Search

Porphyrias are rare inherited disorders caused by specific enzyme dysfunctions in the haem synthesis pathway, which result in abnormal accumulation of specific pathway intermediates. The symptoms depend upon the chemical characteristics of these substances. Porphyrins are photoreactive and cause photocutaneous lesions on sunlight-exposed areas, whereas accumulation of porphyrin precursors is related to acute neurovisceral attacks. Current therapies are suboptimal and mostly address symptoms rather than underlying disease mechanisms. Advances in the understanding of the molecular bases and pathogenesis of porphyrias have paved the way for the development of new therapeutic strategies. In this Clinical Trial Watch we summarise the basic principles of these emerging approaches and what is currently known about their application to porphyrias of hepatic origin or with hepatic involvement.

Erythropoietic protoporphyria (EPP) is a hereditary disease characterized by a deficiency in ferrochelatase (FECH) activity. FECH activity is responsible for the accumulation of protoporphyrin IX (PPIX). Without etiopathogenic treatment, EPP manifests as severe photosensitivity. 95% of affected individuals present a hypomorphic FECH allele trans to a loss-of-function (LOF) FECH mutation, resulting in a reduction in FECH activity in erythroblasts below a critical threshold. The hypomorphic allele promotes the use of a cryptic acceptor splice site, generating an aberrant FECH mRNA, which is responsible for the reduced level of wild-type FECH mRNA and, ultimately, FECH activity. We have previously identified an antisense oligonucleotide (AON), AON-V1 (V1), that redirects splicing to the physiological acceptor site and reduces the accumulation of PPIX. Here, we developed a specific strategy that uses transferrin receptor 1 (TRF1) as a Trojan horse to deliver V1 to erythroid progenitors. We designed a bifunctional peptide (P(1)-9R) including a TFR1-targeting peptide coupled to a nine-arginine cell-penetrating peptide (CPP) that facilitates the release of the AON from TFR1 in endosomal vesicles. We demonstrated that the P(1)-9R/V1 nanocomplex promotes the efficient and prolonged redirection of splicing towards the physiological splice site and subsequent normalization of WT FECH mRNA and protein levels. Finally, the P(1)-9R/V1 nanocomplex increases WT FECH mRNA production and significantly decreases PPIX accumulation in primary cultures of differentiating erythroid progenitors from an overt EPP-affected individual. P(1)-9R is a method designed to target erythroid progenitors and represents a potentially powerful tool for the in vivo delivery of therapeutic DNA in many erythroid disorders.

To assess the performance of 405 nm-induced autofluorescence for the characterization of primary liver nodules on ex vivo resected specimens.Forty resected liver specimens bearing 53 primary liver nodules were included in this IRB-approved prospective study. Intratissular spectroscopic measurements were performed using a 25-G fibered-needle on all ex vivo specimens: 5 autofluorescence measurements were performed in both nodules and adjacent parenchyma. The spectra derivatives of the 635 and 670 nm autofluorescence peaks observed in nodules and in adjacent liver parenchyma were compared (Kruskal-Wallis and Mann-Whitney when appropriate).A total of 42 potentially evolutive primary liver nodules-34 hepatocellular carcinomas, 4 intrahepatic cholangiocarcinomas, 4 hepatocellular adenomas-and 11 benign nodules-5 focal nodular hyperplasias, 6 regenerative nodules-were included. Both 635 and 670 nm Δderivatives were significantly higher in benign as compared to potentially evolutive (PEV) nodules (respectively 32.9 ± 4.5 vs 15.3 ± 1.4; p 0.0001 and 5.7 ± 0.6 vs 2.5 ± 0.1; p 0.0001) with respective sensitivity and specificity of 78% and 91% for distinguishing PEV from benign nodules.405 nm-induced autofluorescence enables the discrimination of benign from PEV primary liver nodules, suggesting that autofluorescence imaging could be used to optimize US targeted liver biopsies.• 405 nm-induced autofluorescence can distinguish liver tumors from the adjacent liver parenchyma. • The analysis of autofluorescence imaging observed within primary liver tumors can discriminate benign tumors from those requiring follow-up or targeted liver biopsy. • In current practice, autofluorescence imaging could be embedded within biopsy needle, to enable, in addition to ultrasound guidance, optimal targeting of liver nodules which could optimize tissue sampling.

Acute hepatic porphyria comprises a group of rare, genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long-term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients. EXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months prior to the study, patients reported a median (range) of 6 (0-52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a healthcare facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0-37.0]). Elevated levels of hepatic δ-aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ-aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased healthcare utilization. Conclusions: Patients experienced attacks often requiring treatment in a healthcare facility and/or with hemin, as well as chronic symptoms that adversely influence day-to-day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies. This article is protected by copyright. All rights reserved.

