Iron Refractory Iron Deficiency Anemia Patients Research Articles

Preliminary Results of a Phase-2 Clinical Trial of the ALK-2 Inhibitor Ker-047 for Treatment of Iron Refractory Iron Deficiency Anemia

Background: Iron refractory iron deficiency anemia (IRIDA) is a rare autosomal recessive disorder, caused by a genetic defect in TMPRSS6. This results in increased signaling through the bone morphogenic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway, leading to disproportionally high levels of hepcidin relative to body iron status. Hepcidin reduces iron transport to the plasma by degrading the cellular iron exporter ferroportin, resulting in systemic iron deficiency and microcytic anemia. Symptomatic interventions such as intravenous iron administration results in iron sequestration in the reticular endothelial system (RES) and generally only a partial response of hemoglobin concentrations. Further, the potential long-term adverse effects of chronic intravenous iron therapy and associated elevated RES-iron levels are unknown in these patients. Blood transfusion can be administered in cases of severe symptomatic anemia but is generally avoided due to transfusion-related risks. Currently, there is an unmet need for more effective, safe, convenient, and direct-acting treatments for IRIDA. Given the central role of aberrant BMP/SMAD signaling in hepcidin production, selective inhibition of the BMP receptor ALK2 to reduce hepcidin represents a rational approach to target a precipitating factor of IRIDA. Preclinical studies in the TMPRSS6 siRNA knockdown mouse model of IRIDA demonstrated that treatment with a selective ALK2 inhibitor reduced hepcidin, increased serum iron, and ameliorated anemia. KER-047 is a novel, oral, investigational, small-molecule kinase inhibitor with potent and selective inhibitory activity against the ALK2 receptor. In a Phase 1 clinical trial, single-ascending and multiple-ascending doses of KER-047 administered to healthy participants resulted in robust decreases from baseline in hepcidin levels, increases in serum iron and transferrin saturation (TSAT), decreases in serum ferritin, and increases in reticulocyte hemoglobin content (Ret-He). Based on these observations, KER-047 was advanced to Phase 2 clinical trials. Aims: The primary objective of this ongoing Phase 2 clinical trial is to evaluate the safety and tolerability of ascending doses of KER-047 in participants with IRIDA. Secondary objectives include evaluation of pharmacokinetic (PK) and pharmacodynamic (PD) parameters. Methods: This is a 2-part open label, dose escalation and dose expansion trial. Part 1 (dose escalation) consists of up to 4 ascending dose cohorts starting with 25 mg q.d. Part 2 will be the dose expansion phase with safety as the primary focus. Patients diagnosed with IRIDA may enroll in all cohorts in Parts 1 and 2. Key inclusion criteria include diagnosis of IRIDA based on a homozygous or compound heterozygous TMPRSS6 genotype, a TSAT <15%, a ferritin level 50-700 µg/L and hemoglobin <13.8 g/dL (males) or <12.7g/dL (females) and signed informed consent. Patients are excluded if they have received IV iron treatment within 28 days prior to trial entry. Participants will be treated once daily with KER-047 for a 2-week period, followed by a 2-week washout period. Data are analyzed by using descriptive statistics. Results: At the time of this abstract submission, one participant had enrolled in the first dose-expansion cohort (KER-047 25mg q.d.). This participant reported intermittent dizziness, however no serious adverse events were observed during treatment. Hemoglobin concentration and mean corpuscular volume remained stable. Ret-He increased by day 5 of treatment. Ferritin levels decreased during treatment and returned to baseline levels after the 2-week treatment period. TSAT/hepcidin ratio, which is characteristically below reference intervals in IRIDA patients, was slightly increased but returned to baseline levels after 2-weeks treatment. Conclusion: Data are preliminary and limited to a single participant thus far in whom KER-047 25mg q.d. was well tolerated. Changes in red cell and iron parameters at this low dose support development of KER-047 as an agent to potentially address an underlying cause of IRIDA. At the time of this abstract submission, two additional patients are scheduled for screening to complete the first dose cohort. We anticipate data from the total of three patients in time for the presentation to further characterize safety of KER-047 and substantiate effects on iron metabolism. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Response to Prolonged Duration of Therapeutic Dose Oral Iron Therapy in a Girl With Novel TMPRSS6 Gene Variants: A Case Report and Review Literature.

