RESUMEN
We describe developments in understanding of the porphyrias associated with each step in the haem biosynthesis pathway and the role of individuals whose contributions led to major advances over the past 150 years. The first case of erythropoietic porphyria was reported in 1870, and the first with acute porphyria in 1889. Photosensitisation by porphyrin was confirmed by Meyer-Betz, who self-injected haematoporphyrin. Günther classified porphyrias into haematoporphyria acuta, acuta toxica, congenita and chronica. This was revised by Waldenström into porphyria congenita, acuta and cutanea tarda, with the latter describing those with late-onset skin lesions. Waldenström was the first to recognise porphobilinogen's association with acute porphyria, although its structure was not solved until 1953. Hans Fischer was awarded the Nobel prize in 1930 for solving the structure of porphyrins and the synthesis of haemin. After 1945, research by several groups elucidated the pathway of haem biosynthesis and its negative feedback regulation by haem. By 1961, following the work of Watson, Schmid, Rimington, Goldberg, Dean, Magnus and others, aided by the availability of modern techniques of porphyrin separation, six of the porphyrias were identified and classified as erythropoietic or hepatic. The seventh, 5-aminolaevulinate dehydratase deficiency porphyria, was described by Doss in 1979. The discovery of increased hepatic 5-aminolaevulinate synthase activity in acute porphyria led to development of haematin as a treatment for acute attacks. By 2000, all the haem biosynthesis genes were cloned, sequenced and assigned to chromosomes and disease-specific mutations identified in all inherited porphyrias. These advances have allowed definitive family studies and development of new treatments.
Asunto(s)
Genómica , Hemo , Porfirias , Historia del Siglo XX , Humanos , Porfirias/genética , Porfirias/historia , Historia del Siglo XIX , Hemo/biosíntesis , Hemo/metabolismo , Historia del Siglo XXI , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismoRESUMEN
BACKGROUND: The porphyrias are a group of rare metabolic disorders whose diagnosis depends on identification of specific patterns of porphyrin precursor and porphyrin accumulation in urine, blood, and feces. Diagnostic tests for porphyria are performed by specialized laboratories in many countries. Data regarding the analytical and diagnostic performance of these laboratories are scarce. METHODS: We distributed 5 sets of multispecimen samples from different porphyria patients accompanied by clinical case histories to 18-21 European specialist porphyria laboratories/centers as part of a European Porphyria Network organized external analytical and postanalytical quality assessment (EQA) program. The laboratories stated which analyses they would normally have performed given the case histories and reported results of all porphyria-related analyses available, interpretative comments, and diagnoses. RESULTS: Reported diagnostic strategies initially showed considerable diversity, but the number of laboratories applying adequate diagnostic strategies increased during the study period. We found an average interlaboratory CV of 50% (range 12%-152%) for analytes in absolute concentrations. Result normalization by forming ratios to the upper reference limits did not reduce this variation. Sixty-five percent of reported results were within biological variation-based analytical quality specifications. Clinical interpretation of the obtained analytical results was accurate, and most laboratories established the correct diagnosis in all distributions. CONCLUSIONS: Based on a case-based EQA scheme, variations were apparent in analytical and diagnostic performance between European specialist porphyria laboratories. Our findings reinforce the use of EQA schemes as an essential tool to assess both analytical and diagnostic processes and thereby to improve patient care in rare diseases.
