Demystification of Chester porphyria: a nonsense mutation in the Porphobilinogen Deaminase gene

The porphyrias arise from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings, and specific enzyme assays. In some cases however, these diagnostic tools reveal overlapping findings, indicating the existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultaneously. Recently, it was reported that the so-called Chester porphyria shows features of both variegate porphyria and acute intermittent porphyria. Linkage analysis revealed a novel chromosomal locus on chromosome 11 for the underlying genetic defect in this disease, suggesting that a gene that does not encode one of the enzymes of heme biosynthesis might be involved in the pathogenesis of the porphyrias. After excluding candidate genes within the linkage interval, we identified a nonsense mutation in the porphobilinogen deaminase gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria. However, we could not detect a mutation in the coding or the promotor region of the protoporphyrinogen oxidase gene that is mutated in variegate porphyria. Our results indicate that Chester porphyria is neither a dual porphyria, nor a separate type of porphyria, but rather a variant of acute intermittent porphyria. Further, our findings largely exclude the possibility that a hitherto unknown gene is involved in the pathogenesis of the porphyrias.     

Molecular characterization of porphyrias in Italy: a diagnostic flow-chart.

The porphyrias are disorders associated with inherited or acquired enzyme deficiencies in the heme biosynthetic pathway. The differential diagnosis is often difficult since the phenotype is very similar in some forms and the biochemical tests are not commonly available. Here we provide an update on the molecular diagnosis of porphyrias in Italy and a flow-chart to facilitate the identification of mutations in heme biosynthetic genes. The molecular analysis has allowed us to identify the molecular defect underlying the disease in 66 probands with different porphyrias [acute intermittent porphyria (AIP), variegate porphyria (VP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP)]. No Italian patients with defects in coproporphyrinogen oxidise (CPOX) gene, responsible for hereditary coproporphyria (HCP), have been detected. The molecular characterization has been extended to 115 relatives with the identification of 55 asymptomatic mutation carriers and 60 normal subjects. We have so far identified 50 different mutations among 4 genes associated with the most common porphyrias showing a high molecular heterogeneity: 22 in the hydroxymethylbilane synthase (HMBS) gene (AIP), 7 in the protoporphyrinogen oxidase (PPOX) gene (VP), 16 in the uroporphyrinogen decarboxylase (UROD) gene (PCT) and 5 in the ferrochelatase (FECH) gene (EPP). Among the 50 molecular defects, 29 seem to be restricted to the Italian population.     

Acute porphyrias in the Argentinean population: a review.

The porphyrias are a group of inherited metabolic disorders of heme biosynthesis which result from a partial deficiency in one of its seven specific enzymes, after its first and rate limiting enzyme, delta-aminolevulinic acid synthetase. They can be classified on the basis of their clinical manifestations into cutaneous, acute and mixed disorders. Acute intermittent porphyria (AIP) is the most common type of hepatic acute porphyrias, inherited as an autosomal dominant trait, caused by a defect in the gene which codifies for the heme enzyme porphobilinogen deaminase. Its prevalence in the Argentinean population is about 1:125,000. A partial deficiency in another enzyme, protoporphyrinogen oxidase, produces variegate porphyria (VP), the second acute porphyria most frequent in the Argentinean population (1:600,000). Here, we review all the mutations we have found in 46 AIP and 9 VP unrelated Argentinean patients. To screen for mutations in symptomatic patients, we have proposed a geneticresearch strategy.     

De Novo mutation found in the porphobilinogen deaminase gene in Slovak acute intermittent porphyria patient: molecular biochemical study

The porphyrias are group of mostly inherited disorders in which a specific spectrum of accumulated and excreted porphyrins and heme precursors are associated with characteristic clinical features. There are eight enzymes involved in the heme synthesis and defects in seven of them cause porphyria. Four of them are described as acute hepatic porphyrias, which share possible precipitation of acute attacks with symptoms engaging the nervous system. Acute intermittent porphyria (AIP), caused by partial deficiency of the porphobilinogen deaminase (PBGD), is the most frequent among hepatic porphyrias. Clinical expression is highly variable and ~ 90 % of AIP heterozygotes remain asymptomatic throughout life. During systematic genetic analysis of the AIP patients diagnosed in the Czech and Slovak Republics, we found a special case of AIP. In a 15-year-old boy with abdominal and subsequent neurological symptomatology, we identified de novo mutation 966insA within the PBGD gene leading to a stop codon after 36 completely different amino acids compared to the wt-sequence. To establish the effects of this mutation on the protein structure, we expressed mutant constructs with described mutation in E. coli and analyzed their biochemical and enzymatic properties. Moreover, computer-assisted protein structure prediction was performed.     

