SLC40A1 Mutations Research Articles

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

Heterozygous mutations in SLC40A1, encoding a multi-pass membrane protein of the major facilitator superfamily known as ferroportin 1 (FPN1), are responsible for two distinct hereditary iron-overload diseases: ferroportin disease, which is associated with reduced FPN1 activity (i.e., decrease in cellular iron export), and SLC40A1-related hemochromatosis, which is associated with abnormally high FPN1 activity (i.e., resistance to hepcidin). Here, we report three SLC40A1 missense variants with opposite functional consequences. In cultured cells, the p.Arg40Gln and p.Ser47Phe substitutions partially reduced the ability of FPN1 to export iron and also partially reduced its sensitivity to hepcidin. The p.Ala350Val substitution had more profound effects, resulting in low FPN1 iron egress and weak FPN1/hepcidin interaction. Structural analyses helped to differentiate the first two substitutions, which are predicted to cause local instabilities, and the third, which is thought to prevent critical rigid-body movements that are essential to the iron transport cycle. The phenotypic traits observed in a total of 12 affected individuals are highly suggestive of ferroportin disease. Our findings dismantle the classical dualism of FPN1 loss versus gain of function, highlight some specific and unexpected functions of FPN1 transmembrane helices in the molecular mechanism of iron export and its regulation by hepcidin, and extend the spectrum of rare genetic variants that may cause ferroportin disease.

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

SLC40A1 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.

Identification of Novel Mutations by Targeted NGS Panel in Patients with Hyperferritinemia.

Background. Several inherited diseases cause hyperferritinemia with or without iron overload. Differential diagnosis is complex and requires an extensive work-up. Currently, a clinical-guided approach to genetic tests is performed based on gene-by-gene sequencing. Although reasonable, this approach is expensive and time-consuming and Next Generation Sequencing (NGS) technology may provide cheaper and quicker large-scale DNA sequencing. Methods. We analysed 36 patients with non-HFE-related hyperferritinemia. Liver iron concentration was measured in 33 by magnetic resonance. A panel of 25 iron related genes was designed using SureDesign software. Custom libraries were generated and then sequenced using Ion Torrent PGM. Results. We identified six novel mutations in SLC40A1, three novel and one known mutation in TFR2, one known mutation and a de-novo deletion in HJV, and a novel mutation in HAMP in ten patients. In silico analyses supported the pathogenic role of the mutations. Conclusions. Our results support the use of an NGS-based panel in selected patients with hyperferritinemia in a tertiary center for iron metabolism disorders. However, 26 out of 36 patients did not show genetic variants that can individually explain hyperferritinemia and/or iron overload suggesting the existence of other genetic defects or gene-gene and gene-environment interactions needing further studies.

Analysis of HFE and Non-HFE Mutations in a Tibet Cohort with Iron Overload

The explore the molecular basis of iron-overload in Tibet nationality population of Tibet. The inpatients with iron-overload in our department from Dec. 1st 2014 to Jul.31st 2016 were enrolled in this study. Abdominal MRI and the mutation sites C282Y and H63D in HFE exon were examined. For HFE mutation-negative patients, the non-HFE mutation was detected, including 5 HJV mutations of G320V, p.Q312X, p.D249H, p.I281T, p.C321X and 2 TFR2 mutations: (Y250X, I238M), and 2 SLC40A1 mutations: (V162del, N144H). Among 113 iron overload patients, only one showed homozygous p.H63D mutation, and one showed heterozygosis p.H63D mutation. In 73 patients accepted non-HFE gene detection, only one was heterozygosis p.D249N mutation in HJV, and one was heterozygosis p.I238M mutation in TFR2. Currently, the pathogenic gene for Tibetan iron-overload has not yet been found.

Inherited Disorders of Iron Overload.