International audience; Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease caused by impaired activity of uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthetic pathway. Massive accumulation of porphyrins in red blood cells is responsible for hemolysis and porphyrin deposition in the skin, inducing severe bullous lesions and progressive photomutilation. Treatment options are scarce, relying mainly on supportive measures and, for severe cases, on bone marrow transplantation. In CEP, gain-of-function mutations in ALAS2 can represent an aggravating factor, and iron restriction can improve disease symptoms. Herein, we present the first case of a CEP patient significantly improved by iron deficiency induced by iterative phlebotomies for almost two years. We observed discontinuation of hemolysis and a marked decrease in plasma and urine porphyrins. The patient reported a major improvement in photosensitivity. No adverse effects were observed. The characterization of 3 CEP siblings in a consanguineous family with contrasting phenotypes modulated by iron availability highlights the importance of iron metabolism in the disease. Erythroid cultures were performed, demonstrating the role of iron in the rate of porphyrin production. Thus, we propose phlebotomy as an efficient, accessible, inexpensive and well-tolerated treatment for CEP.

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

The hereditary porphyrias comprise a group of eight metabolic disorders of the haem biosynthesis pathway characterised by acute neurovisceral symptoms, skin lesions or both. Each porphyria is caused by abnormal function of a separate enzymatic step resulting in a specific accumulation of haem precursors. Seven porphyrias are the consequence of a partial enzyme deficiency while a gain of function mechanism has been recently characterised in a novel porphyria. Acute porphyrias present with severe abdominal pain, nausea, constipation, confusion and seizure, which may be life threatening. Cutaneous porphyrias can be present with either acute painful photosensitivity or skin fragility and blisters. Rare recessive porphyrias usually manifest in early childhood with either severe chronic neurological symptoms or chronic haemolysis and severe cutaneous photosensitivity. Porphyrias are still underdiagnosed, but once they are suspected, and depending on the clinical presentation, a specific and simple front line test allows the diagnosis in all symptomatic patients. Diagnosis is essential to institute as soon as possible a specific treatment. Screening families to identify presymptomatic carriers is crucial to prevent chronic complications and overt disease by counselling on avoiding potential precipitants.

The potential of mRNA to produce therapeutic and protective protein levels is a promising approach for the treatment of a large number of diseases. In a recent study published in Nature Medicine (Published online October 8, 2018. doi.org/10.1038/s41591-018-0199-z), the intravenous delivery of human porphobilinogen deaminase (PBGD) mRNA, targeting the liver, demonstrated its efficacy and safety to replace the defective PBGD protein in preclinical models of acute intermittent porphyria.

International audience; Porphyrias are inherited diseases with low penetrance affecting the heme biosynthesis pathway. Acute intermittent porphyria (AIP), variegate porphyria (VP) and hereditary coproporphyria (HCP) together constitute the acute hepatic porphyrias (AHP). These diseases have been identified as risk factors for primary liver cancers (PLC), mainly hepatocellular carcinoma (HCC: range 87-100%) but also cholangiocarcinoma, alone or combination with HCC. In AHP, HCC annual incidence rates range from 0.16 to 0.35% according to the populations studied. Annual incidence rates are higher in Swedish and Norwegian patients, due to a founder effect. It increases above age 50. The pathophysiology could include both direct toxic effects of heme precursors, particularly δ-aminolevulinic acid (ALA), compound heterozygosity for genes implied in heme biosynthesis pathway or the loss of oxidative stress homeostasis due to a relative lack of heme. The high HCC incidence justifies radiological surveillance in AHP patients above age 50. Efforts are made to find new biological non-invasive markers. In this respect, we describe here the first report of PIVKA-II clinical utility in the follow-up of an AIP patient that develop an HCC. In this manuscript we reviewed the epidemiology, the physiopathology, and the screening strategy of HCC in AHP.

