Hemochromatosis: Ferroptosis, ROS, Gut Microbiome, and Clinical Challenges with Alcohol as Confounding Variable.

Hemochromatosis represents clinically one of the most important genetic storage diseases of the liver caused by iron overload, which is to be differentiated from hepatic iron overload due to excessive iron release from erythrocytes in patients with genetic hemolytic disorders. This disorder is under recent mechanistic discussion regarding ferroptosis, reactive oxygen species (ROS), the gut microbiome, and alcohol abuse as a risk factor, which are all topics of this review article. Triggered by released intracellular free iron from ferritin via the autophagic process of ferritinophagy, ferroptosis is involved in hemochromatosis as a specific form of iron-dependent regulated cell death. This develops in the course of mitochondrial injury associated with additional iron accumulation, followed by excessive production of ROS and lipid peroxidation. A low fecal iron content during therapeutic iron depletion reduces colonic inflammation and oxidative stress. In clinical terms, iron is an essential trace element required for human health. Humans cannot synthesize iron and must take it up from iron-containing foods and beverages. Under physiological conditions, healthy individuals allow for iron homeostasis by restricting the extent of intestinal iron depending on realistic demand, avoiding uptake of iron in excess. For this condition, the human body has no chance to adequately compensate through removal. In patients with hemochromatosis, the molecular finetuning of intestinal iron uptake is set off due to mutations in the high-FE2+ (HFE) genes that lead to a lack of hepcidin or resistance on the part of ferroportin to hepcidin binding. This is the major mechanism for the increased iron stores in the body. Hepcidin is a liver-derived peptide, which impairs the release of iron from enterocytes and macrophages by interacting with ferroportin. As a result, iron accumulates in various organs including the liver, which is severely injured and causes the clinically important hemochromatosis. This diagnosis is difficult to establish due to uncharacteristic features. Among these are asthenia, joint pain, arthritis, chondrocalcinosis, diabetes mellitus, hypopituitarism, hypogonadotropic hypogonadism, and cardiopathy. Diagnosis is initially suspected by increased serum levels of ferritin, a non-specific parameter also elevated in inflammatory diseases that must be excluded to be on the safer diagnostic side. Diagnosis is facilitated if ferritin is combined with elevated fasting transferrin saturation, genetic testing, and family screening. Various diagnostic attempts were published as algorithms. However, none of these were based on evidence or quantitative results derived from scored key features as opposed to other known complex diseases. Among these are autoimmune hepatitis (AIH) or drug-induced liver injury (DILI). For both diseases, the scored diagnostic algorithms are used in line with artificial intelligence (AI) principles to ascertain the diagnosis. The first-line therapy of hemochromatosis involves regular and life-long phlebotomy to remove iron from the blood, which improves the prognosis and may prevent the development of end-stage liver disease such as cirrhosis and hepatocellular carcinoma. Liver transplantation is rarely performed, confined to acute liver failure. In conclusion, ferroptosis, ROS, the gut microbiome, and concomitant alcohol abuse play a major contributing role in the development and clinical course of genetic hemochromatosis, which requires early diagnosis and therapy initiation through phlebotomy as a first-line treatment.

Ovaries of estrogen receptor 1-deficient mice show iron overload and signs of aging.

