ppt0000000 [lecture seule] - hÔpital necker · sophie saunier tania attié-bitach u781 valérie...
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![Page 1: Ppt0000000 [Lecture seule] - HÔPITAL NECKER · Sophie Saunier Tania Attié-Bitach U781 Valérie Cormier-Daire U781 Jean-Michel Rozet U781 Renal ciliopathies (NPH and associated syndromes)](https://reader033.vdocuments.site/reader033/viewer/2022051510/5babd0a709d3f279368c7d14/html5/thumbnails/1.jpg)
Sophie SAUNIER
Inserm U983Hôpital Necker - Enfants Malades, Paris
CiliopathiesApport des techniques de NGS dans la
compréhension de la néphronophthise et
ciliopathies associées
ActualitActualit éés Ns NééphrologiquesphrologiquesJean Jean HamburgerHamburger––HôpitalHôpital NeckerNecker
22 avril 2013, Institut Pasteur22 avril 2013, Institut Pasteur
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NephronophthisisNephronophthisis
� Tubulo-interstitial nephropathy
� Polyuria/polydipsia
� ESRD in childhood or adolescence
� Autosomal recessive
� Genetic heterogeneity (NPHP genes)
� Extra-renal anomalies (45%)
NPH Cohort-Necker >800 families
Thickening of TBM Interstitial fibrosis and
cysts 40%34%
45%
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An
tero
gra
de
IFT
-B
Kin
esi
nII
Re
trog
rad
eIFT
-A
Dy
ne
in2
Basal
Body
microtubules
Tranzition zone
Transition fibers
IFT particle
Kinesin II
Dynein 2
Ciliary
componentCiliary
membrane
Ax
on
em
e
Primary cilium
Sensor of
extracellular cues
- Growth factors- Morphogens- Light- Fluid flow….
MechanosensorChemosensor
Signaling
platform
Shh, Wnt/PCP
Signaling
platform
Shh, Wnt/PCP
Cil connecteurCellules visuelles
Tubule rénal
Ptch1/Smo
POLYCYSTINS
PrimaryPrimary ciliumcilium –– sensorysensory ciliumcilium
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CiliaryCiliary defectsdefects leadlead to to ciliopathiesciliopathiesA
nte
rog
rad
eIF
T-B
Kin
esin
II
Re
trog
rad
eIFT
-A
Dyn
ein2
Basal
Body
microtubules
IFT particle
Kinesin II
Dynein 2
Ax
on
em
e
Signaling
platform
Shh, Wnt/PCP
Signaling
platform
Shh, Wnt/PCP
Ciliopathies
Transition zone
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CiliopathiesA
nte
rog
rad
eIF
T-B
Kin
esin
II
Re
trog
rad
eIFT
-A
Dyn
ein2
Basal
Body
microtubules
IFT particle
Kinesin II
Dynein 2
Ax
on
em
e
Signaling
platform
Shh, Wnt/PCP
Signaling
platform
Shh, Wnt/PCP
Transition zone
CiliaryCiliary defectsdefects leadlead to to ciliopathiesciliopathies
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Phenotypical variability associated with mutation in Phenotypical variability associated with mutation in ciliopathy genesciliopathy genes
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liverkidney
Nephronophthisis
moderate
Sensenbrenner/JATD
Syndromes
Short rib-
polydactyly
Syndromes Joubert
syndrome
Meckel syndrome
Phenotypical variability associated with mutation in Phenotypical variability associated with mutation in ciliopathy genesciliopathy genes
severesevere
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Occipital encephalocelePolydactylyCystic kidney dysplasiaLiver fibrosis
Lethal Viable
JoubertMeckel
MKS3
TMEM67
JBS6
NPHP6
CEP290
MKS4NPHP8
RPGRIP1L
MKS5/JBS7
Tania Attié-Bitach, Necker Hospital, Paris
Allelism
Cerebellar vermis hypoplasia
NPH
+/- RP
Allelelism of Meckel and Joubert syndromesBaala et al, Am J Hum Genet, 2007
Identification of novel MKS and JBS genesBaala et al, Am J Hum Genet, 2007Delous et al, Nat Genet, 2007
Meckel syndromeMeckel syndrome: Extreme phenotype of : Extreme phenotype of Joubert Joubert syndrome syndrome
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Occipital encephalocelePolydactylyCystic kidney dysplasiaLiver fibrosis
Lethal Viable
JoubertMeckel
Allelism
