Y. Deugnier

Current approach to hemochromatosis

Iron overload diseases of genetic origin are an ever changing world, due to major advances in genetics and molecular biology. Five major categories are now established: HFE-related or type1 hemochromatosis, frequently found in Caucasians, and four rarer diseases which are type 2 (A and B) hemochromatosis (juvenile hemochromatosis), type 3 hemochromatosis (transferrin receptor 2 hemochromatosis), type 4 (A and B) hemochromatosis (ferroportin disease), and a(hypo)ceruloplasminemia. Increased duodenal iron absorption and enhanced macrophagic iron recycling, both due to an impairment of hepcidin synthesis, account for the development of cellular excess in types 1, 2, 3, and 4B hemochromatosis whereas decreased cellular iron egress is involved in the main form of type 4A) hemochromatosis and in aceruloplasminemia. Non-transferrin bound iron plays an important role in cellular iron excess and damage. The combination of magnetic resonance imaging (for diagnosing visceral iron overload) and of genetic testing has drastically reduced the need for liver biopsy. Phlebotomies remain an essential therapeutic tool but the improved understanding of the intimate mechanisms underlying these diseases paves the road for innovative therapeutic approaches.

Iron and the liver: Update 2008 ☆

The cross-talk which has taken place in recent years between clinicians and scientists has resulted in a greater understanding of iron metabolism with the discovery of new iron-related genes including the hepcidin gene which plays a critical role in regulating systemic iron homeostasis. Consequently, the distinction between (a) genetic iron-overload disorders including haemochromatosis related to mutations in the HFE, hemojuvelin, transferrin receptor 2 and hepcidin genes and (b) non-haemochromatotic conditions related to mutations in the ferroportin, ceruloplasmin, transferrin and di-metal transporter 1 genes, and (c) acquired iron-overload syndromes has become easier. However, major challenges still remain which include our understanding of the regulation of hepcidin production, the identification of environmental and genetic modifiers of iron burden and organ damage in iron-overload syndromes, especially HFE haemochromatosis, indications regarding the new oral chelator, deferasirox, and the development of new therapeutic tools interacting with the regulation of iron metabolism.

Modifying factors of the HFE hemochromatosis phenotype

C282Y homozygosity is the only common HFE genotype able to produce a complete hemochromatosis phenotype. However, its biochemical penetrance is incomplete (75% in men and 50% in women) and its clinical penetrance is low, especially in women (1 vs 25% in men). Environmental (e.g., diet, alcohol, drugs and metabolic syndrome) and genetic (digenism, common polymorphisms in the bone morphogenetic protein pathway involved in the regulation of hepcidin synthesis) explain a part of the variability of the C282Y homozygous phenotype. All other common HFE genotypes – including C282Y–H63D compound heterozygosity – are not associated with significant biochemical and clinical expression in the absence of comorbid factors (e.g., alcohol, diabetes or steatohepatitis). Better identification of acquired and genetic modifiers of iron burden and iron-related organ damage is needed to improve the preventive, diagnostic and therapeutic management of HFE hemochromatosis.

Non-invasive assessment of liver fibrosis in C282Y homozygous HFE hemochromatosis.

C282Y homozygotes with serum ferritin (SF) levels >1000 μg/L and/or increased serum transaminase levels are at risk of severe F3/F4 fibrosis. Current practical guidelines recommend liver biopsy in such individuals. This prospective observational cohort study aimed to evaluate non‐invasive alternative means such as hyaluronic acid (HA) and transient elastography (TE) for the assessment of severe fibrosis in patients with SF >1000 μg/L or elevated transaminases.

Hyperferritinémies non hémochromatosiques

À tort ou à raison, le dosage de la ferritine sérique est de plus en plus fréquemment demandé dans le cadre de bilans biologiques de routine ou devant des signes peu spécifiques et fort prévalents comme une fatigue, des douleurs articulaires, des anomalies du bilan hépatique ou un trouble du métabolisme des sucres. Il s’ensuit une inflation de cas d’hyperferritinémie dont bien peu, au bout du compte, témoignent d’une surcharge en fer d’origine génétique.

Variable age of onset and clinical severity in transferrin receptor 2 related haemochromatosis: novel observations.