Asunto(s)
Técnicas de Laboratorio Clínico/normas , Porfirias/diagnóstico , Adolescente , Anciano , Biomarcadores/análisis , Preescolar , Europa (Continente) , Femenino , Humanos , Cooperación Internacional , Masculino , Persona de Mediana Edad , Porfirinas/análisis , Control de Calidad , Estándares de Referencia , Proyectos de InvestigaciónAsunto(s)
Síndromes Mielodisplásicos/genética , Porfiria Eritropoyética/genética , Anciano , Exones , Genes Recesivos , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/patología , Polimorfismo de Nucleótido Simple , Porfiria Eritropoyética/patología , Porfirinas/sangre , Porfirinas/orina , Enfermedades Cutáneas Vesiculoampollosas/genética , Uroporfirinas/genéticaRESUMEN
Many toxicological disorders, in common with numerous human diseases, are probably the consequence of multigene interactions with a variety of chemical and physiological factors. The importance of genetic factors may not be obvious initially from association studies because of their complexity and variable penetrance. The human disease, porphyria cutanea tarda (PCT), is a skin disease caused by the photosensitizing action of porphyrins arising secondary to the decreased activity of an enzyme of heme biosynthesis, uroporphyrinogen decarboxylase (UROD), in the liver. It is triggered by idiosyncratic hepatic interaction between genetic factors and chemicals such as alcohol, estrogenic drugs, and polyhalogenated aromatics. PCT and its animal models are known collectively as the hepatic uroporphyrias. There is strong evidence for the participation of iron in the pathogenesis of these conditions. Mouse models have been used to explore the relative importance of a variety of agents such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alcohol, and iron in the development of uroporphyria and to elucidate the mechanism of the depression of hepatic UROD activity. Mutations of the UROD and hemochromatosis (HFE) genes are genetic factors in some PCT patients which can be mimicked in mice heterozygous for the Hfe and Urod null genes. Association studies of uroporphyria induced by TCDD or hexachlorobenzene with DNA markers in mouse intercrosses have shown the participation of other, unknown, genetic factors in addition to the strong influence of the Ahr gene. The pathogenesis of hepatic uroporphyrias exemplifies the complexity of the interactions between chemical and genetic factors that can contribute to the hepatotoxicity of chemicals.
Asunto(s)
Porfirias Hepáticas/genética , Animales , Modelos Animales de Enfermedad , Hemocromatosis/genética , Humanos , Hierro/metabolismo , Hígado/enzimología , Ratones , Fármacos Fotosensibilizantes/toxicidad , Dibenzodioxinas Policloradas/toxicidad , Porfirias Hepáticas/inducido químicamente , Porfirinas/metabolismo , Uroporfirinógeno Descarboxilasa/genéticaRESUMEN
BACKGROUND: Clinically indistinguishable attacks of acute porphyria occur in acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). There are few evidence-based diagnostic strategies for these disorders. METHODS: The diagnostic sensitivity of mutation detection was determined by sequencing and gene-dosage analysis to search for mutations in 467 sequentially referred, unrelated patients. The diagnostic accuracy of plasma fluorescence scanning, fecal porphyrin analysis, and porphobilinogen deaminase (PBGD) assay was assessed in mutation-positive patients (AIP, 260 patients; VP, 152 patients; HCP, 31 patients). RESULTS: Sensitivities (95% CI) for mutation detection were as follows: AIP, 98.1% (95.6%-99.2%); HCP, 96.9% (84.3%-99.5%); VP, 100% (95.7%-100%). We identified 5 large deletions in the HMBS gene (hydroxymethylbilane synthase) and one in the CPOX gene (coproporphyrinogen oxidase). The plasma fluorescence scan was positive more often in VP (99% of patients) than in AIP (68%) or HCP (29%). The wavelength of the fluorescence emission peak and the fecal coproporphyrin isomer ratio had high diagnostic specificity and sensitivity for differentiating between AIP, HCP, and VP. DNA analysis followed by PBGD assay in mutation-negative patients had greater diagnostic accuracy for AIP than either test alone. CONCLUSIONS: When PBG excretion is increased, 2 investigations (plasma fluorescence scanning, the coproporphyrin isomer ratio) are sufficient, with rare exceptions, to identify the type of acute porphyria. When the results of PBG, 5-aminolevulinate, and porphyrin analyses are within reference intervals and clinical suspicion that a past illness was caused by an acute porphyria remains high, mutation analysis of the HMBS gene followed by PBGD assay is an effective strategy for diagnosis or exclusion of AIP.
Asunto(s)
Coproporfirinógeno Oxidasa/genética , Análisis Mutacional de ADN , Genes Dominantes , Hidroximetilbilano Sintasa/genética , Mutación , Porfirias/diagnóstico , Protoporfirinógeno-Oxidasa/genética , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Niño , Cartilla de ADN , Diagnóstico Diferencial , Humanos , Persona de Mediana Edad , Porfirias/genética , Estudios Retrospectivos , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.
