A Porfiria Cutânea Tardia (PCT) é uma desordem dermatológica, caracterizada por fotossensibilidade induzida pela circulação de porfirinas que se depositam na pele. Tanto a forma familial como a esporádica são desordens dependentes do acúmulo de ferro. A presença da mutação do gene da Hemocromatose (HFE) é um importante fator de risco para o acúmulo de ferro e pouco se sabe sobre sua prevalência na população brasileira. Da mesma forma, existem poucos relatos a respeito da associação entre mutação do gene HFE e Porfiria Cutânea Tardia. No presente trabalho descrevemos as frequências dos principais alelos e genótipos do gene da Hemocromatose HFE1 em uma coorte de 25 pacientes brasileiros atendidos no HUPE, com Porfiria Cutânea Tardia, durante o período de janeiro 1990 à dezembro 2012, realizando uma correlação da presença desta mutação com a sobrecarga de ferro neste grupo de pacientes. Neste estudo foi utilizado um grupo controle da população fluminense pareado por idade, sexo e grupo étnico informado, para comparar com os dados avaliados dos pacientes com PCT. A pesquisa das mutações genéticas C282Y e H63D do gene da hemocromatose ocorreu através de técnicas de PCR tempo real e e os resultados ratificados por sequenciamento de Sanger. Dos resultados encontrados, não ocorreram diferenças estatísticas significativas nas frequências alélicas e genotípicas das mutações C282Y e H63D entre a coorte com PCT e a população controle. Entretanto, há um forte indício da participação da mutação H63D em um paciente homozigoto, para desenvolvimento da doença, conforme observado na literatura. Dos ensaios bioquímicos, os níveis de ferritina encontrados entre os pacientes portadores de PCT com a mutação H63D foram maiores que os indivíduos sem a mutação. / Porphyria cutanea tarda (PCT) is a dermatological disorder characterized by photosensitivity induced by circulating porphyrins being deposited on the skin. Both the familial form, as sporadic disorders are dependent on the accumulation of iron. Although the cause of this excess hepatic iron is still unknown, the presence of the mutation of the hemochromatosis gene (HFE) is an important risk factor for iron accumulation. Likewise, little is known about the association between HFE gene mutation and porphyria cutanea tarda. In this paper we describe the main frequencies of genotypes and alleles of the gene for hemochromatosis HFE1 in a cohort of 25 patients treated at a dermatology outpatient clinic with Porphyria Cutaneous Late during the period of 1990-2012, performing a correlation with the presence of this mutation and iron overload in these patients. This study used a control group matched for age, sex and ethnic group reported, to compare with the data evaluated in patients with PCT. The research of genetic mutations C282Y and H63D mutations of the hemochromatosis gene occurred through real-time PCR results and ratified by Sanger sequencing. Results found no statistically significant differences in allele and genotype frequencies of the C282Y and H63D between the PCT cohort and population control. However, there is a strong indication of the participation of the H63D mutation in a patient homozygous for disease development, as noted in the literature. Of biochemical analysis, ferritin levels found among patients with PCT with the H63D mutation were higher than those without the mutation.

A Porfiria Cutânea Tardia (PCT) é uma desordem dermatológica, caracterizada por fotossensibilidade induzida pela circulação de porfirinas que se depositam na pele. Tanto a forma familial como a esporádica são desordens dependentes do acúmulo de ferro. A presença da mutação do gene da Hemocromatose (HFE) é um importante fator de risco para o acúmulo de ferro e pouco se sabe sobre sua prevalência na população brasileira. Da mesma forma, existem poucos relatos a respeito da associação entre mutação do gene HFE e Porfiria Cutânea Tardia. No presente trabalho descrevemos as frequências dos principais alelos e genótipos do gene da Hemocromatose HFE1 em uma coorte de 25 pacientes brasileiros atendidos no HUPE, com Porfiria Cutânea Tardia, durante o período de janeiro 1990 à dezembro 2012, realizando uma correlação da presença desta mutação com a sobrecarga de ferro neste grupo de pacientes. Neste estudo foi utilizado um grupo controle da população fluminense pareado por idade, sexo e grupo étnico informado, para comparar com os dados avaliados dos pacientes com PCT. A pesquisa das mutações genéticas C282Y e H63D do gene da hemocromatose ocorreu através de técnicas de PCR tempo real e e os resultados ratificados por sequenciamento de Sanger. Dos resultados encontrados, não ocorreram diferenças estatísticas significativas nas frequências alélicas e genotípicas das mutações C282Y e H63D entre a coorte com PCT e a população controle. Entretanto, há um forte indício da participação da mutação H63D em um paciente homozigoto, para desenvolvimento da doença, conforme observado na literatura. Dos ensaios bioquímicos, os níveis de ferritina encontrados entre os pacientes portadores de PCT com a mutação H63D foram maiores que os indivíduos sem a mutação. / Porphyria cutanea tarda (PCT) is a dermatological disorder characterized by photosensitivity induced by circulating porphyrins being deposited on the skin. Both the familial form, as sporadic disorders are dependent on the accumulation of iron. Although the cause of this excess hepatic iron is still unknown, the presence of the mutation of the hemochromatosis gene (HFE) is an important risk factor for iron accumulation. Likewise, little is known about the association between HFE gene mutation and porphyria cutanea tarda. In this paper we describe the main frequencies of genotypes and alleles of the gene for hemochromatosis HFE1 in a cohort of 25 patients treated at a dermatology outpatient clinic with Porphyria Cutaneous Late during the period of 1990-2012, performing a correlation with the presence of this mutation and iron overload in these patients. This study used a control group matched for age, sex and ethnic group reported, to compare with the data evaluated in patients with PCT. The research of genetic mutations C282Y and H63D mutations of the hemochromatosis gene occurred through real-time PCR results and ratified by Sanger sequencing. Results found no statistically significant differences in allele and genotype frequencies of the C282Y and H63D between the PCT cohort and population control. However, there is a strong indication of the participation of the H63D mutation in a patient homozygous for disease development, as noted in the literature. Of biochemical analysis, ferritin levels found among patients with PCT with the H63D mutation were higher than those without the mutation.