Since the discovery of the HFE gene and the C282Y mutation (Type 1 haemochromatosis), new genes involved in iron metabolism have been described. Juvenile haemochromatosis has been related to HFE2 and HAMP mutations (Type 2A and 2B) and is described as severe iron overload affecting patients before the age of 30 years (Brissot et al, 2011). Mutations in the TFR2 gene lead to Type 3 haemochromatosis whose clinical picture mimics Type 1. However, rare cases affecting young patients have been reported (Brissot et al, 2011). The ferroportin disease has been linked to SLC40A1 mutation and is described as iron overload affecting patients at any age (Le Lan et al, 2011). The basic mechanism accounting for iron overload in types 1, 2 and 3 haemochromatosis is decreased hepcidin synthesis. TFR2 is also involved in hepcidin synthesis regulation (Wallace et al, 2005; Gao et al, 2010) but its definite mode of action remains to be determined. In Type 1 haemochromatosis, cofactors play an important role because clinical penetrance of C282Y homozygosity is low (Allen et al, 2008). In Type 3 haemochromatosis, clinical expression seems high but reported cases are too scarce to definitely assess penetrance. Here, we report seven new cases of Type 3 haemochromatosis. Transferrin receptor 2 mutations were screened as part of the diagnostic activity of the French Reference Centre for Rare Iron Overload Diseases of Genetic Origin and the associated network of expertise Centres. Written informed consent was obtained and the study was performed in accordance with the Declaration of Helsinki and with the French regulations on medical genetic diagnosis. Patients were tested for HFE mutations (p.Cys282Tyr, p.His63Asp) (Jouanolle et al, 1996; Aguilar Martinez et al, 1997). The entire coding region and intronic flanking sequences of the TFR2 gene (NCBI NM_003227.3, NP_003218.2) were sequenced. To exclude other mutation(s), analyses of the haemochromatosis type 2 (juvenile) (HFE2), hepcidin (HAMP), and ferroportin (SLC40A1) coding sequences were performed. To determine the potential consequences of mutations on the protein, TFR2 amino acid sequence and mutations were input as required into the following algorithms: Scale-Invariant Feature Transform (SIFT; http://sift.jcvi.org/), Polymorphism Phenotyping v2 (Polyphen-2; http://genetics.bwh. harvard.edu/pph2/bgi.shtml), point mutant (Pmut; http:// mmb2.pcb.ub.es:8080/PMut/), SNPs3D (http://www.snps3d. org/), and Scalable Nucleotide Alignment Program (SNAP; http://www.rostlab.org/services/snap/). Seven unrelated patients were diagnosed with Type 3 haemochromatosis. Three were homozygous for the previously undescribed p.Asn412Ile (c.1235A>T), p.Gly430Arg (c.1288G>A), p.Arg678Pro (c.2033G>C) TFR2mutations. Consanguinity was likely only for Patient 1. Four patients were compound heterozygotes for at least one new TFR2 mutation each: p.Leu85_A a96delinsPro (c.254_286 + 9del), p.Met705Hisfs*87 (c.211 2dup), p.Arg730Cys (c.2188C>T), p.Gly735Ser (c.220 3G>A), p.Trp781* (c.2343G>A). One patient, who was a compound heterozygote for two previously described TFR2 mutations (p.Ala444Thr, p.Gly792Arg) (Lee & Barton, 2006; Biasiotto et al, 2008) was found to carry the p.Gly204Ser mutation in the SLC40A1 gene coding for ferroportin. The main clinical and biological features of the patients are summarized in Table I. No mutations were found in the HAMP and HFE2 genes of the patients. Sequencing of the HFE gene revealed no other mutations than the H63D or C282Y (Table I). Patients 1 and 2 were diagnosed earlier than usually described for Type 3 haemochromatosis. Patient 1 was referred for major asthenia at the age of 10 years. Biological workup found high transferrin saturation leading to the diagnosis of haemochromatosis; an echocardiogram revealed no abnormalities. Patient 2 was diagnosed as a result of elevated transferrin saturation in the context of a1-antitrypsin deficiency. Patients 3, 4 and 5 were diagnosed between 20 and 30 years of age. Patient 3 originated from North Africa and was diagnosed upon arrival in France, which could explain the older age at diagnosis. The daughter of Patient 3 was heterozygous for the TFR2:p.Gly430Arg mutation and had normal iron parameters. The parents of Patient 4 were heterozygous for mutation p.Gly735Ser and p.Leu85_Ala96delinsPro respectively, and had normal iron parameters. Patient 6 was diagnosed with non-HFE related haemochromatosis at the age of 28 years. Diagnosis of ferroportin disease was made later by finding the SLC40A1:p.Gly204Ser mutation. The clinical presentation was unusual with elevated transferrin saturation and arthropathy. Moreover, phlebotomies were very well tolerated and removed 19 5 g iron. For these reasons, sequencing of other genes related to iron metabolism was performed and revealed two already described mutations in TFR2. Patient 7 was diagnosed during the diagnostic workup of post-partum infection. She had elevated liver enzymes, and transient elastography (Fibroscan , Echosens, Paris, France)

