Anemia and hyperferritinemia are frequent findings at diagnosis of Gaucher disease (GD). Macrophage-independent dyserythropoiesis and abnormal iron metabolism have been shown. We evaluated hematological and iron status at diagnosis (T0) and the effect of enzyme replacement therapy (ERT) on erythropoiesis and iron utilization over 5-year follow-up in type 1 GD patients and in an ex vivo model of erythropoiesis from CD34 + peripheral blood cells. At T0, 41

Mutations in the ferroportin (FPN) gene SLC40A1 alter iron recycling and cause disturbances in iron homeostasis. The variants of TMPRSS6 contribute to the development of iron deficiencies. In this study, we determined the role of FPN and TMPRSS6 gene polymorphisms in the modulation of iron homeostasis based on biochemical parameters. PCR analysis and sequencing were performed to determine the single nucleotide polymorphisms (SNPs) SLC40A1 c.44–24G>C (rs1439816), SLC40A1 c.663T>C (rs2304704), and TMPRSS6 c.2207T>C (rs855791). Hemoglobin concentration and iron status were determined by standard procedures. We studied 79 iron-loaded individuals for SLC40A1 polymorphisms. Interestingly, 35/79 individuals with SLC40A1 SNPs also carried a TMPRSS6 c.2207T>C polymorphism. The biochemical values of the iron overloaded individuals were compared to those of the individuals carrying TMPRSS6 SNPs and the healthy individuals (wild-type group). The ferritin concentration, transferrin saturation % (TS%), and hemoglobin concentration were significantly higher in the participants with FPN SNPs than in the other three groups. The ferritin concentration and TS% were higher in participants with both SLC40A1 and TMPRSS6 SNPs than in the TMPRSS6 and wild-type groups, while hemoglobin concentration was significantly higher than that in the TMPRSS6 SNP group only. The participants with TMPRSS6 SNPs had significantly lower ferritin concentration, TS%, and hemoglobin concentration than all the other groups. SLC40A1 and TMPRSS6 SNPs might act in the opposite direction, preventing the development of severe iron overload, and the modulation of the iron status by TMPRSS6 SNPs might provide protection.

Partial deficiency of the last enzyme of the heme biosynthetic pathway, namely, ferrochelatase (FECH), is responsible for erythropoietic protoporphyria (EPP) in humans. This disorder is characterized by painful skin photosensitivity, due to excessive protoporphyrin IX (PPIX) production in erythrocytes. Although several papers report the presence of iron deficiency anemia in about 50% of EPP patients, there is still no a conclusive explanation of the why this occurs. In the present work, we explored hematological indices and iron status in 20 unrelated Italian EPP patients in order to propose a new hypothesis. Our data show that microcytosis is present in EPP patients also in the absence of anemia and iron deficiency with a link between PPIX accumulation and reduced MCV, probably indicating an indirect condition of heme deficiency. Patients studied had a downward shift of iron parameters due to increased hepcidin concentrations only in a state of repleted iron stores. Interestingly, hemoglobin synthesis was not limited by iron supply except in cases with further iron loss, in which concomitantly increased soluble transferrin (Tf) receptor (sTfR) levels were detected. The mechanisms involved in the iron uptake downregulation in EPP remain unclear, and the role of PPIX accumulation in microcytosis.