Dietary iron absorption and systemic iron traffic are tightly controlled by hepcidin, a liver-derived peptide hormone. Hepcidin inhibits iron entry into plasma by binding to and inactivating the iron exporter ferroportin in target cells, such as duodenal enterocytes and tissue macrophages. Hepcidin is induced in response to increased body iron stores to inhibit further iron absorption and prevent iron overload. The mechanism involves the BMP/SMAD signaling pathway, which triggers transcriptional hepcidin induction. Inactivating mutations in components of this pathway cause hepcidin deficiency, which allows inappropriately increased iron absorption and efflux into the bloodstream. This leads to hereditary hemochromatosis (HH), a genetically heterogenous autosomal recessive disorder of iron metabolism characterized by gradual buildup of unshielded non-transferrin bound iron (NTBI) in plasma and excessive iron deposition in tissue parenchymal cells. The predominant HH form is linked to mutations in the HFE gene and constitutes the most frequent genetic disorder in Caucasians. Other, more severe and rare variants are caused by inactivating mutations in HJV (hemojuvelin), HAMP (hepcidin) or TFR2 (transferrin receptor 2). Mutations in SLC40A1 (ferroportin) that cause hepcidin resistance recapitulate the biochemical phenotype of HH. However, ferroportin-related hemochromatosis is transmitted in an autosomal dominant manner. Loss-of-function ferroportin mutations lead to ferroportin disease, characterized by iron overload in macrophages and low transferrin saturation. Aceruloplasminemia and atransferrinemia are further inherited disorders of iron overload caused by deficiency in ceruloplasmin or transferrin, the plasma ferroxidase and iron carrier, respectively.

The SLC40A1 R178Q mutation is a recurrent cause of hemochromatosis and is associated with a novel pathogenic mechanism.

Hemochromatosis type 4 is one of the most common causes of primary iron overload, after HFE-related hemochromatosis. It is an autosomal dominant disorder, primarily due to missense mutations in SLC40A1. This gene encodes ferroportin 1 (FPN1), which is the sole iron export protein reported in mammals. Not all heterozygous missense mutations in SLC40A1 are disease-causing. Due to phenocopies and an increased demand for genetic testing, rare SLC40A1 variations are fortuitously observed in patients with a secondary cause of hyperferritinemia. Structure/function analysis is the most effective way of establishing causality when clinical and segregation data are lacking. It can also provide important insights into the mechanism of iron egress and FPN1 regulation by hepcidin. The present study aimed to determine the pathogenicity of the previously reported p.Arg178Gln variant. We present the biological, clinical, histological and radiological findings of 22 patients from six independent families of French, Belgian or Iraqi decent. Despite phenotypic variability, all patients with p.Arg178Gln had elevated serum ferritin concentrations and normal to low transferrin saturation levels. In vitro experiments demonstrated that the p.Arg178Gln mutant reduces the ability of FPN1 to export iron without causing protein mislocalization. Based on a comparative model of the 3D structure of human FPN1 in an outward facing conformation, we argue that p.Arg178 is part of an interaction network modulating the conformational changes required for iron transport. We conclude that p.Arg178Gln represents a new category of loss-of-function mutations and that the study of “gating residues” is necessary in order to fully understand the action mechanism of FPN1.

Haemochromatosis.

Haemochromatosis is defined as systemic iron overload of genetic origin, caused by a reduction in the concentration of the iron regulatory hormone hepcidin, or a reduction in hepcidin-ferroportin binding. Hepcidin regulates the activity of ferroportin, which is the only identified cellular iron exporter. The most common form of haemochromatosis is due to homozygous mutations (specifically, the C282Y mutation) in HFE, which encodes hereditary haemochromatosis protein. Non-HFE forms of haemochromatosis due to mutations in HAMP, HJV or TFR2 are much rarer. Mutations in SLC40A1 (also known as FPN1; encoding ferroportin) that prevent hepcidin-ferroportin binding also cause haemochromatosis. Cellular iron excess in HFE and non-HFE forms of haemochromatosis is caused by increased concentrations of plasma iron, which can lead to the accumulation of iron in parenchymal cells, particularly hepatocytes, pancreatic cells and cardiomyocytes. Diagnosis is noninvasive and includes clinical examination, assessment of plasma iron parameters, imaging and genetic testing. The mainstay therapy is phlebotomy, although iron chelation can be used in some patients. Hepcidin supplementation might be an innovative future approach.

Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as “ferroportin disease”, which is due to “loss of function” mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation.In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.

Identification of a Novel SLC40A1 Arg88Ile Mutation in a Patient with Familial Iron Overload Treated By Phlebotomy

INTRODUCTIONThe cellular iron exporter ferroportin, encoded by the SLC40A1 gene, plays a key role in systemic iron regulation by mediating the absorption of dietary iron from duodenal enterocytes and the release of macrophage iron stores into the plasma. SLC40A1 mutations result in a clinically heterogeneous iron overload disorder exhibiting autosomal dominant transmission. Mutations that impair iron export function result in a classical ferroportin disease phenotype characterized by hyperferritinemia, normal transferrin saturation, and macrophage iron loading, while mutations that impair the regulation of ferroportin by hepcidin result in a non-classical form of disease exhibiting high transferrin saturation and hepatocellular iron loading. Here we report the clinical phenotype of a male patient with a personal and family history of iron overload who was found to harbor a novel SLC40A1 mutation. CLINICAL HISTORYA 39-year-old male of Italian descent came to clinical attention after laboratory evidence of iron overload was detected at the time of a routine physical exam. Serum ferritin was markedly elevated at 5018 ng/mL, while transferrin iron saturation was within the normal range at 42%. A complete blood count revealed hemoglobin 16.1 g/dL, hematocrit 48.7%, and MCV 96.7 fL. Liver function tests revealed mild elevation of transaminases (AST 50 U/L, ALT 114 U/L). HBV and HCV serologies, as well as an anti-nuclear antibody screen, were negative. Rheumatoid factor, ceruloplasmin, and alpha-fetoprotein were within the normal range. Genetic testing for the HFE C282Y, H63D, and S65C variants was negative. Abdominal ultrasound revealed a somewhat course and echogenic liver. The patient's past medical and surgical histories were non-contributory. Family history was notable for a 63-year-old father with non-HFE hemochromatosis treated by phlebotomy, as well as a possible history of iron overload in the paternal grandmother. There was no known family history of hepatocellular carcinoma. The patient reported consuming ≤ 5 alcoholic beverages per week. Review of systems was negative, and no organomegaly was detected on physical exam. The patient began undergoing phlebotomy approximately every 2 weeks, which now has been well tolerated for almost two years. His most recent ferritin level was within the normal range (328 ng/mL). METHODSUsing genomic DNA extracted from peripheral blood as template, all coding regions and intron-exon boundaries of SLC40A1 were amplified by polymerase chain reaction and analyzed by bidirectional Sanger sequencing. Sequence chromatograms were analyzed using Sequencher software. This study was approved by the Yale University Human Investigation Committee (protocol #010412377). RESULTSA heterozygous single nucleotide substitution (c.263G>T, encoding p.Arg88Ile) was identified in exon 3 of the SLC40A1 gene (nomenclature per Ensembl reference transcript ENST00000261024). Ferroportin is a predicted multipass transmembrane protein, and Arg88 resides between two predicted transmembrane domains. Protein sequence alignments reveal that amino acid 88 is conserved as an arginine in ferroportin homologs in species as evolutionarily distant as Xenopus laevis and Danio rerio, suggesting that this residue is required for normal ferroportin function. The p.Arg88Ile variant has not been reported in the 1000 Genomes Project or the Exome Aggregation Consortium, demonstrating that it is not a polymorphism in the general population. The mutation predictor algorithms PolyPhen2, SIFT, and MutationTaster all strongly predicted this mutation to be damaging. DISCUSSIONThe ferroportin variant detected in this case (p.Arg88Ile) represents the third non-synonymous substitution at ferroportin residue 88 detected in patients with iron overload phenotypes. p.Arg88Gly was identified in a 38-year-old male who became anemic under a phlebotomy program (Cunat S., et al., Clin Chem 2007), while p.Arg88Thr was detected in multiple affected members of a single kindred in whom serial phlebotomy was well tolerated (Bach V, et al. Blood Cells Mol Dis 2006). Collectively, these findings suggest that the particular amino acid substitution at residue 88 may influence the degree of cellular iron sequestration. Future work will assess the effect of the Arg88Ile substitution on ferroportin function. DisclosuresNo relevant conflicts of interest to declare.