Introduction: Estrogens are crucial regulators of ovarian function, mediating their signaling through binding to estrogen receptors. The disruption of the estrogen receptor 1 (Esr1) provokes infertility associated with a hemorrhagic, cystic phenotype similar to that seen in diseased or aged ovaries. Our previous study indicated the possibility of altered iron metabolism in Esr1-deficient ovaries showing massive expression of lipocalin 2, a regulator of iron homeostasis. Methods: Therefore, we examined the consequences of depleting Esr1 in mouse ovaries, focusing on iron metabolism. For that reason, we compared ovaries of adult Esr1-deficient animals and age-matched wild type littermates. Results and discussion: We found increased iron accumulation in Esr1-deficient animals by using laser ablation inductively coupled plasma mass spectrometry. Western blot analysis and RT-qPCR confirmed that iron overload alters iron transport, storage and regulation. In addition, trivalent iron deposits in form of hemosiderin were detected in Esr1-deficient ovarian stroma. The depletion of Esr1 was further associated with an aberrant immune cell landscape characterized by the appearance of macrophage-derived multinucleated giant cells (MNGCs) and increased quantities of macrophages, particularly M2-like macrophages. Similar to reproductively aged animals, MNGCs in Esr1-deficient ovaries were characterized by iron accumulation and strong autofluorescence. Finally, deletion of Esr1 led to a significant increase in ovarian mast cells, involved in iron-mediated foam cell formation. Given that these findings are characteristics of ovarian aging, our data suggest that Esr1 deficiency triggers mechanisms similar to those associated with aging.

DMT1-mediated iron overload accelerates cartilage degeneration in Hemophilic Arthropathy through the mtDNA-cGAS-STING axis.

INTRODUCTION: Excess iron contributes to Hemophilic Arthropathy (HA) development. Divalent metal transporter 1 (DMT1) delivers iron into the cytoplasm, thus regulating iron homeostasis. OBJECTIVES: We aimed to investigate whether DMT1-mediated iron homeostasis is involved in bleeding-induced cartilage degeneration and the molecular mechanisms underlying iron overload-induced chondrocyte damage. METHODS: This study established an in vivo HA model by puncturing knee joints of coagulation factor VIII gene knockout mice with a needle, and mimicked iron overload conditions in vitro by treatment of Ferric ammonium citrate (FAC). RESULTS: We demonstrated that blood exposure caused iron overload and cartilage degeneration, as well as elevated expression of DMT1. Furthermore, DMT1 silencing alleviated blood-induced iron overload and cartilage degeneration. In hemophilic mice, articular cartilage degeneration was also suppressed by intro-articularly injection of DMT1 adeno-associated virus 9 (AAV9). Mechanistically, RNA-sequencing analysis indicated the association between iron overload and cGAS-STING pathway. Further, iron overload triggered mtDNA-cGAS-STING pathway activation, which could be effectively mitigated by DMT1 silencing. Additionally, we discovered that RU.521, a potent Cyclic GMP-AMP Synthase (cGAS) inhibitor, successfully suppressed the downward cascades of cGAS-STING, thereby protecting against chondrocyte damage. CONCLUSION: Taken together, DMT1-mediated iron overload promotes chondrocyte damage and murine HA development, and targeted DMT1 may provide therapeutic and preventive approaches in HA.

Coinheritance of hereditary spherocytosis with haemochromatosis: next-generation sequencing reveals.

We report the case of a man in his 50s with extravascular haemolysis, fluctuating indirect hyperbilirubinaemia, elevated transferrin saturation with hyperferritinaemia and normal liver enzymes. Spherocytes were detected in a blood smear and a mutation of unknown significance, c.1626+1G>A p.?, in intron 13 of the SLC4A1 gene, was identified by next-generation sequencing (NGS). The same mutation was found in his daughter, who presented with similar laboratory changes, confirming the diagnosis of hereditary spherocytosis. Abdominal MRI showed hepatosplenomegaly with hepatic iron overload. In this context of haemolysis (without anaemia) and iron overload, a diagnosis of haemochromatosis was presumed. NGS confirmed the presence of the variants p.(His63Asp) and p.(Cys282Tyr) in heterozygosity in the HFE gene. We report this case for the rarity of co-existing two haematological diseases counteracting each other.

Suppression of Growth Differentiation Factor 15 Gene Expression by Curcumin in Patients with Beta-Thalassemia Intermedia.

