María Victoria Rossetti

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%).

δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

BackgroundAcute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines.ResultsWe have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

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.

Acute intermittent porphyria: Characterization of two novel mutations in the porphobilinogen deaminase gene, one amino acid deletion (453‐455delAGC) and one splicing aceptor site mutation (IVS8‐1G>T)

A partial deficiency of Porphobilinogen deaminase (PBG‐D) 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 three previously reported were found in the PBG‐D gene in 12 Argentinean AIP patients corresponding to 5 different families. To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 2 Multiplex PCR reactions, then all coding exons and flanking intronic regions were sequenced. The new mutations are 453‐455delAGC in exon 9 which results in the loss of an alanine residue at position 152, and one new point mutation in the splicing aceptor site in the last position of intron 8 (IVS8‐1G>T) which leds to a 15 bp deletion because a cryptic site (first AG upstream) is used. Both mutations produce amino acid deletion without frameshift effect. To further characterize the 453‐455delAGC mutation, the pKK‐PBGD construct for the mutant allele was expressed in E. coli, the enzymatic activity of the recombinant protein was 1.3% of the mean level expressed by the normal allele. Finally, three missense mutations, previously reported, were identified in three unrelated families. Hum Mutat 14:355, 1999.

Studies on Porphobilinogen-Deaminase from Saccharomyces cerevisiae

Abstract Porphobilinogen-deaminase from Saccharomyces cerevisiae has been isolated and partially purified 80-and 230-fold in the absence or presence of phenylmethylsulphonyl fluoride, respectively. Some properties of the isolated enzyme were studied. Porphyrin formation was linear with time and protein concentration. Optimum pH was about 7.5-7.8. Molecular mass of the protein was 30,000 ± 3000 Dalton when the enzyme was purified in the presence of phenylmethyl sulphonyl fluoride. A less active and unstable 20,000 Da molecular mass species was obtained when purification was performed in the absence of the protease inhibitor. Porphobilinogen-deaminase exhibited classical Michaelis-Menten kinetics. The apparent Km for uroporphyrinogen formation was 19 μм; Vmax was 3.6 nmol uroporphyrin/h and the Hill coefficient was n = 1. Also the action of several reagents on the activity was studied. Protective thiol agents had no effect. Heavy metals inhibited both porphyrin formation and porphobilinogen consumption, but known sulphydryl inactivating chemicals inhibit the former without modifying the latter. Ammonium ions had no effect on the activity while hydroxylamine completely inhibited both porphyrin formation and porphobilinogen consumption.

S-adenosyl-L-methionine and lead intoxication: its therapeutic effect varying the route of administration.

A comparative study on the effect of oral and subcutaneous (sc) or intravenous (iv) administration of S-adenosyl-L-methionine (SAM) in lead poisoning was carried out. SAM was given daily sc (20 mg/kg) and orally (80 mg/kg) to acute lead-intoxicated mice for 20 days. Chronic lead-poisoned patients received SAM, administered intravenously at a daily dose of 12 mg/kg or orally at a dose of 25-30 mg/kg. Independent of the method of administration in either animals or patients, GSH concentration in reduced lead intoxication was increased after SAM dosing. Corresponding blood lead content rapidly decreased and a significant recovery of hepatic and erythrocytic delta-aminolevulinate dehydratase (ALA-D), initially reduced, was clearly produced in the groups receiving SAM, although the response was slightly slower when SAM was given orally. It was found that the bulk of body lead burden was excreted in the feces, showing a peak within the first 24-48 hr and being much greater in animals treated with SAM. In these cases, urinary lead excretion was very low. Lead ALA-D inhibition was also evidenced by elevated urinary excretion of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), and porphyrins. During treatment, precursors and porphyrins elimination declined, reaching normal levels soon after therapy ended. A good correlation between the recovery of both GSH levels and ALA-D activity and decreased lead content was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in familial porphyria cutanea tarda: Two novel and two previously described for hepatoerythropoietic porphyria Communicated by: Mark H. Paalman Online Citation: Human Mutation, Mutation in Brief #354 (2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/354.pdf

Uroporphyrinogen decarboxylase (URO-D) deficiency is responsible for two forms of genetic cutaneous porphyria: familial porphyria cutanea tarda (f-PCT) and hepatoerythropoietic porphyria (HEP). The f-PCT transmitted as an autosomal dominant trait, is characterized by photosensitive cutaneous lesions frequently associated to hepatic dysfunction and is precipitated by various ecogenic factors. The HEP, transmitted as a recessive trait, is more severe than f-PCT and would be considered as the homozygous form of f-PCT. For the mutational analysis of f-PCT patients, the entire URO-D gene was amplified and each exon, intron-exon boundaries and the promoter region were cycle sequenced. Five mutations were found in 6 unrelated families studied, of these, two were new: a nonsense mutation in exon 6 (W159X) and a splice defect in intron 9 (IVS9(-1)G-->C). The other two missense mutations, P62L and A80G, had been previously reported in the homozygous state in HEP families. The g10insA, reported in our laboratory, was again identified in other two unrelated families. In addition 3 novel URO-D polymorphisms in non-coding regions were found. The reverse transcription-PCR and sequencing of the splice mutation carriers RNA did not reveal the presence of an abnormal mRNA, suggesting that no stable transcript from the mutated allele is synthesized. These results increase to 39 the number of mutations identified in the URO-D gene; 4 of them causing both HEP and f-PCT.