The global prevalence of HFE and non-HFE hemochromatosis estimated from analysis of next-generation sequencing data

The prevalence of HFE-related hereditary hemochromatosis (HH) among European populations has been well studied. There are no prevalence data for atypical forms of HH caused by mutations in HFE2, HAMP, TFR2, or SLC40A1. The purpose of this study was to estimate the population prevalence of these non-HFE forms of HH. A list of HH pathogenic variants in publically available next-generation sequence (NGS) databases was compiled and allele frequencies were determined. Of 161 variants previously associated with HH, 43 were represented among the NGS data sets; an additional 40 unreported functional variants also were identified. The predicted prevalence of HFE HH and the p.Cys282Tyr mutation closely matched previous estimates from similar populations. Of the non-HFE forms of iron overload, TFR2-, HFE2-, and HAMP-related forms are predicted to be rare, with pathogenic allele frequencies in the range of 0.00007 to 0.0005. Significantly, SLC40A1 variants that have been previously associated with autosomal-dominant ferroportin disease were identified in several populations (pathogenic allele frequency 0.0004), being most prevalent among Africans. We have, for the first time, estimated the population prevalence of non-HFE HH. This methodology could be applied to estimate the population prevalence of a wide variety of genetic disorders.Genet Med 18 6, 618-626.

Clinical Testing of Five Hereditary Hemochromatosis-Related Genes: Preliminary Evidence for the Benefit of Next Generation Sequencing