BACKGROUND: ß-thalassemia is an inherited disorder caused by defects in the synthesis of the beta-globin chain. One of the significant clinical complications in ß-thalassemia intermedia is iron overload toxicity, which may be attributed to reduced levels of hepcidin. This reduction in hepcidin leads to increased absorption of iron in the intestines, ultimately resulting in iron overload. The objective of this study was to assess the impact of curcumin on the expression of growth differentiating factor-15 (GDF-15) and hepcidin genes in patients with beta-thalassemia intermedia. METHODS: This study was designed as a randomized controlled double-blind clinical trial. Prior to and after the intervention period with curcumin, a blood sample of 5 mL was collected from both the placebo and curcumin-treated groups for the assessment of hepcidin and growth differentiating factor-15 gene expression. RESULTS: This study revealed a significant reduction in the expression of growth differentiating factor-15 in the curcumin group compared to the placebo group during the 3-month treatment period. Furthermore, curcumin supplementation led to a remarkable 10.1-fold increase in the levels of hepcidin in the curcumin group compared to the placebo group. CONCLUSIONS: The results of this study show that curcumin administration increases the mRNA levels of hepcidin in whole blood of thalassemia intermedia patients and supports the idea that curcumin could be a potential treatment to reduce suppression of hepcidin in thalassemias and other iron-loading anemias. CONCLUSIONS: The results of this study show that curcumin administration increases the mRNA levels of hepcidin in whole blood of thalassemia intermedia patients and supports the idea that curcumin could be a potential treatment to reduce suppression of hepcidin in thalassemias and other iron-loading anemias.

Iron overload induces dysplastic erythropoiesis and features of myelodysplasia in Nrf2-deficient mice.

Iron overload (IOL) is hypothesized to contribute to dysplastic erythropoiesis. Several conditions, including myelodysplastic syndrome, thalassemia and sickle cell anemia, are characterized by ineffective erythropoiesis and IOL. Iron is pro-oxidant and may participate in the pathophysiology of these conditions by increasing genomic instability and altering the microenvironment. There is, however, lack of in vivo evidence demonstrating a role of IOL and oxidative damage in dysplastic erythropoiesis. NRF2 transcription factor is the master regulator of antioxidant defenses, playing a crucial role in the cellular response to IOL in the liver. Here, we crossed Nrf2-/- with hemochromatosis (Hfe-/-) or hepcidin-null (Hamp1-/-) mice. Double-knockout mice developed features of ineffective erythropoiesis and myelodysplasia including macrocytic anemia, splenomegaly, and accumulation of immature dysplastic bone marrow (BM) cells. BM cells from Nrf2/Hamp1-/- mice showed increased in vitro clonogenic potential and, upon serial transplantation, recipients disclosed cytopenias, despite normal engraftment, suggesting defective differentiation. Unstimulated karyotype analysis showed increased chromosome instability and aneuploidy in Nrf2/Hamp1-/- BM cells. In HFE-related hemochromatosis patients, NRF2 promoter SNP rs35652124 genotype TT (predicted to decrease NRF2 expression) associated with increased MCV, consistent with erythroid dysplasia. Our results suggest that IOL induces ineffective erythropoiesis and dysplastic hematologic features through oxidative damage in Nrf2-deficient cells.

A newly identified ferritin L-subunit variant results in increased proteasomal subunit degradation, impaired complex assembly, and severe hypoferritinemia.

Ferritin is a hetero-oligomeric nanocage, composed of 24 subunits of two types, FTH1 and FTL. It protects the cell from excess reactive iron, by storing iron in its cavity. FTH1 is essential for the recruitment of iron into the ferritin nanocage and for cellular ferritin trafficking, whereas FTL contributes to nanocage stability and iron nucleation inside the cavity. Here we describe a female patient with a medical history of severe hypoferritinemia without anemia. Following inadequate heavy IV iron supplementation, the patient developed severe iron overload and musculoskeletal manifestations. However, her serum ferritin levels rose only to normal range. Genetic analyses revealed an undescribed homozygous variant of FTL (c.92A > G), which resulted in a Tyr31Cys substitution (FTLY31C ). Analysis of the FTL structure predicted that the Y31C mutation will reduce the variant's stability. Expression of the FTLY31C variant resulted in significantly lower cellular ferritin levels compared with the expression of wild-type FTL (FTLWT ). Proteasomal inhibition significantly increased the initial levels of FTLY31C , but could not protect FTLY31C subunits from successive degradation. Further, variant subunits successfully incorporated into hetero-polymeric nanocages in the presence of sufficient levels of FTH1. However, FTLY31C subunits poorly assembled into nanocages when FTH1 subunit levels were low. These results indicate an increased susceptibility of unassembled monomeric FTLY31C subunits to proteasomal degradation. The decreased cellular assembly of FTLY31C -rich nanocages may explain the low serum ferritin levels in this patient and emphasize the importance of a broader diagnostic approach of hypoferritinemia without anemia, before IV iron supplementation.

