In Vivo Disruption Of The Hepcidin−Ferroportin Regulatory Circuitry Causes Fatal Systemic and Exocrine Pancreatic Iron Overload

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 175-175
Author(s):  
Sandro Altamura ◽  
Hermann Josef Gröne ◽  
Regina Kessler ◽  
Bruno Galy ◽  
Matthias W. Hentze ◽  
...  
Keyword(s):  
Iron Overload ◽  
Molecular Mechanisms ◽  
Histological Analysis ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Transferrin Saturation ◽  
Iron Accumulation ◽  
Blue Staining ◽  
Hepatic Iron

Abstract Systemic iron levels are tightly controlled by the hepatic hormone hepcidin in response to iron availability, inflammation, hypoxia or the iron demand for erythropoiesis. Hepcidin binds to the iron export protein ferroportin (FPN1) to regulate iron release from exporting cells. A mutation of cysteine 326 (C326S) of FPN1 was reported in a patient with non−classical ferroportin disease (Sham et al, 2005) and shown to abrogate hepcidin binding in vitro (Fernandes et al, 2009). To study consequences of the disruption of the hepcidin−ferroportin interaction in vivo, we generated the first knock−in mouse model of C326S non−classical ferroportin disease. Mice with either heterozygous or homozygous C326S FPN alleles are viable and fertile. At 8−weeks of age both heterozygous and homozygous mice show profoundly increased transferrin saturation and serum ferritin levels as well as hepatic iron overload. Histological analysis by Perl’s Prussian blue staining revealed that hepatic iron accumulation is restricted to hepatocytes and that Kupffer cells are spared of iron. In addition, splenic macrophages and duodenal enterocytes are iron−depleted. Macroscopically, C326S homozygous mice show progressive, brown discoloration of the pancreas that correlates with profound iron deposition. Histological analysis reveals that iron localizes exclusively to the exocrine pancreas sparing the islets of Langerhans. Consistently, C326S homozygous mice do not show any signs of diabetes. Pancreatic iron accumulation is closely associated with increased reactive oxygen species (ROS), degeneration of exocrine pancreatic cells, increased plasma lipase and exocrine pancreatic failure. Starting at the age of 33 weeks, pancreatic failure is accompanied by progressive wasting and death. We believe that C326S FPN mice represent the first example of fatal iron overload in an animal model, opening avenues to investigate the underlying molecular mechanisms. Sham R, Phatak PD, West C, et al. Autosomal dominant hereditary hemochromatosis associated with a novel ferroportin mutation and unique clinical features. Blood Cells Mol. Dis. 2005; 34:157−61. Fernandes A, Preza GC, Phung Y, et al. The molecular basis of hepcidin−resistant hereditary hemochromatosis. Blood. 2009;114:437−443. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2096-2096
Author(s):  
Reijane Alves de Assis ◽  
Fernando Uliana Kay ◽  
Paulo Vidal Campregher ◽  
Gilberto Szarf ◽  
Fabiana Mendes Conti ◽  
...  
Keyword(s):  
Iron Overload ◽  
Serum Ferritin ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Transferrin Saturation ◽  
Laboratory Data ◽  
Surrogate Marker ◽  
Control Group ◽  
Double Mutation ◽  
H63d Mutation

Abstract Abstract 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.

PPARα Agonist Fenofibrate Attenuates Iron Induced Liver Injury in Mice by Modulating the Sirt3 and β-catenin Signaling

2021 ◽  
Author(s):  
Ashok Mandala ◽  
William J Chen ◽  
Austin Armstrong ◽  
Milan R Malhotra ◽  
Sanmathi Chavalmane ◽  
...  
Keyword(s):  
Liver Injury ◽  
Iron Overload ◽  
Liver Diseases ◽  
Critical Role ◽  
Chronic Liver ◽  
Iron Accumulation ◽  
Hepatic Iron ◽  
Chronic Liver Diseases ◽  
Pparα Agonist

