Bi-allelic hydroxymethylbilane synthase inactivation defines a homogenous clinico-molecular subtype of hepatocellular carcinoma.

BACKGROUND & AIMS: Acute intermittent porphyria (AIP), caused by heterozygous germline mutations of the heme synthesis pathway enzyme HMBS (hydroxymethylbilane synthase), confers a high risk of hepatocellular carcinoma (HCC) development. Yet, the role of HMBS in liver tumorigenesis remains unclear. METHODS: Herein, we explore HMBS alterations in a large series of 758 HCC cases, including 4 patients with AIP. We quantify the impact of HMBS mutations on heme biosynthesis pathway intermediates and we investigate the molecular and clinical features of HMBS-mutated tumors. RESULTS: We identify recurrent bi-allelic HMBS inactivation, both in patients with AIP acquiring a second somatic HMBS mutation and in sporadic HCC with 2 somatic hits. HMBS alterations are enriched in truncating mutations, in particular in splice regions, leading to abnormal transcript structures. Bi-allelic HMBS inactivation results in a massive accumulation of its toxic substrate porphobilinogen and synergizes with CTNNB1-activating mutations, leading to the development of well-differentiated tumors with a transcriptomic signature of Wnt/ß-catenin pathway activation and a DNA methylation signature related to ageing. HMBS-inactivated HCC mostly affects females, in the absence of fibrosis and classical HCC risk factors. CONCLUSIONS: These data identify HMBS as a tumor suppressor gene whose bi-allelic inactivation defines a homogenous clinical and molecular HCC subtype. LAY SUMMARY: Heme (the precursor to hemoglobin, which plays a key role in oxygen transport around the body) synthesis occurs in the liver and involves several enzymes including hydroxymethylbilane synthase (HMBS). HMBS mutations cause acute intermittent porphyria, a disease caused by the accumulation of toxic porphyrin precursors. Herein, we show that HMBS inactivation is also involved in the development of liver cancers with distinct clinical and molecular characteristics.

Acute Intermittent Porphyria: An Overview of Therapy Developments and Future Perspectives Focusing on Stabilisation of HMBS and Proteostasis Regulators.

Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease with low clinical penetrance, caused by mutations in the hydroxymethylbilane synthase (HMBS) gene, which encodes the third enzyme in the haem biosynthesis pathway. In susceptible HMBS mutation carriers, triggering factors such as hormonal changes and commonly used drugs induce an overproduction and accumulation of toxic haem precursors in the liver. Clinically, this presents as acute attacks characterised by severe abdominal pain and a wide array of neurological and psychiatric symptoms, and, in the long-term setting, the development of primary liver cancer, hypertension and kidney failure. Treatment options are few, and therapies preventing the development of symptomatic disease and long-term complications are non-existent. Here, we provide an overview of the disorder and treatments already in use in clinical practice, in addition to other therapies under development or in the pipeline. We also introduce the pathomechanistic effects of HMBS mutations, and present and discuss emerging therapeutic options based on HMBS stabilisation and the regulation of proteostasis. These are novel mechanistic therapeutic approaches with the potential of prophylactic correction of the disease by totally or partially recovering the enzyme functionality. The present scenario appears promising for upcoming patient-tailored interventions in AIP.

Molecular Analysis of 55 Spanish Patients with Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) results from a decreased activity of hepatic hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. AIP is an autosomal dominant disorder with incomplete penetrance, characterized by acute neurovisceral attacks precipitated by several factors that induce the hepatic 5-aminolevulinic acid synthase, the first enzyme in the heme biosynthesis. Thus, a deficiency in HMBS activity results in an overproduction of porphyrin precursors and the clinical manifestation of the disease. Early diagnosis and counselling are essential to prevent attacks, and mutation analysis is the most accurate method to identify asymptomatic carriers in AIP families. In the present study, we have investigated the molecular defects in 55 unrelated Spanish patients with AIP, identifying 32 HMBS gene mutations, of which six were novel and ten were found in more than one patient. The novel mutations included a missense, an insertion, two deletions, and two splice site variants. Prokaryotic expression studies demonstrated the detrimental effect for the missense mutation, whereas reverse transcription-PCR and sequencing showed aberrant splicing caused by each splice site mutation. These results will allow for an accurate diagnosis of carriers of the disease in these families. Furthermore, they increase the knowledge about the molecular heterogeneity of AIP in Spain.

A Pharmacological Chaperone Therapy for Acute Intermittent Porphyria.

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.

Acute Intermittent Porphyria - an Unexpected Association in a Patient with Newly Diagnosed Crohn's Disease.