Introduction La porphyrie aigue intermittente (PAI) est une maladie genetique rare due a un deficit enzymatique en activite PBGD caracterisee par des crises neuroviscerales aigues comprenant de violentes douleurs abdominales, des signes dysautonomiques et des signes neurologiques centraux et peripheriques sensitivomoteurs [1] . La penetrance des mutations PBGD est faible de l’ordre de 20 % dans les familles ou il existe deja un cas index et de l’ordre de 1 % en population generale. La PAI se complique frequemment d’une insuffisance renale chronique et il existe un risque majore de cancer du foie. L’objectif de cette etude est d’evaluer la prevalence des signes aigus chez les patients symptomatiques (PS, c.-a-d. ayant fait au moins une crise neuroviscerale documentee) et l’existence de symptomes douloureux chroniques chez les PS et chez les patients porteurs sains (PoS, c.-a-d. n’ayant jamais presente de crise neuroviscerale). Patients et methodes L’enquete « PAI-France » est une etude observationnelle prospective, monocentrique des patients PAI du CRMR porphyries. Des questionnaires recherchant les symptomes chroniques et evaluant la qualite de vie (echelle EQ5D) ont ete envoyes aux patients. Un bilan biologique evaluant la fonction renale etait egalement propose. Ont ete consideres comme symptomatiques les patients ayant un antecedent documente de crise neuroviscerale de porphyrie. Resultats Les questionnaires ont ete envoyes a 833 patients (502 PoS, 331 PS). Au total, 388 patients les ont completes (197 PoS et 191 PS), dont 106 hommes (79 PoS et 27 PS) et 282 femmes (118 PoS et 164 PS). Parmi les 191 PS, 93 (49 %) ont declare des symptomes aigus identiques a leur crise inaugurale au moins 1 fois par an. Parmi les hommes et les femmes PS, 5/23 (22 %) et 63/139 (45 %) declarent respectivement des episodes reguliers de douleurs abdominales moderees, d’une duree de plusieurs jours. Parmi les hommes et les femmes PoS, 10/76 (13 %) et 28/116 (24 %) declarent respectivement des episodes reguliers de douleurs abdominales moderees d’une duree de plusieurs jours. Quatorze sur 197 (8 %) des PS declare que leur statut a affecte leur activite professionnelle contre 101/191 (62,7 %) des PoS. Cela s’est traduit par des arrets de travail chez 6/197 (3 %) PoS et 40/191 (21 %) des PS (dont 37 femmes). Quarante sur 197 (20 %) des PS et 68/191 (36 %) des PoS se decrivaient comme moderement ou severement anxieux ou deprime. Discussion La PAI est classiquement decrite comme une pathologie evoluant par crises aigues. L’etude de Schmitt et al. [2] rapporte que parmi les PS, seul 12 % d’entre eux presentent plusieurs crises durant leur vie. Cette nouvelle etude a montre une forte proportion de patients rapportant une symptomatologie aigue compatible avec des crises neuroviscerales dont la frequence pourrait etre donc sous-estimee. L’etude EXPLORE [3] montre que 55 % des patients PAI ayant une forme recurrente severe presentent des symptomes chroniques entre les episodes aigus. Cette nouvelle etude montre que parmi les PS, 22 % chez les hommes et 45 % chez les femmes et parmi les PoS, 13 % chez les hommes et 24 % chez les femmes, presentent des douleurs abdominales moderees de longue duree compatible avec des crises neuroviscerales moderees. Cela pose la question du statut des patients consideres comme asymptomatiques et necessite de revisiter le champ des symptomes chroniques chez ces patients. La proportion elevee (36 %) de patients declarant une symptomatologie a type d’anxiete ou de depression dans le groupe des patients symptomatiques souligne le poids de la maladie. De meme dans le groupe asymptomatique, 20 % des patients rapportent des symptomes d’anxiete ou de depression moderes a severes ce qui pourrait etre le reflet d’une symptomatologie chronique sous-jacente. Il est a noter que 22 des patients connus pour presenter des crises invalidantes frequentes n’ont pas repondu a cette enquete. Conclusion Cette etude montre une probable sous-evaluation du nombre de crises aigues chez les PS et l’existence de douleurs abdominales chroniques en dehors de toute crise aigue chez 42 % des PS mais aussi chez 20 % des PoS ayant un retentissement important sur la qualite de vie des patients. Malgre sa denomination, la PAI pourrait etre une maladie associee a une symptomatologie douloureuse chronique qui a ce jour est peu ou pas prise en charge.

Introduction Les porphyries hepatiques aigues sont des maladies genetiques rares, souvent mal diagnostiquees, provoquees par une mutation de l’une des enzymes de la biosynthese de l’heme. Objectifs L’etude EXPLORE est une etude de l’histoire naturelle et de la prise en charge clinique des patients atteints de porphyrie hepatique avec des crises recurrentes (≥ 3 crises/an) ou sous traitement prophylactique afin de prevenir les crises. Patients et methodes Etude internationale prospective, observationnelle. Antecedents medicaux, examens cliniques, biomarqueurs et des questionnaires sur l’activite de la porphyrie ont ete recueillis. Nous presenterons les donnees pour 112 patients recrutes dans 13 pays. Âge moyen des patients : 39 ans, 89 % de femmes, 93 % presentant une porphyrie aigue intermittente. Les patients ont signale une moyenne de 9,3 crises dans l’annee precedant l’etude, la douleur etant le symptome le plus courant. Duree moyenne des crises 7 jours. Resultats Au total, 65 % des patients ont rapporte des symptomes chroniques, le plus frequent etant la douleur. Les patients ont presente en moyenne 3,7crises/personne/an, 69 % ont necessite un traitement par hemin ou une consultation medicale. Les patients sous traitement prophylactique par hemin ont presente en moyenne 3,5 crises/personne/an. Les dosages moyens d’ALA et de PBG a la selection etaient 9 et 23 fois superieurs a la limite superieure de la normale, respectivement. Discussion EXPLORE, la premiere etude internationale de l’histoire naturelle des porphyries hepatiques avec crises recurrentes demontre que les patients presentent des crises et des symptomes chroniques. Conclusion Il demeure un besoin non satisfait de nouveaux traitements pour prevenir les crises et traiter les symptomes chroniques.

The hereditary porphyrias comprise a group of eight metabolic disorders of the heme biosynthesis pathway. Each porphyria is caused by abnormal function at a separate enzymatic step resulting in a specific accumulation of heme precursors. Porphyrias are classified as hepatic or erythropoietic, based on the organ system in which heme precursors (δ-aminolevulinic acid [ALA], porphobilinogen and porphyrins) are overproduced. Clinically, porphyrias are characterized by acute neurovisceral symptoms, skin lesions or both. However, most if not all the porphyrias impair hepatic or gastrointestinal function. Acute hepatic porphyrias present with severe abdominal pain, nausea, constipation, confusion and seizure, which may be life threatening, and patients are at risk of hepatocellular carcinoma without cirrhosis. Porphyria Cutanea presents with skin fragility and blisters, and patients are at risk of hepatocellular carcinoma with liver iron overload. Erythropoietic protoporphyria and X-linked protoporphyria present with acute painful photosensitivity, and patients are at risk of acute liver failure. Altogether, porphyrias are still underdiagnosed, but once they are suspected, early diagnosis based on measurement of biochemical metabolites that accumulate in the blood, urine, or feces is essential so specific treatment can be started as soon as possible and long-term liver complications are prevented. Screening families to identify presymptomatic carriers is also crucial to prevent overt disease and chronic hepatic complications.