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by mutations in the TMPRSS6 gene, which impair iron homeostasis. We reported a 4-year-old girl who presented with a 1-year history of iron deficiency anemia. Her hemoglobin level increased from 6.5 g/dL to 12.6 g/dL with a prolonged duration of therapeutic dose oral iron therapy (5 mg/kg/d), and the level remained quite stable during the therapy. Genetic analysis of the TMPRSS6 gene revealed compound heterozygotes of 2 novel pathogenic variants: c.811C> T (NM_153609.3) in exon 7 (NP_705837: p.R271Ter) and c.1254C> G in exon 11 (p.Y418Ter). The results highlight the significance of genetic investigation and long-term iron therapy in iron-refractory iron deficiency anemia patients.

TMPRSS6 gene mutations in six Saudi families with iron refractory iron deficiency anemia

Iron-refractory iron deficiency anemia (IRIDA) is considered an autosomal recessive iron deficiency anemia due to mutations in the transmembrane protease serine 6 (TMPRSS6) gene. Variations in iron parameters and a higher risk of iron deficiency have been linked to the TMPRSS6 mutations. Furthermore, human genome-wide association studies (GWAS) identified a common mutation (rs855791) linked to abnormal hematological parameters, highlighting the importance of the TMPRSS6 gene in the regulation of iron homeostasis. This is the first study to investigate TMPRSS6 gene mutation in six Saudi families of probands with iron deficiency anemia unresponsive to oral iron and partially responsive to parenteral iron administration. Each participant provided a vacutainer tube with three blood samples (2.5 ml each) and analyzed based on hematological, biochemical iron profiles, and followed by genotyping by PCR. The TMPRSS6 gene was amplified, sequenced, and analyzed in all probands and family members. Statistical analysis was done using SPSS and SHEsis software. Few functional mutations in these families were suggested (p.W73X, p.E523K and p.V736A). The proband of family 6 presented numerous hematological abnormalities upon initial consultation, including normocytic anemia accompanied by low Hb, normal MCV, low serum iron, low serum ferritin, and normal TIBC. While the p.W73X variant was only found in 2 families, the p.V736A variant was found in all examined Saudi families with IRIDA. Given the evidence outlined for these six cases, future genotype-phenotype correlation studies in a large number of IRIDA patients in Saudi Arabia may be very informative for patient management, in addition to increasing knowledge of TMPRSS6 function during development as well as factors in the regulation of TMPRSS6 and its effect on iron levels in the body.

P1557: DISTINGUISHING OF IRON REFRACTORY IRON DEFICIENCY ANEMIA FROM IRON DEFICIENCY ANEMIA AND THALASSEMIA TRAIT BY SYSMEX RESEARCH RED CELL PARAMETERS

