Neurological manifestations and molecular genetics of acute intermittent porphyria in North Western Russia

Acute intermittent porphyria (AIP, MIM #176000) is an inherited metabolic disease due to a partial deficiency of the third enzyme, hydroxymethylbilane synthase (HMBS, EC: 4.3.1.8), in the haem biosynthesis. Neurological symptoms during an acute attack, which is the major manifestation of AIP, are variable and relatively rare, but may endanger a patient's life. In the present study, 12 Russian and two Finnish AIP patients with severe neurological manifestations during an acute attack were studied prospectively from 1995 to 2006. Autonomic neuropathy manifested as abdominal pain (88%), tachycardia (94%), hypertension (75%) and constipation (88%). The most common neurological sign was acute motor peripheral neuropathy (PNP, 81%) often associated with neuropathic sensory loss (54%) and CNS involvement (85%). Despite heterogeneity of the neurological manifestations in our patients with acute porphyria, the major pattern of PNP associated with abdominal pain, dysautonomia, CNS involvement and mild hepatopathy could be demonstrated. If more strict inclusion criteria for biochemical abnormalities (>10-fold increase in excretion of urinary PBG) are applied, neurological manifestations in an acute attack are probably more homogeneous than described previously, which suggests that some of the neurological patients described previously may not have acute porphyria but rather secondary porphyrinuria. Screening for acute porphyria using urinary PBG is useful in a selected group of neurological patients with acute PNP or encephalopathy and seizures associated with pain and dysautonomia. Clinical manifestations and the outcome of acute attacks were used as a basis for developing a 30-score scale of the severity of an acute attack. This scale can easily be used in clinical practice and to standardise the outcome of an attack. Degree of muscle weakness scored by MRC, prolonged mechanical ventilation, bulbar paralysis, impairment of consciousness and hyponatraemia were important signs of a poor prognosis. Arrhythmia was less important and autonomic dysfunction, severity of pain and mental symptoms did not affect the outcome. The delay in the diagnosis and repeated administrations of precipitating factors were the main cause of proceeding of an acute attack into pareses and severe CNS involvement and a fatal outcome in two patients. Nerve conduction studies and needle EMG were performed in eleven AIP patients during an acute attack and/or in remission. Nine patients had severe PNP and two patients had an acute encephalopathy but no clinically evident PNP. In addition to axonopathy, features suggestive of demyelination could be demonstrated in patients with severe PNP during an acute attack. PNP with a moderate muscle weakness was mainly pure axonal. Sensory involvement was common in acute PNP and could be subclinical. Decreased conduction velocities with normal amplitudes of evoked potentials during acute attacks with no clinically evident PNP indicated subclinical polyneuropathy. Reversible symmetrical lesions comparable with posterior reversible encephalopathy syndrome (PRES) were revealed in two patients' brain CT or MRI during an acute attack. In other five patients brain MRI during or soon after the symptoms was normal. The frequency of reversible brain oedema in AIP is probably under-estimated since it may be short-lasting and often indistinguishable on CT or MRI. In the present study, nine different mutations were identified in the HMBS gene in 11 unrelated Russian AIP patients from North Western Russia and their 32 relatives. AIP was diagnosed in nine symptom-free relatives. The majority of the mutations were family-specific and confirmed allelic heterogeneity also among Russian AIP patients. Three mutations, c.825+5G>C, c.825+3_825+6del and c.770T>C, were novel. Six mutations, c.77G>A (p.R26H), c.517C>T (p.R173W), c.583C>T (p.R195C), c.673C>T (p.R225X), c.739T>C (p.C247R) and c.748G>C (p.E250A), have previously been identified in AIP patients from Western and other Eastern European populations. The effects of novel mutations were studied by amplification and sequencing of the reverse-transcribed total RNA obtained from the patients' lymphoblastoid or fibroblast cell lines. The mutations c.825+5G>C and c.770T>C resulted in varyable amounts of abnormal transcripts, r.822_825del (p.C275fsX2) and [r.770u>c, r.652_771del, r.613_771del (p.L257P, p.G218_L257del, p.I205_L257del)]. All mutations demonstrated low residual activities (0.1-1.3 %) when expressed in COS-1 cells confirming the causality of the mutations and the enzymatic defect of the disease. The clinical outcome, prognosis and correlation between the HMBS genotype and phenotype were studied in 143 Finnish and Russian AIP patients with ten mutations (c.33G>T, c.97delA, InsAlu333, p.R149X, p.R167W, p.R173W, p.R173Q, p.R225G, p.R225X, c.1073delA) and more than six patients in each group. The patients were selected from the pool of 287 Finnish AIP patients presented in a Finnish Porphyria Register (1966-2003) and 23 Russian AIP patients (diagnosed 1995-2003). Patients with the p.R167W and p.R225G mutations showed lower penetrance (19% and 11%) and the recurrence rate (33% and 0%) in comparison to the patients with other mutations (range 36 to 67% and 0 to 66%, respectively), as well as milder biochemical abnormalities [urinary porphobilinogen 47±10 vs. 163±21 mol/L, p<0.001; uroporphyrin 130±40 vs. 942±183 nmol/L, p<0.001] suggesting a milder form of AIP in these patients. Erythrocyte HMBS activity did not correlate with the porphobilinogen excretion in remission or the clinical of the disease. In all AIP severity patients, normal PBG excretion predicted freedom from acute attacks. Urinary PBG excretion together with gender, age at the time of diagnosis and mutation type could predict the likelihood of acute attacks in AIP patients.

