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. © 2016 Wiley Periodicals, Inc.

Heterozygous Mutations in BMP6 Pro-peptide Lead to Inappropriate Hepcidin Synthesis and Moderate Iron Overload in Humans.

BACKGROUND & AIMS: Hereditary hemochromatosis is a heterogeneous group of genetic disorders characterized by parenchymal iron overload. It is caused by defective expression of liver hepcidin, the main regulator of iron homeostasis. Iron stimulates the gene encoding hepcidin (HAMP) via the bone morphogenetic protein (BMP)6 signaling to SMAD. Although several genetic factors have been found to cause late-onset hemochromatosis, many patients have unexplained signs of iron overload. We investigated BMP6 function in these individuals. METHODS: We sequenced the BMP6 gene in 70 consecutive patients with a moderate increase in serum ferritin and liver iron levels who did not carry genetic variants associated with hemochromatosis. We searched for BMP6 mutations in relatives of 5 probands and in 200 healthy individuals (controls), as well as in 2 other independent cohorts of hyperferritinemia patients. We measured serum levels of hepcidin by liquid chromatography-tandem mass spectrometry and analyzed BMP6 in liver biopsy specimens from patients by immunohistochemistry. The functions of mutant and normal BMP6 were assessed in transfected cells using immunofluorescence, real-time quantitative polymerase chain reaction, and immunoblot analyses. RESULTS: We identified 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with unexplained iron overload (9% of our cohort). These mutations were detected in less than 1% of controls. p.Leu96Pro also was found in 2 patients from the additional cohorts. Family studies indicated dominant transmission. Serum levels of hepcidin were inappropriately low in patients. A low level of BMP6, compared with controls, was found in a biopsy specimen from 1 patient. In cell lines, the mutated residues in the BMP6 propeptide resulted in defective secretion of BMP6; reduced signaling via SMAD1, SMAD5, and SMAD8; and loss of hepcidin production. CONCLUSIONS: We identified 3 heterozygous missense mutations in BMP6 in patients with unexplained iron overload. These mutations lead to loss of signaling to SMAD proteins and reduced hepcidin production. These mutations might increase susceptibility to mild-to-moderate late-onset iron overload.

Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis.

BACKGROUND & AIMS: Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. METHODS: We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. RESULTS: One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(-13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). CONCLUSIONS: This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely.

Decreased cardiovascular and extrahepatic cancer-related mortality in treated patients with mild HFE hemochromatosis.

BACKGROUND & AIMS: Mortality studies in patients with hemochromatosis give conflicting results especially with respect to extrahepatic causes of death. Our objective was to assess mortality and causes of death in a cohort of patients homozygous for the C282Y mutation in the HFE gene, diagnosed since the availability of HFE testing. METHODS: We studied 1085 C282Y homozygotes, consecutively diagnosed from 1996 to 2009, and treated according to current recommendations. Mortality and causes of death were obtained from death certificates and compared to those of the general population. Standardized mortality ratios (SMRs) were used to assess specific causes of death and the Cox model was used to identify prognostic factors for death. RESULTS: Patients were followed for 8.3±3.9 years. Overall the SMR was the same as in the general population (0.94 CI: 0.71-1.22). Patients with serum ferritin⩾2000 µg/L had increased liver-related deaths (SMR: 23.9 CI: 13.9-38.2), especially due to hepatic cancer (SMR: 49.1 CI: 24.5-87.9). Patients with serum ferritin between normal and 1000 µg/L had a lower mortality than the general population (SMR: 0.27 CI: 0.1-0.5), due to a decreased mortality, related to reduced cardiovascular events and extrahepatic cancers in the absence of increased liver-related mortality. Age, diabetes, alcohol consumption, and hepatic fibrosis were independent prognostic factors of death. CONCLUSIONS: In treated HFE hemochromatosis, only patients with serum ferritin higher than 2000 µg/L have an increased mortality, mainly related to liver diseases. Those with mild iron burden have a decreased overall mortality in relation to reduced cardiovascular and extrahepatic cancer-related events. These results support a beneficial effect of early and sustained management of patients with iron excess, even when mild.

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.

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.

Ferroportin diseases: functional studies, a link between genetic and clinical phenotype.

Ferroportin (FPN) mediates iron export from cells and this function is modulated by serum hepcidin. Mutations in the FPN gene (SLC40A1) lead to autosomal dominant iron overload diseases related either to loss or to gain of function, and usually characterized by normal or low transferrin saturation versus elevated transferrin saturation, respectively. However, for the same mutation, the phenotypic expression may vary from one patient to another. Using in vitro overexpression of wild-type or mutant FPN proteins, we characterized the functional impact of five recently identified FPN gene mutations regarding FPN localization, cell iron status, and hepcidin sensitivity. Our aim was to integrate functional results and biological findings in probands and relatives. We show that while the p.Arg371Gln (R371Q) mutation had no impact on studied parameters, the p.Trp158Leu (W158L), p.Arg88Gly (R88G), and p.Asn185Asp (N185D) mutations caused an iron export defect and were classified as loss-of-function mutations. The p.Gly204Ser (G204S) mutation induced a gain of FPN function. Functional studies are useful to determine whether or not a FPN gene mutation found in an iron overloaded patient is deleterious and to characterize its biological impact, especially when family studies are not fully informative and/or additional confounding factors may affect bio-clinical expression.

