exclude the possibility that a hitherto unknown gene is involved in the pathogenesis of the porphyrias.     

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