Edouard Bardou-Jacquet

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 disorders of genetic origin: a changing world

Iron disorders of genetic origin are mainly composed of iron overload diseases, the most frequent being HFE-related hemochromatosis. Hepcidin deficiency underlies iron overload in HFE-hemochromatosis as well as in several other genetic iron excess disorders, such as hemojuvelin or hepcidin-related hemochromatosis and transferrin receptor 2-related hemochromatosis. Deficiency of ferroportin, the only known cellular protein iron exporter, produces iron overload in the typical form of ferroportin disease. By contrast, genetically enhanced hepcidin production, as observed in matriptase-2 deficiency, generates iron-refractory iron deficiency anemia. Diagnosis of these iron storage disorders is usually established noninvasively through combined biochemical, imaging and genetic approaches. Moreover, improved knowledge of the molecular mechanisms accounting for the variations of iron stores opens the way of novel therapeutic approaches aiming to restore normal iron homeostasis. In this review, we will summarize recent findings about these various genetic entities that have been identified owing to an exemplary interplay between clinicians and basic scientists.

A novel N491S mutation in the human SLC11A2 gene impairs protein trafficking and in association with the G212V mutation leads to microcytic anemia and liver iron overload

BACKGROUND DMT1 is a transmembrane iron transporter involved in iron duodenal absorption and cellular iron uptake. Mutations in the human SLC11A2 gene coding DMT1 lead to microcytic anemia and hepatic iron overload, with unexpectedly low levels of plasma ferritin in the presence of iron stores. DESIGN AND METHODS We report a patient with a similar phenotype due to two mutations in the SLC11A2 gene, the known p.Gly212Val (G212V) mutation and a novel one, p.Asn491Ser (N491S). To assess the expression of DMT1 in human liver, we studied the expression of the four DMT1 mRNA isoforms by real-time quantitative PCR in control human liver samples. We also studied the effect of G212V and N491S DMT1 mutations on RNA splicing in blood leukocytes and cellular trafficking of dsRed2-tagged-DMT1 protein in the human hepatic cell line HuH7. RESULTS Our results showed that i) only the isoforms 1B-IRE and 1B-nonIRE were significantly expressed in human liver; ii) the G212V mutation did not seem to affect mRNA splicing and the N491S mutation induced a splicing alteration leading to a truncated protein, which seemed quantitatively of low relevance; and iii) the N491S mutation, in contrast to the G212V mutation, led to abnormal protein trafficking. CONCLUSIONS Our data confirm the major role of DMT1 in the maintenance of iron homeostasis in humans and demonstrate that the N491S mutation, through its deleterious effect on protein trafficking, contributes together with the G212V mutation to the development of anemia and hepatic iron overload.

Non-HFE hemochromatosis: Pathophysiological and diagnostic aspects

Rare genetic iron overload diseases are an evolving field due to major advances in genetics and molecular biology. Genetic iron overload has long been confined to the classical type 1 hemochromatosis related to the HFE C282Y mutation. Breakthroughs in the understanding of iron metabolism biology and molecular mechanisms led to the discovery of new genes and subsequently, new types of hemochromatosis. To date, four types of hemochromatosis have been identified: HFE-related or type1 hemochromatosis, the most frequent form in Caucasians, and four rare types, named type 2 (A and B) hemochromatosis (juvenile hemochromatosis due to hemojuvelin and hepcidin mutation), type 3 hemochromatosis (related to transferrin receptor 2 mutation), and type 4 (A and B) hemochromatosis (ferroportin disease). The diagnosis relies on the comprehension of the involved physiological defect that can now be explored by biological and imaging tools, which allow non-invasive assessment of iron metabolism. A multidisciplinary approach is essential to support the physicians in the diagnosis and management of those rare diseases.

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)

Rare HFE variants are the most frequent cause of hemochromatosis in non‐c282y homozygous patients with hemochromatosis

p.Cys282Tyr (C282Y) homozygosity explains most cases of HFE‐related hemochromatosis, but a significant number of patients presenting with typical type I hemochromatosis phenotype remain unexplained. We sought to describe the clinical relevance of rare HFE variants in non‐C282Y homozygotes. Patients referred for hemochromatosis to the National Reference Centre for Rare Iron Overload Diseases from 2004 to 2010 were studied. Sequencing was performed for coding region and intronic flanking sequences of HFE, HAMP, HFE2, TFR2, and SLC40A1. Nine private HFE variants were identified in 13 of 206 unrelated patients. Among those, five have not been previously described: p.Leu270Argfs*4, p.Ala271Valfs*25, p.Tyr52*, p.Lys166Asn, and p.Asp141Tyr. Our results show that rare HFE variants are identified more frequently than variants in the other genes associated with iron overload. Rare HFE variants are therefore the most frequent cause of hemochromatosis in non‐C282Y homozygote HFE patients. Am. J. Hematol. 91:1202–1205, 2016.