The association of Crohn's disease (CD) with acute intermittent porphyria (AIP), both without a family or personal pathological history, is a very rare clinical possibility. We present the case of a 23-year-old male diagnosed on the same admission with ileal CD and with an AIP crisis. The diagnosis was challenging as the symptoms overlapped. Crohn's disease was complicated with intestinal occlusion and sepsis; the inflammatory, metabolic and septic changes represented the trigger factor for the first AIP seizure. The neurological symptoms were the key element for AIP diagnosis. The presence of atypical extra-intestinal manifestations in CD patients should raise also the possibility of AIP.

Inborn Errors of Metabolism and the Gastrointestinal Tract.

Inborn errors of metabolism (IEMs) are usually recognized by characteristic neurologic and metabolic manifestations and sometimes by dysmorphism. However, IEMs can present with a wide variety of gastrointestinal manifestations, whether as the primary or a minor clinical symptom. Regardless, gastrointestinal and hepatic manifestations of IEMs are important clinical features that can help identify an underlying defect; these disorders should be taken into consideration as part of a patient's clinical assessment. It is prudent to include metabolic disorders in the differential diagnosis because in some cases, gastrointestinal symptoms may be the only presenting feature in a patient with an underlying IEM.

Lifestyle factors including diet and biochemical biomarkers in acute intermittent porphyria: Results from a case-control study in northern Norway.

BACKGROUND: Lifestyle factors, including a low intake of carbohydrates, dieting, alcohol consumption, cigarette smoking and stress are some of the possible triggers of attacks in acute intermittent porphyria (AIP). The influence of lifestyle factors, including energy intake, diet and alcohol consumption on the biochemical disease activity in AIP and biochemical nutritional markers were examined. METHODS: A case-control study with 50 AIP cases and 50 controls matched for age, sex and place of residence was performed. Dietary intake was registered using a food diary in 46 matched pairs. Symptoms, alcohol intake, stress and other triggering factors of the last AIP attack were recorded on questionnaires. Porphyrin precursors, liver and kidney function markers, vitamins, diabetogenic hormones and other nutritional biomarkers were analyzed by routine methods. The Wilcoxon matched-pairs signed rank test was used to compare the cases vs. controls. The Spearman's rank correlation coefficient was used on the cases. RESULTS: Increasing total energy intake was negatively correlated with the biochemical disease activity. The intake of carbohydrates was lower than recommended, i.e., 40 and 39% of total energy intake in the AIP cases and controls, respectively. The plasma resistin level was significantly higher (p = .03) in the symptomatic than asymptomatic cases. Plasma insulin was lower in those with high porphobilinogen levels. The intake of sugar and candies were higher in the AIP cases with low U-delta aminolevulinic acid (ALA) levels (p = .04). Attacks were triggered by psychological stress (62%), physical strain (38%), food items (24%) and alcohol (32%) in the 34 symptomatic cases. Alcohol was used regularly by 88% of the cases (3.2 g ethanol/day) and 90% of the controls (6.3 g/day), but the intake was significantly lower in symptomatic than in asymptomatic cases (p = .045). CONCLUSION: A high intake of energy, sugar and candies and a higher insulin level were associated with a lower biochemical disease activity. The resistin level was higher in the symptomatic than the asymptomatic cases. AIP patients drink alcohol regularly, but the intake was significantly lower in the symptomatic cases. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01617642.

Emerging therapies for acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disease caused by hepatic deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme of the heme synthesis pathway. The dominant clinical feature is acute neurovisceral attack associated with high production of potentially neurotoxic porphyrin precursors due to increased hepatic heme consumption. Current Standard of Care is based on a down-regulation of hepatic heme synthesis using heme therapy. Recurrent hyper-activation of the hepatic heme synthesis pathway affects about 5% of patients and can be associated with neurological and metabolic manifestations and long-term complications including chronic kidney disease and increased risk of hepatocellular carcinoma. Prophylactic heme infusion is an effective strategy in some of these patients, but it induces tolerance and its frequent application may be associated with thromboembolic disease and hepatic siderosis. Orthotopic liver transplantation is the only curative treatment in patients with recurrent acute attacks. Emerging therapies including replacement enzyme therapy or gene therapies (HMBS-gene transfer and ALAS1-gene expression inhibition) are being developed to improve quality of life, reduce the significant morbidity associated with current therapies and prevent late complications such as hepatocellular cancer or kidney failure in HMBS mutation carriers with long-standing high production of noxious heme precursors. Herein, we provide a critical digest of the recent literature on the topic and a summary of recently developed approaches to AIP treatment and their clinical implications.

Acute intermittent porphyria exacerbation following in vitro fertilization treatment.

