.

Due Casi Clinici:- Caso di bambino con ittero franco a bilirubina indiretta ( diagnosi di Crigler-Najiar di tipo II)

- Un caso di bambino pallido con segni di carenza di ferro

I1 I2

II1 II2

Paziente maschio,11 anni

Anamnesi familiare:I.1: Colecistectomia all’età di 34 aaI.2: Iperbilirubinemia indirettaI.3: Anemia di ndd

Anamnesi personale:Nato da gravidanza normocondotta, a termine, da parto cesareoIttero neonatale (bilirubina 18 mg/dl) trattato con fenobarbital

I3

Anamnesi infantile:A 5 anni ittero sclerale e cutaneo importante (bilirubina indiretta 8.8 mg/dL)

•Analisi delle regioni codificanti del gene UGT1A1: Assenza di mutazioni causative•Analisi delle regioni non codificanti del gene UGT1A1

Pazie

nte

(TA)7

/7

Ctrl (T

A)6/7

Ctrl

(TA)7

/7

Ctrl

(TA)6

/6

Bilirubina Totale:8.28 mg/dLBilirubina diretta:0.93 mg/dLBilirubina Indiretta:7.35 mg/dL

Albumina: 4.3 g/dLQPE: nella normaAST:24 UI/LALT:17 UI/L

Aptoglobina:<6.56mg/dLLDH: 440 UI/L

Analita v.n.

RBC 4-5.2 *106/uL 3.67

Hb 13-16 g/dL 11.3

Hct 36-49 % 34.4

MCV 80-98 fL 93.7

MCH 27-31 pg 30.8

MCHC 32-36 g/dL 32.9

RDW 11-14% 14.1

PLT 150-450*103/uL 300

Ret % 0.5-2 % 6.5

Ret abs *103/uL 238.5

WBC 4.5-8.5 *103/uL 4.37

Ecografia addominaleFegato: ecostruttura nella normaDimensioni ai limiti sup.Milza: diametro bipolare 15 cm

Test di Coombs diretto e indiretto: NEGATIVOPink Test: 55% (v.n. <30%)AGLT50: dimezzamento dopo 1’ (v.n. dimezzamento dopo 3’)Osservazione dello striscio periferico: presenza di sferocitiEktacitometria:

Erythrocyte Membrane ProteinsErythrocyte Membrane Proteins

Horizontal Interaction

Vert

ical In

tera

ctio

n

HS HE HPP HSt

Source of Bilirubin

• Metabolism of heme. 6-10 mg/kg/day. (adults 3-4mg/kg/day)– 75%: from hemoglobin of old RBCs

released from RES. 1gr produces 34mg of bilirubin.

– 25%: from ineffective erythropoyesis, myoglobine, cytochromes, catalase, peroxidase.

Albumin- BilirubinAlbumin- Bilirubin

Endoplasm. Retic.

BMG and BDG

UDPGA

UCB

UGT

Biliary ducts

UCB

UGT geneUGT gene

4 5

UGT 1 locusmRNA

Enzyme UGT

5’ 3’

NH2 COOH

Substrate binding UDPGA binding

1*7 1*6 1*4 1*1 2 3

285 aa 246 aa

Radioactive PCR of UGT1A promoter

5’ 3’

Exon 1 2 3 4 5

(TA)6 TAA

- 53 - 39C

D

98-100 bp

98 bp100 bp

(TA

)7 /(T

A)

7

(TA

)6 /(T

A)

6

(TA

)6 /(T

A)

7

(TA

)6 /(T

A)

6

1 2 3 4

Transcriptional evaluation of the UGT promoter by luciferase

assay

0

10

20

30

40

50

60

70

80

1 2 3

Serie1UGT1A1

activity

TA6 TA7 TA8

UGT1A1 activity during perinatal period

Hereditary spherocytosis and Gilbert

UGT1

Promoter jaundiced Not jaundiced

A(TA)7TAA/A(TA)7TAA 29(97%) 1(3%)

A(TA)7TAA/A(TA)6TAA 83(56%) 65(44%)

A(TA)6TAA/A(TA)6TAA

Total 112(63%) 66(37%)

Gallstones and UGT1AGallstones and UGT1A

Genotype UGT1A Gallstones NO gallst.