International audience; 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.

Hereditary porphyrias comprise a group of eight metabolic disorders of the haem biosynthesis pathway, characterised by acute neurovisceral symptoms and/or skin lesions. Each porphyria is caused by abnormal functioning of a particular enzymatic step, resulting in specific accumulation of heme precursors. Seven porphyrias are due to a partial enzyme deficiency, while a gain-of-function mechanism has recently been identify in a novel porphyria. Acute porphyrias present with severe abdominal pain, nausea, constipation and confusion, and are sometimes complicated by seizures and severe neurological disorders, which may be life-threatening. Cutaneous porphyrias can also be present, with either acute painful photosensitivity or skin fragility and blisters. Rare recessive porphyrias usually manifest in early childhood with either severe chronic neurological symptoms or chronic haemolysis and severe cutaneous photosensitivity. Porphyrias are still under-diagnosed, but recent advances in the pathogenesis and genetics of human porphyrias are leading to better care of these patients and their families.

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315−48T>C) localized in intron 3. The 315−48C allele increases usage of the 3′ cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

Introduction Les porphyries hepatiques aigues sont des maladies genetiques rares, souvent mal diagnostiquees, provoquees par une mutation de l’une des enzymes de la biosynthese de l’heme. Cela se traduit par l’accumulation de precurseurs neurotoxiques de l’heme : acide aminolevulinique (ALA) et porphobilinogene (PBG) qui peuvent provoquer des douleurs neuroviscerales severes, des crises engageant le pronostic vital et des symptomes invalidants chroniques (douleur, nausee et fatigue). Nous presenterons les donnees mises a jour ≥ 12 mois. Patients et methodes L’etude EXPLORE est une etude internationale prospective, observationnelle de l’histoire naturelle et de la prise en charge clinique des patients atteints de porphyrie hepatique avec des crises recurrentes (≥ 3 crises/an) ou sous traitement prophylactique afin de prevenir les crises. Les antecedents medicaux des patients ainsi que leurs examens cliniques, les biomarqueurs et les questionnaires sur l’activite de la porphyrie ont ete recueillis. Resultats 112 patients recrutes dans 13 pays ont ete suivis pendant 12 mois. Âge moyen des patients : 39 ans, 89 % de femmes, 93 % presentant une porphyrie aigue intermittente, 4 % une porphyrie variegata et 3 % une coproporphyrie hereditaire. Les patients ont signale une moyenne de 9,3 crises au cours des 12 mois precedant l’etude, la douleur etant le symptome le plus courant, survenant dans 99 % des crises. La duree moyenne des crises etait de 7 jours. 65 % des patients ont rapporte des symptomes chroniques, la douleur etant le symptome le plus frequent. Le taux de crises pendant l’etude etait de 3,7crises/personne/an, dont 69 % ont necessite un traitement par hemin ou une consultation medicale. Pour les patients sous traitement prophylactique par hemin, le taux de crises moyen etait de 3,5 crises/personne/an. Les dosages moyens d’ALA et de PBG a la selection (en dehors d’une crise) etaient considerablement eleves jusqu’a un niveau 9 et 23 fois superieur a la limite superieure de la normale, respectivement. Conclusion EXPLORE, la premiere etude internationale de l’histoire naturelle chez des patients atteints de porphyrie hepatique avec des crises recurrentes demontre que les patients presentent des crises et des symptomes chroniques. Compte tenu de la morbidite et de la mortalite, il demeure un besoin non satisfait de nouveaux traitements pour prevenir les crises et traiter les symptomes chroniques.

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.

Hereditary porphyrias comprise a group of eight metabolic disorders of the haem biosynthesis pathway, characterised by acute neurovisceral symptoms and/or skin lesions. Each porphyria is caused by abnormal functioning of a particular enzymatic step, resulting in specific accumulation of heme precursors. Seven porphyrias are due to a partial enzyme deficiency, while a gain-of-function mechanism has recently been identify in a novel porphyria. Acute porphyrias present with severe abdominal pain, nausea, constipation and confusion, and are sometimes complicated by seizures and severe neurological disorders, which may be life-threatening. Cutaneous porphyrias can also be present, with either acute painful photosensitivity or skin fragility and blisters. Rare recessive porphyrias usually manifest in early childhood with either severe chronic neurological symptoms or chronic haemolysis and severe cutaneous photosensitivity. Porphyrias are still under-diagnosed, but recent advances in the pathogenesis and genetics of human porphyrias are leading to better care of these patients and their families.