Dysregulation of iron metabolism and hyper-inflammation are two key points in the pathogenesis of coronavirus disease 2019 (COVID-19). Since high hepcidin levels and low serum iron can predict COVID-19 severity and mortality, we decided to investigate iron metabolism and inflammatory response in 32 COVID-19 adult patients with a diagnosis of COVID-19 defined by a positive result of RT-PCR nasopharyngeal swab, and admitted to an Italian emergency department for acute respiratory failure at different degree. Patients were stratified in 3 groups based on PaO 2 /FiO 2 ratio at admission: 13 (41%) were normoxemic at rest and suffered from exertional dyspnea (group 1); 14 (44%) had a mild respiratory failure (group 2), and 5 (15%) a severe hypoxiemia (group 3). White blood cells were significantly higher in group 3, while lymphocytes and hemoglobin were significantly reduced. Serum iron, transferrin saturation, non-transferrin-bound iron (NTBI) and ferritin were significantly increased in group 2. All the groups showed high hepcidin levels, but in group 3 this parameter was significantly altered. It is noteworthy that in group 1 inflammatory and oxidative indices were both within the normal range. We are aware that our study has some limitations, the small number of enrolled patients and the short period of data collection, but few works have been performed in the Emergency Room. However, we strongly believe that our results confirm the pivotal role of both iron metabolism dysregulation and hyper-inflammatory response in the pathogenesis of tissue and organ damage in COVID-19 patients.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by mutations in G6PD gene. The distribution and frequency of genetic variants differ depending on ethnicity and geographical areas. Because of new migrations different variants are now present in Europe. This retrospective study aims to identify variants among the G6PD deficient subjects referred since 2004 to IRCCS Ca’ Granda Foundation Hospital in Milan. The subjects were divided into 3 groups: group 1 (2004–2008), group 2 (2009–2013), and group 3 (2014–2018). During 15 years a significant decrease of the Mediterranean and an important increase of the African, Asian, and uncommon variants (classified as Others) have been observed. Three new mutations were found: in group 2 heterozygosity for c.[1454G > A] (Gly485Asp) in an adult female with severe anemia, high bilirubin levels and G6PD activity of 0,69 (IU/gHb) and heterozygosity for c.[584A > G] (Gln195Arg) in an elderly woman of Italian origin showing only anemia and enzymatic activity of 1,54 (IU/gHb) were detected. In group 3 hemizygosity for c.[670A > T] (Ile224Phe) in an adult Chinese man without anemia but with total absence of G6PD activity was found. These data reflect the appearance of uncommon G6PD mutations in northern Italy, probably due to new migrations, as consequence G6PD characterization becomes a diagnostic issue.

β-thalassemia is a hereditary disorder caused by defective production of β-globin chains of hemoglobin (Hb) that leads to an increased α/β globins ratio with subsequent free α-globins. Alpha globin excess causes oxidative stress, red blood cells membrane damage, premature death of late-stage erythroid precursors, resulting in ineffective erythropoiesis. The transforming growth factor β (TGF-β) superfamily signaling acts on biological processes, such as cell quiescence, apoptosis, proliferation, differentiation, and migration, and plays an essential role in regulating the hematopoiesis. This pathway can lose its physiologic regulation in pathologic conditions, leading to anemia and ineffective erythropoiesis. Activin receptor-ligand trap molecules such as Sotatercept and Luspatercept downregulate the TGF-β pathway, thus inhibiting the Smad2/3 cascade and alleviating anemia in patients with β-thalassemia and myelodysplastic syndromes. In this review, we describe in extenso the TGF-β pathway, as well as the molecular and biological basis of activin receptors ligand traps, focusing on their role in various β-thalassemia experimental models. The most recent results from clinical trials on sotatercept and luspatercept will also be reviewed.

b-thalassemia is a hereditary disorder caused by defective production of b-globin chains of hemoglobin (Hb) that leads to an increased a/b globins ratio with subsequent free a-globins. Alpha globin excess causes oxidative stress, RBCs membrane damage, premature death of late-stage erythroid precursors, resulting in ineffective erythropoiesis. The transforming growth factor b (TGF-b) superfamily signaling acts on biological processes, such as cell quiescence, apoptosis, proliferation, differentiation, and migration, and also plays an important role in the regulation of hematopoiesis. This pathway can lose its physiologic regulation in pathologic conditions, leading to anemia and ineffective erythropoiesis. Activin receptor ligand trap molecules such as Sotatercept and Luspatercept downregulate the TGF-b pathway by inhibiting the Smad2/3 cascade, thus alleviating anemia in patients with b-thalassemia and myelodysplastic syndromes. In this review, we describe in extenso the TGF-b pathway, as well as the molecular and biological basis of activin receptors ligand traps, focusing on their role in various b-thalassemia experimental models. The most recent results from clinical trials on sotatercept and luspatercept will also be reviewed.