Porphyrias: Animal models and prospects for cellular and gene therapy

The rapid progress in the development of molecular technology has resulted in the identification of most of the genes of the heme biosynthesis pathway. Important problems in the pathogenesis and treatment of porphyrias now seem likely to be solved by the possibility of creating animal models and by the transfer of normal genes or cDNAs to target cells. Animal models of porphyrias naturally occur for erythropoietic protoporphyria and congenital erythropoietic porphyria, and different murine models have been or are being created for erythropoietic and hepatic porphyrias. The PBGD knock-out mouse will be useful for the understanding of nervous system dysfunction in acute porphyrias. Murine models of erythropoietic porphyrias are being used for bone-marrow transplantation experiments to study the features of erythropoietic and hepatic abnormalities. Gene transfer experiments have been startedin vitro to look at the feasibility of somatic gene therapy in erythropoietic porphyrias. In particular, we have documented sufficient gene transfer rate and metabolic correction in different CEP disease cells to indicate that this porphyria is a good candidate for treatment by gene therapy in hematopoietic stem cells. With the rapid advancement of methods that may allow more precise and/or efficient gene targeting, gene therapy will become a new therapeutic option for porphyrias.     

Survivors of Myocardial Infarction due to “Essential” Atherosclerosis

Currently, the porphyrias are classified in four main groups: congenital porphyria, acute intermittent porphyria, porphyria cutanea tarda hereditaria, and porphyria cutanea tarda symptomatica. The acquired form of porphyria (porphyria cutanea tarda symptomatica) occurs in older males and is nearly always associated with chronic alcoholism and hepatic cirrhosis. The main clinical changes are dermatological, with excessive skin fragility and photosensitivity resulting in erosions and bullae. Biochemically, high levels of uroporphyrin are found in the urine and stools. Treatment to date has been symptomatic and usually unsuccessful.A case of porphyria cutanea tarda symptomatica is presented showing dramatic improvement of both the skin lesions and porphyrin levels in urine and blood following repeated phlebotomy.Possible mechanisms of action of phlebotomy on porphyria cutanea tarda symptomatica are discussed.     

Establishing a network of specialist Porphyria centres - effects on diagnostic activities and services

Background

The porphyrias are a heterogeneous group of rare metabolic diseases. The full spectrum of porphyria diagnostics is usually performed by specialized porphyria laboratories or centres. The European Porphyria Initiative (EPI), a collaborative network of porphyria centres formed in 2001, evolved in 2007 into the European Porphyria Network (EPNET), where participating centres are required to adhere to agreed quality criteria. The aim of this study was to examine the state and distribution of porphyria diagnostic services in 2009 and to explore potential effects of increased international collaboration in the field of these rare diseases in the period 2006–2009.

Methods

Data on laboratory, diagnostic and clinical activities and services reported to EPI/EPNET in yearly activity reports during 2006 through 2009 were compared between reporting centres, and possible time trends explored.

Results

Thirty-five porphyria centres from 22 countries, five of which were non-European associate EPNET members, filed one or more activity reports to EPI/EPNET during the study period. Large variations between centres were observed in the analytical repertoire offered, numbers of analyses performed and type and number of staff engaged. The proportion of centres fulfilling the minimum criteria set by EPNET to be classified as a specialist porphyria centre increased from 80% to 94% during the study period.

Conclusions

Porphyria services are unevenly distributed, and some areas are probably still lacking in specialized porphyria services altogether. However, improvements in the quality of diagnostic services provided by porphyria centres participating in EPI/EPNET were observed during 2006 through 2009.

     

Late-onset porphyrias: what are they?