SummaryBackground Erythropoietic protoporphyria (EPP) is a rare metabolic disorder, characterized by photosensitivity, caused by errors of the haem biosynthetic pathway. Avoidance of sun exposure is recommended; however, some patients suggested a paradoxical improvement of symptoms when they move to sunny areas. Objectives In a national French study, we sought to investigate the influence of sun exposure on EPP symptoms. Materials and methods We used a national transversal observational study by questionnaire. Patients were selected from the national record of the Centre Francais des Porphyries (French Porphyrias referral centre). Sun exposure level by geographic area was assessed using climate data provided by the French national meteorological service (Meteo France). Results Eighty-nine patients were included. We notably observed that 40% of patients declared an improvement in their tolerance of sun exposure after repeated sun exposures. In the more sunny areas, the intensity of the pain was lower (r = −0·26) and the duration of the sun exposure responsible for flares was longer (r = 0·39) than in the areas that were less sunny (P < 0·05). Conclusions This study proposes a benefit of natural progressive sun exposure for patients with EPP.

Retrospective estimates of the prevalence of porphyrias have been reported but there has been no large scale prospective study of their incidence. The European Porphyria Network collected information prospectively over a 3 year period about the number of newly diagnosed symptomatic patients with an inherited porphyria (335 patients from 11 countries). Prevalence was calculated from the incidence and mean disease duration. The incidence of hepato-cellular carcinoma (HCC) in acute hepatic porphyria and the prevalence of patients with recurrent acute attacks of porphyria were also investigated. The incidence of symptomatic acute intermittent porphyria (AIP) was similar in all countries (0.13 per million per year; 95 % CI: 0.10 - 0.14) except Sweden (0.51; 95 % CI: 0.28-0.86). The incidence ratio for symptomatic AIP: variegate porphyria: hereditary coproporphyria was 1.00:0.62: 0.15. The prevalence of AIP (5.4 per million; 95 % CI: 4.5-6.3) was about half that previously reported. The prevalence of erythropoietic protoporphyria (EPP) was less uniform between countries and, in some countries, exceeded previous estimates. Fourteen new cases of HCC (11 from Sweden) were reported in patients with acute porphyria. Sixty seven patients (3 VP; 64 AIP: 53 females, 11 males) with recurrent attacks of acute porphyria were identified. The estimated percentage of patients with AIP that will develop recurrent acute attacks was 3-5 %. In conclusion, the prevalence of symptomatic acute porphyria may be decreasing, possibly due to improved management, whereas the prevalence of EPP may be increasing due to improved diagnosis and its greater recognition as a cause of photosensitivity.

Background Congenital erythropoietic porphyria (CEP) is an autosomal recessive photomutilating porphyria with onset usually in childhood, where haematological complications determine prognosis. Due to its extreme rarity and clinical heterogeneity, management decisions in CEP are often difficult. Objectives To develop a management algorithm for patients with CEP based on data from carefully characterized historical cases. Methods A single investigator collated data related to treatments and their outcomes in 29 patients with CEP from the U.K., France, Germany and Switzerland. Results Six children were treated with bone marrow transplantation (BMT); five have remained symptomatically cured up to 11AE5 years post-transplantation. Treatments such as oral charcoal, splenectomy and chronic hypertransfusion were either of no benefit or were associated with complications and negative impact on health-related quality of life. Lack of consistent genotype–phenotype correlation meant that this could not be used to predict disease prognosis. The main poor prognostic factors were early age of disease onset and severity of haematological manifestations. Conclusions A management algorithm is proposed where every patient, irrespective of disease severity at presentation, should receive a comprehensive, multidisciplinary clinical assessment and should then be reviewed at intervals based on their predicted prognosis, and the rate of onset of complications. A BMT should be considered in those with progressive, symptomatic haemolytic anaemia and ⁄or thrombocytopenia. Uroporphyrinogen III synthase genotypes associated with poor prognosis would additionally justify consideration for a BMT. Rigorous photoprotection of the skin and eyes from visible light is essential in all patients.

Background: Congenital erythropoietic porphyria (CEP) is an autosomal recessive cutaneous porphyria caused by decreased activity of uroporphyrinogen III synthase (UROS). Its predominant characteristics include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. Due to its rarity and genetic heterogeneity, clinical phenotypes are unclear and its impact on health-related quality of life (HRQoL) has not been previously assessed. Objectives To define comprehensively CEP phenotypes and assess their impact on HRQoL, and to correlate these factors with laboratory parameters. Methods: A single observer assessed patients with CEP from four European countries. Results: Twenty-seven unrelated patients with CEP, aged between 7AE6 and 65 years, participated in the study. The patients came from the U.K. (17), France (4), Switzerland (4) and Germany (2). Additional data were obtained for two deceased patients. Newly characterized features of CEP include acute-onset cutaneous and noncutaneous symptoms immediately following sunlight exposure, and pink erythematous facial papules. There was a lack of consistent genotype– phenotype correlation in CEP. The main poor prognostic factors in CEP are the early age of disease onset and haematological complications. Conclusions CEP is a multisystem disease; cutaneous, ocular, oral and skeletal manifestations also contribute to disease severity and impact on HRQoL, in addition to the haematological complications. The rarity of the disease can lead to delayed diagnosis. The lack of consistent genotype–phenotype correlation in CEP suggests a contribution to phenotype from other factors, such as environment, patients’ photoprotective behaviour and genes other than UROS. There is currently an unmet need for multidisciplinary management of patients with CEP.

Lionel Arnaud and colleagues identify ABCB6 as the genetic basis of the novel Lan blood group system. The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis1, but it is also suspected to contribute to anticancer drug resistance2,3,4, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(−) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(−) (ABCB6−/−) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.

