Functional associations of genetic variants involved in the clinical manifestation of erythropoietic protoporphyria in the Argentinean population.

BACKGROUND: Combined inheritance of genetic variants in ferrochelatase gene (FECH) are implicated in clinical manifestation of Erythropoietic Protoporphyria (EPP). OBJECTIVE: Identify the genetic variants in FECH gene and their associations in the expression of EPP in Argentina. Determine the allelic frequency of polymorphic variants, associations in cis and its linkage disequilibrium. METHODS: The FECH gene was PCR-amplified and sequenced. Allelic variants of intragenic polymorphisms were identified by PCR followed by sequencing or restriction digestion analysis. Residual FECH activity was determined by prokaryotic expression in Escherichia coli JM109. Data were analyzed using Haploview and Statistix 9. RESULTS: Ten mutations were identified: three novel (p.S222N; p.R298X and p.R367X) and seven already known (g.12490_18067del; p.R115X; p.I186T; c.580_584delTACAG; c.598 + 1 G>T; p.Y209X and p.W310X). The p.R115X mutation was found in two families. The p.S222N mutation expressed 5% of normal activity. Only individuals who inherited a mutation combined in trans to a low expression allele c.1-251G, c.68-23T, and c.315-48C, showed clinical symptoms. The absence of c.315-48C variant was sufficient for not triggering EPP. However, these variants showed high levels of cosegregation and GTC haplotype is over-represented in EPP patients. CONCLUSION: In the dominant inheritance form of EPP, c.315-48C variant in trans to the mutated allele is sufficient to trigger the disease. The presence of GTC haplotype in all patients with dominant EPP could be due to the high level of cosegregation of c.315-48C with c.1-251G and c.68-23T variants in our population.

Scavengers protection of cells against ALA-based photodynamic therapy-induced damage.

The exogenously stimulated formation of intracellularly generated protoporphyrin IX, a precursor of haem, is becoming one of the fastest developing areas in the field of photodynamic therapy (PDT). We tested the action of several free radical scavengers, amino acids, antioxidants and sulphur-containing compounds as protectors from photodamage induced by 5-aminolaevulinic acid (ALA)-mediated PDT, employing the LM2 cell line, derived from a mammary murine adenocarcinoma. We exposed the cells to different concentrations of the compounds, 24 h before PDT, during PDT, and 19 h after treatment. We defined the protection grade (PG) as the ratio between cell survival after ALA-PDT treatment in the presence of the protector and cell survival of ALA-PDT treatment alone. We found that l -tryptophan (PG=9.2 at 2 m m ), reduced glutathione (GSH) (PG=5.8 at 0.8 m m ), N-acetyl- l -cysteine (PG=4.86 at 30 m m ), melatonin (PG=4.5 at 8 m m ) and l -methionine (PG=4.0 at 0.8 m m ) are the best protectors from PDT damage, followed by l -cysteine (PG=2.8 at 0.8 m m ), mannitol (PG=2.6 at 20 m m ) and glycine (PG=2.4 at 40 m m ) whereas oxidised glutathione and S-adenosyl- l -methionine do not exert any protection. We did not found any photoactive action of the protectors in absence of ALA. These results can be considered to modulate the photodamage induced by ALA-PDT.

Photodynamic therapy of activated and resting lymphocytes and its antioxidant adaptive response.

In this work we have studied the effects of ALA-mediated photodynamic therapy (PDT) on resting and mitogen-activated murine splenic lymphocytes, evaluating its impact on cell viability. We have also characterised the stress response, measuring the levels of antioxidant enzymes. A 2h exposure to ALA produced 50% lethality upon irradiation of activated cells with 2.1J/cm2. The decrease in cell survival with increasing time exposure to ALA, correlated well with the higher porphyrin accumulation. In resting lymphocytes, in spite of the low amount of porphyrins formed during 2h incubation with ALA, 40% of the cells died after irradiation, this response was not further increased when higher amounts of porphyrins were synthesised. Superoxide dismutase was impaired by light treatment independently of ALA exposure in activated lymphocytes and, to a lesser extent, in resting lymphocytes. PDT induced an antioxidant adaptive response in activated cells 19 h after irradiation, reflected as a net increase in GSHPx activity and a slight reversion of the catalase (CAT) activity already impaired by light treatment. PDT treatment of activated cells also produced a diminution in the GSH/GSSG ratio. Only activated cells are capable of developing an antioxidant adaptive response to PDT treatment.