(TA)6/(TA)6

(TA)6/(TA)78(24%) 25(76%)

(TA)7/(TA)7 7 (87,5%) 1 (12,5%)

Total 15 (37%) 26 (63%)

Genotype Phenotype

Monogenic diseases

Modifier genes

?

Environment

I1 I2

II1

Femmina, 7 aa

Anamnesi familiare:Negativa per anemiaNegata consanguineità

Anamnesi personale:All’età di 3 aa osservato pallore

Anemia microcitica associata a riduzione dell’indice di saturazione della transferrina

Analita v.n.

RBC 4-5.2 *106/uL 2.9

Hb 13-16 g/dL 6.7

Hct 36-49 % 20

MCV 80-98 fL 65

Ferritina 10-300 ng/mL 25

Sideremia 60-180 ug/dL 14

Transferrina 200-360 mg/dL 290

IS transferrina 15-45% 3.7

•Elettroforesi dell’Hb: assenza di Hb patologiche

•HbF e HbA2: non elevate

•Sangue occulto nelle feci: negativo

•Calprotectina fecale: nella norma

•EGDS: nulla da segnalare

Prima del trattamento

Hb 6.7

MCV 65

Ferritina 25

Sideremia 14

Transferrina 290

IS transferrina 3.7

Dopo il trattamento

6.6

66

24

13

291

3.7

Prima del trattamento

RBC 2.9

Hb 6.7

MCV 65

Ferritina 25

Sideremia 14

Transferrina 290

IS transferrina 3.7

Dopo il trattamento

4.7

9.5

63

46

16

250

6.4

•Analisi delle regioni codificanti del gene TMPRRS6

*c.749T>C p.I212T * c.926G>A p.R271Q* *

(Andrews, NEJM, 1999)

IronIron metabolismmetabolism

The total body iron content of an average The total body iron content of an average male adult is about 4 g;male adult is about 4 g;Total iron:Total iron:

– Red cell mass as haemoglobin Red cell mass as haemoglobin – 65%-– 65%-75%75%

– Muscles as myoglobin Muscles as myoglobin – 10%– 10%– Storage as ferritinStorage as ferritin - 10% - 10%

Bone marrow Bone marrow Reticulo-endothelial cells Reticulo-endothelial cells Liver (0.5-1 g)Liver (0.5-1 g)

– Other Haem proteins Other Haem proteins - 5%- 5%Cytochromes, othersCytochromes, others

In Serum In Serum - 0.1%- 0.1%

Iron balance is maintained by the meticulous regulation of iron absorption from the intestine because there is no regulated pathway for iron excretion

1kg body weight= 50 mg Fe

 Newborn (3,300

Kg)Children(35 Kg) Adult (75 Kg)

Total iron 240-250 mg 1,5 – 2 g 3 -4 g

HB132 – 137,5 mg

(55%)1 – 1,4 g (68%)

2,04 – 2,72 g (68%)

Ferritin101 – 105 mg

(42%)400 – 500 mg

(27%)0,81 -1,08 g (27%)

Myoglobin

7 -7,5 mg (3%)

60 – 80 mg (4%) 120 – 160 mg (4%)

Enzyme 9 – 12 mg (0,6%) 18 – 24 mg (0,6%)

Transferrin 15 – 20 mg (0,1%) 3 – 4 mg (0,1%)

Iron content in the body in different ageIron content in the body in different age

Iolascon A et al.,2013

Transport

Use

Recycling

Storage

Ferritin

Erythroid precursors

Iolascon A, De Falco L Semin Hematol. 2009 Oct;46(4):358-70.

Absorption and metabolism of ironAbsorption and metabolism of iron

Sistemic regulation- Hepcidin, a key regulator of iron Sistemic regulation- Hepcidin, a key regulator of iron homeostasishomeostasis

Nemeth et al., Science 2004

The liver peptide hepcidin is the main regulator of systemic iron homeostasis, since it influences the macrophages and in duodenal activity of the iron exporter ferroportin though its internalization and degradation.

