Erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited disorder of the haem metabolic pathway characterised by accumulation of protoporphyrin in blood, erythrocytes and tissues, and cutaneous manifestations of photosensitivity. EPP has been reported worldwide, with prevalence between 1:75,000 and 1:200,000. It usually manifests in early infancy upon the first sun exposures. EPP is characterised by cutaneous manifestations of acute painful photosensitivity with erythema and oedema, sometimes with petechiae, together with stinging and burning sensations upon exposure to sunlight, without blisters. These episodes have a variable severity depending on the exposure duration and may result in chronic permanent lesions on exposed skin. As protoporphyrin is a lipophilic molecule that is excreted by the liver, EPP patients are at risk of cholelithiasis with obstructive episodes, and chronic liver disease that might evolve to rapid acute liver failure. In most patients, EPP results from a partial deficiency of the last enzyme of the haem biosynthetic pathway, ferrochelatase, EC 4.99.1.1/FECH (encoded by the FECH gene). EPP appears to be inherited as an autosomal dominant disease, the clinical expression of which is modulated by the presence of the hypomorphic FECH IVS3-48C allele trans, but recessive inheritance with two mutated FECH alleles has also been described. In about 2% of patients, overt disease was recently shown to be caused by gain-of-function mutations in the erythroid-specific aminolevulinic acid synthase 2 (ALAS2/ALAS, EC 2.3.1.27) gene and named X-linked dominant protoporphyria. Diagnosis is established by finding increased levels of protoporphyrin in plasma and red blood cells, and detection of a plasma fluorescence peak at 634 nm. Investigations for hepatic involvement, ferrochelatase activity level, genetic analysis (FECH mutations, presence of the hypomorphic FECH IVS3-48C allele trans and ALAS2 mutations) and family studies are advisable. Differential diagnosis includes phototoxic drug reactions, hydroa vacciniforme, solar urticaria, contact dermatitis, angio-oedema and, in some cases, other types of porphyria. Management includes avoidance of exposure to light, reduction of protoporphyrin levels and prevention of progression of possible liver disease to liver failure. As the major risk in EPP patients is liver disease, a regular follow-up of hepatic involvement is essential. Sequential hepatic and bone marrow transplantation should be considered as a suitable treatment for most severe cases of EPP with hepatic involvement. EPP is a lifelong disorder whose prognosis depends on the evolution of the hepatic disease. However, photosensitivity may have a significant impact on quality of life of EPP patients

Efficacy of the melanocortin analogue Nle4-D-Phe7-α-melanocyte-stimulating hormone in the treatment of patients with Hailey-Hailey disease

BackgroundHailey-Hailey disease (HHD) is a rare, chronic and recurrent blistering disorder, which is characterized clinically by erosions occurring primarily in intertriginous regions, and histologically by suprabasal acantholysis. Oxidative stress plays a specific role in the pathogenesis of HHD, by regulating the expression of factors playing an important role in keratinocyte proliferation and differentiation. AimGiven the significance of oxidative stress in HHD, we investigated the potential effects of the antioxidant properties of an -MSH analogue, Nle4-D-Phe7--MSH (afamelanotide), in HHD lesion-derived keratinocytes. ResultsTreatment of HHD-derived keratinocytes with afamelanotide contributed to upregulation of Nrf2 [nuclear factor (erythroid-derived 2)-like 2], a redox-sensitive transcription factor that plays a pivotal role in redox homeostasis during oxidative stress. Additionally, afamelanotide treatment restored the defective proliferative capability of lesion-derived keratinocytes. Our results show that Nrf2 is an important target of the afamelanotide signalling that reduces oxidative stress. Because afamelanotide possesses antioxidant effects, we also assessed the clinical potential of this -MSH analogue in the treatment of patients with HHD. In a phase II open-label pilot study, afamelanotide 16mg was administered subcutaneously as a sustained-release resorbable implant formulation to two patients with HHD, who had a number of long-standing skin lesions. For both patients, their scores on the Short Form-36 improved 30days after the first injection of afamelanotide, and both had 100% clearance of HHD lesions 60days after the first injection, independently of the lesion location. ConclusionsAfamelanotide is effective for the treatment of skin lesions in HHD