Identification and characterization of two novel mutations that produce acute intermittent porphyria: A 3-base deletion (841-843delGGA) and a missense mutation (T35M).

A partial deficiency of Porphobilinogen deaminase (PBGD) is responsible for acute intermittent porphyria (AIP). AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000. Here, two new mutations and two previously reported were found in the PBGD gene in 22 Argentinean AIP patients corresponding to 8 different families. To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 6 PCR reactions, then all coding exons and flanking intronic regions were sequenced. The novel mutations are 841-843delGGA in exon 14, which results in the loss of glycine-281 (G281del), and one 104C>T point mutation in the exon 4 (T35M). To further characterize both novel mutations, the pKK-PBGD construct for the mutant alleles were expressed in E. coli, the enzymatic activity of the recombinant proteins were 1% and 4% of the mean level expressed by the normal allele for 841-843delGGA and T35M, respectively. Hum Mutat 16:373, 2000.

Porfiria variegata: signos cutáneos, agudos y bioquímicos. Primer estudio genético en pacientes Argentinos / Variegate porphyria: cutaneus sign, biochemistry and sharp. Genetic first study in Argentinean patients

Las porfirias son enfermedades metabólicas que surgen como consecuencia de una deficiencia enzimática parcial de alguna de las enzimas del hemo. En la porfiria Variegata (PV) este defecto se encuentra a nivel de la Protoporfirinógeno oxidasa (PPOX) quwe transforma el Protoporfirinógeno IX en Protoporfirina IX. Se caracteriza por presentar el síndromeagudo y cutáneo. Es una patología genéticamente heterogénea, al presente se han detectado 77 mutaciones diferentes en el gen de la PPOX, responsables de esta porfiria. Nuestro objetivo es el estudio bioquímico y molecular de pacientes con sintomatología de PV para llegar a un dignóstico certero de la enfermedad y hacerlo extensivo a sus familiares con el fin de identificar a los portadores asintomáticos y asesorarlos acerca del contacto con los factores desencadenantes de esta porfiria. Hasta el momento el estudio genético nos permitió detectar 2 mutaciones, una mutación puntual recientemente descripta que produce cambio de aminoácido, R168H en el exón 6 y una inserción nueva, 1320InT, que produce corrimiento del marco de lectura. Estos resultados han permitido confirmar el diagnóstico de PV en estos pacientes

Porfiria variegata: signos cutáneos, agudos y bioquímicos / Primer estudio genético en pacientes Argentinos / Variegate porphyria: cutaneus sign, biochemistry and sharp. Genetic first study in Argentinean patients

Las porfirias son enfermedades metabólicas que surgen como consecuencia de una deficiencia enzimática parcial de alguna de las enzimas del hemo. En la porfiria Variegata (PV) este defecto se encuentra a nivel de la Protoporfirinógeno oxidasa (PPOX) quwe transforma el Protoporfirinógeno IX en Protoporfirina IX. Se caracteriza por presentar el síndromeagudo y cutáneo. Es una patología genéticamente heterogénea, al presente se han detectado 77 mutaciones diferentes en el gen de la PPOX, responsables de esta porfiria. Nuestro objetivo es el estudio bioquímico y molecular de pacientes con sintomatología de PV para llegar a un dignóstico certero de la enfermedad y hacerlo extensivo a sus familiares con el fin de identificar a los portadores asintomáticos y asesorarlos acerca del contacto con los factores desencadenantes de esta porfiria. Hasta el momento el estudio genético nos permitió detectar 2 mutaciones, una mutación puntual recientemente descripta que produce cambio de aminoácido, R168H en el exón 6 y una inserción nueva, 1320InT, que produce corrimiento del marco de lectura. Estos resultados han permitido confirmar el diagnóstico de PV en estos pacientes(AU)

Acute intermittent porphyria: biochemical and clinical analysis in the Argentinean population.

