Acute Intermittent Porphyria Patients Research Articles

Anthropometric and Quality-of-Life Parameters in Acute Intermittent Porphyria Patients.

The porphyrias are a group of rare metabolic disorders. The incidence and prevalence are low because the acute intermittent porphyria (AIP) is rare. Our aim was to assess the use of anthropometric and quality-of-life parameters in porphyric patients in order to identify predictor factors that might help in characterizing AIP patients.Sixteen AIP patients from Murcia (Spain) were recruited from local health centers in 2008 and 2009. A control group of 16 healthy people was established. Body composition was assessed by bioelectrical impedance analysis (BIA) and anthropometric measurements: body weight; height; knee-heel height; waist, hip, upper arm and calf circumferences (CCs); biacromion and biiliac diameters; bicondylar and biepicondylar width; and triceps, subscapular, supraspinale, and calf skinfold thickness. Anthropometric indicators were obtained from anthropometric measurements. A quality-of-life evaluation was carried out using the EuroQol-5D (EQ-5D) questionnaire and Barthel and Katz indexes. Significant differences in means were tested by unpaired Student t test. Group differences in anthropometric measurements were tested with a 2-way analysis of variance (group × condition: age group, overweight, and adiposity degree). Relative frequencies were obtained for noncontinuous variables. Significant differences in prevalence were calculated by means of χ2.AIP patients showed statistically significant differences in terms of knee-heel height, biiliac diameter, CC, triceps skinfold thickness, BIA, ponderal index, endomorphy, and ectomorphy. Only 1 quality-of-life indicator, visual analog scale, in the EQ-5D questionnaire showed significant differences between porphyric and control groups.Some anthropometric parameters and the EQ-5D questionnaire could be used to appreciate the presence or follow the evolution of the disease in AIP patients.

Acute intermittent porphyria symptoms during the menstrual cycle.

Acute intermittent porphyria (AIP), a life-threatening form of the disease, is accompanied by several pain, mental and physical symptoms. In this study, we evaluated the cyclicity of AIP and premenstrual syndrome (PMS) symptoms in 32 women with DNA-diagnosed AIP during their menstrual cycles, in northern Sweden. The cyclicity of AIP symptoms and differences in them between the follicular and luteal phases, and the cyclicity of each symptom in each individual woman in different phases of her menstrual cycle were analysed with a prospective daily rating questionnaire. PMS symptoms were also evaluated in the patients on a daily rating scale. Of the 32 women, 30 showed significant cyclicity in at least one AIP or PMS symptom (P < 0.05-0.001). Back pain (10/32) was the most frequent AIP pain symptom and sweet craving (10/15) was the most frequent PMS symptom. Pelvic pain (F = 4.823, P = 0.036), irritability (F = 7.399, P = 0.011), cheerfulness (F = 5.563, P = 0.025), sexual desire (F = 8.298, P = 0.007), friendliness (F = 6.157, P = 0.019), breast tenderness (F = 21.888, P = 0.000) and abdominal swelling (F = 16.982, P = 0.000) showed significant cyclicity. Pelvic pain and abdominal swelling (rs = 0.337, P < 0.001) showed the strongest correlation. The age of women with latent AIP was strongly correlated with abdominal swelling during the luteal phase (rs = 0.493, P < 0.01). Our results suggest that the symptoms of AIP patients change during their menstrual cycles.

Mass spectrometric characterisation of a condensation product between porphobilinogen and indolyl-3-acryloylglycine in urine of patients with acute intermittent porphyria.