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by G6PD gene mutations which affect 400 million people worldwide. To date, more than 140 G6PD mutations have been reported. The distribution and frequency of genetic variants differ depending on ethnicity and geographical areas. Because of new migrations different variants are now present in Europe. Aims: This retrospective study aims to identify variants among the G6PD deficient subjects referred since 2004 to “Ca’ Granda” Foundation Hospital “in Milan. The subjects were divided into 3 groups: group 1 (2004–2008), group 2 (2009–2013) and group 3 (2014–2018).These subjects were studied because of known or suspected G6PD deficiency or for genetic counselling. Methods: The molecular analysis has been carried out by restriction fragments length polymorphisms (RLFP) and direct DNA sequencing. Results: Three hundred and thirty nine G6PD mutated subjects were identified, classified and divided as shown in table 1:Table 1 - G6PD variants identified in glucose 6-phosphate dehydrogenase-deficient subjects diagnosed since 2004 (number of subjects with G6PD mutations in italic, percentage % in brackets). During 12 years a significant decrease of the Mediterranean and an important increase of the African, Asian and uncommon variants (classified as Others) have been observed. In the first eight years Cassano and Chatham variants have an increase that does not change in the last four years. During each period new mutations were found: in group 1 the Gly485Asp (c.[1454G> A]) in homozygosity (female), in group 2 the Glu195Arg (c.[584A> G]) in heterozygosity and in group 3 the Ile224Phe (c.[670A>T]) in hemizygosity. Referring to the entire period of the study 2004–2018, in 3 subjects (1.1%) were found Portici variant, responsible for chronic non-spherocytic haemolytic anaemia, as well as Puerto Limon and Georgia (0,4%). Other variants identified in less than 1% of subjects studied were: Cairo, Mahidol, Ludhiana, Rignano, Santamaria, Sant’Antioco, Canton, Sibari, Cosenza, Abruzzo, Viangchan, Orissa, and Union. Two cases showed the 1365–13T>C polymorphism coupled with 1311C>T variation, generally associated with Mediterranean variant. Summary/Conclusion: These data reflect the appearance of uncommon G6PD mutations in Northern Italy, probably due to new migrations, as consequence G6PD characterization becomes a diagnostic issue. The evidence of polymorphisms in G6PD deficient subjects suggests that not only single variations in the exonic/intronic boundaries, but also specific haplotypes could be causative for G6PD deficiency.

Gaucher disease type 1 is the most common inherited lysosomal storage disorder caused by the deficiency of lysosomal β-glucocerebrosidase (GBA, acid-β-glucosidase), required for the degradation of glycosphingolipids. The deficiency of the enzyme results in the widespread accumulation of glucosylceramide in macrophages, leading to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). However, GD manifestations are caused not only by the burden of glucosylceramide storage, but also by macrophage activation. It seems that GD reflects the downstream consequences of inappropriate macrophage activation, with the release of pro-inflammatory cytokines and other responses to storage material. The aim of this study is to assess in vitro phenotypic characterization, functional properties and gene expression anaysis of GD1 macrophages in order to understand their possible role in inflammation and in impairment of iron metabolism occurring in GD patients. Monocytes were isolated from buffy coats of GD patients (n=3) and controls (n=3) by applying an in vitro model protocol. Monocytes were expanded in ImmunoCult™ medium and 1% ZellShield® antibiotic cocktail. Differentiation was induced in ImmunoCult™ medium, 50 ng/mL M-CSF and 50 ng/mL GM-CSF. To mimic the in vivo condition, macrophage population was loaded with erythrocytes ghosts isolated from GD patients. Cell morphology was analyzed on cytocentrifuged preparations stained with May-Grünwald Giemsa (MGG). Surface marker expression (CD11, CD33, CD68, CD64) were examined by flow cytometry to evaluate macrophages differentiation and phenotype. Gene expression analysis of iron metabolism-related genes was evaluated through Real-Time Quantitative PCR. Biochemical indices (NTBI, GDF15, sTfR and chitotriosidase) were analyzed in supernatant through ELISA assay. Flow cytometry analysis (Fig.1) revealed that without erythrocytes ghosts (preM∅), the proportion of CD11+/CD68+ macrophage population was similar between GD patients and control. However, in GD macrophages, when loaded with Gaucher erythrocytes ghosts (M∅+ghosts), the proportion of CD11++/CD68++ cells increased (36.4%), as a reflection of a more pro-inflammatory phenotype. Treated controls showed no differences. To further characterize these different macrophages subpopulations in GD patients, we used an additional parameter, CD64, because CD64/CD68 markers are specific for M1/M2 polarization. GD M∅+ghosts showed an higher percentage of CD64++/CD68++ population (78%) in comparison of GD pre-M∅ (45%) and controls (36%), confirming a M1 proinflammatory phenotype of GD macrophages loaded with erythrocytes ghosts. Under microscope evaluation, GD M∅+ghosts presented high number of spindle-shaped fibroblastoid, typically M1 phenotype cells, rather than large flat-round cells (M2 cells). Moreover, morphological staining of these cells confirmed the typical features of GD cells with basophilic cytoplasm with characteristic crinkles. Controls showed no differences in macrophage features when loaded with erythrocytes ghosts. To confirm the pro-inflammatory potential of GD M∅+ghosts, high levels of pro-inflammatory mediators TNF-α, IL-1β and (s)TfR) were found in supernatant of GD M∅+ghosts (72,9±8,5, 24,7±2,7 and 3,1±4,4, respectively) compared to pre-M∅ and controls. High GDF15 expression in GD M∅+ghosts (14,77±5,87) respect to preM∅ (1,80±1,1) and controls (1,67±0,8) was observed. By gene expression analysis we observed in GD M∅+ghosts an higher HAMP expression (28,13±5,63) compared to preM∅ (2,49±1,5) and controls (0,71±0,02), and a lower SLC40A1 expression (0,01±0,00) compared to GD preM∅ (0,16±0,05) and controls (0,79±0,25). No significant differences in TFRC and GDF11 expression between GD preM∅ and M∅+ghosts in GD patients were observed. These preliminary data suggest that GD macrophages, when stimulated, display a proinflammatory potential. These activated M1 macrophages could contribute to an inflammatory-producing environment, triggering hepcidin and ferroportin expression by an autocrine/paracrine mechanism and leading to dysregulation of iron distribution. Disclosures Cappellini: CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

In normal subjects serum transferrin saturation is 20%-35%, and “free-iron” better defined as non-transferrin-bound-iron (NTBI) is undetectable. The presence of NTBI has been reported in disorders associated with iron overload such as thalassemia and in serum of patients after intensive cytotoxic chemotherapy for the treatment of hematological malignancies1. Hematopoietic cell transplantation (HCT) is increasingly used as a curative therapy of a variety of malignant and nonmalignant diseases. Transplant conditioning with high doses of cytostatic drugs is associated with rapid transferrin saturation, changes in ferritin levels and accumulation of NTBI.2 The aim of our study was to evaluate the NTBI behavior in transfusion-dependent iron-overloaded thalassemia and non-iron-overloaded non-thalassemia (leukemia) patients during HCT and to verify its relationship to oxidative stress as expression of cellular damage.3 Eighteen patients (median age 8.8 years; range 4-16 years) affected by β-thalassemia major (8 female, 10 male) and 9 patients (median age 27.4 years; range 10-44 years) affected by different types of hematological malignancies (6 female, 3 male) were recruited for this study. There was only one splenectomized patient in each group. The control group consisted of 17 healthy subjects (9 female and 8 male aged 23-40 years). Institutional review board of Pesaro Hospital Hematology Unit approved this prospective observational non-interventional study as part of the ancillary research on HCT in iron loaded patients. All thalassemia patients were transfusion dependent and on deferoxamine subcutaneous infusion (20-40 mg/kg) according to the iron status. All patients stopped deferoxamine infusion at least 4 weeks before transplantation. Leukemia patients received a limited number of red blood cell units according to clinical management of their disease. No leukemia patient received chelation. All patients were conditioned with high dose oral Busulfan (14 mg/kg) from day −9 to −6, followed by cyclophosphamide (120-200 mg/kg) from day −5 to day −2 being day 0 the day of hemopoietic cell infusion. Starting from day −1 all patients were treated with intravenous Cyclosporine (3 mg/kg). NTBI and serum malondyaldehyde (MDA) as marker of lipid peroxidation that indirectly represent the tissue damage were analyzed prior to conditioning therapy (−9 “baseline values”), during therapy (day −5,−2), on the day of HCT (day 0) and up to 4 weeks later (day +7, +14 and + 28). Serum ferritin and hepatic iron concentration (HIC) were obtained from clinical laboratory procedures. Serum NTBI was assayed by high performance liquid chromatography (HPLC) after nitrilotriacetic acid (NTA) chelation. The solution was ultrafiltered and directly injected into the HPLC system with a titanium pump module (PerkinElmer S200, Boston, Massachusetts). NTBI in normal subjects is undetectable or even negative, because samples are measured in parallel