MELD-based graft allocation system fails to improve liver transplantation efficacy in a single-center intent-to-treat analysis

BACKGROUND Since March 2007, priority access to liver transplantation in France has been given to patients with the highest MELD scores. OBJECTIVE To undertake an intent-to-treat comparison of center-based vs. MELD-based liver graft allocation. METHODS Retrospective cohort analysis (patients listed 6th March 2007 to 5th March 2009; MELD period) with a matched historical cohort (patients listed 6th March 2005 to 5th March 2007; pre-MELD period) in a single high-volume center. Analysis was on an intent-to-treat basis, i.e. starting on the day of wait listing. RESULTS Compared to pre-MELD, fewer patients with a MELD score less or equal to 14 (P=0.002), and more patients with a MELD greater or equal to 24 (P<0.05) were transplanted during the MELD period. For HCC candidates, median waiting time increased (121 vs. 54 days, P=0.01), transplantation rate halved (35% vs. 73.5%, P<0.001) and dropouts due to tumor progression increased (16% vs. 0%, P<0.001). Moreover, postoperative course did not change significantly except for infectious complications (35% vs. 24%, P=0.02); overall patient survival was 69.8 ± 3.1% vs. 76 ± 2.9% (P=0.29) and overall graft survival was 77.6 ± 3.4% vs. 82.8 ± 2.9% (P=0.29). Transplant failures were mainly due to deaths on the waiting list in the previous system, but to dropouts related to disease progression in the new system. Cirrhotic patient survival rate did not change (78.1 ± 4.4% vs. 73.5 ± 4.5%, P=0.42), while that of HCC patients decreased (65.3 ± 5.3% vs. 86.8 ± 4.4%, P=0.01). Post-transplant survival worsened significantly according to pre-transplant MELD score (P=0.009). CONCLUSION The MELD-based graft allocation system introduced discrimination against HCC patients, whose incidence has increased dramatically, and should be reevaluated.

Portable hemoglobinometer is a reliable technology for the follow-up of venesections tolerance in hemochromatosis

BACKGROUND The treatment of HFE-related hemochromatosis, one of the most common genetic diseases, is based on phlebotomies whose tolerance is evaluated by regular monitoring of hemoglobin. Using a portable hemoglobinometer (PH) could provide an easy and fast determination of hemoglobin. Therefore, the aim of the present study was to compare, in hemochromatosis patients treated by bloodletting, the hemoglobin concentrations as assayed, on capillary blood, by a PH device and, on venous blood, by a cell counter (CC) device. METHODS For a total period of 12 weeks duration, all patients undergoing phlebotomies in the same hospital outpatient unit had hemoglobin determinations both by the HemoCue and by the laboratorys DxH 800 Coulter. To evaluate the sensitivity and specificity of the HemoCue, patients were classified as presenting or not anemia as defined by hemoglobin level below 11 g/dl. RESULTS Measurements of hemoglobin were performed in 122 patients. The sensitivity and specificity of PH compared to CC were 100 and 98.1%, respectively. Capillary hemoglobin by PH slightly underestimated venous hemoglobin by CC. The Pearsons correlation coefficient between PH and CC was 0.80 (P<0.0001). CONCLUSION PH is a reliable, quick and easy technology, which can be proposed to follow-up the tolerance of venesections in hemochromatosis patients.

Métabolisme du fer en 2012

Resume Le fer intervient dans de nombreuses fonctions biologiques et est donc necessaire. Cependant, en exces, il est toxique. Le maintien d’un niveau adequat de fer ainsi que sa bonne repartition dans l’organisme et dans les cellules sont possibles grâce a des mecanismes de regulation systemiques et cellulaires. Le controle systemique du metabolisme du fer fait intervenir l’hepcidine, peptide secrete par l’hepatocyte, et la ferroportine, proteine membranaire exportant le fer des macrophages et enterocytes vers le plasma. L’hepcidine interagit avec la ferroportine et provoque sa degradation, controlant ainsi le niveau de fer plasmatique. Des niveaux anormaux d’hepcidine sont impliques dans l’apparition de surcharges ou de carences en fer. La comprehension des mecanismes controlant les niveaux d’hepcidine est donc un enjeu majeur. Le controle cellulaire du fer fait intervenir le systeme iron regulatory protein/iron responsive element qui permet d’adapter l’entree du fer lie a la transferrine dans la cellule et la capacite de stockage cellulaire du fer au niveau de fer intracellulaire, permettant ainsi d’eviter l’apparition de situations deleteres pour la cellule. Les nouvelles connaissances acquises sur le metabolisme du fer permettent de mieux comprendre les mecanismes en cause dans les pathologies du metabolisme du fer. De nouvelles strategies diagnostiques et therapeutiques, issues de ces connaissances, permettront d’ameliorer la prise en charge des patients.

Chronic hepatic cytolysis revealing a pheochromocytoma

We report here the first case of chronic cytolysis that led to the diagnosis of pheochromocytoma, in a 48-year-old woman with a recent onset of hypertension. The etiological research ruled out the common causes of raised transaminase levels, and led to the discovery of a left adrenal pheochromocytoma. The sustained normalization of liver function tests after the removal of the tumour strongly suggests that hepatocyte injury was due to catecholamine hyperproduction. The present original clinical case, linking pheochromocytoma and liver dysfunction, raises important mechanistic questions concerning the relationship between catecholamines and liver function. It may also have clinical implications. Indeed, pheochromocytoma should be considered as a possible cause in case of unexplained transaminase increase associated with the recent onset of hypertension.