HFE-Related Hemochromatosis: The Haptoglobin 2-2 Type Has a Significant but Limited Influence on Phenotypic Expression of the Predominant p.C282Y Homozygous Genotype

Phenotypic expression of the common p.C282Y/p.C282Y HFE-related hemochromatosis genotype is heterogeneous and depends on a complex interplay of genetic and non-genetic factors. Haptoglobin has a crucial role in free hemoglobin iron recovery, and exists as three major types: Hp1-1, Hp2-1 and Hp2-2. Hp2-2 favors endocytosis of hemoglobin iron in monocytes/macrophages, resulting in partial iron retention and increased intracellular ferritin levels. This situation is generally not expected to severely affect iron homeostasis, but was found to correlate with elevated serum iron indices in healthy men. Whether the Hp2-2 genotype acts as a modifier in HFE-related hemochromatosis is unclear. In this study we investigated influence of Hp2-2 and of potential confounders on the iron indices of 351 p.C282Y homozygous patients. We conclude that there is a cause-and-effect relationship between the Hp2-2 genotype and increased iron indices in p.C282Y homozygous patients. The Hp2-2 effect is, however, limited and only apparent in males

Impact of HFE genetic testing on clinical presentation of hereditary hemochromatosis: new epidemiological data

BACKGROUND: Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism in Northern European populations. The discovery of a candidate gene in 1996 (HFE), and of its main mutation (C282Y), has radically altered the way to diagnose this disease. The aim of this study was to assess the impact of the HFE gene discovery on the clinical presentation and epidemiology of HH. METHODS: We studied our cohort of 415 patients homozygous for the C282Y allele and included in a phlebotomy program in a blood centre in western Brittany, France. RESULTS: In this cohort, 56.9% of the patients were male and 21.9% began their phlebotomy program before the implementation of the genetic test. A significant decrease in the sex ratio was noticed following implementation of this DNA test, from 3.79 to 1.03 (p < 10(-5)), meaning that the proportion of diagnosed females relatives to males greatly increased. The profile of HH patients at diagnosis changed after the DNA test became available. Serum ferritin and iron values were lower and there was a reduced frequency of clinical signs displayed at diagnosis, particularly skin pigmentation (20.1 vs. 40.4%, OR = 0.37, p < 0.001) and hepatomegaly (11.0 vs. 22.7%, OR = 0.42, p = 0.006). In contrast, fatigue became a more common symptom at diagnosis (68.0 vs. 51.2%, OR = 2.03, p = 0.004). CONCLUSION: This study highlights the importance of the HFE gene discovery, which has simplified the diagnosis of HH and modified its clinical presentation and epidemiology. This study precisely measures these changes. Enhanced diagnosis of HFE-related HH at an early stage and implementation of phlebotomy treatment are anticipated to maintain normal life expectancy for these patients

A novel hypomorphic splice variant in EIF2B5 gene is associated with mild ovarioleukodystrophy

Objective: To identify the genetic cause in an adult ovarioleukodystrophy patient resistant to diagnosis. Methods: We applied whole-exome sequencing (WES) to a vanishing white matter disease patient associated with premature ovarian failure at 26 years of age. We functionally tested an intronic variant by RT-PCR on patient's peripheral blood mononuclear cells (PBMC) and by minigene splicing assay. Results: WES analysis identified two novel variants in the EIF2B5 gene: c.725A > G (p.Tyr242Cys) and an intronic noncanonical mutation (c.1156 + 13G>A). This intronic mutation resulted into generation of various isoforms both in patient's PBMC and in the minigene splicing assay, showing that ~20% residual wild-type isoform is still expressed by the intronic-mutated allele alone, concordant with an hypomorphic effect of this variant. Conclusion: We report two novel variants in EIF2B5, one of them a noncanonical intronic splice variant, located at a +13 intronic position. This position is mutated only in 0.05% of ClinVar intronic mutations described so far. Furthermore, we illustrate how minigene splicing assay may be advantageous when validating splice-altering variants, in this case highlighting the coexistence of wild-type and mutated forms, probably explaining this patient's milder, late-onset phenotype

Insights into the Role of the Discontinuous TM7 Helix of Human Ferroportin through the Prism of the Asp325 Residue

International audienceThe negatively charged Asp325 residue has proved to be essential for iron export by human (HsFPN1) and primate Philippine tarsier (TsFpn) ferroportin, but its exact role during the iron transport cycle is still to be elucidated. It has been posited as being functionally equivalent to the metal ion-coordinating residue His261 in the C-lobe of the bacterial homolog BbFpn, but the two residues arise in different sequence motifs of the discontinuous TM7 transmembrane helix. Furthermore, BbFpn is not subject to extracellular regulation, contrary to its mammalian orthologues which are downregulated by hepcidin. To get further insight into the molecular mechanisms related to iron export in mammals in which Asp325 is involved, we investigated the behavior of the Asp325Ala, Asp325His, and Asp325Asn mutants in transiently transfected HEK293T cells, and performed a comparative structural analysis. Our biochemical studies clearly distinguished between the Asp325Ala and Asp325His mutants, which result in a dramatic decrease in plasma membrane expression of FPN1, and the Asp325Asn mutant, which alters iron egress without affecting protein localization. Analysis of the 3D structures of HsFPN1 and TsFpn in the outward-facing (OF) state indicated that Asp325 does not interact directly with metal ions but is involved in the modulation of Cys326 metal-binding capacity. Moreover, models of the architecture of mammalian proteins in the inward-facing (IF) state suggested that Asp325 may form an inter-lobe salt-bridge with Arg40 (TM1) when not interacting with Cys326. These findings allow to suggest that Asp325 may be important for fine-tuning iron recognition in the C-lobe, as well as for local structural changes during the IF-to-OF transition at the extracellular gate level. Inability to form a salt-bridge between TM1 and TM7b during iron translocation could lead to protein instability, as shown by the Asp325Ala and Asp325His mutants