THE GLUTAMATE-BASED ACID RESISTANCE SYSTEM IN ESCHERICHIA COLI: CHARACTERIZATION OF THE TWO MAJOR STRUCTURAL COMPONENTS

The glutamate-based (gad) system is by far the most efficient acid resistance system in commensal and pathogenic enteric bacteria, like E. coli, L. monocytogenes and S. flexneri [1]. It ensures survival for more than a 2-h exposure to a strongly acidic environment (pH < 2.5), such as that of the stomach. The structural components of the system are the enzyme glutamate decarboxylase (Gad) and its cognate glutamate/-aminobutyrate (GABA) antiporter (GadC). The Gad reaction contributes to pH homeostasis by consuming intracellular H+ while GABA export, via GadC, contributes to the local alkalinization of the extracellular environment. The E. coli chromosome contains two distantly located genes, gadA and gadB, encoding biochemically undistinguishable isoforms of Gad. The gadC gene, located downstream of gadB, is co-transcribed with it [2]. E. coli GadA/B have an acidic pH optimum for activity, which dramatically drops above pH 5.0. Moreover, upon changing the pH from 5 to 6, the enzyme undergoes a pH-dependent conformational change involving the reversible uptake of protons upon acidification [3, 4]. We are currently carrying out a broad-scope biochemical characterization of the gad system. In particular our interest is focused on establishing how the the structural components of this system work in the cell. As a first step, we successfully crystallized and solved the GadB structure both at pH 7.6 (neutral-pH form) and at pH 4.6 (low-pH form) [5]. GadB is a hexameric protein (a trimer of dimers with P32 symmetry). Comparison of the low-pH and the neutral-pH forms shows that the overall structure of the hexamer remains the same, but very significant changes occur at the N- and C-termini and in a -hairpin region spanning residues 300-313. The N-terminal region of each of the six subunits, disordered at neutral pH, assumes a -helical conformation at low pH, forming two short three-helical bundles parallel to the three-fold axis of the hexamer. In the neutral-pH form, the C-terminus of each GadB subunit ends into the active site and blocks it. In the low-pH form, the C-terminus is disordered and the active site is freely accessible to the substrate. Upon exposure to strong acids, the E. coli cell membrane becomes leaky to H+[1]. We demonstrated that under these circumstances GadB, typically localized in the cytoplasm at neutral pH, is detected and assayed (>50 %) in the membrane fraction at acidic pH [5]. The structural basis for this behaviour resides in the N-terminal region of GadB. In the present work we provide structural and biochemical evidence for the existence of a binding site for anions, such as chloride, known to affect significantly GadB activity [3]. The physiological relevance of this binding will be discussed. As a second step of our program, we undertook the large-scale production and characterization of GadC. We purified GadC by affinity chromatography. The protein was purified from the membrane fraction with a yield of more than 1 mg per L of bacterial culture. Biophysical characterization of GadC will be presented

Novel mutation of PPOX gene in a patient with abdominal pain and syndrome of inappropriate antidiuresis

Purpose: Acute porphyrias are metabolic disorders of heme biosynthesis characterized by acute life-threatening attacks. The diagnosis is often missed since clinical presentation is aspecific mimicking other medical and surgical conditions. Variegate porphyria (VP) is an autosomal dominant inherited disease with incomplete penetrance due to decreased activity of the Protoporphyrinogen Oxydase (PPOX) gene; most VP mutations are family specific. We report the case of a 40 year-old woman who presented many times to the emergency department complaining of unexplained abdominal pain and laboratory investigations showed repeatedly hyponatremia. Syndrome of inappropriate antidiuresis (SIAD) was confirmed and measurement of urine porphobilinogen and delta-aminolevulinic acid disclosed the diagnosis of acute porphyria. The genetic analysis of PPOX gene was performed. Methods: The entire coding sequence and exon/intron boundaries of PPOX gene were amplified in 5 different Polymerase Chain Reaction (PCR) fragments. In silico prediction of the pathogenicity of the mutation was determined by using different tools, Polyphen2, SNPs&GO, SNPs3D. Results: The genetic analysis of PPOX gene revealed a novel missense variant c.1376 G > A (p.Cys459Tyr) in heterozygous state. The same variant was later found in one of her cousins with skin lesions and other three younger asymptomatic relatives. We provided evidence that this novel mutation is likely to be pathogenetic. Conclusions: Our case highlights the importance of considering VP in the differential diagnosis of SIAD and underlines the role of genetic screening in the management of such patients. The finding of a novel mutation of PPOX gene in our index case has allowed to recognize an affected family

