Iron Overload-related Diseases Research Articles

Ferroptosis Altered microRNAs Expression in HT-1080 Fibrosarcoma Cells Based on Small RNA Sequencing and Bioinformatics Analysis.

Iron is an essential trace element in the human body. However, excess iron is harmful and may cause ferroptosis. The expression and role of microRNAs (miRNAs) in ferroptosis remain largely unknown. A model of ferroptosis induced by ferric ammonium citrate in HT-1080 cells was established in this study. The miRNAs expression profiles of the control and iron groups were obtained using small RNA sequencing and verified using qRT-PCR. A total of 1346 known miRNAs and 80 novel miRNAs were identified, including 12 up-regulated differentially expressed miRNAs (DE-miRNAs) and 16 down-regulated DE-miRNAs. SP1 was the most important upstream transcription factor regulating DE-miRNAs. The downstream target genes of DE-miRNAs were predicted based on miRDB, TargetScan, and miRBase databases, and 403 common target genes were screened. GO annotation and KEGG analysis revealed that the target genes were mainly involved in various biological processes and regulatory pathways, especially the MAPK signaling pathway and PI3K-Akt signaling pathway. Afterwards, a target genes network was constructed using STRING and Cytoscape, and the hub genes were compared with the ferroptosis database (FerrDb V2) to discover the hub genes related to ferroptosis. EGFR, GSK3B, PARP1, VCP, and SNCA were screened out. Furthermore, a DE-miRNAs-target genes network was constructed to explore key DE-miRNAs. hsa-miR-200c-3p, hsa-miR-26b-5p, and hsa-miR-7-5p were filtered out. Comprehensive bioinformatics analysis of miRNAs and its upstream and downstream regulation in ferroptosis in HT-1080 cells using small RNA sequencing is helpful for understanding the role of miRNAs in iron overload-related diseases and ferroptosis-targeted therapy for cancer.

Natural polyphenol-based nanoparticles for the treatment of iron-overload disease.

Iron-overload diseases are characterized by a variety of symptoms resulting from excessive iron stores, oxidative stress and consequent end-organ damage. Deferoxamine (DFO) is an iron-chelator that can protect tissues from iron-induced damage. However, its application is limited due to its low stability and weak free radical scavenging ability. Herein, natural polyphenols have been employed to enhance the protective efficacy of DFO through the construction of supramolecular dynamic amphiphiles, which self-assemble into spherical nanoparticles with excellent scavenging capacity against both iron (III) and reactive oxygen species (ROS). This class of natural polyphenols-assisted nanoparticles was found to exhibit enhanced protective efficacy both in vitro in an iron-overload cell model and in vivo in an intracerebral hemorrhage model. This strategy of constructing natural polyphenols- assisted nanoparticles could benefit the treatment of iron-overload related diseases with excessive accumulation of toxic or harmful substances.

HFE hemochromatosis in African Americans: Prevalence estimates of iron overload and iron overload-related disease

BackgroundLittle is known about the prevalence of HFE (homeostatic iron regulator) hemochromatosis in African Americans (AA). MethodsWe defined AA as self-identified AA, blacks, or non-Hispanic blacks. We defined hemochromatosis-associated HFE genotypes as p.C282Y/p.C282Y and p.C282Y/p.H63D. We compiled prevalences of these genotypes in AA using published population and cohort data and numbers of men and women ≥18 y‬ in 2018 U.S. Census estimates. We defined iron overload (IO) and IO-related disease by genotype as previously reported in population and cohort studies of hemochromatosis in whites of European ancestry. We used these definitions to estimate prevalences and numbers of AA with IO and IO-related disease associated with hemochromatosis-associated HFE genotypes. ResultsThere were ∼16,287,599 men and ∼17,644,898 women. HFE genotypes and their respective prevalences were: p.C282Y/p.C282Y, 0.00017 (6/34,905) [95% confidence interval 0.000034, 0.00031] and p.C282Y/p.H63D, 0.0012 (41/33,596) [0.000084, 0.0016]. IO prevalences were: men 0.000076 [0.000072, 0.000081] and women 0.0000061 [0.0000050, 0.0000073]. IO-related disease prevalences were: men 0.000063 [0.000059, 0.000067] and women 0.0000021 [0.0000014, 0.0000027]. There were ∼1021 [961, 1091] men and ∼36 [25, 48] women with IO-related disease. ConclusionsWe conclude that ∼1/25,061 AA >18 y have a hemochromatosis-associated HFE genotype and IO and that ∼1/32,103 AA >18 y have a hemochromatosis-associated HFE genotype and IO-related disease.