Porphyrias are inherited disorders of heme biosynthesis. ALA dehydratase porphyria (ADP) and congenital erythropoietic porphyria (CEP) are autosomal recessive porphyrias, and are typically expressed at birth or in childhood. However, a few cases of late-onset recessive porphyrias have been reported. Recently we encountered a late-onset ADP patient who developed symptoms of acute porphyria when he was 63 years old. This was accompanied by polycythemia vera. It was concluded that he developed the porphyria because an abnormal ALAD allele was clonally expanded by polycythemia vera. Upon reviewing the literature, a few cases of late-onset CEP were found to be also associated with hematologic abnormalities suggestive of myelodysplastic syndrome (MDS), another clonal disorder. These findings suggest that these late-onset porphyrias may be heterozygous for their gene defects, but clinical expression may be elicited if there is a loss of heterozygosity, either by a clonal expansion of the porphyric allele or by a loss of function mutation in the other allele.     

一例急性间歇性血卟啉病病例报道及文献回顾

目的:对患有急性间歇性血卟啉病先证者及其两位直系亲属进行基因突变的分析。方法:采用PCR和一代测序技术分别对患者的HMBS基因的外显子及其旁翼区进行序列分析。结果:检测出先证者HMBS基因11号外显子的旁翼区发生杂合突变c.651+2AG,为剪切突变;从先证者母亲以及女儿的HMBS基因上检测出同样的突变位点。结论:根据先证者的家族史、临床表现及相关代谢检查结果诊断为血卟啉病;基因检测结果提示先证者为急性间歇性血卟啉病;先证者的母亲和女儿存在同样的突变位点,提示先证者母亲及其女儿均患有急性间歇性血卟啉病。     

Evidence for involvement of a second genetic locus on chromosome 11q in porphyrin metabolism

Chester porphyria is a distinct type of acute porphyria, which shows a biochemical overlap with acute intermittent and variegate porphyrias and has a dual enzyme deficiency of porphobilinogen deaminase (PBGD) and protoporphyrinogen oxidase. Linkage analysis in an extensive family with Chester porphyria was undertaken using multiple polymorphic markers. A maximum two point Lod score of 5.25 at 0.07 recombination (confidence interval 0.01 to 0.14) was observed with D11S351, which has been localised to 11q23.1. Multipoint linkage analysis confirmed the two point results and gave a maximum Lod score of 7.33 at a distance less than 1 cM proximal to D11S351. PBGD also maps to 11q but four recombinants could be identified from ten informative meioses in this family using a PBGD DNA polymorphism. Thus, a separate locus on 11q appears to be the basis of Chester porphyria.     

European porphyria initiative (EPI): a platform to develop a common approach to the management of porphyrias and to promote research in the field

Porphyrias are uncommon inherited diseases of haem biosynthesis for which the diagnosis and treatment varies in individual countries. Despite the existence of guidelines recommended by porphyria experts concerning the diagnosis and management of the acute porphyrias, and of specialist centres in most European countries, many clinicians still do not apply these guidelines. The European Porphyia Initiative (EPI) network was formed in 2001 in order to compare experience among countries to attempt to develop a common approach to the management of the porphyrias, particularly concerning recommendation of safe and unsafe drugs, and to facilitate international collaborative clinical and biological research. The main achievements of EPI during this period have been: * Drafting and agreeing to consensus protocols for the diagnosis and management of acute hepatic porphyrias. * Creation of a multilingual website, particularly focusing on guidelines for common prescribing problems in acute porphyria and on providing information for patients that is now available in 10 languages: (www.porphyria-europe.org). EPI's current objectives are to develop the EPI platform, expand to new countries, extend to non-acute porphyrias and design European research and clinical trials in porphyria. The project will focus on: 1. Setting up a European laboratory external quality assurance scheme (EQAS) for biochemical and molecular investigations and their interpretation 2. Establishing a consensus drug list in collaboration with the Nordic porphyria network 3. Improving patient counseling 4. Developing large multi-centre, multi-national research projects. Due to the rarity of the porphyrias, it would be very difficult for any one country to provide this data with a sufficient number of patients and within a reasonable timescale. The progress achieved will facilitate improvements in the treatment and development of new therapeutic strategies. It will set a pattern for establishing, and subsequently harmonising, between countries best clinical practice for a rare but important group of diseases, and will help to develop the optimal therapy and ensure its cost effectiveness.     

