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Fourth European Symposium on Rare Anaemias

Clara CamaschellaClara CamaschellaVita-Salute University - San Raffaele Scientific Institute, Milano

Sofia, Bulgaria, November 19-20, 2011

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The iron cycle Hepcidin

Liver secreted peptide

(Jordan et al,JBC 2009)

(Andrews, NEJM1 999)

Active peptide: 25 C-terminal aacleaved from a 84 aa precursor

Hairpin structureAcute phase reactant

(Ganz, Blood 2003)

Hepcidin: the key iron regulator

LiverFe

hepcidin

FeFe

macrophagesenterocytes

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Systemic iron regulation

(Hentze et al, Cell 2010)

Disorders of hepcidin deficiency

Genetic disordersHereditary hemochromatosis type 1,2,3

due to mutations of HFE, Hepcidin and HJV, TFR2

Acquired disordersAcquired disorderssecondary iron overload in iron loading anemias

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Disorders of hepcidin excess

Genetic disordersIron-refractory iron deficiency anemia

Acquired disordersAnemnia of chronic diseasesAnemnia of chronic diseases

Microcytosis - Hypochromia

Reduced size and reduced Hb content of red blood cells, i f d b th t i das inferred by erythrocyte indexes:

MCH < 26 pg (normal values 27-30 pg)MCV < 80 fl (normal values 82-98 fl)MCHC < 30 g/dl (normal values 31-37 g/dl)

Peripheral blood smear

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Microcytic anemia: relevance

Most common form of anemia worldwide:

• Iron deficiency anemia ( heme)

• Thalassemia syndromes ( globin)

Disorder Gene OMIM nDefects of iron transport

Inherited microcytic anemias

Defects of iron transport Hypotransferrinemia TF #209300DMT1 mutations DMT1 #206100

Defects of cellular iron utilizationSideroblastic anemiaX-linked sid. anemia ALAS2 +301300AR sideroblastic anemia SLC25A38 #205950

GLRX5X li k d id i / i ABCB7 #30131X-linked sid. anemia/ataxia ABCB7 #30131

Defect of iron absorptionIRIDA TMPRSS6 #206200

Defects of iron recycling(Aceruloplasminemia CP #604290)

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Iron for erythropoiesis

Daily iron needs for Hb synthesis of maturing erythroblasts: 25 mg

Autosomal recessive, extremely rareFirst description 1961

(A)Hypo-transferrinemia

First description 1961Plasma transferrin nearly absentSevere microcytic anemia since birthLiver iron overloadLow urinary hepcidin levels(Responsive to plasma infusions)( p p )

Hpx mice

Similar phenotypeSplicing mutations of TF

Liver hepcidin RNA low/absent

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Hypotransferrinemia: lesson from patients

hepcidin

Iron-deficienterythropoiesis

transferrin

Microcytic anemia

100% Tf saturation

NTBILiver, pancreas iron overload

hepcidin

Hepcidin suppression by the iron-deficient erythropoiesis increases iron absorption

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New rare disorders of iron utilization:DMT1 deficiency

DMT1: Transporter of divalent metal cations: Mn 2+ Cu 2+ Zn 2+ Fe2+DMT1: Transporter of divalent metal cations: Mn 2+ Cu 2+ Zn 2+ Fe2+

Erythroblasts: endosomal transferrin cycle

Duodenal cell: luminal non heme iron transporter

DMT1 deficiency (OMIM #206100)

mk mouse and Belgrade ratf Gsevere iron-deficient anemia due to G185R

homozygous Dmt1 mutationDmt1 -/- mice even more severe

Patients with homozygous or compound heterozygous DMT1 mutationsheterozygous DMT1 mutationsMicrocytic hypochromic anemia and liver iron overload

(Iolascon et al, J Pediatr. 2008;152:136-9)

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Figure 1. Response of hematologic parameters to darbepoetin

Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

Pospisilova, D. et al. Blood 2006;108:404-405

Reduced iron supply to erythropoiesis

Suppression of hepcidinproduction

(Andrews, NEJM, 1999)

Increased duodenal iron absorption

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Atransferrinemia DMT1 t ti

IDA

Differential diagnosis of iron-related inherited anemias

Atransferrinemia mutationsHb low low low

MCV/MCH low low low

Fe low high low

Tf Low/absent low high

Tf sat high high low

ferritin high high low

hepcidin low low low

Genetic defects of iron absorption

IRIDA: iron refractory-iron deficiency anemiay y(OMIM #206200)

Inappropriately high hepcidin production

(Ganz, T. et al. Blood 2008;112:4292-4297)

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Inappropriate hepcidin activation: IRIDA

Autosomal recessive disorder due to TMPRSS6(matriptase 2) mutations IRIDA = iron refractory iron deficiency anemiaModerate anemia, severe microcytosis Extremely low iron and transferrin saturationNormal serum ferritinNormal serum ferritinHigh serum (and urinary) hepcidin levelsRefractory to oral and partially refractory to iv iron

(Finberg et al, Nat Genet 2008, Sem Hematol 2009)

Hepcidin activation in IRIDA: molecular mechanism

IRIDAIDA

SMADs

BMP

BMPR

m-HJV

TMPRSS6

SMADs

BMP

BMPR

m-HJV

TMPRSS6

↓HEPC↑ serum iron

↑HEPC

S s

↓ serum iron

(Silvestri et al, Cell Met 2008;8:502-11.)