Haemochromatosis in children: A national retrospective cohort promoted by the A.I.E.O.P. (Associazione Italiana Emato-Oncologia Pediatrica) study group.

Haemochromatosis (HC) encompasses a range of genetic disorders. HFE-HC is by far the most common in adults, while non-HFE types are rare due to mutations of HJV, HAMP, TFR2 and gain-of-function mutations of SLC40A1. HC is often unknown to paediatricians as it is usually asymptomatic in childhood. We report clinical and biochemical data from 24 paediatric cases of HC (10 cases of HFE-, 5 TFR2-, 9 HJV-HC), with a median follow-up of 9.6 years. Unlike in the adult population, non-HFE-HC constitutes 58% (14/24) of the population in our series. Transferrin saturation was significantly higher in TFR2- and HJV-HC compared to HFE-HC, and serum ferritin and LIC were higher in HJV-HC compared to TFR2- and HFE-HC. Most HFE-HC subjects had relatively low ferritin and LIC at the time of diagnosis, so therapy could be postponed for most of them after the age of 18. Our results confirm that HJV-HC is a severe form already in childhood, emphasizing the importance of early diagnosis and treatment to avoid the development of organ damage and reduce morbidity and mortality. Although phlebotomies were tolerated by most patients, oral iron chelators could be a valid option in early-onset HC.

The interplay of miRNAs and ferroptosis in diseases related to iron overload.

Ferroptosis has been conceptualized as a novel cell death modality distinct from apoptosis, necroptosis, pyroptosis and autophagic cell death. The sensitivity of cellular ferroptosis is regulated at multiple layers, including polyunsaturated fatty acid metabolism, glutathione-GPX4 axis, iron homeostasis, mitochondria and other parallel pathways. In addition, microRNAs (miRNAs) have been implicated in modulating ferroptosis susceptibility through targeting different players involved in the execution or avoidance of ferroptosis. A growing body of evidence pinpoints the deregulation of miRNA-regulated ferroptosis as a critical factor in the development and progression of various pathophysiological conditions related to iron overload. The revelation of mechanisms of miRNA-dependent ferroptosis provides novel insights into the etiology of diseases and offers opportunities for therapeutic intervention. In this review, we discuss the interplay of emerging miRNA regulators and ferroptosis players under different pathological conditions, such as cancers, ischemia/reperfusion, neurodegenerative diseases, acute kidney injury and cardiomyopathy. We emphasize on the relevance of miRNA-regulated ferroptosis to disease progression and the targetability for therapeutic interventions.

UCHL1 deficiency upon HCMV infection induces vascular endothelial inflammatory injury mediated by mitochondrial iron overload.