▪IntroductionHereditary hemochromatosis (HH) is a genetic form of iron overload. In cases of excessive iron deposition, serious clinical manifestations may occur, such as liver damage, cardiomyopathy, diabetes, and arthritis. First described in 1996, the HFE gene leads to autosomal recessive HH with reduced penetrance. In other words, two mutations in the HFE gene need to be present in a patient in order to develop symptoms of HFE-related HH, but not all patients with two mutations are affected. In the last 15 years, 4 additional genes were discovered that cause HH: HAMP (hepcidin), HFE2 (hemojuvelin), SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2). HAMP, HFE2 and TFR2 mutations are inherited in a recessive pattern, whereas SLC40A1 mutations are inherited in a dominant pattern. HAMP and HFE2 mutations cause a severe, early-onset form of HH. There is some evidence that sequence changes in HAMP, HFE2 and TFR2 may interact with homozygous HFE mutations, causing a more severe phenotype. Current HH testing guidelines only exist for the most common HFE mutations (C282Y and H63D), with no specific recommendations regarding full gene sequencing for any of the HH genes. Recent research suggests that sequential sequencing may be beneficial in patients who test negative for the most common HFE mutations, exhibit a more severe or early-onset phenotype compared to what is normally seen in HFE-related HH, and/or are of non-Caucasian ethnicity. Next Generation Sequencing (NGS) is a new high-throughput sequencing technology that allows testing of multiple genes concurrently and can detect rare and novel HH-causing mutations that are not typically assayed using targeted methods. However, sequencing can also identify sequence changes known as variants of uncertain significance (VUS) - changes that have not yet been characterized as disease-causing or benign. This abstract summarizes the results of clinical NGS for the five HH-related genes, and shows preliminary evidence as to its’ increased diagnostic yield for HH diagnosis.MethodsPatients were referred for clinical full gene sequencing of HFE, HAMP, HFE2, SLC40A1, and/or TFR2 using Next Generation Sequencing (Illumina MiSeq). Results from patients with a clinical indication of iron overload or HH who were tested from 9/2013 - 7/2014 were reviewed. The diagnostic yield of sequencing for all five HH genes was determined. Patients who only had sequencing for a subset of the five genes were analyzed separately. Patients who had testing for a familial mutation were excluded from the review.ResultsIn total, 56 patients underwent HH-related NGS. Thirty-five (62.5%) were males and 21 (37.5%) were females. Ages ranged from 3-77yrs (avg. 40.9yrs). Fifty-one percent were Caucasian, 9% Hispanic, 4% African American, 16% Asian, and 20% not specified.Forty-one patients were tested for all five genes. HH-causing mutations were found in 9 patients: 5 (12.2%) were c.187C>G (p.H63D) HFE homozygous, 1 (2.4%) was c.845G>A (p.C282Y) HFE homozygous, and 3 (7.3%) had mutations in non-HFE genes: SLC40A1 c.430A>T (p.N144Y) heterozygous, SLC40A1 c.533G>A (p.R178Q) heterozygous, and HFE2 c.959G>T (p.R178Q) homozygous. Nine patients (22%) were heterozygous carriers of an HFE mutation. One or more VUSes were found in 5 patients (12.2%). In 18 patients (43.9%), no pathogenic mutations or VUSes were found. There were 15 additional patients who only had sequencing of 1-3 of the available genes. Results for those patients consisted of 1 H63D HFE homozygote, 3 HFE heterozygotes (2 H63D and 1 C282Y) and 1 VUS.ConclusionsThe new sequencing technology of NGS makes it possible to test multiple genes at the same time. In this sample, sequencing of HFE, HAMP, HFE2, SLC40A1, and TFR2 genes resulted in an additional diagnostic yield compared to HFE C282Y and H63D testing alone. In patients who have a genetic explanation for their HH, management can be personalized based on genotype-phenotype correlation (e.g. N144Y SLC40A1 mutations may lead to reduced phlebotomy tolerance) and at-risk family members can be screened. In addition, all patients in this sample with non-HFE positive results were reportedly Caucasian, highlighting the benefit of sequencing regardless of ethnic background. This preliminary study is an important step toward gaining a better understanding of the genetics of HH. Ultimately, NGS data may make it possible to update current clinical guidelines for HH. DisclosuresRabideau:Invitae: Employment, Equity Ownership.

Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.

Control of iron metabolism – Lessons from neonatal hemochromatosis

When hepatologists begin their investigations at the scene of liver damage, iron is certainly on the list of ‘‘usual suspects’’ to be rounded up. Hemochromatosis, a well-known cause of chronic liver disease, is the most prevalent genetic disorder in adults [1]. Serum iron parameters are therefore included in most guidelines on how to investigate patients with elevated transaminases or chronic liver disease [2]. The hemochromatosis (HFE) genotype is an important piece of evidence in patients with liver disease and elevated transferrin saturation with hyperferritinemia, because homozygosity for the C282Y allele of HFE is considered sufficient to diagnose hemochromatosis under these circumstances [1]. Compound heterozygosity for C282Y and H63D has been almost removed from the watch-list and is currently considered a mere risk factor for liver diseases – sufficient to cause hemochromatosis or even cirrhosis only in combination with other health burdens. Hence, a partner in crime for HFE mutation must be found in patients with liver disease and C282Y/H63D compound heterozygosity [3]. If there is evidence for iron overload in the absence of hemochromatosis-associated HFE genotypes and the hepatopathologist finds iron confined to hepatocytes, sharp investigators are well advised to search for the culprit in the genes encoding transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) or even to hunt for combined mutations in these genes if a patient presents with severe iron overload or early-onset disease [4]. If the patient is adolescent or before the 3rd decade of life and presents with heart failure or hypogonadism in addition to – often overlooked – liver disease, other genes will be committed in custody. Mutations in HJV and HAMP, which respectively encode hemojuvelin and hepcidin, will be sufficient to identify juvenile hemochromatosis [5]. Through the power of genetics, iron-related diseases are now subject to close scrutiny. In addition, genetic studies have greatly advanced our understanding of hemochromatosis. This progress is reflected in the proposed re-classification of human iron