C282Y/H63D Compound Heterozygosity Is a Low Penetrance Genotype for Iron Overload-related Disease

BackgroundHereditary hemochromatosis (HH) occurs due to mutations in the HFE gene. While the C282Y mutation is the most common genotype reported in HH, other genotypes are found less frequently, indicating variable degrees of penetrance. We studied the penetrance of the C282Y/H63D compound heterozygote genotype in developing clinically significant iron overload.MethodsWe have completed a retrospective analysis on every individual within Newfoundland & Labrador who were diagnosed as C282Y/H63D compound heterozygote between 1996 and 2009 through a molecular genetics study. We collected data for up to 10 years following the initial genotyping using electronic health records, including laboratory values, phlebotomy status, radiologic reports and clinic records. Iron overload status was classified based on the HealthIron study.ResultsBetween 1996 and 2009, 247 individuals with available health records tested positive for C282Y/H63D compound heterozygosity. Over the 10 years of our study, 5.3% of patients exhibited iron overload-related disease on the background of documented iron overload. Including these individuals, 10.1% of patients had documented iron overload, 23.1% of patients had a provisional iron overload and the remaining 66.8% of patients had no evidence of iron overload. Only 44 patients had documented phlebotomies, likely based on their severe phenotype at baseline. Despite phlebotomy, the prevalence of iron overload was higher among these patients. The penetrance of compound heterozygosity was also significantly higher among men (P < 0.01).ConclusionC282Y/H63D compound heterozygosity is a low penetrance genotype in HH. This is the largest reported cohort of C282Y/H63D compound heterozygotes in North America with an extended follow-up.

Interplay Between Iron Overload and Osteoarthritis: Clinical Significance and Cellular Mechanisms.

There are multiple diseases or conditions such as hereditary hemochromatosis, hemophilia, thalassemia, sickle cell disease, aging, and estrogen deficiency that can cause iron overload in the human body. These diseases or conditions are frequently associated with osteoarthritic phenotypes, such as progressive cartilage degradation, alterations in the microarchitecture and biomechanics of the subchondral bone, persistent joint inflammation, proliferative synovitis, and synovial pannus. Growing evidences suggest that the conditions of pathological iron overload are associated with these osteoarthritic phenotypes. Osteoarthritis (OA) is an important complication in patients suffering from iron overload-related diseases and conditions. This review aims to summarize the findings and observations made in the field of iron overload-related OA while conducting clinical and basic research works. OA is a whole-joint disease that affects the articular cartilage lining surfaces of bones, subchondral bones, and synovial tissues in the joint cavity. Chondrocytes, osteoclasts, osteoblasts, and synovial-derived cells are involved in the disease. In this review, we will elucidate the cellular and molecular mechanisms associated with iron overload and the negative influence that iron overload has on joint homeostasis. The promising value of interrupting the pathologic effects of iron overload is also well discussed for the development of improved therapeutics that can be used in the field of OA.

Network pharmacology-based identification of the key mechanism of quercetin acting on hemochromatosis.

Hemochromatosis is an iron overload disease, which lacks nutritional intervention strategies. This study explored the protective effect of quercetin on hemochromatosis and its possible mechanism through network pharmacology. We used Online Mendelian Inheritance in Man to screen the disease targets of hemochromatosis, and further constructed a potential protein interaction network through STITCH. The above-mentioned targets revealed by Gene enrichment analysis have played a significant role in ferroptosis, mineral absorption, basal cell carcinoma, and related signal pathways. Besides, the drug likeness of quercetin obtained by Comparative Toxicogenomics Database was evaluated by Traditional Chinese Medicine Systems Pharmacology, and potential drug targets identified by PharmMapper and similar compounds identified by PubChem were selected for further research. Moreover, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed the relationship between quercetin and glycosylation. Furthermore, we performed experiments to verify that the protective effect of quercetin on iron overload cells is to inhibit the production of reactive oxygen species, limit intracellular iron, and degrade glycosaminoglycans. Finally, iron-induced intracellular iron overload caused ferroptosis, and quercetin and fisetin were potential ferroptosis inhibitors. In conclusion, our study revealed the correlation between hemochromatosis and ferroptosis, provided the relationship between the target of quercetin and glycosylation, and verified that quercetin and its similar compounds interfere with iron overload related disease. Our research may provide novel insights for quercetin and its structurally similar compounds as a potential nutritional supplement for iron overload related diseases.

