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

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%

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

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

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

Phenotypical variability associated with mutation in Phenotypical variability associated with mutation in ciliopathy genesciliopathy genes

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

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

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

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

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

«« 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

«« 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

• 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

• 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

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

• 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

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

• 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

Delous et al, 2009

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%

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