HFE Mutations Research Articles

Relevance of dietary iron intake and bioavailability in the management of HFE hemochromatosis: a systematic review

Hereditary hemochromatosis (HH) leads to iron loading because of a disturbance in the negative-feedback mechanism between dietary iron absorption and iron status. The management of HH is achieved by repeated phlebotomies. We investigated whether HH patients would benefit from a diet with low iron intake and bioavailability. We performed a systematic review of studies that linked iron bioavailability and status with dietary factors in subjects with diagnosed HH. Studies on heterozygotes for the HFE mutation were excluded. No prospective, randomized study was reported. Nine studies that directly measured iron bioavailability from test meals in HH patients have been described as well as 3 small, prospective, longitudinal studies in HH patients. Eight cross-sectional studies were identified that investigated the effect of dietary composition on iron status. Calculations of iron bioavailability in HH were made by extrapolating data on hepcidin concentrations and their association with iron bioavailability. The potential reduction in the yearly amount of blood to be phlebotomized when restricting dietary iron absorbed was estimated in the 3 longitudinal studies and ranged between 0.5 and 1.5 L. This amount would be dependent on individual disease penetrance as well as the dietary intervention. Despite the limited quantitative evidence and the lack of randomized, prospective trials, dietary interventions that modify iron intake and bioavailability may affect iron accumulation in HH patients. Although this measure may be welcome in patients willing to contribute to their disease management, limited data exist on the clinical and quality-of-life benefit.

Impact of hemochromatosis gene mutations on cardiac status in doxorubicin-treated survivors of childhood high-risk leukemia.

Doxorubicin is associated with progressive cardiac dysfunction, possibly through the formation of doxorubicin-iron complexes leading to free-radical injury. The authors determined the frequency of hemochromatosis (HFE) gene mutations associated with hereditary hemochromatosis and their relationship with doxorubicin-associated cardiotoxicity in survivors of childhood high-risk acute lymphoblastic leukemia. Peripheral blood was tested for 2 common HFE allelic variants: C282Y and H63D. Serum cardiac troponin-T (cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP), which are biomarkers of cardiac injury and cardiomyopathy, respectively, were assayed during therapy. Left ventricular (LV) structure and function were assessed with echocardiography. A total of 184 patients had DNA results for at least 1 variant, and 167 had DNA results for both: 24% carried H63D and 10% carried C282Y. Heterozygous C282Y genotype was associated with multiple elevations in cTnT concentrations (P = .039), but not NT-proBNP. At a median of 2.2 years (range, 1.0 years-3.6 years) after diagnosis, the mean Z-scores for LV fractional shortening (-0.71 [standard error (SE), 0.25]; P = .008), mass (-0.84 [SE, 0.17]; P < .001), and end-systolic (-4.36 [SE, 0.26], P < .001) and end-diastolic (-0.68 [SE, 0.25]; P = .01) posterior wall thickness were found to be abnormal in children with either allele (n = 32). Noncarriers (n = 63) also were found to have below-normal LV mass (-0.45 [SE, 0.15]; P = .006) and end-systolic posterior wall thickness (-4.06 [SE, 0.17]; P < .001). Later follow-up demonstrated similar results. Doxorubicin-associated myocardial injury was associated with C282Y HFE carriers. Although LV mass and wall thickness were found to be abnormally low overall, they were even lower in HFE carriers, who also had reduced LV function. Screening newly diagnosed cancer patients for HFE mutations may identify those at risk for doxorubicin-induced cardiotoxicity.