C. Beaumont

Iron deficiency and iron deficiency anemiaIron deficiency and iron deficiency anemia

Iolascon A et al.,2013

Peripheral blood smear

Characters of this storyCharacters of this story

Normal values for age

   Age MCV (fl)

At born 110-128

5-24 months 80-85

2-6 years 75-90

6-12 years 78-95

>12 years 80-100

RBC: Microcytosis hypochromia reduced size and reduced Hb content of red blood cells, as inferred by erythrocyte indexes

MCH: <26 pg (n.v 27-30)MCHC: <30 g/dl (n.v.31-37)

Characters of this storyCharacters of this story

RDW: red cell distribution width

(measure of anysocytosis, e.g. dual populations)

HRC: % hypochromic red cells

CHr: reticulocyte Hb content

Serum iron

Transferrin

Transferrin saturation

Serum ferritin

Soluble transferiin receptor

Hepcidin assay

• Heme synthesis– Porphyrias

• Erythropoietic porphyria

– Sideroblastic anaemias• X-linked• X-linked with ataxia• Autosomal recessive (due to

glutaredoxin 5 or to Gly transporter deficiency)

• Globin synthesis– Thalassaemias– Hemoglobinopathies

• Iron metabolism– Hereditary hypotransferrinaemia– Aceruloplasminaemia– Divalent metal transporter 1 (DMT1) disease– Ferroportin disease– TMPRSS6 deficiency

Microcytic anemias: ClassificationMicrocytic anemias: Classification

Iolascon A et al.,2013

Hereditary haemochromatosis

Iron-loading Anaemias

Anaemia of Inflammation

Iron-refractory iron-deficiency anaemia

Hepcidin-secreting tumors

HepcidinIron Normal homeostasis

Ganz T. J Am Soc Nephol. 2007;18:394-400.Ganz T, Nemeth E. Am J Physiol Gastrointest Liver Physiol. 2006;290:G199-G203.Courtesy of Tomas Ganz, PhD, MD.

Diseases of Hepcidin DysregulationDiseases of Hepcidin Dysregulation

Iron deficiency anemia

 Nutritional deficiency

Deficit of absorption

Thalassemia heterozygotes

ACDACD+iron deficiency

Hb - - = / - - --

MCV - - - - -

GR - - + - --

RDW = = = / + = / + +

Reticulocytes - - = / + = / + = / + / -

IS - / -- - / -- = = / - -

Ferritin = / - = / + = = = / -

FEP = / + = / + = = = / +

sTfR + + + = = / +

CHr - - = / - - --

Oral response YES NO NONot to be expected

Partial

Iv response YES YES NONot to be expected

Partial

Inheritance AcquiredAcquired /

multifactorialAR Multifactorial Multifactorial

Suggested therapy

Oral iron

Etiological therapy / iv injection if

severe anemia

Not required

Etiological therap yif

possible (EPO, iv iron)

Etiological therap + oral iron

Differential diagnosis of the most common Differential diagnosis of the most common forms of microcytosisforms of microcytosis

Iolascon A et al.,2013

Differential diagnosis of the less common Differential diagnosis of the less common forms of microcytosisforms of microcytosis

  IRIDA Erythropoietic protoporphyria

Sideroblastic anemia  X-linked

Sideroblastic anemia  X-linked with  

ataxia

Microcytic anemia sideroblastic-like 

(GLRX5)