Iron accumulation is frequently associated with chronic liver diseases. However, our knowledge on how iron contributes to the liver injury is limited. Aberrant Wnt/β-catenin signaling is a hallmark of several hepatic pathologies. We recently reported that peroxisome proliferator activated receptor alpha (PPARα) agonist, fenofibrate prevents iron induced oxidative stress and β-catenin signaling by chelating the iron. Sirtuin3 (Sirt3), a type of NAD+-dependent deacetylase that plays a critical role in metabolic regulation was found to prevent ischemia reperfusion injury by normalizing the Wnt/β-catenin pathway. In the present study, we explored if fenofibrate prevents iron induced liver injury by regulating the Sirt3 and β-catenin signaling. In-vitro and in-vivo iron treatment resulted in the downregulation of PPARα, Sirt3, active β-catenin and its downstream target gene c-Myc in the mouse liver. Pharmacological activation of Sirt3, both invitro and in vivo, by Honokiol (HK), a known activator of Sirt3, abrogated the inhibitory effect of iron overload on active β-catenin expression and prevented the iron induced upregulation of αSMA and TGFβ expression. Intrinsically, PPARα KO mice showed significant downregulation of hepatic Sirt3 levels. In addition, treatment of iron overload mice with PPARα agonist fenofibrate reduced hepatic iron accumulation and prevented iron induced downregulation of liver Sirt3 and active β-catenin, mitigating the progression of fibrosis. Thus, our results establish a novel link between hepatic iron and PPARα, Sirt3 and β-catenin signaling. Further exploration on the mechanisms by which fenofibrate ameliorates iron induced liver injury likely has significant therapeutic impact on iron associated chronic liver diseases.

scholarly journals Long-term phlebotomy successfully alleviated hepatic iron accumulation in a ferroportin disease patient with a mutation in SLC40A1: a case report

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sohji Nishina ◽  
Yasuyuki Tomiyama ◽  
Katsuya Ikuta ◽  
Yasuaki Tatsumi ◽  
Yasumichi Toki ◽  
...  
Keyword(s):  
Ferritin Level ◽  
Hereditary Hemochromatosis ◽  
Transferrin Saturation ◽  
Elevated Serum ◽  
Disease Patient ◽  
Iron Accumulation ◽  
Hepatic Iron ◽  
Ferroportin 1 ◽  
Serum Hepcidin

Abstract Background Hereditary hemochromatosis is a heterogenous group of inherited iron-overload conditions that is characterized by increased intestinal absorption and deposition in vital organs. Hepcidin is a soluble regulator that acts to attenuate both intestinal iron absorption and iron release from reticuloendothelial macrophages through internalization of ferroportin-1, an iron exporter. Ferroportin disease is hereditary hemochromatosis which is affected by SLC40A1, a gene coding ferroportin-1, and phenotypically classified into two forms (classical and nonclassical). In nonclassical form, ferroportin mutations are responsible for a gain of function with full iron export capability but insensitivity to downregulation by hepcidin. Here, we report a case of nonclassical ferroportin disease. Case presentation A 46-year-old Japanese man showed elevated serum iron (284 μg/dl), ferritin (1722 ng/ml), transferrin saturation ratio (91.3%), and hepcidin-25 level (139.6 ng/ml). Magnetic resonance imaging (MRI) demonstrated a marked reduction in the signal intensity of the liver in T1- and T2-weighted images. The liver histology exhibited a large amount of iron that had accumulated predominantly in hepatocytes. We identified a heterozygous 1520A > G (p.H507R) mutation in the SLC40A1 gene. Phlebotomy (400 ml at a time) was monthly performed for 3 years in this patient. Importantly, the serum hepcidin level (1.0 ng/ml) was normal when the serum ferritin level was normal and hepatic iron accumulation was remarkably reduced after 3 years of phlebotomy. Conclusions The present case demonstrated for the first time that there was a correlation between hepatic iron levels as measured by MRI and serum hepcidin levels through long-term phlebotomy in a patient with ferroportin disease with the p.H507R mutation of in SLC40A1.

scholarly journals Iron accumulation in the oculomotor nerve of the progressive supranuclear palsy brain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hansol Lee ◽  
Myung Jun Lee ◽  
Eun-Joo Kim ◽  
Gi Yeong Huh ◽  
Jae-Hyeok Lee ◽  
...  
Keyword(s):  
High Resolution ◽  
Progressive Supranuclear Palsy ◽  
Oculomotor Nerve ◽  
Iron Accumulation ◽  
Blue Staining ◽  
Normal Brain ◽  
Myelinated Fiber ◽  
Healthy Controls ◽  
Icp Ms