Acute intermittent porphyria (AIP) is the most common type of hepatic acute porphyria. In this work, we have analyzed the biochemical data of all Argentinean AIP families studied in the Porphyrins and Porphyrias Research Centre (CIPYP). We have shown that: (i) the prevalence for this population is about 1:125,000; (ii) the disease is more frequent in women than in men (7:3); (iii) about 60% are latent carriers; (iv) 15% of patients with symptomatic AIP died during an acute attack; (v) the most important precipitating factors of acute attacks in our population were the ingestion of therapeutic drugs (25%), anesthetics in surgical interventions (25%) and infections (20%); (vi) the initial symptom in Argentinean AIP individuals is severe abdominal pain (100%), and it is often accompanied by constipation (37%), anorexia (37%) and tachycardia (30%); and (vii) the percentage of recurrence of the acute attacks is high (81%).

Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles.

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Regulatory role of ALA-S and ALA-D in a haem-deficient mutant of Saccharomyces cerevisiae.

The biochemical characteristics of a haem-deficient mutant strain B231 of Saccharomyces cerevisiae isolated from D273-10B strain are described. B231 strain accumulates substantial amounts of 5-aminolevulinic acid (ALA) and uroporphyrin III (uro III). This pattern of porphyrins accumulation is due to both a defect in uroporphyrinogen decarboxylase (Uro-D) activity and an enhancement of porphobilinogenase (PBGase) activity. ALA accumulation would indicate that feed-back control by haem is not operating in this strain. ALA synthase (ALA-S), ALA-dehydrase (ALA-D) and PBGase activities; intracellular content of ALA (I-ALA) and porphyrins (I-porphyrins) were examined during the different phases of growth. Both accumulation of metabolites and enzyme activities reached their maximum values at 20 hrs. of growth, when glucose concentration in the medium fell to zero. Evidence for negative feed-back control on ALA-S and ALA-D by heme are provided by the observations of both enhanced I-ALA accumulation and increased ALA-D activity (2.5 times) in the mutant strain. Our results would indicate that both ALA-S and ALA-D can be considered regulatory enzymes in yeast.

Metabolic changes in the heme pathway driven by cyclophosphamide treatment in mice.

In previous work we found a 30% increase in the effectiveness of the photodynamic treatment of cancer when combined with the administration of cyclophosphamide (CPM). Here we have tried to elucidate the mechanism responsible for such potentiation. Male Balb/C mice bearing a transplantable adenocarcinoma were given 2 or 3 doses of 150 mg of CPM/kg weight intraperitoneally. At 16 and 40 hrs. after the last injection the animals were sacrificed. Tumor and liver were excised and 5-aminolevulinic acid dehydratase and porphobilinogen deaminase activities were determined. Intracellular levels of glutathione and cytochrome P450 were also measured. A 15 to 30% decrease in liver 5-aminolevulinic acid dehydratase activity was observed 40 hrs. after the last injection. The tumor enzyme was 30 to 40% inhibited. The activity of liver porphobilinogen deaminase in CPM treated mice decreased to a minimum (15% below the control) at 16 hrs. after administration of the drug and in tumors a decrease of 20% was shown 40 hrs. post CPM injection. The greater the number of CPM doses administered the higher the decrease in the enzymatic activities. CPM treatment did not change total tumor glutathione levels but the reduced/oxidized glutathione ratio was significantly modified in the tumoral tissue. Cytochrome P450 levels were not increased. These data indicate that CPM-induced potentiation of the photodynamic damage of tumoral tissue is mediated by a mechanism other than that of increased porphyrin synthesis.

Enhancement of aminolevulinic acid based photodynamic therapy by adriamycin.

This paper reports on studies that evaluate the interaction between delta-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) and adriamycin (ADM) in an animal model system. Two groups of mice bearing a transplantable mammary adenocarcinoma received ADM i.p. in a single dose of 5 mg (low dose) and 30 mg (high dose) per kg body weight. Sixteen or 40 h after administration of the drug, mice were sacrificed, tumours, livers and hearts were removed and porphyrins, enzyme activities and malondialdehyde content were determined. Tumour explants of ADM-treated mice were incubated with ALA and irradiated with an He-Ne laser. Re-implantation of these in vitro PDT-treated explants into test animals showed that inhibition of tumour growth was significantly enhanced by combined treatment when the low dose of ADM was used. There were no significant changes in porphyrin content, ALA dehydratase and porphobilinogenase activities in the tissues analyzed after ADM treatment as compared with control values. ADM toxicity is thought to be related to semiquinone free radical formation with subsequent generation of reactive oxygen species such as peroxide and hydroxyl radical. These species are considered to initiate lipid peroxidation (LPO) and cause DNA damage. In the case of low-dose treatment with ADM a significant increase in the LPO product, malondialdehyde, was observed after PDT whereas with the high-dose regimen no changes were observed. In the case of explants of (non-irradiated) cardiac tissue malondialdehyde production was also found to be dependent on the dose and time of administration of adriamycin. In our in vivo/in vitro model system we have shown that pre-treatment with ADM increased the cytotoxicity of ALA-PDT at a dosage level of ADM which did not raise LPO levels in heart tissue. The mechanism of this effect has not been clearly elucidated but our data suggest that the observed enhancement of PDT may be attributed in part to the weakening of cellular defence mechanisms by the pre-treatment involving free radical generation by ADM.