[Molecular diagnosis of HFE mutations in routine laboratories. Results of a survey from reference laboratories in France]. / Recommandations pour la (bonne) pratique du diagnostic moléculaire de l'hémochromatose liée au gène HFE. Synthesed'une enquete realisee aupres du reseau national des laboratoires pratiquant le disgnostic de maladies rares du metabolisme du fer.

HFE-related hemochromatosis (HFE hemochromatosis) or type 1 hemochromatosis is an autosomal recessive disease characterized by progressive iron overload usually expressed in adulthood. The HFE gene, located on the short arm of chromosome 6 (6p21.3), encodes a protein that plays a crucial role in iron metabolism by modulating hepcidin synthesis in the liver. Homozygosity for the p.Cys282Tyr mutation accounts for nearly 80% of cases of hemochromatosis in France. Genetic testing is the key investigation to confirm the diagnosis of HFE hemochromatosis. A survey on routine practices was carried out among the eight reference laboratories of the French national network on genetic iron disorders. The main findings from this survey are as follows: 1) the p.Cys282Tyr mutation must be searched for as an initial step to establish the diagnosis of HFE hemochromatosis. This is in agreement with the recommendations of the French Health Authority (HAS) published in 2005. In these recommendations, homozygosity for the p.Cys282Tyr mutation with at least elevated transferrin saturation, is considered the only genotype that confirms of the diagnosis of HFE hemochromatosis; 2) in combination with the p.Cys282Tyr mutation (compound heterozygous genotypes), the p.Ser65Cys and the p.His63Asp variants may contribute to the occurrence of mild iron overload; 3) family screening is mandatory following the detection of homozygous individuals for the p.Cys282Tyr mutation.

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.

Sex and acquired cofactors determine phenotypes of ferroportin disease.

BACKGROUND & AIMS: Ferroportin disease is characterized by iron overload. It has an autosomal-dominant pattern of inheritance and has been associated with mutations in the SLC40A1 gene, which encodes the cellular iron exporter ferroportin. Since the first description in 2001, about 30 mutations have been reported; the heterogeneity of ferroportin disease phenotypes has led to the hypothesis that the nature of the mutation affects the function of the protein in different ways. We studied genotypes and phenotypes of a large cohort of patients with ferroportin disease. METHODS: We studied clinical, biochemical, imaging, histologic, and genetic data from 70 affected subjects from 33 families with 19 mutations. RESULTS: We found that ferroportin disease, at the time of diagnosis, has limited consequences in the absence of cofactors. Data indicated that transferrin saturation, which correlated with fibrosis and levels of alanine aminotransferase, might be a marker of disease severity. Although the study was performed in a large number of families, we observed incomplete penetrance and no correlation between genotypes and phenotypes. CONCLUSIONS: Members of families with ferroportin disease should be screened for biochemical parameters of iron metabolism as well as genotype to detect silent mutations that might cause disease with acquired or genetic cofactors. Patients should be followed up long term to identify potential complications of the disease.

Molecular diagnosis of genetic iron-overload disorders.

Genetic iron overload has long been confined to the picture of classical hemochromatosis related to the HFE C282Y mutation (type 1 hemochromatosis). C282Y homozygosity affects approximately three people out of 1000 of the Caucasian population, representing one of the most frequent genetic predispositions. It has, however, rapidly become clear that the HFE C282Y mutation is not the sole culprit in genetic iron overload. Several novel mutations in HFE and other genes have been discovered and related to various entities, which are now known as types 2, 3 and 4 hemochromatosis. These diseases are far less frequent than the classical type 1 hemochromatosis but, by contrast, are not limited to the Caucasian population. Molecular diagnosis obviously plays a key role in the diagnostic strategy. In the future, it will undoubtedly enable not only identification of new diagnostic markers, but also provide potential molecular targets for pathophysiologically based innovative therapeutic approaches.

A new mutation in the hepcidin promoter impairs its BMP response and contributes to a severe phenotype in HFE related hemochromatosis.