S2545 Hypothyroidism Heralding Hereditary Hemochromatosis (HH): An Unusual Initial Manifestation

INTRODUCTION: HH is an autoimmune inherited disorder of iron metabolism causing unregulated iron absorption resulting in organ damage via iron overload. However, despite the iron excess, thyroid dysfunction is rarely seen. Instead, HH has strong association with autoimmune (AI) thyroiditis and is usually after diagnosis of HH in men. We describe a case of hypothyroidism in female caused by iron overload heralding manifestation of HH which is yet to be reported. CASE DESCRIPTION/METHODS: 30-year-old female, with history significant for hypothyroidism diagnosed 2 years prior, presented with progressively worsening abdominal distension for 1 week. She took laxatives without relief. She denied fever, chills , diarrhea, use of alcohol, smoking, Tylenol, or any illicit drug. Physical examination revealed scleral icterus, distended abdomen with shifting dullness. Lab work revealed elevated transaminases, bilirubin, & INR. Abdominal ultrasound showed ascites, micronodular cirrhosis with signs of portal hypertension (Figure 1). Patient underwent paracentesis which revealed transudative effusion. Cirrhosis investigation revealed homozygous for HFE C282Y gene mutation, consistent with type 1 HH. Due to Hemochromatosis, hypothyroidism secondary to iron deposition was suspected. Records revealed normal-high ferritin, iron saturation 50%, decreased T4, elevated TSH, normal TRH, & negative anti-TPO antibodies. FNAC showed lymphocytic infiltration. Additionally, excessive hemosiderin deposits were also visible (Figure 2). She responded to diuretics and fluid restriction. Hematology follow-up was planned for phlebotomy to prevent further iron overload and patient did well on 6-month follow up. DISCUSSION: HH associated with Autoimmune thyroid disease is well studied, followed by hypothyroidism due to iron deposition in anterior pituitary (seen with decreased TRH). However, no cases have been reported of hypothyroidism due to direct deposition of iron in thyroid gland. Secondly, hypothyroidism in HH occurs classically in men at a late/end stage after development of liver cirrhosis and/or other endocrinopathies. Women are less likely to develop iron overload related diseases due to iron loss through menstruation. Our case is unique because the patient was a young female who developed hypothyroidism prior to other manifestations of HH. Our case highlights the possible benefit of testing for deposition related diseases, particularly hemochromatosis, in case of unknown etiology for hypothyroidism.Figure 1.: Ultrasound abdomen demonstrating coarse echogenic liver, secondary to micronodular cirrhosis.Figure 2.: Fine Needle Aspiration cytology showing dense lymphocytic infiltrate with effacement of thyroid architecture. Hemosiderin deposits also visible (red arrow).

Curculigoside Protects against Excess-Iron-Induced Bone Loss by Attenuating Akt-FoxO1-Dependent Oxidative Damage to Mice and Osteoblastic MC3T3-E1 Cells.