Summary Background Porphyria cutanea tarda (PCT), the most frequent type of porphyria, results from decreased uroporphyrinogen decarboxylase (UROD) activity. Two forms of PCT have been described: a familial form (fPCT) characterized by the inherited decrease of UROD activity in all tissues and a sporadic form (sPCT) characterized by decreased UROD activity in the liver. Cytochrome P450 CYP1A2 plays a major role in triggering experimental uroporphyria in rodents. It has been suggested that the highly inducible -163A/A genotype of the CYP1A2 gene could confer a heightened risk of PCT in patients. Objectives To examine the impact of CYP1A2 polymorphisms on the clinical course of PCT. Methods We performed an extensive CYP1A2 gene analysis in 96 (48 fPCT and 48 sPCT) unrelated French caucasian patients with PCT and in 99 healthy volunteers of similar ethnic origin. Results We did not observe any difference in CYP1A2 allele distribution, including a novel and rare CYP1A2 c.1063C>T (p.R355W) single nucleotide polymorphism. In addition, we compared the frequency of the -163A highly inducible allele both in patients with symptomatic fPCT (n = 48) and in asymptomatic UROD gene mutations carrier relatives (n = 54). This variant was not over-represented in patients with PCT vs. either healthy volunteers or asymptomatic UROD gene mutation carriers. Conclusions The CYP1A2 genotype does not appear to be a major susceptibility factor in the development of fPCT or sPCT in the French population.

Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis that results from an accumulation of protoporphyrin IX in erythroid cells, plasma, skin and liver. EPP leads to acute photosensitivity and, in about 2% of patients, liver disease. EPP is a complex syndrome in which two genes are independently involved: FECH and ALAS2. More than 96% of unrelated EPP patients have ferrochelatase (FECH) deficiency (MIM 177000). Four percent of them present with autosomal recessive inheritance with two mutated FECH alleles. In dominant cases (95%) the inheritance of a common hypomorphic IVS3-48C FECH allele trans to a deleterious FECH mutation reduces FECH activity below a critical threshold. The frequency of the IVS3-48C allele differs widely from the Japanese (45%), to Black West Africans (

Background The gene coding for the D2 dopamine receptor ( DRD2 ) is considered to be one of the most pertinent candidate genes in schizophrenia. However, genetic studies have yielded conflicting results whereas the promising TaqIA variant/rs1800497 has been mapped in a novel gene, ANKK1 . Methods We investigated eleven single nucleotide polymorphisms (SNPs) spanning the DRD2 and ANKK1 genes, using both a case–control association study comparing 144 independent patients to 142 matched healthy subjects, and a transmission disequilibrium test in 108 trios. This classical genetic study was coupled with a cladistic phylogeny-based association test of human variants, and with an interspecies evolution study of ANKK1. Results Case–control study, followed by a 108 trios family-based association analysis for replication, revealed an association between schizophrenia and the ANKK1 rs1800497 ( p = 0.01, Odds Ratio = 1.5, 95% Confidence Interval = 1.1–2.2), and the intergenic rs2242592 ( p = 2 · 10 − 4 , OR = 1.8, 95%CI = 1.3–2.5). A significant SNP–SNP interaction was also found ( p − 5 , OR = 2.0, 95%CI = 1.6–2.5). The phylogeny-based association test also identified an association between both these polymorphisms and schizophrenia. Finally, interspecies comparison of the sequences from chimpanzee, orangutan, rhesus macaque and human species suggested specific involvement of ANKK1 in the human lineage. Conclusions Intergenic rs2242592 appears to be involved in the genetic vulnerability to schizophrenia, whereas the ANKK1 rs1800497 appears to have a modifying rather than causative effect. Finally, ANKK1 may be a specific human lineage-trait involved in a specific human disease, schizophrenia.

Summary Since the elucidation of the genetic code approximately 40 years ago, the role of RNA in biology has gained increasing attention. Now, study of RNA is one of the fastest developing area in molecular and cellular biology. For a long time, biologists have been focused on DNA and proteins with little interest for RNA. The growing interest in genome-wide transcriptional profiling has allowed new developments in RNA research and brought important attention to the RNA world, its incredible complexity and its huge functional role within the cell and its environment. The RNA world with its remarkable range of biological processes have important implications in human health that begins only to be sketched out. In this article in two parts, we will describe the diversity of the RNA world and potential consequences in medicine.

Summary Colorectal cancer is the third cause of cancer and the second most common cause of death from cancer in France. Owing to important advances in our understanding of molecular and biochemical events involved in the process of carcinogenesis and tumor growth, cancer therapy, and especially colorectal cancer therapy, has entered a new era. In this cancer, targeted therapy has appeared the last past few years with two antibodies to epidermal growth factor receptor (EGFR): cetuximab and panitumumab. Numerous studies have shown the benefit of these antibodies in patients with metastatic colorectal cancer. However, this benefit is limited to patients with wild-type KRAS gene only. Actually, mutated KRAS gene is associated with anti-EGFR resistance. Therefore, KRAS mutation studies are mandatory before adding anti-EGFR to chemotherapy. These findings represent an important step forward in the field of personalized medicine which is defined as the determination of the optimal treatment for each patient in order to avoid overtreatment with drugs that have potentially toxic adverse effects but little benefit. A new branch of medicine is rising, personalized medicine.