Background: Iron refractory iron deficiency anemia (IRIDA), iron deficiency anemia (IDA), and Thalassemia trait (TT) are the most common causes of microcytic anemia. It is possible to discriminate between IDA and TT by routine complete blood count (CBC), hemoglobin (Hb) electrophoresis and iron parameters; however, it is very difficult to distinguish IRIDA from IDA and TT using only routine CBC and iron parameters. Aims: The aim was to distinguish IRIDA from IDA and TT using Sysmex research red cell parameters. Methods: The study included a total of 25 patients (n=10, TT; n=10, true IDA; n= 5, genetically confirmed IRIDA). Traditional [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell count (RBC), and red cell distribution volume (%RDW)] and new red cell parameters [percentage hypochromic RBCs (%Hypo-He); percentage microcytic RBCs (%Micro-R)] were measured in a Sysmex-XN®2000 (Sysmex Corporation, Kobe, Japan), along with iron parameters. Participants’ disorders were categorized based on patients’ ferritin, Hb electrophoresis, genetic results, and CBC values. Results: Compared with the IDA patients, IRIDA patients had significantly higher mean levels of ferritin, RBC, %Hypo-He, %Micro-R and significantly lower mean levels of serum iron level MCV, MCH, and MCHC values (p<0.05 for all). Compared with TT patients, IRIDA patients had significantly higher mean levels of ferritin, %Hypo-He and %RD,W and significantly lower mean levels of serum iron level and MCH values (p<0.05 for all). The RBC, Micro-R, Hypo-He, MCV and MCH cut-off values for discriminating IRIDA from IDA patients were 4.9µL, %60, 50%, 60fL, and 16.0pg, respectively. The RDW, Hypo-He, and MCH cut-off values for discriminating IRIDA from TT patients were %19, %30, and 17.0pg, respectively. Summary/Conclusion: This first study has examined the utility of new red cell parameters in patients with IRIDA. Our findings should be confirmed in larger studies; however, we believe that higher percentage of hypochromic microcytic erythrocytes with an elevated RBC value may be useful predictors for distinguishing IRIDA from IDA patients. As well, a moderate percentage of hypochromic erythrocytes with high RDW value can guide physicians in discriminating IRIDA from TT patients

Iron distribution in different tissues of homozygous Mask (msk/msk) mice and the effects of oral iron treatments

Iron‐refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by genetic mutations on TMPRSS6 gene which encodes Matriptase2 (MT2). An altered MT2 cannot appropriately suppress hepatic BMP6/SMAD signaling in case of low iron, hence hepcidin excess blocks dietary iron absorption, leading to a form of anemia resistant to oral iron supplementation. In this study, using the IRIDA mouse model Mask, we characterized homozygous (msk/msk) compared to asymptomatic heterozygous (msk/wt) mice, assessing the major parameters of iron status in different organs, at different ages in both sexes. The effect of carbonyl iron diet was analyzed as control iron supplementation being used for many studies in mice. It resulted effective in both anemic control and msk/msk mice, as expected, even if there is no information about its mechanism of absorption. Then, we mainly compared two forms of oral iron supplement, largely used for humans: ferrous sulfate and Sucrosomial iron. In anemic control mice, the two oral formulations corrected hemoglobin levels from 11.40 ± 0.60 to 15.38 ± 1.71 g/dl in 2–4 weeks. Interestingly, in msk/msk mice, ferrous sulfate did not increase hemoglobin likely due to ferroportin/hepcidin‐dependent absorption, whereas Sucrosomial iron increased it from 11.50 ± 0.60 to 13.53 ± 0.64 g/dl mainly in the first week followed by a minor increase at 4 weeks with a stable level of 13.30 ± 0.80 g/dl, probably because of alternative absorption. Thus, Sucrosomial iron, already used in other conditions of iron deficiency, may represent a promising option for oral iron supplementation in IRIDA patients.

Selective Inhibition of ALK2 Signaling Suppresses Serum Hepcidin and Increases Serum Iron