Summary The present investigation found that curculigoside (CUR) can prevent excess-iron-induced bone loss in mice and cells through antioxidation and inhibiting excess-iron-induced phosphorylation of the Akt-FoxO1 pathway. CUR can attenuate the decreasing of cell viability, enhance autophagy, potentiate the antioxidant effect, and reduce apoptosis in MC3T3-E1 cells treated with excess iron through regulating the expression of FoxO1 target gene. Introduction Oxidative stress induced by iron overload is an important factor involved in primary osteoporosis disease and iron overload-related diseases. Curculigoside (CUR), a phenolic glycoside found abundantly in Curculigo orchioides Gaertn., has been demonstrated to possess antioxidant and antiosteoporotic properties. The aim of the present study is to explore the underlying molecular mechanism of CUR on excess-iron-induced bone loss in mice and osteoblastic MC3T3-E1 cells. Methods An iron-overload mice model was used to study the protective effects of CUR on bone loss induced by oxidative stress. Serum bone metabolism markers and antioxidant enzymes were also measured. To explore the antioxidant mechanism of CUR, the MC3T3-E1 osteoblastic cell line was used. Results In vivo studies showed that BMD and microarchitectural parameters were improved after a 3-month administration of CUR. CUR improved the biochemical parameters related to bone metabolism and the expressions of Runx2, OCN, and type 1 collagen and increased the formation of bone-mineralized nodules in vitro. CUR also inhibited ROS generation and increased the activities of antioxidant enzymes both in vivo and in vitro treated with excess iron. CUR can upregulate the level of FoxO1 and Nrf2, downregulate the level of p53 and the phosphorylation level of FoxO1, improve nuclear translocation of FoxO1, probably by inhibiting the IGFR/AKT signaling pathway, then increased cell viability and autophagy, and reduced apoptosis of MC3T3-E1 cells treated with excess iron by regulating the expression of FoxO1 target genes MnSOD, Gadd45a, Bim, FasL, and Rab7. Conclusions These results demonstrated that CUR was able to alleviate bone loss induced by oxidative stress resulting from iron overload, suggesting its potential use for the treatment of primary osteoporosis and bone loss in iron-overload-related diseases.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases.

ROS-triggered degradable iron-chelating nanogels: Safely improving iron elimination in vivo

Iron-mediated generation of highly toxic Reactive Oxygen Species (ROS) plays a major role in the process leading to iron overload-related diseases. The long-term subcutaneous administration of Deferoxamine (DFO) is currently clinically-approved to improve patient symptoms and survival. However, non-specific toxicity and short circulation times of the drug in humans often leads to poor patient compliance. Herein, thioketal-based ROS-responsive polymeric nanogels containing DFO moieties (rNG-DFO) were designed to chelate iron and to degrade under oxidative stimuli into fragments <10 nm to enhance excretion of iron-bound chelates. Serum ferritin levels and iron concentrations in major organs of IO mice decreased following treatment with rNG-DFO, and fecal elimination of iron-bound chelates increased compared to free DFO. Furthermore, rNG-DFO decreased iron mediated oxidative stress levels in vitro and reduced iron-mediated inflammation in the liver of IO mice. The study confirms that ROS-responsive nanogels may serve as a promising alternative to DFO for safer and more efficient iron chelation therapy.

HFE p.C282Y homozygosity predisposes to rapid serum ferritin rise after menopause: A genotype-stratified cohort study of hemochromatosis in Australian women.

Women who are homozygous for the p.C282Y mutation in the HFE gene are at much lower risk of iron overload-related disease than p.C282Y homozygous men, presumably because of the iron-depleting effects of menstruation and pregnancy. We used data from a population cohort study to model the impact of menstruation cessation at menopause on serum ferritin (SF) levels in female p.C282Y homozygotes, with p.C282Y/p.H63D simple or compound heterozygotes and those with neither p.C282Y nor p.H63D mutations (HFE wild types) as comparison groups. A sample of the Melbourne Collaborative Cohort Study was selected for the "HealthIron" study (n=1438) including all HFE p.C282Y homozygotes plus a random sample stratified by HFE-genotype (p.C282Y and p.H63D). The relationship between the natural logarithm of SF and time since menopause was examined using linear mixed models incorporating spline smoothing. For p.C282Y homozygotes, SF increased by a factor of 3.6 (95% CI (1.8, 7.0), P<0.001) during the first 10years postmenopause, after which SF continued to increase but at less than half the previous rate. In contrast, SF profiles for other HFE genotype groups increase more gradually and did not show a distinction between premenopausal and postmenopausal SF levels. Only p.C282Y homozygotes had predicted SF exceeding 200μg/L postmenopause, but the projected SF did not increase the risk of iron overload-related disease. These data provide the first documented evidence that physiological blood loss is a major factor in determining the marked gender difference in expression of p.C282Y homozygosity.

