Sophie SAUNIER Inserm U983 Hôpital Necker - Enfants Malades, Paris Ciliopathies Apport des techniques de NGS dans la compréhension de la néphronophthise et ciliopathies associées Actualit Actualit é é s N s N é é phrologiques phrologiques Jean Jean Hamburger Hamburger – – Hôpital Hôpital Necker Necker 22 avril 2013, Institut Pasteur 22 avril 2013, Institut Pasteur
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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
- 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
• 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
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
• 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
• 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
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)
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
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
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
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
• 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
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
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
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
• 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
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