Multiple roles of haem in cystathionine ß-synthase activity: implications for hemin and other therapies of acute hepatic porphyria.

The role of haem in the activity of cystathionine ß-synthase (CBS) is reviewed and a hypothesis postulating multiple effects of haem on enzyme activity under conditions of haem excess or deficiency is proposed, with implications for some therapies of acute hepatic porphyrias. CBS utilises both haem and pyridoxal 5'-phosphate (PLP) as cofactors. Although haem does not participate directly in the catalytic process, it is vital for PLP binding to the enzyme and potentially also for CBS stability. Haem deficiency can therefore undermine CBS activity by impairing PLP binding and facilitating CBS degradation. Excess haem can also impair CBS activity by inhibiting it via CO resulting from haem induction of haem oxygenase 1 (HO 1), and by induction of a functional vitamin B6 deficiency following activation of hepatic tryptophan 2,3-dioxygenase (TDO) and subsequent utilisation of PLP by enhanced kynurenine aminotransferase (KAT) and kynureninase (Kynase) activities. CBS inhibition results in accumulation of the cardiovascular risk factor homocysteine (Hcy) and evidence is emerging for plasma Hcy elevation in patients with acute hepatic porphyrias. Decreased CBS activity may also induce a proinflammatory state, inhibit expression of haem oxygenase and activate the extrahepatic kynurenine pathway (KP) thereby further contributing to the Hcy elevation. The hypothesis predicts likely changes in CBS activity and plasma Hcy levels in untreated hepatic porphyria patients and in those receiving hemin or certain gene-based therapies. In the present review, these aspects are discussed, means of testing the hypothesis in preclinical experimental settings and porphyric patients are suggested and potential nutritional and other therapies are proposed.

Heme acts through the Bach1b/Nrf2a-MafK pathway to regulate exocrine peptidase precursor genes in porphyric zebrafish.

Using a zebrafish model of hepatoerythropoietic porphyria (HEP), we identify a previously unknown mechanism underlying heme-mediated regulation of exocrine zymogens. Zebrafish bach1b, nrf2a and mafK are all expressed in the zebrafish exocrine pancreas. Overexpression of bach1b or knockdown of nrf2a result in the downregulation of the expression of the exocrine zymogens, whereas overexpression of nrf2a or knockdown of bach1b cause their upregulation. In vitro luciferase assays demonstrate that heme activates the zymogens in a dosage-dependent manner and that the zymogen promoter activities require the integral Maf recognition element (MARE) motif. The Bach1b-MafK heterodimer represses the zymogen promoters, whereas the Nrf2a-MafK heterodimer activates them. Furthermore, chromatin immunoprecipitation (ChIP) assays show that MafK binds to the MARE sites in the 5' regulatory regions of the zymogens. Taken together, these data indicate that heme stimulates the exchange of Bach1b for Nrf2a at MafK-occupied MARE sites and that, particularly in heme-deficient porphyria, the repressive Bach1b-MafK heterodimer dominates, which can be exchanged for the activating Nrf2a-MafK heterodimer upon treatment with hemin. These results provide novel insights into the regulation of exocrine function, as well as the pathogenesis of porphyria, and should be useful for designing new therapies for both types of disease.

Sustained enzymatic correction by rAAV-mediated liver gene therapy protects against induced motor neuropathy in acute porphyria mice.

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

Liver transplantation for porphyria: who, when, and how?

Porphyrias are a heterogenous group of diseases that may result in disabling or life threatening neurovisceral symptoms and/or cutaneous photosensitivity. In acute intermittent porphyria, the clinical features, particularly neurological symptoms, may be life-threatening and disabling. Conventional treatment with human hemin, though effective in reducing symptoms, does not reverse neuropathy when structural nerve damage has occurred and may cause intense phlebitis. Liver transplantation (LT) may be considered as treatment for those with repeated life-threatening acute attacks resulting in poor quality of life, requirement of ventilatory support, and progressive loss of venous access due to hemin infusion. Patients with variegate porphyria (VP) present after puberty with neurovisceral symptoms and skin manifestations. LT resolved VP in the 1 patient reported in the literature. Aminolaevulinic acid dehydratase deficient porphyria is a rare autosomal recessive disorder and a child who presented with failure to thrive and required transfusions and parenteral nutrition did not improve with LT. In erythropoietic protoporphyria (EPP), there is excessive production of protoporphyrin in the bone marrow. Protoporphyrin is hepatotoxic and pigment loading of hepatocytes and bile canalicular sludging may result in progressive cholestasis and cirrhosis. LT is beneficial for such patients with end-stage liver disease. Perioperative management includes use of filters on operative lights to prevent skin burns and intestinal perforation. Other concerns include development of neuropathy, biliary complications, and recurrent liver disease. This review addresses the rationale, patient selection, evaluation, management issues, and technique of performing LT in various types of porphyria.