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IRIDA: hematological data (32 published cases)

Mean±SDHb g/dl (at presentation) 7.7±1.3Hb g/dl (at diagnosis) 9.21±1.8MCV fl 55.47±7.6Transferrin saturation % 5.03±2.3Ferritin ng/ml 126±82Serum hepcidin nM 257±157*Urin. hepcidin ng/mg creat 4113±3089*

Atransferrinemia DMT1 m tations

Tmprss6 m tations

Differential diagnosis of iron-related inherited anemias

mutations mutationsHb low low low

MCV/MCH low low low

Fe low high low

Tf Low/absent low high

Tf sat high high low

ferritin high high normal/high

hepcidin low low high

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Disorders of mitochondrial iron metabolism: sideroblastic anemias

Perl’s staining Anti-MT-ferritin

(Courtesy of R. Invernizzi, Pavia)

Mitochondrial iron metabolism

(modified from Blood 105;1867-1874, 2005)

Heme

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Defect of heme synthesis

X-linked sideroblastic anemiaThe commonest formDeficiency of ALAS2 reduced heme synthesisAffects males (rarely females) - Variable severityPiridoxin (Vitamin B6)-responsive (some cases)

Autosomal recessive sideroblastic anemiaPhenotype identical to XLSAMutations in SLC25A38, an erythroid specific mitochondrialaminoacid transporter: involved in glycinetransport into mitochondria?Non piridoxin responsive (Guernsey et al, Nat Genet. 2009;41:651-3

Kannengiesser et al. Haematologica 2011;96:808-13)

Defects of Fe/S clusters biogenesis

X-Linked SA with Ataxia (OMIM 301310A d d ib d i 1985 F f ili ld idA syndrome described in 1985. Few families worldwideMild sideroblastic anemia - Late onset of ataxiamissense mutations of ABCB7, a transporter involved in Fe/S cluster biogenesis

GLRX5 deficiencyGLRX5 deficiencyThe human counterpart of zebrafish shiraz shows sideroblasticanemia and iron overload due to an homozygous splicingmutation of GLRX5 (a gene of Fe/S cluster

(Camaschella et al Blood 2007)

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How to suspect an atypical microcytic anemia

Microcytic anemia since childhood (or birth)

Iron parameters not congruous: microcytosis +high transferrin saturation and high serum ferritin high serum ferritin and low transferrin saturation

Ringed sideroblasts (any percentage)

R f t ( ti ll f t ) i ti iRefractory (or partially refractory) microcytic anemia

High hepcidin (Tmprss6 mutations)

Familial cases

Increased ring sideroblastsNormal e-ALAS sequence

GRX 5Enzymes of the ISC assembly

Signs of mitochondrial dysfunction

No signs of mitochondrial

ataxia ABC7

Decisional tree for the candidate gene strategy

No signs of mitochondrial dysfunction

Microcytic hypochromic anemia with no ring sideroblasts

Low/normalNo response to oral ironResponse to i.v. iron

DMT1 isoform IA DctybHephaestinFerroportin

No obvious candidate

sTfR

elevated

No response to iron TfR (expression defect)IRP2

DMT1Steap3Sec15l1MitoferrineTfR (non functional mutant)

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E‐RARE project on microcytic anemias (ERARE‐115, HMA‐IRON)

l ( )Carole Beaumont (France)

Clara Camaschella (Italy)

Martina Muckenthaler (Germany)Martina Muckenthaler (Germany)

Mayka Sanchez (Spain)

Acknowledgements

Vita-Salute University & San Raffaele Scientific Institute

Antonella Nai, Alessia PaganigLaura Silvestri

Alessandro CampanellaMarco Rausa

University of NaplesAchille Iolascon Luigia De Falco

University of VeronaDomenico Girelli Natascia Campostrini

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Fifth Meeting of the International BioIron SocietyBioIron 2013: April 14 – 18, 2013University College London UK

Three siblings of Pakistani origin with transfusion dependent

A novel type of congenital hypochromic anemia associated with a nonsense mutation in the STEAP3/TSAP6 gene

Three siblings of Pakistani origin with transfusion-dependenthypochromic, poorly regenerative anemia and iron overload.

A nonsense heterozygous mutation (p.Cys100Stop) in STEAP3, inherited from the father, no mutation in the mother

Example of combination of a mutated allele and a weaklyExample of combination of a mutated allele and a weakly expressed allele

(Grandchamp B et al online)

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Phenotype similar to non-syndromiccongenital sideroblastic anemia