Effects of Phenyl-1-Pyridin-2yl-Ethanone (PPY)-Based Iron Chelators on the Formation of Methemoglobin

BackgroundIron chelators are used in the treatment of iron overload related diseases and are currently receiving a major attention as potential antitumor drugs. In recent studies, the antitumor activity of thiosemicarbazones-class of iron chelator, including Di-2-pyridilketone-4,4- dimethyl-3-thiosemicarbazone (Dp44mT) has been investigated in over 20 phase I and II clinical trials [1, 2,3]. Iron chelators were also considered as anti-HIV-1 agents. However, the main obstacle to using iron chelators in vivois the deleterious side effect of methemoglobinemia induced by some iron chelators that are able to scavenge electrons from the heme-bound iron in hemoglobin. In our previous studies, we developed novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators that we showed to increase IKBα expression, modulate CDK2 and CDK9 activities and inhibit HIV-1 [4].ObjectiveOur objective was to test the effect of PPYeT iron chelator for methemoglobin induction. The methemoglobin induction effect was compared with several additional iron chelators including Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and PPY analogues.MethodsFluorometric analysis was carried out in promonocytic THP-1 cells to evaluate the ability of our novel PPYeT iron chelator to reduce labile iron pool (LIP). The effect of PPYet on LIP was compared to the effect to SIH. Subsequently, spectrophotometric analysis was used to measure and quantify the production of methemoglobin in human red blood cells lysates and in isolated intact human red blood cells treated with PPYeT and various other iron chelators including DP44mT and DP4mT.ResultsPPYeT significantly reduced LIP in THP-1 cells overloaded with iron comparing to the cells treated with SIH. In RBC lysates and in intact RBC, PPYeT treatment showed notably lesser production of methemoglobin in comparison to DP44mT and DP4mT chelators. In RBC lysates, PPYeT produced about four-fold less methemoglobin than Dp44mT and ten-fold less than Dp4mT.ConclusionThe novel compound, PPYeT, shows a remarkably low ability to catalyze the formation of methemoglobin in human RBC lysates and also in intact RBCs as compared to Dp44mT. These findings indicate that PPYeT may be useful for future in vivo studies as it produces less methemoglobinemia. Further studies will evaluate the effect PPYeT as anti-cancer or anti HIV-1 inhibitor in vivo.AcknowledgmentsThis work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.

Icariin regulates systemic iron metabolism by increasing hepatic hepcidin expression through Stat3 and Smad1/5/8 signaling.

Systemic iron homeostasis is strictly controlled under normal conditions to ensure a balance between the absorption, utilization, storage and recycling of iron. The hepcidin-ferroportin (FPN) axis is of critical importance in the maintenance of iron homeostasis. Hepcidin deficiency gives rise to enhanced dietary iron absorption, as well as to increased iron release from macrophages, and this in turn results in iron accumulation in the plasma and organs, and is associated with a range of tissue pathologies. Low hepcidin levels have been demonstrated in most forms of hereditary hemochromatosis (HH), as well as in β-thalassemia. Therapies that increase hepcidin concentrations may potentially play a role in the treatment of these iron overload-related diseases. To date, natural compounds have not been extensively investigated for this purpose, to the best of our knowledge. Thus, in the present study, we screened natural compounds that have the potential to regulate hepcidin expression. By performing hepcidin promoter-luciferase assay, RT-qPCR and animal experiments, we demonstrated that icariin and berberine were potent stimulators of hepcidin transcription. Mechanistic experiments indicated that icariin and berberine increased hepcidin expression by activating the signal transducer and activator of transcription 3 (Stat3) and Smad1/5/8 signaling pathways. The induction of hepcidin was confirmed in mice following icariin administration, coupled with associated changes in serum and tissue iron concentrations. In support of these findings, the icariin analogues, epimedin A, B and C, also increased hepatic hepcidin expression. However, these changes were not observed in hepcidin-deficient [Hamp1-/- or Hamp1‑knockout (KO)] mice following icariin administration, thereby verifying hepatic hepcidin as the target of icariin. Although berberine exhibited a robust capacity to promote hepcidin expression in vitro, it failed to alter hepcidin expression in mice. Taken together, the findings of the present study suggest that icariin exhibits a robust capacity to increase hepatic hepcidin expression and to modulate systemic iron homeostasis. The present study therefore highlights the significance of using natural compounds to ameliorate iron disorders through the regulation of hepcidin expression.