We document the presence of a previously unknown species in the urine of patients with acute intermittent porphyria (AIP). The compound was fully characterised by liquid chromatography tandem mass spectrometry. Interpretation of both full spectrum acquisition and product ion spectra acquired in positive and negative ionisation modes by quadrupole time of flight MS allowed for the identification of a condensation product arising from porphobilinogen (PBG, increased in the urine of AIP patients) and indolyl-3-acryloylglycine (IAG, derived from indolylacrylic acid and present in human urine). The structure was unequivocally confirmed through comparison between the selected reaction monitoring chromatograms obtained from the urinary species and the condensation product qualitatively synthesised in the laboratory. Owing to the large amounts of both PBG and IAG in urine of AIP patients, the possible ex vivo formation of PBG-IAG in urine samples was evaluated. The product was spontaneously formed at room temperature, at 4 °C and even during storage at -20 °C when spiking a control sample with PBG. A positive correlation was found between PBG and PBG-IAG in samples collected from AIP patients. However, no correlation was found between PBG-IAG and IAG. Purified PBG-IAG did not form the characteristic chromogen after application of p-dimethylaminobenzaldehyde in HCl, thus suggesting that the current techniques used to measure PBG in urine of AIP patients based on Ehlrich's reaction do not detect this newly characterised PBG-IAG fraction.

Vitamin D-binding protein as a biomarker of active disease in acute intermittent porphyria

Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder caused by a deficiency of hepatic porphobilinogen deaminase (PBGD). The disease is characterized by life threatening acute neurovisceral attacks. The aim of this study was to identify metabolites secreted by the hepatocytes that reflect differential metabolic status in the liver and that may predict response to the acute attack treatment. Plasma vitamin D binding protein (VDBP) from a mouse model of AIP displayed an abnormal migration in 2D-electrophoresis that is efficiently recovered upon gene therapy leading to liver specific over-expression of the PBGD protein. The change in VDBP mobility results from a differential isoelectric point suggesting a post-translational modification that takes place preferably in the liver. Liquid chromatography–mass spectrometry (LC–MS) analysis of human samples before and after glycosidase treatment revealed glycosylated plasma VDBP specifically in patients with recurrent attacks of AIP. Glycosylated VDBP recovered normal values in three severely afflicted AIP patients submitted to therapeutic liver transplantation. Our findings suggest that post-translational modification of VDBP might be considered as a promising biomarker to study and monitor the liver metabolic status in patients with AIP. SignificanceWe describe an increased glycosylation of VDBP in porphyric livers. Normal glycosylation was recovered upon liver gene therapy in a mouse model of porphyria or after liver transplantation in severely afflicted patients with AIP. Moreover, quantification of glycosylated VDBP by our ELISA immunoassay or LC–MS protocol in patients undergoing PBGD-gene therapy (www.aipgene.org) may be used as a marker indicating improvement or normalization of the patient's hepatic metabolism.This article is part of a Special Issue entitled: HUPO 2014.

Hydroxymethylbilane synthase gene mutations and polymorphisms in Brazilian families with acute intermittent porphyria.

Acute intermittent porphyria (AIP), an autosomal dominant disorder, is caused by a deficiency of hydroxymethylbilane synthase (HMBS). In the present study, we sought to establish a correlation between HMBS activity with the presence of mutations and polymorphisms. Enzyme activity was measured in red blood cells of four Brazilian unrelated AIP families (n = 124) and in blood donors (n = 80). The HMBS mutations in AIP family members were studied by PCR-SSCP followed by direct sequencing. Six intragenic SNPs (1345 G>A, 1500 T>C, 2377 C>A, 2478 A>G, 3581 A>G, and 7064 C>A) were determined by PCR-RFLP. Abnormal SSCP patterns in exons 7, 9, 12, and 15 were observed. DNA sequencing analysis revealed one nonsense mutation, R149X, two missense mutations, G111R and L338P, and one deletion, CT 730-731. All mutation carriers had lower enzyme activity. All polymorphisms, except 2377 C>A and 7064 C>A, showed no significant differences compared with previous reports. Mutation screening allowed the detection of the missense mutation, L338P, and the 730_731delCT deletion, two as yet unreported mutations in Brazilian AIP patients. Our findings also showed a high frequency of 2478 A>G and 3581 A>G polymorphism combinations suggesting that these polymorphisms contributed to enzymatic activity reduction in our study population.

Liver Fibrosis Associated with Iron Accumulation Due to Long-Term Heme-Arginate Treatment in Acute Intermittent Porphyria: A Case Series.