Hepcidin is an endocrine regulator of iron metabolism that, when elevated, can decrease levels of iron available for erythropoiesis and, as a result, decrease red blood cell production. Signaling though activin-like kinase-2 (ALK2), a TGFβ type 1 receptor, has been implicated in regulation of hepcidin-mediated iron regulation and mobilization; however, to date, ALK2's specific degree of involvement has not been convincingly elucidated, mainly due to the redundant effects of the type 1 receptors ALK3 and ALK5. Activation of type 1 receptors including ALK2, ALK3, and ALK5 via ligand BMPs and co-receptor hemojuvelin (m-HJV), results in downstream SMAD phosphorylation, increased hepcidin, and decreased serum iron while suppression of the receptor signaling would have the opposite effects. In order to assess the specific effect of ALK2 inhibition on hepcidin and iron mobilization, we utilized multiple modalities of inhibition and tested inhibitors in both naive and diseased animals. KTI-2338, a small molecule ALK2 kinase inhibitor, has been characterized to inhibit ALK2 signaling potently and selectively in in-vitro assays. To further assess the specific contribution of ALK2 to hepcidin expression and iron mobilization, we evaluated the effect of the novel neutralizing antibody KTI-A2.0MAb. This fully human antibody is targeted against the extracellular domain of ALK2 with no affinity for the other type 1 receptors and provides a unique tool for understanding ALK2 involvement in this system. In wild-type animals, targeting ALK2 signaling with either a small molecule or biologic therapeutic leads to decreased serum hepcidin and increased serum iron. To assess the efficacy of ALK2 inhibition in a disease state, we utilized an siRNA-based model of Iron Refractory Iron Deficiency Anemia (IRIDA). In IRIDA, patients exhibit a loss of functional TMPRSS6, a gene that encodes the transmembrane type II serine protease Matriptase-2 (MT-2). MT-2 suppresses hepcidin secretion by cleaving m-HJV, interrupting ALK2 signaling and downstream SMAD activation. Failure to cleave m-HJV allows continued activation of BMPRs, increased hepcidin, and decreased serum iron. Phenocopying what is observed in IRIDA patients, intravenous dosing of TMPRSS6 targeted siRNA results in suppressed TMPRSS6 expression and functional MT-2, increases in serum hepcidin, and decreases in serum iron. Therapeutic dosing of either a small molecule or biologic ALK2 inhibitor in the siRNA based IRIDA model resulted in rescue of hemoglobin, hematocrit, serum hepcidin, and serum iron in the disease state. Following treatment, hemoglobin, hematocrit, and serum iron were increased and serum hepcidin was decreased in treated groups compared to control cohorts receiving vehicle. Herein, we have evaluated multiple modalities of ALK2 inhibition in both healthy and disease states. We have characterized that inhibition of ALK2 signaling via either modality in both naïve and anemic mice contributes to a decrease in serum hepcidin and increase in serum iron levels. Though the use of a selective ALK2 targeted biologic does not completely preclude involvement of other BMP receptors such as ALK3, these data support our assertion that ALK2 signaling is an integral part of hepcidin-mediated iron mobilization, and illustrate the potential therapeutic benefit of ALK2 inhibition (with a small molecule inhibitor or a neutralizing monoclonal antibody) in anemia of high hepcidin including IRIDA and anemia of inflammation. Disclosures Backus: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Medeiros:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Effect of Oral Iron Treatment in Tmprss6 Knock-out Mouse Model