Polymorphisms in the Gene That Encodes the Iron Transport Protein Ferroportin 1 Influence Susceptibility to Tuberculosis

We studied the association between iron intake and polymorphisms in the iron transporter gene SLC40A1 and the risk of tuberculosis. We compared iron intake, the frequency of SLC40A1 mutations, and interactions among these variables among 98 tuberculosis patients and 125 controls in Kwazulu-Natal, South Africa. Four SLC40A1 single-nucleotide polymorphisms (SNPs) were associated with an increased risk of tuberculosis and 1 SNP with reduced risk. We also found a gene-environment interaction for 4 nonexonic SNPs and iron intake. This pilot study demonstrated an association between polymorphisms in SLC40A1 and tuberculosis and provided evidence of an interaction between dietary iron and SLC40A1.

Sideroblastic anemia, iron overload, andALAS2R452S in African-American males: Phenotype and genotype features of five unrelated patients

Sideroblastic anemia, iron overload, and<i>ALAS2</i>R452S in African-American males: Phenotype and genotype features of five unrelated patients

Identification of Mutations in SLC40A1 That Affect Ferroportin Function and Phenotype of Human Ferroportin Iron Overload

Patients with ferroportin iron overload due to loss-of-function mutations in SLC40A1 have macrophage iron overload, hyperferritinemia, and normal transferrin saturation. In contrast, hepatocellular iron storage, hyperferritinemia, and increased saturation of transferrin are a distinct clinical presentation of ferroportin iron overload that results from SLC40A1 mutations that confer resistance of ferroportin to hepcidin-mediated inactivation. SLC40A1 was sequenced in patients from 2 independent pedigrees affected by hepatic iron overload unrelated to HFE. Functions of the ferroportin variants were tested in vitro. A patient heterozygous for the variant p.W158C in SLC40A1 presented with macrophage iron overload, hyperferritinemia, and normal transferrin saturation. A patient with hepatocellular iron storage, hyperferritinemia, and increased transferrin saturation was heterozygous for p.H507R. Expression of the p.W158C form of ferroportin in 293T cells resulted in defective trafficking to the plasma membrane and reduced iron export activity; the iron export activity of cells that expressed the p.H507R form of ferroportin did not differ from cells that expressed ferroportin without this mutation. The p.H507R of ferroportin localizes normally to the plasma membrane but is resistant to hepcidin-mediated inactivation. Addition of a synthetic peptide derived from ferroportin without these mutations (amino acids 500-518) decreased the inhibitory activity of hepcidin in cells, whereas a peptide from the same region, with p.H507R, had no effect on hepcidin activity. The variant p.W158C in SLC40A1 impairs intracellular trafficking of ferroportin, resulting in reduced iron export. The variant p.H507R does not bind hepcidin in vitro and results in apparent hepcidin resistance.

Global Sequencing for the Molecular Background of Hereditary Hemochomatosis In Brazilian Patients