Le réseau des Centres de Ressources Biologiques Humains

Les decouvertes de cibles therapeutiques et doutils diagnostiques nouveaux sont aujourdhui le fruit du developpement considerable de la biologie cellulaire et moleculaire et des biotechnologies. Apres le sequencage du genome humain, la pertinence des donnees obtenues par les techniques a haut debit, notamment celles issues de la genomique et de la proteomique, depend en premier lieu de lassurance-qualite et de la tracabilite du materiel biologique a partir duquel sont effectuees les recherches, ainsi que des annotations qui qualifient les echantillons. Les Centres de Ressources Biologiques, qui incluent les biobanques, les cryobanques et les tumorotheques sont donc des infrastructures essentielles pour la decouverte doutils innovants pour la prevention, le diagnostic et le traitement des maladies, et la mise au point de nouveaux medicaments. La constitution dun reseau de Centres de Ressources Biologiques Humains sur des bases ethiques et juridiques harmonisees constitue aujourdhui un enjeu majeur pour le developpement de la recherche medicale en Europe.

In patients with cirrhosis, serum albumin determination should be carried out by immunonephelometry rather than by protein electrophoresis.

OBJECTIVEnSerum albumin is a key parameter for prognosis in cirrhosis. We compared levels of serum albumin determined by both protein electrophoresis and immunonephelometry, with special reference to the Child-Pugh classification.nnnDESIGN AND METHODSnOne hundred and thirty-one patients, including 39 with cirrhosis, were included prospectively during 2 months. The aetiology of cirrhosis was mainly alcoholism (67%) and hepatitis C virus (HCV) (18%). Serum albumin was determined simultaneously by electrophoresis (Hydrasys SEBIA following protein determination by the biuret reaction) and by immunonephelometry (BECKMAN Nephelometer). Values were compared by non-parametric tests.nnnRESULTSnFor the whole population, electrophoretic and immunonephelometric values correlated (p = 0.85; P < 0.0001), but electrophoresis significantly overestimated serum albumin by a median 1.6 g/l (P < 0.0001) with a large spread in values (range, -3.9 to 12.7). Median overestimation in cirrhosis was 2.6 g/l (P < 0.0001; range, -2.0 to 10.2) and 1.0 g/l (P < 0.0001; range, -3.9 to 12.7) in patients without cirrhosis (difference, P < 0.02). For 6/39 (15.4%) patients with cirrhosis, this overestimation led to an underestimation in the Child-Pugh classification.nnnCONCLUSIONnIn our experience, electrophoresis can lead to serum albumin values which are significantly different compared to those obtained by immunonephelometry. This discrepancy may lead to an incorrect Child-Pugh classification. Therefore, in the follow-up of cirrhotic patients, serum albumin should be determined by immunonephelometry.

Association sarcoïdose et cirrhose biliaire primitive : revue de la littérature à propos d'un nouveau cas

Resume Lassociation dune sarcoidose et dune cirrhose biliaire primitive chez une patiente de 56 ans avec suivi sur 5 ans est rapportee. La sarcoidose a ete affirmee sur les images mediastino-pulmonaires, la scintigraphie au gallium, le lavage bronchiolo-alveolaire, les donnees de la ponction biopsie hepatique (presence de granulomes epithelioides intralobulaires). Le diagnostic de cirrhose biliaire primitive a repose sur la cholestase intra-hepatique, laugmentation du taux des IgM a 4,4 g/l, la forte positivite des anticorps anti-mitochondries au 1/1000 et les donnees histopathologiques (cholangite chronique destructive). Une telle association est rare (12 cas detailles dans la litterature), souleve des problemes physiopathologiques et allonge la liste des mecanismes de cholestase a evoquer en situation de sarcoidose.

Hereditary hypotransferrinemia can lead to elevated transferrin saturation and, when associated to HFE or HAMP mutations, to iron overload.

As our understanding of iron metabolism improves through the more accurate description of iron metabolism actors, new causes of iron overload are identified. We, here, report 16 cases of hereditary hypotransferrinemia related to 4 previously undescribed TF (transferrin) mutations (p.Val221Gly, p.Arg609Trp, p.Glu370Lys, p.Tyr533X and p.Cys421Arg). We show that, besides increasing serum transferrin saturation without iron overload, hypotransferrinemia, when associated to mutations in HFE or HAMP or to acquired factors, can lead to clinically relevant iron burden. These cases emphasize the usefulness of serum transferrin determination in the diagnostic evaluation of iron overload and the importance for clinicians to be aware of this syndrome.