Introduction: Iron Refractory Iron Deficiency Anemia (IRIDA) is an autosomal recessive iron metabolism disorder caused by mutations in Tmprss6 gene which encodes for Matriptase2 (MT2) that, by activating hemojuvelin (HJV), regulates the production of hepcidin, the master iron regulatory hormone. Altered MT2 cannot suppress hepatic BMP6/SMAD signaling in low iron condition, hence the resulting hepcidin excess blocks dietary iron absorption and cells release, leading to a form of iron deficiency that is typically refractory to oral iron supplementation. IRIDA is characterized by moderate/severe microcytic anemia (Hemoglobin 6-9 g/dL; MCV 45-65 fL); low transferrin saturation (&lt;5%); impaired oral iron absorption and only a transient response to parenteral iron. Nonetheless, the current treatment is mainly based on parenteral iron therapy. A case study on a child with IRIDA showed for the first time the ability of Sucrosomial® Iron, to increase hemoglobin and MCV values over time (Capra et al., 2017). This oral iron formulation is an innovative preparation of ferric pyrophosphate, covered by a phospholipids plus sucrester matrix, with gastro-resistance properties, high bioavailability and tolerability due to alternative absorption pathways as endocytosis and M cells mediated route (Gomez-Ramirez et al., 2018). Moreover, Sucrosomial® Iron has been successfully used to treat iron deficiency in various clinical conditions, including inflammatory bowel diseases (Abbati et al., 2019). To confirm and characterize the ability of Sucrosomial® Iron to increase Hb in IRIDA disease we studied the response to Sucrosomial® Iron in a IRIDA mouse model (Mask) comparing the efficacy of Sucrosomial® Iron and Sulfate Iron at two different doses and in chronic treatment. Aim: to study Sucrosomial® Iron effect in IRIDA using the Tmprss6 knock-out mouse model Material and Methods: m/m homozygous mice (9-weeks old male mice, four mice per experimental group) were kept at iron balance diet and treated with 0.5 or 4 mg/Kg of Ferrous sulfate, Sucrosomial® Iron (patent n° PCT/IB2013/001659 owned by Alesco s.r.l, Italy), or vehicle by gavage for 35 days. Four 9-weeks old m/- male mice per experimental group were daily treated and Hb and Ht were monitored weekly. Mice were sacrificed at the end of treatments; blood, and different organs were collected for analysis. Total RNA was isolated from tissues using TRIzol Reagent (Ambion), cDNA was generated by Reverse transcription (Promega, Milan, Italy) and samples were analyzed for Hepcidin and Socs3 mRNA levels by qRT-PCR using PowerUp SYBR Green Master Mix (Life Technologies). Results: we analyzed the iron status of anemic homozygous Mask mice from 3 to 35 weeks of age by studying serological and tissue iron content. Interestingly only Sucrosomial® Iron (not Ferrous Sulfate), increased hemoglobin level from 11-12 to 13-14 g/dL in the first week with a tendency to increase until the fourth week, when it stabilized at 13 g/dL (Figure 1A-B). Serum iron concentration was higher in the Sucrosomial® Iron treated animals than in those treated with vehicle, while was lower in the Ferrous sulfate treated animals. Similar pattern was observed for spleen iron content that increased in mice treated with Sucrosomial® Iron but not in those receiving Ferrous sulfate. Liver iron concentration did not apparently varied after the treatments, but duodenal iron increased significantly only in the mice treated with the higher dose of Ferrous sulfate (Figure 1 C-F). Interestingly, we found that the mice treated with both doses of Ferrous sulfate, but not those treated with Sucrosomial® Iron, had a higher mRNA levels of hepcidin and of the inflammatory marker Socs3 (Figure 1 G-H). Conclusion: this study showed for the first time that Sucrosomial® Iron is able to increase hemoglobin level in a mouse model of IRIDA, probably due to its alternative absorption pathway. Sucrosomial® Iron could be used as effective iron supplement to improve iron status in IRIDA patients. Disclosures Girelli: La Jolla Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Vifor Pharma: Other: honoraria for lectures; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees.

