The expression of protoporphyrinogen oxidase in human tissues.

Protoporphyrinogen oxidase is the penultimate enzyme in the haem biosynthetic pathway. In this study, the expression of protoporphyrinogen oxidase in a variety of human organs has been documented by immunohistochemical means at the light microscopy level in order to shed light on its inter- and intra-organ distribution. The expression varied amongst organs and the various cell types within an organ. The pattern of staining generally reflected presumed metabolic functionality and haem demand. Strongest staining was noted in hepatocytes, proximal convoluted tubules of the kidney, serous cells of the peribronchial gland in the lung, parietal cells of the stomach, tips of the villi in the small intestine and interstitial cells of the testis. Our results suggest that there are some significant sites of haem synthesis in addition to the liver and bone marrow, and should be borne in mind in studies related to haem or porphyrin dynamics and flux.

Molecular characterization of erythropoietic protoporphyria in South Africa.

BACKGROUND: Erythropoietic protoporphyria (EPP) results from a partial deficiency of ferrochelatase (FECH). Clinical expression normally requires coinheritance of a common hypomorphic FECH allele (IVS3-48C) in trans to a deleterious (primary) FECH mutation. OBJECTIVES: To characterize South African subjects with EPP, by identification and assessment of FECH sequence variations, including the IVS3-48C polymorphism. METHODS: Polymerase chain reaction amplification, single-strand conformational polymorphism analysis and restriction endonuclease analysis were employed to identify and determine the frequencies of FECH sequence variations, including the IVS3-48C polymorphism, in a study cohort of symptomatic and asymptomatic South African EPP family members, and a matched control cohort. RESULTS: We identified 29 patients from 18 families. With the exception of one family, who may represent a phenocopy of EPP, the presentation of EPP was typical. All were of European immigrant stock, and we have not identified EPP in other ethnic groups. Ten sequence variations were identified, including four apparent disease-causing mutations, the IVS3-48T/C polymorphism and five further polymorphisms. The molecular basis of EPP was established for 15 of the 17 families. A 5-bp deletion in exon 7 (757_761delAGAAG) was present in 12 of these families and haplotype studies in these families suggested a single mutational event and thus a local founder effect for this deletion. The other mutations were family specific and included two previously described splice-site mutations (IVS3+2T>G and IVS7+1G>A) and a novel 7-bp deletion in exon 4 (356_362delTTCAAGA). CONCLUSIONS: The IVS3-48C allele appears to modulate the phenotypic expression of EPP in the South African EPP cohort as observed in other populations.

A systematic study of the clinical and biochemical expression of variegate porphyria in a large South African family.

BACKGROUND: Variegate porphyria (VP) is an autosomal dominant disorder associated with deficient haem synthesis. Recent reports indicate that the clinical penetrance of VP may have been overestimated in studies which predated the availability of DNA-based testing for VP. OBJECTIVES: To undertake a study specifically designed to assess the clinical and biochemical penetrance of VP in a kindred characterized by gene status. METHODS: We studied a large family carrying the South African founder mutation which is known to result in almost complete haplodeficiency. All informative members were tested for the R59W mutation. Biochemical evidence of porphyria was sought by porphyrin analysis and by plasma fluorescence scanning. The presence of clinically expressed porphyria was assessed using a structured questionnaire and telephone or personal interview. RESULTS: Of 62 informative subjects, 33 had inherited the mutation. Of 28 adults, one subject had experienced a single acute attack. She and a further 10 subjects had experienced photosensitivity. The frequency of acute attacks in this family is therefore 4% (95% confidence interval, CI 1-18%), and of photosensitivity is 39.3% (95% CI 24-58%). The sensitivity and specificity of porphyrin analysis in this family were 0.46 (95% CI 0.30-0.64) and 1.00 (95% CI 0.85-1.00), respectively, and for plasma scanning the values were 0.85 (95% CI 0.58-0.96) and 1.00 (95% CI 0.72-1.00), respectively. CONCLUSIONS: The clinical penetrance of VP in our family is approximately 40%. Many more subjects with VP are diagnosed in an asymptomatic phase than previously, and the acute attack is now an uncommon manifestation of VP. Plasma scanning is more sensitive than faecal porphyrin analysis, but neither is sufficiently sensitive for the detection of carrier status.

Administration of oral activated charcoal in variegate porphyria results in a paradoxical clinical and biochemical deterioration.

BACKGROUND: Porphyrinogens are the obligate intracellular precursors of haem. These compounds are, however, unstable and are easily oxidized to the corresponding porphyrins, which are the form in which they are usually measured in the laboratory. A substantial enterohepatic cycling of porphyrins has been shown. Administration of oral activated charcoal, by interrupting this cycle, may reduce plasma and urine porphyrin levels in patients with some forms of porphyria. The effect of charcoal in subjects with variegate porphyria (VP) has not been reported. OBJECTIVES: To determine the clinical and biochemical effects of the administration of oral activated charcoal in patients with VP. METHODS: Oral activated charcoal was administered to eight subjects with VP. Clinical activity was assessed by skin lesion counts fortnightly for 6 weeks, 6 weeks after cessation of therapy, and during a subsequent 6-week control period during which no charcoal was taken. Urine and plasma porphyrins and urine precursors were measured by standard techniques. RESULTS: Treatment resulted in a significant increase in skin disease, urine and plasma porphyrins. CONCLUSIONS: Oral charcoal administration results in a paradoxical aggravation of VP, suggesting a complex and as yet undefined interaction of hepatic porphyrin metabolism and bowel porphyrin reabsorption. Oral sorbents should not be prescribed to subjects with VP.

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.

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.

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.

Identification and characterisation of a deletion (537delAT) in the protoporphyrinogen oxidase gene in a South African variegate porphyria family.