Mutations in human CPO gene predict clinical expression of either hepatic hereditary coproporphyria or erythropoietic harderoporphyria.

Hereditary coproporphyria (HCP), an autosomal dominant acute hepatic porphyria, results from mutations in the gene that encodes coproporphyrinogen III oxidase (CPO). HCP (heterozygous or rarely homozygous) patients present with an acute neurovisceral crisis, sometimes associated with skin lesions. Four patients (two families) have been reported with a clinically distinct variant form of HCP. In such patients, the presence of a specific mutation (K404E) on both alleles or associated with a null allele, produces a unifying syndrome in which hematological disorders predominate: 'harderoporphyria'. Here, we report the fifth case (from a third family) with harderoporphyria. In addition, we show that harderoporphyric patients exhibit iron overload secondary to dyserythropoiesis. To investigate the molecular basis of this peculiar phenotype, we first studied the secondary structure of the human CPO by a predictive method, the hydrophobic cluster analysis (HCA) which allowed us to focus on a region of the enzyme. We then expressed mutant enzymes for each amino acid of the region of interest, as well as all missense mutations reported so far in HCP patients and evaluated the amount of harderoporphyrin in each mutant. Our results strongly suggest that only a few missense mutations, restricted to five amino acids encoded by exon 6, may accumulate significant amounts of harderoporphyrin: D400-K404. Moreover, all other type of mutations or missense mutations mapped elsewhere throughout the CPO gene, lead to coproporphyrin accumulation and subsequently typical HCP. Our findings, reinforced by recent crystallographic results of yeast CPO, shed new light on the genetic predisposition to HCP. It represents a first monogenic metabolic disorder where clinical expression of overt disease is dependent upon the location and type of mutation, resulting either in acute hepatic or in erythropoietic porphyria.

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse.

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.

Experimental hepatic uroporphyria induced by the diphenyl-ether herbicide fomesafen in male DBA/2 mice.

Hepatic uroporphyria can be readily induced by a variety of treatments in mice of the C57BL strains, whereas DBA/2 mice are almost completely resistant. However, feeding of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen (0.25% in the diet for 18 weeks) induced hepatic uroporphyria in male DBA/2N mice (liver porphyrin content up to 150 nmol/g, control animals 1 nmol/g), whereas fomesafen-treated male C57BL/6N mice displayed only a slight elevation of liver porphyrins (approximately 5 nmol/g). The profile of accumulated hepatic porphyrins in fomesafen-treated DBA/2N mice resembled the well-characterised uroporphyria induced by polyhalogenated aromatic hydrocarbons, while histological examination confirmed the presence of uroporphyria-specific cytoplasmic inclusions in the hepatocytes. Uroporphyrinogen decarboxylase activity decreased to about 30% of control values in fomesafen-treated DBA/2N mice; microsomal methoxyresorufin O-dealkylase activity was slightly reduced. The amount of CYP1A1 and CYP1A2 mRNA, as determined by real-time PCR, was not significantly changed; mRNA encoding the housekeeping 5-aminolevulinic acid synthase was elevated 10-fold. Total liver iron was slightly increased. A similar uroporphyria was induced by the herbicide formulation Blazer, containing a structurally related herbicide acifluorfen, when fed to DBA/2N mice at a dose corresponding to 0.25% of acifluorfen in the diet. Since DBA/2 mice are almost completely resistant to all well-characterised porphyrogenic chemicals, the results suggest the possible existence of a yet unknown mechanism of uroporphyria induction, to which the DBA/2 mouse strain is more sensitive than the C57BL strain.

Genetic analysis of variegate porphyria (VP) in Italy: identification of six novel mutations in the protoporphyrinogen oxidase (PPOX) gene.

