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GENETIC/METABOLIC EFFECT OF IRON METABOLISM & RARE ANEMIAS 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012 Clara Camaschella Università Vita-Salute San Raffaele IRCCS San Raffaele, Milano, Italy DISCLOSURE Clara Camaschella Università Vita-Salute - IRCCS San Raffaele, Milano NO DISCLOSURE Iron for erythropoiesis Daily iron needs for Hb synthesis of maturing erythroblasts: 25 mg Advances in iron metabolism research…. Systemic iron regulation (Hentze et al, Cell 2010) Iron and hepcidin levels in Val Borbera individuals (Traglia et al, J Med Genet, 2011) Hepcidin inhibition in iron deficiency, hypoxia and erythropoiesis expansion Proposed inhibitors: Epo, Hif1-alpha, s-HJV, GDF15/TWSG1 TMPRSS6/Matriptase 2 (Hentze et al, Cell 2010) 1. Defects of iron absorption IRIDA - OMIM #206200 Iron refractory iron deficiency anemia: Autosomal recessive disorder due to TMPRSS6 (matriptase-2) mutations Moderate anemia since childhood, severe microcytosis Extremely low iron and transferrin saturation Normal serum ferritin Inappropriately high hepcidin levels Refractory to oral and partially refractory to iv iron (Finberg et al, Nat Genet 2008, Sem Hematol 2009) TMPRSS6/Matriptase-2: the hepcidin inhibitor Matriptase-2 is encoded by TMPRSS6 gene on chr 22 RNA expression: liver (kidney, olfactory epithelium) Protein: 811 amino acid type II transmembrane serine protease synthesized as an inactive zymogen (TTPS family: enteropeptidase, hepsin,corin, matriptase 1…) N TM SEA CUB CUB L L L SERINE PROTEASE C Y141C L166fs I212T R774C A605fs G603R K636fs L674F P686fs S561X Mask S570fs R599X G442R E461fs E486D D521N C510S E522K Y335X Y393X Q229fs W247fs K253EQ S304L R271Q A118D Mutations associated with IRIDA (Silvestri et al Blood 2009 De Falco et al, Hum Mut 2010) Hepcidin activation in IRIDA: molecular mechanism IRIDA IDA m-HJV BMP TMPRSS6 m-HJV BMP BMPR SMADs HEPC serum iron TMPRSS6 BMPR SMADs HEPC serum iron (Silvestri et al, Cell Met 2008;8:502-11.) Hepcidin: the key iron regulator Liver Fe hepcidin Fe enterocytes macrophages IRIDA: hematological data Mean±SD Hb g/dl (at presentation) 7.7±1.3 Hb g/dl (at diagnosis) 9.21±1.8 MCV fl 55.47±7.6 Transferrin saturation 5.03±2.3 % Ferritin ng/ml 126±82 Serum hepcidin nM 257±157* Urin. hepcidin ng/mg creat 4113±3089 * (Camaschella and Poggiali Curr Op Ped, 2010) How to diagnose IRIDA Evidence of microcytic anemia since the first months of life Moderate degree of anemia, more severe in children (increased requests) Familial cases (autosomal recessive) Discrepancy between ferritin and Tf saturation levels Exclude celiac disease and other absorption disorders (Normal/high serum hepcidin in the presence of IDA and normal CRP) Refractory to oral (control dose, type of iron and compliance) and partially refractory to iv iron DNA sequence of TMPRSS6 gene (common SNP excluded) How to treat IRIDA Oral iron ineffective (at least two cycles) I.V. iron: partial or slow response Epo: a single case reported with positive results (Ramsey et al, Hum Mol Genet 2009). Iron must be added A recent report suggest some effect of ascorbic acid: Cau M, Galanello R, Giagu N, Melis MA. Responsiveness to oral iron and ascorbic acid in a patient with IRIDA. (BCMD 2011) 2. Defects of TfRC cycle 1. Defects of transferrin (the ligand) 1. Defects of TfRC are not described! 2. Defects of TfRC components: DMT1, STEAP3 (Camaschella C, Br J Haematol, in press) Atransferrinemia (OMIM #209300) Autosomal recessive, extremely rare Plasma transferrin nearly absent Severe microcytic anemia and liver iron overload Low urinary hepcidin levels Responds to plasma infusions Hpx mice Similar phenotype Splicing mutations of transferrin Hepcidin low/undetectable Hypotransferrinemia: lesson from patients hepcidin transferrin 100% Tf saturation Iron-deficient erythropoiesis NTBI Liver, pancreas iron overload Microcytic anemia Transferrin (and TFR cycle) are indispensable for erythropoiesis but not for liver iron uptake (NTBI) Hepcidin suppression by the iron-deficient erythropoiesis increases iron absorption DMT1 deficiency (OMIM #206100) mk mouse and Belgrade rat severe iron-deficient anemia due to G185R homozygous Dmt1 mutation Dmt1 -/- mice even more severe Patients with homozygous or compound heterozygous DMT1 mutations Microcytic hypochromic anemia and liver iron overload (less severe than atransferrinemia) (Iolascon et al, J Pediatr. 