Synthesis and evaluation of oxidation-responsive alginate-deferoxamine conjugates with increased stability and low toxicity

Deferoxamine is commonly used for iron-overload related diseases, its drawbacks such as instability and toxicity, however, significantly limited its utility in clinic. To address these issues, oxidation-responsive alginate-deferoxamine (Alg-DFO) conjugates were synthesized and their structure was characterized. The metabolism studies shown the conjugation of alginate significantly increased the stability of the DFO, with half-life more than 10 times longer than that of the free DFO. Moreover, the conjugates could not only quickly respond to oxidative stimuli and degradation, suggesting their potential to be cleared from the body by responding to iron-overload associated oxidative environment to avoid its accumulation and safety concern, but also protect iron binding capacity of the attached DFO from oxidation. The degradation mechanism for oxidative-response was proposed. In addition, the conjugates shown lower cytotoxicity compared to the free DFO. Taken together, the Alg-DFO conjugates synthesized in this work has promise for treating iron-overload related conditions.

Homozygosity For The C282Y Substitution In The HFE Gene: The Incomplete Penetrance And Variable Expressivity

The syndrome of hepatic cirrhosis diabetes and skin pigmentation (‘Bronze diabetes’) has been well documented, including its propensity to lead to hepatocellular cancer. However, this picture of advanced disease is much less common nowadays with increased awareness and early diagnosis. However, in addition to this, it has been increasingly recognised that in contrast to other diseases inherited as autosomal recessive traits, subjects carrying the genetic predisposition infrequently develop overt disease. This is due only in part to physiological and pathological blood loss, and further relevant genetic mutations have been anticipated. Indeed, an international consortium has recently identified that the genetic variant ( GNPAT ) has been identified as predisposing to iron overload related disease. Further mutations can be anticipated and will assist in early diagnosis and treatment as well as identifying subjects predisposed to significant iron overload.

Iron overload is rare in patients homozygous for the H63D mutation.

Previous research has suggested that the H63D HFE mutation is associated with elevated iron indexes. However, the true penetrance of this mutation remains unclear. To assess the proportion of H63D homozygotes with laboratory abnormalities consistent with iron overload. The present study was a retrospective analysis of all individuals referred for HFE genotyping in Newfoundland and Labrador between 1999 and 2009, who were found to be homozygous for the H63D mutation. Using electronic health records, results of ferritin, transferrin saturation, aspartate aminotransferase and alanine aminotransferase testing performed closest to the time of genetic testing were recorded for each patient. Iron overload was classified using previously published definitions from the HealthIron study. SPSS version 17.0 (IBM Corporation, USA) was used for descriptive statistics and to compare means using one-way ANOVA. Between 1999 and 2009, 170 individuals tested positive for H63D⁄H63D. At the time of genotyping, 28.8% had an elevated mean (± SD) ferritin level of 501±829 μg⁄L and 15.9% had an elevated transferrin saturation of 0.45±0.18. At genotyping, 94 individuals had sufficient data available to classify iron overload status. Only three (3.2%) had documented iron overload while the majority (85.1%) had no evidence of iron overload. Sixty individuals had follow-up data available and, of these, only four (6.7%) had documented iron overload, while 45 (75.0%) had no evidence of iron overload. Only one individual had evidence of iron overload-related disease at genotyping and at follow-up. H63D homozygosity was associated with an elevated mean ferritin level, but only 6.7% had documented iron overload at follow-up. The penetrance of the H63D mutation appeared to be low.