Regular heme-arginate treatment in AIP does not only lead to increased serum ferritin but may also induce liver fibrosis. This should be taken into account, when weighing the risks and benefits of repeated heme-arginate treatment against the risk and benefits of treating refractory AIP by liver transplantation.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Porphobilinogen deaminase gene mutations in Polish patients with non-erythroid acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an metabolic disorder characterized by a partial deficiency of the porphobilinogen deaminase, the enzyme of heme biosynthesis. The metabolic defect in AIP involves an approximately half-normal activity of porphobilinogen deaminase (PBGD, EC 4.3.1.8), the enzyme catalyzing condensation of four porphobilinogen molecules to hydroxymethylbilane. Due to tissue-specific alternative transcript splicing, the PBGD gene mutations within the range of exons 3-15 may lead to classical AIP involving erythrocytes and all the other tissues. Mutations within intron and exon 1 may result in the so-called non-erythroid AIP in which the PBGD activity is normal in erythrocytes and diminished in other tissues. The aim of the present study was to characterise molecular errors in the PBGD gene in Polish patients with non-erythroid AIP and to evaluate the efficacy of the DNA sequencing method in the early diagnosis of this disorder. Twenty five members of nine non-erythroid AIP families were assessed. In each of them DNA sequencing was performed using the Big Dye Terminator Cycle Sequencing Kit v.1.1 on the Hitachi 3730 Analyzer (Applied Biosystem, USA). Four mutations were detected in intron 1 of the PBGD gene, including one unreported novel mutation, 33+(4-12) del AGTGCTGAG, of an unknown biological mechanism, and three previously described mutations, i.e. 33+1 G > A, 33+2 T > C, 33+5 G > C, responsible for abnormal transcript splicing in the area of exon 1. Of 14 asymptomatic members of proband families in 6 subjects were diagnosed with AIP, and in 8 the AIP was excluded based on the DNA sequencing method. DNA sequencing based analysis is the only reliable method for correct diagnosis of asymptomatic non-erythroid AIP patients with normal urinary excretion of heme precursors. The mutations found in Polish patients with non-erythroid AIP represented those of splice defect and resulted in abnormal exon 1 splicing.

Gene therapy for rare diseases – the case for acute intermittent Porphyria

It could be argued that gene therapy is the ultimate personalised medicine and potentially preventive medicine too. There is little closer to the personalisation of medicine than the ability to repair or restore the function of a person’s own faulty genes, the core principle of gene therapy. With the first approval of gene therapy products in both the western and eastern world, this is increasingly close to becoming a reality in clinical practice. At the same time, the advances in scientific understanding and technological ability to analyse the human genome imply that gene therapy could also be used to prevent the development of disease. Many rare diseases are monogenic diseases and as such are ideal candidates for gene therapy. One example is Acute Intermittent Porphyria (AIP). The disease is caused by mutations in the porphobilinogen deaminase (pbgd) gene which encodes one of the enzymes involved in the haem biosynthesis pathway. This gives rise to enzyme deficiency and in turn accumulation of intermediate toxic metabolites in the heam pathway. The disease which has prevalence in Europe of approximately 5.9 per million population, manifests as severe abdominal pain, mental symptoms and peripheral neuropathy leading to poor life quality. Current therapy targets the symptoms of the disease without being able to modify the pathology, let alone provide a cure. AMT-021 is an adeno-associate virus 5 (AAV5)-based gene therapy product that aims to specifically deliver and express PBGD in the liver cells of affected patients therefore restoring the defect in haem biosynthesis and avoiding the accumulation of associated toxic metabolites. Following a series of preclinical development programme and pivotal toxicology and safety studies AMT-021 entered clinical trials (Phase I) in December 2012. This was the first time a potentially curative treatment was tested in AIP patients and from a scientific perspective this was the first time an AAV-5 based gene therapy product was administered in humans. The trial is expected to be completed in the second half of 2014. Outside the significance of this drug development programme for the treatment of patients with AIP and gene therapy for rare diseases, it also represents a successful example of an academic-industrial partnership that has been made possible by funding from the European Commission’s Framework Programme 7. The AIPGENE consortium right from the beginning aimed to harness excellent pre-clinical research and by following a very pragmatic and translational approach focused it on developing a much needed therapy for a rare disease while extending the scientific frontiers of gene therapy as well.