Identification of CYP3A5 and CYP2B6 Polymorphisms in Porphyria Cutanea Tarda Associated to Human Immunodeficiency Virus

To date, few or no data concerning the prevalence of polymorphisms in drug metabolism genes of antiretroviral drugs have been reported in the Argentinean population or in porphyric individuals worldwide. The purpose of the current investigation was to determine whether interindividual differences in cytochrome P450 3A5 (CYP3A) and 2B6 (CYP2B6) genes could influence the triggering of Porphyria Cutanea Tarda (PCT) in subjects with human immunodeficiency virus (HIV) after antiretroviral exposure. A total of 141 subjects, 60 control volunteers and 81 unrelated individuals with PCT were included in the study. In the porphyric group, 21 individuals were HIV positive. To evaluate the presence of the alleles CYP3A5*3, CYP3A5*6 and CYP2B6*6 a polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis was performed. The frequencies of CYP3A5*3 were 0.91 in control group, 0.89 in PCT patients and 0.89 in PCT-HIV. CYP2B6*6 frequencies were 0.31 in control group, 0.34 in PCT group and 0.30 in PCT-HIV group. We have shown that the allelic frequencies of CYP3A5*3 or CYP2B6*6 in our population were similar to those reported for other Caucasian populations. Although, we have not found significant differences in polymorphisms of CYP3A5 and CYP2B6 between the different groups analyzed, there are an enormous number of biological variables that may influence antiretroviral treatment, like other genetic polymorphisms of phase I or phase II enzymes, or transporters like multidrug resistance transporter gene (MDR1), which can contribute to antiretroviral drug toxicities and response or even it is possible that the PCT-HIV association has more than one factor responsible for the onset of PCT symptoms.

Diagnosis of latent acute intermittent porphyria by genetic analysis

Porphyrias, a group of inherited enzyme disorders of the haem biosynthetic pathway, can be clinically classi®ed into acute and non-acute types. Acute intermittent porphyria (AIP), the most common acute type, is an autosomal dominant disorder caused by a partial de®ciency of porphobilinogen deaminase (PBG-D). Acute attacks of this disease are often precipitated by factors such as drugs, alcohol and caloric deprivation, and early detection is important for the prevention of acute attacks. In asymptomatic carriers the excretion of haem precursors is often normal in between attacks, and diagnosis is based on the determination of erythrocyte PBG-D activity. There is, however, some overlap in erythrocyte PBG-D activity between normal individuals and AIP patients. These limitations prompted us to undertake the study of the mutations responsible for AIP at the DNA level. To date, about 159 mutations in the PBG-D gene have been reported, including missense, non-sense, frameshift and splice site mutations. These may lead to a decrease in PBG-D levels, the complete absence of detectable protein, the production of an altered but stable protein with abnormal catalytic properties, or an absolutely unstable protein. In this study we compared the biochemical results with the genetic data of a group of patients with AIP.

Novel Null-Allele Mutations and Genotype-Phenotype Correlation in Argentinean Patients with Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited disorder of porphyrin metabolism in which decreased activity of ferrochelatase (FECH) leads to accumulation of protoporphyrin IX (PP IX) in red blood cells, plasma, liver, and bile, and increased PP IX excretion in feces. Clinically, EPP is characterized by photosensitivity that begins in early childhood and includes burning, swelling, itching, and painful erythema in sun-exposed areas. Chronic liver disease is an important complication in a minority of EPP patients, and in some cases liver transplantation has been performed. So far, about 110 different mutations and several polymorphisms have been characterized in the human FECH gene. The relationship between mutations, polymorphisms, and porphyria development in Argentinean patients was investigated. This is the first genetic study carried out in the Argentinean population. In five Argentinean EPP families we detected three novel mutations: a deletion (451delT) producing a stop codon located 18 codons downstream from the mutation and two splicing mutations: IVS1-2A>G leading to exon 2 skipping and IVS4-2A>G, which causes the loss of the first 48 bp of exon 5. We also found two previously described mutations: C343T and 400delA, which produce stop codons. All patients had an FECH activity 25% of normal and also had the polymorphisms −251A>G in the promoter region and IVS1-23 C>T and IVS3-48 T>C. Our findings provide supporting evidence for the concept that the inheritance of the low expression allele IVS3-48C in trans with a mutation in the FECH gene is necessary for EPP to become clinically manifest.