AbstractAbstract 5146 INTRODUTION:Most hereditary hemochromatosis (HH) patients are homozygous for the p.C282Y mutation in the HFE gene. But rare HFE variants have been shown to be associated with HH. In addition, four main types of non-HFE HH are caused by mutations in the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) genes. The main aim of this study was to screen for HFE, HJV, HAMP, TFR2 and SLC40A1 mutations and to investigate their relationship with HH. MATERIAL E METHODS:Fifty-one Brazilian patients with primary iron overload (transferrin saturation > 50% in females and 60% in males) were eligible. Subsequent bidirectional sequencing for each exon of HFE, HJV, HAMP, TFR2 and SLC40A1 genes was performed. The effect of HFE p.V256I novel mutations on protein structure was analyzed by in silico molecular dynamic and free energy calculations. RESULTS:Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, the p.S65C mutation was found in heterozygosis with p.H63D in two patients and the homozygous genotype for the p.H63D was found in two patients. One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior suggested that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. Sequencing HJV revealed one patient presenting Juvenile hemochromatosis (JH) (homozygous genotype for the HJV p.G320V mutation); two patients carrying heterozygous genotype for the p.E302K mutation; and one patient with heterozygosis p.A310G polymorphism. Sequencing HAMP revealed one patient carrying p.P48G novel mutation in the heterozygous form. Three and five non-pathogenic polymorphisms were observed in the TFR2 and SLC40A1 genes, respectively. Sequencing SLC40A1 also identified one patient with homozygous genotype for the p.R561G described mutation; and one patient with homozygous genotype for the p.G204S novel mutation. CONCLUSION:The HFE p.C282Y in homozygosis or in heterozygosis with p.H63D was the most frequent mutation associated with HH in our sample population. The novel HFE p.V256I mutation could not be implicated in the molecular basis of the HH phenotype. Two described mutations, HJV p.E302K and SLC40A1 p.R561G, could have functional consequences according to previously studies contributing to HH phenotype. Two novel mutations, HAMP p.R48G and SLC40A1 p.G204S, may be implicated with iron overload in these patients, but further studies are need to explain their impact on proteins. Disclosures:No relevant conflicts of interest to declare.

Congenital Erythropoietic Porphyria Due to Co-Inheritance of GATA1 and UROS Gene Mutations

AbstractAbstract 3208The Congenital Erythropoietic Porphyria (CEP) is a rare form of Chronic Porphyria characterized by severe photosensivity. Secondary infections of cutaneous lesions may lead to scarring, deformities and disfigurement of the light-exposed parts of the body such as hands, ears, nose, and eyelids. Erythrodontia, osteodystrophia, combining osteolysis and osteoporosis, hypercellular bone marrow, hemolytic anemia, hypersplenism, splenomegaly and red-coloured urine are present in almost all patients. Generally, CEP is an autosomic recessive disease caused by mutations in homozygosis or compound heterozygosis in UROS gene coding for the fourth enzyme of the heme biosynthesis pathway. At present a new form of CEP with an X-linked inheritance associated with R216W mutation in the GATA 1 gene has been described only in one French-English family by Philips JD et al. GATA1 is a key zinc finger transcription factor that coordinates hematopoietic cell differentiation. We described a Turkish family where a severe CEP phenotype segregates with thalassemia and thrombocytopenia. The propositus is an 4 year old boy who presented photosensitive bullous dermatosis, splenomegaly, short stature and pallor. His mother was chronically anemic (Hb 10.9 g/dL) and thrombocytopenic (96×109/L) known as chronic immune thrombocytopenic purpura, whereas the father and the brother were asymptomatic. At birth, the boy had hypochromic, microcytic anemia, thrombocytopenia and thrombasthenia. Reticulocyte counts slightly increased. At 2 months he was transfused, splenomegaly and heart murmur were detected on physical examination. At this point his Hb level was 6,9 g/dl, WBC 7,400×109/L, platelets 64–106×109/L, MCV 70 flL, RDW 28%. The patient's peripheral-blood smear revealed microcytic, hypochromic red cells, target cells, basophilic stippling, and rare nucleated red cells, findings compatible with thalassemia but DNA analysis for beta and alpha thalassemia was normal. A bone marrow aspirate revealed a hypercellular marrow and erythroid hyperplasia. Dyserythropoiesis was noted with nuclear budding, nuclear bridging, and occasional multinucleation. Megakaryocytes were decreased in number, Hb electrophoresis revealed increased levels of fetal hemoglobin (HbA2 2%; Hb F 68%). At 3 years liver MRI detected iron overload. Ferritin level was 329ng/dL. Because of some skin lesions on his body he was then tested with urine and plasma analysis and the results were consistent with the diagnosis of congenital erythropoietic porphyria. Urine total porphyrins were 5414 nmol/mmol creatinin (N<35) with an excess of isomer I of both Uroporphyrins and Coproporhyrins. Total plasma porhyrins were 312 nmol/L (N<10). We analyzed the UROS gene and we identified a new mutation c.338A>T (D113V) in heterozygosis in the propositus, in his father and in his brother. On the basis of hematological status we decided to analyze also GATA1 gene and the mutation c.646C>T (R216W) has been detected in the child and on one allele of his mother who presents a balanced pattern of X-inactivation. The patient is similar to the case of association of CEP with an hematologic phenotype of beta-thalassemia intermedia. Markedly elevated Hb F levels, and thrombocytopenia result from mutations in the transacting factor GATA-1 presented by Philips JD et al. This patient additionally has a severe liver iron overload. A newborn sibling died at birth because of sepsis and neonatal hepatitis. Liver iron overload was also detected in the sibling's autopsy. All components of family were heterozygous for the H63D mutation in HFE gene but no other HAMP, HJV and SLC40A1 mutations were found. The patient started a regular transfusion program and Deferasirox was given for hemosiderosis after Desferroxamine uncompliance. This is the first report of the association of GATA1 and UROS gene mutations that could explain the very severe hematological phenotype. Disclosures:No relevant conflicts of interest to declare.