AbstractAbnormal iron accumulation around the substantia nigra (SN) is a diagnostic indicator of Parkinsonism. This study aimed to identify iron-related microarchitectural changes around the SN of brains with progressive supranuclear palsy (PSP) via postmortem validations and in vivo magnetic resonance imaging (MRI). 7 T high-resolution MRI was applied to two postmortem brain tissues, from one normal brain and one PSP brain. Histopathological examinations were performed to demonstrate the molecular origin of the high-resolution postmortem MRI findings, by using ferric iron staining, myelin staining, and two-dimensional laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging. In vivo iron-related MRI was performed on five healthy controls, five patients with Parkinson’s disease (PD), and five patients with PSP. In the postmortem examination, excessive iron deposition along the myelinated fiber at the anterior SN and third cranial nerve (oculomotor nerve) fascicles of the PSP brain was verified by LA-ICP-MS. This region corresponded to those with high R2* values and positive susceptibility from quantitative susceptibility mapping (QSM), but was less sensitive in Perls’ Prussian blue staining. In in vivo susceptibility-weighted imaging, hypointense pixels were observed in the region between the SN and red nucleus (RN) in patients with PSP, but not in healthy controls and patients with PD. R2* and QSM values of such region were significantly higher in patients with PSP compared to those in healthy controls and patients with PD as well (vs. healthy control: p = 0.008; vs. PD: p = 0.008). Thus, excessive iron accumulation along the myelinated fibers at the anterior SN and oculomotor nerve fascicles may be a pathological characteristic and crucial MR biomarker in a brain with PSP.

scholarly journals Ultrashort echo time imaging for quantification of hepatic iron overload: Comparison of acquisition and fitting methods via simulations, phantoms, and in vivo data

2018 ◽  
Vol 49 (5) ◽  
pp. 1475-1488 ◽  
Author(s):  
Aaryani Tipirneni-Sajja ◽  
Ralf B. Loeffler ◽  
Axel J. Krafft ◽  
Andrea N. Sajewski ◽  
Robert J. Ogg ◽  
...  
Keyword(s):  
Iron Overload ◽  
Hepatic Iron ◽  
Ultrashort Echo Time ◽  
Hepatic Iron Overload ◽  
Echo Time

scholarly journals Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 49-49
Author(s):  
Qiuling Chen ◽  
Yuelong Jiang ◽  
Qinwei Chen ◽  
Long Liu ◽  
Bing Xu
Keyword(s):  
B Cell ◽  
Solid Tumors ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Lymphocytic Leukemia ◽  
Advanced Lung Cancer ◽  
Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

Clinical, Pathological, and Molecular Correlates in Ferroportin Disease. A Study of Two Novel Mutations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 706-706
Author(s):  
Domenico Girelli ◽  
Ivana De Domenico ◽  
Claudia Bozzini ◽  
Ilaria Tenuti ◽  
Nadia Soriani ◽  
...  
Keyword(s):  
Iron Overload ◽  
Cellular Localization ◽  
Transferrin Saturation ◽  
Cell Types ◽  
Mouse Bone Marrow ◽  
Pathological Features ◽  
Functional Studies ◽  
Type Iv