TMPRSS6 Gene Polymorphism and Serum Hepcidin in Iron Deficiency Anemia

Background: Iron deficiency anemia (IDA) is a global health problem and common medical condition seen in everyday clinical practice. Hepcidin which is the key regulator of iron homeostasis, that down-regulates iron export by binding to ferroportin which is expressed on the surface of iron-releasing cells, leading to its degradation thus reducing plasma iron levels. Overexpression of hepcidin leads to iron deficiency anemia. Matriptase-2 (MT-2) which is encoded by transmembrane protease serine 6 (TMPRSS6) gene regulates hepcidin expression. Objective: Was to evaluate the role of TMPRSS6 gene polymorphisms and serum hepcidin level in IDA patients. Subjects and methods: This study was carried out on 30 patients with iron deficiency anemia and 30 age and sex matched individuals as control group. Patients were subdivided into (group 1) 14 patients with acquired iron deficiency anemia (IDA) and (group 2)16 patients with iron refractory iron deficiency anemia(IRIDA).TMPRSS6 gene single nucleotide polymorphisms(SNPS), (rs4820268)and(rs855791)were evaluated using real time – polymerase chain reaction (RT-PCR) while serum hepcidin level was measured by enzyme linked immunosorbent assay (ELISA). Results: There was a significant increase in frequency of the TMPRSS6 SNPs rs855791 and rs 4820268 separately in IRIDA group compared to IDA group,(P=0.003),(P=0.007) respectivelyand to control group(P=0.000) and (P=0.000) respectively.Also, there was highly significant increase in frequency of both mutations together in IRIDA group compared to IDA group (P=0.000) and to control group (P=0.000). In IDA group there was a significant increase in the frequency of TMPRSS6 SNPs rs 855791 when compared to control group (P=0.013), and a non-significant difference in frequency of SNPs rs 4820268 when compared to control group (P=0.092).Also, there was a non-significant difference in frequency of both mutations together in IDA group in comparison to control group (P> 0.05).There was a highly significant decrease in serum hepcidin level in IDA group and highly significant increase in hepcidin levels in IRIDA group compared to control group (P=0.000).In IRIDA patients group only there was highly significant increase in hepcidin level in those with mutation in both SNPs together (rs4820268 and rs855791) than patients with one mutation or with wild type(P=0.000).There was a significant decrease in Hb and iron and highly significant decrease in MCV, MCH and ferritin in IRIDA patients with mutation of both SNPs together (rs 4820268 and rs855791) compared to one mutation or wild type (P=0.015,0.016,0.002 ,0.000, 0.006 respectively).Also, there was highly significant decrease in Hb, MCV, MCH and ferritin in IDA patients with mutation of both SNPs together(rs 4820268 and rs 855791) compared to one mutation or wild type (P= 0.002,0.002 ,0.004, 0.009 respectively). In IDA patients there was a significant positive correlation between hepcidin and ferritin (P =0.034, r = 0.279),while there was a significant negative correlation between hepcidin and MCH in IRIDA group (P =0.032, r = - 0.536).Conclusion: In IRIDA the rs4820268 and rs855791 mutations separately or in combination lack inhibitory effect on hepcidin and consequently iron profile and hemoglobin while in IDA although there was significant increase in frequency of rs855791 mutations, there was no effect on hepcidin which suggests that the rs 4820268 mutation is necessary for affecting hepcidin either alone or in combination with rs 855791 mutation. In IDA, there may be other mechanisms or mutations causing anemia.

Iron refractory iron deficiency anemia: a heterogeneous disease that is not always iron refractory

TMPRSS6 variants that affect protein function result in impaired matriptase‐2 function and consequently uninhibited hepcidin production, leading to iron refractory iron deficiency anemia (IRIDA). This disease is characterized by microcytic, hypochromic anemia and serum hepcidin values that are inappropriately high for body iron levels. Much is still unknown about its pathophysiology, genotype–phenotype correlation, and optimal clinical management. We describe 14 different TMPRSS6 variants, of which 9 are novel, in 21 phenotypically affected IRIDA patients from 20 families living in the Netherlands; 16 out of 21 patients were female. In 7 out of 21 cases DNA sequencing and multiplex ligation dependent probe amplification demonstrated only heterozygous TMPRSS6 variants. The age at presentation, disease severity, and response to iron supplementation were highly variable, even for patients and relatives with similar TMPRSS6 genotypes. Mono‐allelic IRIDA patients had a milder phenotype with respect to hemoglobin and MCV and presented significantly later in life with anemia than bi‐allelic patients. Transferrin saturation (TSAT)/hepcidin ratios were lower in IRIDA probands than in healthy relatives. Most patients required parenteral iron. Genotype alone was not predictive for the response to oral iron. We conclude that IRIDA is a genotypically and phenotypically heterogeneous disease. The high proportion of female patients and the discrepancy between phenotypes of probands and relatives with the same genotype, suggest a complex interplay between genetic and acquired factors in the pathogenesis of IRIDA. In the absence of inflammation, the TSAT/hepcidin ratio is a promising diagnostic tool, even after iron supplementation has been given. Am. J. Hematol. 91:E482–E490, 2016. © 2016 Wiley Periodicals, Inc.