Ferroportin disease: A systematic meta-analysis of clinical and molecular findings

Background & AimsClassical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies.MethodsWe conducted a systematic review of the literature and meta-analysis of the biochemical presentation, genetics, and pathology of ferroportin disease.ResultsOf the 176 individuals reported with SLC40A1 mutations, 80 were classified as classical phenotype with hyperferritinemia and normal transferrin saturation. The non-classical phenotype with hyperferritinemia and elevated transferrin saturation was present in 53 patients. The remaining patients had normal serum ferritin or the data were reported incompletely. Despite an increased hepatic iron concentration in all biopsied patients, significant fibrosis or cirrhosis was present in only 11%. Hyperferritinemia was present in 86% of individuals with ferroportin mutations. Bio-informatic analysis of ferroportin mutations showed that the PolyPhen score has a sensitivity of 99% and a specificity of 67% for the discrimination between ferroportin mutations and polymorphisms.ConclusionsIn contrast to HFE hemochromatosis, ferroportin disease has a high penetrance, is genetically heterogeneous and is rarely associated with fibrosis. Non-classical ferroportin disease is associated with a higher risk of fibrosis and a more severe overload of hepatic iron.

Clinical Presentation and Molecular Pathophysiology of Autosomal Dominant Hemochromatosis Caused by A Novel Ferroportin Mutation

Mutations in the SLC40A1 gene, which encodes ferroportin, are associated with autosomal dominant hemochromatosis. Ferroportin is inhibited directly by hepcidin, a key iron-regulatory peptide, and functional consequences of SLC40A1 mutations account for observed phenotypic differences in patients with ferroportin disease. We describe a large pedigree with a novel SLC40A1 mutation and, through in vitro analysis, elucidate the associated molecular mechanism of iron overload. The entire coding sequence of the SLC40A1 gene was sequenced in a pedigree, presenting with autosomal dominant hyperferritinemia. The functional effects of a novel SLC40A1 mutation were studied by overexpression of wild-type and mutant ferroportin fusion proteins in human embryonic kidney cells. Iron export was studied in these cells using (59)Fe transport assays; subcellular localization of ferroportin was examined by way of confocal microscopy. A novel SLC40A1 mutation p.R489K segregated with iron overload in a family with clinical and histopathological signs of macrophage-type ferroportin disease. Human embryonic kidney cells overexpressing p.R489K ferroportin showed decreased iron export capacity when compared with wild-type ferroportin overexpressing cells. Subcellular localization studies demonstrated that p.R489K ferroportin was retained abnormally within an intracellular compartment. We report a novel pathological SLC40A1 variant associated with abnormal cell surface expression of ferroportin due to intracellular retention of the mutant protein. These findings predict macrophage-type ferroportin disease, the phenotype observed in this kindred. Study of the molecular cell biology of ferroportin and its mutants is key to understanding the pathogenesis of this increasingly recognized form of hemochromatosis, which responds poorly to conventional therapy.