Iron storage disease in Asia‐Pacific populations: The importance of non‐HFE mutations

Hereditary hemochromatosis (HH) is a widely recognized and well-studied condition in European populations. This is largely due to the high prevalence of the C282Y mutation of HFE. Although less common than in Europe, HH cases have been reported in the Asia-Pacific region because of mutations in both HFE and non-HFE genes. Mutations in all of the currently known genes implicated in non-HFE HH (hemojuvelin, hepcidin, transferrin receptor 2, and ferroportin) have been reported in patients from the Asia-Pacific region. This review discusses the molecular basis of HH and the genes and mutations known to cause non-HFE HH with particular reference to the Asia-Pacific region. Challenges in the genetic diagnosis of non-HFE HH are also discussed and how new technologies such as next generation sequencing may be informative in the future.

Telomere length and elevated iron: the influence of phenotype and HFE genotype.

Elevated body iron stores are associated with morbidity and mortality due to oxidative stress. Hereditary hemochromatosis, a common condition caused by HFE gene mutations, can lead to excess iron storage and disease but clinical penetrance of HFE gene mutations is low and many people with elevated iron stores lack HFE mutations. We analyzed data from the Hemochromatosis and Iron Overload Screening Study to assess the relationship among HFE genotype (individuals with either homozygous or compound heterozygous status for C282Y and/or H63D HFE mutations were defined as genotype positive, or G+), elevated iron phenotype (individuals exceeding gender-specific transferrin saturation and serum ferritin threshold levels were considered phenotype positive, or P+), and leukocyte telomere length, a marker of biological aging and cumulative oxidative stress. In unadjusted analyses in comparison to individuals who were G-P-, G+P- were not significantly different (OR 0.74; 95% CI 0.26-2.04), while the G+P+ (OR 2.03; 95% CI 1.15-3.56), and G-P+ (OR 2.24; 95% CI 1.5-3.29) had increased risk of short telomeres (<=25th percentile) rather than long telomeres (>=75th percentile). In analyses adjusting for age, gender, and race/ethnicity, the effect of individuals with elevated iron phenotypes having short telomeres persisted with G+P+ individuals (OR 1.94; 95% CI 1.02-3.72), and G-P+ individuals (OR 2.17; 95% CI 1.39-3.39) being significantly different from the G-P- group. In conclusion, elevated iron phenotype, but not HFE genotype, was associated with shortened telomeres. Further studies will be needed to determine whether telomere length provides a marker for morbidities specifically associated with iron overload.

Correlation ofhemochromatosisgene mutations and cardiovascular disease in hemodialysis patients

BACKGROUND AND OBJECTIVESCardiovascular disease (CVD) is a major cause of death in hemodialysis (HD) patients. Hemochromatosis (HFE) gene mutations are reported to be associated with CVD. The present study aims to investigate the association of HFE gene polymorphism with CVD in HD patients.DESIGN AND SETTINGSCross-sectional case-control.METHODSC282Y/H63D mutations of HFE gene were evaluated in 560 HD patients and 480 healthy controls from 4 HD centers in North China. The results obtained from this evaluation process were correlated with biochemical parameters including iron status (serum iron, ferritin, and transferrin concentration), cardiovascular disease, and inflammation marker CRP, IL-6, TNF-α.RESULTSNo C282Y mutations were detected in HD patients or healthy controls in this study. The genotype of H63D heterozygous mutation was similar in HD patients with CVD, HD patients without CVD, and controls. H63D homozygous mutation was 7.4% (19/257), 3.1% (9/303), and 1.0% (5/480) for the 3 groups, respectively. Compound heterozygosity was not found in this study. The relative risk for CVD in HD patients with H63D homozygous mutation was 2.59 (95% CI: 1.15–5.84). H63D homozygous mutation had significantly higher serum ferritin concentrations compared with wild-type individuals. Moreover, HD patients had significantly higher levels of inflammatory biomarkers such as CRP, IL-6, and TNF-α. The multivariate logistic regression analysis revealed that H63D mutation instead of ferritin level was an independent risk factor of CVD for HD patients.CONCLUSIONSOur study demonstrates for the first time that there was an association between H63D homozygous mutations and CVD in HD patients. Elevated serum CRP, IL-6, and TNF-α levels were also related to CVD in HD patients.