Variegate porphyria is an autosomal dominant disorder of haem metabolism resulting from a partial decrease in protoporphyrinogen oxidase activity. Variegate porphyria is highly prevalent in South Africa, the result of a founder effect now confirmed genetically as a single point mutation (R59W) which has been described in nearly all South African variegate porphyria patients studied. Only two other mutations (H20P, R168C) have been reported in South Africa. We utilised simultaneous, single-stranded conformational polymorphism and heteroduplex analysis, and direct sequencing to identify a further mutation; a 2 bp deletion in exon 6 which results in a premature stop codon 11 codons downstream from the mutation and is the first reported deletion in the protoporphyrinogen oxidase gene in a South African family. The familial segregation of this mutation strongly suggests that it is the disease causing mutation for variegate porphyria in this family. This further evidence for allelic heterogeneity limits the utility of tests for the R59W mutation in the diagnosis of variegate porphyria in South Africa.

Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis.

The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 microM, respectively. Polyclonal antibody to B. subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 microM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.

Variegate porphyria.

Variegate porphyria is an autosomal dominant inherited trait resulting in decreased activity of protoporphyrinogen oxidase. It is characterized clinically by photosensitive skin disease and a propensity to acute neurovisceral crises. The disease is found worldwide but has an exceptionally high frequency in South Africa. The gene for human protoporphyrinogen oxidase has been identified and sequenced, and several mutations in the protoporphyrinogen oxidase gene sequence have been identified. In South Africa, fewer patients now present with acute attacks, leaving a greater proportion with skin disease or asymptomatic disease. Acute attacks of variegate porphyria appear to be less easily provoked and to be milder than those associated with acute intermittent porphyria.

Variegate porphyria in South Africa, 1688-1996--new developments in an old disease.

Variegate porphyria, an autosomal dominant inherited trait resulting in decreased activity of protoporphyrinogen oxidase, the penultimate haem biosynthetic enzyme, is characterised clinically by photosensitive skin disease and a propensity to acute neurovisceral crises. The disease has an exceptionally high frequency in South Africa, owing to a founder effect. The specific mutation in the protoporphyrinogen oxidase gene sequence which represents this founder gene has been identified. Genetic diagnosis is therefore now possible in families in whom the gene defect is known. However, the exact nature and degree of activity of the porphyria can only be determined by detailed quantitative biochemical analysis of excreted porphyrins. The relative contributions of the acute attack and the skin disease to the total disease burden of patients with variegate porphyria is not static, and in South Africa there have been significant changes over the past 25 years, with fewer patients presenting with acute attacks, leaving a greater proportion to present with skin disease or to remain asymptomatic with the diagnosis being made in the laboratory. The most common precipitating cause of the acute attack of VP is administration of porphyrinogenic drugs. Specific suppression of haem synthesis with intravenous haem arginate is the most useful treatment of a moderate or severe acute attack. Although cutaneous lesions are limited to the sun-exposed areas, management of the skin disease of VP remains inadequate.

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.

Mapping of the variegate porphyria (VP) gene: contradictory evidence for linkage between VP and microsatellite markers at chromosome 14q32.

The gene for variegate porphyria (VP), an autosomal dominant disease with a high prevalance in South Africa, evidently due to a founder effect, was previously mapped to chromosome 14q32. In the current study this localization was evaluated by linkage and haplotype analyses using microsatellite markers spanning a region of more than 20 cM on chromosome 14q32. In many recent studies linkage disequilibrium between disease and marker loci has been utilized to map genes in founder populations, but we could not find any association between VP and the markers used in this study. Our data suggest that the allocation of VP to chromosome 14q32 may be incorrect.

Hepatoerythropoietic porphyria precipitated by viral hepatitis.

Porphyria cutanea tarda (PCT), the condition resulting from a deficiency of hepatic uroporphyrinogen decarboxylase activity, is the commonest form of porphyria. Both acquired and familial form exist and are commonly associated in adults with liver disease and hepatic iron overload. The condition is extremely rare in children; most cases of childhood PCT are familial and some particularly severe cases have been shown to have a hepatoerythropoietic porphyria or homozygous uroporphyrinogen decarboxylase deficiency. A case is described of hepatoerythropoietic porphyria in which the disease was first precipitated at the age of two by a coincidental hepatitis A infection and improved as the hepatitis cleared. This paper reviews the evidence that viral hepatitis may precipitate overt PCT in children in a manner analogous to the precipitation of PCT in adults by alcohol associated liver disease.

Homozygous variegate porphyria: an evolving clinical syndrome.

Variegate porphyria is one of the most frequently encountered genetic conditions in South Africa. It is inherited as an autosomal dominant disease and in excess of 300 heterozygous cases have been studied by the Cape Town unit. Despite this, the homozygous condition has not previously been encountered in South Africa. We report two cases of homozygous variegate porphyria, one of whom represents the first South African case. We delineate a syndrome principally characterized by growth retardation, developmental delay, epileptic seizures, photosensitivity and an abnormal porphyrin excretion pattern. In addition we describe, in one case, two features not previously reported: skin disease in areas unexposed to light and a severe sensory neuropathy which may account at least in part for the hand deformities of this disorder.

The effect of oral activated charcoal on the course of congenital erythropoietic porphyria.

The administration of oral activated charcoal to two patients with congenital erythropoietic porphyria has previously been reported to result in a marked reduction in plasma and urinary porphyrin concentrations and in one case, clinical remission. We describe an additional case in which the use of charcoal was associated with an apparent exacerbation of the biochemical activity of the disease following an initial period of remission. This result is unexpected, and currently unexplained. We conclude that charcoal therapy in porphyria may not be without risk, and should be used with caution.