with a corresponding blank formed by NTA and water which contains small amounts of iron which is not bound by transferrin; therefore the removal of blank makes normal samples negative. Free MDA was evaluated by means of a colorimetric assay for lipid peroxidation using a Bioxytech LPO-586 kit (Oxis International Inc, Portland, Oregon). Continuous variables were expressed as mean ± SD unless specifically indicated. Statistical differences among the groups were evaluated by Mann-Whitney test. Linear-regression analysis was performed for the correlation between 2 variables. At baseline the median ferritin in thalassemia patients was 1580 ng/mL (range 759-6360 ng/mL), while in non-thalassemia patients was 789 ng/mL (range 27-3916 ng/mL). The median HIC in thalassemia patients was 12.9 mg/g/dw (range 4.09-40.00 mg/g/dw, normal value <1.6 mg/g/dw), in non-thalassemia patients HIC was not measured. NTBI values were significantly higher in all patients compared with controls (−0.80 ± 0.70 μmol/L) pre and post HCT (thalassemia 1.76 ± 1.38 μmol/L P < .0001; leukemia 0.55 ± 1.45 μmol/L P = .013), although leukemia patients had overlapping NTBI values with respect to controls at baseline and at day 14. Significantly higher levels of NTBI were detected in thalassemia compared with leukemia patients at days −9 (baseline), 0 and 14 but not at the other time points during the study and particularly during and immediately after chemotherapy. NTBI levels were constantly higher in the thalassemia group but the relative increase compared to baseline was higher in leukemia patients (between 2.2- and 3-fold) than in thalassemia patients (between 1.6- and 2.5-fold). Early after transplant, consistently with erythropoietic recovery, NTBI returned to respective baseline values (Figure 1A). MDA values significantly correlated with NTBI values in leukemia patients independently from distance from transplant or chemotherapy (Figure 1B). No significant correlation was found in thalassemia group between MDA and NTBI values (data not shown). The markedly increase of NTBI in serum after myeloablative chemotherapy was originally attributed to suppression of erythropoietic activity. Other possible sources of extracellular iron after myeloablative conditioning include lysis of erythroid bone marrow cells by cytotoxic injury.4 Our study confirms reduction of the iron uptake by erythroid precursors during and immediately after chemotherapy. NTBI decrement in all the patients after HCT could be explained by high iron requirement by the reconstituting allogeneic erythroid marrow. The correlation between NTBI and MDA in leukemia and not in thalassemia patients may suggest that the previous iron overload in thalassemia and previous already elevated NTBI and MDA levels are able to attenuate changes in the oxidative state before and after transplantation. High levels of iron overload, in the absence of chelation, are responsible for the persistence of NTBI high levels even after marrow reconstitution in thalassemia patients. These data indicate that, although iron overload per se may lead to NTBI presence, the bone marrow ablation by cytotoxic drugs, in preparation of HCT is responsible for the appearance and increasing of NTBI in plasma, which may have a toxic effect causing organ damage even in non-thalassemia, non-iron loaded, patients. In conclusion these data support the concept of iron release from erythroid cells during bone marrow ablation by cytotoxic drugs even in the absence of systemic iron overload. The “free-iron” form may contribute to organ toxicity detectable during chemotherapy in preparation of HCT.5 In iron-overloaded patients with high NTBI levels a chelation period pre and immediately after bone marrow ablation and HCT could be beneficial in terms of organ toxicity and transplantation outcome.6 The possible clinical benefit must be demonstrated in a prospective controlled clinical trial. Dr Angelucci is Chair of the steering committee of the TELESTO trial by Novartis Oncology on iron chelation therapy in MDS.

Elli, Luca MD; Poggiali, Erika MD; Tomba, Carolina MD; Andreozzi, Fabio MD; Nava, Isabella PhD; Bardella, Maria Teresa MD; Campostrini, Natascia MD; Girelli, Domenico MD; Conte, Dario MD; Cappellini, Maria Domenica MD Author Information

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.

Objectives: Thrombocytopenia and splenomegaly are common features in several haematological disorders. Gaucher disease (GD) is a rare lysosomal storage disorder frequently characterized by thrombocytopenia and splenomegaly, which represents a clinical challenge for haematologists and internists. Case: We describe the case of a 37-year-old patient with a diagnosis of spherocytosis since childhood, who developed hepatic failure and presented striking features of GD including hepatosplenomegaly, bone fractures and post-partum bleeding. We reconsidered the diagnosis of spherocytosis and investigated Gaucher disease. Conclusion: GD should be considered in the differential diagnosis of thrombocytopenia and splenomegaly.