Summary Understanding on human inheritance has significantly improved these last 20 years. Even if monogenic diseases study brought important informations on the functionning of genes and deciphering of human genome on its organization, knowledge has evolved and has changed our view of heredity. Mendel laws applied to monogenic disorders are not any more sufficient to explain transmission of our characters. Genome regulations and its interactions included with environnement demonstrate the complexity of transmission of our traits and the resulting adjustments acquired and hereditary alike for each individual. Moreover, human genetic is inadequately familiar to physicians and biologists and is insufficiently taught in medical universities. In this paper, we remind some important and current notions on human molecular genetic in order to help the non specialist physician or biologist to answer genetic questions to a consultant.

Résumé Le séquençage d’ADN est devenu un outil essentiel en biologie moléculaire tant en médecine que dans de nombreuses autres disciplines des sciences de la vie. Le séquençage a été décrit il y a environ 30 ans et n’a cessé d’évoluer depuis cette période. Cette méthode est devenue une technique courante dans les laboratoires de biologie moléculaire. Les connaissances acquises grâce à cette méthode et la possibilité de séquencer des génomes de grande taille, tel que le génome humain, ont amené les chercheurs à développer des techniques de séquençage de plus en plus sophistiquées. Cet article, composé de deux parties, présente les techniques actuellement utilisées pour séquencer l’ADN, qu’il soit humain ou d’autre origine, et les méthodes de séquençage en développement. Ces dernières constituent un réel bouleversement. Le séquençage à l’échelle individuelle n’est plus loin. En dehors des problèmes éthiques qu’elle soulève, cette révolution pose de nouvelles questions, par exemple : comment interpréterons-nous les nombreuses variations génétiques observées chez un individu, quelles en seront les conséquences sur ses prédispositions génétiques aux maladies et autres risques, quels en seront les retentissements sur le phénotype ? De nombreuses études en cours cherchent les réponses. Dans tous les cas, la révolution est en marche.

Summary Mrs H.D. has the hereditary feature of porphyria and presents diffuse, intense abdominal pains coming along with vomiting and nausea. In the examination, the temperature is 38.3 °C, the belly is flexible and the rest of the examination is normal. The doctor evokes then two diagnoses.

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19–20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

Objectives CX3CR1, the receptor of CX3CL1/fractalkine, is involved in regulation of inflammatory response and the CX3CR1-I249-M280 naturally occurring mutants are associated with altered binding to the ligand. Our aim was to evaluate the frequency of CX3CR1 V249I and T280M polymorphisms and NOD2/CARD15 mutations in Crohn's disease patients and to search for a relationship with phenotype. Methods Clinical data were retrospectively collected. V249I and T280M polymorphisms of CX3CR1 gene and NOD2/CARD15 mutations (R702W, G908R, 3020InsC) were identified. Results Two hundred and thirty-nine patients (140 females, 39.7±14.1 years) were included. About 37.4% were heterozygous and 8.8% were homozygous for the V249I CX3CR1 polymorphism, 18.1% were heterozygous and 1.3% homozygous for the T280M CX3CR1 polymorphism and 35.9% had at least one of the three mutations of NOD2/CARD15. The T280M CX3CR1 polymorphism was not associated with any phenotype. In univariate analysis, stenosis was significantly associated with both V249I CX3CR1 polymorphism and 3020InsC NOD2/CARD15 mutations. In smoker patients carrying the CX3CR1 allele I249, there was a significant increase in the frequency of fibrostenosing disease [P=0.005, odds ratio (OR): 3.25] whereas this relationship disappeared in the group of nonsmokers (P=0.72). In multivariate analysis, 3020InsC NOD2/CARD15 mutations and the V249I CX3CR1 polymorphism were independent risk factors for intestinal stenosis (P=0.046, OR: 1.8 and P=0.044, OR: 2.4, respectively). Conclusion In Crohn's disease, V249I CX3CR1 polymorphism is associated with intestinal strictures, particularly in smokers. This association is independent of CARD15 mutations.

Resume La trisomie 21 (ou syndrome de Down), presence de trois chromosomes 21 au lieu de deux, est l’anomalie chromosomique la plus frequente. Une legislation specifique a ete mise en place en 1997 pour un depistage au cours de la grossesse a l’aide de l’echographie et de marqueurs seriques doses chez la femme enceinte. En cas de suspicion de trisomie 21, une amniocentese ou une choriocentese est proposee, afin de realiser un caryotype. La Haute Autorite de sante a recemment publie un rapport proposant la modification de cette legislation. Bien que la place de la biologie moleculaire ne soit pas definie dans le diagnostic de la trisomie 21, des tests existent depuis plusieurs annees tant en France qu’a l’etranger. Ces tests appeles aussi tests rapides permettent d’obtenir une reponse en 24 a 48 heures. Le resultat est ensuite confirme par le caryotype. Cet article rapporte les principes du diagnostic moleculaire et discute de la place que pourrait avoir la biologie moleculaire dans le diagnostic de la trisomie 21, ces tests etant surtout utilises dans les pays anglo-saxons.