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

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

Abstract 5313: The potential role of cellular iron in head and neck squamous cell carcinoma.

Abstract Introduction. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Half of the patients present with advanced disease, which despite aggressive treatments, achieves five-year survival rates of only 50%, underscoring a need to better understand the biology of this disease. One approach would be to examine the role of cellular iron in HNSCC. We are interested in the role of iron as a result of: (1) A previous study in our lab in which uroporphyrinogen decarboxylase was identified as a radiosensitizing target for HNSCC. The radiosentization was due to alterations in iron homeostasis which elevated reactive oxygen species (ROS) and enhance tumor oxidative stress and cytotoxicity. (2) The requirement of iron in a rate limiting step with ribonucleotide reductase for DNA synthesis, and hence cell proliferation. (3) The deregulation of iron in many cancers. Experimental design. A list of proteins involved in iron homeostasis was generate from the literature and evaluated using qRT-PCR in 3 HNSCC cells (FaDu, UTSCC 42a, UTSCC 8) compare to a Normal Oral Epithelial (NOE) cell line. The most highly over expressed iron protein across all cell lines compared to the NOE was hemochromatosis (HFE), thus was selected for further evaluation. Knockdown of HFE was achieved using a siRNA based approach and cellular effects were determined using MTS, clonogenic, BRDU and flow cytometry assays with or without 4 Gy of radiation. Iron rescue experiments where preformed using Ferric ammonium citrate (FACs). Iron chelation was accomplished in HNSCC cells lines using ciclopirox olamine (CPX), a clinical approved iron chelating chemotherapeutic. Results. HFE was selected for further evaluation based on the expression in HNSCC cell lines versus the NOE cell line. Mutations in HFE are linked to the genetic condition hemochromatosis, which is characterized by elevated hepcidin levels and iron accumulation in peripheral tissues. Cell proliferation assays (MTS, clonogenic, BRDU assay) after HFE knockdown with or without RT in HNSCC cells demonstrated a significant decrease in proliferation across all cancer cells with negligible effects on NOE cells. Furthermore, we observed a significant decrease in the Labile iron pool and cellular ROS levels, following HFE knockdown. Re-introduction of iron into the cell after HFE knockdown rescued our phenotype, suggesting that this process is indeed mediated by cellular iron levels. Next, we treated HNSCC cell lines CPX and observed a significant decrease in cell viability compared to control treated cells. Conclusion. HFE appears to be an important protein in controlling cellular iron. HFE knockdown resulted in a reduction in cellular iron which decreases the amount of iron available for DNA synthesis and hence cell proliferation. Therefore, elevated cellular iron appears to be an important factor for the progression of HNSCC, thus iron chelation strategies may valuable in the context of this disease. Citation Format: Michelle Lenarduzzi, Angela Hui, Winnie Yue, Justin Williams, Fei Fei Liu. The potential role of cellular iron in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5313. doi:10.1158/1538-7445.AM2013-5313

Is myocardial iron content associated with left atrial systolic function in asymptomatic hereditary hemochromatosis?

We previously reported that left atrial (LA) systolic function is augmented in asymptomatic hereditary hemochromatosis subjects (HH) as compared to the age/gender‐matched volunteer control subjects (VC). In this study, we analyzed the relationship between elevated myocardial iron content (MIC) and LA systolic function in these subjects. All HH were NYHA functional class I and homozygous for the HFE gene C282Y mutation under standard therapy. VC did not have HFE mutations. 29 HH (ages; 56±10, 9 females) and 18 VC (ages; 55±8, 5 females) underwent LA function assessment by echocardiography and MRI‐based MIC assessment with T2*. LA ejection force (LAF) was significantly elevated in HH as to VC (14.0±8.3 vs. 9.4±4.7 kdyne, P=0.016) indicating augmented LA systolic function in HH. However, MIC did not significantly differ between the groups (27.6±7.8 msec vs. 28.6±5.5 msec, HH vs. VC, the values were in the normal range) and MIC failed to correlate with LAF or the other parameters of LA systolic function. Our findings suggest that the augmented LA function in asymptomatic HH subjects is independent from MIC and additional factors associated with HH, such as elevated oxidative stress may be responsible for this. However, this study does not address the possibility of elevated MIC to induce abnormal diastolic function in iron overload populations. (This research was supported by the intramural research program of the NHLBI, NIH.)