Variegate Porphyria (VP) is one of the acute hepatic porphyrias, and is clinically characterised by skin lesions and acute neuropsychiatric/visceral attacks that occur separately or together. The disorder is caused by a partial deficiency of protoporphyrinogen oxidase, the penultimate enzyme in the heme biosynthetic pathway, and a number of mutations have been described for the corresponding gene (PPOX). Here we report a genetic analysis of VP in Italy, and the identification of six novel and three previously characterised mutations from nine affected individuals and families. Among those newly identified, two mutations were small deletions (c.418_419delAA; c.759delA), leading to the formation of premature stop codons, two were splicing defects (IVS10+2T>G; IVS12+1G>C), one was a nonsense (c.384G>A=p.W128X) and one a missense mutation (c.848T>A=I283N). This is the first study of the molecular genetics of Variegate Porphyria in patients of Italian origin, and the finding of six novel mutations out of nine identified confirms the genetic heterogeneity observed for this disorder.

Two novel mutations and coexistence of the 991C>T and the 1339C>T mutation on a single allele in the coproporphyrinogen oxidase gene in Swedish patients with hereditary coproporphyria.

Hereditary coproporphyria (HCP) is an autosomal dominant disorder, resulting from a partial deficiency of the enzyme coproporphyrinogen oxidase (CPO). This enzyme catalyzes the sixth step of the heme biosynthetic pathway, and mutations in the CPO gene have been coupled to HCP. The present study was undertaken to identify disease-producing mutations in the CPOgene in nine Swedish families with HCP. Exon 1 of the CPO gene of the nine probands was analyzed directly by sequencing, and exons 2-7 were screened by denaturating gradient gel electrophoresis, followed by sequencing of exons showing abnormal band pattern. Mutations were detected in five of the nine families. In two of these families, the novel mutations 623C>T (S208F, exon 2) and 982C>T (R328C, exon 5) were identified, respectively. In the affected members of the other three families, the previously reported mutations 991C>T (R331W, exon 5) and 1339C>T (R447C, exon 7) were shown to coexist on one allele. The present study contributes 2 novel mutations to the 34 that have been previously reported to cause HCP. In addition, this is the first report on patients carrying two HCP-coupled mutations on one allele.

Molecular, immunological, enzymatic and biochemical studies of coproporphyrinogen oxidase deficiency in a family with hereditary coproporphyria.

A 27-year-old woman who had recurrent pain in renal bed since 1998 with increasing character, was stationary admitted. The patient showed dark urine, complained of hair loss and took since 1994 a hormonal oral contraceptive. No photosensitivity was observed. Determinations of urinary porphyrin metabolites in 1998 revealed a porphyria cutanea tarda like excretion pattern with elevations of uro- (1767 nmol/24 hr, normal <29 nmol/24 hr) and heptacarboxyporphyrin (568 nmol/24 hr; normal <4 nmol/24 hr). Follow-up studies in feces showed the characteristics of a hereditary coproporphyria with dominance of coproporphyrin isomer III (total= 1470 nmol/g, isomer III= 93%), (normal: <37 nmol/g, isomer III = 25-35%). The excretion of porphyrin precursors (delta-aminolevulinic acid and porphobilinogen) was increased by taking an ethinylestradiol-cyproteronacetate-preparation, but acute and/or chronic manifestations were not observed. Coproporphyrinogen oxidase activity was decreased to 35% in the patient (normal=138+/-21 pkat/g protein; x+/-s), whereas the activity of red cell uroporphyrinogen decarboxylase was normal. Her mother and both sisters could be verified as heterozygous gene carriers of hereditary coproporphyria by their urinary and fecal excretion parameters and because of reduced coproporphyrinogen oxidase activity up to 50%. The father was normal with respect to his genotype. Molecular analysis revealed a hitherto unknown mutation with the transversion of a cytosine to thymine at nucleotide position 854 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by DGGE in the mother and her three daughters. The investigation of the immunological nature of the defective coproporphyrinogen oxidase gene from the whole family revealed decreased concentrations of coproporphyrinogen oxidase protein in the patient, her mother and her two sisters.

Functional studies of mutations in the human protoporphyrinogen oxidase gene in variegate porphyria.

The autosomal dominant disorder, variegate porphyria (VP), results from mutations in the protoporphyrinogen oxidase (PPOX) gene. We have investigated the effects of 22 disease-associated missense mutations in this gene on enzyme activity. Mutants were generated in the expression plasmid pHPPOX by site-directed mutagenesis. They were screened for PPOX activity by complementation of the Escherischia coli strain SAS38X which lacks PPOX activity. Ten mutants (G40E, L85P, G232R, de1281H, V282D, L295P, V335G, S350P, L444P, G453V) had no detectable PPOX activity. PPOX activity of the remaining 12 mutants (L15F, R38P, L73P, V84G, D143V, R152C, L154P, V158M, R168H, A172V, V290L, G453R) ranged from less than 1% to 9.2% of wild-type activity. Our findings show that all 22 mutations substantially impair or abolish PPOX activity in a prokaryotic expression system and add to the evidence that they cause VP.

