GENETIC/METABOLIC EFFECT OF IRON METABOLISM & RARE ANEMIAS

3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias

Limassol, 24 – 26 October 2012

Clara CamaschellaUniversità 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)

(Traglia et al, J Med Genet, 2011)

Iron and hepcidin levels in Val Borbera individuals

Hepcidin inhibition in iron deficiency, hypoxia and erythropoiesis expansion

(Hentze et al, Cell 2010)

Proposed inhibitors:

Epo, Hif1-alpha, s-HJV,GDF15/TWSG1TMPRSS6/Matriptase 2

1. Defects of iron absorptionIRIDA - 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

CN TM CUB LCUB 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…)

Y1

41

CL

16

6fs

I21

2T

Q2

29

fsW

24

7fs

R2

71

Q

C5

10

S

S5

61

XS

57

0fs

E4

86

D

S3

04

L

A1

18

D

Y3

35

XY

39

3X

G4

42

R

E4

61

fs

D5

21

NE

52

2K

Ma

sk

R5

99

X

A6

05

fs

K6

36

fs

P6

86

fs

R7

74

C

Mutations associated with IRIDA

L6

74

F

(Silvestri et al Blood 2009De Falco et al, Hum Mut 2010)

K2

53

EQ

G6

03

R

Hepcidin activation in IRIDA: molecular mechanism

HEPC

SMADs

BMP

BMPR

m-HJV

TMPRSS6

serum ironHEPC

SMADs

BMP

BMPR

m-HJV

TMPRSS6

serum iron

IRIDA IDA

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

Hepcidin: the key iron regulator

hepcidin

Fe

Liver

macrophagesenterocytes

Fe

Fe

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*

IRIDA: hematological data

(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)

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 miceSimilar phenotype

Splicing mutations of transferrin

Hepcidin low/undetectable

Hypotransferrinemia: lesson from patients

Iron-deficienterythropoiesis

transferrin

Microcytic anemia

100% Tf saturation

NTBILiver, pancreas iron overload

Transferrin (and TFR cycle) are indispensable for erythropoiesisbut not for liver iron uptake (NTBI)

hepcidin

Hepcidin suppression by the iron-deficient erythropoiesis increases iron absorption

DMT1 deficiency (OMIM #206100)

mk mouse and Belgrade ratsevere iron-deficient anemia due to G185R homozygous Dmt1 mutationDmt1 -/- 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 oflow 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)

Atransferrinemia

DMT1 mutations

IRIDA IDA

Hb low low low low

MCV low low low low

Fe low high low low

Tf Low/absent low high high

Tf sat high high low low

ferritin high high normal/high low

hepcidin low low high low

Differential diagnosis of iron-related inherited anemias

Perl’s staining Anti-MT-ferritin(Courtesy of R. Invernizzi, Pavia)

3. Defects of iron utilization: sideroblastic anemias

Mitochondrial iron metabolism

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

Heme

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 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)

GLRX5-mutant patient follow up

Hb

g/d

L

Fer

ritin

ng/

mL

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 edsGeneReviews University of Washington, Seattle)

ACERULOPLASMINEMIA: pathogenesis

liver iron overload

Low serum FeIncreased Fe absorption

Mild “iron deficiency”anemia

FPN

CP

Fe2+

(

(

Fe3+

ferroxidase activity -- cellular iron efflux !

Acp -/- mouse

Aceruloplasminemia: diagnosis

Microcytic/normocytic anemia

High serum ferritin, low transferrin saturationLow serum copper (< 10g/dL; nv 70-125g/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 anemiaDMT1 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)

Disorder Gene OMIM n

Defect of iron absorption IRIDA TMPRSS6 #206200 Defects of iron transport/erythroid uptake

Hypotransferrinemia TF #209300DMT1 mutations DMT1 #206100STEAP3 mutations STEAP3

Defects of cellular iron utilizationSideroblastic anemiaX-linked sid. anemia ALAS2 +301300X-linked sid. anemia/ataxia ABCB7 #30131AR sideroblastic anemia SLC25A38 #205950

GLRX5Defects of iron recycling Aceruloplasminemia CP #604290

Inherited iron-metabolism related anemias

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 PaganiLaura Silvestri

Alessandro CampanellaMarco Rausa

University of NaplesAchille Iolascon Luigia De Falco

University of VeronaDomenico Girelli Natascia Campostrini

Fifth Meeting of the International BioIron Society 

BioIron 2013: April 14 – 18, 2013University College London UK

www.bioiron.org