HFE MUTATIONS AND IRON OVERLOAD IN PATIENTS WITH ALCOHOLIC LIVER DISEASE

Alcoholic liver disease (ALD) is generally associated with iron overload, which may contribute to its pathogenesis, through increased oxidative stress and cellular damage. There are conflicting reports in literature about hemochromatosis (HFE) gene mutations and the severity of liver disease in alcoholic patients. To compare the prevalence of mutations in the hemochromatosis (HFE) gene between patients with ALD and healthy controls; to assess the relation of HFE mutations with liver iron stores and liver disease severity. Liver biopsy specimens were obtained from 63 ALD patients (during routine treatment) and 52 healthy controls (during elective cholecystectomy). All individuals underwent routine liver function tests and HFE genotyping (to detect wild-type sequences and C282Y, H63D, S65C, E168Q, E168X, V59M, H63H, P160delC, Q127H, Q283P, V53M and W164X mutations). Associations between HFE mutations and risk of excessive liver iron stores, abnormal serum ferritin, liver fibrosis, or necroinflammatory activity were assessed by multivariate logistic regression analysis. ALD patients had significantly higher serum ferritin and transferrin saturation than controls (both P<0.05), but the distribution of HFE mutations was similar between the two groups. For ALD patients, the odds ratio for having at least one HFE mutation and excessive liver iron stores was 17.23 (95% confidence interval (CI): 2.09-142.34, P = 0.008). However, the presence of at least one HFE mutation was not associated with an increased risk of liver fibrosis or necroinflammatory activity. Active alcohol ingestion showed the strongest association to increased serum ferritin (OR = 8.87, 95% CI: 2.11-34.78, P = 0.003). s ALD patients do not present with a differential profile of HFE mutations from healthy controls. In ALD patients, however, the presence of at least one HFE mutation increases the risk of having excessive liver iron stores but has no detectable effects on liver disease activity or severity.

A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice

Because of the increasing evidence that H63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences of H63D HFE in vivo using mice that carry H67D HFE (homologous to human H63D). Although total brain iron concentration did not change significantly in the H67D mice, brain iron management proteins expressions were altered significantly. The 6-month-old H67D mice had increased HFE and H-ferritin expression. At 12months, H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia in H67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-old H67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in the H67D mice. These findings demonstrate that the H63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. The H67D knock-in mouse can be used as a model to evaluate how the H63D HFE mutation contributes to neurodegenerative diseases.

Loss ofHfeLeads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells

Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe(-/-) mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated. Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4. Hfe(-/-) RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe(-/-) cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe(-/-) RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe(-/-) cells. RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe(-/-) RPE cells as well as in Hjv(-/-) RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe(-/-) and Hjv(-/-) RPE cells.

HFE Gene Mutation Associated with the Severity of Gestational Diabetes Mellitus in Belarusian Women

To determine whether the H63D and C282Y mutations in HFE (hemochromatosis) gene are associated with the risk of gestational diabetes mellitus (GDM), we conducted the study of 65 incident cases. The class of gestational diabetes (A1, A2, B) in pregnant women was defined based on the results of glycemic profile and 75-g oral glucose tolerance test. Two single nucleotide polymorphisms (H63D and C282Y) in HFE gene were genotyped by PCR and RFLP (Restriction Fragment Length Polymorphism). The frequencies of mutations in patients cohort were: 0.14 for H63D and 0.02 for C282Y, which are similar to the data reported for Belarusian population (0.16 and 0.04 respectively). The detailed analysis of case subjects indicated association of H63D mutation with the severity of gestational diabetes mellitus. In the frequencies of H63D mutation and genotypes between the case subjects with A1 and B gestational diabetes were detected significant differences. Our data indicated that the presence of H63D mutation in pregnant women with GDM aggravates the disease—odds ratio 7.4 (95% CI 1.8 - 30.5). Women with gestational diabetes have severe increased risk for illness progressing to class B if they are H63D mutation carriers.