A mouse model for South African (R59W) variegate porphyria: construction and initial characterization.

Variegate porphyria is inherited as an autosomal dominant disease with variable penetrance. It is characterized clinically by photocutaneous sensitivity and acute neurovisceral attacks, and biochemically by abnormal porphyrin excretion in the urine and feces. While the world-wide incidence of variegate porphyria is relatively low, in South Africa it is one of the most common genetic diseases in humans. Due to the large number of patients with variegate porphyria in South Africa, and the fact that variegate porphyria is representative of both the so-called "acute" and the "photocutaneous" porphyrias, it would be valuable to have an animal model in which to study the disease. In this study we have produced a mouse model of "South African" variegate porphyria with the R59W mutation in C57/BL6 mice via targeted gene replacement. Hepatic protoporphyrinogen oxidase activity was reduced by approximately 50% in mice heterozygous for the mutation. Urine and fecal samples from these mice, in the absence of exogenous inducers of hepatic haem synthesis, contain elevated concentrations of porphyrins and porphyrin precursors in a pattern similar to that found in human variegate porphyric subjects. Bypassing the rate-limiting step in haem biosynthesis by feeding 5-aminolevulinic acid to these mice, results in an accentuated porphyrin excretory pattern characteristic of the variegate porphyric phenotype and urinary porphobilinogen is increased significantly. This initial characterization of these mice suggest that they are a good model for variegate porphyria at the biochemical level.

Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the liver of C57BL/6J mice is a model for clinical sporadic porphyria cutanea tarda (PCT). There is massive uroporphyria, inhibition of uroporphyrinogen decarboxylase (UROD) activity, and hepatocellular damage. A variety of evidence implicates the CYP1A2 enzyme as necessary for mouse uroporphyria. Here we report that, 5 weeks after a single oral dose of TCDD (75 microg/kg), Cyp1a2(+/+) wild-type mice showed severe uroporphyria and greater than 90% decreases in UROD activity; in contrast, despite exposure to this potent agent Cyp1a2(-/-) knockout mice displayed absolutely no increases in hepatic porphyrin levels, even after prior iron overload, and no detectable inhibition of UROD activity. Plasma levels of alanine-aminotransferase (ALT) and aspartate aminotransferase (AST)-although elevated in both genotypes after TCDD exposure-were significantly less in Cyp1a2(-/-) than in Cyp1a2(+/+) mice, suggesting that the absence of CYP1A2 also affords partial protection against TCDD-induced liver toxicity. Histological examination confirmed a decrease in hepatocellular damage in TCDD-treated Cyp1a2(-/-) mice; in particular, there was no bile duct damage or proliferation that in the Cyp1a2(+/+) mice might be caused by uroporphyrin. We conclude that CYP1A2 is both necessary and essential for the potent uroporphyrinogenic effects of TCDD in mice, and that CYP1A2 also plays a role in contributing to TCDD-induced hepatocellular injury. This study has implications for both the toxicity assessment of TCDD and the hepatic injury seen in PCT patients.

Identification of the first variegate porphyria mutation in an indigenous black South African and further evidence for heterogeneity in variegate porphyria.

Variegate porphyria is an autosomal dominant disorder of haem metabolism resulting from reduced levels of the penultimate enzyme in the pathway, protoporphyrinogen oxidase. Here we investigate the molecular basis of variegate porphyria in four non-R59W South African families. We report the identification of the first mutation in the protoporphyrinogen oxidase gene in a black South African individual (V290M). In addition, we document three further mutations, a missense mutation (L15F), a deletion followed by a substitution [c769delG;770T > A], and a nonsense mutation (Q375X), in individuals of European or mixed ancestry. Our data provide further evidence of genetic heterogeneity in South Africa.

Identification of a founder mutation in the protoporphyrinogen oxidase gene in variegate porphyria patients from chile.