Urinary Metabolic Fingerprint of Acute Intermittent Porphyria Analyzed by 1H NMR Spectroscopy

(1)H NMR is a nonbiased technique for the quantification of small molecules that could result in the identification and characterization of potential biomarkers with prognostic value and contribute to better understand pathophysiology of diseases. In this study, we used (1)H NMR spectroscopy to analyze the urinary metabolome of patients with acute intermittent porphyria (AIP), an inherited metabolic disorder of heme biosynthesis in which an accumulation of the heme precursors 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) promotes sudden neurovisceral attacks, which can be life-threatening. Our objectives were (1) to demonstrate the usefulness of (1)H NMR to identify and quantify ALA and PBG in urines from AIP patients and (2) to identify metabolites that would predict the response to AIP crisis treatment and reflect differential metabolic reprogramming. Our results indicate that (1)H NMR can help to diagnose AIP attacks based on the identification of ALA and PBG. We also show that glycin concentration increases in urines from patients with frequent recurrences at the end of the treatment, after an initial decrease, whereas PBG concentration remains low. Although the reasons for this altered are elusive, these findings indicate that a glycin metabolic reprogramming occurs in AIPr patients and is associated with recurrence. Our results validate the proof of concept of the usefulness of (1)H NMR spectroscopy in clinical chemistry for the diagnosis of acute attack of AIP and identify urinary glycin as a potential marker of recurrence of AIP acute attacks.

Adrenal hormonal imbalance in acute intermittent porphyria patients: results of a case control study.

BackgroundAcute Intermittent Porphyria (AIP) is a rare disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers are at risk of presenting acute life-threatening neurovisceral attacks. The disease induces overproduction of heme precursors in the liver and long-lasting deregulation of metabolic networks. The clinical history of AIP suggests a strong endocrine influence, being neurovisceral attacks more common in women than in men and very rare before puberty. To asses the hypothesis that steroidogenesis may be modified in AIP patients with biochemically active disease, we undertook a comprehensive analysis of the urinary steroid metabolome.MethodsA case–control study was performed by collecting spot morning urine from 24 AIP patients and 24 healthy controls. Steroids in urine were quantified by liquid chromatography-tandem mass spectrometry. Parent steroids (17-hydroxyprogesterone; deoxycorticosterone; corticoesterone; 11-dehydrocorticosterone; cortisol and cortisone) and a large number of metabolites (N = 55) were investigated. Correlations between the different steroids analyzed and biomarkers of porphyria biochemical status (urinary heme precursors) were also evaluated. The Mann–Whitney U test and Spearman’s correlation with a two tailed test were used for statistical analyses.ResultsForty-one steroids were found to be decreased in the urine of AIP patients (P < 0.05), the decrease being more significant for steroids with a high degree of hydroxylation. Remarkably, 13 cortisol metabolites presented lower concentrations among AIP patients (P < 0.01) whereas no significant differences were found in the main metabolites of cortisol precursors. Nine cortisol metabolites showed a significant negative correlation with heme precursors (p < 0.05). Ratios between the main metabolites of 17-hydroxyprogesterone and cortisol showed positive correlations with heme-precursors (correlation coefficient > 0.51, P < 0.01).ConclusionsComprehensive study of the urinary steroid metabolome showed that AIP patients present an imbalance in adrenal steroidogenesis, affecting the biosynthesis of cortisol and resulting in decreased out-put of cortisol and metabolites. This may result from alterations of central origin and/or may originate in specific decreased enzymatic activity in the adrenal gland. An imbalance in steroidogenesis may be related to the maintenance of an active disease state among AIP patients.