OBJECTIVES: Assisted reproductive technology is commonly used for women with infertility. We report a case of acute intermittent porphyria associated with in vitro fertilization treatment. CASE REPORT: A 35-year-old woman with tubal factor infertility presented to our clinic with persistent low abdominal pain and hyponatremia after transvaginal oocyte retrieval. During admission, she experienced a generalized tonic-clonic seizure attacked following by dark brown color urine. Urinary tests showed elevated porphobilinogen, 5-aminolevulinic acid, uroporphyrin, and coproporphyrin, confirming the diagnosis of acute intermittent porphyria. The patient's condition continued to improve after hemin treatment and rehabilitation. CONCLUSION: Newly onset acute intermittent porphyria during the course of controlled ovarian hyperstimulation for in vitro fertilization is a rare but possible complication. Acute intermittent porphyria should be taken into consideration for persisted unexplained abdominal pain and seriously alerted if accompanied with neurological symptoms. Special tests for acute intermittent porphyria should be taken into consideration for the differential diagnosis of lower abdominal pain after oocyte retrieval.

[Porphyrias]. / Porphyrien.

Porphyrias are metabolic disorders of the heme biosynthesis. Clinically, they can be differentiated into acute and non-acute porphyrias. The symptomatic phase of acute hepatic porphyrias is characterized by overproduction of neurotoxic porphyrin precursors and porphyrins. Acute intermittent porphyria, Variegate porphyria, Hereditary coproporphyria and Doss porphyria belong to this group of metabolic disorders. The clinical presentation of the acute hepatic porphyria syndrome includes abdominal, psychiatric, neurological and cardiovascular symptoms. The diagnosis is based on a tenfold increased urinary excretion of porphobilinogen (apart from Doss porphyria). Besides symptomatic therapy with non-porphyrinogenic drugs, electrolyte compensation and intensive monitoring, intravenous administration of glucose and heme arginate is established for treatment. Among the non-acute types like Porphyria cutanea tarda, Erythropoietic protoporphyria and Congenital erythropoietic porphyria, the accumulated porphyrins cause photosensitivity of the skin up to severe liver damage. The location of the deficient enzyme within the heme biosynthesic pathway determines the pattern of the accumulated porphyrins. Besides light protection, there are different therapies depending on the type of non-acute porphyria. Ultimately, liver transplantation may be considered in therapy-resistant cases of acute hepatic porphyrias and bone marrow transplantation in severe cases of erythropoietic porphyrias.

Acute intermittent porphyria as a cause of respiratory failure: case report.

A 45-year-old man with end-stage renal disease due to polycystic kidney disease was admitted to the hospital because of recurrent abdominal pain, progressive peripheral motor neuron neuropathy, and respiratory failure. The diagnosis of acute intermittent porphyria was confirmed by an elevated porphyrin concentration in the urine and the presence of an R167Q mutation in the porphobilinogen deaminase gene. Use of hydroxyzine, weight loss, and/or a mild upper respiratory viral infection might have been the provoking factor of the acute intermittent porphyria. Treatment with intravenous hemin (3 mg/kg) and a high-carbohydrate diet (3000 kcal/d) had no clinical effect. Tetraplegia and chronic respiratory insufficiency developed, and the patient needed a pacemaker because of a symptomatic sinus bradycardia due to autonomic dysfunction. The patient died 10 months after the first manifestation of acute intermittent porphyria.

Structure of human porphobilinogen deaminase at 2.8 A: the molecular basis of acute intermittent porphyria.

Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.

Drug use in porphyria: a therapeutic dilemma.

One of the most frequent precipitating factors for attacks of porphyria is the administration of drugs. Use of drugs with porphyrinogenic potential often worsens the condition and often poses a therapeutic dilemma. A 23-year-old female patient presented to the casualty room with abdominal pain, chest pain and vomiting. Her past medical history was significant with episodes of generalised abdominal pain. The patient was initially treated for her abdominal pain and vomiting. She developed seizures and was treated with diazepam and phenytoin. Based on the positive investigation reports (positive urine porphyrins, elevated urine ALA and positive porphobilinogen) and symptoms, a diagnosis of acute intermittent porphyria (AIP) was done. Before the diagnosis of AIP was made, the patient was treated with drugs which are not considered to be safe in porphyric patients, such as phenytoin, metoclopramide, and diclofenac. The use of these drugs probably contributed to the initial worsening of the patient's clinical condition. After the diagnosis of AIP was made, the patient was treated with safer alternatives; gabapentin as the antiepileptic agent, promethazine as antiemetic, and propanalol as the antihypertensive agent. Withdrawal of the unsafe agents and symptomatic management with the safer alternatives contributed to the recovery of the patient. Along with the case report and the observations made on the various drugs used in the patient, the importance of the various information sources available on the safety potential of these agents is discussed. The observations with the drugs used in our case will be a useful addition to the existing information on the safety of these agents.

Manifestaciones neurológicas asociadas a porfiria aguda intermitente / Neurologic disorders associated with acute intermittent porphyria

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