Meckel syndromeMeckel syndrome: Extreme phenotype of : Extreme phenotype of Joubert Joubert syndrome syndrome
Cerebellar vermis hypoplasia
NPH
+/- RP
hypomorphic
mutationsloss-of-function
mutations
Nature of the mutation
Modifier genes
« Mutation load » N. Katsanis
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NPHP3
NEK8NPHP2
MKS1
TMEM17
TMEM67
TMEM216
TMEM231
CC2D2A
TCTN1
TCTN2
TCNT3
ARL13B
AHI1
B9D1
B9D2
Sang et al., Cell 2011Garcia-Gonzalo et al., Nat Genet 2011Chi et al., Nat Cell Biol, 2011
RPGRIP1L
NPHPNPHP--JBTSJBTS--MKS modules at the transition zoneMKS modules at the transition zone
An
tero
gra
de
IFT
-B
Kin
esin
II
Re
trog
rad
eIFT
-A
Dyn
ein2
Basal
Body
Ax
on
em
e
Inversin
compartment
Zone de transition = « gatekeeper »
Régule l’entrée et la sortie des protéines ciliaires
NPHP-JBTS-MKS complex
NPHPNPHP--JBTSJBTS--MKS MKS complexcomplex
Primary cilium
Mollet et al., 2005
NPHP4/ac.tubulin
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An
tero
gra
de
IFT
-B
Kin
esin
II
Re
trog
rad
eIFT
-A
Dyn
ein2
Ax
on
em
e
Inversin
compartment
A. Bizet
Alteration of ciliary membrane protein composition in Alteration of ciliary membrane protein composition in NPHPNPHP--KD IMCD cells KD IMCD cells
control NPHP1-KD NPHP4-KD
control NPHP1-KD
Basal
Body
Ax
on
em
e
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«« CiliomeCiliome »» sequencingsequencing in patients in patients withwith ciliopathiesciliopathies
1210 ciliary candidate genes
J. Cohen, CNRS Gif s/YvetteB. Durand, Université LyonP. Jackson, Genentech, CA
Cohorte-Necker
>800 familles
~50% patients
sans mutations
identifiées
Avantages: - exome ciblé (5,3 Mb)- multiplexage des échantillons (128 ech/run)> coût réduit- Identification mutations causales et gènes modificateurs
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«« CiliomeCiliome »» sequencingsequencing in patients in patients withwith ciliopathiesciliopathies
Sophie Saunier Tania Attié-Bitach U781 Valérie Cormier-Daire U781 Jean-Michel Rozet U781
Renal ciliopathies (NPH and associatedsyndromes)
Fetal ciliopathies (Meckel, Hydrolethalus)
SRP syndromes (EVC, JATD) Retinal ciliopathies (LCA)
1210 ciliary candidate genes
J. Cohen, CNRS Gif s/YvetteB. Durand, Université LyonP. Jackson, Genentech, CA
Cohorte-Necker
>800 familles
~50% patients
sans mutations
identifiées
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• 113 patients avec NPH +/- atteintes extra rénales
• 11 patients avec données de liaison
• Pas de délétion du gène NPHP1
• 21 patients issus familles consanguines
ResultsResults of of «« CiliomeCiliome »» in 113 NPH patientsin 113 NPH patients
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• 113 patients avec NPH +/- atteintes extra rénales
• 11 patients avec données de liaison
• Pas de délétion du gène NPHP1
Identification de deux mutations causales dans un même gène dans 67% des patients
• 21 patients issus familles consanguines
Séquençage NGS multiplex
75%
34%
NPH +
Atteintes
extra-rénales
NPH
isolée
ResultsResults of of «« CiliomeCiliome »» in 113 NPH patientsin 113 NPH patients
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Transition zone
BBSomeBBSomeBBSome
IFT-AComplex
IFTIFT--AAComplexComplex
Répartition des mutations
• Diagnostic in 45% of NPH cases• Identification of new genes (22%)• Pas de mutations causales (33%)
18
19
8
625
37
Re
trog
rad
eIFT
-A
Dyn
ein2
Basal
Body
Ax
on
em
e
NPHP-JBTS-MKS complex
NPHPNPHP--JBTSJBTS--MKS MKS complexcomplex
ResultsResults of of «« CiliomeCiliome »» in 113 NPH patientsin 113 NPH patients
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• Fréquence des mutations dans les gènes du complexe IFT-A
> 17% patients avec mutations causales> 11% patients avec une seule mutation (effet modificateur?)