Analysis of long-range interactions in primary human cells identifies cooperative CFTR regulatory elements

International audienceA mechanism by which control DNA elements regulate transcription over large linear genomic distances is by achieving close physical proximity with genes, and looping of the intervening chromatin paths. Alterations of such regulatory 'chromatin looping' systems are likely to play a critical role in human genetic disease at large. Here, we studied the spatial organization of a ≈790 kb locus encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysregulation of CFTR is responsible for cystic fibrosis, which is the most common lethal genetic disorder in Caucasian populations. CFTR is a relatively large gene of 189 kb with a rather complex tissue-specific and temporal expression profile. We used chromatin conformation at the CFTR locus to identify new DNA sequences that regulate its transcription. By comparing 5C chromatin interaction maps of the CFTR locus in expressing and non-expressing human primary cells, we identified several new contact points between the CFTR promoter and its surroundings, in addition to regions featuring previously described regulatory elements. We demonstrate that two of these novel interacting regions cooperatively increase CFTR expression, and suggest that the new enhancer elements located on either side of the gene are brought together through chromatin looping via CTCF

Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease

International audienceAbstract SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on protein stability and its conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in membrane bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366, and Arg371) that are located at the membrane‐cytosol interface and consistently interact with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE) molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward‐facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid polar heads. This results in a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt‐bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease

Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A

International audienceFerroportin 1 (FPN1) is a major facilitator superfamily transporter that is essential for proper maintenance of human iron homeostasis at the systemic and cellular level. FPN1 dysfunction leads to the progressive accumulation of iron in reticuloendothelial cells, causing hemochromatosis type 4A (or ferroportin disease), an autosomal dominant disorder that displays large phenotypic heterogeneity. Although crystal structures have unveiled the outward- and inward-facing conformations of the bacterial homolog Bdellovibrio bacteriovorus Fpn (or Bd2019) and calcium has recently been identified as an essential cofactor, our molecular understanding of the iron transport mechanism remains incomplete. Here, we used a combination of molecular modeling, molecular dynamics simulations, and Ala site-directed mutagenesis, followed by complementary in vitro functional analyses, to explore the structural architecture of the human FPN1 intracellular gate. We reveal an interdomain network that involves 5 key amino acids and is likely very important for stability of the iron exporter facing the extracellular milieu. We also identify inter- and intradomain interactions that rely on the 2 Asp84 and Asn174 critical residues and do not exist in the bacterial homolog. These interactions are thought to play an important role in the modulation of conformational changes during the transport cycle. We interpret these results in the context of hemochromatosis type 4A, reinforcing the idea that different categories of loss-of-function mutations exist. Our findings provide an unprecedented view of the human FPN1 outward-facing structure and the particular function of the so-called "gating residues" in the mechanism of iron export.-Guellec, J., Elbahnsi, A., Le Tertre, M., Uguen, K., Gourlaouen, I., Férec, C., Ka, C., Callebaut, I., Le Gac, G. Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A

The Spectra of Disease-Causing Mutations in the Ferroportin 1 (SLC40A1) Encoding Gene and Related Iron Overload Phenotypes (Hemochromatosis Type 4 and Ferroportin Disease)

International audienceSLC40A1 is the sole iron export protein reported in mammals and is a key player in both cellular and systemic iron homeostasis. This unique iron exporter, which belongs to the major facilitator superfamily, is predominantly regulated by the hyposideremic hormone hepcidin. SLC40A1 dysfunction causes ferroportin disease, and autosomal dominant iron overload disorder characterized by cellular iron retention, principally in reticuloendothelial cells, correlating with high serum ferritin and low to normal transferrin saturation. Resistant to hepcidin, SLC40A1 mutations are rather associated with elevated plasma iron and parenchymal iron deposition, a condition that resembles HFE-related hemochromatosis and is associated with more clinical complications. With very few exceptions, only missense variations are reported at the SLC40A1 locus; this situation increasingly limits the establishment of pathogenicity. In this mutation update, we provide a comprehensive review of all the pathogenic or likely pathogenic variants, variants of unknown significance, and benign or likely benign SLC40A1 variants. The classification is essentially determined using functional, structural, segregation, and recurrence data. We furnish new information on genotype-phenotype correlations for loss-of-function, gain-of-function, and other SLC40A1 variants, confirming the existence of wide clinical heterogeneity and the potential for misdiagnosis. All information is recorded in a locus-specific online database

The dual loss and gain of function of the FPN1 iron exporter results in the ferroportin disease phenotype

International audienc