Safety and Liver Transduction Efficacy of rAAV5-cohPBGDin Nonhuman Primates: A Potential Therapy for Acute Intermittent Porphyria

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

Pseudoexon Activation in the HMBS Gene as a Cause of the Nonerythroid Form of Acute Intermittent Porphyria

To the Editor: Acute intermittent porphyria (AIP)1 (MIM 176000) is a low-penetrance, autosomal dominant disorder caused by decreased activity of the third enzyme in the pathway of heme biosynthesis, hydroxymethylbilane synthase (HMBS). Partial deficiency of HMBS can lead to episodic life-threatening neurovisceral attacks of acute porphyria, which are often provoked by drugs, alcohol, or hormonal factors (1). Biochemical analyses of urinary porphobilinogen and porphyrins (fecal porphyrins and plasma porphyrins) can provide the diagnosis of AIP in symptomatic patients, but genetic testing for a defect in the HMBS gene (1) is required to accurately identify asymptomatic affected family members, who can then be counseled on the lifestyle modifications required to limit the risk of attacks. We previously reported on our experience with using direct genomic sequencing and dosage analysis of the HMBS gene for genetic analysis of AIP (2). We found a 98% sensitivity for detecting disease-causing mutations in unrelated symptomatic patients with a biochemical diagnosis of AIP (2), and we speculated that the cause in AIP patients in whom no mutation was found was likely to be mutations in unexplored parts of the gene, possible regulatory variants, or defects at another locus. We investigated a …

Renal Failure Affects the Enzymatic Activities of the Three First Steps in Hepatic Heme Biosynthesis in the Acute Intermittent Porphyria Mouse

Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males) and over 13 (females) in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.

Hepatocellular carcinoma without cirrhosis: think acute hepatic porphyrias and vice versa

Hepatocellular carcinoma without cirrhosis: think acute hepatic porphyrias and vice versa

Circulating fluorocytes at the first attack of acute intermittent porphyria: A missing link in the pathogenesis

Background Acute intermittent porphyria (AIP) is an autosomal dominant disorder of the haem biosynthesis resulting from a partial deficiency of hydroxymethylbilane synthase (HMBS) with incomplete penetrance. By conventional means, it is able to identify asymptomatic mutation carrier by molecular diagnosis, but one cannot reliably predict an acute porphyric attack. The presence of fluorescent red cells (fluorocytes) in AIP is probably under-recognized since AIP is a hepatic porphyria and not associated with photosensitivity. Methods We used an automatic image acquisition platform to detect the circulating fluorocytes at 700 nm emission in a diabetic AIP patient during acute attack. We screened the patient and her family members for the mutation on HMBS, urine porphobilinogen and circulating fluorocytes. Results The patient was heterozygous for a disease-causing mutation on HMBS and several bright circulating fluorocytes were detected. We showed evidence that protoporphyrin contributed to the erythrocyte auto-fluorescence. Interestingly, asymptomatic mutation carriers with increased urine porphobilinogen did not have circulating fluorocytes. All mutation-negative family members revealed no circulating fluorocytes. Conclusion Sudden decrease in plasma glucose concentration might invoke acute attack of AIP and appearance of circulatory fluorocytes. Potential of detecting fluorocytes as screening test or for predicting an acute attack of AIP in diabetes is worth investigating.

Identification and Characterization of Feline Acute Intermittent Porphyria: The First Naturally-Occurring Animal Model.

