A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria.

Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig. 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia. Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal. While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4). VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant. PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed. Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein. The human PPO gene contains thirteen exons, spanning approximately 4.5 kb. We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W). A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type. The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency. Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

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.

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.

Allosteric inhibition of human lymphoblast and purified porphobilinogen deaminase by protoporphyrinogen and coproporphyrinogen. A possible mechanism for the acute attack of variegate porphyria.

Variegate porphyria (VP) is characterized by photocutaneous lesions and acute neuropsychiatric attacks. Decreased protoporphyrinogen oxidase activity results in accumulation of protoporphyrin (ogen) IX and coproporphyrin (ogen) III. During acute attacks delta-aminolevulinic acid and porphobilinogen also increase, suggesting that porphobilinogen deaminase (PBG-D) may be rate limiting. We have examined the effects of porphyrinogens accumulating in VP on PBG-D activity in Epstein-Barr virus-transformed lymphoblast sonicates from 12 VP and 12 control subjects. Protoporphyrinogen oxidase activity was decreased and protoporphyrin increased in VP lymphoblasts. PBG-D in control lymphoblasts obeyed Michaelis-Menten kinetics (Vmax 28.7 +/- 1.8 pmol/mg per h, Hill coefficient 0.83 +/- 0.07). VP sonicates yielded sigmoidal substrate-velocity curves that did not obey Michaelis-Menten kinetics. Vmax was decreased (21.2 +/- 2.0 pmol/mg per h) and the Hill coefficient was 1.78 +/- 0.17. Addition of protoporphyrinogen IX and coproporphyrinogen III to control sonicates yielded sigmoidal PBG-D substrate-velocity curves and decreased PBG-D Vmax. Addition of porphyrins or uroporphyrinogen III did not affect PBG-D activity. Removal of endogenous porphyrin (ogens) from VP sonicates restored normal PBG-D kinetics. Purified human erythrocyte PBG-D obeyed Michaelis-Menten kinetics (Vmax 249 +/- 36 nmol/mg per h, Km 8.9 +/- 1.5 microM, Hill coefficient 0.93 +/- 0.14). Addition of protoporphyrinogen yielded a sigmoidal curve with decreased Vmax. The Hill coefficient approached 4. These findings provide a rational explanation for the increased delta-aminolevulinic acid and porphobilinogen during acute attacks of VP.

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.

Effects of heme arginate on cytochrome P450-mediated metabolism of drugs in patients with variegate porphyria and in healthy men.

We investigated the effects of heme on metabolism of coumarin, debrisoquin, caffeine, and lidocaine in seven female patients with variegate porphyria and in 10 healthy men. During baseline conditions metabolism of the drugs was identical in the two groups. Compared with the results without heme, a single infusion of heme arginate (3 mg/kg heme) significantly decreased the debrisoquin/4-hydroxy-debrisoquin metabolic ratio in subjects with porphyria (p = 0.016) and in the control subjects (p = 0.016) and increased formation of monoethylglycinexylidide from lidocaine (p = 0.016 and p = 0.004, respectively). Metabolism of coumarin and caffeine was not affected by heme. Our results show that, in patients with porphyria and in healthy subjects, exogenous heme is able to accelerate the reactions mediated by the cytochrome isozymes CYP2D6 (debrisoquin) and CYP3A4 (lidocaine) but not reactions mediated by CYP1A2 (caffeine) and CYP2A6 (coumarin). This suggests that influence of heme on drug metabolism is P450 isozyme-specific.

Recurrent missense mutation in the protoporphyrinogen oxidase gene underlies variegate porphyria.

The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway. Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO). Recently, VP has been linked to the PPO gene on chromosome 1q22-23, and several disease-causing mutations have been described. In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H). This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function. Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families.

Identification of a novel mutation of the CPO gene in a Japanese hereditary coproporphyria family.

Hereditary coproporphyria (HCP) is an autosomal dominant disease characterized by a deficiency of coproporphyrinogen oxidase (CPO) caused by a mutation in the CPO gene. Only 11 mutations of the gene have been reported in HCP patients. We report another mutation in a Japanese family. Polymerase chain reaction-single strand conformational polymorphism and direct sequence analyses demonstrated a C to T substitution in exon 1 of the CPO gene at nucleotide position 85, which lies in the putative presequence for targeting to mitochondria. This mutation changes the codon for glutamine to a termination codon at amino acid position 29. MaeI restriction analysis showed two other carriers in the family. The C-T mutation is located within a recently proposed putative alternative translation initiation codon (TIC-1), supporting that TIC-1 is the real TIC rather than TIC-2.

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.

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.

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.

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.

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.

A novel missense mutation in exon 4 of the human coproporphyrinogen oxidase gene in two patients with hereditary coproporphyria.

Hereditary coproporphyria (HCP) is an autosomal dominant disease characterized by a deficiency of coproporphyrinogen oxidase. To date, four mutations of the gene have been reported. We report here another mutation in two Japanese families with HCP, which was revealed by analysis of polymerase chain reaction (PCR)-amplified DNA fragments of the gene by a direct-sequencing method. A point mutation, G to A, was found in exon 4 of the gene at position 538 of the cDNA from the reported putative translation initiation codon ATG. This mutation results in a glycine to arginine substitution at amino acid 180. Two carriers in the family were successfully diagnosed by detecting the mutation using restriction analysis of the PCR products.

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.