• Tous les gènes du complexe sont impliqués: identification des premières mutations dans IFT140 et IFT144
• Corrélation génotype-Phénotype: NPH +/-atteintes du squelette (Syndromes de Jeune/ Sensenbrenner)
Bredrup et al., 2011; Perrault et al., 2012; Davis et al., 2011
Mutations in IFTMutations in IFT--A A genesgenes in NPH patientsin NPH patients
IFT
-B K
ines
inII IF
T-A
Dynein
2
Basal Body
Axo
nem
eIFTA
IFT144IFT140IFT139IFT122IFT121IFT43
DYNC2H1
IFTBIFT172IFT88IFT81IFT80IFT74IFT57IFT54IFT52IFT46IFT27IFT20
KIF3A/B/C
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syndrome de Saldino-Mainzer (MSS)
- Épiphyses en cônes (phalanges)
- Insuffisance rénal chronique(NPH)
- Dystrophie rétinienne
- Anomalies du fémorales
- Petite taille- Ataxie cérébelleuse- Fibrose hépatique
Pt1 Pt 2 Pt3
Total variants called 6158 6437 7208
Non-synonymous + SpliceSites + Frameshifts
1123 510 639
Unknown SNP variants(dbSNP132 1K genome)
150 112 201
Unknown variants(in-house database)
48 52 114
IFT140
3 patients MSS
Total de 6 familles avec SSM + 1
famille avec JATD
Perrault et al., Am J Hum, 2012
Mutations of Mutations of IFT140 IFT140 genegene in in MainzerMainzer--SaldinoSaldino syndromesyndrome
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• Défauts de ciliogénèse et délocalisation
des IFT-B dans les fibroblastes de patients
•Delocalisation des mutants IFT140
dans les cellules RPE1 cells
IFT
46
/
ac-
αα ααtu
bu
lin
Perrault et al., Am J Hum, 2012
Mutations of Mutations of IFT140 IFT140 genegene in in MainzerMainzer--SaldinoSaldino syndromesyndrome
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NPH MSS JATD Sensenbrenner
High High phenotypicalphenotypical variabilityvariability associatedassociated withwithmutations in IFTmutations in IFT--A A genegene
• Corrélation génotype-phénotype
Stop +faux-sens
Bredrup et al., 2011; Perrault et al., 2012;
Faux-sens+ faux-sens
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• 5 familles avec NPH isolée, HTA et protéinurie (0.25-1.5g/24h)
• 7 familles avec SNCR, lésions de HSF (C. Antignac)• Age IRT moyen: 20 ans
TTC21B/IFT139/NPHP12 TTC21B/IFT139/NPHP12 recurrentrecurrent mutation P209L mutation P209L isisassociatedassociated withwith NPH and FSGSNPH and FSGS
Lésions tubulaires type « NPH » Lésions glomérulaires type HSF
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• 5 familles avec NPH isolée, HTA et protéinurie (0.25-1.5g/24h)
• 7 familles avec SNCR, lésions de HSF (C. Antignac)• Age IRT moyen: 20 ans
Autre fonctions des IFTs en dehors de leur rôle dans le transport ciliaire essentielles au maintien du rein
A. Bizet, E. Huynh Cong
TTC21B/IFT139/NPHP12 TTC21B/IFT139/NPHP12 recurrentrecurrent mutation P209L mutation P209L isisassociatedassociated withwith NPH and FSGSNPH and FSGS
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Identification of mutations in 20 Identification of mutations in 20 novelnovel candidate candidate genesgenes
Nb
de
pa
tie
nts
mu
tés
• Mutations pathogènes (hétérozygotes composites ou homozygotes) chez 25 patients (4 NPH isolées versus 19 NPH+ atteintes extrarénales)
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Patient NPH2218
Patient NPH2161
a b
c
d
e
• late-onset retinitis pigmentosa• NPH with late-onset ESRD (34 y. old)• cholestasis• brachydactyly
• short long bones >severe dwarfism, narrow thorax, brachydactyly• obesity• liver failure• retinal degeneration, • severe intellectual deficiency (says a few words at the age of 10)• cerebellar ataxia, partial agenesis of the vermis.• NPHP (6 y. old)
Patients Patients mutatedmutated in a new in a new IntraFlagellarTransportIntraFlagellarTransport genegene
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Patient NPH2218
Patient NPH2161
a b
c
d
e
p.C1727R, hetp.D464_I465del, het
5’ Splice sitep.K144Nfs*76
Mutations hypomorphes
Mutations tronquantes
Patients Patients mutatedmutated in a new in a new IntraFlagellarTransportIntraFlagellarTransport genegene
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Patient NPH2218
Patient NPH2161
a b
c
d
e
Patients Patients mutatedmutated in a new in a new IntraFlagellarTransportIntraFlagellarTransport genegene
5’ Splice sitep.K144Nfs*76
Mutations tronquantes
