.
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
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