A rare disease is a disease that occurs in less than one patient in 2000. It is a real public health issue for which sudden awareness has been increasing since the end of the 1980's and begun to be politically and medically structured in the 1990's. About 10 years ago, in order to improve research, health and social care of these diseases, France and Europe begun to create organisms with the help of associations of patients, physicians, researchers and pharmaceutical industries to fight again the estimated 7000 rares disorders for 27 millions patients (about 8% of european population). In this article, we summarize the actual organisation again these diseases and the main policies to help patients and relatives now and in the near future.

Resume La maladie de Crohn et la rectocolite hemorragique sont les maladies inflammatoires chroniques de l'intestin les plus frequentes dans les pays occidentaux avec une frequence de 1/250. La morbidite de ces deux maladies est importante. La maladie de Crohn a ete recemment mise en avant par la decouverte en 2001 d'un gene implique dans sa pathogenese, le gene CARD15 codant pour la proteine NOD2. Cette decouverte majeure a joue un role important dans la mise en evidence du role fondamental joue par l'immunite innee (ou acquise) dans les defenses immunitaires du tube digestif et a abouti a la decouverte de nouvelles molecules therapeutiques. Cet article rapporte les connaissances recentes sur la physiopathologie de la maladie de Crohn et le role joue par les mutations dans le gene CARD15. Il souligne l'influence tant polygenique qu'environnementale sur la physiopathologie de la maladie.

Resume La biologie moleculaire est omnipresente en biologie medicale et plus particulierement en microbiologie. De nombreux articles demontrent son importance tant dans le domaine du diagnostic que du pronostic, de l'evaluation therapeutique, de l'epidemiologie ou des risques biologiques naturels ou non. La quantite considerable d'articles sur ce sujet n'apporte pas toujours une reponse evidente sur le role de la biologie moleculaire dans un laboratoire de microbiologie qu'il soit hospitalier ou non. Cette revue constitue une synthese des apports de cette discipline en microbiologie. A partir de cet etat des lieux, certaines questions se posent, par exemple : la biologie moleculaire constitue-t-elle un reel apport en microbiologie ? Dans quelles indications prescrire un examen de biologie moleculaire ? Les reponses ne sont pas toujours simples. Elles sont evidentes dans certains cas (l'hepatite C par exemple) et le sont moins dans d'autres, la tuberculose par exemple. Dans la premiere partie de l'article, nous avons parle des generalites appliquees a la microbiologie. Dans cette deuxieme partie, nous abordons certaines applications, reflets de l'importance prise par la biologie moleculaire en microbiologie.

Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5'-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.

BACKGROUND Erythropoietic protoporphyria is a severe photodermatosis that is associated with acute phototoxicity. Patients with this condition have excruciating pain and a markedly reduced quality of life. We evaluated the safety and efficacy of an alpha-melanocyte-stimulating hormone analogue, afamelanotide, to decrease pain and improve quality of life. METHODS We conducted two multicenter, randomized, double-blind, placebo-controlled trials of subcutaneous implants containing 16 mg of afamelanotide. Patients in the European Union (74 patients) and the United States (94 patients) were randomly assigned, in a 1:1 ratio, to receive a subcutaneous implant containing either afamelanotide or placebo every 60 days (a total of five implants in the European Union study and three in the U.S study). The type and duration of sun exposure, number and severity of phototoxic reactions, and adverse events were recorded over the respective 180-day and 270-day study periods. Quality of life was assessed with the use of validated questionnaires. A subgroup of U.S. patients underwent photoprovocation testing. The primary efficacy end point was the number of hours of direct exposure to sunlight without pain. RESULTS In the U.S. study, the duration of pain-free time after 6 months was longer in the afamelanotide group (median, 69.4 hours, vs. 40.8 hours in the placebo group; P = 0.04). In the European Union study, the duration of pain-free time after 9 months was also longer in the afamelanotide group than in the placebo group (median, 6.0 hours vs. 0.8 hours; P = 0.005), and the number of phototoxic reactions was lower in the the afamelanotide group (77 vs. 146, P = 0.04). In both trials, quality of life improved with afamelanotide therapy. Adverse events were mostly mild; serious adverse events were not thought to be related to the study drug. CONCLUSIONS Afamelanotide had an acceptable side-effect and adverse-event profile and was associated with an increased duration of sun exposure without pain and improved quality of life in patients with erythropoietic protoporphyria. (Funded by Clinuvel Pharmaceuticals and others; ClinicalTrials.gov numbers, NCT01605136 and NCT00979745.)

Many red cell polymorphisms are a result of selective pressure by the malarial parasite. Here, we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of Plasmodium falciparum malarial parasites. This phenomenon is due to the absence of ferrochelatase and not an accumulation of substrate, as demonstrated by the normal growth of P falciparum parasites in the EPP phenocopy, X-linked dominant protoporphyria, which has elevated substrate, and normal ferrochelatase levels. This observation was replicated in a mouse strain with a hypomorphic mutation in the murine ferrochelatase gene. The parasite enzyme is not essential for parasite growth as Plasmodium berghei parasites carrying a complete deletion of the ferrochelatase gene grow normally in erythrocytes, which confirms previous studies. That ferrochelatase is essential to parasite growth was confirmed by showing that inhibition of ferrochelatase using the specific competitive inhibitor, N-methylprotoporphyrin, produced a potent growth inhibition effect against cultures of P falciparum. This raises the possibility of targeting human ferrochelatase in a host-directed antimalarial strategy.