2008;152:136-9) Lesson from DMT1 human mutants DMT1 is essential in erythropoiesis DMT1 is not essential for liver iron uptake DMT1 is not essential for duodenal iron absorption (alternative pathways?heme absorption?) Increased iron absorption occurs because of low hepcidin levels Partial response of anemia to erythropoietin treatment A novel type of hypochromic anemia associated with a nonsense mutation in the STEAP3 gene (Grandchamps et al, Blood 2011) Differential diagnosis of iron-related inherited anemias Atransferrine mia DMT1 mutations IRIDA IDA Hb low low low low MCV low low low low Fe low high low low low high high Tf Low/absent Tf sat high high low low ferritin high high normal/high low hepcidin low low high low 3. Defects of iron utilization: sideroblastic anemias Perl’s staining Anti-MT-ferritin (Courtesy of R. Invernizzi, Pavia) Mitochondrial iron metabolism Heme (modified from Blood 105;1867-1874, 2005) Defects of heme synthesis X-linked sideroblastic anemia (OMIM #300751) The commonest form Deficiency of ALAS2 reduced heme synthesis Affects males (rarely females) - Variable severity Piridoxin (Vitamin B6)-responsive (some cases) Autosomal recessive sideroblastic anemia (OMIM 301310) Phenotype more severe than XLSA Mutations in SLC25A38, an erythroid mitochondrial aminoacid transporter: involved in mitochondria glycine transport (?) Piridoxin unresponsive (Guernsey et al, Nat Genet. 2009;41:651-3) Defects of Fe/S clusters biogenesis X-Linked SA with Ataxia (OMIM 301310) A syndrome described in 1985. Few families worldwide Mild sideroblastic anemia - Late onset of ataxia missense mutations of ABCB7, a transporter involved in Fe/S export from mitochondria GLRX5 deficiency The human counterpart of zebrafish shiraz shows sideroblastic anemia and iron overload due to an homozygous splicing mutation of GLRX5 (a gene of Fe/S cluster) (Camaschella et al Blood 2007) GLRX5-mutant patient follow up 12 2500 2000 1500 6 1000 4 haemoglobin ferritin 500 2 20 07 La te 20 07 20 06 Ea rly de c Ea rly 20 04 La te 20 04 Ea rly 20 05 La te 20 05 m ay 20 03 jan 20 03 20 03 20 02 0 20 01 0 20 00 ferritin ng/ml 8 Ferritin ng/mL Hb g/dL Haemoglobin g/dl 10 Ye a rs Correlation Hb/ferritin: r = -0.79 start transfusions; stop transfusions: start DFO: stop DFO 4. Defects of iron recycling: aceruloplasminemia AR (OMIM #604290) - Mutations of Ceruloplasmin (Cp) Iron overload in liver, RE cells, pancreas, basal ganglia. Clinical triad in midlle age: 1. Diabetes 2. Neurological disease (ataxia,dementia), 3. Retinal degeneration (Miyaijma H. in: Pagonet al eds GeneReviews University of Washington, Seattle) ACERULOPLASMINEMIA: pathogenesis Acp -/- mouse ( ( ferroxidase activity -- cellular iron efflux ! FPN Fe2+ liver iron overload CP Fe3+ Low serum Fe Increased Fe absorption Mild “iron deficiency” anemia Aceruloplasminemia: diagnosis Microcytic/normocytic anemia High serum ferritin, low transferrin saturation Low serum copper (< 10mg/dL; nv 70-125mg/dL) (Low ferroxidase plasma activity) Undosable Ceruloplasmin (Cp gene mutations) MRI of liver, pancreas and basal ganglia (striatum thalamus and dentate nucleus) How to recognize an atypical microcytosis 1. Refractory (or partially refractory) microcytic anemia DMT1 deficiency: no response to i.v. iron 2. Iron parameters not congruous: high transferrin saturation and high serum ferritin high serum ferritin and low transferrin saturation 3. Ringed sideroblasts (any percentage) 4. Familial cases 5. (High hepcidin) TMPRSS6 mutations (Camaschella C Br J Haematol, in press) Inherited iron-metabolism related anemias Disorder Gene Defect of iron absorption IRIDA TMPRSS6 Defects of iron transport/erythroid uptake Hypotransferrinemia TF DMT1 mutations DMT1 STEAP3 mutations STEAP3 Defects of cellular iron utilization Sideroblastic anemia X-linked sid. anemia ALAS2 X-linked sid. anemia/ataxia ABCB7 AR sideroblastic anemia SLC25A38 GLRX5 Defects of iron recycling Aceruloplasminemia CP OMIM n #206200 #209300 #206100 +301300 #30131 #205950 #604290 Camaschella C, Br J Haematol, 2012 online E-RARE project on microcytic anemias (ERARE-115, HMA-IRON) Carole Beaumont (France) Clara Camaschella (Italy) Martina Muckenthaler (Germany) Mayka Sanchez (Spain) Acknowledgements Vita-Salute University & San Raffaele Scientific Institute Antonella Nai, Alessia Pagani Laura Silvestri Alessandro Campanella Marco Rausa University of Naples Achille Iolascon Luigia De Falco University of Verona Domenico Girelli Natascia Campostrini Fifth Meeting of the International BioIron Society BioIron 2013: April 14 – 18, 2013 University College London UK www.bioiron.org