ABSTRACT The porphyrias are a diverse group of metabolic disorders arising from diminished activity of enzymes in the heme biosynthetic pathway. They can present with acute neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these disorders is demonstrated by the fact that some acute porphyria patients with the underlying genetic defect(s) are latent and asymptomatic while others present with severe symptoms. This indicates that there is at least one other risk factor required in addition to the genetic defect for symptom manifestation. A systematic review of the heme biosynthetic pathway highlighted the involvement of a number of micronutrient cofactors. An exhaustive review of the medical literature uncovered numerous reports of micronutrient deficiencies in the porphyrias as well as successful case reports of treatments with micronutrients. Many micronutrient deficiencies present with symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized that a vitamin B6 deficiency and related micronutrient deficiencies may play a major role in the pathogenesis of the acute porphyrias. In order to further investigate the porphyrias, a computational model of the heme biosynthetic pathway was developed based on kinetic parameters derived from a careful analysis of the literature. This model demonstrated aspects of normal heme biosynthesis and illustrated some of the disordered biochemistry of acute intermittent porphyria (AIP). The testing of this model highlighted the modifications necessary to develop a more comprehensive model with the potential to investigated hypotheses of the disordered biochemistry of the porphyrias as well as the discovery of new methods of treatment and symptom control. It is concluded that vitamin B6 deficiency might be the risk factor necessary in conjunction with the genetic defect to trigger porphyria symptoms.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: With knowledge of the human genome increasing constantly we are continually faced with new and potentially groundbreaking methods for managing, treating and/or identifying diseases and predisposition to diseases and conditions at a genetic level. The human cytochrome P450 (CYP) super-family of genes code for enzymes that can participate in metabolism of drugs and foreign chemicals and in steroid synthesis and metabolism. Mutations in these genes may contribute to clinically relevant diseases. In this study, the effects of mutations within four CYP genes were evaluated in two South African disease groups - variegate porphyria and breast cancer. Variegate porphyria (VP) has an unusually high incidence in South Africa due to the R59W founder mutation in the protoporphyrinogen oxidase (PPOX) gene that causes a disruption in the haem biosynthetic pathway. VP presents with variable clinical symptoms and has a relatively low penetrance. It is expected that environmental factors and modifier genes play a role in the clinical expression of VP. CYP genes are implicated as candidate modifier genes for the expression of VP due to the function they have in metabolising many drugs contraindicated in porphyria patients, and the necessity of haem binding to the apoprotein to produce a functional CYP enzyme. This is the first study to investigate CYPs as possible modifier genes for VP clinical expression. Six CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 - 734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4), associated with four CYP loci, were genotyped in a VP population and a suitable control population. The results observed are suggestive of CYPIAlml and CYPIBI playing a role as modifiers for the clinical expression of VP as they were significantly associated (P<O.05) with the presence ofVP related symptoms. Breast cancer is one of the leading causes of death in women due to cancer. It is believed that breast cancer may be the result of co-participation between common DNA alterations and environmental exposures. Polymorphisms in enzymes involved with the metabolism of environmental exposures, such as the cytochrome P450 enzymes, are therefore expected to play an aetiological role in breast cancer. Two groups of South African breast cancer patients (Caucasian and of mixed ancestry) and