Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP.

Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human HMBS (hHMBS) cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme's domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP.

From a dominant to an oligogenic model of inheritance with environmental modifiers in acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a disease affecting the heme biosynthesis pathway caused by mutations of the hydroxymethylbilane synthase (HMBS) gene. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. We evaluated the prevalence, penetrance and heritability of AIP, in families with the disease from the French reference center for porphyria (CFP) (602 overt patients; 1968 relatives) and the general population, using Exome Variant Server (EVS; 12 990 alleles) data. The pathogenicity of the 42 missense variants identified was assessed in silico, and in vitro, by measuring residual HMBS activity of the recombinant protein. The minimal estimated prevalence of AIP in the general population was 1/1299. Thus, 50 000 subjects would be expected to carry the AIP genetic trait in France. Penetrance was estimated at 22.9% in families with AIP, but at only 0.5-1% in the general population. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the area in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles. In conclusion, the striking difference in the penetrance of HMBS mutations between the general population and the French AIP families suggests that AIP inheritance does not follow the classical autosomal dominant model, instead of being modulated by strong environmental and genetic factors independent from HMBS. An oligogenic inheritance model with environmental modifiers might better explain AIP penetrance and heritability.

Haem Biosynthesis and Antioxidant Enzymes in Circulating Cells of Acute Intermittent Porphyria Patients.

The aims of the present study were to explore the expression pattern of haem biosynthesis enzymes in circulating cells of patients affected by two types of porphyria (acute intermittent, AIP, and variegate porphyria, VP), together with the antioxidant enzyme pattern in AIP in order to identify a possible situation of oxidative stress. Sixteen and twelve patients affected by AIP and VP, respectively, were analysed with the same numbers of healthy matched controls. Erythrocytes, neutrophils and peripheral blood mononuclear cells (PBMCs) were purified from blood, and RNA and proteins were extracted for quantitative real time PCR (qRT-PCR) and Western-blot analysis, respectively. Porhobilinogen deaminase (PBGD) and protoporphyrinogen oxidase (PPOX) gene and protein expression was analysed. Antioxidant enzyme activity and gene expression were additionally determined in blood cells, together with protein carbonyl content in plasma. PBMCs isolated from AIP patients presented low mRNA levels of PBGD when compared to controls, while PBMCs isolated from VP patients presented a decrease in PPOX mRNA. PPOX protein content was higher in AIP patients and lower in VP patients, compared to healthy controls. Regarding antioxidant enzymes, PBMCs and erythrocyte superoxide dismutase (SOD) presented statistically significant higher activity in AIP patients compared to controls, while catalase activity tended to be lower in these patients. No differences were observed regarding antioxidant gene expression in white blood cells. Circulating cells in AIP and VP patients present altered expression of haem biosynthetic enzymes, which could be useful for the differential diagnosis of these two types of porphyria in certain difficult cases. AIP patients present a condition of potential oxidative stress similar to VP patients, evidenced by the post-transcriptional activation of SOD and possible catalase impairment.

Porfiria aguda intermitente en población pediátrica de la región de Murcia: fenotipo y prevalencia / Acute intermittent porphyria in a paediatric population in the region of Murcia: Phenotype and prevalence

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Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver.

Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.

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

BACKGROUND: 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. OBJECTIVES: 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. MATERIAL AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Biallelic inactivation of protoporphyrinogen oxidase and hydroxymethylbilane synthase is associated with liver cancer in acute porphyrias.