Frequency of human hemochromatosis (HFE) gene mutations in Egyptians with β-thalassemia

Background Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Two point mutations have been described and are referred to as H63D and C282Y. On the other hand, iron overload is a well-documented complication in thalassemia syndromes. Interactions between thalassemia and hemochromatosis may further increase iron overload. This work aimed at studying the frequency of the H63D and C282Y mutations of the HFE gene in an Egyptian population with β-thalassemia (thalassemia major, intermedia, and minor) by comparing it with normal individuals without hemoglobinopathies. Participants and methods This study included 86 patients with β-thalassemia; 40 of these patients had β-thalassemia major and intermedia and the other 46 patients had β-thalassemia minor (carriers). In addition, 70 individuals were included in the study and served as controls. All the populations studied were screened for H63D and C282Y mutations of the HFE gene using the PCR-restriction fragment length polymorphism (PCR-RFLP) method. Results The allelic frequencies found for H63D and C282Y mutations in this study were 18.6 and 0%, respectively, among the total alleles of individuals with β-thalassemia and 12.8 and 1.4% among controls without hemoglobinopathies. On comparing thalassemia cases, thalassemia carriers, and the control group, the allele frequency of the H63D mutation was significantly higher among β-thalassemia carriers (24%) compared with patients with β-thalassemia (12.5%) and controls (12.8%) (P=0.049). Our study also found an almost significantly higher frequency of the heterozygote genotype for the H63D mutation in patients with β-thalassemia (30.2%) than the controls (17.1%) (Fisher’s test, P=0.06). Compound heterozygosis for H63D and C282Y was found in one of the individuals in our control group. Conclusion The frequent occurrence of β-thalassemia disease with the H63D gene mutation raises the possibility of genetic interactions and emphasizes the value of screening for HFE mutations in thalassemias to detect early cases of iron overload and to modify its treatment modalities.

Predictors of haemoconcentration at delivery: association with low birth weight

[corrected] To assess the factors associated with risk of haemoconcentration at delivery, such as initial haemoglobin levels and alterations in the HFE gene, and its effect on low birth weight in pregnant women supplemented with moderate doses of iron. Case-control study nested in a longitudinal study conducted on 217 healthy pregnant women taking moderate iron supplementation and their newborns. Women were classified according to the risk of haemoconcentration at delivery, defined as Hb > 130 g/L. Each subject's obstetric and clinical history, smoking habit, and iron biochemical parameters (haemoglobin (Hb), serum ferritin and transferrin saturation) were recorded at 1st, 2nd and 3rd trimester and at delivery. Polymorphisms of the HFE gene (C282Y, H63D and S65C) were also measured. The average of iron supplementation of all the women was 43.9 mg/dia (geometric mean, 95 % CI: 43.6-44.1). Higher levels of Hb at early gestation and the presence of HFE mutations were associated with greater risk of haemoconcentration at delivery, adjusted odds ratios of 1.14 (95 % CI: 1.05-1.25) and 5.35 (95 % CI: 1.6-17.8). Haemoconcentration at delivery was associated with a greater risk of low birth weight, adjusted odd ratio of 11.48 (95 % CI: 1.13-116.6). Moderate daily doses of supplementary iron may be harmful for foetal growth in women with alterations in HFE gene and who started pregnancy with good haemoglobin levels. Overall, this suggests the importance of determining a woman's iron status early in her pregnancy in order to establish a more appropriate pattern of supplementation.