Genetic investigation of the porphobilinogen deaminase gene in Swedish acute intermittent porphyria families

A total of 12 mutations associated with acute intermittent porphyria (AIP) have been detected in the porphobilinogen deaminase gene in Swedish AIP families. Three of them are newly discovered and unique to the Swedish population: a splice mutation in intron 6 (int6+1), a missense mutation in exon 11 (Gly216Asp) and a TG deletion in exon 14. Received: 23 December 1996 / Accepted: 17 February 1997     

Detection of DNA variations in the polymorphic hydroxymethylbilane synthase gene by high-resolution melting analysis

Acute intermittent porphyria (AIP) represents the most frequent type of acute porphyria. The underlying cause is a defect in the hydroxymethylbilane synthase (HMBS) gene. Diagnosis of AIP is crucial for preventing life-threatening, acute attacks among both symptomatic and asymptomatic carriers. We established the diagnostic tool, high-resolution melting (HRM), for diagnosing AIP. Of 13 amplicons amplified by PCR in the presence of the LCGreen Plus dye, 4 showed polymorphic backgrounds. The ability of the HRM method to detect DNA variations in the HMBS gene was tested on a DNA sample with 10 known mutations by a curve shape scan using the LightScanner instrument. Furthermore, genomic DNA (gDNA) samples from 97 individuals with suspected hepatic porphyria were tested. All possible genotypes from each of four polymorphic amplicons were detected. Each of the 10 mutations tested had an altered melting profile compared with the melting profile of the controls. Screening the group of subjects with suspected hepatic porphyria revealed nine different DNA variations, four of which were novel. In conclusion, HRM is a fast, cost-effective prescreening method for detecting DNA variations in the HMBS gene. Therefore, the screening can be easily applied to a porphyria family if misdiagnosis or rare dual porphyria is suspected.     

Identification of a mutation in the ovine uroporphyrinogen decarboxylase (UROD) gene associated with a type of porphyria

Porphyria is a group of at least eight diseases caused by abnormalities in the chemical steps that lead to haeme production. The different types of porphyria show different signs and symptoms and can be strongly influenced by environmental factors. Mutations of the uroporphyrinogen decarboxylase (UROD) gene have been shown to be causative for porphyria cutanea tarda (PCT) in humans. Porphyria is a rare disorder in livestock. Although disorders of haeme biosynthesis have been described in cattle, pigs, sheep and cats, PCT has only been reported in pigs. We observed typical signs of porphyria (photosensitivity and porphyrinuria) in a flock of German Blackface sheep and postulated that the porphyria could be caused by a mutation in the UROD gene. To investigate this, we cloned and sequenced the ovine UROD gene. We identified a single point mutation (C --> T) in UROD which leads to an amino acid substitution at Leu 131 Pro, which is located within the active cleft site of the UROD protein.     

Analytical review enzymatic defects of hereditary porphyrias: An explanation of dominance at the molecular level

Summary In four of the five autosomal dominant porphyrias four different partial enzymatic defects of the porphyrin biosynthetic pathway have been discovered in the last few years. With the exception of protoporphyria, the residual enzymatic activity in carriers of these defects is approximately equal to 50% of that found in controls. In each case the pattern of excretion of porphyrin and/or porphyrin precursors reflects the site of the partial metabolic block. There are indications, at least in intermittent acute porphyria, that the degree of penetrance of the disorder varies according to the level of phenotypic expression, being highest for the enzyme deficiency, lower for the excretion of precursors and lowest for the clinical symptoms. It is proposed that environmental factors, and probably also gene interaction, are the cause of the different degrees of penetrance.On leave from the University of Naples, Italy     

DNA polymorphisms within the porphobilinogen deaminase gene in two Swedish families with acute intermittent porphyria

Summary Two unrelated families with acute intermittent porphyria (AIP), an autosomal dominant disease related to a defect in porphobilinogen deaminase (PBG-D, EC 4.1.3.8.), were studied with regard to three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, BstNI) within the PBG-D gene. The results indicate that linkage analysis of RFLPs within the gene can be used as a complement to PBG-D analysis for the diagnosis of gene carriers in families with AIP.     