Variegate porphyria (VP) and acute intermittent porphyria (AIP), the two most common types of acute porphyrias (AHPs), result from a partial deficiency of protoporphyrinogen oxidase (PPOX) and hydroxymethylbilane synthase (HMBS), respectively. A rare but serious complication in the AHPs is hepatocellular carcinoma (HCC). However, the underlying pathomechanisms are yet unknown. We performed DNA sequence analysis in cancerous and non-cancerous liver tissue of a VP and an AIP patient, both with HCC. In samples of both cancerous and non-cancerous liver tissues from the patients, we identified the underlying PPOX and HMBS germline mutations, c.1082dupC and p.G111R, respectively. Additionally, we detected a second somatic mutation, only in the cancer tissue i.e., p.L416X in the PPOX gene of the VP patient and p.L220X in the HMBS gene of the AIP patient, both located in trans to the respective germline mutations. Both somatic mutations were not detected in 10 non-porphyria-associated HCCs. Our data demonstrate that in the hepatic cancer tissue of AHP patients, somatic second-hit mutations result in nearly complete inactivation of the enzymes catalyzing major steps in the heme biosynthetic pathway. Both PPOX and HMBS, which might act as tumor suppressors, play a crucial role in the development of HCC in these individuals.

Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria.

The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype-phenotype relations for AIP.

Helper-dependent adenoviral liver gene therapy protects against induced attacks and corrects protein folding stress in acute intermittent porphyria mice.

Acute intermittent porphyria (AIP) is a hepatic metabolic disease that results from haplo-insufficient activity of porphobilinogen deaminase (PBGD). The dominant clinical feature is acute intermittent attacks when hepatic heme synthesis is activated by endocrine or exogenous factors. Gene therapy vectors over-expressing PBGD protein in the liver offers potential as a cure for AIP. Here, we developed a helper-dependent adenovirus (HDA) encoding human PBGD (hPBGD) and assessed its therapeutic efficacy in a murine model of AIP. Intravenous or intrahepatic administration of HDA-hPBGD to AIP mice resulted in a sustained hepatic hPBGD expression in a dose-dependent manner. Intrahepatic administration conveyed full protection against induced porphyria attacks at a significantly lower viral dose than intravenous injection. Transgenic hPBGD accumulated only in the cytosol of hepatocytes as the endogenous protein. Characterization of PBGD-deficient mouse strains revealed that a strong PBGD deficiency causes the chronic disturbance of cytosolic and endoplasmic reticulum folding machineries. This disturbance was completely restored over time by the over-expression of hPBGD. HDA-hPBGD is a promising vector that protects against porphyria attacks and resolves the chronic folding stress associated with low levels of PBGD activity.

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.

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.

Marked geographic aggregation of acute intermittent porphyria families carrying mutation Q180X in Venezuelan populations, with description of further mutations.

Acute intermittent porphyria (AIP) caused by mutations in the hydroxymethylbilane synthase gene (HMBS), has been reported in almost all human populations, with varying frequencies. A founder effect for a few specific mutations in geographic regions where prevalence is high (Sweden, The Netherlands, Switzerland) has been established through haplotype analyses, while some other mutations (R26H, R26C) have been repeatedly reported in many populations with different genetic backgrounds. Epidemiological, biochemical and molecular data on AIP in Venezuela were gathered during the last two decades; 24 independent families with AIP were ascertained, based on a deficient HMBS activity and increased porphobilinogen (PBG) urinary excretion. Molecular analyses of coding and splicing regions were performed in 23 families, to establish disease-causing changes, and haplotype analyses were used to assess ancestral kinships between them. Changes were detected in 16 out of 23 families, 9 of them being different: R26H, R26C, c.87+5G>A, c.267-54_61delgaaggggt, R116W, Q180X, c.825+1G>A, c.913-1delG, and 3' UTR *277G>A. Seven mutations were found, each one in a single family; one mutation was present in two unrelated families, whereas mutation Q180X was shared by 7 independent kindreds, all of which had the same haplotype (-);T;A;T;G;T;A;G (3167delG; 3530T>C; 3581A>G; 3982T>C; 6479G>T; 7052T>C; 7064A>C; 7779G>A). Six out of seven different Q180X carrier families came from the same geographic focus (Santa Lucía, Miranda State). Dense geographic aggregation with one identical haplotype strongly suggests a remote founder phenomenon for these Venezuelan AIP families, carrying an unreported but most frequent mutation.

[Molecular genetic study of acute intermittent porphyria in Russia: mutation analysis and functional polymorphism search in porphobilinogen deaminase gene].