Porphyrin synthesis in drug induced hepatic porphyria

Liver δ-aminolaevulate (ALA) synthetase and ALA dehydratase are induced to a greater extent in 3,5-diethoxy carbonyl-1,4-dihydrocollidine (DDC) injected mice as compared to the allyl isopropyl acetamide (AIA) injected rats. DDC treated mice do not show an increase in porphobilinogen (PEG) levels commensurate with the increase in ALA levels and the two enzyme activities, but accumulate enormous quantities of protoporphyrin in the liver. Normal mouse liver has an inherent greater capacity to convert PBG to porphyrins as compared to that of the rat. This together with the inhibition of iron incorporation into protoporphyrin in vivo at later stages of DDC administration can account for the large accumulation of protoporphyrin in these animals.

Angiosarcoma, porphyria cutanea tarda, and probable chloracne in a worker exposed to waste oil contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

A worker developed angiosarcoma, porphyria cutanea tarda, and skin lesions characteristic of mild chloracne. About 10 years earlier he had been employed at a truck terminal in Saint Louis, Missouri, at a time when it was sprayed with waste oil contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The occurrence of these three rare conditions in a single exposed worker supports the aetiological relation between environmental exposure to TCDD and the subsequent development of soft tissue sarcoma and porphyria cutanea tarda.

Molecular forms of porphobilinogen deaminase in acute intermittent porphyria. A study by Western immunoblotting

Acute intermittent porphyria is an inborn error of haem synthesis which is transmitted as a dominant character with variable phenotypic expression. The disorder is caused by a partial deficiency of porphobilinogen deaminase in all tissues so far studied. The nature of the enzymatic deficiency of porphobilinogen deaminase in haemolysates from patients with acute intermittent porphyria was examined by the use of monospecific antibody probes. In affected heterozygotes from three British pedigrees of diverse ancestry, the catalytic deficiency of porphobilinogen deaminase was accompanied by diminished enzyme protein, as determined by radial immunodiffusion. No evidence of functionally attenuated enzyme was demonstrable by kinetic studies. The molecular forms of the residual enzyme were investigated in red cell extracts and in lysed preparations of reticulocytes by a sensitive Western blotting procedure. This revealed the presence of reduced amounts of porphobilinogen deaminase polypeptide co-migrating with wild type enzyme (Mr approximately 40,000), and no evidence of variant forms in situ. The studies show that porphobilinogen deaminase deficiency in acute intermittent porphyria is commonly associated with a CRM-phenotype. The residual activity under these circumstances is thus related to expression of a single normal allele, since sensitive techniques detected neither aberrant nor degraded forms of the enzyme in erythroid tissues.

Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients

BACKGROUND: Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. OBJECTIVES: Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. METHODS: An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. RESULTS: Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. CONCLUSIONS: Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients

Cutaneous porphyrias : Clinical and histopathological study

The prevalence of variegate porphyria (VP) (2.1:100 000, in 2006 n=108) was higher in Finland than elsewhere in European countries due to a founder effect (R152C). The incidence of VP was estimated at 0.2:1 000 000 based on the number of new symptomatic patients yearly. The prevalence of porphyria cutanea tarda (PCT) was 1.2:100 000 (in 2006 n=63), which is only one fourth of the numbers reported from other European countries. The estimated incidence of PCT was 0.5:1 000 000. Based on measurements of the uroporphyrinogen decarboxylase activity in erythrocytes, the proportion of familial PCT was 49% of the cases. The prevalence of erythropoietic protoporphyria (EPP) was at 0.8:100 000 (in 2006 n=39) including asymptomatic carriers of a mutation in the ferrochelatase (FECH) gene. The incidence of EPP was estimated at 0.1:1 000 000. After 1980 the penetrance was 37% among patients with VP. Of the mutation carriers (n=57) 30% manifested with skin symptoms. Frequency of skin symptom as only clinical sign was stable before or after 1980 (22% vs. 21%), but acute attacks became infrequent (29% vs. 7%). Of the symptomatic patients 30% had both acute attacks and skin symptoms and 80% had skin symptoms. Fragility (95%) and blistering (46%) of the skin in the backs of the hands were the most common skin symptoms. Transient correction of porphyrin metabolism using eight haem arginate infusions within five weeks had no effect on the skin symptoms in three of four patients with VP. In one case skin symptoms disappeared transiently. One patient with homozygous VP had severe photosensitivity since birth. Sensory polyneuropathy, glaucoma and renal failure developed during the 25-year follow-up without the presence of acute attacks. The I12T mutation was detected in both of his alleles in the protoporphyrinogen oxidase gene. Lack of skin symptoms and infrequency of acute attacks (1/9) in the patients with I12T mutation at the heterozygous stage indicate a mild phenotype (the penetrance 11%). Four mutations (751delGAGAA, 1122delT, C286T, C343T) in the FECH gene were characterised in four of 15 families with EPP. Burning pain (96%) and swelling (92%) of the sun-exposed skin were the major skin symptoms. Hepatopathy appeared in one of 25 symptomatic patients (4%). Clinical manifestations and associated factors of PCT were similar in the sporadic and familial types of PCT. The majority of the patients with PCT had one to three precipitating factors: alcohol intake (78%), mutations in hemochromatosis associated gene (50%), use of oestrogen (25% of women) and hepatitis B or C infections (25 %). Fatty liver disease (67%) and siderosis (67%) were commonly found in their liver biopsies. The major histopathological change of the sun-exposed skin in the patients with VP (n=20), EPP (n=8) and PCT (n=5) was thickening of the vessel walls of the upper dermis suggesting that the vessel wall is the primary site of the phototoxic reaction in each type of porphyria. The fine structure of the vessel walls was similar in VP, EPP and PCT consisting of the multilayered basement membrane and excess of finely granular substance between the layers which were surrounded by the band of homogenous material. EPP was characterised by amorphous perivascular deposits extending also to the extravascular space. In direct immunofluorescence study homogenous IgG deposits in the vessel walls of the upper dermis of the sun-exposed skin were demonstrated in each type of porphyria. In EPP the excess material around vessel walls consisted of other proteins such as serum amyloid protein, and kappa and lambda light chains in addition to the basement membrane constituents such as collagen IV and laminin. These results suggest that the alterations of the vessel walls are a consequence of the repeated damage and the repairing process in the vessel wall. The microscopic alterations could be demonstrated even in the normal looking but sun-exposed skin of the patients with EPP during the symptom-free phase suggesting that vascular change can be chronic. The stability of vascular changes in the patients with PCT after treatment indicates that circulating porphyrins are not important for the maintenance of the changes.

Prevalência da mutação do gene HFE em pacientes com porfiria cutânea tardia do Hospital Universitário Pedro Ernesto- UERJ

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.

Pseudoporfiria induzida pela diálise tratada com N-acetilcisteína oral

Pseudoporphyria is a rare bullous dermatosis that clinically and histopathologically is similar to porphyria cutanea tarda. It mainly affects patients with chronic renal failure on peritoneal dialysis or hemodialysis. Medications can also be involved in the etiology. Diagnosis and management of this condition is a challenge for dermatologists. The authors report a case of pseudoporphyria related to dialysis with favorable outcome after the use of oral N-acetylcysteine.