Clinical characteristics and analysis of HFE gene variants (C282Y and H63D) in Jordanian Arab patients with age-related macular degeneration

Age-related macular degeneration (AMD) is a complex genetic disorder with multiple etiologies. Multiple genes as well as environmental effects are thought to play a role in causing AMD. Recent evidence pointed that elevated iron overload, resulting from hereditary defects of iron homeostasis, is associated with retinal degeneration and consequently plays a role in the pathogenesis of AMD. Hemochromatosis is a genetic disorder in which excess iron is absorbed from the diet and deposited in different tissues, primarily caused by mutations in HFE gene. Two major mutations in HFE are responsible for most hemochromatosis cases, namely, C282Y and H63D.In this work we gathered information relating to 37 AMD patients from Jordan, and investigated the potential association between hemochromatosis, or more specifically, carrier state for a mutation in HFE gene (which may moderately increase dietary iron absorption) and AMD, given the effect of elevated iron levels on AMD occurrence. Questionnaires and blood samples were collected from patients visiting the eye care clinic in the King Abdullah hospital in Jordan. DNA was extracted from patient samples and mutations in HFE were genotyped (using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and DNA sequencing) and compared to 106 control samples.We could not detect C282Y (rs1800562) variant in our patient population or in the controls. For carrier status with H63D (rs1799945) we had 30.3% compared to a frequency of 22.7% in the controls (p=0.37). H63D allele frequency was 15.2% in our patients compared to 11.8% in the controls (p=0.30).H63D variant seems to be more frequent in AMD patients though not reaching a significance of p=0.05. To date, this is the first attempt to link HFE (particularly, H63D) mutation to AMD.

RNAi-Mediated Inhibition of Tmprss6 Ameliorates Anemia and Secondary Iron Overload in a Mouse Model of β-Thalassemia Intermedia and Decreases Iron Overload in Hfe−/− Mice

AbstractAbstract 1018β-Thalassemia intermedia (TI), an inherited hemoglobinopathy caused by partial loss of β-globin synthesis, is characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis as well as secondary iron overload. Hereditary hemochromatosis (HH) is most frequently caused by mutations in HFE and is marked by excess uptake of dietary iron with concomitant tissue iron overload. In both diseases, increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (encoded by Hamp1). The membrane serine protease Matriptase-2 (encoded by Tmprss6) attenuates BMP-mediated Hamp1 induction by cleaving the BMP co-receptor, hemojuvelin. Previously, it has been shown that elevating Hamp1 expression by genetic inactivation of Tmprss6 reduces disease severity in the Hbbth3/+ mouse model of TI and prevents iron overload in Hfe−/− mice. Therefore, a therapeutic approach comprising specific inhibition of Tmprss6 could prove efficacious in TI and HH.Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to >80% inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ and Hfe−/− mice with concomitant >2-fold elevation in Hamp1 expression. In the TI model, Tmprss6 silencing leads to ∼30% reductions in serum iron and non-heme liver iron. In Hfe−/− mice, serum iron and non-heme liver iron are similarly reduced, and Perls staining of peri-portal iron is diminished. Remarkably, the partial iron restriction induced by Tmprss6 inhibition in Hbbth3/+ mice leads to dramatic improvements in the hematological aspects of the disease phenotype: the severity of the anemia is decreased as evidenced by an approximately 1 g/dL increase in total hemoglobin and a 50% decrease in circulating erythropoietin levels. As in the human disease, Hbbth3/+ mice exhibit the hallmarks of ineffective erythropoiesis including splenomegaly, decreased erythrocyte survival and marked reticulocytosis. Treatment with LNP formulated Tmprss6 siRNA leads to a dramatic 2–3 fold decrease in spleen size, a 3–4 fold decrease in reticulocyte counts and a >7-day increase in RBC half-life. Histological analysis of spleens from Tmprss6 siRNA treated animals demonstrates restoration of normal splenic architecture, as well as a reduction in the number of Tfr1-positive erythrocyte precursors in the spleen. Furthermore, as evidenced by the near normalization of blood smears, the overall quality of erythropoiesis in treated animals is vastly improved.Taken together, these data demonstrate that RNAi-mediated silencing of liver Tmprss6 elevates Hamp1 expression and reduces iron overload in both TI and HH model mice. More significantly, Tmprss6 siRNA treatment ameliorates all aspects of the disease phenotype in the TI mouse model. These results support the development of an RNAi therapeutic targeting TMPRSS6 for the treatment of TI, HH and potentially other disorders characterized by excess iron absorption due to physiologically inappropriately low levels of hepcidin. Disclosures:Racie:Alnylam Pharmaceuticals: Employment. Butler:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership.