The Prevalence and Incidence of Iron Overload in C282Y Homozygotes

Purpose: Hereditary Hemochromatosis (HH) is a genetic disease of unregulated iron absorption via the GI tract that may result in inappropriate deposition and damage to various organs, including the liver and heart. The most common mutation implicated in HH is homozygosity for a Cysteine-Tyrosine substitution at position 282 on chromosome 6 (C282Y). It has been recognized that this genotype is incompletely penetrant, so that the risk of iron overload in C282Y homozygotes is not clear. The purpose of this study was to determine the risk of iron overload in patients homozygous for the C282Y mutation. Methods: This was a retrospective study of all persons in NL who had tested positive for C282Y homozygosity since test inception in 1999 to January of 2009. The study received full approval from the local ethics board. Electronic medical records were reviewed to obtain indices of iron overload (ferritin and transferrin saturation) and liver function tests, which were then recorded annually for as long as such data were available for each patient. Age, gender, and any available clinical data (such as liver biopsy, CT scan results, and need for phlebotomy) were also recorded. Patients were classified at each time point as having iron overload-related disease, iron overload, provisional iron overload, or no iron overload using the same definitions proposed by the HealthIron investigators. Microsoft Excel® 2010 was used to analyze the results. Results: A total of 360 patients tested positive for the C282Y mutation. Thus far, the records of 92 C282Y homozygotes have been reviewed. Nineteen cases had insufficient data available and were excluded from the analysis. The remaining 73 patients had mean follow-up of 6.7 years. Of these, 13 (18%) fit the criteria for documented iron overload, 38 (52%) were classified as provisional iron overload, and 22 (30%) had no evidence of iron overload. Eleven of the 13 patients (85%) with documented iron overload were were male, and the average age at genotyping was 49.4 years. No patients in the provisional or no evidence categories at baseline developed documented iron overload by the end of the study period. Conclusion: In this study, 18% of C282Y homozygotes satisfied the criteria for documented iron overload at the time of genotyping. No C282Y homozygotes without iron overload at diagnosis developed it during the study period.

Monitoring intracellular labile iron pools: A novel fluorescent iron(iii) sensor as a potential non-invasive diagnosis tool

The physiological and pathophysiological importance of intracellular redox active "labile" iron has created a significant need for improved noninvasive diagnostic tools to reliably monitor iron metabolism in living cells. In this context, fluorescent iron-sensitive chemosensors in combination with digital fluorescence spectroscopic methods have proven to be highly sensitive and indispensable tools to determine cellular iron homeostasis. Recently, application of fluorescent iron sensors has led to the identification of a complex sub-cellular iron compartmentation. Cell organelle-specific iron sensors will significantly contribute to enhance fundamental knowledge of cellular iron trafficking, representing a crucial prerequisite for the future development of therapeutic strategies in iron dysregulatory diseases. Here we present physicochemical characterization and functional investigation of a new 3-hydroxypyridin-4-one based fluorescent iron(III) sensor, exclusively monitoring labile iron pools in the endosomal/lysosomal compartments. In vitro studies of the fluorescein labeled probe were carried out in murine bone marrow derived macrophages. Endosomal/lysosomal accumulation of the probe was revealed by confocal microscopy. Flow cytometry analyses demonstrated high sensitivity of the probe towards exogenous alterations of intracellular iron concentrations as well as in response to the chelation potency of iron chelators, clinically approved for treatment of iron-overload related diseases.

Chloroquine Modulates Arginase Activity by Limiting the Availability of Intracellular Mobile Iron Pool in Human Erythroid Cells.