Acute intermittent porphyria (AIP) is an autosomal dominant hereditary disease, caused by partial deficiency of porphobilinogen deaminase (PBGD), one of the key enzymes ofheme biosynthesis. This study describes molecular genetics of AIP in Russia. Mutation analysis of PBGD gene in 70 unrelated patients revealed 47 various genetic defects, 28 of which had not been described previously. Mutations 53delT and Argl 73 Trp (recorded 8 times, in total 23%) proved to be the most common in Russia. Microdeletion 53delThas monophyletic origin and was found only in Russia. Molecular genetic examination of 132 relatives of AIP patients from 40 families revealed 52 latent carriers of the disease. Low (about 10%) AIP penetrance indicates that a mutation in the PBGD gene is an important but not sufficient prerequisite for clinical manifestation of the disease. Modulation of penetrance in erythropoietic protoporphyria by coinheritance of a mutant allele and a functionally defective wild type allele of ferrochetalase gene has been shown previously. We hypothesized that similar mechanism works in AIP. Sequencing of the full length PBGD genes from unrelated AIP patients as well as SN P analysis, and the analysis of abnormal PBGD mRNA splicing showed that in case ofAIP, this hypothesis is not true and some other factors are responsible for the penetrance of this disease.

Feline acute intermittent porphyria: a phenocopy masquerading as an erythropoietic porphyria due to dominant and recessive hydroxymethylbilane synthase mutations.

Human acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn error of heme biosynthesis due to the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Here, we describe the first naturally occurring animal model of AIP in four unrelated cat lines who presented phenotypically as congenital erythropoietic porphyria (CEP). Affected cats had erythrodontia, brownish urine, fluorescent bones, and markedly elevated urinary uroporphyrin (URO) and coproporphyrin (COPRO) consistent with CEP. However, their uroporphyrinogen-III-synthase (URO-synthase) activities (deficient in CEP) were normal. Notably, affected cats had half-normal HMB-synthase activities and elevated urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), the deficient enzyme and accumulated metabolites in human AIP. Sequencing the feline HMB-synthase gene revealed different mutations in each line: a duplication (c.189dupT), an in-frame 3 bp deletion (c.842_844delGAG) identical to that causing human AIP and two missense mutations, c.250G>A (p.A84T) and c.445C>T (p.R149W). Prokaryotic expression of mutations c.842_844delGAG and c.445C>T resulted in mutant enzymes with <1% wild-type activity, whereas c.250G>A expressed a stable enzyme with approximately 35% of wild-type activity. The discolored teeth from the affected cats contained markedly elevated URO I and III, accounting for the CEP-like phenocopy. In three lines, the phenotype was an autosomal dominant trait, while affected cats with the c.250G>A (p.A84T) mutation were homozygous, a unique recessive form of AIP. These animal models may permit further investigation of the pathogenesis of the acute, life-threatening neurological attacks in human AIP and the evaluation of therapeutic strategies. GenBank Accession Numbers: GQ850461-GQ850464.

Identification and characterization of HMBS gene mutations in Spanish patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant disorder with low penetrance that results from a partial deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. The disease is clinically characterized by acute neurovisceral attacks that are precipitated by several factors including certain drugs, steroid hormones, alcohol and fasting. Early diagnosis and counselling are essential to prevent attacks, being mutation analysis the most reliable method to identify asymptomatic carriers in AIP families. In this study we have investigated the molecular defect in 15 unrelated Spanish AIP patients. Mutation analysis of the HMBS gene revealed a total of fourteen mutations including six novel ones, two of them were on the same allele in one patient. The novel mutations were three missense (R26L, R173G and D178H), two frameshift (c.749_765dup and c.874insC) and one intronic deletion (IVS12+3_+11delAGGGCCTGT). RT-PCR and sequencing demonstrated that the intronic mutation caused abnormal splicing and exon 12 skipping. Prokaryotic expression of the novel missense mutations showed that only D178H had significant residual activity. These findings will facilitate the accurate identification of presymptomatic AIP carriers in these families and they further emphasize the molecular heterogeneity of AIP in Spain.