The role of TMPRSS6 polymorphisms in iron deficiency anemia partially responsive to oral iron treatment

Iron refractory iron deficiency anemia (IRIDA) is a rare hereditary disease caused by mutations in TMPRSS6 gene encoding Matriptase-2, a negative regulator of hepcidin transcription. Up to now, 53 IRIDA patients from 35 families with different ethnic origins have been reported and 41 TMPRSS6 mutations have been identified. TMPRSS6 polymorphisms are more frequent than mutations, and have been associated with variation in iron and hematologic parameters. Our study evaluated their presence in 113 subjects with iron deficiency anemia (IDA) partially responsive to oral iron therapy and in 50 healthy blood donors. Thalassemic trait was diagnosed in 38 patients. Sequencing analysis of TMPRSS6 gene revealed that the frequency of several polymorphisms was markedly different between IDA subjects and controls. In particular, the V736A TMPRSS6 polymorphism was associated to moderately lower hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin levels, and in thalassemia carriers with marked anemia and microcytosis. A new variant-H448R- and two uncommon polymorphisms -A719T and V795I- were also identified. These results indicate that TMPRSS6 polymorphisms are more frequent in subjects with persistent IDA than in healthy controls, and in thalassemia carriers V736A variant may account for lower hemoglobin and MCV levels. Further studies in larger court of patients are necessary to identify potential haplotypes and polymorphisms responsible for low response to oral iron treatment and may be useful for planning a correct iron supplementation.

Functional and clinical impact of novel TMPRSS6 variants in iron-refractory iron-deficiency anemia patients and genotype-phenotype studies.

Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation analysis demonstrates that patients carrying two nonsense mutations present a more severe anemia and microcytosis and higher hepcidin levels than the other patients. We confirm that TMPRSS6 mutations are spread along the gene and that mechanistically they fully or partially abrogate hepcidin inhibition. Genotyping IRIDA patients help in predicting IRIDA severity and may be useful for predicting response to iron treatment.

Matriptase-2 is essential for hepcidin repression during fetal life and postnatal development in mice to maintain iron homeostasis

AbstractIron is an essential element required for development and survival of all living organisms. In fetuses, maternofetal iron transfer across the placenta is essential for growth and development. In neonates, efficient intestinal iron absorption is required to scavenge as much iron as possible from the low-iron-content milk. During these periods, efficient iron mobilization is ensured by the downregulation of the iron regulatory hormone hepcidin by as-yet uncharacterized molecular mechanisms. Here we demonstrate that the recently described hepcidin repressor—the serine protease matriptase-2 (encoded by Tmprss6)—is responsible for this repression throughout development, with its deficiency leading to increased hepcidin levels triggering iron deficiency and anemia starting in utero. This result might have implications for a better understanding of iron homeostasis during early development in iron-refractory iron deficiency anemia patients, who present with microcytic anemia caused by hyperhepcidinemia, and of questions about the role of matriptase-2 in human neonates.

Novel TMPRSS6 mutations associated with iron-refractory iron deficiency anemia (IRIDA)

Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. In this paper we describe 12 IRIDA patients belonging to 7 unrelated families and identify 10 (9 novel) TMPRSS6 mutations spread along the gene sequence: 5 missense, 1 non sense and 4 frameshift. The frameshift and non sense mutations are predict to result in truncated protein lacking the catalytic domain. The causal role of missense mutations (Y141C, I212T, R271Q, S304L and C510S) is demonstrated by in silico analysis, their absence in 100 control chromosomes and the high conservation of the involved residues. The C510S mutation in the LDLRA domain in silico model causes an intra-molecular structural imbalance that impairs matriptase-2 activation. We also assessed the in vitro effect on hepcidin promoter and the proteolytic activity of I212T and R271Q variants demonstrating a reduced inhibitory effect for the former mutation, but surprisingly a normal function for R271Q which appears a silent mutation in vitro. Based on mRNA expression studies I212T could also decrease the total amount of protein produced, likely interfering with mRNA stability. Collectively, our results extend the pattern of TMPRSS6 mutations associated with IRIDA and propose a model of causality for some of the novel missense mutation.

Matriptase-2, A Novel Suppressor of Hepcidin.