Abstract Background: Mutations in the iron exporter Ferroportin (Fpn) lead to type IV hemochromatosis (Ferroportin Disease, FD), a dominantly inherited disorder with heterogeneous clinical and biochemical patterns. Some patients present with predominant macrophage iron overload (M), marked elevation of serum ferritin, normal-to-low transferrin saturation (TS), and, possibly, iron restricted erythropoiesis. Others present with a phenotype resembling classical HFE-related hemochromatosis, i.e. characterized by high TS and predominant hepatocyte iron overload (H). These differences are thought to reflect heterogeneity in the functional behaviour of Fpn mutant proteins. Methods: Two unrelated probands referring to the Centre for Iron Overload Disorders in Verona because of non-HFE hemochromatosis were screened for Fpn mutations by DHPLC (Cremonesi L, Br J Haematol 2005). The functional behaviour of mutants Fpn was studied by generating Fpn-GFP constructs transfected into different cell types (HEK293T, Cos7, and mouse bone marrow macrophages), and analyzing their cellular localization, as well as their capabilities to bind hepcidin and export iron (De Domenico I, PNAS 2005). The two mutations were also expressed in zebrafish, to evaluate their impact on iron-dependent erythropoiesis. Results: Patient 1, a 59 year old male, had clinical, biochemical (TS 74.8%, ferritin 9,000 μg/l), and pathological features (marked iron overload in either macrophages and hepatocytes, absence of overt cirrhosis) somewhat ambiguous, possibly suggesting a type M Fpn variant with late secondary hepatocyte overload. He was found to be heterozygous for the new L233P mutation. Functional studies revealed that Fpn L233P does not appropriately traffic to the cell surface, resulting in inappropriate inhibition by hepcidin. Fpn L233P expression in vivo in zebrafish resulted in iron limited erythropoiesis, consistent with a type M mutation leading to macrophage iron retention. Patient 2, a 59 year old female, had features more clearly suggesting a type M Fpn variant (TS 22.7%, ferritin 1,771 μg/l, macrophage iron load), but tolerated very well phlebotomies without developing signs of anemia. She was found to be heterozygous for the new I152F mutation. Functional studies revealed a unique pattern (never observed until now), since Fpn I152F localized appropriately on cell membrane, bound near normally to hepcidin, but showed a “primary” deficit of iron export capability. I152F expression in zebrafish resulted in a trend towards iron limited erythropoiesis, though quantitatively less clear than L223P. Conclusions: FD is a heterogeneous disease caused by generally “private” mutations in Fpn. The clinical, biochemical, and pathological features vary depending on the different behaviour of mutant Fpn. In vitro and in vivo molecular expression studies are very useful to clarify the pathophysiogical spectrum of this disease.

Iron Overload in C282Y Heterozygotes: Identification of New Rare HFE Gene Mutants and a Step Strategy for Diagnosis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1859-1859
Author(s):  
Patricia Aguilar-Martinez ◽  
Severine Cunat ◽  
Fabienne Becker ◽  
Francois Blanc ◽  
Marlene Nourrit ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Status ◽  
Hereditary Hemochromatosis ◽  
Genetic Defect ◽  
Transferrin Saturation ◽  
Hfe Gene ◽  
Compound Heterozygous ◽  
Exon 2 ◽  
High Iron ◽  
Iron Parameters

Abstract Introduction: Homozygozity for the p.Cys282Tyr (C282Y) mutation of the HFE gene is the main genotype associated with the common form of adult hereditary hemochromatosis. C282Y carriers do not usually develop iron overload, unless they have additional risk factors such as liver diseases, a dysmetabolic syndrome or an associated genetic defect. The commonest is the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele. However, a few rare HFE mutations can be found on the 6th chromosome in trans, some of which are of clinical interest to fully understand the disorder. Patients and Methods: We recently investigated four C282Y carrier patients with unusually high iron parameters, including increased levels of serum ferritin (SF), high transferrin saturation (TS) and high iron liver content measured by MRI. They were males, aged 37, 40, 42, 47 at diagnosis. Two brothers (aged 40 and 42) were referred separately. The HFE genotype, including the determination of the C282Y, H63D and S65C mutations was performed using PCR-RFLP. HFE sequencing was undertaken using the previously described SCA method (1). Sequencing of other genes (namely, HAMP, HJV/HFE2, SLC40A1, TFR2) was possibly performed in a last step using the same method. Results: We identified three rare HFE mutant alleles, two of which are undescribed, in the four studied patients. One patient bore a 13 nucleotide-deletion in exon 6 (c.[1022_1034del13], p.His341_Ala345>LeufsX119), which is predicted to lead to an abnormal, elongated protein. The two brothers had a substitution of the last nucleotide of exon 2 (c.[340G>A], p.Glu114Lys) that may modify the splicing of the 2d intron. The third patient, who bore an insertion of a A in exon 4 (c.[794dupA],p.[trp267LeufsX80]), has already been reported (1). Discussion: A vast majority of C282Y carriers will not develop iron overload and can be reassured. However, a careful step by step strategy at the clinical and genetic levels may allow to correctly identify those patients deserving further investigation. First, clinical examination and the assessment of iron parameters (SF and TS) allow identifying C282Y heterozygotes with an abnormal iron status. Once extrinsic factors such as heavy alcohol intake, virus or a dysmetabolic syndrome have been excluded, MRI is very useful to authenticate a high liver iron content. Second, HFE genotype must first exclude the presence of the H63D mutation. Compound heterozygozity for C282Y and H63D, a very widespread condition in our area, is usually associated with mild iron overload. Third, HFE sequencing can be undertaken and may identify new HFE variants as described here. The two novel mutations, a frameshift modifying the composition and the length of the C terminal end of the HFE protein and a substitution located at the last base of an exon, are likely to lead to an impaired function of HFE in association with the C282Y mutant. However, it is noteworthy that three of the four patients were diagnosed relatively late, after the 4th decade, as it is the case for C282Y homozygotes. Three further unrelated patients are currently under investigation in our laboratory for a similar clinical presentation. Finally, it can be noted that in those patients who will not have a HFE gene mutant identified, analysis of other genes implicated in iron overload must be performed to search for digenism or multigenism. None of our investigated patients had an additional gene abnormality.