Disturbed iron metabolism in erythropoietic protoporphyria and association of GDF15 and gender with disease severity

Patients with erythropoietic protoporphyria (EPP) have reduced activity of the enzyme ferrochelatase that catalyzes the insertion of iron into protoporphyrin IX (PPIX) to form heme. As the result of ferrochelatase deficiency, PPIX accumulates and causes severe photosensitivity. Among different patients, the concentration of PPIX varies considerably. In addition to photosensitivity, patients frequently exhibit low serum iron and a microcytic hypochromic anemia. The aims of this study were to (1) search for factors related to PPIX concentration in EPP, and (2) characterize anemia in EPP, i.e., whether it is the result of an absolute iron deficiency or the anemia of chronic disease (ACD). Blood samples from 67 EPP patients (51 Italian and 16 Swiss) and 21 healthy volunteers were analyzed. EPP patients had lower ferritin (p = 0.021) and hepcidin (p = 0.031) concentrations and higher zinc\u2013protoporphyrin (p < 0.0001) and soluble-transferrinreceptor (p = 0.0007) concentrations compared with controls. This indicated that anemia in EPP resulted from an absolute iron deficiency. Among EPP patients, PPIX concentrations correlated with both growth differentiation factor (GDF) 15 (p = 0.012) and male gender (p = 0.015). Among a subgroup of patients who were iron replete, hemoglobin levels were normal, which suggested that iron but not ferrochelatase is the limiting factor in heme synthesis of individuals with EPP

Screening genetico per l'emocromatosi ereditaria nei donatori del Lazio

L’emocromatosi ereditaria (EE) è una malattia a trasmissione generalmente autosomica recessiva, caratterizzata da aumentato assorbimento del ferro ed accumulo in diversi organi. Diagnosi e intervento precoce consentono di evitare tale accumulo, con una normale qualità della vita mediante salasso-terapia periodica. La mutazione genetica più frequentemente responsabile dell’EE è la C282Y-gene HFE. Sono inoltre coinvolte altre mutazioni: l’omozigosi C282Y, H63D o l’eterozigosi composta C282Y/H63D sono responsabili della maggioranza dei casi di EE. La maggior parte degli screening genetici per EE sono stati preceduti da indagini biochimiche (% sat. transferrina; ferritina). Lo studio si propone di individuare portatori di mutazioni per EE, senza effettuare una pre-selezione di coloro con sovraccarico di ferro. Sono stati centralizzati per la ricerca di 18 mutazioni dei 3 geni coinvolti nell’EE 6832 donatori provenienti da 9 SIMTI del Lazio. L’analisi è stata effettuata mediante estrazione di DNA da sangue, amplificazione per PCR e ibridazione inversa. Per i donatori omo- e/o eterozigoti l’indagine è stata estesa a 253 familiari; a tutti è stato chiesto il consenso informato. Attualmente sono stati analizzati 3370 individui e identificati 67 (2,0%) donatori e 17 (6,7%) familiari con genotipo patologico per EE. I risultati sono riportati in Figura. In caso di genotipo a rischio per EE è stato studiato il metabolismo del ferro ed accertata l’assenza di sofferenza/danno parenchimale; i donatori senza danno d’organo sono stati riammessi a donare. Il test genetico ha consentito di identificare soggetti geneticamente a rischio, ma con valori di sat. transferrina e ferritina nella norma per età e sesso. La diagnosi precoce e le donazioni regolari consentono interventi di prevenzione primaria più vantaggiosi, anche in termini costi-benefici, rispetto alla gestione di pazienti che abbiano sviluppato danno d’organo; in aggiunta l’incremento atteso del numero di donazioni potrebbe contribuire al raggiungimento dell’autosufficienza regionale. Riteniamo inoltre che il completamento dei test di screening genetico possa fornire un campionamento rappresentativo per lo studio dell’incidenza dell’EE nel Lazio