population-matched controls were genotyped for four CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A and CYPIBI 8372 A>C). This represents the first investigation of the potential role of CYPs as breast cancer risk modifiers in the two South African populations. Significant differences were observed (P<O.003) in genotype and allele frequencies between the breast cancer patients and controls for the CYPIBI 8372 A>C polymorphism in the population of mixed ancestry. Vast differences in allele frequencies were also observed between the two groups of breast cancer populations. These results emphasize the importance of population-based risk assessment when genetic testing and counselling for complex disease susceptibility is offered. The results of this study provide the first evidence suggesting a role for CYPs in modifying the clinical expression of VP and in acting as risk factors for developing breast cancer in a South African population. / AFRIKAANSE OPSOMMING: Met die konstante toename van kennis oor die mensgenoom kom ons voortdurend te staan voor nuwe metodes vir die beheer, behandeling en/of identifikasie van siektes en vatbaarheid vir siektes op 'n genetiese vlak. Die mens sitochroom P450 geensuperfamilie kodeer vir ensieme betrokke in die metabolisme van medisyne en ander chemiese stowwe en steroïed-sintese en -metabolisme. Mutasies in hierdie gene kan 'n bydrae lewer tot kliniese relevante siektes. In hierdie studie is die effek van mutasies in vier sitochroom gene bestudeer in twee Suid-Afrikaanse siekte groepe, variegate porfirie en borskanker. Variegate porfirie (VP) het 'n besonderse hoë frekwensie in Suid-Afrika as gevolg van die R59W stigter-mutasie in die protoporfirinogeen oksidase (PPOX) geen. Hierdie mutasie lei tot 'n versteuring in die heem biosintese padweg. VP presenteer met variërende kliniese simptome en het 'n betreklike lae penetrasie. Daar word vermoed dat omgewingsfaktore en kandidaat modifiserende gene 'n rol speel in die kliniese beeld van VP. Sitochroom P450 gene is geïdentifiseer as kandidaat modifiserende gene as gevolg van hulle rol in die metabolisme van verbode medikasie vir porfirie pasiënte, asook die binding van heem aan die apoproteïen wat noodsaaklik is vir die produksie van funksionele sitochroom P450 ensiem. Hierdie is die eerste studie wat sitochroom P450 gene as moontlike modifiserende gene vir die kliniese uitdrukking van VP ondersoek. Ses sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4) is ondersoek in beide 'n VP populasie en 'n geskikte kontrole populasie. Die resultate suggereer 'n rol vir CYPIAlml en CYPIBI in die modifisering van die kliniese uitdrukking van VP aangesien hulle betekenisvolle assosiasie (P<O.05) met die voorkoms van VP-verwante simptome getoon het. Borskanker IS een van die vernaamste oorsake van kankersterftes onder vroue. Borskanker IS waarskynlik die resultaat van interaksie tussen algemene DNSveranderinge en omgewings-blootstelling. Daar word dus verwag dat polimorfismes in ensieme betrokke by die metabolisme van omgewingselemente, soos byvoorbeeld die sitochroom P450 ensieme, 'n etiolgiese rol in borskanker sal speel. Die genotipes van twee groepe Suid-Afrikaanse borskanker lyers (Kaukasiërs en van gemengde herkoms) en populasie-passende kontroles is bepaal vir vier sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C). Hierdie studie verteenwordig die eerste ondersoek na die potensiële rol van sitochroom P450s as risiko-modifiserende faktore vir borskanker in die twee populasies. Betekenisvolle verskille (P<O.003) in genotipe en allele frekwensies is waargeneem tussen die borskanker lyers en kontroles vir die CYPIBI 8372 A>C polimorfisme in die gemengde herkoms populasie. Beduidende verskille in alleel frekwensies is ook waargeneem tussen die twee borskanker populasies. Hierdie resultate beklemtoon die belangrikheid van populasie gebaseerde risiko-beraming wanneer genetiese toetse en voorligting vir komplekse siekte-vatbaarheid aangebied word. Die resultate van hierdie studie bied die eerste getuienis dat sitochroom P450s 'n rol kan speel in die modifisering van die kliniese beeld van VP en ook kan optree as as risiko faktore vir die ontwikkeling van borskanker in 'n Suid-Afrikaanse populasie.

