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La nuova celiachia ?
Diagnosi Serologica, Istologica
o… molecolare!
Discutiamone con luigi greco
L’Anno del Signore 2013
European Laboratory for Food Induced Diseases
Univ. Federico II
1
Child / Adolescent with Symptoms suggestive of CD
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Anti-TG2 IgA & total IgA*
Anti-TG2
positive
Anti-TG2
negative
Not CD
Transfer to Paediatric GI
Paed. Gi discusses with family the 2 diagnostic pathways
and consequences considering patient’s history &
anti-TG2 titers
Anti-TG2 >10 x normal
Anti-TG2 <10 x normal
EMA & HLA DQ8/DQ2
EMA pos
HLA pos
EMA pos
HLA neg
EMA neg
HLA neg
EMA neg
HLA pos
OEGD & biopsies
Marsh 0 -1
Unclear case
CD+
GFD
& F/u
Not
available
Consider further diagnostic
testing if:
IgA deficiency
Age:
< 2 years
History: - low gluten intake
- drug pretreatment
- severe symptoms
- associated diseases
Consider
false neg.
HLA test,
Consider
biopsies
Consider
false pos.
anti-TG2
Consider:
false positive serology
false negative biopsy
or potential CD
Extended evaluation of
HLA/;serology/biopsies
Marsh 2 or 3
CD+
GFD
& F/u
* Or specific IgG based tests
2
Asymptomatic person at genetic risk for CD
explain implication
of positive
test result(s)
and get consent for testing
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HLA DQ2 / DQ8 (+/- TG2)
HLA positive
DQ2 and/or DQ8
HLA negative
DQ2 and DQ8
Consider retesting in
intervals or if symptomatic
TG2 & total IgA*
Titer > 3 x normal
No CD,
no riks for CD
Titer < 3 x normal
TG2 Negative
Not CD
EMA
EMA positive
OEGD & Biopsies
EMA negative
from Bulbus & 4 x pars descendens,
proper histological work up
Marsh 2 or 3
x
CD+
x
GFD & F/u
Consider:
False negative results,
exclude IgA deficiency
and history of low gluten
intake or drugs
Marsh 0 or 1
Unclear case
F/u on normal diet Consider:
false pos serology, false neg
biopsy or potential CD
Consider:
Transient / false positive Anti-TG2
F/u on normal diet with further
serological testing
* Or specific IgG based tests
3
Anticorpi dellaFree
celiachia:
raccomandazioni
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Come primo test si raccomanda la ricerca di IgA anti-TG2
Gli anticorpi anti-endomisio sono di conferma per la diagnosi
I kit rapidi non sono destinati a sostituire le prove di laboratorio o a fornire
una diagnosi definitiva. Devono sempre essere confermati.
I test per la rilevazione di anticorpi IgG o IgA antigliadina non devono
essere utilizzati per la diagnosi di malattia celiaca.
I test che misurano gli anticorpi sierici contro peptidi deamidati della
gliadina non aggiungono alcuna precisione meglio degli Anti-TGASI
4
Correlazione tra sierologia e istologia
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• La severità della lesione istologica correla con i
valori di IgA anti-tTG
• La specificità degli anticorpi IgA anti-tTG per la
diagnosi della celiachia è del 93% (92-95); ed è
più alta nel caso degli EMA (96%; 94-98).
• In queste circostanze la biopsia è ancora
necessaria?
5
Il Network MEDICEL – Mediterranean Celiac
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6
Tutto si basa sulla relazione tra livello di TTGASI e
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Danno Istologico (Marsh stage) - Italia
7
Country
Spearman Rho
Spain
-0.09
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France
0.062
Correlation
between tTG-log
and Marsh stage
Ma questa base
di sicurezza non
c’è dovunque !
p value
0.5
0.6
Naples
0.53
0.0001
Sicily
0.37
0.008
Malta
0.18
0.9
Slovenia
0.13
0.33
Croatia
-0.07
0.6
Bosnia
0.09
0.58
Albania
0.42
0.1
Montenegro
0.25
0.079
Greece
0.007
0.9
Turkey
0.201
0.16
Egypt
0.17
0.3
Tunisia
0.194
0.17
Algeria
-0.141
0.32
Morocco
0.33
0.018
8
Carmela è …..
DR3-7
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DQ2-8
Chrom 5q31-33
HLA DR Extended
Chrom 19p13.1
MIC-A-B / TNF
Chrom 11, 9, 15
CTLA4
E centinaia? di
altri geni ad
effetto minore
…ggeniale !!
9
Quali geni
spiegano la fortissima
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predisposizione genetica ?
Ereditarietà
?????
54%
50 Geni non
HLA
6%
HLA
40%
10
Manca almeno metà della ereditarietà :
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Chi
spiegaTemplates
il resto ??