Altered 5-aminolevulinic acid metabolism leading to pseudoporphyria in hemodialysed patients.

Hemodialysed patients with no history of porphyria may present neurological symptoms similar to those seen in acute porphyrias. Porphyria has been associated with an increase in plasma levels of 5-aminolevulinic acid and porphobilinogen. Our aim was to evaluate these parameters and the activities of the enzymes involved in the first steps of heme metabolism in non-porphyric hemodialysed patients. The activities of 5-aminolevulinate dehydratase and deaminase were determined in red blood cells (RBC) from 78 hemodialysed patients, before and after dialysis. Plasma levels of 5-aminolevulinic acid, porphobilinogen and zinc were also measured. These parameters were also measured in 40 volunteers to obtain controls levels. The levels of 5-aminolevulinic acid (0.98 +/- 0.09 microgram/ml) and porphobilinogen (1.32 +/- 0.13 micrograms/ml) were raised in non-porphyric patients prior to hemodialysis (P < 0.001) compared with controls (5-aminolevulinic acid 0.13 +/- 0.02 microgram/ml; porphobilinogen 0.90 +/- 0.09 microgram/ml). After dialysis there was a decrease in both 5-aminolevulinic acid (to 0.61 +/- 0.05 microgram/ml) and porphobilinogen (to 1.10 +/- 0.16 micrograms/ml) although both parameters remained higher than controls (P < 0.001). The activities of both 5-aminolevulinate dehydratase (0.550 +/- 0.095 U/ml RBC), and deaminase (54.13 +/- 9.13 U/ml RBC) were diminished in blood samples of patients before dialysis (P < 0.001) compared to controls (dehydratase 0.975 +/- 0.115 U/ml RBC; deaminase 77.32 +/- 10.00 U/ml RBC). After dialysis 5-aminolevulinate dehydratase activity was partially recovered (to 0.666 +/- 0.100 U/ml RBC) while deaminase returned to normal values (73.45 +/- 9.46 U/ml RBC). The plasma zinc concentration in hemodialysed patients (44 +/- 12 micrograms/100 ml) was significantly lower than controls (105 +/- 30 micrograms/100 ml, P < 0.001). Addition of 22.5 mM zinc to the dehydratase reaction mixture raised the activity of 5-aminolevulinate dehydratase in blood samples of hemodialysed patients taken before and after dialysis. The study reports a partial loss of activity of 5-aminolevulinate dehydratase and deaminase activities in red blood cells from non-porphyric patients undergoing hemodialysis. Since plasma zinc levels were below normal in hemodialysed patients, and the activity of 5-aminolevulinate dehydratase could be restored by the addition of zinc, it is suggested that these abnormalities in heme metabolism may be explained by altered zinc and associated antioxidant status following dialysis.

Zinc aminolevulinic acid dehydratase reactivation index as a tool for diagnosis of lead exposure.

The correlation between blood lead level (BLL), delta-aminolevulinate dehydratase (ALA-D) activity, and other common biochemical parameters used to assess a plumbism diagnosis have been carefully analyzed, with the aim of correctly interpreting the data handled in the laboratory. No correlation was observed between BLL and free erythrocyte porphyrins. In the case of ALA-D or Zn-reactivated ALA-D despite the direct correlation with BLL, the curve follows a potential or a logarithmic line, which is not the best to calculate BLL. The so-called Zn-ALA-D-reactivation index (iZn) has been defined as the ratio between the activity of Zn-reactivated ALA-D and the activity of ALA-D. The plot of BLL against the iZn revealed a very good linear relationship which allows an estimate of BLL with reasonable accuracy within a very wide range.