Role of Magnetic Ressonance Imaging – T2* in Hereditary Hemochromatosis.

AbstractAbstract 2096 Introduction:Hereditary hemochromatosis (HH) is an autossomic recessive disorder characterized by increased iron absorption. Magnetic resonance imaging – T2* (MRI-T2*) has become a reliable and robust methodology to directly assess the iron burden, with better results in transfusional hemosiderosis compared to indirect methods, such as serum ferritin and transferrin saturation (TS). However, little is known about its role in HH. Objectives:Describe the demographic profile of HH type 1 patients as to the type of the HFE mutation and correlate laboratory parameters to MRI-T2*results. Methods:We collected data from patients with a positive HFE gene mutation who performed abdominal and/or cardiac MRI-T2* in our institution from 2004 to 2011. Images retrieved from the digital archive were analyzed by two blinded independent radiologists using the Thalassemia-Tools software (Cardiovascular Imaging Solutions, London, UK). Laboratory data available within 6 months before or after the MRI study were analyzed using the t-Student test, Exact Fisher's test analysis and multivariate analyses. Results:We analyzed 81 patients, 76 (93%) males and 5 (6.2%) females, with a median age of 48 years (21–80). Liver, pancreatic and splenic MRI-T2*values and LIC calculation were performed in 80 patients, and cardiac T2* assessment in 57 patients. The inter-observer T2* variation coefficient was 5%. Serum ferritin was abnormal in 70 patients (90.9%), while TS was abnormal in 34% of the tests.In our study sample, the H63D mutation was present in 70 patients (86.4%): 11 (13.6%) were homozygous, 59 (72.8%) heterozygous and 7 (8.6%) double heterozygous for C282Y/H63D. Only three patients (3.7%) were homozygous and 6 (7.4%) were heterozygous only for the C282Y mutation. The S65C mutation was detected in heterozygous state in 2 (2.5%) of cases.Two out 57 cases had a positive T2* result and were classified as light cardiac overload (T2*:18.98 e 19.14 ms). Both had the H63D mutation (1 homozygous and 1 heterozygous). Thirty seven out of 80 patients (46.3%) had liver overload in abdominal MRI (T2*: 3.8–11.4ms), being 33 (41.3%) light overload and four (5%) moderate overload (T2*:1.8–3.8ms). We found that 77.8% of patients with liver overload were C282Y carriers, of which 57.2% had double mutation and 40.3% had H63D mutation in hetero or homozigosity. Pancreatic overload was found in 20 patients (25.1%), while 30 patients (37.5%) had splenic overload. There was a slight correlation (r: 0.365) between liver T2* and splenic T2* (p=0.001).The presence of C282Y and H63D mutations was statistically associated with a higher frequency of abnormal liver T2* (p=0.017 and p=0.042, respectively). The H63D mutation was associated with iron accumulation in the liver (p=0,037) and homozygous carriers showed higher levels of liver overload (p=0,038). Conclusion:In our study, serum ferritin was a better surrogate marker for iron overload than ST. In addition, up to 40.3% of patients with H63D mutation had evidence of hepatic iron overload by MRI. These findings differ from the currente literature. The higher RMI positivity might be due to a higher sensitivity to detect lower levels of organic iron. Despite the lack of a control group and laboratory tests or MRI in all the cases studied, our results suggest that RMI-T2* is a promising methodology to guide the therapeutic management of HH patients. The clinical impact of this finding must be investigated in further studies. Disclosures:No relevant conflicts of interest to declare.