Abstract SCI-26Hepcidin inhibition is a process essential to increase iron release from duodenal cells and macrophages to plasma in conditions of high iron requests such as iron deficiency, hypoxia and erythropoietic expansion. Among the reported potential inhibitors of hepcidin the serine protease matriptase-2 has a strong effect in vivo both in mice and in humans. Matriptase-2 is a member of the transmembrane serine protease (TTPS) family, encoded by TMPRSS6 gene, whose expression is tissue restricted, mainly to the liver and to a lesser extent to kidney and olfactory epithelium. The role of matriptase-2 in iron metabolism was first defined in the Mask mouse, which after birth develops a type of iron deficiency anemia refractory to oral iron and a peculiar pattern of hair loss, because of inappropriate overexpression of hepcidin and impaired iron absorption, a phenotype due to the deletion of the matriptase-2 serine protease domain. An identical phenotype is reported in Tmprss6 -/- mice and a correspondent phenotype is observed in iron-refractory iron-deficiency anemia (IRIDA) patients. We have shown that matriptase-2 cleaves through a proteolytic processing the bone morphogenetic protein (BMP) co-receptor hemojuvelin from plasma membrane in vitro, indicating that the suppression of the BMP signaling is essential for hepcidin inhibition. The finding also suggests that the BMP pathway requiring hemojuvelin as coreceptor is the main iron-dependent pathway of hepcidin regulation. The hepcidin inhibitory effect is observed also in zebrafish and is abolished in the human homologue of Mask. We have investigated several missense TMPRSS6 mutants affecting different domains of the protein, reported in IRIDA patients, in comparison with wild type matriptase-2 and Mask. Transient overexpression in hepatoma cells shows that all mutants are deficient in the ability to inhibit the hepcidin promoter in a classic luciferase-based assay and that the decreased hepcidin inactivation broadly corresponds to the inability to cleave hemojuvelin from plasma membrane. Studies of the in vitro processing of the mutants indicate that determinants of the pathogenic effect others than the protease activity are the intracellular processing and the ability of self-activation of matriptase-2. These results have implications for the molecular pathogenesis of IRIDA. DisclosuresNo relevant conflicts of interest to declare.

Iron-Refractory Iron Deficiency Anemia

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder characterized by iron deficiency anemia unresponsive to oral iron treatment but partially responsive to parenteral iron therapy. IRIDA has recently been shown to be caused by mutations in the gene TMPRSS6, which encodes a transmembrane serine protease (also known as matriptase-2) expressed by the liver. IRIDA patients show inappropriately elevated levels of hepcidin, a circulating hormone produced by the liver that inhibits both iron absorption from the intestine and iron release from macrophage stores. Recent studies suggest that TMPRSS6 normally acts to downregulate hepcidin expression by cleaving hemojuvelin, a membrane-bound protein that promotes hepcidin signaling in hepatocytes. A discussion of the clinical presentation of IRIDA, the molecular genetics of this disorder, and recent studies elucidating the underlying pathophysiology are presented.

Matriptase-2 mutations in iron-refractory iron deficiency anemia patients provide new insights into protease activation mechanisms

Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. Here we describe two novel heterozygous mutations within the matriptase-2 (TMPRSS6) gene of monozygotic twin girls exhibiting an IRIDA phenotype. The first is the frameshift mutation (P686fs) caused by the insertion of the four nucleotides CCCC in exon 16 (2172_2173insCCCC) that is predicted to terminate translation before the catalytic serine. The second mutation is the di-nucleotide substitution c.467C>A and c.468C>T in exon 3 that causes the missense mutation A118D in the SEA domain of the extracellular stem region of matriptase-2. Functional analysis of both variant matriptase-2 proteases has revealed that they lead to ineffective suppression of hepcidin transcription. We also demonstrate that the A118D SEA domain mutation causes an intra-molecular structural imbalance that impairs matriptase-2 activation. Collectively, these results extend the pattern of TMPRSS6 mutations associated with IRIDA and functionally demonstrate that mutations affecting protease regions other than the catalytic domain may have a profound impact in the regulatory role of matriptase-2 during iron deficiency.