.
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
Paziente maschio,11 anni
I1
I2
II1
II2
Anamnesi familiare:
I.1: Colecistectomia all’età di 34 aa
I3 I.2: Iperbilirubinemia indiretta
I.3: Anemia di ndd
Anamnesi personale:
Nato da gravidanza normocondotta,
a termine, da parto cesareo
Ittero neonatale (bilirubina 18 mg/dl)
trattato con fenobarbital
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
Bilirubina Totale:8.28 mg/dL
Bilirubina diretta:0.93 mg/dL
Bilirubina Indiretta:7.35 mg/dL
Albumina: 4.3 g/dL
QPE: nella norma
AST:24 UI/L
ALT:17 UI/L
Aptoglobina:<6.56mg/dL
LDH: 440 UI/L
Ecografia addominale
Fegato: ecostruttura nella norma
Dimensioni ai limiti sup.
Milza: diametro bipolare 15 cm
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
WBC
*103/uL
4.5-8.5 *103/uL
238.5
4.37
Test di Coombs diretto e indiretto: NEGATIVO
Pink Test: 55% (v.n. <30%)
AGLT50: dimezzamento dopo 1’
(v.n. dimezzamento dopo 3’)
Osservazione dello striscio periferico:
presenza di sferociti
Ektacitometria:
Vertical Interaction
Erythrocyte Membrane Proteins
Horizontal Interaction
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- Bilirubin
UDPGA
UCB
UCB
BMG and
BDG
UGT
Biliary ducts
Endoplasm. Retic.
UGT gene
5’
3’
1*7
1*6
1*4
1*1
2
3
4
5
UGT 1 locus
mRNA
Enzyme UGT
NH2
COOH
Substrate binding
285 aa
UDPGA binding
246 aa
Radioactive PCR of UGT1A promoter
2
Exon 1
3
4
5
3
’
5
’
C
- 53
- 39
(TA)6 TAA
D
98-100 bp
100 bp
98 bp
3
4
(TA)6/(TA)6
(TA)7/(TA)7
(TA)6/(TA)7
2
(TA)6/(TA)6
1
Transcriptional evaluation of the
UGT promoter by luciferase
assay
80
70
60
UGT1A1
50
40
activity
Serie1
30
20
10
0
1
2
TA6
TA7
3
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%)
112(63%)
66(37%)
A(TA)6TAA/A(TA)6TAA
Total
Gallstones and UGT1A
Genotype UGT1A
(TA)6/(TA)6
(TA)6/(TA)7
(TA)7/(TA)7
Total
Gallstones
NO gallst.
8(24%)
25(76%)
7 (87,5%)
1 (12,5%)
15 (37%)
26 (63%)
Monogenic diseases
?
Genotype
Environment
Modifier genes
Phenotype
Femmina, 7 aa
I1
I2
Anamnesi familiare:
Negativa per anemia
Negata consanguineità
Anamnesi personale:
All’età di 3 aa osservato pallore
II1
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
Dopo il
trattamento
Hb
6.7
6.6
MCV
65
66
Ferritina
25
24
Sideremia
14
13
Transferrina
290
291
IS transferrina
3.7
3.7
Prima del
trattamento
Dopo il
trattamento
RBC
2.9
4.7
Hb
6.7
9.5
MCV
65
63
Ferritina
25
46
Sideremia
14
16
Transferrina
290
250
IS transferrina
3.7
6.4
•Analisi delle regioni codificanti del gene TMPRRS6
*c.749T>C p.I212T
*
* c.926G>A p.R271Q
*
Iron metabolism
The total body iron content of an
average male adult is about 4 g;
Total iron:
– Red cell mass as haemoglobin – 65%75%
– Muscles as myoglobin – 10%
– Storage as ferritin - 10%
Bone marrow
Reticulo-endothelial cells
Liver (0.5-1 g)
– Other Haem proteins - 5%
Cytochromes, others
– In Serum - 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
(Andrews, NEJM, 1999)
Iron content in the body in different age
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
HB
132 – 137,5 mg
(55%)
1 – 1,4 g (68%)
2,04 – 2,72 g
(68%)
Ferritin
101 – 105 mg
(42%)
400 – 500 mg
(27%)
0,81 -1,08 g (27%)
60 – 80 mg (4%) 120 – 160 mg (4%)
Myoglobin
Enzyme
Transferrin
Iolascon A et al.,2013
7 -7,5 mg (3%)
9 – 12 mg (0,6%) 18 – 24 mg (0,6%)
15 – 20 mg (0,1%)
3 – 4 mg (0,1%)
Absorption and metabolism of iron
Ferritin
Use
Transport
Erythroid precursors
Storage
Recycling
Iolascon A, De Falco L Semin Hematol. 2009 Oct;46(4):358-70.
Sistemic regulation- Hepcidin, a key regulator of iron
homeostasis
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.
Nemeth et al., Science 2004
C. Beaumont
Iron deficiency and iron deficiency anemia
Iolascon A et al.,2013
Characters of this story
RBC: Microcytosis hypochromia
reduced size and reduced Hb content of red blood cells,
as inferred by erythrocyte indexes
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
MCH:
<26 pg (n.v 27-30)
MCHC: <30 g/dl (n.v.31-37)
Peripheral blood smear
Characters 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
Microcytic anemias: Classification
• 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
Iolascon A et al.,20
Diseases of Hepcidin Dysregulation
Iron deficiency
anemia
Iron
Normal homeostasis
Hereditary haemochromatosis
Iron-loading Anaemias
Hepcidin
Anaemia of Inflammation
Iron-refractory iron-deficiency anaemia
Hepcidin-secreting tumors
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.