Deficiency of DMT1

Hypotransferrinemia

Aceruloplasminemia

Deficiency of Steap3

Hb - /-- - - - --- (età dipendente) -- - - ---

MCV -- -- - - -- --- -- - -

GR -- - - - - - - - --

RDW = = = = = = = = =

Reticulocytes - - - - - - - - ---

SI -- /--- + + + + ++ 100% + ++

Ferritin = / - = = = = + = + +++

FEP ++ +++ = / - = / - = + = = +

Oral response NO NO NO NO NO NO NO YES NO

Iv responseYES, not

long-lasting

NO NO NO NO NO NO YES NO

Inheritance AR AD/AR X- linked X- linked AR AR AR AR/AD AR

Suggested therapy

not possible -carotene Vit B6 Vit B6 Iron chelation EPO Plasma /

apotransferrinIron

chelationEPO, iron chelation

Iolascon A et al.,2013

Treatment Treatment

YES

IRON WITH meals

NO

TO evaluate the response to treatment

continue treatment with oral iron

-LOW COMPLIANCE-malabsorption-BLOOD loss

YES

Iv therapy reassessmentdiagnostic

Adverse effects

Iv therapy ORAL IRON

YES NO

NO

NO YES

Iolascon A et al.,2013

ORAL TREATMENT

ADVERS EFFECTS OF ORAL TREATMENT

• Defective iron transport or utilization DMT1 deficiency, Hypo-transferrinemia

• Defects of iron absorption IRIDA (Iron-Refractory Iron Deficiency Anemia)

• Defects of mitochondrial iron utilization Inherited (and acquired) Sideroblastic Anemias

• Defects of iron recycling usually normocytic-normochromic anemias Aceruloplasmina, ACD (some cases)

Defects of iron MetabolismDefects of iron Metabolism

BMPRs

BMP

TMPRSS6SMAD 1-5-8

complex

SMAD 4

m-HJV

SMAD 1-5-8complex

P

SMAD 1-5-8complex

P

P mRNA HAMP

s-HJV

Adapted from Silvestri L, et al. Blood. 2009;113:5605-5608

The role of TMPRSS6 in the hepcidin regulatory The role of TMPRSS6 in the hepcidin regulatory pathwaypathway

CN TM CUB LCUB LL SERINE PROTEASE

ATG

ATG

STOP

I212T

Y141C S304L

R271Q

Q229fsL166fs

W247fs

S561X

SEA

C510S

S570fs

Iolascon et al.2010

TMPRSS6 gene and proteinTMPRSS6 gene and protein

N: .amino-terminus C1r/C1s, urchin embryonic growth factor and bone morphogenic protein 1 domainC: carboxy-terminus L: low density lipoprotein receptor clas A domain (LDLR)TM: transmembrane domain Serine Protease: serine protease domainSEA: sea urchin sperm protein, enteropeptidase agrin Black oval: cleavage activation siteCUB: complement protein subcomponents

MCV 47-60 fL

Serum Iron -

Tf saturation -

sTfR ++

BM sideroblasts -

FEP +

Liver Iron n

Neonatal appearance +/-

Effect oral /iv Fe +/-

Serum or urinary Hepcidin

+

Inheritance AR

Therapy -

Laboratory findings of Laboratory findings of IRIDA-TMPRSS6 mutationsIRIDA-TMPRSS6 mutations

Iolascon A et al.,2013

Aknowledgements:Aknowledgements:

my coworkers at CEINGE , Naplesmy coworkers at CEINGE , Naples

Prevalence of Iron Deficiency and Iron Prevalence of Iron Deficiency and Iron Deficiency Anemia Deficiency Anemia

Iron nutrition and iron status changes in Italian ininfants in the last decade (ISS), 1995

4% 2%

59%36%

20%10%

28%16%

USA

Côte-d'Ivoire

Asie du Sud-Est

Philippine Islands

UK

7% 2% 8% 2%

France

?20.7%5%

1983

Breastfeeding and IronBreastfeeding and Iron

•The initiation of solid food should not be delayed 4-6 months after birth (Boyce at al, 2011)

•Weaning food should be initiated 4 months after birth for infants without risk for atopic dermatitis (Schoetzau et al, 2002)

•Children need at least 1 month to adapt to solid food (Kang et al, 2006)

•When nutrition is provided only by breastfeeding for more than 6 months iron intake is insufficient (Hyung etal,2013)

Birth

4 m

onth

s

6 m

onth

s

Wea

ning

food

(low ri

sk)

Wea

ning

food

(lhigh

risk

)

ID if only breastfeeding

Red Meat: -beef, horse , heep, duck…

White Meat: - rabbit, chicken, turkey, pig…

Meat and IronMeat and Iron

Iron content

Horse 3.2 mg/100g

Turkey 2.5 mg/100g

Beef 2.1 mg/100g

Pig 1.5 mg/100g

Chicken 1.5 mg/100g

meat products red meat

poultry meat sausages

Child

Adolescent

Adult

Indagine INRAN-SCAI 2005-2006