Increased iron absorption associated with intensive erythropoiesis and iron deposition as a result of continuous hemolysis contribute to cellular injuries in sickle cell disease (SCD). Iron overload seems to be a predisposing factor of disease severity and mortality in significant number of adult SCD and β-thalassemia patients. Indeed, there is now an impressive body of evidence indicating that increased intracellular iron pool might be the hallmark of mammalian cell susceptibility to oxidative stress. Recently, we showed that cysteine, in the presence of molecular iron, promotes arginase activity by driving the Fenton reaction in sickle erythrocytes (unpublished report). It has been suggested that the reduction of arginine bioavailability for nitric oxide (NO) production secondary to increased plasma arginase levels in SCD may contribute to severe pathophysiological derangements and increase mortality. We have previously shown that chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a linear competitive mode of inhibition on sickle erythrocyte arginase. In the present report we showed that treatment of cultured human erythroleukemic cells with 100 or 200 μM iron (ferric ammonium citrate) induced a significant increase in cell proliferation with corresponding increase in arginase activity. By using specific antibodies it was demonstrated that the increased arginase activity correlated with an increase in arginase-1 levels in the cells. Upon treatment of cells with 10 μM CQ, in the presence or absence of ferric ammonium citrate (FAC), the levels of arginase enzyme decreased by 33.5 % and 36.7 % respectively. In an effort to expand our understanding of the link between intracellular iron and CQ-mediated modulation of arginase activity, FAC-treated cells were co-incubated with 10 μM CQ for 48 hrs and the intracellular and membrane associated iron levels were determined by nitrilotriacetic acid (NTA)-ultracentrifugation assay. Our results indicate that CQ maximally enhanced the sequestration of intracellular iron by 39.3 % and that no effects of CQ were noted on the membrane associated iron levels. Further the level of ferritin was reduced by 29.4 % in CQ-treated cells in comparison with the FAC-treated cells alone as determined by immunosorbent assay. This data suggests that the observed diminished ferritin levels in the presence of CQ may be due to repression by translational regulators of ferritin function secondary to low intracellular iron levels, as evidenced in this study. Because treatment of cells with an intracellular iron chelator deferoxamine, in combination with CQ, caused a significant decrease in the levels of ferritin (60 % vs control; p = 0.006) and consequent reduction in ARG-1 levels (58.3 % vs control), we conclude that the sequestration of intracellular iron contributes in part to the CQ-mediated inhibition of arginase activity. We further investigated the effects of CQ on ferritin-Fe in a cell free system, reasoning that the effects of CQ on the intracellular iron pool may involve the modulation of iron mobilization from iron sources, such as ferritin (the main intracellular iron storage protein). To this end, purified human ferritin was incubated at predetermined time intervals in the presence or absence of 10–100 μM CQ at 37°C and the extent of NTA-mediated iron mobilization from ferritin was determined. Our results showed that CQ limits the mobilization of iron from ferritin in a dose-dependent manner. Our presented data demonstrate, for the first time, that CQ-mediated limitation of intracellular mobile iron pool modulates arginase activity as well as arginase-1 levels. We therefore speculate that an extreme increase in intracellular iron pool may result in the stimulation of arginase activity as observed in hemolytic diseases. In conclusion, we believe that our results represent a new and important improvement in understanding the pleiotropic effects of CQ on arginase activity, with specific reference to its participation on the limitation of intracellular iron availability in erythroid cells. Hence incorporating this template into structure-based enzyme design studies could lead to the production of new forms of efficient arginase inhibitors, which could be useful as therapeutic regimen in hemoglobinopathies and other iron overload related diseases.

Determinants for Iron Overload-Related Disease in Siblings of Probands with Clinically Detected HFE Hereditary Hemochromatosis: The Hemochromatosis Family Study.

The clinical expression of HFE-associated hereditary hemochromatosis (HH) gen is highly variable and may be influenced by nongenetic factors and coinherited genetic modifiers, complicating early screening options to prevent iron-overload related disease. The aim of this study was to verify the existence of HH-related disease in C282Y homozygous siblings of C282Y homozygous probands with clinically detected HFE-related HH and to identify factors predictive for the iron-related disease within these siblings. To this end, C282Y homozygous (n=110, males n=53) and non-homozygous siblings (n=318, males n=145) of 224 probands were compared for levels of serum iron parameters, and self-reported environmental and lifestyle factors and previously diagnosed HH-related diseases. Compared to non-homozygous C282Y siblings, C282Y homozygous siblings more often mentioned to have been diagnosed with arthropathy (Odds Ratio [OR] 2.76, 95% Confidential Interval [CI] 1.71–4.46) and liver disease (OR 2.90, 95%CI 1.27– 6.62). Using multivariate logistic regression modelling, genotype (OR 2.29, 95%CI 1.04– 5.02), age (OR 1.07, 95% CI 1.04–1.09) and gender (OR 1.71, 95%CI 1.04–2.80) were found predictive for the development of iron-associated organ disease. With genotype in the model, there was neither an additive predictive value of the serum iron parameters, nor of body mass index (BMI) or alcohol intake. However, when the predictive value of the iron parameters was analyzed in siblings above 55 yrs, the input of the serum ferritin levels was also significant, with a less prominent influence of gender. In conclusion, our results show that the prevalence of hemochromatosis-attributed morbid conditions is increased in the C282Y homozygous siblings compared to their non-homozygous counterparts. Results furthermore suggest that age and gender, but not BMI and alcohol intake, add to the identification of C282Y homozygous siblings most at risk to develop hemochromatosis-associated disease. These findings will be instrumental in the definition of a high-risk group for iron overload-related disease among siblings of clinically detected C282Y homozygous probands and may contribute to the cost-effectiveness of family screening.