Nature Genetics, May 2012, 483
Malattia
Ereditarietà
% Varianza
spiegata
N. di
Polimorfismi
SNPs
Celiachia
0,5-0,87
44% (40-47)
2550
Diabete
0,2-0,69
49% (46-53)
2919
Artrite
Reumatoide
0,53-0,68
18% (15-20)
2231
Infarto
0,3-0,63
48% (43-54)
1766
Polimorfismi in geni molto comuni con rischio bassissimo : da
1,001 a 1,1 : cioè quasi indistinguibile dal non malato , ma
speculabile con enormi 10.000 campioni !
11
Siete pronti
???
Fisiologia
???
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Dobbiamo
sceglierne
qualcuno !!
European Laboratory for
Food Induced Diseases
12
Ci sarà un 1% di differenza anche tra di loro !!!!
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13
COSA CI
Free DICONO
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• Non vi sono geni ‘deficitari’ : tutto funziona
come nei normali
• Vi sono geni di vantaggio per le difese da
virus e batteri : I nostri ‘antibiotici’ naturali
• I geni della ‘celiachia’ sono quelli
comunemente usati nella complessa
reazione immunitaria
• Condividiamo molti geni con altre
patologie immunitarie
European Laboratory for Food Induced Research Federico II
14
cREL AND TNFAIP3…REGULATORS
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OF NFkB –
Maiuri & Greco 2993
•Just 6 hours after gluten
exposure the NF-kB
complex is fully activated.
TNFAIP3
cREL
•cREL is one of the subunit
of the complex
•TNFAIP3 acts in a negative
feedback loop to control NFkB-dependent
gene
expression
15
IL-21: A CRUCIAL ROLE IN THE ACTIVATION OF
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THE GLUTEN-INDUCED CD4 CELLS
IL-21
IL-21
IL-21
16
RGS1:
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REGULATES THE HOMING OF I.E.L
RGS1
•Elevated RGS1 levels profoundly reduce T cell migration to lymphoid-homing
chemokines, whereas RGS-1 depletion selectively enhances such hemotaxis in
gut T cells.
•Its capacity to limit egress of inflammatory and/or autoimmune cells could clearly
promote immunopathology.[
17
LPP: AN INTEGRAL COMPONENT OF CELL
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MIGRATION.
•LPP gene has an activity as
cell adhesion and is an integral
component of cell migration.
•Regulation of LPP expression
results in a increase or
decrease cell migration.
•Over-expression
of
LPP
increased Epidermal Growth
Factor-stimulated migration of
vascular SMCs induced by
TGF-β1, confirming the role of
LPP in cell motility.
18
KIAA1109:
TH17 CELLS
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DIFFERENTIATION
The KIAA1109 gene is in the
region of /Tenr/IL2/IL21 in
chromosome 4q27, many
times replicated in association
studies OF CD.
KIAA1109/Tenr/IL2/
IL21
This region is involved in
differentiation of naïve human
CD4+ T cells into Th17 cells,
which produce a variety of
cytokines including IL-17A, IL17F, IL-21 and IL-22.
Genetic alterations in the 4q27
locus could results in nonfunctional IL-21 and hence
lack of IL-17A or vice versa.
19
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0,2
0
0,04
20
Rischio a posteriori per classe HLA
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sommando
gli alleli di rischio “A”
0.35
•DQ2 omozigote: 21%  30%
0.30
•DQ2 in trans: 17%  26%
0.25
•DQ2 eterozigote: 6%  17%
DQ22
0.20
Score
DQ2T
0.15
DQ2
•DQ8: 5%  13%
•DQ negativi: 0.6%  5%
DQ8
0.10
DQ--
0.05
0.00
Percentuali di rischio in base
all’HLA rispetto all’aggiunta degli
alleli di rischio per i tre
SNP associati.
2.00 3.00 3.00 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.50 6.00
N° di alleli di rischio “A”
21
Aumento del rischio a priori per classe HLA sommando
gli alleli di rischio
“A” per
i 3 SNP analizzati
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350.00
300.00
250.00
No Dq2 or DQ8
DQ22
200.00
DQ2T
DQ2
150.00
DQ8
NODQ
100.00
DQ8
50.00
Double DQB1*02
0.00
2
3
3
3.5
3.75
4
4.25
4.5
4.75
5
N° di alleli di rischio “A”
5.5
6
22
Risultati dell’applicazione del modello predittivo
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101 fratelli
Soggetti in esame
Rischio HLA
INTERMEDIO
Classe G1-G2
Classi G3-G4
Classe G5
33
48
20
ALTO
Rischio NON-HLA
Combinazione rischio
HLA + NON-HLA
BASSO
ALTO
BASSO
Score bayes ≥ mediana
Score bayes ≤ mediana
26
22
ALTO
33+26=
59
BASSO
20+22=
42
23
ROC - Performance of the predicting model
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24
STUDI DI ESPRESSIONE GENICA
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La genomica da sola è in grado di
spiegare solo una quota della
componente genetica della celiachia,
la rimanente percentuale è attribuibile
a meccanismi di epigenetica:
•Regolazione
genica
dell’espressione
•Metilazione del DNA
•Azione di micro-RNA
I Step:
Analisi dell’espressione genica dei geni candidati in pazienti celiaci e
controlli sani in diversi tessuti umani:
•Mucosa intestinale
•Linee cellulari di linfociti T
•Monociti isolati da sangue periferico
25
DUODENAL BIOPSIY
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•
•
•
•
SH2B3 expression was higher in CD mucosa than in controls
TAGAP was significantly up-regolated in CD mucosa
TNFSF14 levels were higher in CD patients and in CD-GFD patients
TNFRSF14.TNFAIP3, and RGS1mRNA levels show a modest upregulation in CD patients versus controls also if this difference is not
statistically significant
26
DUODENAL
BIOPSY
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•IL-21 are more elevated in CD than in control
•IL-2, KIAA1109 mRNA do not show significantly variations among the two
groups but they seem to have an up-regulating trend in CD
•cREL expression was lower in CD monocytes compared with controls
27
DISCRIMINANT ANALYSIS
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IN Free
DUODENAL
BIOPSY
The aim of the discriminant analysis is to weigh the discriminating capacity of each
single gene to obtain a single new composite variable.