Abstract 3014Poster Board II-990Porphyrias are disorders of heme biosynthesis and are clinically classified as either erythroid with cutaneous involvement or hepatic with acute neurovisceral attacks in human patients. The clinical features, specific enzyme defects, biochemical abnormalities, and gene mutations have been extensively studied in humans. Here we describe clinical, biochemical, and molecular genetic studies of four unrelated lines of cats that appeared clinically to have a congenital erythropoietic porphyria (CEP), but were ultimately identified to have acute intermittent porphyria (AIP). The porphyric cats had erythrodontia, porphyrinuria, and mild anemia, but no evidence of acute life-threatening neurovisceral attacks or cutaneous lesions. Their urinary uroporphyrin I and coproporphyrin I concentrations were markedly increased (up to 650-fold and ∼10-fold, respectively). Their erythrocyte and hepatic uroporphyrinogen III synthase activities (deficient in CEP) were normal. However, all porphyric cats had '50% of normal hydroxymethylbilane synthase (HMBS) activity in erythrocytes and tissues, and increased levels of urinary 5-aminolevulinic acid and porphobilinogen. Moreover, porphyrin analysis of the discolored teeth from the porphyric cats revealed markedly elevated uroporphyrin I and coproporphyrin I isomers, thereby explaining their CEP-like phenotype. Sequencing the feline HMBS gene revealed different mutations in each of the four lines of porphyric cats: a T duplication in exon 5, c.189dupT (p.Leu64SerfsX65), which predicts a frameshift and premature stop codon, an in-frame 3 bp deletion in exon 14, c.842_844delGAG (p.delGly281), which is identical to a mutation causing AIP in a human patient, and two missense mutations: in exon 6,c.250G>A (p.Ala84Thr) and in exon 9, c.445C>T (p.Arg149Trp), the latter was also reported in a human AIP patient. Prokaryotic expression of the feline p.delGly281 and p.Arg149Trp mutations resulted in <1% residual activity, while the p.Ala84Thr missense mutation had ∼34% of the expressed feline HMB-synthase wild-type activity. Family studies further confirmed that the p.Ala84Thr missense mutation was inherited as an autosomal recessive trait with the porphyric cats being homozygous for the mutation and the parents and littermates being asymptomatic heterozygotes. In contrast, the porphyric cats from the other three lines were heterozygous for their mutations, thus reflecting the autosomal dominant inheritance observed in humans with AIP. While genetically-engineered mice with AIP have been generated, we describe here the first naturally-occurring animal models of dominant and recessive forms of AIP. Although the cats' phenotype is more similar to that of human patients with CEP, these cats may permit further characterization of the disease pathogenesis as well as the evaluation of therapeutic strategies for human patients with AIP including pharmacologic chaperone, gene replacement, transplantation, and stem cell therapies. Disclosures:No relevant conflicts of interest to declare.

Role of two nutritional hepatic markers (insulin‐like growth factor 1 and transthyretin) in the clinical assessment and follow‐up of acute intermittent porphyria patients

Acute intermittent porphyria (AIP) is caused by a deficiency of hydroxymethylbilane synthase. Clinical manifestations are abdominal pain and neurovisceral symptoms, accompanied by overproduction of heme-precursors in the liver, which frequently remains long-lasting in AIP patients. We tested the hypothesis that this condition may be associated with alterations of hepatic proteins known to be either increased or decreased in serum according to diverse pathological conditions including malnutrition, inflammation or liver disease. Serum proteins were analyzed in 26 biochemically active AIP patients that were classified according to the EPI (European Porphyria Initiative) guidelines as follows: (i) patients who presented a single acute attack having remained so far free of clinical symptoms; (ii) patients who present recurrent attacks or chronic symptoms associated with exacerbations of AIP. Most of the serum proteins were within normal limits, however insulin-like growth factor 1 (IGF-1) was decreased in 53.8% of AIP patients (z-score = -2.86 +/- 0.37) and transthyretin (prealbumin) was found significantly decreased in 38.5% of them. The IGF-1 z-score was lower in group B versus group A patients (-2.66 vs. -1.43; P = 0.024). The coincident decrease of both IGF-1 and transthyretin was associated with worsening of the clinical condition. This first study in humans suggests that the clinical expression AIP is associated with a state of under-nutrition and/or with hepatic inflammation due to the sustained accumulation of heme-precursors. We propose the use of both IGF-1 and transthyretin as biomarkers of disease morbidity/severity for the clinical follow-up of AIP patients.

Identification of Three Novel Substitutions in the Erythroid Promoter of the HMBS Gene: A New Erythroid Variant of Human Acute Intermittent Porphyria?