Human cytomeglovirus (HCMV) infection predisposes blood vessels to atherosclerosis (AS) and post-transplantation restenosis, but the underlying molecular basis remains elusive. Here, we found that HCMV infection activates AIM2 inflammasome and pyroptosis in vascular endothelial cells by inducing mitochondrial iron overload. Mechanistically, under normal conditions, ubiquitin carboxyl terminal hydrolase-L1 (UCHL1) was identified as a DUB enzyme that interacts with, deubiquitylates, and stabilizes ferredoxin reductase (FDXR), an important mitochondrial protein that regulates mitochondral iron homeostasis. However, HCMV infection induces the aberrantly elevated m6A modification and R-loops, the three-stranded DNA-DNA:RNA hybrid structures. The expression of UCHL1 was remarkably reduced by m6A modification-mediated mRNA decay and R-loop-dependent transcriptional termination after HCMV infection. Deficiency of UCHL1 causes ubiquitination and degradation of FDXR. Loss of FDXR induces the mitochondrial iron overload, which consequently leads to AIM2 inflammasome activation and endothelial injury. Moreover, both downregulation expression of UCHL1 and related inflammatory injury in vascular endothelium was observed in MCMV-infected mice. Notably, STM2457, a METTL3 specific inhibitor, restores the expression of UCHL1 upon HCMV infection, thereby inhibiting the inflammatory injury of vascular endothelial cells. Our findings delineate a novel mechnism involved in HCMV-induced inflammatory injury to vascular endothelium and implicate the role of METTL3 inhibitor as a potential therapeutic approach.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Testing and Management of Iron Overload After Genetic Screening-Identified Hemochromatosis.

Importance: HFE gene-associated hereditary hemochromatosis type 1 (HH1) is underdiagnosed, resulting in missed opportunities for preventing morbidity and mortality. Objective: To assess whether screening for p.Cys282Tyr homozygosity is associated with recognition and management of asymptomatic iron overload. Design, Setting, and Participants: This cross-sectional study obtained data from the Geisinger MyCode Community Health Initiative, a biobank of biological samples and linked electronic health record data from a rural, integrated health care system. Participants included those who received a p.Cys282Tyr homozygous result via genomic screening (MyCode identified), had previously diagnosed HH1 (clinically identified), and those negative for p.Cys282Tyr homozygosity between 2017 and 2018. Data were analyzed from April 2020 to August 2023. Exposure: Disclosure of a p.Cys282Tyr homozygous result. Main Outcomes and Measures: Postdisclosure management and HFE-associated phenotypes in MyCode-identified participants were analyzed. Rates of HFE-associated phenotypes in MyCode-identified participants were compared with those of clinically identified participants. Relevant laboratory values and rates of laboratory iron overload among participants negative for p.Cys282Tyr homozygosity were compared with those of MyCode-identified participants. Results: A total of 86 601 participants had available exome sequences at the time of analysis, of whom 52 994 (61.4%) were assigned female at birth, and the median (IQR) age was 62.0 (47.0-73.0) years. HFE p.Cys282Tyr homozygosity was disclosed to 201 participants, of whom 57 (28.4%) had a prior clinical HH1 diagnosis, leaving 144 participants who learned of their status through screening. There were 86 300 individuals negative for p.Cys282Tyr homozygosity. After result disclosure, among MyCode-identified participants, 99 (68.8%) had a recommended laboratory test and 36 (69.2%) with laboratory or liver biopsy evidence of iron overload began phlebotomy or chelation. Fifty-three (36.8%) had iron overload; rates of laboratory iron overload were higher in MyCode-identified participants than participants negative for p.Cys282Tyr homozygosity (females: 34.1% vs 2.1%, P < .001; males: 39.0% vs 2.9%, P < .001). Iron overload (females: 34.1% vs 79.3%, P < .001; males: 40.7% vs 67.9%, P = .02) and some liver-associated phenotypes were observed at lower frequencies in MyCode-identified participants compared with clinically identified individuals. Conclusions and Relevance: Results of this cross-sectional study showed the ability of genomic screening to identify undiagnosed iron overload and encourage relevant management, suggesting the potential benefit of population screening for HFE p.Cys282Tyr homozygosity. Further studies are needed to examine the implications of genomic screening for health outcomes and cost-effectiveness.

Revealing the contribution of iron overload-brown adipocytes to iron overload cardiomyopathy: Insights from RNA-seq and exosomes coculture technology.