Asunto(s)
Ferroquelatasa/genética , Genes Recesivos , Queratodermia Palmoplantar/complicaciones , Queratodermia Palmoplantar/genética , Protoporfiria Eritropoyética/complicaciones , Protoporfiria Eritropoyética/genética , Adolescente , Adulto , Niño , Femenino , Ferroquelatasa/fisiología , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Estaciones del AñoRESUMEN
Neurological complications are important components of an acute attack of porphyria, and early diagnosis and treatment of porphyria could prevent the development of these complications. Pischik et al. investigated the frequency of acute porphyria among patients admitted to neurological wards in Russia. The investigators identified 108 patients with acute polyneuropathy or encephalopathy, along with abdominal pain, back pain and/or dysautonomia. Urine samples were screened for acute porphyria by use of the qualitative Watson-Schwartz test for porphobilinogen and through measurement of coproporphyrin. Twelve patients had acute intermittent porphyria, and 11 had false-positive results. The specificity of the screen would have been improved by omission of the coproporphyrin test. The Watson-Schwartz test is, by itself, unreliable, and any positive test should be confirmed quantitatively. Improved identification of acute porphyria requires heightened clinical awareness and access to urinary porphobilinogen measurement. We suggest that all hospitals that admit acutely ill patients should be able to provide a validated determination of porphobilinogen within 24 h.
RESUMEN
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.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Cromosomas Humanos X/genética , Porfirias Hepáticas/patología , Eritrocitos/metabolismo , Femenino , Hemo/metabolismo , Humanos , Masculino , Mutación , Porfirias Hepáticas/genética , Protoporfirinas/sangreRESUMEN
Erythropoietic protoporphyria (EPP) results from deficiency of ferrochelatase (FECH). Accumulation of protoporphyrin IX causes life-long acute photosensitivity. Microcytic anemia occurs in 20% to 60% of patients. We investigated 178 patients with dominant EPP confirmed by molecular analysis. Erythropoiesis was impaired in all patients; all had a downward shift in hemoglobin (Hb), and the mean decreased in males by 12 g/L (1.2 g/dL). By World Health Organization criteria, 48% of women and 33% of men were anemic. Iron stores, assessed by serum ferritin (sFn), were decreased by two-thirds, but normal serum soluble transferrin receptor-1 and iron concentrations suggested that erythropoiesis was not limited by iron supply. FECH deficiency in EPP appears to lead to a steady state in which decreased erythropoiesis is matched by reduced iron absorption and supply. This response may in part be mediated by protoporphyrin, but we found no correlation between erythrocyte protoporphyrin and Hb, sFn, total iron-binding capacity, or transferrin saturation.
Asunto(s)
Antígenos CD/sangre , Eritropoyesis , Ferritinas/sangre , Hemoglobinas/análisis , Hierro/sangre , Protoporfiria Eritropoyética/sangre , Protoporfirinas/sangre , Receptores de Transferrina/sangre , Anemia/sangre , Anemia/enzimología , Estudios Transversales , Eritrocitos/enzimología , Eritrocitos/metabolismo , Femenino , Ferroquelatasa , Humanos , Masculino , Trastornos por Fotosensibilidad/sangre , Trastornos por Fotosensibilidad/enzimología , Protoporfiria Eritropoyética/enzimología , Factores SexualesRESUMEN
Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.
Asunto(s)
Ferroquelatasa/genética , Ferroquelatasa/fisiología , Eliminación de Gen , Dosificación de Gen , Protoporfiria Eritropoyética/genética , Análisis Mutacional de ADN , Exones , Salud de la Familia , Haplotipos , Humanos , Repeticiones de Microsatélite , Mutación , Reacción en Cadena de la Polimerasa , Protoporfirinas/metabolismo , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Late-onset erythropoietic protoporphyria (EPP) is a rare complication of myelodysplastic syndrome (MDS) but has not been described in association with a myeloproliferative disorder (MPD). EPP is normally an inherited disorder characterized by photosensitivity that starts in early childhood and results from overproduction of protoporphyrin secondary to ferrochelatase (FECH) deficiency. Severe liver disease occurs in 1% to 2% of patients. Here we report that severe photosensitivity and cholestatic liver disease in a patient with a myeloproliferative disorder was caused by excess protoporphyrin production from a clone of hematopoietic cells in which one FECH allele had been deleted. Our observations suggest that the usual explanation for the association of late-onset EPP with MPD and MDS is acquired somatic mutation of one FECH allele in bone marrow and show for the first time that the consequent overproduction of protoporphyrin may be severe enough to cause acute liver damage.
