Peter N. Meissner

C-Terminal Deletions in the ALAS2 Gene Lead to Gain of Function and Cause X-linked Dominant Protoporphyria without Anemia or Iron Overload

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

The acute porphyrias

Public attention has been drawn to the acute porphyrias in the past few years by speculation that they affected the character of George III and the creative genius of Vincent van Gogh. During the same period, there have been important advances in the understanding of the molecular basis of the acute porphyrias and in diagnosis and the clinical management of patients and their families. Four types of porphyria are classified as acute because they produce acute neurovisceral crises (panel). Here, we outline current knowledge of the three autosomal dominant types: acute intermittent porphyria, variegate porphyria, and hereditary coproporphyria.

An analysis of 112 acute porphyric attacks in Cape Town, South Africa: Evidence that acute intermittent porphyria and variegate porphyria differ in susceptibility and severity.

Abstract: Four forms of porphyria may present clinically with the acute attack, an episodic, severe, and potentially life-threatening manifestation characterized by abdominal and neurologic symptoms. We describe our experience with 112 consecutive attacks observed and treated in 25 patients with the 2 most common forms of acute porphyria in Cape Town, South Africa; 25 attacks in 10 patients with variegate porphyria and 87 attacks in 14 patients with acute intermittent porphyria. The remaining patient experienced more than 100 sequential, severe, and poorly remitting attacks, which are not included in our analysis. In our population, the relative risk of an acute attack in acute intermittent porphyria compared with that in variegate porphyria was 14.3 (confidence intervals, 6.3-32.7). Patients with variegate porphyria were significantly older (median age at first attack, 30 yr) than those with acute intermittent porphyria (median age at first attack, 23.5 yr; p < 0.0001), and demonstrated an equal sex ratio, whereas the male:female ratio in acute intermittent porphyria was 2:12 (p < 0.0001). There was a significant difference in the incidence of factors precipitating the acute attack. Drug exposure was a frequent precipitant of the acute attack in variegate porphyria, whereas hormonal factors were more important in acute intermittent porphyria (p < 0.00001). Patients with acute intermittent porphyria also showed a trend to earlier and more frequent recurrent acute attacks following the initial admission. Mean urine precursor levels, blood pressure, pulse rate, and heme arginate requirement were all significantly higher in patients with acute intermittent porphyria. No significant difference in the frequency of serious complications or in outcome could be shown. We describe our experience with treatment with heme arginate, and provide evidence that heme arginate results in a prompt and statistically significant improvement in symptoms. The incidence of serious complications and mortality in this series was low, confirming a trend to an increasingly good prognosis for patients with acute porphyria who receive expert treatment. Abbreviations: AIP = acute intermittent porphyria, ALA = aminolevulinic acid, CT = computed tomography, PBG = porphobilinogen, VP = variegate porphyria.

Crystal Structure of Protoporphyrinogen Oxidase from Myxococcus xanthus and Its Complex with the Inhibitor Acifluorfen

Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.

Erythroid Heme Biosynthesis and Its Disorders

Heme, which is composed of iron and the small organic molecule protoporphyrin, is an essential component of hemoglobin as well as a variety of physiologically important hemoproteins. During erythropoiesis, heme synthesis is induced before, and is essential for, globin synthesis. Although all cells possess the ability to synthesize heme, there are distinct differences between regulation of the pathway in developing erythroid cells and all other types of cells. Disorders that compromise the ability of the developing red cell to synthesize heme can have profound medical implications. The biosynthetic pathway for heme and key regulatory features are reviewed herein, along with specific human genetic disorders that arise from defective heme synthesis such as X-linked sideroblastic anemia and erythropoietic protoporphyria.

Protoporphyrinogen oxidase and porphobilinogen deaminase in variegate porphyria.

Abstract. Two enzymes of the haem biosynthetic pathway were investigated in patients with variegate porphyria. Protoporphyrinogen oxidase in cultures of Epstein‐Barr virus transformed lymphoblasts from twenty‐seven patients showed a mean maximal velocity (Vmax) of 0·39 ± 0·08+ nmol of protoporphyrin mg protein‐1 h‐1, a 52% reduction (P < 0·001) from a non‐porphyric control group (0·82 ± 0·10). Km values (1·00 ± 0·27 μM) did not differ significantly (P > 0·05) from control values in any of the patients. The mean Vmax of porphobilinogen deaminase in the cultures was 1·50 ± 0·18 nmol of uroporphyrin mg protein‐1 min‐1, a 24% reduction (P < 0·001) from controls (1·94 ± 0·14). Mean porphobilinogen deaminase activity in the erythrocytes of twenty‐one patients with variegate porphyria was 8·37 ± 1·99 nmol of uroporphyrin 1 erythrocytes‐1 s‐1, a 28% reduction (P < 0·001) from normal (11·98 ± 2·11). The reduced activities of these two enzymes comply with the expression of variegate porphyria during its quiescent and acute phases.

Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases

The effects of various protoporphyrinogen oxidase (PPOX) mutations responsible for variegate porphyria (VP), the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant human PPOX and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The K(m)s for the arginine-59 mutants suggested a substrate-binding problem. T(1/2) indicated that arginine-59 is required for the integrity of the active site. The dominant alpha-helical content was decreased in the mutants. The degree of alpha-helix did not correlate linearly with T(1/2) nor T(m) values, supporting the suggestion that arginine-59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two-state transition suggesting formation of a multi-domain structure. All mutants studied were more resistant to thermal denaturation compared to wild type, except for R168C.

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.

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.

A review of the clinical presentation, natural history and inheritance of variegate porphyria: its implausibility as the source of the ‘Royal Malady’

It has been suggested that King George III of Great Britain suffered from the haem biosynthetic disorder, variegate porphyria. This diagnosis is pervasive throughout the scientific and popular literature, and is often referred to as the ‘Royal Malady.’ The authors believe it inappropriate to view the case for porphyria purely in terms of symptoms, as has generally been the case in his presumptive acute porphyria diagnosis. Accordingly, this review provides a current description of the natural history and clinical presentation of the porphyrias, against which we measure the case for porphyria in George III and his relatives. The authors have critically assessed the prevalence of porphyria in a population, the expected patterns and frequency of inheritance, its penetrance and its expected natural history in affected individuals, and conclude that neither George nor his relatives had porphyria, based on four principal reasons. First, the rarity of the disease mandates a very low prior probability, and therefore implies a vanishingly low positive predictive value for any diagnostic indicator of low specificity, such as a historical reading of the symptoms. Second, penetrance of this autosomal dominant disorder is approximately 40%, and one may expect to have identified characteristic clinical features of porphyria in a large number of descendants without difficulty. Third, the symptoms of both George III and his relatives are highly atypical for porphyria and are more appropriately explained by other much commoner conditions. Finally, the natural history of the illnesses reported in this family is as atypical for variegate porphyria as are their symptoms.