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PlanarPlanar CellCell PolarityPolarity (PCP) (PCP) isis criticalcritical for for renalrenal tubulartubularDevelopmentDevelopment
An
tero
gra
de
IFT
-B
Kin
esin
II
Re
trog
rad
eIFT
-A
Dyn
ein2
Basal
Body
microtubules
Transition zone:
NPHP1/4/8/
NPHP5/6
IFT particle
Kinesin II
Dynein 2
Ax
on
em
e
Inversin compartment
NPHP2/NPHP3/NPHP9
Signaling
platform
Shh, Wnt/PCP
Signaling
platform
Shh, Wnt/PCP
Planar cell Polarity (PCP)
cystogenesis
Loss of orientated cell division
in PCP and PKD mutants
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• Convergence extension defect
2.5 dpf
• Anomalies du pronéphros
N4-morphantcontrol
Burcklé C., et al., HMG 2011
Cla
ud
inG
FP
fis
hli
ne
Coll. S. Schneider-Maunoury (Paris)
Nphp8 MorphantsNphp4 Morphants
• Anomalies du cerveau
Mahuzier A *, Gaudé H*, et al. J Cell Biol 2012
N4-morphantcontrol
NPHP4 et NPHP8 required for control of Wnt/PCP and NPHP4 et NPHP8 required for control of Wnt/PCP and Wnt/canonical pathways during zebrafish developmentWnt/canonical pathways during zebrafish development
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Dvl
ββββcat
Wnt-PCP
Dvl
DvlDvl
Wnt-ββββcatenin
Proliferation
Cell migration
Polarity
Adhesion
Convergence-
Extension
NPHP4 et NPHP8 required for control of Wnt/PCP and NPHP4 et NPHP8 required for control of Wnt/PCP and Wnt/canonical pathways during zebrafish developmentWnt/canonical pathways during zebrafish development
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Dvl
ββββcatX
Wnt-PCP
DvlDvl
Wnt-ββββcatenin
from Simmons et al., 2005
Dvl
• NPHP4 induit la dégradation de dishevelled
(Dvl) par le proteasome
Proteasome
inhibitor
MD
CK
cells
control NPHP4-KD
Burcklé C., et al., HMG 2011
Represseur
NPHP4
NPHP4 et NPHP8 required for control of Wnt/PCP and NPHP4 et NPHP8 required for control of Wnt/PCP and Wnt/canonical pathways during zebrafish developmentWnt/canonical pathways during zebrafish development
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Dvl
ββββcat
Represseur
NPHP4
Activateur
X
Wnt-PCP
DvlDvl
Wnt-ββββcatenin
from Simmons et al., 2005
MDCK cells
Control NPHP8-KD
Mahuzier A *, Gaudé H*, et al. J Cell Biol 2012
Nphp8 MorphantsControl
NPHP8
NPHP4
NPHP8
Dvl
Zebrafish floor plate cells
• NPHP8/NPHP4 stabilisent Dvl à la base du
cil pour favoriser Wnt/PCP
NPHP4 et NPHP8 required for control of Wnt/PCP and NPHP4 et NPHP8 required for control of Wnt/PCP and Wnt/canonical pathways during zebrafish developmentWnt/canonical pathways during zebrafish development
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• 9 NPH familles > 7 nouveaux
gènes candidats
Exometotal
25%
66%
NPH +
Atteintes
extra-rénales
NPH
isolée
33%
Abnormal 3D structures
Nephrocystins and epithelial morphogenesisNephrocystins and epithelial morphogenesis
Delous et al, 2009
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genes
candidats
Composés chimiques(Biophenics)
cohort of
NPH
patients
Patient fibroblasts
3D cultures
Zebrafish TALEN models
Mécanismes pathophysiologiques
Zebrafish mutants
NovelNovel NPH NPH genegene identification identification strategystrategy
Outils diagnostiques
Thérapeutique
Ciliome v3
1222 gènes, 5.3 Mb
Ciliome v3
1222 gènes, 5.3 Mb
exome total v5
> 20 000 gènes, 51
Mb
exome total v5
> 20 000 gènes, 51
Mb
34%41%
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Titre
E. Filhol
P. Krug
C. Jeanpierre
A. Bizet
V. Grampa
F. Legendre
R. Montjean
H. Gaudé
C. Burcklé
M. Delous
AcknowledgementsAcknowledgements
K. ToryC. HumbertA. BenmerahM. FaillerM. Macia
M.C. Gubler
C. Antignac
Imagine/Necker:
T. Attié (U871)JM Rozet (U871)V. Cormier-Daire (U871)F. Terzi (U845)N. Garcelon (IUH)
Necker Hospital:
Services de NéphrologieService de GénétiqueReference center MARHEA
France
S. Schneider-Maunoury (UPMC, Paris)P. Bastin (Pasteur Institute, Paris)J. Cohen (Gif s/Yvette)B. Durand (Université de Lyon)Club « Cilia, Flagella and centrosomes
GDR3581 - CILIA CNRS
SOFFOET –Société française de feotopathologie
International:F. Hildebrandt (Boston, USA)P. Jackson (San Francisco, USA)H. Arts (Nijmegen, Netherlands)P. Beales (London, UK)
R. Salomon
L. Heidet
U983-Inserm
Plateforme Génomique
Christine Bole, M. ParisotPlateforme Bioinformatique
P. Nitschké, C. Masson
B. Knebelman
S. Burtey
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