1. 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

2. DISCLOSURE Clara Camaschella Università Vita-Salute - IRCCS San Raffaele, Milano NO DISCLOSURE

3. Iron for erythropoiesis Daily iron needs for Hb synthesis of maturing erythroblasts: 25 mg

4. Advances in iron metabolism research….

5. Systemic iron regulation (Hentze et al, Cell 2010)

6. (Traglia et al, J Med Genet, 2011) Iron and hepcidin levels in Val Borbera individuals

7. Hepcidin inhibition in iron deficiency, hypoxia and erythropoiesis expansion (Hentze et al, Cell 2010) Proposed inhibitors: Epo, Hif1-alpha, s-HJV, GDF15/TWSG1 TMPRSS6/Matriptase 2

8. 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)

9. TMPRSS6/Matriptase-2: the hepcidin inhibitor CN TMCUB L LL SERINE PROTEASESEA 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…)

10. Y141C L166fs I212T Q229fs W247fs R271Q C510S S561X S570fs E486D S304LA118D Y335X Y393XG442RE461fs D521N E522K Mask R599X A605fsK636fs P686fs R774C Mutations associated with IRIDA L674F (Silvestri et al Blood 2009 De Falco et al, Hum Mut 2010) K253EQ G603R

11. Hepcidin activation in IRIDA: molecular mechanism  HEPC SMADs BMP BMPR m-HJV TMPRSS6  serum iron  HEPC SMADs BMP BMPR m-HJV TMPRSS6  serum iron IRIDA IDA (Silvestri et al, Cell Met 2008;8:502-11.)

12. Hepcidin: the key iron regulator hepcidin Fe Liver macrophagesenterocytes Fe

13. Mean±SD Hb g/dl (at presentation)7.7±1.3 Hb g/dl (at diagnosis)9.21±1.8 MCV fl55.47±7.6 Transferrin saturation % 5.03±2.3 Ferritin ng/ml126±82 Serum hepcidin nM257±157* Urin. hepcidin ng/mg creat 4113±3089 * IRIDA: hematological data (Camaschella and Poggiali Curr Op Ped, 2010)

14. 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)

15. 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)

16. 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)

17. Autosomal recessive, extremely rare Plasma transferrin nearly absent Severe microcytic anemia and liver iron overload Low urinary hepcidin levels Responds to plasma infusions Atransferrinemia (OMIM #209300) Hpx mice Similar phenotype Splicing mutations of transferrin Hepcidin low/undetectable

18. Hypotransferrinemia: lesson from patients Iron-deficient erythropoiesis transferrin Microcytic anemia 100% Tf saturation NTBI Liver, pancreas iron overload Transferrin (and TFR cycle) are indispensable for erythropoiesis but not for liver iron uptake (NTBI) hepcidin Hepcidin suppression by the iron-deficient erythropoiesis increases iron absorption

19. 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)

20. 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

21. A novel type of hypochromic anemia associated with a nonsense mutation in the STEAP3 gene (Grandchamps et al, Blood 2011)

22. Atransferrine mia DMT1 mutations IRIDAIDA Hblow MCVlow Felowhighlow TfLow/absentlowhigh Tf sathigh low ferritinhigh normal/highlow hepcidinlow highlow Differential diagnosis of iron-related inherited anemias

23. Perl’s stainingAnti-MT-ferritin (Courtesy of R. Invernizzi, Pavia) 3. Defects of iron utilization: sideroblastic anemias

24. Mitochondrial iron metabolism (modified from Blood 105;1867-1874, 2005) Heme

25. 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 B 6 )-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)

26. 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)

27. GLRX5-mutant patient follow up Hb g/dL Ferritin ng/mL Correlation Hb/ferritin: r = -0.79 start transfusions; stop transfusions: start DFO: stop DFO

28. 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)

29. ACERULOPLASMINEMIA: pathogenesis liver iron overload Low serum Fe Increased Fe absorption Mild “iron deficiency” anemia FPN CP Fe 2+ ( ( Fe 3+  ferroxidase activity --   cellular iron efflux ! Acp -/- mouse

30. Aceruloplasminemia: diagnosis Microcytic/normocytic anemia High serum ferritin, low transferrin saturation Low serum copper (< 10  g/dL; nv 70-125  g/dL) (Low ferroxidase plasma activity) Undosable Ceruloplasmin (Cp gene mutations) MRI of liver, pancreas and basal ganglia (striatum thalamus and dentate nucleus)

31. 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)

32. DisorderGene OMIM n Defect of iron absorption IRIDATMPRSS6 #206200 Defects of iron transport/erythroid uptake HypotransferrinemiaTF #209300 DMT1 mutationsDMT1 #206100 STEAP3 mutationsSTEAP3 Defects of cellular iron utilization Sideroblastic anemia X-linked sid. anemiaALAS2 +301300 X-linked sid. anemia/ataxia ABCB7 #30131 AR sideroblastic anemiaSLC25A38 #205950 GLRX5 Defects of iron recycling Aceruloplasminemia CP #604290 Inherited iron-metabolism related anemias Camaschella C, Br J Haematol, 2012 online

33. E-RARE project on microcytic anemias (ERARE-115, HMA-IRON) Carole Beaumont (France) Clara Camaschella (Italy) Martina Muckenthaler (Germany) Mayka Sanchez (Spain)

34. 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

35. Fifth Meeting of the International BioIron Society BioIron 2013: April 14 – 18, 2013 University College London UK www.bioiron.org