Volatile anaesthetics induce biochemical alterations in the heme pathway in a B-lymphocyte cell line established from hepatoerythropoietic porphyria patients (LBHEP) and in mice inoculated with LBHEP cells

Hepatoerythropoietic porphyria (HEP) is the homozygous form of Porphyria Cutanea Tarda (PCT), characterized by an accumulation of porphyrins due to uroporphyrinogen decarboxylase deficiency.Fluorinated volatile anaesthetics are often used to produce general anaesthesia. Anaesthesia has certainly been implicated in the triggering of acute porphyria crisis.The effects of volatile anaesthetics in a B-lymphocyte cell line established from HEP patients (LBHEP) on heme metabolism have been investigated.LBHEP cells were exposed to sodium phosphate buffer containing dissolved Enflurane, Isoflurane or Sevoflurane (10mM) during 20min.Aminolevulinate synthase (ALA-S) activity, the regulatory enzyme of heme synthesis, was 300% induced by the anaesthetics. A 25-30% diminution of porphobilinogenase (PBG-ase) activity was found when Isoflurane or Sevoflurane were added to the cells, while no significant changes were detected after Enflurane treatment.Although some oxidative stress has been induced by the anaesthetics, reflected by the 35% diminution of glutathione (GSH), no alteration in heme oxygenase (HO) activity, the enzyme involved in heme breakdown and frequently induced as a response to stress stimuli, was observed.Studies using animals inoculated with LBHEP cells were also performed. Findings here described mimic biochemical alterations in the heme pathway, which are characteristic of another hepatic porphyria, similar to those previously reported when these anaesthetics were administered to animals, and they also advertise about the possible unsafe use of these drugs in the case of hepatic non-acute porphyrias.     

Uroporphyrinogen synthase in human blood: developmental studies.

Uroporphyrinogen synthase (URO-S), the enzyme that catalyzes the conversion of porphobilinogen to uroporphyrinogen I, has been measured in whole blood lysates by a fluorometric microassay. Cord and fetal bloods have 3 and 6 times the specific activity, respectively, of adult control subjects. The three groups seem to present a similar genetic heterogenity with ratios of highest to lowest URO-S specific activity close to 2. These results establish normal ranges for URO-S activity in human blood, which may be useful for the early detection of carriers of a gene for acute intermittent porphyria.     

In inherited porphyrias, lead intoxication is a toxogenetic disorder

1. delta-Aminolevulinic acid dehydratase (ALA-D), blood lead and several enzymes and metabolites of the heme biosynthetic pathway were measured in a number of symptomatic porphyric patients, 22 with acute intermittent porphyria, three with hereditary hepatic coproporphyria, 10 with hereditary porphyria cutanea tarda, two with erythropoietic protoporphyria and two with congenital erythropoietic porphyria and in 84 lead intoxicated persons. 2. In the 39 individuals suffering from the inherited porphyrias and in 32 lead poisoned patients with a 30-50% reduced deaminase, blood lead content was not sufficiently increased (average 28 micrograms%) to account for the greatly decreased activity of ALA-D (average 36% of controls). 3. After a relatively trifling lead exposure they developed the signs of acute lead intoxication. 4. A second group of lead intoxicated patients showing low ALA-D activity and corresponding high concentration of lead in blood, exhibited no other physiologic deviation in the enzymes and metabolites of porphyrin biosynthesis. 5. Individuals with inherited porphyrias are ultrasensitive to low level lead exposure and that lead would also act as a triggering factor. In these patients, lead intoxication can be considered a toxogenetic disorder. 6. An inversely linear correlation between ALA-D activity and blood lead content was obtained for both groups of lead intoxicated patients, however, a different constant (k) for each was obtained, which we have taken as a measure of lead toxogeneticity: k = 10 +/- 1 for lead intoxicated individuals with otherwise normal heme metabolism and k = 5 +/- 0.5 for lead intoxicated symptomatic porphyric patients. 7. Determination of erythrocytic ALA-D, besides blood lead, will be a valuable indicator for preventive medical care for these patients, when they are expected to be exposed to lead either environmentally or in their professional life.     

Uroporphyrinogen-I-synthetase in erythrocytes in acute intermittent porphyria

The primary genetic defect in acute intermittent porphyria is a decreased uroporphyrinogen I-synthetase [EC.4.3.1.8] activity. As a beginning of a genealogical study of the known families with members suffering from this disease in the People's Republic of Bulgaria, the red cell uroporphyrinogen I-synthetase was determined in 3 families by the method of Mandel et al [8]. Except for the three propositi, an enzyme deficiency was established in 3 latent carriers of the pathological gene, two of whom had normal values of the urinary epsilon-aminolevulinic acid and porphobilinogen. The determination of red cell uroporphyrinogen I-synthetase proved to be a valuable parameter for revealing the latent AIP.