A porfiria aguda intermitente é uma doença autossômica dominante, decorrente de um distúrbio na via biossintética do heme, causado pela redução dos níveis da enzima uroporfirinogênio-I-sintetase. As manifestações clínicas envolvem o sistema nervoso periférico e o central. O diagnóstico baseia-se na excreção urinária elevada dos precursores das porfirinas ácido d-aminolevulínico e porfobilinogênio. O objetivo deste estudo foi analisar o quadro clínico apresentado por pacientes do Hospital das Clínicas de São Paulo com porfiria aguda intermitente, atendidos no período de janeiro de 1979 a dezembro de 1999. Foram avaliados 37 pacientes, com idades entre 6 e 48 anos, na proporção de 2,7 mulheres:1 homem. A faixa etária na qual ocorreu o maior número de crises foi a terceira década. Os pacientes apresentaram 63 crises, sendo que 13 deles também tiveram manifestações de caráter crônico. As manifestações clínicas mais freqüentes foram: dor abdominal, alteração da cor da urina, mudança no ritmo intestinal, déficit motor ou sensitivo-motor, vômitos, alteração do nível de consciência ou confusão mental, crises convulsivas, quadros disautonômicos cardio-vasculares e distúrbios psiquiátricos. As crises foram classificadas em leves, moderadas e graves, segundo critérios previamente estabelecidos. Todas as manifestações crônicas foram caracterizadas como leves. A neuropatia periférica motora ou sensitivo-motora nunca foi a manifestação inicial da crise de porfiria aguda intermitente. Houve correlação entre o tipo e o número de fatores precipitantes e a manifestação da neuropatia periférica motora ou sensitivo-motora, verificando-se que as crises nas quais ela está ausente foram em geral desencadeadas por apenas um fator, mais comumente de origem endócrina ou metabólica endógena, como período menstrual e jejum, enquanto que nas crises com neuropatia periférica houve a participação de vários fatores concomitantemente, sendo estes principalmente de origem exógena, como medicamentos. Os tratamentos mais utilizados nos surtos foram a administração de glicose, aumento da ingestão de carboidratos e o uso de fenotiazínicos. / Acute intermittent porphyria is an autosomal dominant disease, caused by a disturbance in the heme biosynthetic pathway, secondary to the reduction on the levels of uroporphyrinogen-I-synthetase enzyme. Clinical manifestations involve central and peripheral nervous system. The diagnosis is based on the elevated urinary excretion of porphyrins precursors d-aminolevulinic acid and porphobilinogen. The aim of this study was to analyze the clinical manifestations of acute intermittent porphyria in patients of the Hospital das Clínicas of São Paulo, seen between January 1979 and December 1999. 37 patients were studied, from 6 to 48 years old, with a rate of 2,7 women: 1 man. The age in which most of the crisis occurred was the third decade. The patients presented 63 crisis, and 13 of them presented also with chronic manifestations. The commonest clinical presentations were: abdominal pain, change in urine color, change in bowel habits, motor or sensory-motor deficit, vomiting, alteration of consciousness or mental confusion, convulsions, dysautonomic cardiovascular signs and psychiatric disorders. The crisis were classified as mild, moderate and severe, following criteria previously established. All chronic manifestations were characterized as mild. The peripheral motor or sensory-motor neuropathy was never the initial manifestation. Correlation was found between the kind and the number of precipitating factors, and the absence of peripheral neuropathy was in general related to just one factor, more commonly of endogenous endocrine or metabolic origin, like menstrual period and starvation, while in the crisis with peripheral neuropathy multiple factors were involved at the same time, these being of exogenous origin, like drugs. The most commonly used treatments were glucose administration, elevation of carbohydrate intake, and phenothiazines use.

La porphyrie aiguë intermittente (PAI) est la plus fréquente des porphyries hépatiques aiguës. Elle est décrite comme une maladie autosomique dominante dont le trait génétique est estimé à 1/1675 en France avec une pénétrance faible et variable allant de 10% à 50% dans les familles connues de PAI. La PAI est due à des mutations réduisant le niveau d’activité de l’hydroxyméthylbilane-synthase (HMBS). Son déficit entraîne l’accumulation de précurseurs neurotoxiques responsables de la symptomatologie clinique. Dans le foie, la synthèse d’hème est contrôlée par l’enzyme ALA-Synthétase 1 (ALAS1) dont l’activité est régulée par un rétrocontrôle négatif par le produit final : l’hème. Le traitement consiste à freiner l’induction d’ALAS1 induit par la carence en hème, par l’administration d’hème exogène. Ce traitement de la crise aiguë est très efficace mais génère rapidement une dépendance physique avec apparition de crises récurrentes nécessitant l’administration chronique d’hème exogène. L’objectif principal de ce projet a été d’étudier les mécanismes physiopathologiques et génétiques liés à cette pathologie afin de traiter et conseiller au mieux les patients. Une partie du projet a consisté à explorer les facteurs génétiques modulateurs de la pénétrance de la maladie. Tout d’abord, une prévalence minimale du trait génétique dans la population générale a été estimée à 1/1299 permettant d’en déduire une pénétrance de l’ordre de 1% alors que celle dans les familles PAI suivies par le CFP est estimée à 22,9 %. Ensuite, concernant les facteurs pouvant expliquer cette différence, la présence d’une mutation type non-sens est plus fréquemment associée aux formes sévères et à une pénétrance plus élevée. De plus, les études de corrélation et d’héritabilité suggèrent plutôt une transmission de type oligogénique associée à des facteurs épigénétiques modulateurs de la pénétrance dont le facteur environnemental. Une autre partie a consisté à explorer les effets de l’administration d’hème exogène sur les patients et un modèle murin de PAI créé génétiquement. Chez l’homme, le traitement est associé à une augmentation des formes chroniques (1,7 % avant vs 7,5 % après l’introduction du celui-ci). Dans le modèle murin de PAI, les