Hemocromatosis: etiopatogenia, diagnóstico y estrategia terapéutica

Hereditary hemochromatosis (HH) constitutes several inherited disorders characterized by an increased intestinal absorption of iron with its subsequent accumulation in tissues. Most (approximately 90%) patients with HH have mutations in HFE. Aproximately 95% of persons with HFE-related HH are homozygous for the C282Y mutation. Population studies indicate that the penetrane of the C282Y mutation is incomplete. Mutations in the iron-related genes encoding for hemojuvelin, hepcidin, ferroportin, trasnferrin receptor 2 and ferritin result in non-HFE related HH. In HFE-related HH the excess iron is preferentially deposited in the cytoplasm of parenhymal cells of varios organs, including the liver, pancreas, heart, endocrine glands, skin and joints. Symptoms are related to damage of this organs. The diagnosis includes iron studies, genetic testing and liver biopsy to assess the hepatic iron concentration and degree of liver injury. Because phlebotomy prevents and reverses the accumulation of excess iron, is the treatment of choice.

Liver hepcidin mRNA expression is inappropriately low in alcoholic patients compared with healthy controls

Hepcidin plays a crucial role in iron metabolism, preventing its absorption at the basolateral enterocyte membrane. Hepcidin regulation is complex and regulated at the transcriptional level. The relation between iron overload and alcoholic liver disease is well known, but its mechanism is not clear. We present an observational, case-control study, aimed at evaluating the effects of alcohol on the expression of hepcidin in human participants. We intended to assess whether iron overload related to alcohol ingestion was caused by hepcidin-impaired expression by determining hepcidin mRNA expression and relating it to iron stores, both in alcoholic patients and in normal controls. We compared liver hepcidin mRNA expression between 25 active drinkers with alcoholic liver disease, without cirrhosis, and 20 healthy controls. All individuals were evaluated for HFE mutations, complete blood count, coagulation, glucose, kidney function, liver function, viral hepatitis, C-reactive protein, interleukin 6, tumor necrosis factor α, and serum iron, ferritin, and transferrin saturation. Total RNA was isolated from liver samples, cDNA was obtained by reverse transcription, and hepatic expression levels of hepcidin were determined by real-time PCR using the comparative Ct method (2(-ΔΔCt)). Serum ferritin and transferrin saturation were significantly higher in patients. Hepcidin was downregulated in patients compared with the controls by a mean factor of -0.44 (log10 2(-ΔΔCt)) (P=0.009). Hepcidin expression was not significantly different between the several grades of fibrosis, necroinflammatory activity, and liver iron stores. Heavy alcohol consumption caused the highest hepcidin mRNA suppression. The hepcidin mRNA expression/serum ferritin ratio was significantly lower in alcoholic patients (P<0.0001). Hepcidin liver expression is inappropriately low in alcoholic patients with active alcoholism and preserved hepatic function, and we conclude that this is the mechanism for alcohol consumption-associated iron overload in humans.

Identification and characterization of the first SLC11A2 isoform 1a mutation causing a defect in splicing process and an hypomorphic allele expression of the SLC11A2 gene

Identification and characterization of the first <scp>SLC</scp>11<scp>A</scp>2 isoform 1a mutation causing a defect in splicing process and an hypomorphic allele expression of the <i><scp>SLC</scp>11<scp>A</scp>2</i> gene