Acquired and inherited porphyria: clinical and biochemical features.

Inheritance in 30 cases of porphyria cutanea tarda (PCT) and their relatives was investigated. Seventeen families were studied using the clinical criteria, quantitation, and thin layer chromatography of urinary porphyrins. Thirteen families (13 propositus and 48 relatives) were investigated by using the above criteria and in vitro porphyrin biosynthesis by erythrocytes from delta-aminolevulinic acid. Three different types of PCT were identified: overt, subclinical, and latent. Among 61 members examined, 13 had overt PCT. In six families, ten members had subclinical PCT and six latent PCT showing that in these six families PCT was a hereditary disorder. In seven other families inheritance could not be demonstrated.

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells.

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.

Porphyrin biosynthesis. Immobilized enzymes. IV. Studies on aminolaevulate dehydratase attached to Sepharose.

Bovine liver aminolaevulate dehydratase (ALA-D) has been chemically attached to Sepharose 4B and its properties have been studied. The optimal conditions for coupling have been determined. It was found that the immobilized enzyme retained a significant percentage of the activity of the free enzyme. The coupling yield was rather high. The insolubilized enzyme requires both anaerobiosis and a thiol activator for maximal activity. It can be stored at 4 degrees C for long periods with little loss of activity and it can be repeatedly used without alteration of its enzymic capacity. Attachment of ALA-D to the gel has led to an enhanced thermal stability. pH optima of free and bound enzyme was the same while a small decrease in the Km of the matrix bonded ALA-D as compared to that of the soluble enzyme was observed. The use of the fixed-ALA-D for the preparation of PBG is described.

Porphyrin biosynthesis. Immobilized enzymes and ligands. VII. Studies on bovine liver porphobilinogenase.

Bovine liver porphobilinogenase (PBGase) has been covalently attached to Sepharose, and some of their properties have been studied. The optimal conditions for binding have been determined. The water-insoluble PBGase retained a high percentage of the activity of the soluble enzyme; the coupling yield was also high. Sepharose-PBGase could be stored at 4 C for periods up to 5 weeks with 40% loss of activity; however, both by storage and repeated use, isomerase was inactivated and the percentage of uroporphyrinogen I formed was increased. Attachment of PBGase to Sepharose has led to enhanced thermal stability. pH optima of the insolubilized enzyme was shifted 0.6 units towards the alkaline side as compared to that of the native enzyme.

Studies on cow liver porphobilinogen deaminase.

Further properties of cow liver deaminase are reported. Highly purified deaminase migrated as a single badn on starch and polycrylamide gels electrophoresis. Molecular weight determinations by means of gel filtration on calibrated columns of Sephadex G-100, Sepharose 4 B and B 10-Gel P-100, gave values of 40 000 +/- 4 000. Data obtained suggest that cow liver deaminase exists as a single polypeptide chain. Heating partially purified preparations of deminase resulted in an enhancement of activity. Added cosynthetase to these fractions increased the percentage of uroporphyrinogen III formed but also diminished total uroporphyrinogens synthesis. The action of several compounds added to the system was studied. Thiol reagents and divalent metals as Hg 2+ , Pb2+, (d2+ and Zn2+ inhibited deaminase, indicating the presence of thiol groups essential for activity, probably involved in the cyclization step. Certain concentrations of sodium, potassium and magnesium salts enhanced activity. Several chelators tested were without effect on the deminase. Some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the enzyme.

Porphyrin biosynthesis. Immobilized enzymes and ligands. VII. Studies on bovine liver porphobilinogenase.

Bovine liver porphobilinogenase (PBGase) has been covalently attached to Sepharose, and some of their properties have been studied. The optimal conditions for binding have been determined. The water-insoluble PBGase retained a high percentage of the activity of the soluble enzyme; the coupling yield was also high. Sepharose-PBGase could be stored at 4 C for periods up to 5 weeks with 40

loss of activity; however, both by storage and repeated use, isomerase was inactivated and the percentage of uroporphyrinogen I formed was increased. Attachment of PBGase to Sepharose has led to enhanced thermal stability. pH optima of the insolubilized enzyme was shifted 0.6 units towards the alkaline side as compared to that of the native enzyme.