injections intrapéritonéales répétées induisent une augmentation paradoxale d’ALAS1 (3 fois), une augmentation de l’hème oxygénase 1 qui catabolise l’hème (HMOX1, 9 fois) ainsi que des voies de l’inflammation (analyse transcriptomique et protéomique hépatique) et une surcharge en fer. De plus, cette administration induit une altération des complexes de la chaine respiratoire mitochondriale responsable d’anomalies du métabolisme énergétique au niveau hépatique, cérébral et musculaire pouvant expliquer la symptomatologie neuroviscérale. En conclusion, ce travail a permis d’explorer les caractéristiques génétiques de la maladie (prévalence, pénétrance) en remettant en cause le mode de transmission autosomique dominant jusqu’ici admis, et d’explorer les mécanismes physiopathologiques associées à l’administration d’hème exogène faisant de cette thérapeutique un pharmakon / The biosynthesis of porphyrins is one of the most conserved pathways known. By associating different metals, porphyrins give rise to the "pigments of life". The formation of haem is accomplished by a sequence of eight dedicated enzymes encoded by different genes, some being active in ubiquitous as well as in erythroid isoforms. In humans, the genes for each of the haem synthetic enzymes may become the target of mutations that give rise to an impaired cellular enzyme activity called porphyrias. The acute porphyrias are characterized by attacks of neuropsychiatric symptoms, which may be due to a toxic surplus of the porphyrin precursor 5-aminolevulinic acid, or a consequence of a deficit of vital hemoproteins. Mutations of the gene encoding the third enzyme: hydroxymethylbilane synthase, are associated with the most frequent type of acute hepatic porphyria, acute intermittent porphyria. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. In the classical form of AIP, HMBS activity is about 50% lower than normal in all tissues. These levels of activity in basal conditions are not sufficiently low to cause symptoms. However, factors increasing hepatic heme demand, resulting in an upregulation of hepatic aminolevulinate synthase (ALAS1, the first enzyme of the heme biosynthesis pathway), precipitate acute attacks. The treatment of the attack of AIP consists to repress ALAS1 and restores metabolic equilibrium. But this treatment leads side effects and dependency. The pathophysiological mechanism of the disease is partially known and difficult to explore because there is not an AIP model or prediction model of porphyrogenicity. We aimed to obtain further insight into the pathophysiological mechanism of AIP and into the genetic (prevalence and penetrance) of AIP, and the contribution of genetic factors to the variable clinical expression of HMBS mutations.We first calculated the penetrance of HMBS mutations in AIP patients seen at the French reference center for porphyria: 22.9%. We then used the Exome Variant Server (EVS) to estimate the prevalence of deleterious HMBS mutations in the general population: 1/1299; and the penetrance of the AIP genetic trait in France: 1%. Finally, we investigated further the genetic factors underlying the penetrance of AIP by analyzing genotype/phenotype correlations, and the pattern of familial correlations for the symptoms of the acute crises of AIP. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the era in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance suggesting that AIP inheritance does not follow the classical autosomal dominant model. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles.On the other hand, we explored the effect of heme administration. In human, the introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. We show that repeated hemin infusions in mice trigger a high level heme oxygenase 1 response, induce a pro-oxidative iron accumulation, a complex pattern of liver inflammation with macrophage infiltration and an alteration of oxidative phosphorylation

The biosynthesis of tetrapyrolles in eukaryotes and the alpha-subclass of purple photosynthetic bacteria is controlled by the pyridoxal 5?-phosphate (PLP)-dependent enzyme, 5-aminolevulinate synthase (ALAS). Aminolevulinate, the universal building block of these macromolecules, is produced together with Coenzyme A (CoA) and carbon dioxide from the condensation of glycine and succinyl-CoA. The three-dimensional structures of Rhodobacter capsulatus ALAS reveal a conserved active site serine that moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed, substrate-bound enzyme conformation, and simultaneously to within 3-4 angstroms of the thioester sulfur atom of bound succinyl-CoA. To elucidate the role(s) this residue play(s) in enzyme activity, the equivalent serine in murineerythroid ALAS was mutated to threonine or alanine. The S254A variant was active, but both the KmSCoA and kcat values were increased, by 25- and 2-fold, respectively, suggesting the increase in turnover is independent of succinyl-CoA-binding. In contrast, substitution of S254 with threonine results in a decreased kcat, however the Km for succinyl-CoA is unaltered. Removal of the side chain hydroxyl group in the S254A variant notably changes the spectroscopic properties of the PLP cofactor and the architecture of the PLP-binding site as inferred from circular dichroism spectra. Experiments examining the rates associated with intrinsic protein fluorescence quenching of the variant enzymes in response to ALA binding show that S254 affects product dissociation. Together, the data led us to suggest that succinyl-CoA binding in concert with the hydrogen bonding state of S254 governs enzyme conformational equilibria. As a member of the alpha-oxoamine synthase family, ALAS shares a high degree of structural similarity and reaction chemistry with the other enzymes in the group. Crystallographic studies of the R. capsulatus ALAS structure show that the alkanoate component of succinyl-CoA is bound by a conserved arginine and a threonine. To examine acyl-CoA-binding and substrate discrimination in murine erythroid ALAS, the corresponding residues (R85 and T430) were mutated and a series of CoA substrate analogs were tested. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that calculated for the wild-type enzyme, suggesting this residue is strategic in substrate binding. Hydrophobic substitutions of the residues that coordinate acyl-CoA-binding produce ligand-induced changes in the CD spectra, indicating that these amino acids affect substrate-mediated changes to the microenvironment of the chromophore. Pre-steady-state kinetic analyses of the R85K variant-catalyzed reaction show that both the rates associated with product-binding and the parameters that define quinonoid intermediate lifetime are dependent on the chemical composition of the acyl-CoA tail. Each of the results in this study emphasizes the importance of the relationship between the bifurcate interaction of the alkanoic acid component of succinyl-CoA and the side chains of R85 and T430. From the X-ray crystal structures of Escherichia coli 8-amino-7-oxonoanoate synthase and R. capsulatus ALAS, it was inferred that a loop covering the active site moved 3-6 Å between the holoenzymic and acyl-CoA-bound conformations. To elucidate the role that the active site lid plays in enzyme function, we shuffled the portion of the murine erythroid ALAS cDNA corresponding to the lid sequence (Y422-R439), and isolated functional variants based on genetic complementation in an ALA-deficient strain. Variants with potentially greater enzymatic activity than the wild-type enzyme were screened for increased porphyrin overproduction. Turnover number and the catalytic efficiency of selected functional variants with both substrates were increased for each of the enzyme variants tested, suggesting that increased activity is linked to alterations of the loop motif. The results of transient kinetics experiments for three isolated variants when compared to wild-type ALAS showed notable differences in the pre-steady-state rates that define the kinetic mechanism, indicating that the rate of ALA release is not rate-limiting for these enzymes. The thermodynamic parameters for a selected variant-catalyzed reaction indicated a reduction in the amount of energy required for catalysis. This finding is consistent with the proposal that, in contrast to the wild-type ALAS reaction, a protein conformational change associated with ALA release no longer limits turnover for this variant enzyme.

A porfiria aguda intermitente é uma doença autossômica dominante, decorrente de um distúrbio na via biossintética do heme, causado pela redução dos níveis da enzima uroporfirinogênio-I-sintetase. As manifestações clínicas envolvem o sistema nervoso periférico e o central. O diagnóstico baseia-se na excreção urinária elevada dos precursores das porfirinas ácido d-aminolevulínico e porfobilinogênio. O objetivo deste estudo foi analisar o quadro clínico apresentado por pacientes do Hospital das Clínicas de São Paulo com porfiria aguda intermitente, atendidos no período de janeiro de 1979 a dezembro de 1999. Foram avaliados 37 pacientes, com idades entre 6 e 48 anos, na proporção de 2,7 mulheres:1 homem. A faixa etária na qual ocorreu o maior número de crises foi a terceira década. Os pacientes apresentaram 63 crises, sendo que 13 deles também tiveram manifestações de caráter crônico. As manifestações clínicas mais freqüentes foram: dor abdominal, alteração da cor da urina, mudança no ritmo intestinal, déficit motor ou sensitivo-motor, vômitos, alteração do nível de consciência ou confusão mental, crises convulsivas, quadros disautonômicos cardio-vasculares e distúrbios psiquiátricos. As crises foram classificadas em leves, moderadas e graves, segundo critérios previamente estabelecidos. Todas as manifestações crônicas foram caracterizadas como leves. A neuropatia periférica motora ou sensitivo-motora nunca foi a manifestação inicial da crise de porfiria aguda intermitente. Houve correlação entre o tipo e o número de fatores precipitantes e a manifestação da neuropatia periférica motora ou sensitivo-motora, verificando-se que as crises nas quais ela está ausente foram em geral desencadeadas por apenas um fator, mais comumente de origem endócrina ou metabólica endógena, como período menstrual e jejum, enquanto que nas crises com neuropatia periférica houve a participação de vários fatores concomitantemente, sendo estes principalmente de origem exógena, como medicamentos. Os tratamentos mais utilizados nos surtos foram a administração de glicose, aumento da ingestão de carboidratos e o uso de fenotiazínicos. / Acute intermittent porphyria is an autosomal dominant disease, caused by a disturbance in the heme biosynthetic pathway, secondary to the reduction on the levels of uroporphyrinogen-I-synthetase enzyme. Clinical manifestations involve central and peripheral nervous system. The diagnosis is based on the elevated urinary excretion of porphyrins precursors d-aminolevulinic acid and porphobilinogen. The aim of this study was to analyze the clinical manifestations of acute intermittent porphyria in patients of the Hospital das Clínicas of São Paulo, seen between January 1979 and December 1999. 37 patients were studied, from 6 to 48 years old, with a rate of 2,7 women: 1 man. The age in which most of the crisis occurred was the third decade. The patients presented 63 crisis, and 13 of them presented also with chronic manifestations. The commonest clinical presentations were: abdominal pain, change in urine color, change in bowel habits, motor or sensory-motor deficit, vomiting, alteration of consciousness or mental confusion, convulsions, dysautonomic cardiovascular signs and psychiatric disorders. The crisis were classified as mild, moderate and severe, following criteria previously established. All chronic manifestations were characterized as mild. The peripheral motor or sensory-motor neuropathy was never the initial manifestation. Correlation was found between the kind and the number of precipitating factors, and the absence of peripheral neuropathy was in general related to just one factor, more commonly of endogenous endocrine or metabolic origin, like menstrual period and starvation, while in the crisis with peripheral neuropathy multiple factors were involved at the same time, these being of exogenous origin, like drugs. The most commonly used treatments were glucose administration, elevation of carbohydrate intake, and phenothiazines use.