Porphyrias are inherited disorders in the biosynthesis of heme. Acute intermittent porphyria (AIP), the most common form, is autosomal dominant and it is characterized by recurrent attacks of abdominal pain, gastrointestinal dysfunction, and neurologic disturbances. AIP is caused by molecular defects in the hydroxymethylbilane synthase gene (HMBS) causing a partial deficiency of the third enzyme of the heme biosynthetic pathway. This gene maps to chromosome 11q23.2 with a total of 15 exons. Two distinct promoters direct the synthesis of housekeeping and erythroid specific mRNAs by alternative splicing. The housekeeping promoter is located upstream the exon 1 while the erythroid promoter include a portion of intron 1, the exon 2 and the intron 2. The exon 1 and the exons 3 to 15 generate the housekeeping mRNA while the exons 2 to 15 generate the erythroid mRNA giving rise to the housekeeping HMBS isoform and to the erythroid HMBS isoform respectively. In the classic form of AIP, both the housekeeping and the erythroid HMBS isoforms are deficient since the must common molecular defects involve the common region of the gene from exon 3 to exon 15. Approximately 5% of AIP patients have normal levels of the erythroid HMBS isoform and defects in the housekeeping promoter or in the exon 1 which is specific for the housekeeping HMBS isoform, causing the ‘non-erythroid variant’ of AIP. So far no mutations are known in the erythroid promoter. In this study we searched for molecular defects in HMBS gene in three Italian patients with typical clinical and biochemical signs of AIP. The diagnosis was based on elevated urinary excretion of porphyrinic precursors and reduced erythrocyte HMBS activity. The entire HMBS gene has been amplified by PCR and submitted to direct automated sequencing: no mutations known as responsible for classic form of AIP have been identified. However, each of three patients had a substitutions in the erythroid promoter of HMBS gene that could justify the reduced erythrocyte HMBS activity: c.34–115 C>A, c.34–126 G>C and c.34–156G>A. In order to establish if these substitutions were polymorphisms, more than 200 alleles from Italian normal subjects were sequenced and none of them revealed the substitutions. These three mutations, located specifically in the intron 2, could affect the normal splicing of exon 3 causing an abnormal mRNA, to give rise to the classical form of AIP. The mRNA analysis, however, didn't reveal any abnormal mRNA. Moreover real time experiments in the lymphocytes showed a 100% expression of housekeeping HMBS mRNA excluding the non-sense mediated decay mechanism. These data suggest that these mutations could affect the normal function of the erythroid promoter only, causing an erythroid variant of human acute intermittent porphyria. Further expression studies are in progress.

Nerve function and dysfunction in acute intermittent porphyria

Acute intermittent porphyria (AIP) is a rare metabolic disorder characterized by mutations of the porphobilinogen deaminase gene. Clinical manifestations of AIP are caused by the neurotoxic effects of increased porphyrin precursors, although the underlying pathophysiology of porphyric neuropathy remains unclear. To further investigate the neurotoxic effect of porphyrins, excitability measurements (stimulus-response, threshold electrotonus, current-threshold relationship and recovery cycle) of peripheral motor axons were undertaken in 20 AIP subjects combined with the results of genetic screening, biochemical and conventional nerve conduction studies. Compared with controls, excitability measurements from five latent AIP patients were normal, while 13 patients who experienced acute porphyric episodes without clinical neuropathy (AIPWN) showed clear differences in their responses to hyperpolarizing currents (e.g. reduced hyperpolarizing I/V slope, P < 0.01). Subsequent mathematical simulation using a model of human axons indicated that this change could be modelled by a reduction in the hyperpolarization-activated, cyclic nucleotide-dependent current (I(H)). In contrast, in one patient tested during an acute neuropathic episode, axons of high threshold with reduced superexcitability, consistent with membrane depolarization and reminiscent of ischemic changes. It is proposed that porphyrin neurotoxicity causes a subclinical reduction in I(H) in AIPWN axons, whereas porphyric neuropathy may develop when reduced activity of the Na(+)/K(+) pump results in membrane depolarization.