Asunto(s)
Ferroquelatasa/genética , Células Madre Hematopoyéticas/metabolismo , Hepatopatías/etiología , Trastornos Mieloproliferativos/complicaciones , Trastornos por Fotosensibilidad/etiología , Protoporfiria Eritropoyética/complicaciones , Protoporfiria Eritropoyética/genética , Enfermedad Aguda , Edad de Inicio , Colestasis/etiología , Células Clonales/patología , Eliminación de Gen , Células Madre Hematopoyéticas/patología , Humanos , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Protoporfiria Eritropoyética/etiología , Protoporfirinas/biosíntesisRESUMEN
Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.
Asunto(s)
Consumo de Bebidas Alcohólicas/genética , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana/genética , Porfiria Cutánea Tardía/genética , 5-Aminolevulinato Sintetasa/metabolismo , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Citocromo P-450 CYP1A2/metabolismo , Proteína de la Hemocromatosis , Hierro/metabolismo , Complejo Hierro-Dextran/farmacología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Porfiria Cutánea Tardía/etiología , Porfiria Cutánea Tardía/metabolismo , Uroporfirinas/metabolismoRESUMEN
Acute intermittent porphyria occasionally causes frequent and crippling acute neurovisceral attacks associated with increased hepatic production of porphyrin precursors, resulting in long-term damage, poor quality of life, and shortened life expectancy. There has been no cure for this condition, but replacement of deficient hepatic enzymes might restore excretion of porphyrin precursors to normal and prevent acute attacks. We aimed to treat severe acute intermittent porphyria in a 19-year-old woman by liver transplantation. After the transplant, concentrations of haem precursors in the patient's urine returned to normal, and 1.5 years later her quality of life was good. Our report suggests some hope of cure for selected patients with severe forms of this disease.
Asunto(s)
Trasplante de Hígado , Porfiria Intermitente Aguda/cirugía , Adulto , Ácido Aminolevulínico/orina , Femenino , Estudios de Seguimiento , Humanos , Trasplante de Hígado/métodos , Porfiria Intermitente Aguda/orina , Calidad de Vida , Resultado del Tratamiento , Uroporfirinógenos/orinaRESUMEN
Protoporphyrinogen oxidase (PPOX; EC 1.3.3.4), the penultimate enzyme of haem biosynthesis, is a nucleus-encoded flavoprotein strongly associated with the outer surface of the inner mitochondrial membrane. It is attached to this membrane by an unknown mechanism that appears not to involve a membrane-spanning domain. The pathway for its import to mitochondria and insertion into the inner membrane has not been established. We have fused human PPOXs containing N-terminal deletions, C-terminal deletions or missense mutations to yellow fluorescent protein (YFP) and have used these constructs to investigate the mitochondrial import of PPOX in human cells. We show that all the information required for efficient import is contained within the first 250 amino acid residues of human PPOX and that targeting to mitochondria is prevented by fusion of YFP to the N-terminus. Deletion of between 151 and 175 residues from the N-terminus is required to abolish import, whereas shorter deletions impair its efficiency. Fully efficient targeting appears to require both a major targeting signal, the whole or part of which is contained between residues 151 and 175, and which may be involved in anchoring to the inner mitochondrial membrane, together with interaction between this region and a sequence(s) within the first 150 residues. These features suggest that the mechanism for import of human PPOX to mitochondria differs from those identified for the translocation of nucleus-encoded, membrane-spanning, inner membrane proteins. In addition, a missense mutation outside this region (Val(335)-->Gly) prevented targeting to mitochondria and delayed the appearance of YFP fluorescence. This mutation appeared to prevent import by a direct effect on protein folding rather than by altering a sequence required for targeting. It may lead to sequestration of the PPOX-YFP construct in an unfolded conformation, followed by proteolytic degradation, possibly through enhanced binding to a cytosolic chaperone protein.
Asunto(s)
Mitocondrias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Secuencia de Aminoácidos , Línea Celular , Flavoproteínas , Humanos , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Transporte de Proteínas , Protoporfirinógeno-Oxidasa , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.
Asunto(s)
Etanol/farmacología , Proteínas de la Membrana/deficiencia , Porfiria Cutánea Tardía/inducido químicamente , Porfiria Cutánea Tardía/metabolismo , Uroporfirinas/orina , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Citocromo P-450 CYP1A2/metabolismo , Modelos Animales de Enfermedad , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Hierro/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados/genética , Distribución TisularRESUMEN
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.