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Background: Acute Hepatic Porphyrias (AHPs) are characterized by an acute neuroabdominal syndrome including both neuropsychiatric symptoms and neurodegenerative changes. Two main hypotheses explain the pathogenesis of nervous system dysfunction: (a) the ROS generation by autooxidation of 5-aminolevulinic acid accumulated in liver and brain; (b) liver heme deficiency and in neural tissues that generate an oxidative status, a component of the neurodegenerative process., Methods: We review results obtained from Acute Intermittent Porphyria (AIP) and Variegate Porphyria (VP) families studied at clinical, biochemical, and molecular level at the CIPYP in Argentina. The relationship between the porphyric attack and oxidative stress was also evaluated in AHP patients and controls, to identify a marker of neurological dysfunction., Results: We studied 116 AIP families and 30 VP families, 609 and 132 individuals, respectively. Genotype/phenotype relation was studied. Oxidative stress parameters and plasma homocysteine levels were measured in 20 healthy volunteers, 22 AIP and 12 VP individuals., Conclusion: No significant difference in oxidative stress parameters and homocysteine levels between the analyzed groups were found., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

In Argentina, porphyria cutanea tarda (PCT) is strongly associated with infection with human immunodeficiency virus (HIV); however, whether the onset of this disease is associated with HIV infection and/or the antiretroviral therapy has not been determined. The ABCB1 gene variants c.1236C>T, c.2677G>T/A and c.3435C>T affect drug efflux. The GSTT1 null, GSTM1 null and GSTP1 (c.313A>G) gene variants alter Glutathione S-transferase (GST) activity, modifying the levels of xenobiotics. The aim of the present study was to evaluate the role of genetic variants in initiation of PCT and to analyze the genetic basis of the PCT-HIV association. Control individuals, and HIV, PCT and PCT-HIV patients were recruited, PCR-restriction fragment length polymorphism was used to genotype the ABCB1 and GSTP1 variants, and multiplex PCR was used to study the GSTM1 and GSTT1 variants. The high frequency of c.3435C>T (PCT and PCT-HIV) and c.1236C>T (PCT) suggested that the onset of PCT were not specifically related to HIV infection or antiretroviral therapy for these variants. c.2677G>T/A frequencies in the PCT-HIV patients were higher compared with the other groups, suggesting that a mechanism involving antiretroviral therapy served a role in this association. PCT-HIV patients also had a high frequency of GSTT1 null and low frequency for GSTM1 null variants; thus, the genetic basis for PCT onset may involve a combination between the absence of GSTT1 and the presence of GSTM1 . In conclusion, genes encoding for proteins involved in the flow and metabolism of xenobiotics may influence the PCT-HIV association. The present study is the first to investigate the possible role of GST and ABCB1 gene variants in the triggering of PCT in HIV-infected individuals, to the best of our knowledge, and may provide novel insights into the molecular basis of the association between PCT and HIV., (Copyright: © Pagnotta et al.)

Porphyrias are a heterogeneous group of metabolic disorders that result from the altered activity of specific enzymes of the heme biosynthetic pathway and are characterized by accumulation of pathway intermediates. Porphyria cutanea tarda (PCT) is the most common porphyria and is due to deficient activity of uroporphyrinogen decarboxylase (UROD). Acute intermittent porphyria (AIP) is the most common of the acute hepatic porphyrias, caused by decreased activity of hydroxymethylbilane synthase (HMBS). An Argentinean man with a family history of PCT who carried the UROD variant c.10_11insA suffered severe abdominal pain. Biochemical testing was consistent with AIP, and molecular analysis of HMBS revealed a de novo variant: c.344 + 2_ + 5delTAAG. This is one of the few cases of porphyria identified with both UROD and HMBS mutations and the first confirmed case of porphyria with dual enzyme deficiencies in Argentina., Competing Interests: GN Cerbino is a postgraduate student from the University of Buenos Aires and a member at the Technical Career from CONICET. L Abou Assali is a postdoctoral fellow from the University of Buenos Aires. A Batlle, VE Parera, and MV Rossetti are Superior, Principal, and Independent researchers from CONICET. LS Varela is a Principal Member at the Technical Career from CONICET. Lucia Tomassi is a clinic doctor at Hospital General de Agudos “José María Ramos Mejía.”, (Copyright © 2020 G. N. Cerbino et al.)