Wilks' Lambda df1 Exact F
1
2
3
4
5
TNFAIP3
IL21
REL
RGS1
LPP
,404
,300
,261
,235
,222
1
2
3
4
5
59,002
45,521
35,809
30,143
25,272
Sig.
,000
,000
,000
,000
,000
Wilk’s Lambda shows the ability to discriminate between Coeliac and Controls
1-------------------------------0
Wilks’ Lambda
28
CLASSIFICATION
RESULTS
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Predicted Group Membership
Total
STATUS
Count Control
Celiac
%
Control
Celiac
Control
19
2
Celiac
1
20
20
22
95
10
5
90
100
100
92,9% OF ORIGINAL GROUPED CASES CORRECTLY CLASSIFIED.
29
DIFFERENTFree
EXPRESSION
PROFILES
IN
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CASES AND CONTROLS
DISCRIMINANTION BY GENE EXPRESSION IN MUCOSAL TISSUE
6,000
Diagnostic Probability x 5
Discriminant Score
4,000
2,000
0,000
1
4
7
10
13
16 19
22 25
28
31 34
37 40
43
46 49
-2,000
-4,000
-6,000
-8,000
CELIACS
CONTROLS
30
PERIPHERAL BLOOD MONOCYTES
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•
•
•
•
•
KIAA1109 gene was over-expressed in CD, Crohn and CD-GFD vs controls
c-REL was lower in CD monocytes
SH2B3 was lower in CD monocytes
LPP expression was lower in CD monocytes
TNFAI3 mRNA showed a modest diminution in CD patient among control and
CD-GFD
• RGS1 was lower in CD monocytes
31
DISCRIMINANT
ANALYSIS IN MONOCYTES
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we developed a similar linear discriminant analysis for the gene expression in
peripheral blood monocytes. By stepwise statistic entered the expression of four
candidate genes were selected by multivariate analysis of CD, similar to the results
obtained in the duodenal tissue.
Wilks’ Lambda
Candidate
Step
Exact F
Genes
Statistic
Statistic
Sig.
1
c-REL
0.138
68.711
<0.001
2
LPP
0.090
50.848
<0.001
3
TNFAIP3
0.062
45.461
<0.001
4
KIAA1109
0.048
39.597
<0.001
By computing the discriminant score and
the relative membership probability, 95.5%
of patients (91% controls and 100% celiac
patients) were correctly classified.
Predicted Group Membership
Control
Celiac
Total
Real Group
Control
10 (91%)
1 (9%)
11
Membership
Celiac
0 (0%)
9 (100%)
9
10
10
32
20
HOW MUCH GENE
EXPRESSION
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TemplatesIN MONOCYTES
SEPARATE CELIACS FROM CONTROLS ?
Discriminant Score
DISCRIMINANT SCORE IN MONOCYTES
10
8
6
4
2
0
-2
-4
-6
-8
-10
COELIACS
1
2
3
4
5
6
7
8
9
10
11
12
CONTROLS
CELIACS
CONTROLS
33
DISCRIMINANT
ANALYSIS IN PERIPHERAL
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BLOOD MONOCYTES
The D-Score for active celiac patients was negative in all cases, while it was
positive for all the other groups on differentiated clusters.
34
OUR BIOLOGICAL HYPOTHESIS!
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cREL/TNFAIP
3
RGS1
LPP
???
KIAA110
9
IL-21
RGS1
RGS1
IL-21
IL-21
RGS1
35
CONCLUSIONS
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•We obtained a stimulating suggestion of the possible relationships
among the expression of candidate genes in the target mucosa, and in
the peripheral blood monocytes.
• The analysis of the expression of each single genes does not permit to
identified an overview of pathogenic mechanism of CD, whereas with a
discriminant analisys approach we identified different functional
pathways may be
involved in the complex gluten induced
abnormal responses!
•We now suggest that the expression of a small set of candidate genes
identified CD patients without considering clinical data, HLA and anti-tTG
antibodies.
36
Per Free
quanto
ancora ?
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FAREMO UNA BIOPSIA ‘VIRTUALE PER
ESPLORARE LO STATO FUNZIONALE
DELLA MUCOSA INTESTINALE ?
37