O ácido 5-aminolevulínico (ALA) é o primeiro precursor do grupo heme acumulado, principalmente no fígado, em alguns tipos de porfirias hepáticas hereditárias (porfiria aguda intermitente-AIP e tirosinemia) ou adquiridas (intoxicação por chumbo) devido à diminuição da atividade da enzima porfobilinogênio deaminase. Amostras de biópsias de fígado de pacientes portadores de AIP revelaram alterações estruturais nas mitocôndrias e no retículo endoplasmático, acúmulo de lipofuscina, gordura e corpúsculos de ferritina. Têm sido demonstrado que mutações mitocondriais induzidas por pró-oxidantes também contribuem para o envelhecimento celular e para o desenvolvimento do câncer. Esses dados podem estar relacionados à maior incidência de carcinoma hepatocelular (HCC) em pacientes sintomáticos de AIP. In vitro, ALA produz espécies reativas de oxigênio (ROS), através da oxidação catalisada por metais, e pode ser visto como uma fonte endógena de ROS, iniciando danos oxidativos a estruturas celulares como o DNA, podendo estar envolvido na iniciação e promoção do câncer. Além disso, o produto final de oxidação do ALA, o ácido 4,5-dioxovalérico (DOVA) é capaz de induzir modificações nas bases do DNA como outros derivados carbonílicos reativos. Neste estudo, demonstramos que o ALA é capaz de produzir danos ao DNA como quebras de fita de DNA plasmidial, aumento do nível de 8-oxo-7,8- dihidro-2\'-desoxiguanosina e 5-hidroxi-2´-desoxicitidina em DNA de órgãos de ratos tratados com ALA e aumento da formação de diversas bases modificadas em DNA de timo de bezerro. O DOVA reagiu com 2´-desoxiguanosina e DNA de timo de bezerro isolado produzindo dois adutos diastereoisômeros. O ALA e o DOVA foram capazes de aumentar a mutagenicidade em S. typhimurium TA104 e induzir resposta SOS em E. coli PQ37. Danos ao DNA mitocondrial e nuclear também foram detectados através da técnica de reação quantitativa em cadeia da polimerase em fibroblastos humanos transformados tratados com ALA. Todos esses dados fornecem informações referentes ao potencial genotóxico do ALA e permitem estabelecer uma proposta de bases moleculares para conectar as lesões ao DNA promovidas pelo ALA com a maior incidência de carcinoma hepatocelular em pacientes sintomáticos de AIP. / 5-Aminolevulinic acid (ALA) is a heme precursor accumulated in some inborn (acute intermittent porphyria-AIP and tyrosinosis) or acquired (lead poisoning) types of hepatic porphyria. In AIP patients, ALA is overproduced and accumulated in the liver. Liver biopsy samples of AIP patients revealed mitochondrial and endoplasmic reticulum structural alterations and accumulation of lipofucsin, fat and ferritin bodies. Mitochondrial mutations induced by pro- oxidants were suggested to contribute to cellular aging and cancer. These findings may be connected to the higher frequency of hepatocellular carcinoma (HCC) associated to symptomatic AIP patients. In vitro, ALA produces reactive oxygen species (ROS) upon metal- catalyzed oxidation and can be viewed as a deleterious endogenous source of ROS, triggering oxidative damage to cell structures and organs and being involved in the initiation and promotion of cancer. Besides, the final oxidation product of ALA, the 4,5-dioxovaleric acid (DOVA) is expect to induce DNA base modifications as already shown for other reactive carbonyl derivatives. In this study we demonstrated that ALA is able to produce DNA lesions such as strand breaks in plasmid DNA, increased steady state level of 8-oxo-7,8- dihydro-2\'-deoxyguanosine and 5-hydroxy-2´-deoxycytidine in rat organs DNA of ALA-treated rats and increased formation of several modified DNA bases in calf thymus DNA. 4,5-Dioxovaleric acid was showed to react with 2\'-deoxyguanosine and isolated calf thymus DNA through Schiff?s base formation to produce two diastereisomeric adducts. Aminolevulinic acid and DOVA were able to increase mutagenicity of the S. typhimurim strain TA104 and induce SOS response in E. coli PQ37. The mitochondrial and nuclear DNA damage were also detected by quantitative polymerase chain reaction technique in transformed human fibroblasts treated with ALA. All these data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that ALA may be involved in the induction of HCC in symptomatic AIP patients.