Target TMPRSS6 for the Treatment of Hereditary Hemochromatosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1047-1047
Author(s):  
Sheri Booten ◽  
Daniel Knox ◽  
Luis Alvarado ◽  
Shuling Guo ◽  
Brett P. Monia
Keyword(s):  
Iron Overload ◽  
Genetic Disorder ◽  
Hereditary Hemochromatosis ◽  
Genetic Defect ◽  
Transferrin Saturation ◽  
Common Mutation ◽  
C282y Mutation ◽  
Iron Storage ◽  
Hepcidin Expression ◽  
Iron Deficient

Abstract Abstract 1047 Hereditary hemochromatosis (HH) is a genetic disorder in which hyperabsorption of dietary iron leads to accumulation of iron in multiple tissues including liver and heart. A common clinical manifestation in HH patients is cirrhosis and hepatocellular carcinoma as a result of iron-mediated injury in liver. The most prevalent genetic defect for HH is the failure to up-regulate hepcidin, a peptide hormone that inhibits the absorption of iron in duodenum and the release of iron from intracellular iron storage such as macrophages. Mutations in a number of genes have been identified as the cause for HH, including hepcidin itself. However, the most common mutation is C282Y mutation in HFE, which is a positive regulator for hepcidin expression. C282Y mutation represents about 85% of the HH population. HFE C282Y HH is an autosomal recessive disease with a ∼50% penetrance. Currently, the only treatment available for iron overload is phlebotomy which will continue throughout the patient's life. Hepcidin is mainly expressed and secreted by the liver and its expression is regulated predominantly at the transcription level. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor for hepcidin expression. It's been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of TMPRSS6 in Hfe−/− mice could ameliorate the iron overload phenotype (Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg KE et al., Blood, 2011). Using second generation antisense technology, we identified antisense oligonucleotides (ASOs) targeting mouse TMPRSS6 for the treatment of HH. These compounds were first identified through in vitro screens in mouse primary hepatocytes. After 4 weeks of treatment in C57BL/6 mice on normal chow, we observed an 80% to 90% reduction of liver TMPRSS6 mRNA with a subsequent 2–3 fold induction of liver hepcidin mRNA. Serum iron and transferrin saturation levels were reduced by ∼50%. These ASOs are currently being evaluated in a diet-induced iron overload model and an Hfe−/− iron overload model. Our preliminary results demonstrate that targeting TMPRSS6 is a viable approach for the treatment of hereditary hemochromatosis and possibly other iron-loading diseases associated with suppressed hepcidin levels. Disclosures: Booten: Isis Pharmaceuticals: Employment. Knox:Isis Pharmaceuticals: Summer Intern. Alvarado:Isis Pharmaceuticals: Employment. Guo:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals: Employment.

Target TMPRSS6 Using Antisense Technology for the Treatment of Hereditary Hemochromatosis and β-Thalassemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 481-481 ◽  
Author(s):  
Shuling Guo ◽  
Carla Casu ◽  
Sara Gardenghi ◽  
Sheri Booten ◽  
Andy Watt ◽  
...  
Keyword(s):  
Treatment Group ◽  
Iron Overload ◽  
Hereditary Hemochromatosis ◽  
Iron Concentration ◽  
Transferrin Saturation ◽  
Control Group ◽  
Mrna Levels ◽  
Liver Iron ◽  
Hepcidin Expression ◽  
Antisense Technology

Abstract Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to <150ug/dl in treatment group), as well as liver iron accumulation. Histopathological evaluation and Prussian's Perl Blue staining indicated that iron was sequestered by macrophages, which led to an increase in spleen iron concentration. The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

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