Porphyrias are a group of metabolic diseases that arise from deficiencies in the heme biosynthetic pathway. A partial deficiency in hydroxymethylbilane synthase (HMBS) produces a hepatic disorder named Acute Intermittent Porphyria (AIP); the acute porphyria is more frequent in Argentina. In this paper we review the results obtained for 101 Argentinean AIP families and 6 AIP families from foreign neighbour countries studied at molecular level at Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP). Thirty-five different mutations were found, of which 14 were described for the first time in our population. The most prevalent type of mutations was the missense mutations (43%) followed by splice defects (26%) and small deletions (20%). An odd case of a double heterozygous presentation of AIP in a foreign family from Paraguay is discussed. Moreover, it can be noted that 38 new families were found carrying the most frequent mutation in Argentina (p.G111R), increasing to 55.66% the prevalence of this genetic change in our population and adding further support to our previous hypothesis of a founder effect for this mutation in Argentina. Identification of patients with an overt AIP is important because treatment depends on an accurate diagnosis, but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death.

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008, three unrelated patients were diagnosed with variegate porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the protoporphyrinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338+3insT, c.807G>A, and c.808-1G>C. As these mutations do not affect the protein sequence, we hypothesized that they might be splicing mutations. RT-PCRs performed on the patient's mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patient's disease by causing aberrant splicing, we utilized a minigene approach. We found that the three mutations lead to exon skipping; therefore, the abnormal mRNAs are most likely degraded by a mechanism such as nonsense-mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.

A partial deficiency in protoporphyrinogen oxidase (PPOX) produces the acute/cutaneous (or mixed) variegate porphyria (VP), the third most frequent porphyria in Argentina. This autosomal dominant disorder is clinically characterized by skin lesions and/or acute neurovisceral attacks. The precise diagnosis of patients with a symptomatic VP is essential to provide accurate treatment. It is also critical to identify asymptomatic relatives to avoid precipitating factors and prevent acute attacks.Functional consequences of five PPOX missense mutations were evaluated in a prokaryotic expression system. Three mutations were found in families previously reported c.101A>T (p.E34V), c.670T>G (W224G), c.995G>C (G332A) and two were novel findings c.227C>T (p.S76F), c.1265A>G (p.Y422C). All mutations were identified in heterozygotes with reduced PPOX activity and variable clinical expression of the disease, including asymptomatic cases. Prokaryotic expression showed that all five missense mutations decreased the PPOX activity, demonstrating their detrimental effect on enzyme function, and thus, providing evidence for their causative role in VP. These results reinforce the importance of molecular genetic analysis for VP diagnosis and especially the usefulness of prokaryotic expression of missense mutations to assess their deleterious effect on PPOX activity.MM and BXG contributed equally to the publication. RES and MVR share senior authorship.

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.

Background: A partial deficiency in Protoporphyrinogen oxidase (PPOX) produces the mixed disorder Variegate Porphyria (VP), the second acute porphyria more frequent in Argentina. Identification of patients with an overt VP is absolutely important because treatment depends on an accurate diagnosis but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death., Methods: We have studied at molecular level 18 new Argentinean patients biochemically diagnosed as VP. PPOX gene was amplified in one or in twelve PCR reactions. All coding exons, flanking intronic and promoter regions were manual or automatically sequenced. For RT-PCR studies RNA was retrotranscripted, amplified and sequenced. PPOX activity in those families carrying a new and uncharacterized mutation was performed., Results: All affected individuals harboured mutations in heterozygous state. Nine novel mutations and 3 already reported mutations were identified. Six of the novel mutations were single nucleotide substitutions, 2 were small deletions and one a small insertion. Three single nucleotide substitutions and the insertion were at exon-intron boundaries. Two of the single nucleotide substitutions, c.471G>A and c.807G>A and the insertion (c.388+3insT) were close to the splice donor sites in exons 5, 7 and intron 4 respectively. The other single nucleotide substitution was a transversion in the last base of intron 7, g.3912G>C (c.808-1G>C) so altering the consensus acceptor splice site. However, only in the first case the abnormal band showing the skipping of exon 5 was detected. The other single nucleotide substitutions were transversions: c.101A>T, c.995G>C and c.670 T>G that result in p.E34V, p.G332A and W224G aminoacid substitutions in exons 3, 10 and 7 respectively. Activity measurements indicate that these mutations reduced about 50% PPOX activity and also that they co-segregate with this reduced activity value. Two frameshift mutations, c.133delT and c.925delA, were detected in exons 3 and 9 respectively. The first leads to an early termination signal 22 codons downstream (p.S45fsX67) and the second leads to a stop codon 5 codons downstream (p.I309fsX314). One reported mutation was a missense mutation (p.G232R) and 2 were frameshift mutations: c.1082insC and 1043insT. The last mutation was detected in six new apparently unrelated Argentinean families., Conclusion: Molecular analysis in available family members revealed 14 individuals who were silent carriers of VP. Molecular techniques represent the most accurate approach to identify unaffected carriers and to provide accurate genetic counselling for asymptomatic individuals. The initial screening includes the insertion search.

Background: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, delta-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., Results: We 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.

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.

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.

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)