Differential diagnosis of the most common
forms of microcytosis
Nutritional
deficiency
Deficit of
absorption
Thalassemia
heterozygotes
ACD
ACD+iron
deficiency
Hb
MCV
GR
RDW
Reticulocytes
IS
Ferritin
FEP
sTfR
CHr
=
- / -=/=/+
+
-
=
- / -=/+
=/+
+
-
=/+
=/+
=/+
=
=
=
+
=/-
=/+
=/+
=/=
=
=
-
--+
=/+/=/=/+
=/+
--
Oral response
YES
NO
NO
Not to be
expected
Partial
Iv response
YES
YES
NO
Not to be
expected
Partial
Inheritance
Acquired
Acquired /
multifactorial
AR
Multifactorial
Multifactorial
Suggested
therapy
Oral iron
Etiological
Etiological
therapy / iv
therap yif
Etiological therap +
Not required
injection if
possible (EPO,
oral iron
severe anemia
iv iron)
Iolascon A et al.,2013
Differential diagnosis of the less common
forms of microcytosis
IRIDA
Sideroblastic
Sideroblastic
Microcytic anemia
Erythropoietic
anemia XDeficiency Hypotransferri Acerulopla Deficiency
anemia Xsideroblastic-like
protoporphyria
linked with
of DMT1
nemia
sminemia of Steap3
linked
(GLRX5)
ataxia
Hb
- /--
-
-
-
--- (età
dipendente)
--
-
-
---
MCV
--
--
-
-
--
---
--
-
-
GR
--
-
-
-
-
-
-
-
--
RDW
=
=
=
=
=
=
=
=
=
Reticulocytes
-
-
-
-
-
-
-
-
---
SI
-- /---
+
+
+
+
++
100%
+
++
Ferritin
=/-
=
=
=
=
+
=
+
+++
FEP
++
+++
=/-
=/-
=
+
=
=
+
Oral response
NO
NO
NO
NO
NO
NO
NO
YES
NO
Iv response
YES, not
longlasting
NO
NO
NO
NO
NO
NO
YES
NO
Inheritance
AR
AD/AR
X- linked
X- linked
AR
AR
AR
AR/AD
AR
not
possible
b-carotene
Vit B6
Vit B6
Iron chelation
EPO
Suggested
therapy
Plasma /
Iron
apotransferrin chelation
EPO, iron
chelation
Iolascon A et al.,2013
Treatment
ORAL TREATMENT
ADVERS EFFECTS OF
ORAL TREATMENT
YES
NO
TO EVALUATE
THE RESPONSE
TO TREATMENT
IRON WITH MEALS
ADVERSE EFFECTS
YES
IV THERAPY
YES
NO
NO
ORAL IRON
-LOW COMPLIANCE
-MALABSORPTION
-BLOOD LOSS
YES
Iolascon A et al.,2013
NO
CONTINUE
TREATMENT
WITH ORAL
IRON
IV THERAPY REASSESSMENT
DIAGNOSTIC
Defects of iron Metabolism
• 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)
The role of TMPRSS6 in the hepcidin
regulatory pathway
BMP
s-HJV
m-HJV
BMPRs
P
SMAD 1-5-8
complex
SMAD 1-5-8
complex
TMPRSS6
P
SMAD 1-5-8
complex
SMAD 4
P
mRNA HAMP
Adapted from Silvestri L, et al. Blood. 2009;113:5605-5608
TMPRSS6 gene and protein
N
TM
SEA
CUB
CUB
L
L
SERINE PROTEASE
L
C
ATG
STOP
ATG
R271Q
I212T
Y141C
S304L
S570fs
W247fs
L166fs
Q229fs
N: .amino-terminus
C: carboxy-terminus
TM: transmembrane domain
SEA: sea urchin sperm protein, enteropeptidase agrin
CUB: complement protein subcomponents
C510S
S561X
C1r/C1s, urchin embryonic growth factor and bone morphogenic protein 1 domain
L: low density lipoprotein receptor clas A domain (LDLR)
Serine Protease: serine protease domain
Black oval: cleavage activation site
Iolascon et al.2010
Laboratory findings of
IRIDA-TMPRSS6 mutations
Iolascon A et al.,2013
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
-
Aknowledgements:
my coworkers at CEINGE , Naples
Prevalence of Iron Deficiency and Iron
Deficiency Anemia
UK
France
USA
Asie du Sud-Est
7% 2% 8% 2%
4% 2%
20%10%
20.7%
5%
1983
?
28%16%
59%36%
Côte-d'Ivoire
Iron nutrition and iron status changes in Italian in
infants in the last decade (ISS), 1995
Philippine
Islands
Breastfeeding 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)
ID if only breastfeeding
Meat and Iron
Red Meat: -beef, horse , heep,
duck…
White Meat: - rabbit, chicken,
turkey, pig…
poultry meat
sausages
meat products
red meat
Indagine INRAN-SCAI 2005-2006
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