Iron refractory iron deficiency anaemia (IRIDA) is caused by mutations in the TMPRSS6 gene, which encodes transmembrane protease, serine 6 (TMPRSS6, also known as matriptase-2) expressed by the liver. TMPRSS6 belongs to the family of type II serine proteases (IISPs) that are involved in the crucial processes of remodelling extracellular matrix components and regulating cell-surface receptors, adhesion molecules and proteolytic processes (Finberg et al, 2008; Ramsay et al, 2009). It has been recently demonstrated that TMPRSS6 is an essential regulator of iron homeostasis, as it is a physiological suppressor of hepcidin. TMPRSS6 mutations cause IRIDA, as increased hepcidin concentrations degrade intestinal ferroportin and do not allow normal iron absorption (Finberg et al, 2008; Ramsay et al, 2009). Familial and sporadic cases of IRIDA have been described with different type of TMPRRS mutations and different clinical presentations at various ages. These may range from severe anaemia with parenteral iron requirement in infancy up to isolated microcytosis and low transferrin saturation with unremarkable anaemia in adult age (Tchou et al, 2009; Pellegrino et al, 2012). Polyendocrine autoimmune syndromes (PAS) are acquired diseases with highly variable clinical phenotype. Patients develop several autoantibodies, eventually leading to the immune-mediated destruction of endocrine organs (Eisenbarth & Gottlieb, 2004). The main recognized categories are the rare monogenic Type I, which starts in childhood with a highly variable clinical phenotype encompassing hypoparathyroidism and adrenocortical failure, and the most frequent polygenic type II, which has variable onset from infancy to adulthood, includes autoimmune Addison disease plus type 1A diabetes or thyroid autoimmunity (TA) and is more varied in its manifestations. The coexistence of more than one endocrinopathy presumably results from shared genetic susceptibility leading to loss of tolerance towards multiple tissues. Therefore, according to the ‘splitters’ who consider each of the combination of disorders as a separate syndrome, there are also PAS type III, referring to thyroid autoimmunity plus another autoimmunity (except Addison and type 1A diabetes) and PAS type IV, referring to two or more other organ-specific autoimmune diseases (Betterle & Zanchetta, 2003; Eisenbarth & Gottlieb, 2004). In more detail, PAS type III encompasses type IIIa, the combination of TA plus type 1A diabetes; type IIIb, the combination of TA plus anti-gastric parietal-cell (anti-GPC), also reported as ‘thyreogastric syndrome’; and type IIIc, the combination of TA plus coeliac disease (Sterzl et al, 2008; Checchi et al,2010). A clear association between iron deficiency, autoimmune gastritis and Helicobacter pylori (HP) infection has been demonstrated (Hershko & Skikne, 2009; Tozzoli et al, 2010). However, to the best of our knowledge, the association of TMPRSS6 mutations and PAS had never been reported. Following the observation of TMPRSS6 mutation in one of our patients diagnosed with IgA glomerulonephritis in the setting of iron deficiency anaemia (IDA) and PAS type IIIb, we looked for TMPRSS6 mutation in other patients with IDA and PAS type III. Patients were selected on the basis of the following criteria: (i) presence of unexplained iron deficiency leading to diagnosis of IRIDA, documented by personal history and failure of iron oral supplement the improve the status of IDA, (ii) diagnosis of PAS type III by the occurrence of TA plus type IA diabetes (Type IIIa), anti-GPC (type IIIb), or coeliac disease (type IIIc) and (iii) absence of other autoimmune diseases. All patients were enrolled after giving written informed consent and management of personal data was approved by the Local Ethics Committee as requested by the Italian Privacy Safeguard Act. Genomic DNA was isolated from peripheral blood lymphocytes and sequence variations in TMPRSS6 were evaluated by polymerase chain reaction (PCR) and direct sequencing. Direct sequencing was performed using a fluorescence-tagged dideoxy chain terminator method in an ABI BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. Eight patients fulfilled the inclusion criteria (Table 1). They were all Caucasian Italian women, seven with PAS type IIIb and one with PAS type IIIc; five of them had clinically evident autoimmune thyroid disease needing drug therapy. Gastritis was diagnosed in five patients, with previous HP infection in three of them. Age (years) at genetic diagnosis Anti-thyroglobulin titre (normal range <60 IU/l) Anti-gastric mucosal antibodies titre (normal range <10 IU/l) Hetero-zygous 2207 T>C (variant V736A)Exon 17 Homo-zygous 2207 T>C (variant V736A) Exon 17 Homo- zygous 2207 T>C (variant V736A) Exon 17 Homo- zygous 2207 T>C (variant V736A) Exon 17 Homo- zygous 2207 T>C (variant V736A) Exon 17 Hetero-zygous Deletion 1813G (variant