Variegate porphyria (VP; OMIM 176200) is characterized by a partial defect in the activity of protoporphyrinogen oxidase (PPO), the seventh enzyme of the porphyrin-heme biosynthetic pathway. The disease is usually inherited as an autosomal dominant trait displaying incomplete penetrance. In an effort to characterize the spectrum of molecular defects in VP, we identified 3 distinct mutations in 6 VP families from Chile by PCR, heteroduplex analysis, automated sequencing, restriction enzyme digestion and haplotyping analysis. The mutations consisted of 2 deletions and 1 missense mutation, designated 1239delTACAC, 1330delT and R168H. The occurrence of the missense mutation R168H had been reported previously in American, German and Dutch VP families, suggesting that this may represent a frequent recurrent mutation. Interestingly, the mutation 1239delTACAC was found in patients from 4 unrelated families living in different parts of Chile, suggesting that it might represent a common mutation in Chile. Haplotype analysis using 15 microsatellite markers which closely flank the PPO gene on chromosome 1q22, spanning approximately 21 cM, revealed the presence of R168H on different haplotypes in 6 VP patients from 3 unrelated families. In contrast, we found the occurrence of 1239delTACAC on the same chromosome 1 haplotype in 11 mutation carriers from 4 unrelated families with VP. These findings are consistent with R168H representing a hotspot mutation and 1239delTACAC existing as a founder mutation in the PPO gene. Our data comprise the first genetic studies of the porphyrias in South America and will streamline the elucidation of the genetic defects in VP patients from Chile by allowing an initial screening for the founder mutation 1239delTACAC.

Variegate porphyria with coexistent decrease in porphobilinogen deaminase activity.

Variegate porphyria is a rare disease caused by a deficiency of protoporphyrinogen oxidase. In most cases, the clinical findings are a combination of systemic symptoms similar to those occurring in acute intermittent porphyria and cutaneous lesions indistinguishable from those of porphyria cutanea tarda. We report on a 24-year-old woman with variegate porphyria who, after intake of lynestrenol, developed typical cutaneous lesions but no viscero-neurological symptoms. The diagnosis was based on the characteristic urinary coproporphyrin and faecal protoporphyrin excretion patterns, and the specific peak of plasma fluorescence at 626 nm in spectrofluorometry. Biochemical analysis revealed that most of the family members, though free of clinical symptoms, excrete porphyrin metabolites in urine and stool similar to variegate porphyria, accompanied by a significant decrease of porphobilinogen deaminase activity of a range which is ordinarily found in patients with acute intermittent porphyria only (approximately 50%). These data first led to the assumption of two separate and independently inherited genetic defects, similar to the dual porphyria of Chester. Molecular analysis, however, revealed only a missense mutation of the protoporphyrinogen oxidase gene, but not of the porphobilinogen deaminase gene. Thus, in the family presented, porphobilinogen deaminase deficiency is likely to be a phenomenon secondary to the genetic defect of protoporphyrinogen oxidase.

Homozygous variegate porphyria in South Africa: genotypic analysis in two cases.

Variegate porphyria is an autosomal dominant disorder of heme metabolism which results from decreased activity of the enzyme protoporphyrinogen oxidase. Clinically, the disease manifests postpubertally and is characterized by photocutaneous sensitivity and/or acute neurovisceral crises. However, in homozygous variegate porphyria, onset of the disease usually occurs in infancy with severe skin manifestations. The molecular basis of variegate porphyria in two severely affected probands in two South African families is described. Mutation detection included combined SSCP-heteroduplex analysis followed by direct sequencing. The unrelated probands both had the common R59W mutation while the other lesion was Y348C or R138P (both novel mutations), causing homozygous variegate porphyria.

Novel molecular defects of the delta-aminolevulinate dehydratase gene in a patient with inherited acute hepatic porphyria.

Cloning and expression of the defective gene for delta-aminolevulinate dehydratase (ALAD) from the second of 2 German patients with ALAD deficiency porphyria (ADP), who had been originally reported by Doss et al. in 1979, were performed. Cloning of cDNAs for the defective ALAD were performed using Epstein-Barr virus (EBV)-transformed lymphoblastoid cells of the proband, and nucleotide sequences of cloned cDNA were determined. Two separate mutations of ALAD cDNA were identified in each ALAD allele. One was G457A, termed "H1," resulting in V153M substitution, while the other was a deletion of 2 sequential bases at T(818) and C(819), termed "H2," resulting in a frame shift with a premature stop codon at the amino acid position of 294. Using allele-specific oligonucleotide hybridization, the mother of the proband was shown to have an H1 defect, while using genomic DNA analysis, the father was shown to have an H2 defect. Expression of H1 cDNA in Chinese hamster ovary cells produced an ALAD protein with only a partial activity (10.65% +/- 1.80% of the normal), while H2 cDNA encoded no significant protein. These data thus demonstrate that the proband was associated with 2 novel molecular defects of the ALAD gene, 1 in each allele, and account for the extremely low ALAD activity in his erythrocytes ( approximately 1% of normal).