Low levels of hepcidin are responsible for the development of iron overload in p.Cys282Tyr HFE related hemochromatosis. Every genetic factor lowering the hepcidin gene expression could contribute to a more severe phenotype in HFE hemochromatosis. Based on this hypothesis, we identified a heterozygous nc.-153 C>T mutation in the hepcidin gene promoter sequence in a patient homozygous for the p.Cys282Tyr HFE mutation who presented massive iron overload, resisting to well conducted iron depletive treatment. Our results demonstrate that the nc.-153 C>T mutation, located within a BMP-RE (Bone Morphogenetic Protein-Responsive Element): i) decreases the transcriptional activity of the hepcidin promoter, ii) alters its IL-6 (Interleukin-6) total responsiveness, and iii) prevents the binding of the SMAD protein complex (1/5/8 and 4) to the BPM-RE. In conclusion, our results suggest that a mutation in the BMP-RE of hepcidin promoter may impact on human iron metabolism.

A new missense mutation in the L ferritin coding sequence associated with elevated levels of glycosylated ferritin in serum and absence of iron overload.

BACKGROUND: Elevated serum ferritin levels are frequently encountered in clinical situations and once iron overload or inflammation has been ruled out, many cases remain unexplained. Genetic causes of hyperferritinemia associated to early cataract include mutations in the iron responsive element in the 5' untranslated region of the L ferritin mRNA, responsible for the hereditary hyperferritinemia cataract syndrome. DESIGN AND METHODS: We studied 91 probands with hyperferritinemia comprising 25 family cases belonging to families with at least two cases of unexplained hyperferritinemia, and 66 isolated cases. In the families, we also analyzed 30 relatives. Hyperferritinemia was considered as unexplained when transferrin saturation was below 45% and/or serum iron below 25 mumol/L and/or no tissue iron excess was detected, when inflammation had been ruled out and when iron responsive element mutation was absent. We carried out sequencing analysis of the FTL gene coding the L ferritin. RESULTS: A novel heterozygous p.Thr30Ile mutation in the NH2 terminus of L ferritin subunit was identified in 17 probands out of the cohort. The mutation was shown to cosegregate with hyperferritinemia in all the 10 families studied. No obvious clinical symptom was found associated with the presence of the mutation. This unique mutation is associated with an unusually high percentage of ferritin glycosylation. CONCLUSIONS: This missense mutation of FTL represents a new cause of genetic hyperferritinemia without iron overload. We hypothesized that the mutation increases the efficacy of L ferritin secretion by increasing the hydrophobicity of the N terminal "A" alpha helix.

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.

Common variants in the BMP2, BMP4, and HJV genes of the hepcidin regulation pathway modulate HFE hemochromatosis penetrance.

Most cases of genetic hemochromatosis (GH) are associated with the HFE C282Y/C282Y (p.Cys282Tyr/p.Cys282Tyr) genotype in white populations. The symptoms expressed by C282Y homozygotes are extremely variable. Only a few suffer from an overt disease. Several studies have suggested that, in addition to environmental factors, a genetic component could explain a substantial part of this phenotypic variation, although very few genetic factors have been identified so far. In the present study, we tested the association between common variants in candidate genes and hemochromatosis penetrance, in a large sample of C282Y homozygotes, using pretherapeutic serum ferritin level as marker of hemochromatosis penetrance. We focused on two biologically relevant gene categories: genes involved in non-HFE GH (TFR2, HAMP, and SLC40A1) and genes involved in the regulation of hepcidin expression, including genes from the bone morphogenetic protein (BMP) regulatory pathway (BMP2, BMP4, HJV, SMAD1, SMAD4, and SMAD5) and the IL6 gene from the inflammation-mediated regulation pathway. A significant association was detected between serum ferritin level and rs235756, a common single-nucleotide polymorphism (SNP) in the BMP2 genic region (P=4.42x10-5). Mean ferritin level, adjusted for age and sex, is 655 ng/ml among TT genotypes, 516 ng/ml in TC genotypes, and 349 ng/ml in CC genotypes. Our results further suggest an interactive effect on serum ferritin level of rs235756 in BMP2 and a SNP in HJV, with a small additive effect of a SNP in BMP4. This first reported association between common variants in the BMP pathway and iron burden suggests that full expression of HFE hemochromatosis is linked to abnormal liver expression of hepcidin, not only through impairment in the HFE function but also through functional modulation in the BMP pathway. Our results also highlight the BMP regulation pathway as a good candidate for identification of new modifier genes.

[Screening for hereditary HFE hemochromatosis]. / Dépistage de l'hémochromatose génétique HFE.

Screening for hereditary HFE hemochromatosis in the general population, by either phenotype or genotype, is not currently recommended by the French High Health Authority. Targeted screening for hereditary HFE hemochromatosis in groups with specific diseases (people with asthenia, arthropathic disorders, liver or heart disease, etc.) has not been shown to be effective. Family screening in first-degree relatives of any proband homozygous for C282Y is strongly advised. This should involve both phenotypic screening, that is, testing for serum iron markers and, if possible, a genotype study of siblings and adult children, conducted according to the rules for genetic counseling and testing. This type of screening is cost-effective. One obstacle today is that the national health insurance fund does not reimburse the HFE test.