A605fs) Exon 15 The patients had variable degrees of anaemia with microcytosis (mean cell volume, 59·6–88·9 fl), transferrin saturation 2–18% and serum ferritin 2–14 μg/l and all needed intravenous iron infusion. In six of the eight patients, TMPRSS6 variations were found: The single nucleotide polymorphism rs855791 in exon 17, resulting in non-synonymous (V736A) change in the serineprotease domain of PMPRSS6, was found in five patients (four homozygous and one heterozygous), while one patient showed a heterozygous G deletion (1813G) in exon 15 (A605fs). Our data first demonstrate that TMPRSS6 variations are very frequently associated with IDA in patients suffering from PAS type III. Both TMPRSS6 mutations reported here have been previously associated with IRIDA (Tchou et al, 2009), but never in the setting of PAS. All our patients fulfilled the criteria for IRIDA and PAS type III. A complex genetic influence in the pathogenesis of autoimmune diseases is well known, as demonstrated in coeliac disease or, for instance, for specific genetic polymorphisms of the insulin gene (INS) in type 1A diabetes (Eisenbarth & Gottlieb, 2004). Based on the results of our study we speculate that mutations or polymorphisms modulating TMPRSS6 expression, which consequently alter iron absorption at the gastrointestinal mucosal level, might be linked to acquired changes in gastrointestinal molecules/peptides. These could make them more likely to be targets when the immune system fails to maintain self-tolerance, as in PAS (Fig 1). The main limits of these preliminary observations are the lack of a control group of healthy people, which could help establish the usual incidence of TMPRSS6 mutations within the normal population, and the lack of a control group of patients with PAS but without IDA, in order to confirm and assess the strength of the reported association between TMPRSS6 and PAS. We are currently working on both of these aspects, systematically analysing PAS patients for IRIDA. Despite all these limits, our findings could provide new insight into the genetic factors influencing anaemia, PAS and gastrointestinal-related disorders. All authors declare that they have participated in writing the paper and have seen and approved the final version of the paper. Caterina Canavese: Designed the research study, drafted the paper, revised and approved the final version of the paper. Piero Stratta: Contributed to the designs of the study, critically revised the paper and approved the final version. Isabella Nava: Performed the research, contributed essential reagents or tools and approved the final version. Lorena Duca: Performed the research, contributed essential reagents or tools and approved the final version. MDomenica Cappellini: Interpretated the results, crtitically revised the paper and approved the final version. Cristina Izzo: Performed the research, analysed the data and approved the final version. Marco Quaglia: Designed the research study, wrote the paper and approved the final version. All authors state that there are no conflicts of interest. The authors declare no funding sources.

Abstract Introduction Iron Refractory Iron Deficiency Anemia (IRIDA) is an autosomal recessive form of iron deficiency anemia (IDA) caused by mutations in TMPRSS6 gene, and characterized by unresponsiveness to oral iron supplementation and low effectiveness of parenteral iron administration (Finberg 2009). So far 50 cases from 32 families have been reported and 40 mutations have been identified (De Falco 2013). Although mutations are extremely rare, recent insights have revealed that highly frequent polymorphisms of TMPRSS6 gene may influence iron absorption, being associated with increased risk of IDA (An 2012). Patients and Methods Between January 2009 and May 2013, 88 subjects (11 males, 77 females) with mean age 39+/-14 years were referred to the Hereditary Anemia Centre of “Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico di Milano” for persistent IDA poorly responsive to oral iron. All the patients (pts) were investigated for celiac disease, gastrointestinal bleeding, and HP infection. Hematological parameters, iron status, inflammatory markers, and thyroid function were tested. Sequence variation in TMPRSS6 gene was evaluated by PCR and direct sequencing in genomic DNA isolated from peripheral lymphocytes. Thalassemia trait was suspected and investigated in 27/88 (31%) pts (3 males, 24 females) using HPLC and genetic analysis of globin chains. Fifty healthy donors (15 females, 35 males) with mean age 28±9 yrs were used as control group. Results Frequency of SNP-120, SNP-113, P33P, K253E, Y418Y, D521D, Δ15accc and V739Y results significantly different between pts and controls. Association study revealed that in pts homozygosis for V736A is frequently associated with homozygosis for D521D and Y739Y, while polymorphic alleles