Fundamentos: A porfiria cutânea tardia é a forma mais comum das porfirias e caracteriza-se pela diminuição da atividade da enzima uroporfirinogênio descarboxilase. Há vários relatos da associação das mutações do gene HFE da hemocromatose hereditária com porfiria cutânea tardia no mundo, mas até hoje apenas um estudo foi realizado no Brasil. Objetivo: Estudar a associação da porfiria cutânea tardia com as mutações C282Y e H63D do gene HFE da hemocromatose hereditária. Identificar a associação com etilismo, hepatite C, hepatite B e infecção pelo HIV e relacioná-los com a presença ou não das mutações do gene HFE e estudar retrospectivamente a resposta terapêutica à cloroquina. Métodos: Estudo ambispectivo para detectar as mutações C282Y e H63D em 60 pacientes com porfiria cutânea tardia no período de 2003 até 2012. O histórico familiar, etilismo, hepatite C, hepatite B e anti-HIV foram investigados. O estudo das mutações HFE foi realizado com PCR em tempo real. A resposta terapêutica foi avaliada utilizando a dosagem das porfirinas urinárias (urina de 24 horas), o perfil de ferro (ferro sérico, ferritina e saturação de transferrina) e as enzimas hepáticas antes e após a remissão bioquímica. Resultados: A frequência dos alelos das mutações foi significativamente mais elevada nos pacientes com PCT para C282Y (8,3% versus 1,77%, odds ratio 5,02, IC [95%] = [4,1%; 14,8%], p=0,0001) e H63D (27,5% versus 14,05, odds ratio 2,32, IC [95%] = [19,7%; 36,4%], p=0,0004) em relação à população grupo controle. A hepatite C estava presente em 41,7% dos pacientes e estava associada à ingestão de álcool em 71,7% dos casos. Conclusões: As mutações HFE e a expressão clínica da hemocromatose hereditária podem contribuir isoladamente para o desencadeamento da PCT, independente-mente da presença de outros fatores precipitantes; o que torna a pesquisa das mutações HFE um exame necessário nos pacientes com PCT. Nos pacientes homozigotos para C282Y e heterozigotos compostos (C282Y/H63D) a flebotomia é o tratamento de primeira escolha. A porfiria cutânea tardia pode ser um marcador cutâneo para a hemocromatose e o dermatologista pode auxiliar no seu diagnóstico e tratamento precoce. / Background: Porphyria cutanea tarda (PCT) is the most common form of porphyria and is characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with PCT worldwide, although up to date only one study has been conducted in Brazil. Objective: Study the association between porphyria cutanea tarda and C282Y and H63D mutations in the HFE gene of hereditary hemochromatosis. Identify the association with alcoholism, hepatitis C, hepatitis B and HIV infection and relate them with the presence or absence of the HFE gene mutations and study retrospectively the therapeutic response to chloroquine. Methods: Ambispective study in the period from 2003 to 2012 to detect the C282Y and H63D mutations in 60 patients with porphyria cutanea tarda. The family history, alcoholism, hepatitis C, hepatitis B and HIV were investigated. HFE mutations were held with real-time PCR. The therapeutic response was assessed using the urinary porphyrins (24h urine), the iron profile (serum iron, ferritin and transferrin saturation) and the liver enzymes, before and after biochemical remission. Results: The frequency of alleles of the mutations were significantly higher in patients with PCT for C282Y (8.3% vs. 1.77%, odds ratio 5.02, CI [95%] = [4.1%; 14.8%], p = 0.0001) and H63D (27.5% vs. 14.05, odds ratio 2.32, CI [95%] = [19.7% and 36.4%], p = 0.0004) in relation to group control population. Hepatitis C was found in 41.7% of the patients and was associated with the ingestion of alcohol in 71.7% of cases. Conclusions: The HFE mutations and clinical expression of hereditary hemochromatosis can contribute in an isolated manner to the outbreak of PCT, independently of the existence of other precipitating factors. This makes the search for HFE mutations necessary in patients with PCT. In patients who are homozygous for C282Y and compound heterozygotes (C282Y/H63D) phlebotomy is the treatment of first choice. Porphyria cutanea tarda can be a cutaneous marker for hemochromatosis and the dermatologist can help in its diagnosis and early treatment.