Iron overload has been demonstrated to be associated with insulin resistance, iron overload cardiomyopathy (IOC). Brown adipose tissue (BAT) is emerging as a novel therapeutic target for the treatment of various diseases, not only because of its capacity for dissipating excess energy via non-shivering thermogenesis, but also because of its implication in physiological and pathophysiological processes. However, little attention has been devoted to the precise alterations and impacts of iron overload-BAT. We conducted RNA-Seq analysis on BAT samples obtained from mice subjected to a high iron diet (HID) or a normal chow diet (CON), respectively. The RNA-seq transcriptomic analysis revealed that 1,289 differentially expressed RNAs (DEGs) were identified, with a higher number of the downregulated genes (910 genes) compared to the upregulated genes (379 genes). The results of Gene Ontology (GO) and The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the downregulated DEGs were primarily involved in hypertrophic cardiomyopathy, dilated cardiomyopathy, which were defined as IOC under the iron overload condition. The association between iron overload-BAT with cardiomyopathy was further investigated using exosome coculture technology. Our results demonstrated that the exosomes derived from ferric citrate treated-mature HIB 1B brown adipocytes, could be internalized by HL-1 cardiomyocytes, and contributed to the dysfunction in these cells. The present study has revealed the alterations and impacts of iron overload-BAT, particularly on the onset of IOC via not only RNA-seq but also exosomes coculture technology. The outputs might shed light on the novel therapeutic strategies for the treatment of IOC.

Synovium is a sensitive tissue for mapping the negative effects of systemic iron overload in osteoarthritis: identification and validation of two potential targets.

BACKGROUND: The prevention and treatment of osteoarthritis (OA) pose a major challenge in its research. The synovium is a critical tissue in the systematic treatment of OA. The present study aimed to investigate potential target genes and their correlation with iron overload in OA patients. METHODS: The internal datasets for analysis included the microarray datasets GSE46750, GSE55457, and GSE56409, while the external datasets for validation included GSE12021 and GSE55235. The GSE176308 dataset was used to generate single-cell RNA sequencing profiles. To investigate the expression of the target genes in synovial samples, quantitative reverse transcription-PCR, western blotting, and immunohistochemical assay were conducted. ELISA was used to detect the levels of ferritin and Fe2+ in both serum and synovium. RESULTS: JUN and ZFP36 were screened from the differentially expressed genes, and their mRNA were significantly reduced in the OA synovium compared to that in normal synovium. Subsequently, complex and dynamically evolving cellular components were observed in the OA synovium. The mRNA level of JUN and ZFP36 differed across various cell clusters of OA synovium and correlated with immune cell infiltration. Moreover, ferritin and Fe2+ were significantly increased in the serum and synovium of OA patients. Further, we found that JUN elevated and ZFP36 decreased at protein level. CONCLUSIONS: The synovium is a sensitive tissue for mapping the adverse effects of systemic iron overload in OA. JUN and ZFP36 represent potential target genes for attenuating iron overload during OA treatment. Some discrepancies between the transcription and protein levels of JUN suggest that post-transcriptional modifications may be implicated. Future studies should also focus on the roles of JUN and ZFP36 in inducing changes in cellular components in the synovium during OA pathogenesis.

REPIN1 regulates iron metabolism and osteoblast apoptosis in osteoporosis.

Osteoporosis is not well treated due to the difficulty of finding commonalities between the various types of it. Iron homeostasis is a vital component in supporting biochemical functions, and iron overload is recognized as a common risk factor for osteoporosis. In this research, we found that there is indeed evidence of iron accumulation in the bone tissue of patients with osteoporosis and REPIN1, as an origin specific DNA binding protein, may play a key role in this process. We revealed that sh-Repin1 therapy can rescue bone loss in an iron-overload-induced osteoporosis mouse model. Knockdown of Repin1 can inhibit apoptosis and enhance the resistance of osteoblasts to iron overload toxicity. REPIN1 promoted apoptosis by regulating iron metabolism in osteoblasts. Mechanistically, knockdown of Repin1 decreased the expression of Lcn2, which ameliorated the toxic effects of intracellular iron overload. The anti-iron effect of lentivirus sh-Repin1 was partially reversed or replicated by changing LCN2 expression level via si-RNA or plasmid, which indirectly verified the key regulatory role of LCN2 as a downstream target. Furthermore, the levels of BCL2 and BAX, which play a key role in the mitochondrial apoptosis pathway, were affected. In summary, based on the results of clinical specimens, animal models and in vitro experiments, for the first time, we proved the key role of REPIN1 in iron metabolism-related osteoporosis.