F5F, P33P, K253E, S361S, Δ15accc are linked to V736A trans-allele. Based on the observation that homozygosis for V736A is not present in healthy control, we analyzed the hematological parameters in anemic pts homozygotes, heterozygotes, and wild type for V736A. No significant differences were found (table 1). Considering only the thalassemia pts, the combination of thalassemia trait and V736A is associated with a more severe anemia (Hb 10.3±1.4 g/dL, MCV 62.9±6.7 fL median ferritin 30 ng/mL), requiring blood transfusion in particular circumstances (pregnancy, surgery). Moreover, one new variant (H448R) was identified in a pt with IDA requiring parenteral iron supplementation. Two rare variants, A719T and V795I (estimated frequency 0.000/1 and 0.004/9), were detected respectively in two sisters, causing the IRIDA phenotype only in one, and in two patients, who require parenteral iron therapy. Discussion and Conclusion Several TMPRSS6 polymorphisms are more frequent in anemic pts than in healthy donors, suggesting their role in the refractoriness to oral iron. No significant differences were observed in hematological data related to V736A genotype. This is not surprising because all the pts were previously treated with iron therapy, which contribute to partially reduce the degree of anemia. In this study we found peculiar haplotypes, a new variant (H448R) and two rare variants (A719T and V795I), which may account for impairment in TMPRSS6 activity. Further studies are necessary to clarify the role of TMPRSS6 polymorphysms, which will allow identifying individuals at risk for more severe IDA, particularly in thalassemia pts, driving to correct diagnosis and management of iron supplementation, sparing to the patient inadequate therapeutic choices and diagnostic procedures. Disclosures: Cappellini: NOVARTIS: Membership on an entity’s Board of Directors or advisory committees.

Abstract Abstract 5285 INTRODUCTION: The growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to be related to ineffective or apoptotic erythropoiesis. GDF-15 levels were found to be significantly elevated in sera of patients with β thalassemia major (TM), however little is known about the GDF15 expression during thalassemic erythropoiesis. Non-transfusion dependent thalassemia intermedia (NTDT) represents the model of thalassemic erythropoiesis not affected by transfusions. AIM: To determine the GDF15 gene expression profile during normal and thalassemic erythroid differentiation in standard cultures and under different iron conditions, from CD34+ of normal and thalassemia intermedia subjects. METHODS: After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with NTDT by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. Each culture was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4 mM) as iron chelating agent and ferric ammonium citrate (FAC 100 mM) at day 0 of culture. The expression profiling of GDF15 gene was evaluated at baseline, day 7 and day 14 by real-time PCR (2̂-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit (R&D Systems, Minneapolis, MN). RESULTS: GDF15 expression and secretion increased significantly during erythroid differentiation either in normal and in NTDT cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were higher (althoug not statistically significant) compared to control (Table 1). At day 14 in control cultures GDF15 expression is up-regulated by DFO and down-regulated by FAC addition. In NTDT GDF15 expression was influenced by iron addition in cultures, but was not increased by iron depletion (Table 2). CONCLUSIONS: GDF15 levels in erythroid cultures are related to the erythropoietic stage of differentiation. In NTDT cultures the GDF15 gene profile and protein levels in supernatants are higher than in normal cultures. GDF15 levels seem to be modulated by iron in normal cultures whereas in NTDT cultures they seem to be independent from iron availability. This in vitro study supports that signals different than iron, such as erythropoietic stress, could be the major factor regulating GDF15 expression. Disclosures: No relevant conflicts of interest to declare.

SummaryTransmembrane Protease, Serine 6 (TMPRSS6) has an important role in iron homeostasis and its mutations, performed in TMPRSS6‐deficient mice, have been recently associated with iron‐refractory iron deficiency anaemia (IRIDA). Several variants of TMPRSS6 have been already identified; however the role of polymorphisms and TMPRSS6 haplotypes, causing iron deficiency anaemia, have not yet been investigated. This study sequenced the TMPRSS6 gene in 16 subjects with IRIDA phenotype and identified 27 DNA polymorphisms. Eight single nucleotide polymorphisms and four haplotypes were significantly associated with iron‐refractory anaemia (P < 0·001). Our preliminary results suggest a possible association between specific haplotypes of TMPRSS6 and IRIDA.