5-AMINOLEVULINIC ACID-INDUCED ALTERATIONS OF OXIDATIVE-METABOLISM IN SEDENTARY AND EXERCISE-TRAINED RATS

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria patients and lead-exposed individuals, has previously been shown to autoxidize with generation of reactive oxygen species and to cause in vitro oxidative damage to rat liver mitochondria. We now demonstrate that chronically ALA-treated rats (40 mg/kg body wt every 2 days for 15 days) exhibit decreased mitochondrial enzymatic activities (superoxide dismutase, citrate synthase) in liver and soleus (type I, red) and gastrocnemius (type IIb, white) muscle fibers. Previous adaptation of rats to endurance exercise, indicated by augmented (cytosolic) CuZn-superoxide dismutase (SOD) and (mitochondrial) Mn-SOD activities in several organs, does not protect the animals against liver and soleus mitochondrial damage promoted by intraperitoneal injections of ALA. This is suggested by loss of citrate synthase and Mn-SOD activities and elevation of serum lactate levels, concomitant to decreased glycogen content in soleus and the red portion of gastrocnemius (type IIa) fibers of both sedentary and swimming-trained ALA-treated rats. In parallel, the type IIb gastrocnemius fibers, which are known to obtain energy mainly by glycolysis, do not undergo these biochemical changes. Consistently, ALA-treated rats under swimming training reach fatigue significantly earlier than the control group. These results indicate that ALA may be an important prooxidant in vivo.

Estresse oxidativo em porfiria hepática experimental disparada por succinilacetona - um inibidor da ácido 5-aminolevulínico desidratase

Para otimizar um modelo experimental para o estudo do desbalanço redox em porfirias relacionadas ao acúmulo de ácido 5-aminolevulínico-(ALA), via inibição da ALA desidratase-(ALA-D), ratos foram tratados com o éster metílico de succinilacetona-(SAME), um catabólito da tirosina que inibe fortemente a ALA-O, mimetízando o estado metabólico observado nos portadores de portirias e tirosinemias. Estabeleceram-se modelos de tratamento agudo por 36 e 18 h. No primeiro, os animais receberam 3 injeções de SAME (10, 40 ou 80 mg/kg, grupos Ali-IV). No segundo, os animais receberam 3 injeções de 40 mg/kg de SAME, ALA ou éster metílico de ALA (grupos BII-IV), ALA:SAME (30: 10 mg/kg, grupo BV), ou 10 mg/kg SAME (grupo BVI). Paralelamente, avaliou-se se os sintomas neurológicos característicos das portirias decorriam de danos oxidativos mitocondriais. Para isso, aplicou-se uma tecnologia óptica para medidas da difusão da depressão cortical que determinou a oxigenação e o estado redox do cit c em mitocôndrias do córtex cerebral de ratos submetidos ao tratamento crônico com ALA (40 mg/kg), SAME (10 e 40 mg/kg) e ALA:SAME (30: 1O mg/kg), a cada 48 h, durante 30 dias. Tratamento agudo/36 h: Os níveis de ALA no plasma, fígado, cérebro e urina e o clearance renal do ALA aumentaram nos grupos tratados. A atividade de ALA-D e a coproporfirina urinária reduziram. A marcação para proteínas carboniladas, ferro e ferritina aumentou no fígado e cérebro dos grupos tratados, especialmente no All. Os níveis de malondialdeído hepático aumentaram no grupo AIV. A razão GSH/GSH+GSSG e a atividade de GPx cerebrais aumentaram nos grupos AIV e AIII, respectivamente. Consistentemente com estes dados indicando um desbalanço oxidativo induzido pelo SAME, alterações mitocondriais e citosólicas ultraestruturais foram reveladas, especialmente no fígado. Tratamento agudo/18 h: Os níveis de ALA plasmáticos aumentaram nos grupos tratados, exceto em BIV. O grupo BII mostrou aumento dos níveis hepáticos de ALA. Interessantemente, a inibição da atividade de ALA-D não foi evidenciada. O conteúdo de ferro plasmático aumentou no grupo BII. Para os grupos tratados com 10 e 40 mg SAME/kg, a atividade de SOD hepática reduziu ~50% com a extensão do tratamento de 18 para 36 h, sugerindo que este último é mais efetivo em promover danos oxidativos induzidos pelo ALA. Tratamento crônico/30 dias: Embora nenhuma alteração tenha sido evidenciada no estado redox dos animais tratados, o tratamento com ALA reduziu o fluxo sanguíneo cerebral (CBF) e o consumo de oxigênio-(CMRO2), sugerindo uma vasoconstrição mediada pelo ALA, efeito este confirmado por ensaios de reatividade vascular conduzidos em anéis de aorta de ratos incubados com ALA. O tratamento com ALA:SAME restaurou os níveis de CBF e CMRO2. Interessantemente, a disponibilidade do radical superóxido-(O2•-) estava reduzida nos anéis de aorta incubados com ALA. Juntos, estes dados: a)validam o modelo de tratamento agudo/36 h para o estudo bioquímico e dos possíveis efeitos fisiológicos induzidos pelo ALA, e b)sugerem que as alterações mediadas pelo ALA exógeno levam à vasoconstrição.