OPG/RANK/RANKL axis relation to cardiac iron-overload in children with transfusion-dependent thalassemia.

OPG/RANK/RANKL axis was reportedly involved in initiating various diseases, especially bone and cardiovascular diseases. This study aimed to assess the relationship between some OPG, RANK, and RANKL polymorphisms and alleles and iron-overload-induced cardiomyopathy in children with transfusion-dependent thalassemia (TDT). This study included 80 TDT children and 80 age and sex-matched controls. Real-time PCR was done for rs207318 polymorphism for the OPG gene and rs1805034, rs1245811, and rs75404003 polymorphisms for the RANK gene, and rs9594782 and rs2277438 polymorphisms for the RANKL gene. Cardiac T2* MRI and ejection fraction (EF) were done to assess the myocardial iron status and cardiac function. In this study, there were no significant differences in frequencies of the studied polymorphisms between cases and controls (p > 0.05 in all). In TDT children, OPG rs2073618 (G > C) had a significant relation to myocardial iron overload (p = 0.02). Its C allele had significantly more frequent normal EF than its G allele (p = 0.04). RANK rs75404403 (C > DEL) had a significant relation to cardiac dysfunction (p = 0.02). Moreover, the C allele of that gene had significantly more frequent affected EF than its DEL allele (p = 0.02). The A allele of RANKL rs2277438 (G > A) had significantly less frequent severe cardiac iron overload than the G allele (p = 0.04). In conclusion, the OPG/ RANK/RANKL genes may act as genetic markers for iron-induced cardiomyopathy in TDT children. Some of the studied genes' polymorphisms and alleles were significantly related to myocardial iron overload and cardiac dysfunction in TDT children.

MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter.

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.

Hyperferritinemia and non-HFE hemochromatosis: differential diagnosis and workup.

Hyperferritinemia is a common reason for referral to a hepatogastroenterologist. The most frequent causes are not associated with iron overload (e.g. inflammatory diseases, alcohol abuse, metabolic syndrome, etc.). However, hyperferritinemia can also be caused by a genetic variant in one of the iron regulatory genes, called hereditary hemochromatosis, often but not always associated with iron overload. A variation in the human Hemostatic Iron Regulator protein (HFE) gene is the most common genotype, but many other variants have been described. In this paper we discuss two cases of rare hyperferritinemia associated disorders, ferroportin disease and hyperferritinemia-cataract syndrome. We also propose an algorithm for evaluating hyperferritinemia, facilitating a correct diagnosis and preventing potentially unnecessary examinations and therapeutic actions.

A form of inherited hyperferritinemia associated with bi-allelic pathogenic variants of STAB1.

Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.

Iron overload due to SLC40A1 mutation of type 4 hereditary hemochromatosis.

Hereditary hemochromatosis type 4 is an autosomal-dominant inherited disease characterized by a mutation in the SLC40A1 gene encoding ferroportin. This condition can be further subdivided into types 4A (loss-of-function mutations) and 4B (gain-of-function mutations). To date, only a few cases of type 4B cases have been reported, and the treatment has not been clearly mentioned. Here, we report a genotype of hereditary hemochromatosis type 4B involving the heterozygous mutation c.997 T > C (p. Tyr333His) in SLC40A1. The patient was treated with red blood cell apheresis every month for 1 year, followed by oral deferasirox, and the combined therapy was found to be effective.